ABSTRACT

Introduction: The objective of the study was to determine the distribution of two morpho-genetic traits namely cerumen type and earlobe attachment among Niger Deltans.

Methods: This observational research embraced descriptive cross-sectional survey method and involved the use of data form, direct observation and interviews. Cluster sampling was employed in this study which involved 2304 subjects. Cerumen types (dry and wet) were determined by visual examination. The two fold classification of earlobe attachment types by Powell and Whitney were followed in the study.

Results: The Niger Deltans displayed a higher frequency of attached ear lobes (58.98%) and a low frequency of free ear lobes (40.97%). The gender difference was not significant (P > 0.05). The wet cerumen was seen to be more frequent. The wet cerumen was seen to be more frequent in the females. The gender difference in the distribution of cerumen type was not significant (P > 0.05).

Conclusion: The distribution of earlobe attachment and cerumen type in the Niger Delta region has been documented. The Niger Deltans possess a higher frequency of attached ear lobes. The wet cerumen was more frequent in the females.

Keywords: Distribution, Earlobe, Attachment, Cerumen, Niger Deltans

Earlobe attachment refers to the way the bottom of the ear is connected to the head. “Free” dominant genes cause the ear to be lobed and the attachment to the head is genetically in an upward direction. The

recessive “attachment” gene results in an upward earlobe and a more horizontal attachment to the head (Lai and Walsh, 1966).

Figure 1: Photograph of attached earlobe

Two genetically determined types of cerumen (earwax) exist: the wet type, which is dominant, and the dry type, which is recessive. The wet type of earwax has a higher concentration of lipid and pigment granules (50% lipid) in the substance than the dry type (30% lipid) (Guest et al.,

Figure 2: Photograph of free earlobe

2004). Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations (Yoshiura et al., 2006). Earwax type has been used by anthropologists to track human migratory patterns in some parts of the world (Bass and Jackson, 1977).

A study was done on the pattern of morphogenetic traits combination amongst the population of Ekpoma in Nigeria (Nwaopara et al., 2009). A remarkable research was done on morphogenetic traits combination pattern amongst the population of Ekpoma, Nigeria: focus on tongue rolling, ear lobe attachment, blood groups and genotypes (Nwaopara et al., 2008). A study considered the o c c u r r e n c e s o f t h e f r e q u e n c y d i s t r i b u t i o n o f morphological, genetical and behavioural traits among the people of Indian region (Bhasin et al., in 1992). The study area is the Niger Delta region in Nigeria. The Niger Delta is the southernmost region of Nigeria. Although it occupies ninety percent of the Nigerian coastline, it is specifically between the Mahin River estuary in the west and the Cross River estuary in the east. The region has been producing oil since 1956 (Enemugwem, 2010).

Figure 3: Photograph of dry ear wax  Figure 4: Photograph of wet ear wax

Literature search reveals dearth of data on the distribution of cerumen type and earlobe attachment in the Niger Delta Region in Nigeria. The objective of the study was to determine the distribution of morpho-genetic traits namely cerumen type and earlobe attachment among Niger Deltans. This study provides a comprehensive data for use in anthropology and forensic medicine.

MATERIALS AND METHODS

The study which is an observational research embraced the descriptive cross-sectional survey method and involved the use of data form, direct observation and interviews. Cluster sampling was employed in this study and the research involved 2304 subjects. The research subjects met the following criteria: Parents and grandparents must be from Niger Delta and subjects had no history of craniofacial trauma or disorder.

Cerumen types (dry and wet) were determined by visual examination. Any cerumen which was grey and flaky was regarded as dry. The wet type was the class for honey-brown to dark-brown and moist cerumen. The two fold classification of earlobe attachment types of Powell and Whitney (1937) were followed in the study. People with earlobes that curve up between the lowest point of the earlobe and the point where the ear joins the head were regarded as having free or unattached earlobes. Others with earlobes that blend in with the side of the head were noted as having attached earlobes. Ethical clearance was sought from the Research and Ethics Committee in the Faculty of Basic Medical Science of the Delta State University prior to the commencement of this research. All subjects were verbally informed about the aim of the study and they agreed to participate.

Data collection was done between the first day and the last day of the months of March and July respectively in the year 2013. Statistical analyses were performed using SPSS version 18. Results were arranged according to gender to ascertain the occurrence of sexual dimorphism. The chi-square test was useful in this regard. The morpho-genetic traits were compared between the genders. Values at P<0.05 were termed significant.

RESULTS

The results revealed that 1147 (49.79%) males and 1157 (50.21%) females participated in the present study.

Table 1: The distribution of earlobe attachment among Niger Deltans.

The Niger Deltans possess a higher frequency of attached ear lobes (58.98%) and a low frequency of free ear lobes (40.97%). The gender difference was not significant (P > 0.05).

Table 2: The distribution of cerumen type among Niger Deltans

Table 2 shows that the wet cerumen was seen to be more frequent. The wet cerumen was seen to be more frequent in the females. The gender difference was not significant (P > 0.05).

DISCUSSION

The frequency of dry cerumen was shown to be low (37.5%) in the sampled Niger Deltan population. This study did not agree with the high frequencies of dry type cerumen found in Eskimos of the Nome and Bethel areas of Alaska (Bass and Jackson, 1977). The frequencies of dry cerumen were also shown to be high in Mongoloid populations and low among Europoids. Intermediate frequencies were found among peoples of subequatorial Africa. Findings from some studies have exposed the potential for using cerumen type as a marker in population and anthropological studies (Ibraimov, 1991).

The present study did not concur with the findings of Yoshiura et al. (2006) who documented that dry earwax is seen in up to 95% of East Asians, but in no more than 3% of people of European and African origin. Yoshiura et al. (2006) also stated that populations in Southern Asia, the Pacific Islands, Central Asia, Asia Minor, and Native North Americans and those of Asian ancestry, fall in the middle with dry earwax incidence ranging from 30 to 50 percent. Petrakis et al. (1967) determined the occurrence of sticky and dry cerumen in 483 Indians from various tribes of the United States and saw elevated frequencies of the dry cerumen in Indians of pure ancestry. Among the Niger Deltans, dry cerumen was seen to have almost equal occurrences in males and females. This did not concur with

Singh and Sengupta who in 2004 did research on the Assamese Sikhs in India. They saw that dry cerumen was highly frequent in the males (61.54%), while the wet type predominated in females (55.77%). The present study concurred with that of Singh and Sengupta ( 2004) with reference to the non-significant gender difference (P > 0.05) seen in the cerumen type.

The present analysis of data on ear lobe attachment revealed an incidence of attached ear lobe of 58.98%. The frequency of attached ear lobes seen did not concur with Nwaopara et al., (2008) who reported 31.61% for attached earlobe and 63.39% for free earlobe. The present study did not concur with Kalia and Gupta (1978) who observed higher incidence of free ear lobes (73.84%). The present study did not concur to that where the frequency of free ear lobes was found to be in the range of 56% to 74% in five endogamous groups of Haryana by Yadav et al., (2000). The present analysis of data on ear lobe attachment differed from that by Singh and Sengupta in 2004 who researched on the Indians and saw that the frequency of free earlobe (83.65%) is higher than attached earlobe (16.35%). They observed that in females, frequency of free earlobe show relatively higher incidence (90.38%) than in the males (76.92%). The sex variation as revealed from their chi-square test was significant (0.01 >P) (Singh and Sengupta, 2004). This study did not concur with theirs as the males in this study had frequency of free earlobe which showed relatively higher incidence (21.66%) than that of the females (19.31%).

CONCLUSION

The distribution of earlobe attachment and cerumen type in the Niger Delta region has been documented. The Niger Deltans possess a higher frequency of attached ear lobes. The wet cerumen was more frequent in the females.

REFERENCES

1. Bass E.J. and Jackson J.F. (1977). Cerumen types in Eskimos. American Journal of Physical Anthropology, 47 (2): 209–210.

2. Bhasin M.K., Walter H. and Danker-Hopfe H. (1992). The distribution of genetical, morphological and behavioural traits among the people of lndian Region. Dehli, Kamla-Raj Enterprises.

3. Enemugwem H.  (2010).  The  Niger  Delta  of Nigeria: A World Class Oil Region in Africa, 2000-2006. Africana Journal, 4 (1) 6: 165-166.

4. Guest J.F., Greener M.J., Robinson A.C. and Smith A.F. (2004). Impacted cerumen: composition, production, epidemiology and management. Quarterly Journal of Medicine, 97 (8): 477–488.

5. Ibraimov A.I. Cerumen (1991). phenotypes in certain populations of Eurasia and Africa. American Journal of Physical Anthropology, 84: 209-211.

6. Kalia V.K. and Gupta A.K. (1978). Earlobe types among the Punjabis. Indian Journal of Physical Anthropology and Human Genetics, 4: 69-74.

7. Lai L.Y.C. and Walsh R.J. (1966). Observations on ear lobe types. Acta Genetica, 16: 250-257.

8. Nwaopara A.O., Anibeze C.I.P., Akpuaka F.C. and Agbontaen O.F. (2008). Morphogenetic Traits Combination Pattern amongst the population of Ekpoma, Nigeria: Focus on Tongue Rolling, Ear Lobe Attachment, Blood groups and Genotypes. African Journal of Biotechnology, 7: 3593-3598.

9. Nwaopara A.O., Anibeze C.I.P., Akpuaka F.C. and Uhumuavbi E. (2009). The Pattern of Morphogenetic Traits Combination amongst the population of Ekpoma, Nigeria: Focus on

10. Dimples, Widows Peak, Blood groups and Genotypes. The Internet Journal of Biological Anthropology, 3 (2).

11. Petrakis N.L., Molohon K.T. and Tepper D.J. (1967). Cerumen in American Indians: genetic implications of sticky and dry types. Science, 158: 1192-1193.

12. Powell E.F. and Whitney D.D. (1937). Ear lobe inheritance: an unusual three-generation photographic pedigree chart. Journal of Heredity, 28: 184-186.

13. Singh J. and Sengupta S. (2004). Some Morpho-genetic and Behavioural Traits among the Assamese Sikhs. Anthropologist, 6 (4): 253-255.

14. Yadav J.S., Yadav A.S. and Chadha P.C. (2000). Studies on Morphogenetic and Behavioural Traits in five endogamous groups of Haryana. Journal of Pan African Studies, 2: 329-332
15. Yoshiura K., Kinoshita A., Ishida T., Ninokata A., Ishikawa T., Kaname T. et al. (2006). (39 co-authors). A SNP in the ABCC11 gene is the determinant of human earwax type. Nature Genetics, 38 (3): 324-330

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ABSTRACT

Introduction: The aim of this study was to determine the tooth occlusion pattern among the Niger Deltans in Nigeria.

Materials and Methods: The study adopted an observational research design. This descriptive cross-sectional survey involved the use of data form and direct observation. The research involved 2304 subjects and multistage sampling; a complex form of cluster sampling was employed. Tooth occlusion pattern were classified following the fourfold models. Subjects were classified as having any of the following: edge to edge, mild overbite, severe overbite and negative overbite. Data obtained were subjected to statistical analyses using chi-square test, with the aid of the Statistical Package for the Social Sciences, version 18. P-value < 0.05 was considered significant.

Results: The results of the study revealed the frequency of mild overbite as the highest (male = 26.13%, female = 25.39%) which was followed by edge to edge type of tooth occlusion (male = 16.28%, female = 18.75%). The incidences of both severe and negative overbite were observed more in males than females (P > 0.05).

Conclusion: This research has elucidated human variability in the tooth occlusion pattern of the Niger Deltans.

Keywords: Tooth, Occlusion, Pattern, Niger Deltans

INTRODUCTION

Dental occlusion refers to the contact between the teeth of the upper jaw (maxillary teeth) and the teeth of the lower jaw (mandibular teeth). The term Malocclusion means the teeth are not aligned properly which  is  most  often  hereditary.  There  are  different categories  of  occlusion.  According  to  Eveleth’s classification  of  tooth  occlusion  pattern  (1972)  the categories are: edge to edge which means the upper teeth and the lower teeth align to each other, mild overbite means the upper teeth slightly overlap the lower teeth and severe overbite is more overlap, negative overbite is when the lower jaw overlap the upper jaw. Tooth occlusion pattern was studied and classified in North Eastern Brazilians [1]. Literature search reveals dearth of data on the distribution of tooth occlusion pattern in the Niger Delta Region in Nigeria. This study provides a comprehensive data for use in orthodontics, anthropology and forensic medicine. The aim of this study was to determine the tooth occlusion pattern among the Niger Deltans. The Niger Delta region consists of present-day Bayelsa, Delta, and Rivers States. The region also includes Abia, Akwa-Ibom, , Edo, Imo and Ondo States. There are about 31 million people of more than 40 ethnic groups including the Bini, Efik, Esan, Ibibio, Igbo, Annang, Oron, Ijaw, Itsekiri, Yoruba, Isoko, Urhobo, Ukwuani and Kalabari, among the inhabitants in the Niger Delta, speaking about 250 different dialects [2].

MATERIALS AND METHODS

The study area is Niger Delta and the study adopted an observational research design. This descriptive cross-sectional survey involved the use of data form and direct observation. The research involved 2304 subjects and multistage sampling; a complex form of cluster sampling was employed. Tooth occlusion pattern were classified following the fourfold models [1]. Subjects were classified as having any of the following: edge to edge, mild overbite, severe overbite and negative overbite (figures 1to 4). Edge to edge occlusion pattern was the impression for an occlusion in which the incisal edges of the maxillary incisors meet the incisal edges of the mandibular incisors. Mild overbite was the class when the upper anterior teeth overlap the lower anterior teeth creating a vertical overlap of the anterior teeth which is 2-3 millimeters. Severe overbite was defined when there was excess amount or percentage of vertical overlap of the lower incisors (greater than 3mm or 30% percent or 1/3 rd the clinical crown height of the mandibular incisors) by the upper incisors. Negative overbite was when the lower anterior teeth overlap the upper anterior teeth. The subjects with history or clinical presentation of pathological conditions, trauma or fracture of the jaw were excluded from the study.

Prior to this study, approval was obtained from the Anatomy Department Research and Ethics Committee in the Delta State University, Abraka. Consent was also obtained from each participant. Statistical analyses were performed using SPSS version 18. The tooth occlusion patterns were compared between the genders. Values at P<0.05 were considered significant. The chi-square test was useful in the bid to achieve this

RESULTS

Table 1: The gender of the Niger Deltans.

Table 2: The tooth occlusion pattern among the Niger Deltans.

Table 2 revealed that the frequency of mild overbite type of tooth occlusion was found to be the highest which was followed by edge to edge bite. The negative overbite was rare among the participants. The gender difference in the tooth occlusion pattern among Niger Deltans was not significant (P > 0.05).

DISCUSSION

The present study among the Niger Deltans in Nigeria saw the frequency of mild overbite as the highest (male = 26.13%, female = 25.39%) which was followed by edge to edge type of tooth occlusion (male = 16.28%, female = 18.75%). The incidences of both severe and negative overbite were observed more in males than females. This study concurred with one done among the Assamese Sikhs in India. They saw that the frequency of mild overbite type of tooth occlusion was found to be the highest (male = 44.90%, female = 54.00%) which was closely followed by edge to edge bite (male = 36.73%, female 32.0%). They noted that the incidence of negative overbite was observed more in males (16.33%) than females (10.00%). Their study revealed that severe overbite type was rare among them (male = 2.04%, female = 4.00%). They documented that bisexual variation had no significant differences (0.80 > P > 0.70) among them [3]. The gender difference in the tooth occlusion pattern among the Niger Deltans was not significant (P > 0.05) just like the study done among the Assamese Sikhs [3].

The Mishing tribals had a predominance of edge to edge bite of dental occlusion pattern and the bisexual differences observed were not significant. The percentage figures with regards to overbitting among them are much lower than the corresponding figures observed in this study [4]. The different studies compared and discussed above portrayed differences in the tooth occlusion considered. There may be reasons for this such as racial origin, methodology and age.

CONCLUSION

This research has elucidated human variability in the tooth occlusion pattern of the Niger Deltans.

REFERENCES

1. Eveleth P.B. (1972). An anthropometric study of North Eastern Brazilians. American Journal of Physical Anthropology, 37:

2. Sandeep G., Sonia G. (2012). Pattern of Dental Malocclusion in Orthodontic Patients in Rwanda: A Retrospective Hospital Based Study. Rwanda Medical Journal, 69 (4): 13-18.

3. Vegesna M., Chandrasekhar R., Chandrappa V. (2014). Occlusal Characteristics and Spacing in Primary Dentition: A Gender Comparative Cross-Sectional Study. International Scholarly Research Notices, 2014.

4. Onyeaso C.O., Sote E.O. (2002). A study of malocclusion in the primary dentition in a population of Nigerian Children. Nigerian Journal of Clinical Practice, 5 (1): 52-56.

5. CRS Report for Congress, Nigeria: Current Issues. Updated 30 January 2008.

6. Singh J., Sengupta S. (2004). Some Morpho-genetic and Behavioural Traits among the Assamese Sikhs. Anthropologist, 6 (4): 253-255.
7. Sengupta S. (1993). Physical Anthropology of the Koch Population of North East India. Mittal Publications, Delhi.

The post The Tooth Occlusion Pattern among the Niger Deltans in Nigeria appeared first on Nigerian Biomedical Science Journal.

Bauchi ZM, Alawa JN, Akpulu SP, Musa SA

Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University, Zaria, Kaduna State, Nigeria

Hambolu JO,

Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna State, Nigeria

Umoh JU,

Department of Veterinary Public Health and Prevention, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Kaduna State, Nigeria

All correspondence to: Zainab Bauchi; zainab_bauchi@yahoo.com

ABSTRACT

This study describes the haematological profile of the African Straw-coloured Fruit Bat experimentally infected with the rabies virus (RABV). 40 African Straw Coloured Fruit bats (Eidolon Helvum) were used for this study. The bats were grouped into A, B, C, D, E, F, G and H. Groups B to H were injected intermuscularly with rabies virus, while A served as control. On day 1, 3, 5, 7, 14, 21 and 28 post-innoculation, blood was obtained through cardiac puncture. Cell counts were performed with an automated haematological analyzer. Haematological parameters investigated were packed cell volume (PCV), erythrocyte counts, leukocyte counts, blood hemoglobin (Hb). Results showed significant increases in Hb count, MCH, MCHC, WBC and neutrophil counts when compared to the control. Significant decreases were observed in lymphocyte count, and no significant changes were observed in eosinopihils, basophils and PCV.

