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.

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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.

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