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Chiroptera Neotropical 15(1), July 2009 Maintenance of the haematophagous bat Desmodus rotundus in captivity for experimental studies on rabies Marilene F. de Almeida¹, Caroline C. Aires² Luzia F. A. Martorelli¹, Rodrigo F. de Barros¹ and Eduardo Massad³ 1. Zoonosis Control Center of São Paulo Municipality. Rua Santa Eulália, 86 – Santana - São Paulo, Brasil – CEP 02031-020. 2. Zoology Museum of São Paulo University - CP 42694- CEP 04299-970. 3. School of Medicine. The University of São Paulo LIM 01 – HC/FMUSP, Av. Dr. Arnando, 455 – Departamento de Informática Médica - São Paulo, Brasil - CEP 01246-903 and Department of Infectious and Tropical Diseases (Hon. Prof.), London School of Hygiene & Tropical Medicine, London, UK. * Corresponding author. Email: [email protected] Abstract Current knowledge concerning rabies transmission by the haematophagous bat Desmodus rotundus is considerable. However, many aspects of the relationship between haematophagous bats and rabies still remain uninvestigated or contradictory. In an attempt to investigate some of these aspects, our group designed a cage system developed for the purpose of experimentally investigating rabies infections and the oral rabies immunization of haematophagous bats. The cages are made of polycarbonate, a transparent material that allows for total visualization of the bats for observation, photos and video monitoring and resistant to effects to bats excreta. Nine cages with independent air supply and exhaust systems are maintained on a stand. A prefilter unit using inner and outer HEPA filters guarantees air supply to bats through the cage interior, and air exhaustion provides for safe conditions for researchers. Drinking bottles are located externally. Adequate consumption of defibrinated blood, weight maintenance, low bat mortality (16.4%) and longevity are demonstrated herein. The maintenance of bats in these cages decreases the necessity of handling them and is consistent with the conclusion that the bats adapt well to captivity in this type of cage. Keywords: cage, captivity, Desmodus rotundus, rabies, vampire bats. health and breeding conditions (Greenhall 1935; Wimsatt and Guerrieri 1961; Wimsatt et al. 1973; Barnard 1995). In previous studies, the purpose of the cage was to facilitate studies investigating reproduction, behaviour and bat biology, but the design of such cages did not consider biosafety issues. These issues are critical, especially for such rabies studies as those attempting to determine the dose of rabies virus lethal to this species, the antibody response following experimental rabies infection, routes for immunization against rabies, and the possibility of rabies virus aerosol formation (Constantine 1962; 1967). The purpose of the present project was to develop a cage system for bats that permits experimental studies of rabies in captivity (Almeida et al. 2005a, b, Almeida et al. 2008), while equally considering the adaptation of the bats to captivity, essential for obtaining reliable results, and the biosafety precautions needed to protect laboratory staff. Introduction Several studies of antigenic typing and phylogenetic analysis of the rabies virus have indicated that bats are one of the most important vectors and/or reservoirs of rabies in the world (Nadin Davis et al. 2001, Krebs et al. 2005, Schaefer et al. 2005, Velasco-Villa et al. 2006, Kobayashi et al. 2007). Whereas rabies viruses associated with insectivorous bats were involved in 32 out of 35 (91%) human rabies cases that occurred in the United States from 1958 to 2000 (Messenger et al. 2002), the haematophagous bat Desmodus rotundus was associated with 136 out of 230 (59%) human rabies deaths reported in the Latin America from 2004 to 2007 (PAHO 2008), including outbreaks in northern Brazil that totalled 64 deaths (da Rosa et al. 2006; Barbosa et al. 2008). Additionally, as a rabies vector, haematophagous bats may cause heavy economic losses in the livestock industry in many Latin American countries (Ito et al. 2001; Favoretto et al. 2002). In 1932, Clark and Dunn maintained D. rotundus in captivity for the first time using defibrinated blood. Many studies have shown that these bats can be introduced successfully into the laboratory and maintained in numerous types of cages for prolonged periods in apparent good Material and methods The D. rotundus bats used in this study were collected in caves and in abandoned buildings from cities in the states of Minas Gerais and São Paulo, southeastern Brazil, between July 2001 434 Chiroptera Neotropical 15(1), July 2009 and February 2004. A total of 195 specimens (80 males and 115 females) were collected. The bats were transported in individual cotton bags measuring 25x30cm. They