KEYWORD: eidolon helvum, rabies virus, hematology, experimental infection

INTRODUCTION
Bats of various species have been identified as reservoir hosts of many viruses that can cross the barrier between species and infect humans. These viruses include the Ebola virus and Marburg viruses, Nipah and Hendra viruses, corona viruses, as well as rabies and other lyssaviruses (Esona et al., 2010). With nearly 1, 150species of bats, these animals represent nearly a quarter of all the species of mammals on earth. The majority of bats
species live in tropical and semitropical regions (Reide, 2004). Bats are the only flying mammals and they have a wide range of feeding and roosting habits, social behaviours, and reproductive strategies. Bats have high ecological and economic importance due to their role in seed dispersal and serve as source of protein when taken as bush meat. Diversity in their biology makes bats not only a fascinating group of animals to study but also a difficult one (Danmaigoro et al., 2014). They are unique in their agility (potential for long distance travel) and often aggregate in
very large colonies, and these activities aid in the spread of diseases (Danmaigoro et al, 2013). The Straw-coloured
Fruit Bat, Eidolon helvum inhabits vast areas of sub- Saharan Africa in colonies of up to 1,000,000 individuals
and yet there is very limited understanding of its ecology and role in disease transmission. The migratory nature,
expansive colonies and preference for urban and suburban roost sites of this species raises concerns as to its potential as reservoir for infectious disease and its spillover into human and domestic animal populations (Torrance, 2009). In recent years, bats have been implicated in numerous emerging infectious diseases and are increasingly
recognized as important reservoir hosts for viruses that can cross species barriers to infect humans and other domestic and wild mammals (Calisher et al., 2006). Several studies have shown bats to be reservoirs of numerous human and animal viruses and although bats are one of the oldest animals, little is known about their immune systems (Virtue et al, 2011). The ability of bats to remain asymptomatic to viral infection may be due to the rapid
control of viral replication very early in the immune response through innate antiviral mechanisms (Zhou et al.,
2011). The ability to control such highly pathogenic viruses such as the rabies virus, raises the question whether
bats might have evolved particularly effective mechanisms of immune control, however, information on the innate
immune response and hematologic parameters of bats infected with the rabies virus (RABV) is particularly
scarce. Studies have reported presence of rabies antibodies in fruit bats in Nigeria, which suggests a possible role of the fruit bat in the maintenance of rabies in Nigeria (Aghomo et al.,1990).
In this study, we examined the hematological profiles of Eidolon Helvum after experimental infection with rabies
virus in order to establish reference values for this species.
METHODOLOGY
Capture and experimental infection 40 African Straw coloured fruit bats were captured from roosts in Samaru, Kaduna State, Nigeria. The animals were identified in the department of Biological sciences,  Ahmadu Bello University, Zaria, Kaduna, Nigeria. The animals were kept in quarantine for 4 – 6 weeks. Oral swabs for rabies virus were negative. The animals were anaesthetized with 0.l ml/10g body weight of ketamine and inoculated 10⁵ median mouse intracerebral lethal dose (MICLD₅₀) (de Almeida et al., 2014) with 0.02ml of the 10% suspension of the rabies virus intermusculary into both left and right masseter muscles (Turmelle et al., 2010). The animals were grouped into A, B, C, D, E, F, G and H with group A serving as control (table 1).

Euthanasia of Animals and Blood Collection

On days 1, 3, 5, 7, 14, 21 and 28 post-innoculation (pi), one group of bats was euthanized using ketamine. The animals were then placed on the dissecting board. Blood samples were collected via cardiac puncture quickly after the animals were anesthetized. For each animal, the samples were dispensed into EDTA coated sample bottles for haematological investigations. The haematological parameters tested were packed cell volume (PCV), haemoglobin concentration (Hb), total red blood cells (RBC) count, mean cell volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and leukocyte count. The analysis was performed using an automatic haematology analyzer (Sysmex KX-21N)

Statistical analysis

Results were expressed as mean ± SEM. One way analysis of variance (ANOVA) was used to compare the mean values between the groups. Statistical analyses were done using Statistical package for social sciences (SPSS, 21 Chicago Illinois). A p value of p<0.05 was considered statistically significant.

Table 1: Experimental animals and days of sacrifice

RESULTS

Changes in red blood cell (RBC) parameters

In this study, we investigated the haematological parameters of African Straw Coloured Fruit Bat (Eidolon Helvum) after experimental infection with rabies virus. The mean values for Hb, PCV, RBC, MCV, MCH and MCHC in the control animals was 12.06 g/dl±0.86, 45.20%±1.77, 7.54±0.11, 59.86±1.85, 15.97±1.03 and 26.65 respectively. Hb concentrations were observed to have significantly increased when compared with the control (p<0.05). Comparison between control and groups 6 and 7 (13.28g/dl±0.25 and 13.96g/dl±0.17) showed the greatest significance. A decrease in PCV levels was observed across the groups, but the decrease was not statistically significant. RBC count showed no statistically significant change across the groups. A significant increase in PCV was observed between control and groups 6 and 7 (18.07%±0.74 and 18.77%±0.24). MCHC also increased significantly in groups 6 and 7 (31.21±0.92 and

30.36±1.80) when compared to the control. MCV increased significantly in group 7 (62.88± 4.25) and decreased in groups 2, 3 and 4 (51.72±11.58, 39.52±11.18, 46.90±0.31 and 46.25±0.21) (table 2).

Changes in white blood cell (WBC) count

WBC counts were significantly increased between the control (7.98±0.30) and groups 1-4 (11.88±1.69, 20.72±0.33, 14.42±1.38 and 11.90±0.31). Lymphocyte counts significantly decreased across all the groups when compared with the control (77.30±1.43). The greatest difference was observed between control and group 1 (19.54±0.65). Neutrophil count increased significantly across all the groups when compared to the control (21.40±1.15). The greatest difference was between control and group 1 (78.74±0.50). There were no significant changes observed in eosinophil and basophil counts (table 3).

Table 2: Red (RBC) parameters. * p<0.05 indicates significance when compared to control,

^a indicates increase between control and group 7, ^b indicates decrease between control and groups 2,3 and 4.

Table 3: White (WBC) parameters

*P<0.05 indicates significance compared to control

DISCUSSION

Bats are reservoirs of several high-impact viruses that cause significant human diseases, including Nipah virus, Marburg virus and rabies virus. They also harbour many other viruses that are thought to have caused disease in humans after spillover into intermediate hosts, including SARS and MERS coronaviruses. As is usual with reservoir hosts, these viruses apparently cause little or no pathology in bats. Despite the importance of bats as reservoir hosts of zoonotic and potentially zoonotic agents, virtually nothing is known about the host/virus relationships (Schountz, 2014).

Experimental infection with RABV did not produce any significant change in RBC counts and in PCV. The values obtained for both RBC count and PCV were within the normal range for the African Straw Coloured fruit bat as reported by Balthazary et al., (2007). The haemoglobin concentrations showed significant increases however the figures obtained were all within the normal range for the species (11.3±2.6 g/dl). The mean values for mean corpuscular volume (MCV) also displayed significant increases although the values were also within the normal range for the African Straw Coloured Fruit Bat (59.6±18.3 mm 3). Mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) also displayed significant increases that were within the normal range (15.5 ± 6.4 pg and 26.3 ± 7.3 g/dl respectively) as reported by Balthazary et al., 2007). These results indicate that the RABV infection did not alter the values of these parameters beyond the normal values.

White blood cell count increased significantly across the groups. These values far exceed the reported normal ranges of WBC counts in as reported by Balthazary et al., 2007 and Torrance, 2009. The white blood cells are the cells of the immune system and involved in protecting the body against infection and foreign invaders (Maton et al., 1997). The increase in the values indicated an immune response was mounted by the immune system of the bats in response to the RABV infection. De Almeida et al., 2014, reported lower WBC values after experimental infection with RABV in hematophagus Desmondus Rotundus bats. However, Gnanadurai et al., (2013) found that experimental infection of dogs with a wt RABV is not invariably lethal, and that survival correlates with the presence of high VNA titers, evidence of WBC infiltration, and elevated levels of protein in the CSF.

There were significant increases in neutrophil count and decreases in lymphocyte count. This disagrees with the work of de Almeida et al., 2014, who reported increases in lymphocytes and decreases with neutrophils in hematophagus Desmondus Rotondus Bats. However, neutrophils are the first line of defence in viral infections and their numbers generally increase within the first hours of viral infection. The cellular innate immune response to microbes consists of two main types of  reactions:  inflammation  and  antiviral  defense.

Inflammation is the process of recruitment of leukocytes and plasma proteins from the blood, their accumulation in tissues, and their activation to destroy the microbes. The major leukocytes that are recruited in inflammation are the phagocytes, neutrophils (which have short life spans in tissues) and monocytes (Abbas et al., 2015).

CONCLUSION

Experimental infection of the African Straw Coloured Fruit Bat (Eidolon helvum) with rabies virus resulted in increased WBC infiltration with increases in neutrophil counts in the early stages of infection. These findings differ from other bat species studied by previous researchers. The RBC counts correspond with previously documented results. Studies on infections in the African Straw Coloured Fruit Bat (Eidolon helvum) are scarce and more work needs to be undertaken to establish the pattern of immune response of this specie.

ACKNOWLEDGMENTS

The authors thank the Ahmadu Bello University, Zaria, Kaduna State, Nigeria for supporting this project. The authors would also like to thank the staff of the Veterinary Teaching Hospital, Ahmadu Bello University, Zaria, Kaduna State, Nigeria for assisting in this project.

REFERENCES

1. Abbas, A.K., Lichtman, A.H and Pillai, S. (2015): Cellular and Molecular Immunology. 8t edition. Elsevier Saunders Philadelphia, PA, USA

2. Aghomo HO, Ako-Nai, AK, Oduye OO, Tomori, O and Rupprecht, CE (1990). Detection of Rabies virus antibody in fruit bat (Eidolon Helvum) from Nigeria. Journal of wildlife diseases. 26(2) pp 258-261

3. Balthazary, S.T., Max, R.A., Mlay, E., Shayo, G., M l a y, P. a n d P h i r i , E . C . ( 2 0 0 7 ) : Some haematological, biochemical and zootechnical parameters of fruit eating bat (Eidolon helvum) in Morogoro Tanzania. Tanzania Veterinary Journal 24 (2): pp. 129-138

4. Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. (2006) Bats: important reservoir hosts of emerging viruses. Clinical Microbiology Reviews 19:531e45.

5. Danmaigoro A., Onu JE, Sonfada ML, Umar AA, O y e l o w o  F O , a n d H e n a S A         ( 2 0 1 3 ) ,“Histomorphometry of the lower respiratory system of Straw Coloured Fruit Bat (Eidolon helvum),” Scientific Journal of Health, Safety and Environment, vol. 1, no. 4, pp. 75–80.

6. Danmaigoro, A,Onu J E, Sonfada M L,Umaru M A, Hena S A and Mahmuda A. (2014). Gross and M o r p h o m e t r i c A n a t o m y o f t h e M a l e Reproductive System of Bats (Eidolon Helvum). Veterinary Medicine International volume 2014, Http :// dx.doi.org/ 10.1155/ 2014/358158

7. de Almeida MF, Trezza-Netto J, Aires CC, de Barros RF , da Rosa AR and Massad E Hematologic       profile      of   hematophagous Desmodus rotundus bats before and after experimental infection with rabies virus. (2014) Revista da Sociedade Brasileira de Medicina Tropical 47(3):371-373, http://dx.doi.org/ 10.1590/0037-8682-0169-2013

8. Gnanadurai, Clement W. Gnanadurai1., Ming Zhou1., Wenqi He1., Christina M. Leyson1, Chien-tsun Huang1, Gregory Salyards1,2, Stephen B. Harvey2, Zhenhai Chen3, Biao He3, Yang Yang1,4, D. C. Hooper5, Berhnard Dietzchold5, Zhen F. Fu1,4* (2013): Presence of Virus Neutralizing Antibodies in Cerebral Spinal Fluid Correlates with Non-Lethal Rabies in Dogs. P L o S N e g l T r o p D i s 7 ( 9 ) : e 2 3 7 5 . doi:10.1371/journal.pntd.0002375

9. Maton, D., Hopkins, J., McLaughlin, Ch. W., Johnson, S., Warner, M. Q., LaHart, D., Wright, J.D., and Kulkarni, D.V. (1997): Human Biology and Health. Englewood Cliffs, New Jersey, USA. Prentice hall. ISBN 0-13-981176-1

10. Mazarakis ND., Azzouz M.and Rohell JB. (2001) Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery. Mol. Genet., 10, 2109-21.

11. Messenger SL., Smits JS. and Rupprecht CE. (2002). Emerging Epidemiology of Bat-associated cryptic cases of rabies in humans in the United States. Infect. Dis., 2002, 35, 738-47.
12. Nuovo GJ, DeFaria DL, Chanona-Vilchi JG and Zhang Y. (2005). Molecular detection of rabies encephalitis and correlation with cytokine expression. Modern Pathology (2005) 18, 62–67. doi:10.1038/ modpathol.3800274

13. Riede K, “Global register of migratory species: from global region,” Final Report of R and D Projekt 808 05 081, Federal Agency of Nature Conservation, 2004.

14. Samuel, C. E. (2001). Antiviral actions of interferons. Microbiol. Rev.14:778–809.

15. Schountz, T. (2014): Immunology of Bats and their Viruses: Challenges and Opportunities. Viruses. 6,4880-4901; doi: 10.3390/v6124880

16. Torrance, A. (2009): Bva Overseas Travel Grant Report 2009. British Veterinary Association
17. Turmelle AS, Jackson FR, Green D, McCracken GF and Rupprecht CE (2010) Host immunity to repeated rabies virus infection in big brown bats Journal of General Virology, 91, 2360–2366 DOI 10.1099/vir.0.020073-0

18. Virtue ER, Marsh GA, Baker ML, Wang L-F (2011) Interferon Production and Signaling Pathways Are Antagonized during Henipavirus Infection of Fruit Bat Cell Lines. PLoS ONE 6(7): e22488. doi:10.1371/journal.pone.0022488
19. Zhou P, Cowled C, Todd S, Crameri G, Virtue ER, et al. (2011) Type III IFNs in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity. J Immunol 186: 3138–3147

The post Haemotological profile of African Straw-Coloured Fruit Bats (Eidolon Helvum) Experimentally Infected with Rabbies Virus appeared first on Nigerian Biomedical Science Journal.

Joseph Emberga TORYILA,

Human Physiology Department, Faculty of Medicine, Ahmadu Bello University, Zaria. Nigeria. Email: joetoryila@gmail.com

Angela DANBORNO,

Human Physiology Department, Faculty of Medicine, Ahmadu Bello University, Zaria. Nigeria. Email: adanborno@ku.edu

Confidence CHRISTOPHER,
Human Physiology Department, Faculty of Medicine, Ahmadu Bello University, Zaria. Nigeria. Email: chistopherch@gmail.com

James TIMBUAK,

Human Anatomy Department, Faculty of Medicine, Ahmadu Bello University, Zaria. Nigeria.Email: jtimbuak@abu.edu.ng

David PAUL

Chemistry Department, Faculty of Sciences, Ahmadu Bello University, Zaria. Nigeria. Email: edpaul@abu.edu.ng

All correspondence to: Joseph Emberga Toryila, Human Physiology Department, Faculty of Medicine,

Ahmadu Bello University, Zaria. Nigeria. Email: joetoryila@gmail.com

ABSTRACT

OBJECTIVE: Garcinia kola seed has been used locally for the treatment of many different disorders including gastrointestinal disorders like diarrhea, but its precise mechanism of action is not well investigated. In this study the effect of aqueous extract of Garcinia kola seed was studied on smooth muscle preparation in vitro.

METHODS: Fresh Garcinia Kola seeds were used in this study. Two different concentrations of the extract were prepared (10mg/ml and 15mg/ml) and their volumes were varied. After about 24 hrs of fasting, each Rabbit was then euthanized. The abdomen was immediately opened using forceps (laparatomy) and the Rabbit’s ileum about 2-3cm in length were excised and washed with tyrode solution in a petri-dish. One end of the segment of the ileum was attached to an isotonic transducer (type F-60), the segment was passively viewed and recorded for further analysis on a 4-channel physiograph (type PMP-4B). The results were expressed as mean ± Standard Deviation. The differences between the means were analyzed using the independent Student t-test. The result was considered significant at P<0.05.

RESULTS: The extract showed a dose dependent response with lower volumes of 0.1, 0.2, 0.4 and 0.6mls of 10mg/ml showing insignificant decrease in strength of contraction with the P-values (0.266, 0.230, 0.309 and 0.272) respectively. Whereas there was progressive increase in decrease of contraction from 0.8ml, 1ml of 10mg/ml, 0.1 to 0.2ml of 15mg/ml with P-values ( 0.036, 0.016, 0.027 and 0.001) respectively. Similar result was seen in decrease rate of contraction. When concurrently administered with Acetylcholine, the extract also showed does dependent decrease in strength of contraction caused by Acetylcholine with P-values of 0.018 for 1ml of 10mg/ml and 0.611 for 0.2ml of 15mg/ml.

CONCLUSION: The inhibitory effect Garcinia kola extract can be said to be mediated through the same mechanism with Acetylcholine. Further investigation should be carried out to establish its mechanism of action on the intestine and other organs.

KEY WORDS: Garcinia kola seed, Rabbit ileum.

INTRODUCTION

Garcinia Kola also called Bitter Kola in English,

Orogbo in Yoruba, Namijin-goro in Hausa and Akuilu in Igbo. This plant is extensively used in herbal medicine and also as food. It is usually found in

tropical rainforest region of West Africa. It is found in places like Benin, Ivory Coast, Ghana, Liberia, Nigeria, Senegal and Sierra Leone, and also in other African nations like Cameroon, Democratic Republic of Congo and Gabon

[1]. In recent times, researchers have been motivated to consider the effects of a number of medicinal plants that are believed to possess therapeutic properties for a number of body tissues, organs and systems. One of such plants that has gained much attention is Garcinia Kola (G. Kola ), commonly known as bitter kola in English

[2]Plants are the bases of traditional medicine in Africa, and have been used for thousands of years. These plants often exhibit a wide range of biological and pharmacological activities; such as anti-inflammatory, anti-bacterial, and anti-fungal properties

[3]. Due to the need for the development of new drugs with better pharmacological activities and maintenance of normal homeostasis,  dependence on plants continue to increase as scientists exploit them to get their bioactive compounds

[4]. There is shifting of attention from synthetic drugs to natural plant products, plants and plant extracts which are known to provide a source of inspiration for novel drug compounds are now been used for enhancing organs and body systems performance in man and animals

[5,6]. In Nigeria, G. kola seeds are used in traditional medicine for the treatment of poisons

[7, 8,9], asthma, diarrhoea, gastroenteritis and menstrual cramps, G. kola seeds have bronchodilating effect on tracheal smooth muscle cells in humans

[8]. Preliminary phytochemical studies had indicated that the seeds of G. kola lack caffeine [10], it contains alkaloids and biflavonoids

[7, 11]. Alkaloid and biflavonoid fractions from these seeds exhibit antagonistic effects on drug-induced spasms on rat duodenum and uterus and on guinea pig ileum

[7], The inhibitory effects of G. kola seeds on tracheal smooth muscle has been demonstrated in humans [8].

