Haemotological profile of African Straw-Coloured Fruit Bats (Eidolon Helvum) Experimentally Infected with Rabbies Virus

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

GROUP	NUMBER	DAY OF SACRIFICE
CONTROL GROUP A	5	Day 28
EXPERIMENTAL GROUP B	5	Day 1 pi (post inoculation)
EXPERIMENTAL GROUP C	5	Day 3 pi (post inoculation)
EXPERIMENTAL GROUP D	5	Day 5 pi (post inoculation)
EXPERIMENTAL GROUP E	5	Day 7 pi (post inoculation)
EXPERIMENTAL GROUP F	5	Day 14 pi (post inoculation)
EXPERIMENTAL GROUP G	5	Day 21 pi (post inoculation)
EXPERIMENTAL GROUP H	5	Day 28 pi (post inoculation)

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.

Control

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group 7

Mean±SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Hb

12.06±0.86*

11.04±0.39

11.14±0.63

11.62±0.38

11.46 ± 0.36

11.66 ± 0.21

13.96

± 0.17*

13.28

± 0.25*

PCV

45.20±1.77

43.60±1.97

41.00±1.18

42.60

± 1.08

47.20

± 0.97

44.60 ± 0.68

44.88

± 1.32

44.44

± 1.99

RBC

7.54 ± 0.11

6.84 ± 0.13

7.54 ± 0.17

7.38 ± 0.21

7.62 ± 0.08

7.60 ± 0.37

8.18 ± 0.32

7.07 ± 0.17

MCV

59.86

± 1.85ab

51.72

± 11.58

39.52

± 11.18b

46.90

± 10.31b

46.25

± 0.21b

56.82 ± 1.68

58.90

± 2.30

62.88

± 4.25a

MCH

15.97

± 1.03*

16.13

± 0.41

14.84

± 1.04

15.75

± 0.22

15.05

± 0.54

15.15 ± 0.51

18.07

± 0.74*

18.77

± 0.24*

MCHC

26.65

± 1.40*

25.50

± 1.36

27.10

± 0.75

27.33

± 1.07

24.26

± 0.34

26.14 ± 0.26

31.21

± 0.92*

30.36

± 1.80*

 

 

Table 3: White (WBC) parameters

Control

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Group 7

Mean±SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

WBC

7.98

± 0.30*

11.88 ± 1.69*

20.72 ± 0.33*

14.42 ± 1.38*

11.90 ± 0.31*

8.18 ± 0.52

7.22 ± 0.28

7.76 ± 0.20

Lymphocyte 77.30

± 1.43*

19.54 ± 0.65**

28.16 ± 0.50*

33.32 ± 2.81*

52.70 ± 2.63*

65.82 ± 0.49*

70.18 ± 1.62*

70.86

± 3.11*

Neutrophil

21.40

± 1.15*

78.74 ± 0.50**

73.16 ± 1.84*

64.84 ± 2.99*

44.78 ± 2.30*

33.72 ± 0.36*

39.24 ± 8.9*1

27.14

± 2.70*

Eosinophil

0.92

± 0.23

0.98 ± 0.33

0.16 ± 0.10

1.44 ± 0.51

0.14

± .10

0.20 ± 0.10

0.34 ± 0.24

1.30 ± 0.57

Basophil

0.36

± 0.25

0.74 ± 0.29

0.50 ± 0.32

0.40 ± 0.24

0.18

± 0.10

0.26 ± 0.18

0.44 ± 0.17

0.70 ± 0.44

 

*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