were weighed using a dynamometer of one Newton. All personnel who handled bats wore individual protection equipment and had been immunized against rabies; moreover, their titre of rabies antibodies was monitored every six months, as recommended in the São Paulo State technical manual (2003). The ethics committee at São Paulo University authorized all experimental procedures and the capture and captivity of bats was authorized by a Brazilian institution responsible for wild animal care (Instituto Brasileiro do Meio Ambiente, IBAMA). Bats were divided by sex and capture location into groups varying from 2 to 15 individuals. The groups were maintained in captivity for a period varying from four months up to one year. Once in captivity, bats were identified using a nylon collar and a designated letter (secur-a-tie®-08350 Avery Dennison, USA) (Figure 1). the upper screen permitted accommodation of the bats in accordance with the behavior of the species. The cages were placed on a rack, which holds nine cages, made of PVC and polypropylene (Figure 2), with two independent air handling systems: one for air supply and the other for air exhaustion. The external air was sucked into an axial motor and passed through a prefilter unit (G3, retention of 97% of particles measuring 3 to 7μm to increase the life span of the main filter). The cages were closed with aluminium lids - the upper lid was doubled so that an air filter unit (ALA 100) could be located between the upper aluminium lids. Outgoing air passed through this filter and was released to the outside. The air turnover rate in the cage was 50 times per hour, according to the manufacturer of the cages (Alesco® São Paulo, Brazil) (Figure 3). Figure 1: Collar and letters used on the bats for identification. The cages were maintained in an airconditioned room. Every day the minimum and maximum temperature of the room were noted. Light was turned on for two or three hours a day whilst the room was cleaned, food was provided and any technical procedures were performed. Once a week the bats were weighed and examined. During the day the room received direct, low-intensity illumination through two windows covered with a screen (mesh size 0.2 cm). The cages were cylindrical (40cm in diameter and 50cm in height) and made of polycarbonate, with reinforcement added to the PVC area in contact with faeces, urine and blood. The cages were provided with aluminium-wire (mesh size of 0.5cm) screens. The lower screen permitted separation of the bats from their excrements and Figure 2: Cylindrical form cages for Desmodus rotundus made of polycarbonate, with aluminium-wire screens at their extremities. Air 435 Chiroptera Neotropical 15(1), July 2009 supply and exhaustion system integrated through the interior of cages. Figure 3: Details of the locations of the ventilation system and the attached feeding bottles. Bats were fed with defibrinated swine blood. The swine blood was obtained weekly from a slaughterhouse, defibrinated and stored at 2-4°C. Under these conditions, the blood remains usable for a week; after this time, any remaining blood was discarded. Frozen blood was also used occasionally. Once a week, the bats were provided defibrinated blood supplemented with vitamins at a concentration of 1ml per litre of blood (Clusivol® Wyeth-Whitehall Ltda.). Once a day, 25 to 30 ml of blood per bat was provided at 25-30°C in the late afternoon. The bats were fed using external plastic bottles, similar to the models used with other laboratory animals (Beiramar, BBP250 São Paulo, Brazil), which were removed during the day, immersed in 1% sodium hypochlorite and then washed in running water. The statistical test applied was the chi square uncorrected test at the 95% confidence interval. The test was run using the software EPI INFO 6.0 to compare the mortality between large groups (more than 8 animals in a cage) and small groups (less than 8 animals in one cage) and to compare the weights of females and males. significant difference occurred in mortality between small and large groups (p=0.35; 95% CI). The average daily consumption of blood was 20.1ml, with a range of variation between 15.1ml and 29.1ml. The average daily consumption among groups of males was 20.3ml (+/- 2.0ml). The average daily consumption among groups of females was 20.0ml (+/- 2.1ml). The average daily consumption was 20.1ml for large groups and 25.3ml for small groups. The average weight of adult males was 28.6 g; with a range of variation between 25.4 and 32.5 g. The average weight of nonpregnant adult females was 33.4 g, with a range of variation between 30.6 and 38.7 g. The average weight of pregnant females was 54 g, with a range of variation between 48 and 60 g. The majority of nonpregnant females (88.9%) had gained weight by