Biflavonoids from G. kola have anti-inflammatory properties [7], is a natural antioxidant [12, 13, 14], and is hepatoprotective [14, 15]. Extracts from G. kola seeds have been reported to alter estrous cycle, inhibit ovulation, induce teratogenicity [16], and to be non-toxic to erythrocytes even at high dose ranges [16]. In the search for alternatives to synthetic hypoglycaemic agents, Adaramoye and Adeyemi reported anti- diabetic and hypolipidaemic effects of fractions of Kolaviron (from G. kola seed extract) in streptozotocin (STZ)-diabetic rats [15]. These reports are indicative of the ability of the active components of this species of kola and of flavonoids from other plants [17] to arrest inflammation and/or smooth muscle hypermotility and of other ailments when used in traditional medicine [18]. The two main types of receptors in the GIT are the excitatory cholinergic and inhibitory adrenergic receptors. The excitatory are stimulated by the parasympathetic nervous system while the inhibitory are stimulated by the sympathetic nervous system [19]. Specifically, the excitatory receptors of the GIT are the Muscarinic receptors the M_3, M₂ receptors, and the neurohumoral transmitter or neurotransmitter that binds to these receptors to elicit contractile response is Acetylcholine (Ach). Drugs or agents that binds to these same receptors to produce effects similar to that of Ach are called cholinergic drugs or agonists, cholinomimetics or parasympathomimetics e.g. Methacholine,while drugs or agents that bind to it to inhibit the parasympathomimetic effects are called cholinergic b l o c k e r s , a n t a g o n i s t s , a n t i c h o l i n e r g i c s o r parasympatholyticse.g. Atropine [20].

Rapid growth in research methods and advancement in sophistication of research equipments warrants that new pharmaceutical outfit will need expertise of physiologists, and ethno medical practices, given that the first stage of drug development which is identification of new chemical substance as a potential target for drug use may need massive data of ethnomedical practice.

Garcinia kola has been said to be used traditionally for the treatment of many disorders including gastrointestinal smooth muscles disorder like diarrhea. But the physiological mechanism through which it acts on the gastrointestinal smooth muscle to elicit it effects has not yet been well investigated.

MATERIALS AND METHODS

Fresh Garcinia Kola seeds were bought from farmer in Samaru Market, Zaria. They were taken to the herbarium of the department of Biological Sciences, Faculty of Science, ABU, Zaria for identification. After that, the seeds were dried under the sun to ease detachment of the thin back or cuticle attached from the epicarp of the seeds. The seeds were then dried under the moderate sun temperature for about 2 weeks. And then taken to the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, ABU, Zaria where they were then pounded into powder using mortar and pestle. The powder was dissolved into three liters of distilled water. The solution was shaken vigorously and allowed to stand on the bench shaking at intervals. The solution was then refrigerated for 24 hours and then sieved using laboratory sieve. This was then allowed to stand for about an hour to allow the heavier particles settle down, after which it was filtered using Whattman filter paper and then transferred into an open tray and dried in an ovum at 40⁰C for 3 days and then scraped with a spatula and then crushed into fine powder using laboratory mortar and pestle [21]. The drugs: Acetylcholine (Ach), Adrenaline (Adr), and Atropine (Atr) were obtained commercially from Zayo- Sigma Chemical Ltd Jos, Nigeria.

EXPERIMENTAL PROCEDURE

The experimental procedure was used as described by Reboller [22]. After about 24 hrs of fasting, each Rabbit was then euthanized. The abdomen was immediately opened using forceps (laparatomy) and the Rabbit’s ileum about 2-3cm in length were excised and washed with tyrode solution in a petri-dish. A segment of the ileum was then suspended in an organ bath (25ml) containing tyrode solution, which was immersed in a thermally controlled organ bath of 60ml capacity, at a temperature of about 37ºC, which was continuous airation by the aerator.One end of the segment of the ileum was attached to an isotonic transducer (type F-60), the segment was passively viewed and recorded for further analysis on a 4-channel physiograph (type PMP-4B), the speed of the machine was set at 0.025cm/sec, with a sensitivity of 50mV/cm.

Post equilibration period was followed by the spontaneous contractions and after taking the reading of the normal contractions, the standard drugs were administered alone and together; initially Ach 0.05ml of (1mg/ml), Adr 0.05ml (20mg/ml), Atr 0.05 (20mg/ml) and Ach 0.05 (1mg/ml) extract 0.1ml of 10mg/ml, extract 0.2ml of (10mg/ml), 0.4ml 0f 10mg/ml, 0.6 of 10 mg/ml, 0.8 of 10mg/ml and 1ml of 10mg/ml, again extract 0.1 of 15mg/ml and 0.2 of 15mg/ml (15mg/ml) were added, and then, extract 1ml (10 mg/ml) and Ach 0 . 05ml (1mg/ml),extract 0.2ml of 15 mg/ml and Ach 0.05 of (1mg/ml), their effects were recorded on the physiograph. Different doses of 10 and 15mg/ml of the extract were administered and the effective doses were interacted with Ach. The tissue was washed three times after each administration and then allowed to rest in order to return to a stable baseline before administration of new volume or dose. The effect of all the administered agents was recorded on the Physiograph.

STATISTICAL ANALYSIS

The results were expressed as mean ± Standard Deviation. The differences between the means were analyzed using the independent Student t-test. The result was considered significant at P<0.05.

RESULT: The results obtained for both strength and rate of contraction of the isolated Rabbit ileum when Standard drugs and aqueous extract of G. kola seed were administered are presented in table 1. Ach at 0.05ml of 1mg/ml gave significant increase in contraction (P<0.00). Adrenaline 0.05ml of 20mg/ml also gave significant decrease in contraction (P<0.00). While Atropine 0.05ml of 20µg/ml was able to block Ach thereby giving an insignificant result (P < 0.28).

Table 1 Effect of Standard Drugs on Strength of Contraction

Ach1mg/ml(0.05ml)

There was a significant decreased in strength of contraction of the ileum at low volumes of Garcinia Kola (0.1, 0.2, 0.4, and 0.6mls of 10mg/ml). There was a significant decreased in strength of contraction at higher volumes of 0.8ml and 1ml of 10mg/ml and also at 0.1and 0.2mls of 15mg/ml (P>0.036, P<0.016, P<0.027, P<0.001) respectively.

Table 2 Effect of Aqueous Extract of Garcinia kola Seed on the Strength of Contraction of Rabbit Ileum

From table 2 above,Garcinia kola Extract was not able to cause significant decrease in strength of contraction of the ileum at low volumes of 0.1, 0.2, 0.4, and 0.6mls of 10mg/ml. However, Garcinia kola Extract gave significant decreased in strength of contraction at higher volumes of 0.8ml and 1ml of 10mg/ml and also at 0.1and 0.2mls of 15mg/ml (P<0.036,P< 0.016, P<0.027,P< 0.001) respectively.

Garcinia kola extract at a lower concentration of 10mg/ml (1ml) was not able to block the effect of Ach, so Ach was still able to cause significant increase in strength of contraction (P< 0.018 P<0.015). But at a higher concentration of 15mg/ml (0.2ml) the extract was able to inhibit the effect of Ach (P< 0.611. P<0.109).

Table 3 Effects of Aqueous Extract Garciniakola Seed and Acetylcholine on the Strength of Contraction of Isolated Rabbit Ileum

NS————– not significant, S————— significant

Ach—— Acetylcholine

Table 4 Effect of Standard Drugs on Rate of Contraction

Table 4; There was no significant increase in the rate of contraction with the concentration of Ach at 1mg/ml P<0.060. Adr 20mg/ml (0.05ml) showed slight insignificant decreased in rate of contraction. While Atropine was able to block Ach thereby giving marked insignificant result (P< 0.423).

Table 5 Effect of Aqueous Extract of Garciniakola Seed on the Rate of Contraction of Rabbit Ileum

Table 5; Garcinia kola extract at various volumes of 0.1, 0.2, 0.4, 0.6, 0.8, and 1mls of 10mg/ml and 0.1ml of 15mg/ml showed insignificant decreased in the rate of contraction. However, 0.2ml of 15mg/ml showed significant decreased in the rate of contraction.

Table 6 Effects of Aqueous Extract Garciniakola Seed and Acetylcholine on the

Rate of Contraction of Isolated Rabbit Ileum

NS————– not significant, S————— significant

Ach—— Acetylcholine

The extract at 1ml of 10mg/ml and 0.2ml of 15mg/ml showed insignificant increase in rate of contraction of caused by Ach.(P<0.05)

DISCUSION

Aqueous extract of Garcinia kola decreased the strength of contraction, the marked inhibition observed attests to the parasympatholytic effect of Garcinia kola extract, given that in atropine and Ach interaction inhibition was observed P=0.27 and Ach single interaction caused contraction P 0.05 as seen in table 1, additionally the mechanism of action of extract could be predicted comparatively from this fact. And again in the experimental procedures involving determination of strength of contraction the volume of the extract was varied between 0.1 to 0.6ml of 10mg/ml, however the results were not statistically significant (P < 0.266, P< 0.230, P< 0.309, P< 0.27) respectively which show that the extract actions may be dose dependent, these observed effects may be interpreted looking at the marked decrease in contraction recorded at high doses of 0.8 and 1ml of 10mg/ml and 0.1, and 0.2ml of 15mg/ml respectively. This corresponds with the findings of (18) on their work on “Antispamodic and Spasmolytic effects of Methanolic Extract from Seed of Garcinia kola on Isolated Rat Small Intestine. Similarly in extract-standard drug interaction (0.05 ml of 1mg/ml of Ach), progressive decrease in strength of contraction was observed, depicting the fact that the extract has anticholinergic and also antispasmodic effect on the intestine. This also agrees with the work of (18) who stated that G. kola Extract exhibited rapid dose-dependent spasmolytic effect on spasms induced by Ach. Result on rate of contraction of extract followed similar pattern, for at low dose decrease in rate was recorded P=1 respectively for 0.4 and 0.6ml 0f 10mg /ml, at high dose slight increase in decreased of rate contraction was seen P .426 and P<.001 for 1ml of 10mg/ml and 0.2ml of 15mg/ml respectively. The extract cause decrease in rate of contraction in combine interaction with Ach the standard drug, on the whole, the extract clearly cause inhibition of GIT movement. The mechanism of action of extract could be empirically inferred from interaction of standard drugs for their mechanism of actions are well established in the literature, therefore, presumably the molecular components of interaction or mechanism of action of the extract perhaps could be that the extract employed parasympatholytic mechanism. Garcinia kola inhibitory effect can be said to be due to the presence of biflavonoids/flavonoids which has inhibitory effect on smooth muscle activity [9]. The effect is more pronounced with high dose than low dose, marked decrease in contraction may have therapeutic application on GIT pathologies and perhaps used in drug development and evolution. Again the nature of the combine interaction results could be linked to concurrent addition of extract and standard drug, since the volume and concentration of the extract was varied unlike the drug’s, time lag and activity affinity of each molecules may have initiated contractile response, also the response may depend on the type of competition between the drug-receptors and extract-receptor interactions for the contraction was slowed and P< respectively for concurrent addition of extract and Ach from tables (3,6).

CONCLUSION

Aqueous extract of Garcinia kola seed showed inhibitor property, it caused decrease in strength of contraction of the Rabbit Ileum which is dose dependent, and also from its interaction with Acetylcholine, it could be said that its effect is mediated through parasympatholytic mechanism. Studies should be carried out using Extract and calcium channel blockers.

REFERENCES

1. Ejele AE, Iwu IC, Enenebeaku CK, Ukiwe LN,Okolue BN. Bioassay-Guided Isolation, Purification and Partial Characterization of Antimicrobial Compound from Basic metabolite of Garcinia Kola.JETEAS 2012: 3(4):668-72.
2. Uko OJ, Usman A,Ataja AM. Vet. Arch 2001; 71: 287-97.

3. Uchiyama T1,Chess WR. Muscarinic receptor subtypes of the bladder and gastrointestinal tract. J Smooth Muscle Res 2006; 40(6):237-47.

4. Ajayi IA, Ajibade O, Oderinde RA.Preliminary Phytochemical Analysis of some Plant Seed. Res. J ChemSc2011; 1(3):58-62.

5. Ghamba PE, Agbo EB, Umar AF, Bukbuk DN. The effects of diethyl ether and aqueous Garcinia kola seeds extracts on some bacterial isolates. Acad.Arena. 2011; 3(2):87-94.

6. Iwu MM, Duncan AR, Okunji CO In: J. Janick (ed). Perspectives on new crops and new uses. Alexandria. ASHS Press 1999; 457-462.

7. Dada AA, Ikuerowo M. Afri. J Agric Res 2009; 4 (4):344-47.

8. Braide VB. Antispasmodic extracts from seeds of Garcinia kola. Fitoterapia 1989; 9:123.

9. Orie NN and Ekon EUA. The bronchodilator effect of Garcinia kola. E Afri.Med J 1993; 70: 143-45.

10. Kabangu L, Galeffi C, Aonzo E, Nicoletti M,Messana L. New biflavone from the bark of G. kola. PlantaMedica 1987; 11:275-77.

11. Eka OU. Isolation of pure caffeine from Cola nitida and chemical composition of Garcinia kola. NigJ PhysiolSc 1986; 10:94-8.

12. Iwu MM. Antihepatotoxic constituents of Garcinia kola seeds. Experientia 1985; 41:699-700.

13. Olatunde FE, Hansen M, Rain-Haren P,Dragsted LO. Commonly consumed and naturally occurring substances affect bio makers of oxidative stress and DNA-damage in healthy rats. FChemToxicol 2002; 42:1315-22.

14. Olatunde FE, Akanni OO,Emerole GO. Antioxidant and scavenging activity of flavonoid extract (kolaviron) of Garcinia kola seeds. Pharm Bio 2002; 40:107-16.

15. Farombi EO, Abarikwu SO, Adedara IA,Oyeyemi MO. Curcumin and kolaviron ameliorate di-n-butylphthalate-induced testicular damage in rats. BasicClin Pharm Toxicol 2007; 100(1):43-8.

16. Adaramoye OA,Adeyemi EO. Hypoglycaemic and hypolipidaemic effects of fractions from Kolaviron, a b i f l a v o n o i d c o m p l e x f r o m G a r c i n i a k o l a instreptozotocin-induced diabetes mellitus rats. J Pham and Pharmacol 2006; 58:121-28.

17. Akpantah AO, Oremosu AA, Noronha CC, et al. Effects of Garcinia Kola seed extract on o v u l a t i o n ,oestrous cycle and foetal development in cyclic female Sprague-Dawley rats. Nig JPhysiolSc2005; 20:58-62.
18.  Agil M, Khan IZ, Ahmed MB, Ishikura NA. Novel Glycoside – 5,6, 7 – Trimethoxy Flavone 4′-B-B-D-Glycoside from Buchnerahispida . Disc and Innos1994; 6:343-45.
19. Udia PM1, Braide VB, Owu DU. Antispasmodic and spasmolytic effects of methanolic extract from seeds of Garcinia kola on isolated rat small intestine. Nig J PhysiolSc 2009; 24(2):111-16.
20. Guyton AC, John EH. Textbook of medical physiology. 11th edition, Elsevier Sanders, Philadelphia, pp 771-75, 2006.
21. Ruffolo RR. Review of Importance Concept in Receptor Theory. JAutPharm 1982; 99:570-82.
22. Nanyak ZG, Ibraheem MG, Ali AH, Ali IS. The Effect of Aqueous Extract of Garcinia Kola Seed on the Liver Histology. J Nat Sc Res2013; 3(1):81-7.
23. Reboller E, Arrubo MP, Murillo MD. Effect of Lipopolysaccharide on Rabbit Small Intestine M u s c u l a r C o n t r a c t i l i t y I n v i v o : R o l e o f Prostaglandings. Neuro-gastr. Motility 2002; 14: 6-33.

The post Effect of Aqueous Extract of Garcinia Kola Seed on Isolated Rabbit Ileum appeared first on Nigerian Biomedical Science Journal.

Adejumo B. I. (Medical Laboratory Sciences Department, University of Benin, Benin City, Nigeria.)

Nwobu G. O. (Medical Laboratory Sciences Department, Igbinedion University, Okada, Nigeria.)

Ewenighi C. O. (Medical Laboratory Sciences Department, Ebonyi State University, Abakaliki, Nigeria)

Dimkpa U. (Physiology Department, Nnewi Campus, Nnamdi Azikiwe University, Awka, Nigeria)

Odionyema U. C. (Department of Medical Laboratory Science, College of Health Technology, Akure, Nigeria)

Ezimokhai T. J. P . (Lulli Diagnostics Limited, Benin City, Nigeria.) 

All correspondence to: Adejumo B.I., Medical Laboratory Sciences Department, University of Benin,
Benin City, Nigeria. Email: babatunde.adejumo@uniben.edu, bigadejumo@yahoo.com

ABSTRACT
Aim: This study aimed at determining the prevalence of Insulin Autoantibody (IAA) and Islets cell Autoantibody (ICA) in Edo State, Nigeria. Method: Insulin Autoantibody (IAA) and Islets cell Autoantibody (ICA) levels were estimated using ELISA reagents from Biomerica Inc. Other parameters such as fasting blood sugar, urine glucose, and urine protein were assessed using standard colorimetric and biochemical techniques. A total number of four hundred and fifty five (455) subjects, 211 (46%) males, and 244 (54%) females with ages ranging between 5 – 76 years were assessed. Subjects were free from long standing illness, endocrine disorder and infectious diseases. Most of the subjects were those attending diabetes clinics in secondary and tertiary Institutions in Edo State, relatives of type I diabetics and volunteered controls. A structured questionnaire was used to collect sociodemographic data. Sociodemographic data included age at diagnosis, age at inclusion, gender, family history of diabetes, drinking and smoking habits, and the duration of diabetes. In cases of patient uncertainty the duration was calculated from the documented date of diagnosis. Other information was obtained from the patients’ files, including the mode of presentation, type of treatment, and course of the disease.
RESULTS: The prevalence of IAA in the study population is 100% in Type I diabetics, 10% in Type II, 26.7% in diabetic relatives, 1.6% in non-diabetic control, while 11. 9% represents the prevalence of IAA in the general population. ICA also has 100% prevalence in Type I diabetics, 10% in Type II, 26.7% in diabetic relatives, 2% in non-diabetic controls, and 12.1% in the general population.
CONCLUSION: The seroprevalence of ICA and IAA is high in this study, public enlightenment, change of life style and early routine check is advocated to prevent diabetes development, complications and reduce mortality rate.

