Abstract

Among the 1,198 species of bats identified worldwide, only three species feed exclusively on blood, namely, the common vampire bat (Desmodus rotundus), the hairy-legged vampire bat (Diphylla ecaudata), and the white-winged vampire bat (Diaemus youngi). D. rotundus is the most common hematophagous bat, is found from Mexico to South America, and feeds on the blood of mammals, while the other two species use avian blood as a source of food¹1. Reis NR, Shibatta AO, Peracchi AL, Pedro WA, Lima IP. Sobre os Morcegos Brasileiros. In: Reis NR, Peracchi AL, Pedro WA, Lima IP, editors. Morcegos do Brasil. Londrina; 2007. p. 17-25..

Because of its food preference, the D. rotundus bat is involved in the rabies cycle and is responsible for significant losses of livestock²2. Pan-American Health Organization. Sistema de Informação Epidemiológica. Históricos Anuais. 2011. [Cited 2014, June 10]. Available from http://siepi.panaftosa.org.br/anuais.aspx
http://siepi.panaftosa.org.br/anuais.asp... . However, the therapeutic proprieties of the anticoagulant in their saliva and its orientation system make them the subject of many investigations³3. Witt W, Maass B, Baldus B, Hildebrand M, Donner P, Schleuning WD. Coronary thrombolysis with Desmodus salivary plasminogen activator in dogs. Fast and persistent recanalization by intravenous bolus administration. Circulation 1994; 90:421-426.,⁴4. Schnitzler HU, Moss CF, Denzinger A. From spatial orientation to food acquisition in echolocating bats. Trends Ecol Evol 2003; 18:386-394..

In this study, we examined the hematological profiles of D. rotundus bats before and after experimental infection with rabies virus in order to establish reference values for this species.

Bats were anesthetized with ketamine hydrochloride (Ketamina®) by injection into the pectoral muscle, considering the weight and volume (0.1ml/10g of body weight). Ninety-six blood samples were collected by intracardiac puncture using surface-heparinized 1ml syringes. Sixty-eight of the blood samples were collected before the experimental infection, and 28 were collected on day 33 following the experimental infection. Blood was taken from bats surviving rabies infection, clarifying the difference between the numbers in the samples.

The bats were euthanized in a CO₂ chamber following blood collection. All procedures were designed to avoid animal suffering, and this research was approved by the Ethics Committee of São Paulo University. The capture of bats was authorized by a Brazilian institution responsible for wild animal care (Instituto Brasileiro do Meio Ambiente; license nr. 107/00).

One hundred ninety-five bats were maintained in captivity in biosafety cages specially designed for rabies experimentation⁵5. Almeida MF, Aires CC, Martorelli LFA, Barros RF, Massad E. Maintenance of the Hematophagous Bat Desmodus rotundus in Captivity for Experimental Studies on Rabies. Chiroptera Neotropical 2009; 15:433-440.. The virus strain used for experimental infection was isolated from a naturally infected D. rotundus bat. The dose used was 10⁵ MICLD₅₀ (Mouse Intracerebral Lethal Dose), which was previously tested in studies of on rabies experimental infection⁶6. Almeida MF, Martorelli LFA, Aires CC, Sallum PC, Durigon EL, Massad E. Experimental rabies infection in hematophagous bats Desmodus rotundus. Epidemiol Infec 2005; 133:523-527..

Before counting, red blood cells were first diluted 1:400 in 0.85% sodium chloride solution and white blood cells were diluted 1:5 in Turk solution. Blood cell impressions in slides were stained using Leishman staining. Cell counts were performed in a Neubauer chamber.

Before the rabies experimental infection, the average red blood cell was 9.97 × 10⁶/mm³ (± 1.83 × 10⁶/mm³) and average white blood cell was 4.80 × 10³/mm³ (± 2.09 × 10³/mm³). The neutrophils represented an average of 69.9% of blood cells, while lymphocytes represented 26.9%. After the experimental infection, the average of red blood cells was 9.43 × 10⁶/mm³ (± 3.05 × 10⁶/mm³), and that of white blood cells was 3.98 × 10³/mm³ (± 2.21 × 10³/mm³). Neutrophils represented an average of 40% of white blood cells and lymphocytes represented 59%.