the end of the captivity period (an average of 1.93 g). In contrast, the majority of males (76.5%) had lost weight (on average 2.42 g) by the end of the captivity period. The difference between the increase in weight of females and the decrease in weight that occurred in males was significant (p= 52.14; 95% CI). The nylon collar used for bat identification caused local inflammation in 11% of the bats. Throughout the experimental period, 33 bats showed signs of being bitten by other bats (17%). The regions of body most frequently affected were the head (34%), the face (27%; eye, chin, lip and nasal region), scapula (17%), ear (15%), the thumb (5%) and the patagium (2%). The wounds Results Of the 195 total bats that had been captured throughout the experimental period, 32 bats died: the majority (23 bats, 72%) during the first 10 days of captivity; seven due to low weight during the winter, and two after fighting with other bats. Total mortality comprised 16.4% and occurred in both large and small groups of animals. No 436 Chiroptera Neotropical 15(1), July 2009 received no medical treatment and healed in two to three days. The average minimum temperature in the room was 20.7ºC and the average maximum temperature in the room was 23.8ºC. The relative humidity in the room ranged from 55% to 65%. blood but did not relate this to the amount of blood consumed; hence it became impossible to compare blood consumption in our captivity study with that of other studies. When bats are maintained in small groups or in isolation, the average daily consumption of blood tends to be higher, as in this case, because there is no competition for food. Under natural conditions, feeding is an individual performance and the competition for food is not a restrictive factor; however, in captivity, the cages are collective and the bats must share food. The inevitable competition, in particular among males, could reduce the average consumption of food per bat. Also, fights among females did not occur with the same frequency that they did among males, and it was observed, on a few occasions, that the females regurgitate blood for other members of the group. The average daily consumption of blood was 20.3ml for males and 20ml for nonpregnant females, representing 71% and 60%, respectively, of their total body weight, considering the average weight of adult males and nonpregnant females. Busch (1988) reported that vampire bats in captivity consumed an average of 60% of their body weight in blood over 30 minutes and Wimsatt and Guerrieri (1962) demonstrated that captive bats ingest large quantities of blood, sometimes exceeding 50% of their body weight. The temperature in the room was chosen based on previous studies (Wimsatt and Guerrieri 1962) using temperatures ranging from 21ºC to 25ºC. It was also considered that the average ambient temperatures of roosts are between 21ºC to 23ºC (Flores-Crespo and Sota 1991). However, it is possible that the room temperature influenced the weight of the bats during the winter. A decrease in weight, along with the death of individuals with low weight was also observed, the majority of cases occurring in males. The capacity for thermoregulation in D. rotundus is marginal (Wimsatt 1969). In the presence of lower temperatures, bats must expend a higher amount of energy than they can afford, simply for the maintenance of normal body temperature; particularly when bats are kept in small groups (Delpietro and Russo 2002). Water was provided for the first 21 days of captivity. However, it was not consumed and was thereafter removed. Results of investigations into the vampires’ water requirements have been variable. Altenbach (1981) reported that captive specimens of the haematophagous bat Diphylla ecaudata drank no water in captivity during 161 days of observation. Wimsatt and Guerrieri (1961) observed captive D. rotundus to drink little free water for a long as two years. Accordingly, it is evident that defibrinated blood provides the bats’ requirements for water; Discussion Air supply and exhaustion through the interior of cages were implemented in consideration of the possibility of aerosol formation by the rabies virus (demonstrated by Constantine 1962; 1967), and the high concentrations of rabies virus used in experimental infection and vaccination studies (Almeida et al. 2005a, b; Almeida et al. 2008). In addition, the system diminishes the characteristic smell of bat faeces and urine. Initially, groups consisting of small numbers of bats were maintained. These numbers were progressively increased in number (2 to 15) to establish the adequate number of bats per cage. Eight to ten bats seemed to be the ideal number per cage, so this range was adopted as the standard. When 8 to 10 bats were housed in a cage, two