Key words: Diabetes mellitus, IAA, ICA, Prevalence, Edo state

INTRODUCTION
Diabetes mellitus is a genetically and clinically heterogeneous group of disorders (1). It is a disease of ß cells of the Islets of Langerhans or disease resulting from defective secretion and utilization of insulin (2). The hyperglycaemia of diabetes mellitus results from abnormalities of insulin secretion (2). The secretion can be delayed, deficient or inappropriate for the blood glucose homeostasis, or its action being affected by circulating antagonists, e.g. antibody or abnormalities of its tissue receptor (3). Diabetes mellitus is broadly classified into two main groups, Insulin Dependent Diabetes Mellitus (IDDM), Type 1 and Non Insulin Dependent Diabetes Mellitus (NIDDM), Type 2. Type 1 is caused by auto destruction of ß cells of Islets of Langerhans

while the Type 2 results from non-production of insulin or insufficient circulating insulin.

Insulin autoantibodies (IAA) are one of several markers for Type 1 (autoimmune) diabetes. Unlike the other markers, their legend is unique to the beta cell (4). IAAs are the first markers to appear during the symptomless period which precedes diabetes and they are present in the vast majority of young children destined to develop diabetes (4). They are present at titres which relates inversely to age at onset of IDDM (5) (6), and are believed to mark for the rapidity of progression to the disease. Thus, IAA are rare in IDDM of adult onset, which is generally of slow progression, but present almost universally in children who develop diabetes below the age of five years (6), where progression is typically rapid. The origins of IAA, and the timing of their appearance in the circulation, are however unknown. Nevertheless, the rare description of congenital IDDM where the infant, but not the mother, is seropositive for islet related autoantibodies including IAA (7) indicate that insulitis can begin during gestation.

Recent studies in humans and in animal models of spontaneous insulin-dependent diabetes have demonstrated the presence of islet cell cytoplasmic antibodies (ICA), islet cell surface antibodies (ICSA) and insulin autoantibodies (IAA) during a prolonged but relatively asymptomatic pre-diabetic period (8)(9)(10)(11)(12)(13)(14). Most studies evaluating immune markers for prediction of Type 1 diabetes mellitus have focused on first degree relatives, although only 10% of newly diagnosed patients have affected first degree relatives. A previous study (15) has reported a significant association of IgG IAA with ICA – positive in first-degree relatives of Type 1 diabetics. Several attempts have been made to determine the prevalence of diabetes by surveys of community. Most surveys have shown that there are about as many unknown diabetes as known cases.
There is paucity of studies in the prevalence of IAA and ICA in Nigerian population. We therefore aimed at determining the prevalence of both IAA and ICA in diabetics compared with their non-diabetic controls. In addition, we determined the prevalence of IAA and ICA in the general population and the relatives of type 1 diabetics. An early detection of circulating IAA and ICA is important in order to identify the
individuals in the general population, the siblings, and families of IDDM patients, who are at a high risk of developing this disease because of their genetic predisposition to diabetes hence the need for this study.

Materials and Methods
Subjects
This study was conducted at Central Hospital, Benin City. The subjects included 55 diabetic patients on appointment visit to diabetic clinic and those on admission; 150 relations of the type 1 diabetic patients; 250 volunteer controls and a general population of 455 participants. Participants’ consents were obtained before participation in the study. Ethical clearance was obtained from the Ethical Committee of Central Hospital, Benin City, Edo State, Nigeria.

Classification of diabetes
Patients were classified as Type 1 or Type 2 according to the clinical criteria recommended in the 1997 World Health Organisation(16). Classifications were based on age at diagnosis, mode of onset (acute versus insidious presentation), duration of disease, current treatment, BMI, waist-to-hip ratio, blood pressure, random or fasting glucose, HbA1c and urine ketones. A classification of Type 1 diabetes was defined by the following criteria: onset in patients aged 30 years or less, presentation of acute classical symptoms, and that required insulin therapy to control hyperglycaemia. A classification of Type 1 diabetes was also designated to patients older than 30 years that required insulin treatment, lacked metabolic control, and were determined to be underweight. A classification of Type 2 diabetes was designated to patients who were older than 30 years at diagnosis and did not need insulin for metabolic control. A Type 2 classification was also designated to patients younger than 30 years who were obese and had diabetes for a long duration without requiring insulin treatment. Patients that did not clearly exhibit the clinical features of either Type were classified as undetermined and excluded from the present analysis.

Sample Collection
Ten millilitres of blood was collected intravenously, five millilitres was dispensed into a plain container, and the other five millilitres glucose and protein were immediately detected qualitatively using Combi – 9 strip.

Data Analysis
Statistics was done using Statistical Package for Social Sciences program (SPSS) version 16.0. Values obtained in this research were represented as mean ± Standard error of mean (SEM) for continuous variables and percentages for categorical variables. Analysis of variance (ANOVA) was used to compare data at 95% confidence interval (p<0.05).
was dispensed into a fluoride oxalate container. The blood samples were spun at 1500rpm for 10minutes and the supernatant serum/plasma were separated into separate tubes. . The serum/plasma samples were stored at -200C for up to 2weeks prior to the analysis of fasting blood glucose and antibodies. IAA and ICA were determined using ELISA reagents from Biomerica Inc., U.S.A. Blood glucose was determined colorimetrically using Randox kits from United Kingdom. Fresh urine samples were voided into clean sterile containers, glucose and protein were immediately detected qualitatively using Combi – 9 strip.

Tables 1 and 2 show the general prevalence of IAA (n = 54) in the general population (n = 455) to be 11.9% while the prevalence of ICA (n = 55) in the general population is (n = 455) is 12.1%. However, in diabetic Type 1 population (n = 5), the prevalence of ICA and IAA is 100%. In Type 2 diabetic population, the prevalence of IAA (n = 50) is 10%, while ICA (n = 50), is 10% (n = 50) respectively. Diabetic relatives population of IAA and ICA (n = 150) have a combined general prevalence of 26.7% (n = 40). However, the general population prevalence of IAA (n = 250) and ICA (n = 250), are 1.6% (n = 4) and 2% (n = 5) respectively.

DISCUSSION
Most studies on the role of autoimmune markers in the pathogenesis of type 1 (insulin – dependent) diabetes mellitus have focused on first degree relative. Although, the risk for type-1 diabetes is increased among relatives, it is likely many of them may have a reduced acute insulin response to glucose (17, 18). The present study indicated 26.7% prevalence of both IAA and ICA in relatives of type 1 diabetic patients. Another study, (19) in London among English school children found prevalence of 2.8% and 6.8% respectively in the population and siblings of the diabetic patients. The work carried out by a researcher (20), at Malmon, Sweden in a study of a heterogenous group of high risk subjects (non-diabetic patients genetically at higher risk for IDDM including discordant monozygotic twins and ICA positive first degree relatives), IAA were detected in 31% of ICA positive individuals.
The present study indicated 100% prevalence of IAA and ICA in the diabetic type 1 patients and 10% prevalence in type 2 diabetics for the two antibodies respectively. In addition, we observed 1.6% prevalence of IAA and 2.0% of ICA in non-diabetic controls. Our finding is not in agreement with some workers (21), who found the prevalence of IAA in 18% of newly diagnosed IDDM patients. But with an improved radiometry antibody assay, they found approximately 40% of fresh onset, untreated IDDM patients. The reason may be due difference in the methodology. Similarly, our study did not agree with a previous study (22) which found 2 out of 68 diabetics and 1 out of 68 controls among Nigerian diabetics have ICA. Some workers (23), (24), (25), (26), (27), (28), have also reported prevalence of 20 – 50% among newly diagnosed IDDM patients in Pasco Country, Florida, USA. However, with an improved radiometry antibody assay, they found approximately 40% of fresh onset, untreated IDDM patients. The reason may be due differences in methodologies. The 100% prevalence observed in IAA and ICA in this study may be due to the fact that the patients have all commence the administration of exogenous insulin.
Our data also indicated 11.9% prevalence of IAA in the general population. This is at variance with the work of some researchers (29), who reported the prevalence of IAA to be 4% among 1031 populace in Sweden. Also the prevalence of ICA in the general population (n = 455) is 12.1%. This is at variance with the work of (30) who found the prevalence of ICA to be 6.8% among Tanzanian Africans. Another previous study (29) also found 1.4% prevalence of ICA in a Swedish population.
It is believed that the variations in prevalence in this work relative to other previous works may be due to differences between the Africans and Caucasians in terms of diet, climate, method of diagnosis and other etiological factors that trigger the onset of the diabetes mellitus. However, to the best of our knowledge, this is the first documented work to evaluate combined IAA and ICA measurements among African populations either with diabetes or without diabetes.

Conclusion
The seroprevalence of ICA and IAA are high in this study and their occurrence are almost at the same rate in all the groups, it shows that categorising diabetes into type 1 or 2 on the basis of ICA may generally not fit the pattern of diabetes as seen in Nigeria and possibly other parts of the tropics. Though there is no plan for follow up on this work, if those positive IAA and ICA individuals are followed up, majority of them may eventually develop the disease, which brings us to the need to advocate for early routine check up and vigorous public enlightenment to avoid development of diabetes mellitus, which may lead to complications and subsequently mortality.

REFERENCES
1. Fajans S.S., Cloutier M.C. and Crowther R.I. Clinical and Etiologic Heterogeneity of idiopathic Diabetes Mellitus. Diabetes; (1978); 27:11- 12.

2. Lynch M.J., Raphael S.S., Mellor L.D., Spare D.D. and Inwood J.H.M. Medical Laboratory Technology and Clinical Pathology 2ndEd. W.B Saunders Co. (Philiadephia), (1969); pp367.

3. Robert H.W. and Daniel P. (Jnr). The Pancreas. In Robert, HW (Ed.).textbook of Endocrinology, 6th Edi,

W.B. Saunders Co.(Philadelphia); (1974):pp 553.

4. Eisenbarth G.S.T. Type 1 diabetes mellitus.A chronic autoimmune disease. N.Engl J. Med. (1986); 313: 1360 – 1368.

5. Vardi P., Ziegler A.G., Matthews J.H., Dib S., Keller R.J., Ricker A.T., Wolsdrof J.H., Herskowitz R.D., Rabizadeh A., and Eisenbarth G.S. The concentration of insulin autoantibodies at onset of Type 1 diabetes; inverse log-linear correlation with age.Diab.Care; (1988); 11:736-739.

6. Karjalainen J., Salmela P., Ilonen J., Surcel H.M. andKmp M.A comparism of childhood and adult Type 1 diabetes mellitus.N.Engl J. Med. (1988); 320: 881-886.

7. Dotta F., Gillio C., Fanilla C., Moretti A., Bosco N., Sulli P., Colarizi C., Tiberti E., Anastasi G. and Di Mario U. Autoimmunity to pancreatic ß cells in congenital diabetes mellitus.Diabetologia. (1996); 39 (suppl 1): A97.

8. Gorsuch A.N., Spencer K.M., and Lister J.The natural history of Type 1 diabetes mellitus: evidence for a long prediabetic period. Lancet. (1981);2: 1363-1365.

9. Betterie C., Zanette F., Tienoo and Trevban A. Five year follow-up of non-diabetes with islet cell antibodies.Lancet II;(1982): 284.

10. Beakkoskov S., Dyrberg T. and Learnmark A. Autoantibodies to a 64-kilodalton islet cell protein precede the onset of spontaneous diabetes in the BB rat. Science (1984); 224:1348.

11. Dyrberg T., Poussier P., Nakhooda F., Marliss E.B. and Lernmark A. Islet cell surface and lymphocyte antibodies often precede the spontaneous diabetes in the BB rat. Diabetologen, (1984); 26: 159.

12. Srikanta S., Ricker A.T., McCulloch D.K., Soeldner J.S., Eisenbarth G.S., and Palmer J.P. Autoimmunity to insulin, beta cell dysfunction, and development of insulin-dependent diabetes mellitus. Diabetes; (1986); 35:139-142.

13. Takei I., Maruyama T., Taniyama M. and Kataoka K. Humoral immunity in the NOD mouse. In insulitis and type 1 diabetes Lesson from the NOD mouse (ed by Tarui Y. Tochino and K. Nonaka). Academic Press. Tokyo. (1986); pp 101.

14. Reddy S., Bibby N.J. and Elliot R.B. Ontogeny of islet cell antibodies, insulin autoantibodies, and insulitis in the non-obese diabetic mouse. Diabetologia, (1988);31:322.

15. Dean B.M., Becker F., McNally J.M., Tarn A.C., Schwartz G., Gale E.A.M. and Bottazzo G.F. Insulin autoantibodies in the pre-diabetic period correlation with islet antibodies and development of diabetes. Diabetologia.(1986);29:339-342.

16. W.H.O. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus: Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 20: 1183–1197, 1997.

17. Srikanta S., Ganda O.P., Jackson R.E., Gleason M.E., Kaldany M.R., Garovoy E.I., Milford C.B., Carpenter B., Soeldner J.S., and Eisenbarth G. S. Type 1 diabetes mellitus in monozygotic twins: chronic progressive beta cell dysfunction. Annals Int. Med. (1983); 99:320-326.

18. McCulloch D.K., Klaff L.J. and Kahn S.E. Non progression of subclinical ß-cell dysfunction among first degree relatives of IDDM patients 5 years follow up of the Seatle family study. Diabetes, (1990); 39:549 – 556.

19. Bingley P.J., Bonifacio E., Shattock M., Gillmor H.A., Sawtell P.A., Dunger D.B., Scott R.D.M., Bottazzo G.F., and Gale E.A.M.Can islet cell antibodyies predict IDDM in the general population? Diabetes Care, (1993); 16:45-50.

20. Osson M. L. Sundkvist G. And Lernmark A. Prolonged incubation in the two colourimmuno fluorescence Test increases the prevalence and titres of islet cell antibodies in Type 1 (insulin-dependent) diabetes mellitus. Diabetologia, (1987); 30(5): 327-332.

21. Palmer J.P., Asplin C.M., Clemons P., Lyen K., Tatpati O., Raghu P.K. and Paquette T.L. Insulin antibodies in insulin dependent diabetics before insulin treatment. Science (Wash DC), (1983); 222:1337-1339.

22. Oli J.M., Bottazzo G.F., and Doniach D. Islet cell antibodies and diabetes in Nigerians.Trop Geogr Med. 1981; 33:161–164.

23. Arslanian S.A., Becker D.J., Rabin B., Atchison R., Eberhardt M., Cavender D., Durman J. and Draish

A.L. Correlates of insulin antibodies in newly diagnosed children with insulin-dependent diabetes before insulin therapy. Diabetes, (1985); 34:926-930.

24. Bergman S., Ludvigsson J., Binder C. and Mandrup – Poulsen T. Insulin antibodies before treatment in ICA – positive children with IDDM. Diabetes Res. Clin Practice Suppl. (1985); 1:45.

25. Wilkin T., Armitage M., Casey C., Pyke D.A., Hoskins P.J., Rodier M., Diaz J.L., and Leslie R.D.G.Value of insulin autoantibodies as serum markers for insulin dependent diabetes mellitus. Lancet. (1985); 1:480-482.

26. Atkinson M.A., Maclaren N.K., Riley W.J., Winter W.E., Fisk D.D. and Spillar R.P. Are insulin autoantibodies markers for insulin-dependent diabetes mellitus? Diabetes.(1986);35:894.

27. Karjalainen J., Kmp M., Mustonen A., Ilonen J. and Akerblom H.K. Relation between antibody and complement fixing islet cell antibody at clinical diagnosis of IDDM. Diabetes, (1986); 35:620.

28. McEvoy R.C., Witt M.E., Ginsberg-Fellner F. and Rubinstein P. Anti-insulin antibodies in children with Type 1 diabetes mellitus: genetic regulation of production and presence at diagnosis before insulin replacement. Diabetes, (1986); 35:634-641.

29. Samuelsson U., Ludivigeon J., and Sundikvist G. Islet cell antibodies (ICA), insulin antibodies (IAA), islet cell surface (ICSA) and C- peptide in 1031 school children in a population with a background incidence of IDDM. Diabetes Res. Clin.Pract. (1994); 26 (3) : 155-162.

30. McLarty D.G., Athaide I., Bottazzo G.F., Swai A.M., and Alberti K.G. Islet cell antibodies are not specifically associated with insulin-dependent diabetes in Tanzanian Africans. Diabetes Res ClinPract.1990; 9:219–224.

The post Seroprevalence of Insulin Autoantibody (IAA) and Islets cell Autoantibody (ICA) In Edo State, Nigeria. appeared first on Nigerian Biomedical Science Journal.

A.A Sadeeq; A.O Ibegbu; J. A Timbuak; M, Tanko; S.P Akpulu; H.R Bello; S.A Musa
Department of Human Anatomy; Faculty of Medicine. Ahmadu Bello University, Zaria. Nigeria

L. H. Adamu: Department of Anatomy; Faculty of Medicine. Bayero University, Kano. Nigeria 

I.S El-Ladan: Department of Anatomy; Faculty of Medicine. Umaru Musa Yar’addua University, Katsina Nigeria

H.O Kwanashie: Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences. Ahmadu Bello University, Zaria. Nigeria.

All correspondence to: Abubakar Sadeeq Adamu; aasadeeq@abu.edu.ng

ABSTRACT
Mercury is a wide spread environmental pollutant that occurs naturally and has been reported to cause some health problems worldwide. Calcium is an essential neurotrace elements that is required for nerve cells to release neurotransmitters. The present work study the effects of mercury toxicity on memory and level of calcium (ca2+) using short-lived method for Neutron Activation Analysis (NAA-1) in the brain tissues of adult Wistar rats. Twenty four (24) adults Wistar of both sexes were used for the studies and randomly divided into four groups of six rats per group. Group 1 was the Control and received normal saline, while Groups 2, 3 and 4 received 12.45mg/kg, 28.9mg/kg and 49.8mg/kg body weight of mercuric Chloride respectively for twenty one (21) days through oral route. Animals were trained for spatial learning and memory using Morris water maze method and latency time to find the flat form was recorded. The animals were anesthetized and humanely sacrificed using chloroform. The brain was fixed in Bouin’s fluid and processed histologically using H and E stain. Moreover, some of the tissues were crushed by using mortar and pestle after oven drying at 100oC for short-live irradiation method which was exposed to characteristic Gamma-ray to reveal the presence of Ca2+ ion. The photomicrograph reveals that, there was distortion of the pyramidal cells, congestion, necrosis and sparse distribution of hippocampal cells. The spatial memory test showed a decreased in latency time among the control group while the treated groups showed an increased in latency time that was statistically significant (P=0.05). NAA-1 shows an increased level of calcium concentration among mercury treated animals (P=0.01) when compared to the control animals in group I. It was concluded from this studies that; mercuric chloride exposure has effects on the cyto-archetecture of the hippocampus and alters the level of neurotrace element (calcium ion) which in turn affects spatial learning and memory in adults Wistar rats.