We observed that the blood of hematophagous D. rotundus bats contains on average, 2-fold more red blood cells than found in human blood. In addition, the bat erythrocytes are smaller than those of humans, which was also observed by Sealander⁷7. Sealander JA. The influence of body size, season, sex, age and other factors upon some blood parameters in small mammals source. J Mammal 1964; 45:598-616. in relation to others small mammals. The reduced size of red blood cells in D. rotundus bats improves oxygen binding and, thus, enhances their ability to transport oxygen. Due to the small size and large number of red cells, the total surface area available for oxygenation is increased⁸8. Neuweiler G. The biology of bats. Oxford, UK: Oxford University Press; 2000.. Besides their ability to fly, bats have also developed a highly specialized circulatory system to meet the needs of flight.

The number of erythrocytes observed in our study (Table 1) with D. rotundus bats is very similar to values reported previously⁹9. Almeida BFM, Barbosa TS, Ciarlini LSRP, Pedro WA, Beluccio ML, Queiroz LH, et al. Valores hematológicos de morcegos hematófagos Desmodus rotundus (E. Geoffroy, 1810) mantidos em cativeiro. Chiroptera Neotropical 2010; 16:780-785.–¹³13. Krutzsch PH, Wimsatt WA. Some normal values of peripheral blood in the vampire bat. J Mammal 1963; 44:547-559.. High erythrocyte counts were also observed in studies with other bat species. Neuweiler⁸8. Neuweiler G. The biology of bats. Oxford, UK: Oxford University Press; 2000. showed values of erythrocytes varying between 9.3 × 10⁶/mm³ and 15.4 × 10⁶/mm³ in 15 species of bats. Baptista and Esberard¹²12. Baptista M, Esbérard C. Valores Hematológicos de Artibeus sp e Desmodus rotundus (Mammalia, Chiroptera). Rev Cient Inst Pesq Gonzaga da Gama Filho 1997; 3:11-22. showed average values of 8.04 × 10⁶ erythrocytes/mm³ in four species of frugivorous bats belonging to the Artibeus genus. Similar results (8.9-10.1 × 10⁶/mm³) were reported by Ratnasooriya¹⁴14. Ratnasooriya WD, Udagama-Randeniya PV, Yapa WB, Digana PMCB, Dharmasini MG. Haematological parameters of three species of wild-caught microchiropteran bats, Miniopterus schreibersii, Taphozous melanopogon and Hipposideros lankadiva in Sri Lanka. J Sci Univ Kelaniya 2005; 2:27-40. for three species of bats from Sri Lanka.