bottles of blood were sufficient to ensure adequate feeding, but for higher densities per cage, three or four bottles were required. External feeding bottles were chosen so that it was not necessary to open the cage to daily feed the bats. Bottles are preferable to open dishes or bowls used in other captivity projects (Wimsatt and Guerrieri 1962) as they limit contamination of the blood with the faeces and urine of the animals. In the captivity system described herein bats fed rapidly, in agreement with previous reports (Wimsatt 1969; Busch 1988). Within 10 to 30 minutes the bottles were emptied. In general, for the first two days, the bats refused to accept food; however, blood consumption increased rapidly, stabilizing after one to two weeks of captivity. Swine blood was chosen as the food source because was most easily obtained in sufficient quantity to keep the animals. Swine blood is among the most preferable for this bat species according Goodwin and Greenhall (1961), and it was used successfully by Setien et al (2002). As bats feed directly from mammals in nature, their food is presumably nearly always at the standard mammalian body temperature. Hence, all blood was warmed to 25° - 30°C before being presented. According to Wimsatt and Guerrieri (1962) a daily blood consumption average of 15 to 16ml is sufficient to maintain vampire bats in captivity. Greenhall (1935) reported that captive vampire bats consume an average of 10 to 25ml of blood each evening; however, they did not relate this value to the volume that was offered. Setien et al (2002) reported that bats received 15 to 20 ml of 437 Chiroptera Neotropical 15(1), July 2009 1962; Barnard 1995; Delpietro and Russo 2002) for defence and in social interactions. Levels of dominance are determined by fights among males for access to the preferred female roosting sites (Wilkinson 1985; Lord 1992). The rates of aggression can reflect this dominant behavior and could explain why the same bats were bitten many times, while others never showed any bites. As expected, the region of the body most frequently affected was the head and face. Another explanation is that due to the cage size restriction, mothers had great difficultly in protecting their young, and babies were bitten by adults, as previously observed by Winsatt and Guerrieri (1961) and Greenhall (1935). Generally the wounds completely healed within two or three days; however, two bats died after fights as a result of their wounds. Some groups showed one dominant bat that refused to share the food with the others in the group. It was not possible to compare the mortality rate obtained in this work with rates found for other captivity studies of D. rotundus due to a lack of previously published data. For Artibeus lituratus and Sturnira lilium, the mortality rate in captivity is 28% and 50%, respectively (Rasweiler and Ishiyama 1973); for Glossophaga soricina the mortality rate is 11% in captivity and in Carolia perspicillata the mortality rate in captivity is 7% (Rasweiler and Bonilla 1972). It is not possible to compare the rates of mortality among species of bats maintained under different captivity conditions. It was not the aim of this study to evaluate reproduction in D. rotundus and the survival of young bats in captivity; however, among the 115 females, 40 (34.8%) were pregnant, all of whom conceived prior to capture. Of these, 6 females died in the adaptation period, 16 pregnancies ended in miscarriage, 6 premature young survived less than 12 hours, 8 survived less than 30 days and 4 lived to adulthood. Most of the miscarriages (91.7%) occurred between one and two months after the introduction of the females into captivity, during the adaptation period. According to Wilkinson (1985), the infant mortality of D. rotundus in nature is 54% (Wilkinson 1985). A major disadvantage of the air supply and exhaustion system of the cages is the cost. The Hepa filters cost approximately 150 dollars each, which is moderately expensive, and have to be changed every six months. However, these cages are extremely suited to use by a group of researchers in many studies; the great durability of the cages, the quality of observation enabled by photos and video monitoring, and the built-in biosafety precautions compensate for this expense. The adequate consumption of defibrinated blood, low bat mortality (16.4%), longevity, however, according to Busch (1988), bats consume 38% less blood when provided with free access to water. In the first week of captivity, the bats would feed neither when the lights were on in the secure environment nor in the presence of humans, but after a few days they accepted food under these conditions. Normally, the bats fed immediately after being provided with blood. Only two groups of seven and