Keywords: NAA-1, short-live analysis, Mercury, memory, calcium ion.

INTRODUCTION
Mercury, a heavy metal is a highly deleterious environmental pollutant that can lead to many health problems in the world (WHO, 2003). Man in his environment is exposed to much potential hazards by heavy metals via bioaccumulation and biodegradation which are transferred to man via food chain due to anthropogenic activities (Wang et al., 2007). Mercury can exist either as elemental, organic and inorganic mercury (Burger et al., 2011). Sources of Mercuric compounds are mostly from Industrial sources, gas, fumes, battery disposals, broken mercury thermometer and coal combustion (Akagi, 1995; Bjomberg et al., 2011). Natural source of Mercury such as Mercury chloride that is found in higher densities in rocks and volcanic activities can give half off of the mercury present in nature (Park, 2000; Booth, 2005). There are many routes of exposure to mercury which include: Oral exposure, inhalational exposure and dermal exposure (Vupputuri et al., 2005: Berlin, 2006). Mercury and its compounds have been shown to also have effects on the growth, weight, renal system, liver, enzymes, memory  and psychological disturbances to mention but a few (Kosan et al., 2001; Rao, 2001; Valera et al., 2008;). Signs and symptoms of mercury poisoning include; Irritability, excitability, restlessness of the skin and eyes, headache, dizziness, difficulty in breathing and frequent urination (ATDRS, 2011). Mercury has no known nutritional or biomedical importance but has various applications and uses such as preservation, employed by pharmaceutical company, agriculture and in cosmetic production (W.H.O, 2005). Calcium is especially important element for signal transduction in cells. For this reason, it is sometimes called a second messenger (Khachaturiazn et al., 1989). Coupled with the fact that calcium is necessary for neurons to release neurotransmitters in the brain, calcium plays an important role in the areas of the brain responsible for storing and retrieving memory (Khachaturiazn et al., 1989). The present work, study the effects of mercury toxicity on memory and level of ca2+ using short-lived method for Neutron Activation Analysis (NAA-1) in the brain tissues of adult Wistar rats.

MATERIALS AND METHOD
Experimental Animals
Twenty four Wistar rats of average weight of 210g was used for this study and were acclimatized for three weeks in the animal house of the Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria. The animals were fed with Grower’s mesh brand of the Animal feed which was prepared in pellet form to reduce spillages. The animals were fed 3 times daily while clean water was provided in plastic drinking bottle and animals were allowed to feed and drink ad-bilitum. The animals were divided into four (4) groups with six animals per group.
Chemical substances
Mercuric chloride (May and Bakers limited, Dagenham England) with batch number XN202 was obtained from Steve Moore chemicals limited Samaru, Zaria. Kaduna State, Nigeria.
Administration of Mercury Chloride
The LD50 of Mercuric chloride was adopted from the Manufacturer (as 166mg/kg body weight. The Concentration of Mercury chloride used was determined using 30%, 15% and 7.5% of the LD50 per kg body weight according to the manufacturer’s instruction. Animals in Group I served as the Control and were given normal saline, while animals in Group II, III and IV were orally administered with mercuric chloride at 12.45 mg/kg, 24.9 mg/kg and 49.8 mg/kg body weight respectively of the LD50 per kg body weight. The administration of mercuric chloride lasted for 21 days through oral route.

Neurobehavioral test
Spatial learning and memory test using Morris water maze
Morris water maze test was used to develop and test spatial learning and memory in animals according to the methods of Morris (1981) which was further developed by Liu et al (2011). Animals were placed in a circular pool of clear transparent water which was partition into four quadrants. According to this method, a platform was submerged beneath the surface of the maze pool; the animal task is to find the hidden platform. The animal starting point was changed from time to time so as to build a cohesive spatial representation of the pool in order to find the platform and the latency time was recorded both during the training and experimental periods.
Animal Sacrifices
After the administration, the animals were humanely sacrificed and incision was made through the skin and muscle of the skull. The skull was opened through a mid-sagittal incision and the brain was removed and fixed in Bouin’s fluid. The tissues were routinely processed and stained using haematoxylin and eosin. And some tissue were processed for short-lived irradiation for neurotrace elements detection.

Tissues Preparation for Neuro-trace Element Analysis Method
Trace element analysis of the brain tissue was carried out with the aid of Nuclear Research Reactor (NNIR-1) using NAA-1 method at the Center for Energy Research and Training Ahmadu Bello University, Zaria, Kaduna, Nigeria according to IAEA (2004). The brain tissue samples were Oven dried at 100oC using Memmert DINI ovum-280 model-KA. After successful drying, tissues were crushed in Agate mortar and piston at zero contaminant level in order to have the tissues in powdered form which were then put in sealed polyethene bag and weighed using Metter AE 240 Electronic Digital balance to prepare tissue for nuclear irradiation. In NAA-1 short-lived irradiation method used, each sample was irradiated for 5 minutes and exposed to a semi-conductor detector for characteristic gamma-rays detection for 10 minutes followed by another 10 minutes for 3 hours after the first count. This is to allow safe handling of the radioactive nuclei formed (Akaho and Nyako, 2002).The short lived analysis revealed the presence of neurotrace element calcium (Ca2+). The samples were counted for short –lived irradiation using Maestro software attached to an Analog to Digital Converter (ADC) card and Multi-channel Analyzer card (MCA) according to method of Jonah (2005).

Statistical analysis
Data obtained was expressed as Mean ± SEM (Standard error of Mean). One Way Analysis of Variance (ANOVA) was used to compare the Means between and within the groups. P-value less than 0.05 was considered statistically significant. Statistical analysis was performed using EZanalyze v3.0 and a post hoc test of Bonferroni was applied. Chart was produce using Microsoft(R) Excel 2007 for windows. The Trace elements present in the tissue were analyzed using WIN SPAN 2004 software for peak analysis and efficient calibration and calculation of elements detected.

RESULTS
The effect of mercury chloride exposure on Morris water maze test.
The results on mercuric chloride exposure on spatial learning and memory test, showed a decreased in the meantime taken for the animals to complete Morris water maze task among the control group throughout the period of administration as shown in Table 1. While animals in group II and III had an increased in latency time to find an escape route during Morris water maze activity, though the increase is between weeks 3 and weeks 2 respectively which was significant (P=0.05). And animals in group IV showed an increased that was statistically significant (P=0.01) throughout the period of administration as shown in table 1 below.

Table 1: Effect of mercury chloride ingestion on spatial learning and memory using Morris water maze test

the hippocampus of group III animals, with degeneration of the pyramidal cell layer, loss of some of the pyramidal cells and clumping of pyramidal cell nuclei. Group IV animals show disorientation of the pyramidal cell layer and degeneration of some pyramidal cells with the pyramidal cells appearing to be smaller than normal as shown in Plate D.

Short-lived analysis for calcium (Ca2+) level in the brain
The results of Neuro-trace element analyses indicates the presence of Calcium (ca2+) in the brain tissues of the animals which showed a significant increase (P =0.01) in concentration of Calcium in Group III and IV animals when compared to the control. While Groups II animals showed an increased in calcium concentration that is not significant as shown in figure 1

DISCUSSION
The pyramidal cells in the hippocampus manifested some changes like necrosis degeneration and loss of neuronal cell fiber compared to the control group which could be as a result of the exposure of mercuric chloride. This implies that the activity of the hippocampus in memory formation and learning will be impaired and the role of the hippocampus that involved storage and retrieval of information will also be lost. The findings in this study agree with the studies of Wolf et al., (2003), who reported that rats exposed to high concentration of mercury vapor, showed neurodegenerative changes in the hippocampus which was responsible for memory deficit in such animals.
Result from the present study shows that, there was an increased in time taken by the experimental rats to find the hidden platform in Morris water maze test which was significant. Conversely, the pyramidal cell layer of the hippocampus appears to be damaged with dead cells, and vacuolated spaces and distortion in the general morphology of the pyramidal cells. These alterations can consequently result to memory impairments which could be as a result of neuronal degeneration. The destruction of the pyramidal cells implies that activity from the brain region that projects into the pyramidal layer of the hippocampus will also be lost such as memory and learning ability (Wolf, 2009; Quirino, 2012). Mutter (2010) had reported that short term occupational exposure to high levels of mercury induced slight cognitive deficits. A memory deficit among animals exposed to methyl mercury was not significant in latency time or swim length between the different groups of animals according to Olson (2005).
The findings of the present study revealed a high level of calcium in the brain due to mercury intoxication. Increased in calcium level among groups that receive medium and high doses of mercuric chloride implies that, the role of calcium as a second messenger in signal transmission between cells will be loss, which is required for nerve cells to release neurotransmitters in the central nervous system. This finding agrees with Bandtlowc (1993) which explained that excess calcium at the surface of nerve cells causes them to ‘fire’ spontaneously sending messages around the brain for no purpose except confusion and cause a ‘short circuit’ allowing messages sent to one destination to get misrouted to another which causes memory loss.

CONCLUSION
It was concluded from the present study that oral administration of mercuric chloride has effects on spatial learning and memory and can cause histopathological assault in the hippocampus like necrosis, loss of nerve fibers of the pyramidal cells and neuronal clumping. The short-lived analysis for neurotrace elements revealed the presence of calcium in the brain which was high in concentration due to mercury exposure.

ACKNOWLEDGMENT
I appreciate the support of Mal. Shehu Shika of Center for Energy Research and Training. Ahmadu Bello University Zaria, Nigeria for a bench space in his Laboratory and Mal Ado Garba of A. B.U for his financial support.

REFERENCES
1. Akagi, A. (1995). Human exposure to mercury due to gold mining in the Tapajo river basin, amazon, Brazil: Specification of mercury in human hair, blood, and urine. Water, Air and Soil Pollution. 80: 85–94.

2. Akaho.E.H and Nyako B.J. (2002). Characteristic of neutron flux in irradiation site of MNSR reactor using Westcott formalism for the neutron activation analysis. Apply radiation isotopy 57,265-273

3. Agency for Toxic Substance Diseases Registry (2011). Exposure to hazardous substances and reproductive health. American Family Physician 48(8):1441-1448.

4. Bandtlowc. E., M. F. Schmidtt,. D. Hassingemr., E. Schwab & S. B. Kater (1993). Role of Intracellular Calcium In Ni-35-Evoked Collapse Of Neuronal Growth Cones. Science 259: Pp80-83

5. Berlin M. (2005).Mercury. In: Friberg L, Nordberg G.R, Vouk V.B, eds. Handbook on the toxicology of metals. 2nd ed. New York, NY: Elsevier Press.

6. Bjornberg.A., M. Vahter, B., Berglund, B., Niklasson, M., Blennow., and G. Sandborgh-Englund, (2011). “Transport of methylmercury and inorganic mercury to the fetus and breast-fed infant,” Environmental Health Perspectives, vol. 113, no. 10, pp. 1381–13

7. Burger, J.,C. Jeitner, and M. Gochfeld, (2001)“Locational differences in mercury and selenium levels in 19 species of saltwater fish from New Jersey,” Journal of oxicology and Environmental Health, vol. 74, no. 13, pp. 863–874.

8. Booth S and D. Zeller. (2005). “Mercury, food webs, and marine mammals: implications of diet and climate change for human health,” Environmental Health Perspectives, vol. 113, no, pp. 521–526
9. IAEA (2004) WINSPAN 2006 a multipurpose gamma ray spectrum analysis software CIAE, Beijing china.

10. Jonah S.A; Balogun G. I.,Umar., I Maiyaki .M.C. (2005). Neutron spectrum parameters in irradiation channels of Nigerian research reactor -1(NNIR-1) for NAA standardization Journal of radio anal nuclear chemistry 266(1) 83-88.

11. Khachaturiazn. S. 1989. The Role Of Calcium Regulation In Brain Aging: Reexamination Of A Hypothesis. Aging 1: 17-34.

12. Kosan, C, A. K. Topaloglu, and B. Ozkan, (2001)“Chronic mercury intoxication simulating pheochromocytoma: effect of captopril on urinary mercury excretion,” Pediatrics International, vol. 43, no. 4, pp. 429–430.

13. Liu. Li., Jiong D., Charles M., Junying G., Gang., Hu, M., Xiao.K (2011). Pretraining affects Morris water maze performance with different patterns between control and ovariectomized plus d-galactose-injected mice. Behavioural Brain Research 217:1, 244-247

14. Morris W (1981) Test For Spatial Learning And Memory.Science.136,

15. Mutter J, Curth A, Naumann J, Deth R, Walach H (2010). Does inorganic mercury play a role in Alzheimer’s disease? A systematic review and an integrated molecular mechanism Journal Alzheimers Disease. 22(2):357-74.

16. Olson K, Boush G.M (2005). Decreased learning capacity in rats exposed prenatally and postnatally to low doses of mercury. Bulletin of Environmental Contaminant Toxicology 13:73-79.

17. Park, S.H, S. Araki, A. Nakata (2000). “Effects of occupational metallic mercury vapor exposure on suppressor-inducer (CD4+CD45RA+) T lymphocytes and CD57+CD16+ natural killer cells,” International Archives of Occupational and Environmental Health, vol. 73, no. 8, pp. 537–542,

18. Quirino Cordeiro Júnior, M.D.; Marcília de Araújo Medrado Faria, M.D.; Renério Fráguas Júnior, M.D. (2012) Depression, Insomnia, and Memory Loss in a Patient With Chronic Intoxication by Inorganic Mercury. The Journal of Neuropsychiatry and Clinical Neurosciences 191-205

19. Rao M.V and P. S. N. Sharma ( 2001). “Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice,” Reproductive Toxicology, vol. 15, no. 6, pp. 705–712,.

20. Valera B, Dewailly E, Poirier P (2008). Cardiac autonomic activity and blood pressure among Nunavik Inuit adults exposed to environmental mercury: a cross-sectional study. Environmental Health. 28:924.

21. W.H.O (World Health Organisation) (2003). Elemental mercury and inorganic mercury compounds: Human health aspects. Concise International Chemical Assessment Document. CICAD 50. Geneva.2003

22. Wang J.S, Huang PM, Liaw WK, (2007). Kinetics of the desorption of mercury from selected fresh water sediments as influenced by chloride. Water, Air, Soil Pollution 56:533-542.

23. Wolf U, Rapoport M.J, Schweizer TA (2009). “Evaluating the affective component of the cerebellar cognitive affective syndrome”. Journal Neuropsychiatry Clinical .Neuroscience. 21 (3) 245–53.

24. Vupputuri S, Longnecker MP, Daniels JL, Guo X, Sandler DP (2000). Blood mercury level and blood pressure among US women: results from the National Health and Nutrition Examination Environmental research. 97:195–200

The post Role of Mercury Toxicity on Memory and Calcium (Ca2+) Level Determination Using NAA-1 in the Brain Tissues of Adult Wistar Rats appeared first on Nigerian Biomedical Science Journal.

Toryila J.E, Amadi K, Odeh S.A, Egesie U.G.: Human Physiology Department, Faculty of Medicine, ABU Zaria.

Adelaiye A.B. Achie L.N: Human Physiology Department, Faculty of Medical Sciences Unijos.

All correspondents to:  Toryila J.E Human Physiology Department, Faculty of Medicine, ABU Zaria. 

ABSTRACT
BACKGROUND: Contraception is an important health issue in preventive medicine because it protects women globally from the effects of unwanted pregnancy and allows them to integrate into society. This research priority included efforts to discover the short and long term effect of combined oral contraceptive (DUOFEM) on some Physiological parameters and possible mechanism of actions in female wistar rats.
Method: Eighty (80) female wistar rats aged 10-12 weeks weighing 180-250 g were used for the study. They were divided into four groups of 20 rats each comprising 10 treated and 10 control rats. The treated rats received 0.6mg/kg body weight of COC intragastically for 36, 48, 60 and 72 days in five-day cycles (four days treatment with one-day break. A haematology analyzer was used to perform a complete blood count (CBC or FBC) .An enzyme-linked immunosorbent assay (ELISA) was used for the quantitative determination of alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT). Prothrombin time (PT) and Activated partial thromboplastin time (APTT) were performed using Sysmex CA-6000 Coagulation Analyzer. Liver function tests, Erythropoietin, interleukin-6 (IL-6) and interleukin-11 (IL-11) were determined using rat ELISA kit (Karmiya Biomedical company, USA).
Results: There were significant decrease in Hb, PCV, RBC and WBC counts, lymphocytes (L), PT, IL-6 and IL-11 in all treated groups compared to controls (P<0.005). There was significant increase in alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), sodium and potassium in all treated groups compared to controls(P<0.05). There were however no significant changes in activated thromboplastin time (APTT) and erythropoietin (EPO). The lowest values of Hb, PCV, RBC count, Lymphocytes, PT, IL-6 and Fibrinogen and the highest values of ALT,ALP and AST were observed in long
term used (72 days).Long term used of combined oral contraceptives may leads to more complications than short time use. Combined oral contraceptives users should be monitored for some Physiological parameters.

KEY WORDS: Combined Oral Contraceptives, Physiological Parameters, Wistar rats.

INTRODUCTION
Birth control is a major factor in public health and welfare, preserving the general and reproductive health of women and allowing them to choose the movement of a planned pregnancy

[1]. The World Health organization (WHO) and other global organizations are seeking ways to increase the amount of information and access people have to contraception and other resources related to family planning all round the world.
Oral contraceptives had been reported to have beneficial effects in reducing the incidence of pelvic inflammatory disease, decrease risk of ectopic pregnancy, benign breast lesions, ovarian and endometrial cancers, protection against osteoporosis and rheumatoid arthritis among the users

[2]. Oral contraceptives are sometimes used to treat heavy or irregular menstruation and endometriosis. Oral contraceptive agents can also be used in hormonal replacement therapy, and in the emergency post-coital contraception

[3]. Oral contraceptive decreases the risk of ectopic pregnancy, benign breast lesions, ovarian and endometrial cancers, and offer protection against osteoporosis and rheumatoid arthritis

[4]. Despite the general acceptability and the obvious advantages that have been attributed to oral contraceptives use, some serious side effects have been reported in women taking them. Studies have indicated a relationship of oral contraceptives use and cardiovascular disease, altered levels of coagulation factors, thrombosis, platelet changes, atherosclerosis and multiple sclerosis. Estrogen has been known to have prothrombin effects and elevates cardiovascular and venous thromboembolism risk

[5]. There is little or no data on the effects of Combined Oral Contraceptives (DUOFEM) on Haematological parameters, especially the growth factors such as the cytokines or interlukin-11 and 6, and erythropoietin. Also little or no data have been established in PT, APTT and Liver Function Test using animal model. It is hoped that the result might throw some light on the need of monitoring coagulopathy in women taking these pills. The world population is now seven billion. Nigeria population is estimated to be 176 million and will reach 400 million by the year 2050 [6]. Rapid population growths would have a detrimental effect on socioeconomic development of Nigeria.
There is a concern over population explosion in Nigeria and the drive to control it is leading to indiscriminate used of oral contraceptives. Unintended pregnancy leads to induced abortion which is not legalized in Nigeria, except to save the woman`s life. This research priority included efforts to discover the short and long term effect of combined oral contraceptive (DUOFEM) on some Physiological parameters and possible mechanism of actions in female wistar rats.The knowledge might provide useful interventions towards solving the problem(s). Thus the safety in the contraceptives use and improvement in the health of the user are assured.