Hematological values for white blood cell counts have shown contradictory results. The number of white blood cells obtained in this study with D. rotundus bats was discordant with those reported by Almeida et al.⁹9. Almeida BFM, Barbosa TS, Ciarlini LSRP, Pedro WA, Beluccio ML, Queiroz LH, et al. Valores hematológicos de morcegos hematófagos Desmodus rotundus (E. Geoffroy, 1810) mantidos em cativeiro. Chiroptera Neotropical 2010; 16:780-785., Santos et al.¹⁰10. Santos AP, Mottin VD, Aita RS, Franciscatto C, Lopes STA, Franco WS, et al. Valores hematológicos e bioquímicos de morcegos hematófagos (Desmodus rotundus rotundus) no sul do Brasil. Acta Scientiae Veterinariae 2007; 35:55-58., Vilar et al.¹¹11. Vilar TD, Volino Vaz FM, Gitti CB, Cordeiro AV, Lobo R, Fonseca URC, et al. Hematologic values of Desmodus rotundus (Geoffroy, 1890) in Rio de Janeiro. Rev Univ Rural 2005; 25:57-58., Baptista¹²12. Baptista M, Esbérard C. Valores Hematológicos de Artibeus sp e Desmodus rotundus (Mammalia, Chiroptera). Rev Cient Inst Pesq Gonzaga da Gama Filho 1997; 3:11-22. and was concordant with those reported by Krutzsch and Winsatt¹³13. Krutzsch PH, Wimsatt WA. Some normal values of peripheral blood in the vampire bat. J Mammal 1963; 44:547-559.. However, in studies performed by Almeida et al.⁹9. Almeida BFM, Barbosa TS, Ciarlini LSRP, Pedro WA, Beluccio ML, Queiroz LH, et al. Valores hematológicos de morcegos hematófagos Desmodus rotundus (E. Geoffroy, 1810) mantidos em cativeiro. Chiroptera Neotropical 2010; 16:780-785., Santos et al.¹⁰10. Santos AP, Mottin VD, Aita RS, Franciscatto C, Lopes STA, Franco WS, et al. Valores hematológicos e bioquímicos de morcegos hematófagos (Desmodus rotundus rotundus) no sul do Brasil. Acta Scientiae Veterinariae 2007; 35:55-58., and Vilar et al.¹²12. Baptista M, Esbérard C. Valores Hematológicos de Artibeus sp e Desmodus rotundus (Mammalia, Chiroptera). Rev Cient Inst Pesq Gonzaga da Gama Filho 1997; 3:11-22., leucograms showed high standard deviations (approximately 3.0 × 10⁶/mm³ to 6.0 × 10⁶/mm³).

In relation to the results for differential white blood cell counts, few basophils and monocytes were evident in the blood smears, while eosinophils were not observed. The proportion of neutrophils to lymphocytes was 7: 3, which is similar to those values reported by Santos et al.¹⁰10. Santos AP, Mottin VD, Aita RS, Franciscatto C, Lopes STA, Franco WS, et al. Valores hematológicos e bioquímicos de morcegos hematófagos (Desmodus rotundus rotundus) no sul do Brasil. Acta Scientiae Veterinariae 2007; 35:55-58., Vilar et al.¹¹11. Vilar TD, Volino Vaz FM, Gitti CB, Cordeiro AV, Lobo R, Fonseca URC, et al. Hematologic values of Desmodus rotundus (Geoffroy, 1890) in Rio de Janeiro. Rev Univ Rural 2005; 25:57-58., and Baptista and Esbérard¹²12. Baptista M, Esbérard C. Valores Hematológicos de Artibeus sp e Desmodus rotundus (Mammalia, Chiroptera). Rev Cient Inst Pesq Gonzaga da Gama Filho 1997; 3:11-22..

As expected after a viral infection, there was a significant increase of lymphocytes and the proportion of neutrophils to lymphocytes changed from 7: 3 to 4: 6. In differential white blood cell counts, few basophils and monocytes were observed in blood smears, and eosinophils were not observed. No change in erythrocyte profiles was observed following experimental infection.

In conclusion, the values of erythrocytes observed in D. rotundus bats are in accord with several studies and may serve as reference values for this species, even considering the differences among methods of analysis used by each author. However, regarding leucocytes, more studies should be undertaken to better understand the wide variations in leucocyte counts observed in this species and to determine whether these differences are related to age, sex, size, or the reproductive state of bats, as observed by Ratnasooriya¹⁴14. Ratnasooriya WD, Udagama-Randeniya PV, Yapa WB, Digana PMCB, Dharmasini MG. Haematological parameters of three species of wild-caught microchiropteran bats, Miniopterus schreibersii, Taphozous melanopogon and Hipposideros lankadiva in Sri Lanka. J Sci Univ Kelaniya 2005; 2:27-40. and Valdivieso and Tamsitt¹⁵15. Valdivieso D, Tamsitt JR. Hematological data from tropical American bats. Can J of Zool 1971; 49:31-36. for other bats species.

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