Materials and methods
DRUGS: The combined oral contraceptive used is DUEFEM®. They were obtained from family clinic, Ahmadu Bello University Teaching Hospital, Shika-Zaria, and from the Society for Family Health (SFH) Abuja, Nigeria. COCs DUOFEM® tablets which combined ethinyl estradiol and Norgestrel were manufactured by Wyeth Ayerst (USA) and packed and marketed by the Society for Family Health, Lagos, Nigeria. DUOFEM® is a child spacing pill containing ferrous fumarate tablets. Each DUOFEM cycle contains 28 pills; each white tablet contains 0.3mg Norgestrel and 0.03mg Ethinglestradiol and each brown tablet contains 75mg ferrous fumarate. DUOFEM® has a molecular weight of 312.4458g/mol [7].
Eighty (80) female wistar rats aged 10-12 weeks weighing 180-250 g were used for the study. They were divided into four groups of 20 rats each comprising 10 treated and 10 control rats. The treated rats received 0.6mg/kg body weight of COC intragastically for 36, 48, 60 and 72 days in five-day cycles (four-days treatment with one-day break). The COC was given intragastically in 5-day cycles (4-day treatment with 1-day break). All controls were given fresh water ad libitum daily for the period of the experiment. Experimental animals in the study were treated in accordance with the National Protection Laws of Animal Welfare [8]. Ethical clearance was obtained from the Ahmadu Bello University Animal Ethical Committee.
Haematology analyzer was used to perform a complete blood count (CBC or FBC) .An enzyme-linked immunosorbent assay (ELISA) was used for the quantitative determination of alkaline phosphate (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) . Prothrombin time (PT) andActivated partial thromboplastin time (APTT) were performed using Sysmex CA-6000 Coagulation Analyzer. Liver function tests, Erythropoietin, interleukin-6 (IL-6) and interleukin-11 (IL-11) were determined using rat ELISA kit ( Karmiya Biomedical company, USA). Fibrinogen was estimated by Clauss Assay [9]

Statistical Analysis: The result obtained from this study was analyzed using SPSS version 20 for windows. Analysis of Variance (ANOVA) was used to compare means, and values were compared at P < 0.05. Post Hoc multiple comparisons for significant differences between groups were established by Turkey’s HSD. All the data are expressed as Mean ± Standard Error of Mean (SEM).

RESULTS:
Table 1: The effects of combined oral contraceptives on some haematological parameters in female wistar rats

A, b, c, and d- significant, N- No significant

Lowest values of Hb, PCV and RBC count were observed in treated group D (72days)

Table 2: The effects of combined oral contraceptives on some haemostatic parameters and cytokines in female wistar rats

A, b, c, and d – significant, N- No significant

The lowest values of PT, IL-6 and Fibrinogen were observed in the treated group D (72 Days).

Figure 1: The effects of combined oral contraceptives on AST in female wistar rats 

 Short and Long-Term Effects of Combined Oral…

DISCUSSION
In this study the effect of COC on haematological and biochemical parameters in female wistar rats were investigated. The result shows a reduction in haemoglobin(Hb), Packed cell volume(PCV), Red blood cell (RBC), White blood cell (WBC) counts and lymphocytes in all the groups that were given COC, compared to the controls in each group (P<0-05). The finding of lower Hb, PCV, RBC, WBC and Lymphocytes agrees with the finding of [10].The findings were however, contrary to that of other investigators like Bulur [11], Babatunde [12], Abdalla [13], who reported no changes in full blood count in women on COC. The least values of Hb, PCV and RBC were found in group D (72 days). It could be that the use of COC for a longer period may lead to anaemia. The finding of lower Hb, PCV and RBC counts in COC treated groups compared to controls may be as a result of the haemodilution effect of estrogen and the effect of estrogen on the cytokines like IL-11 and IL-6. The findings of lower values of WBC counts in COC treated groups suggest that the use of COC may alter immune response.
The findings of lower WBC count in COC treated groups was in agreement with that of Sajida [10], and contrary to the findings of Surasak et al [14] who observed no changes in WBC count and that of Araz et al[15] who observed increase in WBC count in COC treated female wistar rats. The difference may be as a result of the use of different COC with different concentration of estrogen and progesterone and the duration of use. There was no significant changes observed in COC treated groups in platelet counts compared to the controls. This finding agrees with that of Surasak et al. [14], Peter [16] Bulur et al [11] and Babatunde et al [12]. In general, the use of COC in female wistar rats suppressed haemopoiesis in this study resulting in lowering of Hb, PCV, RBC and WBC counts.
There was no significant change in serum erythropoietin level in all the controls. This finding is contrary to that of Prechile et al.[17] who found that estradiol benzoate inhibited the production of erythropoietin (EPO). Erythropoietin promotes the survival, proliferation and differentiation of erythrocytic progenitors. It appears COC has little or no effect in EPO production. Decreased RBC count in this study may not be as a result of COC effect on EPO. There was decrease fibrinogen levels in all the COC treated groups compared to the control. This finding differs from that of Eliana et al [18] and Peter et al. [16] who reported increase in fibrinogen level in women taking COC. The finding is also contrary to that of Akhigbe et al [19] who found no significant change in fibrinogen level in female wistar rats treated with COC. Fibrinogen functions as a messenger molecule that coordinates and regulates the body response to inflammation. The association between increase in plasma fibrinogen and thrombosis and the risk of myocardial infarction, atherosclerosis and other cardiovascular diseases are well established. Higher level of fibrinogen raises the risk of stroke [13] .Low fibrinogen levels is associated with low risk of cardiovascular diseases. Effect of COC on the liver as observed in this study may be the cause of decreased in fibrinogen level by a third generation COC (DUOFEM).
Prothrombin Time (PT) significantly reduce in all the COC treated groups compare to the controls (P<0.001). There was no significant reduction in Activated Prothrombin Time (APTT). This finding agrees with that of Abdalla 13] ,in Sudan found significant reduction in PT and APTT in women taking COC. Babatunde [12] found that there was no significant change in the level of APTT in Nigerian women taking COC for three months. This is in agreement with the finding of this study. Also Eliana [18] found reduced PT and APTT in Albanian women taking COC. Reduction in prothrombin time may be as a result of reduced serum level of fibrinogen. Estrogen is also said to increase coagulation factors leading to decreased prothrombin time. The finding of this study is also in agreement with that of Ahmed [20] and Nasir [21] who reported a significant reduction in APTT and PT in women taking COC. The exogenous estrogen in the COC has been shown to accumulate in the liver stimulate the release of procoagulants (Factors II, VII and X) from the degenerating liver cells. Significant decreased in PT observed in this study is possible due to enhanced coagulation process as a result of increased plasma levels of coagulation factors of extrinsic pathway.
There was a significant decrease in serum level of antithrombin (AT), protein C (PC) and protein S (PS) in COC treated groups compared to the controls. Proteins C and S and antithrombin are components of the anticoagulant system. In haemostasis, the procoagulant
system is in balance with the anticoagulant and fibrinolytic system [22]. It is important to emphasize that a disturbance in normal hemostasis may result in thrombosis. This finding is in agreement with that of Soare [23] and John [24] who observed lower values of PC in women using oral contraceptives. Decreased AT, PC and PS may be responsible for the changes observed in haemostasis. AT, PC and PS are vitamin K-dependent. Effect of COC on the liver may be responsible for the reduction of serum level of AT, PC and PS in COC treated groups.
There was a significant decrease in serum level of interleukin-6 (IL-6) and a slight decrease in serum interleukin-11 (IL-11). Estrogen is able to decrease IL-6 expression by blocking the estroblast’s synthesis of IL-6 receptors [25]. IL-11 and IL-6 are haemopoiesis-promoting factors capable of enhancing the growth of myeloid, erythroid and megakaryocytic progenitor cells. They are capable of mediating a complex array of pro- and anti-inflammatory effects. Reduction in IL-11 and IL-6 may be responsible for decreased RBC, PCV, WBC and platelet counts in this study. IL-6 produces C – reactive protein (CRP) which leads to cardiovascular risk. Experimental studies have shown strong correlations between the risk of cardiovascular diseases and inflammatory markers such as CRP and tissue neurosis factor-a (TNF a) [26].
IL-6 is a pleotropic cytokine which stimulates B-lymphocyte and T-lymphocyte differentiation, and activates macrophages and natural killer cells (NK). IL-6 promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions [27]. The finding of reduced serum IL-6 and11 is in agreement with other finding [28] who reported decrease serum level of IL-6 and 11 in menopausal women taking hormonal replacement therapy.
There were increased values of alkaline phosphate (ALP), Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) in all COC treated groups compared to controls (P<0.001). The liver plays a central role in the metabolism of estrogens and progesterones. COC acts directly or indirectly on the liver to produce a variety of biological effects which have both physiological and pathological significance [28]. Higher levels of liver enzymes in the blood streams are prime indicators of liver damage.
The use of COCs has been rarely associated with liver tumours, both benign (hepaticadeomas and nodular hyperplasia) and malignant (hepatocellular carcinoma). The finding of increased liver enzymes is in agreement with that of Dickerson [29] and contrary to that of Surasak [14] who found no changes in liver enzymes in women on COC. Raised serum level of ALP, AST and ALT in treated groups may be due to functional alterations involving the hepatic excretory mechanism. The effect of the COC (DUOFEM) on the liver may be the cause for haemostatic disorder, since PC, PS and AT were reduced.
There was a significant increase in serum sodium level in group D (72 days, P<0.044) compared to the control. Potassium was significantly increased in groups C (60 days) and D (72 days). Sodium retention has been reported in women taking COC [19]. The result in this study is contrary to that of Taneepanichskul [30] who reported no significant changes in electrolytes in women taking COC.
Hypertension occasionally occurs in women receiving estrogen-progesterone combination for contraception. Estrogen raises the plasma level of rennin and increases the production rate of aldosterone which leads to water and sodium retention [31, 32] reported reduced potassium level and increased sodium level in female rats. There was no significant change in bicarbonate and chloride in treated groups compared to the controls. Body electrolyte balances are critical for normal cellular function and maintaining adequate blood and plasma volume and osmolality.

CONCLUSION
This study confirms findings of previous studies which also reported an increased risk for venous thromboembolism with third generation COC. This research work has confirmed the effect of COC (DUOFEM) on haemopoiesis. There is a potential risk of long term immune suppression in female taking COC (Duofem). Estrogen has shown to have long term effect on liver cell which may affect liver enzymes production and functions. There is the tendency for COC to be used earlier in life and for longer period of time, the number of women entering the high risk group for venous thromboembolism and liver cell adenomas maybe on the increase. Therefore, we recommend that women on COC should be regularly monitored for Physiological parameters.

ACKNOWLEDGMENT
I want to appreciate all staff of Haematology, Immunology, Chemical Pathology departments and Histopatology Dept. of ABUTH Zaria for their assistance in the analysis of the samples. Dr. Olayemi of Society for Family Health Abuja, for supplying the COC (DUOFEM) and all staff of Human Physiology Department, University of Jos.

REFERENCES

1. World Health Organization (2009). Hormonal Contraception and Liver Disease. Contraception 80; 325 –326.

3. Vessy, M. P. (1995). Endometrial and ovarian cancer and oral contraceptives: Findings in a large cohort study. British Journal of Cancer 71, 1340-1342.

4. Rosing, J., and Tans, G. (1999). Effect of oral contraceptives on haemostasis and thrombosis. American Journal of Obstetrics and Gynecology 180(6), 375-197.

5. Margolis, K. L., Adami, H. O., Luo, J., Ye, W., and Weiderpass, E. (2007). A prospective study of oral contraceptive use and risk of myocardial infarction among Swedish women. Fertility and Sterility 88, 310-316..

7. Chitturi S, Farrell GC 2013). Adverse effects of hormones and hormone antagonists on the liver. In, Kaplowitz N, DeLeve LD, eds. Drug-induced liver disease. 3rd ed. Amsterdam: Elsevier, , pp. 605-20. (Review of hepatotoxicity of oral contraceptive steroids including cholestasis, vascular disorders, benign tumors and hepatocellular carcinoma).

8. Akinsanya M A, Adeniyi T T, Ajai GO, Oyedele MA (2010). Effect of Vitamin E and folic acid on some antioxidant activities of female wistar rats administered combined oral contraceptives. African journal of Biochemistry Research , 4 (10):pp 238-242.

9. Clauss, A. (1957). Gerinnung Sphysiologieche schnelinethode zur bestimmung des fibrinogen. Acta Haematol, 17:237. Cited by: Beutler, E., Lichtman, M. A., and Colle .E

10. Sajida, S. H., Al-Chalaby, S. M. T., and Amjad, F. A. (2006). Effect of oral contraceptive pills on haematological indices. Tikrit Medical Journal 12(1), 65-69.

11. Bulur, S., Albayrak, M., Bulur, S., Keskin, F., Köse, S.A., Aslantas, Y., Türker, Y., and Ozhan, H. (2012). Effect of combined oral contraceptive use on platelet volume in women at reproductive age. Clinical. Experimental. Obstetrics and Gynecology 39(3), 314-316.

12. Babatunde, A. S., and Olatunji, P. O. (2004). Short-term effect of oral contraceptive pills on some haemostatic parameters in healthy Nigerian women. Nigerian. Postgraduate Journal 11(4), 246-250.

13. Abdalla, T. M., Kordofani, A. A. Y., and Nimir, A. A. H. (2008). Haemostatic studies in Sudanese women on oral contraceptive pills. Khartoum Medical Journal 1(3), 116-118.

14. Surasak, T. (2007). Effect of a new oral contraceptive with drospirenone on vital signs, complete blood count, glucose, electrolytes, renal and liver function. Journal of Medical Association Thailand 90(3), 426-431.

16. Peter, U. O. (2013). Women use of oral contraceptives – does it have any effect on haematological parameters? Annals of the College of Medicine 33(1 & 2).

17. Prechile, C., Ira, A., Rappaport, L. F. S., Mario, C., and Albert, S. G. (1972). The role of Estrogen in the regulation of Erythropoietin. Endocrinology. Obstetrics and Gynecology 180(6), 5375-5382.renal and liver function. J Med Assoc Thai.; 90: 426 – 31.

19. Akhigbe, R. E., Azeez, M. O., Ige, S. F., Oyeyipo, L. P., Ajao, F. O., and Alade A. O. (2008). Haematological effects of long-term administration of combined oral contraceptive in rats. International Journal of Pharmacology 4(5), 403-406..

20. Ahmed J. Al-Husaynee and Muna A. Kashmona (2007). Effect of combined oral contraceptive pills on some haemostatic parameters. Annals of the College of Medicine Mosul, 33(1 & 2): 66-69.

21. Nasir A, Qamaruddin B, Salman NA. Effect of low dose oral pill on haemostatic parameters in a set of Pakistani population. J. Pakistan Med. Assoc. 2008: 58: 229

22. Karl, M., Bjorn, A., and Bjorn, D. (2008). Human activated protein C variants in a rat model of arterial thrombosis. Thrombosis Journal 6(16), 1477-1560..

23. Soare, A. M., and Popac (2010). Deficiencies of proteins C, S and antithrombin III and activated Protein C Resistance. Their involvement in the occurrence of Arterial thromobosis. Journal of Medical Life 3(4), 412-415.

24. John, E. G., and Emmanuel, C. B. (2012). Protein S deficiency. http://emedicine.medscape .com/article/205582-overview 5-8-2014

25. Subhadeep, C., Olga, L., and Sandra, T. D. (2008). Estrogen is a modulator of vascular inflammation. IUBMB Life 60(6), 376-382.

26. Trussell, J. (2007). “Contraceptive Efficacy”. In Hatcher, Robert, A., et al. Contraceptive Technology (19th rev. ed.). New York: Ardent Media.

27. Dickerson J, Bressler R, Christian CD (1992). Liver function tests and low-dose estrogen oral contraceptives. Contraception; 22(6): 597-603.

28. Taneepanichskul S, Jaisamrarn U, Phupong V (2007). Effect of a new oral contraceptive with drospirenone on vital signs, complete blood count, glucose, electrolytes,

30. Kang, J. H., Wiggs, J. L., Rosner, B. A., Hankinson, S. E., Abdrabou, W., and Fan, B. J. (2010). Endothelial nitric oxide synthase gene variants and primary open-angle glaucoma: Interactions with gender and postmenopausal hormone use. Investigation Ophthalmological Sciences 51, 971–979.

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Oyewopo AO., Lawal IA., Olawepo A., Enaibe BU., Ajao MS

Department of Anatomy, Faculty of Basic Medical Sciences, Colleges of Health Sciences, University of Ilorin, Kwara State, Nigeria.

Oyewopo C.I.: Department of Anaesthesia, University of Ilorin, Teaching Hospital (UITH), Ilorin Kwara State.

Afolabi O.O.: Department of Morbid Anatomy, University of Ilorin, Teaching Hospital (UITH), Ilorin Kwara State.

All correspondence to: Dr. Oyewopo AO, E-mail: wolesake@yahoo.com.

ABSTRACT
Introduction: The objectives of this study is to determine the effects of cryptorchidism and hyperthermia on the testicular cytoarchitecture of adult male rats, quality and quantity of semen and the level of oxidative stress in the testes before and after intervention of Alpha-tocopherol (Vitamin E).
Methods: Twenty four (24) adult Wistar rats were used for this experiment and they were divided into seven groups (n=4). Experimental cryptorchidism was carried out in some animals while others were subjected to hyperthermia from two sources (hot oven and light bulb) for 28 days; followed by intervention of 100mg/kg alpha-tocopherol for fourteen (14) days.
All animals were then euthanized under anaethesia. The testes were excised and fixed and processed using haematoxylin and eosin technique. Plasma was assayed for glutathione, superoxide dismutase (SOD) and malondialdehyde (MDA).
Discussion: Cryptorchidism and hyperthermia caused in cycto-architectural distortion in the testicular structures of exposed animals and significant reductions in caudal epididymal sperm count and motility, morphology and life and death ratio. Plasma SOD and GLU levels also reduced significantly in exposed animals while MDA levels increased significantly, indicating peroxidation of testicular tissue. Alpha-tocopherol due to its anti-oidative properties impeded lipoperoxidation of the membrane, conserving cell union and increasing the amount of germ cells in the seminiferous epithelium.
Conclusion: At doses and durations tested, alpha-tocopherol (aT) reverted to a significant extent the effects of cryptorchidism and hyperthermia by reducing apoptosis in germ cells, promoting cell survival and reducing histological alterations to the seminiferous epithelium.

Keywords: Cryptorchidism, hyperthermia and Alpha-tocopherol.

INTRODUCTION
The testes function in production of the male gametes or spermatozoa and male sexual hormone, (hence referred to as double glands) which stimulates the accessory male sexual organs and causes the development of the male sex characteristics (Mescher, 2010; Lutz, 2009). In most mammals, the temperature around the testicular region is lower than the core body temperature. In man, scrotal temperature is 2-30C lower than rectal temperature and the optimum temperature. Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility (Thundathi et al., 2012). Oyewopo and Togun in 2005 reported that a small increase in the temperature of the testis does not destroy the germinal epithelium; however, it reduces testis weight and sperm production and brings a greater incidence of morphologically abnormal spermatids and spermatozoa (Oyewopo and Togun, 2005; Bedford, 1991). Occupational exposure to high temperatures adversely affects testicular function causing partial or complete spermatogenic arrest, also causes deterioration in sperm morphology and impairs motility. This leads to oligoasthenoteratozoospermia (OAT) and azoospermia (Dada et al., 2001). Cryptorchidism (undescended testes); the absence of one or both testes from the scrotum happens to be the most common birth defect of the male genitalia, which if persists could result in reduced fertility and increased risk of testicular germ cell tumors (Wood and Elder, 2009). Cryptorchidism has been implicated to cause oxidative stress in animals that the process was performed on, it was reported that Cryptorchid rats had lower testicular weight, sperm count, germ cell count, testicular superoxide dismutase (SOD) concentration, testicular total protein and higher testicular malondialdehyde (MDA) concentration compared to animals in control group (Ayobami et al., 2013; Dutta et al., 2013). Alpha-Tocopherol (aT) is an important antioxidant that localizes to cell membranes. The primary function of vitamin E is as an antioxidant; because it helps reduce oxidation of lipid membranes and the unsaturated fatty acids and prevents the breakdown of other nutrients by oxygen. This protective, nutritional antioxidant function is also performed and enhanced by other antioxidants, such as vitamin C, beta-carotene, glutathione (L-cysteine), and the mineral selenium (Laila and Sahar 2009; Aybek et al., 2008; Traber and Kayden 1987). A significant reduction of lipoperoxidation was observed in the cryptorchid group treated with a-Tocopherol compared to the untreated cryptorchid group, at long term, an increase in the area and maturation of the seminiferous epithelium, a decrease in apoptosis and histological alterations and an increase in fertility was observed in animals treated with alpha-Tocopherol (Saalu et al., 2014; Rosa et al., 2011).
The broad aim of this study investigate the protective properties of alpha tocopherol (vitamin E) on cryptorchidic and heat (hyperthermia) induced testicular injury.

MATERIALS AND METHODS
The experiment was performed in conformity with the Rules and Guidelines of the Animal Ethics Committee of University of Ilorin. The experiments were conducted at the Department of Anatomy, College of Health Science, University of Ilorin.
Animals
Twenty four (24) male Wistar rats (150-210g) were purchased from Banky livestock company, Oke-Ose, Ilorin, Kwara State. The animals where kept in the animal house of the Faculty of Clinical Science, University of Ilorin, under light and dark cycle at room temperature. Proper aeration maintained by the use of wire gauze wooden cage. The rats were fed on growers’ feed from Bendel feeds Nigeria Limited, Ilorin, and distilled water throughout the duration of the experiment. The rats were arranged randomly into groups A-F, kept in different compartments of the cage. The rats were left under the above stated condition for two weeks so as to acclimatize with the condition of their new environment.
Grouping of animals
Group A- (n=4) control animals; these experimental animals were given pelleted feed and tap water ad libitum throughout the period of the experiment;
Group B- (n=4) experimental cryptorchidism was carried out this group;
Group C- (n=4) experimental cryptorchidism was carried out and then 100mg alpha tocopherol for 14days;
Group D- (n=4) animals were exposed to heat from hot oven for 28 days;
Group E- (n=4) animals were exposed to heat from hot oven for 28 days then 100mg/kg vitamin E for 14days;
group F- (n=4) animals were exposed to heat from light bulb for 28 days, group G- animals were exposed to heat from light bulb for 28 days then 100mg/kg vitamin E for 14days.
Animal sacrifice and tissue collection
At the end of the substance administration, the rats were sacrificed 24hrs after the last day of the exposure using ketamine (0.2ml). The animals were pinned on the dissecting board. Dissection was done on the anterior abdominal wall (thoraco-abdominal sagittal incision) using the necessary surgical equipment/instruments. Blood was first collected via the apex of the heart with a hypodermic syringe, and the blood was centrifuged, the epididymis was also excised and placed in normal saline for semen analysis. The testis was removed and then fixed Bouin’s fluid.
Semen analysis
The testes from each rat were carefully exposed and removed. They were trimmed free of the epididymides and adjoining tissues. Epididymal sperm concentration: Spermatozoa in the right epididymis were counted by a modified method of Yokoi and Mayi, (2003). Briefly, the epididymis was minced with anatomic scizzors in 5mL physiologic saline, placed in a rocker
for 10 minutes, and allowed to incubate at room temperature for 2 minutes. After incubation, the supernatant fluid was diluted 1:100 with solution containing 5 g sodium bicarbonate and 1mL formalin (35%). Total sperm number was determined by using the new improved Neuber`s counting chamber (haemocytometer). Approximately 10µL of the diluted sperm suspension was transferred to each counting chamber of the haemocytometer and was allowed to stand for 5 minutes. This chamber was then placed under a binocular light microscope using an adjustable light source. The ruled part of the chamber was then focused and the number of spermatozoa counted in five 16-celled squares. The sperm concentration was the calculated multiplied by 5 and expressed as [X] x 106 /ml, where [X] is the number of spermatozoa in a 16-celled square (Oyewopo et al., 2010).
Sperm progressive motility: This was evaluated by an earlier method by Sonmez et al, (2005). The fluid obtained from the left cauda epididymis with a pipette was diluted to 0.5 mL with Tris buffer solution. A slide was placed on light microscope with heater table, an aliqout of this solution was on the slide, and percentage motility was evaluated visually at a magnification of x 400. Motility estimates were performed from three different fields in each sample. The mean of the three estimations was used as the final motility score. Samples for motility evaluation were stored at 350c. Sperm morphology: The sperm cells were evaluated with the aid of light microscope at x 400 magnification. Caudal sperm were taken from the original dilution for motility and diluted 1:20 with 10% neutral buffered formalin (Sigma-Aldrich, Oakville, ON, Canada). Five hundred sperm from the sample were scored for morphological abnormalities (Atessahin et al., 2006). Briefly, in wet preparations using phase-contrast optics, spermatozoa were categorized. In this study a spermatozoon was considered abnormal morphologically if it had one or more of the following features: rudimentary tail, round head and detached head and was expressed as a percentage of morphologically normal sperm (Oyewopo et al., 2010).

Estimation of lipid peroxidation (Malondialdehyde)
Lipid peroxidation in the testicular tissue was estimated calorimetrically by thiobarbituric acid reactive substances TBARS method of Buege and Aust (1 978). A principle component of TBARS being malondialdehyde (MDA), a product of lipid peroxidation. In brief, 0.1 ml of tissue homogenate (Tris-Hcl buffer, pH 7.5) was treated with 2 ml of (1:1:1 ratio) TBA-TCA-HCl reagent (thiobarbituric acid 0.37%, 0.25 N HCl and 1 5% TCA) and placed in water bath for 1 5 min, cooled. The absorbance of clear supernatant was measured against reference blank at 535nm. Concentration was calculated using the molar absorptivity of malondialdehyde which is 1 .56 x1 05 M-1 cm-1 and expressed as nmol/mg protein (Oyewopo et al., 2010).

Estimation of Glutathione level
Total GSH was estimated in various tissues by the method of Sedlak and Lindsay. Briefly, 5% tissue homogenates were prepared in 20 mM EDTA, pH 4.7, and 100 µl of the homogenate or pure GSH was added to 0.2 M Tris-EDTA buffer (1.0 ml, pH 8.2) and 20 mM EDTA, pH 4.7 (0.9 ml) followed by 20 µl of Ellman’s reagent (10 mmol/l DTNB in methanol). After 30 min of incubation at room temperature, absorbance was read at 412 nm in a Beckman DU-640 spectrophotometer. Samples were centrifuged before the absorbance of the supernatants was measured (Sedlak and Lindsay, 1968).

Estimation of Superoxide dismutase (SOD) level
Testicular tissues were transferred into 5ml ice-cold sucrose solution (0.25M) and homogenized. The homogenates were further centrifuged at 3000 rpm for 15 min to obtain the supernatant, which was then aspirated with Pasteur pipette into sample bottle, stored overnight at 4°C before being used for assays. Tissue activities of superoxide dismutase (SOD) were determined by the method of Marklund and Marklund, (1974).
Statistical analysis
Data collected were analysed using Microsoft Excel and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (HSD) with the aid of SPSS V20. Data were presented as means ± SEM (standard error of mean) P value less than 0.05 (p<0.05) was considered statistically significant.

RESULTS
HISTOLOGICAL OBSERVATION
The micrograph from control group (group A) showed normal population of germinal epithelium, intact structure and arrangement of seminiferous tubules, presence of leydig cell, and well vascularized. Micrograph from group B (cryptorchidic animals) showed degeneration of spermatogenic cells and absence of spermatozoa in the lumen. Micrograph from Group C (cryptorchidic animals that were given alpha tocopherol) showed regeneration of spermatogenic cells and evidence of spermatogenesis due to presence of spermatozoa in the lumen. Micrograph from animals exposed to heat from hot oven (Group D), showed degeneration of basement membrane and clustering of spermatogonia cells, but Regeneration of of spermatogenic cells and normal spermatogenesis was evident in animals exposed to heat from hot oven followed by alpha-tocopherol (group E). Micrograph of animals exposed to heat from light bulb (Group D), showed distorted interstitium and cellular hyperplasia, which was reversed in animals that was given alpha-tocopherol.

Figure (A-G): photomicrographs showing the cyto-architecture of the testes across all experimental groups. Stain- Haematoxylin and Eosin (H & E). Mag- 400.
A- Control, B- cryptorchidism, C- crypt + vit E, D- hot oven, E- hot oven + vit E, F- light bulb, G- light bulb + vit E.
L-Lumen, White arrows- Spermatogenic cells, Black arrow- Basement membrane

Semen analysis
Table 1: percentage sperm count, sperm motility, life and ratio and sperm morphology across all groups.

Biochemical analysis
Table 2: malondialdehyde (MDA), glutathione (GLU) and superoxide dismutase (SOD) levels across all groups.

DISCUSSION
Cryptorchidism and hyperthermia caused desquamation and degeneration of germ cells which might be due to the destabilization of Sertoli cell membranes caused by lipoperoxidation, irregular shape of the seminiferous tubule, and degeneration of the interstitial tissues; this was in accordance to the report of Dutta et al., in 2013; Oyewopo and Togun in 2005 and Lee and Coughlin, in 2001. In cryptorchidic and hyperthermic animals exposed to alpha tocopherol (aT), lipoperoxidation of the membrane was impeded,
conserving cell union and increasing the amount of germ cells in the seminiferous epithelium (Shikone et al., 1994).
The reduction in sperm parameters analysed as seen in figures 4.2 to 4.6 (i.e. sperm counts, motility, life and death ratio and morphology) in both cryptorchidic and heat treated animals might be due oxidative stress action that cryptorchidism and hyperthermia cause to testicular tissues. Oyewopo & Togun in 2005 and Lee & Coughlin in 2001 also reported a significant reduction in sperm parameters of cryptorchidic and heat treated animals. But the effects of cryptorchidism and hyperthermia (heat) was reversed in animals treated with alpha tocopherol (Vitamin E) by not only returning sperm parameters to normalcy but also enhance the rate of spermatogenesis in the animals. My finding was corroborated by works of Saalu et al., in 2014 and Rosa et al., in 2011.
Cryptorchidism and hyperthermia cause a decrease in antioxidant enzyme activity or an increase in the production of ROS, (i.e. superoxide anion, hydroxyl radical, nitric oxide and hydrogen peroxide) which stimulates lipoperoxidation which results in increase in malondialdehyde and reduced glutathione and superoxide dismutase levels in exposed animals. The work of Saalu et al., 2014; Rosa et al., 2011; Ishii et al., 2005 corroborate my findings. However administration of alpha tocopherol to the treated groups showed increase in cell’s endogenous antioxidant defence system, inhibiting ROS production and impede lipoperoxidation (as seen in figures 4.8 to 4.10). This result was corroborated by work of Saalu et al., 2014; Ayobami et al., 2013; Rosa et al., 2011 and Laila and Sahar in 2009.
CONCLUSION
Cryptorchidism and hyperthermia resulted in oxidative stress in exposed animals which is evident in distortion seen in the histological slides, reduced sperm parameters and antioxidant activities. This work showed that aT reduced apoptosis in germ cells, promoting cell survival and reducing histological alterations to the seminiferous epithelium.

REFERENCES
1. Aybek H., Aybek Z., Rota S., Sen N. and Akbulut M., (2008). The effects of diabetes mellitus, age, and vitamin E on testicular oxidative stress. Fertil Steril., 90(3): 755-760.

2. Ayobami O. A., Hameed A. A., and Isiaka A. A. (2013). Effects of methanolic extract of moringa oleifera leaves on semen and Biochemical parameters in cryptorchid rats. Afr J Tradit Complement Altern Med. (2013) 10(5):230-235.

3. Bedford J.M. (1991). Effects of elevated temperature on the epididymis and testis: experimental studies. Adv Exp Med Biol. 1991; 286:19-32.

4. Dada R., Gupta, N.P., Kucheria K. (2001). Deterioration of Sperm Morphology In Men Exposed To High Temperature. J Anat. Soc. India 50(2) 107-111 (2001).

5. Dutta S., Joshi K.R., Sengupta P., Bhattacharya K. (2013). Unilateral and bilateral cryptorchidism and its effect on the testicular morphology, histology, accessory sex organs, and sperm count in laboratory mice. J Hum Reprod Sci. 2013 Apr; 6 (2):106-10.

6. Ishii T., Matsuki S., Iuchi Y., Okada F., Toyosaki S., Tomita Y., Ikeda Y., Fujii J., (2005). Accelerated impairment of spermatogenic cells in SOD1-knockout mice under heat stress. Free Radic. Res. 39, 697–705.

7. Laila Sabik M.E. and Sahar S. A. (2009).Alpha-tocopherol and ginger are protective on Cyclophosphamide-induced gonadal toxicity in adult male albino rats. Basic and Applied Pathology 2009; 2: 21–29.

8. Lee P.A. and Coughlin M.T. (2001).Fertility after Bilateral Cryptorchidism Evaluation by Paternity, Hormone, and Semen Data. Horm Res 2001; 55:28–32.

9. Lutz Slomianka (2009). Blue Histology- Male Reproductive System. School of Anatomy and Human Biology – The University of Western Australia.

10. Marklund S. and Marklund G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 47: 469-474.

11. Mescher A.L. (2009). Junquiera’s Basic Histology: Text and Atas. 12th Ed. Pg- ISBN 978-0-07-160431-4; MHID 0-07-160431-6.

12. Oyewopo AO, Saalu LC, Osinubi AA, Imosemi IO, Omotoso GO, Adefolaju GA. (2010). The attenuating effects of zinc on Propoxur-induced oxidative stress, impaired spermatogenesis and deranged steroidogenesis in Wistar rats. Journal of Medicine and Medical Sciences, Vol. 1(5) pp. 178-184. June 2010.

13. Oyewopo O. A. and Togun V. A. (2005) Effect of temperature on motility and concentration on male Sprague Dawley rats epididymal spermatozoa. Sci. Focus. 10: 35-40.

14. Rosa Vigueras-Villaseñor M., Idahue O., Oscar Gutierrez-Pérez, Margarita Chavez-Saldaña, Osvaldo C., Daniel S.M., and Julio Rojas-Castañeda C. (2011). Protective effect of a-tocopherol on damage to rat testes by experimental

cryptorchidism. Int J Exp Pathol. 2011 Apr; 92(2): 131–139.

15. Saalu, L.C., Oluyemi, K.A. and Omotuyi, I.O. (2014). Alpha-Tocopherol (vitamin E) attenuates the testicular toxicity associated with experimental cryptorchidism in rats. African Journal of Anatomy Vol. 1 (1), pp. 001 -005, January, 2014.

16. Sedlak J. and Lindsay R.H. (1968). Estimation of total, protein-bound and non-protein sulfhydryl groups in tissues with Ellman’s reagent. Analytical Biochemistry, 25: 192-205.

17. Shikone T., Billig H. and Hsueh A.J. (1994). Experimentally induced cryptorchidism increases apoptosis in rat testis. Biol Reprod. 1994 Nov; 51(5):865-72.

18 Thundathi J.C., Rajamanickam G.D., Kastelic J.P., Newton L.D. (2012). The effects of increased testicular temperature on testis-specific isoform of Na+/K+ -ATPase in sperm and its role in spermatogenesis and sperm function. Reprod Domest Anim. 2012 Aug; 47 Suppl 4:170-7.

19 Traber M.G. and Kayden H.J. (1987). Tocopherol distribution and intracellular localization in human adipose tissue. The American journal of clinical nutrition.

20. Wood H.M., Elder J.S. (2009). “Cryptorchidism and testicular cancer: separating fact from fiction”. The Journal of urology 181 (2): 452–61.

The post Cryptorchidism And Hyperthermia Induced Testicular Injury In Adult Male Rats; Intervention Of Alpha Tocopherol appeared first on Nigerian Biomedical Science Journal.

Enabulele, O.I.
Faculty of Life Sciences,Department of Microbiology, University of Benin Edo State, Nigeria.

Tchounga, K.S., Ukaji, D.C., Ajugwo’ A.O.
Department of Medical Microbiology/ Medical Mycology, Faculty of Medical Laboratory Science,
Madonna University Elele Campus, Rivers State,Nigeria
All correspondence to: ukajidamian@yahoo.com

ABSTRACT
Serotype and biotype of Vibrio cholerae isolated from stool samples collected from patients with acute diarrhea and water samples were determined during September to November 2004 cholera outbreak in Uzebba in Edo–State, Nigeria. A total of 137 stool samples and 50 water samples were investigated. The samples were subjected to standard recommended microbiological techniques and confirmation of isolates by seroagglutination using Vibrio cholerae polyvalent O1 and O139 antisera and monovalent Ogawa and Inaba antisera, while biotyping was carried out by agglutination test and sensitivity to polymyxin B. Out of 137 stool samples, 63((45.98%) were found to be positive for Vibrio cholerae serogroup O1, and of 63Vibrio cholerae O1, 60(95.24%) were Vibrio cholerae serogroups O1 serotype Inaba, biotype classical and 3(4.76%) were found to be Vibrio cholerae serogroup O1, serotype Ogawa, biotype classical. Only stream water samples yielded growth of Vibrio cholerae serogroup O1, with 3(23.7%) out of 13 stream water samples. Well and bore hole water samples yielded no growth of Vibrio cholerae. Out of three Vibrio cholerae O1 isolated from stream water samples, 2(66.67%) were found to be positive, for Vibrio cholerae serogroup O1, serotype Inaba and classical biotype, while 1(33.33%) was Vibrio cholerae serogroup O1, serotype Ogawa and El -Tor biotype. This study demonstrated that some Vibrio cholerae O1 serogroup O1 isolated from stream water and stool samples had the same serotypes and biotypes. Therefore, there is need to perform a strong epidemiological surveillance for emergence and distribution of Vibrio cholerae O1.

Key words: Vibrio cholerae O1, serotype, biotype, water supplies, stool samples.

INTRODUCTION
Cholera (frequently called Asiatic cholera or epidemic cholera) is an acute diarrheal illness caused by a Gram negative slightly curved rod Vibrio cholerae (Ryan and Ray, 2004). Cholera is a worldwide problem, especially in developing countries. It has been very rare in developed nations for hundred years; however the disease is common today in other parts of the world, including the India sub-Asian continent and sub-Sahara Africa. Cholera is a major health threat in poor nations that frequently results in mortality. Although more than 100 serogroups of Vibrio cholerae exist, only two cause human disease. Vibrio cholerae O1 of which there are two biotypes (Classical and EL Tor); classified into serotypes Ogawa and Inaba and rarely Hikojima and Vibrio cholerae O139, which emerged in 1992. Cholera world wide is usually characterized by painless diarrhea and vomitting.
In 1991, there were more than a million cases in Central and South Canada. Several new cases have since been reported in Louisiana and other Gulf Coast areas (Werdlow
et al., 2002). African countries have in recent years experienced more epidemics and (WHO, 1999). A total of 29321 cholera cases and 10586 deaths were reported to WHO in 1998, with Africa accounting for the largest part with 72% of the global total (WHO, 1999). An explosive outbreak of cholera occurred among Rwanda refugees in Goma. Democratic Republic of Congo involved about 70,000 cases and caused about 12,000 deaths in 1994 (Siddique et al., 1995). In 1995, West Africa reported 64% cholera cases and 61% cases of cholera deaths (WHO, 1999). Between July and November 2001, several outbreaks were reported in the region, including 897 cases with 47 deaths in Cote d’voire and 575 cases with 21 deaths in Kano (WHO, 2001). In 2004, another outbreak involving 1316 cases and 76 deaths were again reported in Kano, while Edo-State (Uzebba) witnessed an outbreak of cholera involving 300 reported cases with 50 deaths (WHO, 2004). The Onslaught of cholera in Africa continent continues unbeaten as another one was reported in Nigeria involving 400 cases in Jigawa State, resulting in 15 deaths in October 2009 (WHO, 2009).

Strategies for prevention and control of this infectious disease depend on understanding the origin, transmission and other characteristics associated with it epidemiology. Therefore, this study was carried out to determine the prevalent serogroup, serotypes and biotypes of Vibrio cholerae responsible for the cholera outbreak in Edo-State, (Uzebba) between September to November 2004.

Materials and Methods
Stool samples were collected from patients with acute diarrihea. During the outbreak, exclusion criteria included patients using antimicrobial agents within the previous two weeks to avoid cases of antibiotic associated diarrhea. Water samples were collected in clean containers containing alkaline peptone water (APW) from various sources of water supply (streams, boreholes and wells).

Sample Processing
Stool samples collected in plain containers were cultured directly onto Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar plates and incubated at 37oc for 24 hours. Water samples inoculated in APW were incubated at 37oc for 4 to 6 hours, after the incubation period, from the inoculated APW, plating was done onto TCBS agar and the plates incubated at 37oc for 24 hours. The isolates were purified by sub-culturing single colonies on sterile Nutrient agar plates which were then incubated at 37oc for 24 hours. Following standard morphological and biochemical tests according to Buchanan and Gibbons (1974), characteristic colonies grown on the selective TCBS agar were then confirmed for identification. The series of biochemical tests commonly used to identify V. cholerae (Baumann and Schubert, 1984; West and Cowell, 1984) were originally designed for clinical samples in order to specifically detect pathogenic vibrios. The series of biochemical tests include: Gram staining, oxidase , glucose and L-arabinose , methyl red , voges-proskauer and ornithine tests were performed.

Serological Test
Serological identification of isolates was done using slide agglutination test (Sakazaki and Donovan, 1984; Shimada et al., 1994), using polyvalent Vibrio cholerae O1 and Vibrio cholerae O139 antisera and monovalent Ogawa and Inaba antisera.

Biotyping
Biotyping of Vibrio cholerae isolates was done by using agglutination with chicken red blood cells and polymixin in B (50 units) sensitivity test (WHO, 1987).

RESULTS
A total of 137 stool samples collected from hospitalized patients during cholera outbreak from September to November 2004 in Uzebba (Edo – State), and water samples 50(13 stream water, 20 borehole water and 17 well water samples) were studied.

Table 1 shows the frequency of isolation, serotyping and distribution of Vibrio cholerae. Isolated from stool and water samples in Uzebba. Out of 137 stool samples, 63(45.98%) were found positive for Vibrio cholerae O1 and 60(95, 24%) out of 63, belonged to Inaba serotype, while 3(4.76%) were Ogawa serotype. None of the samples yielded growth of Vibrio cholerae O139. Water samples were also analyzed bacteriologically, 3((23.07%) out of 13 water samples collected from streams yielded growth of Vibrio cholerae O1 and borehole and well water samples yielded no growth. Table 2 shows the distribution of Vibrio cholerae O1 biotype isolated from stool and water samples. All vibrio cholerae O1 from stool samples both Inaba and Ogawa serotypes were Classical biotype, two Vibrio cholerae O1 Inaba serotype isolated from stream water samples were Classical biotype, while only Vibrio cholerae O1 Ogawa isolated from stream water sample was EL Tor biotype.
DISCUSSION
Cholera continues to be an important pubic Health problem among many poorer and vulnerable communities despite the fact that bacteriology, epidemiology and public health aspects of the disease were described in detail over a centuary (Shears, 2001). In the study, we isolated and identified 66 Vibrio cholerae out of which 63 isolates were from stool samples and 3 isolates from water samples.It was observed after serological test that the isolates were Vibrio cholerae O1 and that there was no serogroup O139 isolates. These findings are in agreement with the report of Tamang et al.,( 2005) reported that Vibrio cholerae O1 were predominant in Nepal, also similar reports were presented by Urassa et al.,( 2000);Inaet al.,(2007) reported that during his study in Manhica District hospital Southern Mozambique, all isolates were Vibrio cholerae O1. During this study, it was noticed that two serotypes Inaba and Ogawa co-existed with Vibrio cholerae O1 serotype Inaba being the most predominant with 62(93.94%) while 4(6.06%) were Vibrio cholerae O1 Ogawa serotype; and these isolates were both from stool and water samples. Our findings are in accordance with reports of Mercy et al.,( 2004) who isolated Vibrio cholerae O1 in Ghana with Inaba serotype having the highest occurrence over Ogawa serotype. Similar report was presented by Shukla et al., ( 2006 ) who recorded an emerged predominance of Vibrio cholerae O1 serotype Inaba over Ogawa between 2004 – 2006 in East Delhi.

The co-existence of the two serotypes recorded during our study was not in conformity with Inacio et al., (2007) who reported that only Vibrio cholerae O1 serotype Ogawa was isolated in Manhica District hospital Southern Mozambique. Our findings also contradicted claims by Hossein et al., ( 2005) that after a six year study on Vibrio cholerae in South Eastern Tran, all Vibrio cholerae O1 isolated were Ogawa serotype. EL Tor was the predominant biotype causing outbreak up till 1998. But during our study in Uzebba, the Classical biotype emerged predominant that although that one of the isolates from water sample was EL Tor biotype. The high emergence of Classical biotype in Uzebba could be justified by the
reports of Rafi et al., ( 2004) also isolated Vibrio cholerae O1 Classical biotype between 2000 – 2001 in Rawalpindi. This high occurrence of Vibrio cholerae O1 Inaba and Classical biotype in our study is not in accordance with the reports of Bradley et al.,(1997), Jacques et al.,( 2002) and Iwanaga et al.,( 2004), who reported that most of the strains of Vibrio cholerae O1 isolated from Madagascar, Bangladesh, Tanzania, Zaire, Latin America, Southern and Eastern regions of India were Ogawa serotype and EL Tor biotype. Our study revealed that high percentage of Vibrio cholerae isolated from both stool and water samples were Vibrio cholerae O1 Inaba and Classical biotype, which is also contrary to the findings of Hossein, et al.,(2005) that all strains of Vibrio cholerae O1 isolated between July and September 1998 in Goa belonged to EL Tor biotype, 53(66%) of them being Ogawa serotype, while 21(26%) were Inaba serotype.

Some isolates obtained from stream water samples when serotype and biotype were reported as Vibrio cholerae O1 Inaba serotype Classical biotype same as some of the isolates obtained from patient’s stool samples during the outbreak. One could be tempted to say that by referring to the phenotypic and genotypic characteristics of the isolates, this stream water that has been one of the sources of water supply in Uzebba community must have played an important role in the spread of cholera outbreak in that region. We can also say that outbreak in Uzebba is defined by social and environmental factors.The importance of aquatic reservoir depends on sanitary conditions of the community(CEDECO,2010. Cholera is spread mainly through drinking fecal – contaminated water. . While contaminated water remains the major route for cholera transmission (Shapiro, et al., 1999) food and utensils are also important (Rabbani and Greenough, 1999), emphasizing the importance of hygiene within the house hold (Fotedal, 2001).

Effective food hygiene measures include cooking food thoroughly and eating it while still hot; preventing cooked foods from being contaminated through contact with raw food or drinking water is important (Seas and Gotuzzo, ensuring proper management of excreta to avoid contamination of other water sources were important measures to reduce cholera transmission.   Education of the population at risk regarding appropriate hygienic practice is always recommended. Identification of local customs that place people at risk is also important in order to eliminate such practices (Sears and Gotuzzo, 2000). A greater understanding of the pathogen, its biology, ecology, epidemiology and strategies for treatment and prevention are essential to guide policies and programmes for the control of cholera. . Adequate measures to improve hygiene and sanitation and supply of safe potable water are needed to prevent any future outbreak of cholera in Uzebba.

The post Serotypes and Biotypes of Vibrio Cholerae O1 Isolated from Stool and Water Samples in Uzebba (Edo – State). appeared first on Nigerian Biomedical Science Journal.

Atiegha C. Igoni M , Victor I.,
Department Of Medical Laboratory Sciences, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Ayaowei I.T.
Department Of Health Information Management, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Eseimokumo Me.
Department Of Pharmarcy Technician Studies, College Of Health Technology Otuogidi-ogbia , Bayelsa State.

All correspondence to: IGONI M ; Bayelsa State College of Health Technology: e mail; achristo40@gmail.com.

ABSTRACT
The treatment of tuberculosis has evolved from streptomycin to the DOTS strategy .Even as at that it is a serious challenge. This retrospective study was carried out to evaluate the effectiveness of the method. A total of sixty seven(67) Tuberculosis cases were evaluated at the Tuberculosis Treatment centre Nembe comprehensive Health Centre, Bayelsa state , it covered a period of four (4) years, from 2009 to 2012. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. It is therefore recommended for community and family based treatment.

Key words: DOTS, Cured, treatment completed and treatment failure.

INTRODUCTION
Tuberculosis, a major health challenge and leading infectious killer is caused mainly in man by Mycobacterium tuberculosis. The bacilli enters the body by inhalation of droplets or dust particles containing it. It occurs as pulmonary tuberculosis or non- pulmonary tuberculosis. In pulmonary tuberculosis the pulmonary aveoli and surrounding lymph glands are lodged by the bacilli resulting in lesion characterized by acute inflammatory reactions with accumulation of fluid and white blood cells around the aveoli, while the non pulmonary tuberculosis includes the renal and urogenital tuberculosis, military tuberculosis and tuberculosis meningitis with varying symptoms1.
In sub-saharan Africa, cases of tuberculosis have increased dramatically, overwhelming control programs 2. This could be as a result of the fact that tuberculosis infection control interventions are not routinely implemented in contrast to what happens in high income countries with low prevalence of tuberculosis where infection control policy is routinely observed3. However the launch of directly observed treatment shortcourse (DOTS) in 1995 by the world health organization (WHO) has shown to be an effective intervention that will lead to reduced tuberculosis transmission and decreasing number of tuberculosis cases4,5. Also it is among the most cost-effective global health interventions available today6,7.
The DOTS strategy is based around a short-course treatment regimen for a minimum of six months of four drugs in combination (2 months of Isoniazide, Rifampicin ,Pyrazinamide and Ethambutol), a good management practice, sputum smear microscopy for diagnosis and the direct observation of doses to ensure adherence. Treatment success under DOTS concerns two outcomes- cured and treatment completed: “cured” if patient finish the treatment regimen with negative bacteriology at the end of the treatment whereas “treatment completed” refers to patients who have finished the regiment in full without showing evidence of treatment failure and without negative bacteriology. This treatment regiment however use to suffer from a number of drawbacks, with the combination of available drugs, the duration of treatment cannot be reduced below six months without a significance in relapses although re-infection with a different strain of mycobacterium tuberculosis can cause tuberculosis recurrence, which is considered to be an important measure of efficacy of tuberculosis treatment8,9. Also when treatment is given under sub-optimal conditions, regiments are associated with high rate of patients non-adherence specially in low income countries which harbor high burden of diseases10. The consequence of this is increased mortality and creation of clinic infections, drug resistant cases. The World Health Organization estimates in 2004, 424,203 cases of multidrug resistant tuberculosis globally among which 181,408 cases occurred in patients who had already been treated with standard (first line) therapy11.
This study was therefore carried out to determine the effectiveness of DOTS (Directly Observed Treatment Short course) on tuberculosis in the Nembe Local Government Area of Bayelsa state south-south Nigeria.

MATERIALS AND METHODS
Setting Comprehensive Health Centre Nembe, is situated in Nembe, the headquarters of Nembe local government area, about eighty kilometers south-east of Yenagoa the capital of Bayelsa State, Nigeria.

Subjects
The subjects of this report are those patients that reported to the TB treatment centre and those patients who were got through surveillance in the surrounding communities. A total of sixty seven positive cases were involved in the study. These cases were got over a four year period (from 2009 – 2012)

Laboratory analysis
Patients were given sterile wide mouthed plastic containers which they voided sputum into. Three specimens were collected from them over a two days period (spot, early morning and spot). Sputum analysis was done using the Ziehl-Neelson technique as described by cheesbrough12.

Method of drug administration
The strategy adopted here is the short course regiment that lasts for six months. Upon visit to the treatment center, Zeihl-Neelson(AFB) test is carried out. Treatment commences on patients who are newly diagnosed i.e. their sputum show smear positive. After the initial test, a second test (Zeihl- Neelson) is conducted upon revisit at the end of the second month of chemotherapy. The same thing applies
upon a second and third revisit which is at the end of the fifth and sixth months respectively.
For category 2 patients i.e. those who defaulted treatment or had a relapse, treatment is for seven months. Treatment on the intensive phase is for three months after which the second AFB test is done. The third and fourth AFB tests are done at the end of the fifth and seventh month of chemotherapy. Patients that absconded during treatment were not included in this work.

In 2009 eighteen new cases of sputum positives were recorded (six scanty and twelve +s). Upon first revisit i.e. after intensive phase, all eighteen persons were tested smear (AFB) negative. On second revisit the same feet was recorded. In the last revisit seventeen tested negative while one patient was AFB positive: table 1.

In 2010, seventeen persons were tested positive with varying degree of intensity of infection ( one scanty, eighty +s, four ++s and four +++s). sixteen persons tested smear negative at the end of the first revisit while person was tested positive. At the end of the fifth month of chemotherapy all fifteen persons tested smear negative (second revisit). After six months of chemotherapy one patient was tested smear positive while the other sixteen persons that continued their treatment to the end were tested negative. Conclusively, eleven persons were cured while one person was termed a “Treatment Failure”: table 2
Table 3 represents the summary of the 2011 treatment outcome. There were ten sputum smear positive cases in
2011. Eight of them were graded +, while one patient had 2+ and another 3+. Seven persons tested AFB negative upon the first revisit and one patients sputum was positive. In the second and third revisits, all eight patients had there AFB test negative. i.e. in 2011 eight persons were cured of tuberculosis in the treatment center.
Two scanty cases, twelve +s and eight cases of 2+s were recorded in the year 2012, twenty two cases in all. Three scanty cases were recorded in the first revisit stage, while nineteen tests were negative. In the second and third revisit all twenty two tests were smear negative. in 2012 twenty two persons were cured of tuberculosis in the treatment center. Table 4

Discussion
In this four years under study, the success of this method of treatment has been so tremendous. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. When the result was subjected to the chi-square test, it shows that there is a significant difference between those that are cured of TB by the dots strategy in the five years under study and those that were not cured by the strategy p<0.05, (234.845, df 3, assymp. Sig (2-sided) 0.000

Conclusion:
Since with the DOTS strategy it is very difficult for patients to skip treatment, (which in most times is the cause of resistance), the researcher recommends that this strategy should be adopted, be it community based or family based)

Acknowledgment
We wish to express our heartfelt thanks to God almighty for his abundance grace to carry out this research and the strength to stand it all, without him nothing would be done. We also greatly appreciate the provost of the Bayelsa State College of Health technology, Dr Adias T. Charles for his inspiration and motivation. Also, the research and manpower development department of the College was involved with the funding. Finally we thank the staffs of the comprehensive health centre Nembe, Bayelsa State.

The post Diagnosis And Treatment Of Tuberculosis By The Directely Observsed Treatment Shortcourse (DOTS): A Case Study Of Nembe Comprehensive Health Centre TB Treatment Unit, South-South Nigeria. appeared first on Nigerian Biomedical Science Journal.