eight males never accepted food in the presence of humans or in a lit environment, even after five and six months of captivity, respectively. When the blood was provided, these groups of bats waited until the lights had been turned off and the people had withdrawn from the room. They then descended to feed, but they always remained alert, and at the slightest sound, they returned to the upper screen. The great majority of bats, however, descended to the lower screen and waited near the drinking place at feeding time. As stated in the literature, on average, mature, nonpregnant females were heavier than adult males. In our captivity their range of weight variation was 30.6 to 38.7g, higher than that observed (29 to 33g) by Wimsatt (1969). Among males, the average body weight was 28.6 g, with a range of variation between 25.4 and 32.5 g. Although the majority of males (76.5%) had lost weight (on average 2.42 g) by the end of the captivity period, their average weight was similar to that observed by Bush (1988) (30.1 g) and Wimsatt (1969) (29.1 to 30.8 g). Polycarbonate was chosen as the cage material because it is transparent, allowing for full visualization of the bats for observation, photos and video monitoring. Also, this material was resistant to the corrosive effects of bat excreta. Faeces and urine from bats were copious. Urine flow begins promptly after the onset of feeding. Ammonia is corrosive to many types of material, but the aluminium screens used in the cages were resistant to the effects of the excreta. The aluminium-wire was easily removable, facilitating cage cleaning. When the lower aluminium screens were immersed in 1% sodium hypochlorite for 20 to 30 minutes for disinfection, the excreta were completely dissolved. The cages were washed with water and washing powder. It has been reported that bats are usually heavily infested by ectoparasites of several species (Wimsatt and Guerrieri 1961; Greenhall 1935). In our captivity study, only one species of parasite was detected during a short period, albeit in large numbers. We identified it as Necrobia rufipes, a beetle (Coleoptera) of the Family Cleridae. N. rufipes is a necrophagous insect that is most likely attracted by the smell of blood. Haematophagous bats present naturally aggressive behaviour (Wimsatt and Guerrieri 438 Chiroptera Neotropical 15(1), July 2009 Barnard S.M. 1995. Bats in captivity. Wild Ones Animal Books. California USA, 194p. Busch C. 1988. Consumption of blood, renal function and utilization of free water by the vampire bat (Desmodus rotundus). Comparative Biochemistry and Physiology 90 A(1): 141-146. Clark H.C. and Dunn L.H. 1932. Experimental studies on Chagas’Disease in Panama. American Journal Tropical Medicine 12(1): 4977. Constantine D.G. 1962. Rabies transmission by nonbite route. Public Health Report 77(4): 287289. Constantine D.G. 1967. Rabies transmission by air in bat caves Public Health Report 82: 867888. da Rosa E.S.; Kotait I.; Barbosa T.F.; Carrieri M.L.; Brandão P.E.; Pinheiro A.S.; Begot A.L.; Wada M.Y.; de Oliveira R.C.; Grisard E.C.; Ferreira M.; Lima R.J.; Montebello L.; Medeiros D.B.; Sousa R.C.; Bensabath G.; Carmo E.H. and Vasconcelos P.F. 2006. Battransmitted human rabies outbreaks, Brazilian Amazon. Emerging Infection Diseases Aug; 12(8): 1197-202. Delpietro V.H.A and Russo R.G. 2002 Observations of commom vampire bats Desmodus rotundus and hairy-legged vampire bat Diphylla ecaudata in captivity. Mammalian Biology 67:65-78 Favoreto S.R.; Carrieri M.L. and Cunha, C.M. 2002. Antigenic typing of Brazilian rabies virus samples isolated from animals and humans, 1989-2000. Revista Instituto Medicina Tropical São Paulo 44(2): 91-95. Flores-Crespo R. and Sota A. 1991. Biology and Control of the Vampire Bat. In: The Natural History of Rabies (edited by Baer G.M.). 2nd Edition CRC Press, INC. Boca Raton, Florida. Goodwin G.C. and Greenhall A.M. 1961. A review of the bats of Trinidad and Tobago. Bulletin of American Museum Natural History. 122:187. Greenhall A. M. 1935. Notes on behavior of captive vampire bats. Mammalia 441-451. Ito M.; Arai Y.T.; Itou T.; Sakai T.; Ito F.H.; Takasaki T. and Kurane I. 2001. Genetic characterization and geographic distribution of rabies virus isolates in Brazil: identification of two reservoir, dogs and vampire bats, Virology 284: 214-222. Kobayashi Y.; Sato G.; Kato M.; Itou T.; Cunha E.M.; Silva M.V.; Mota C.S.; Ito F.H. and Sakai T. 2007. Genetic diversity of bat rabies viruses in Brazil. Archives of Virology 152(11): 1995-2004. Krebs J.W.; Mandel E.J.; Swerdlow D.L. and Rupprecht C.E. 2005. Rabies surveillance in the United States during 2004. Journal of weight maintenance and the overall healthy maintenance of the bats decreased the necessity of handling them, thereby avoiding disturbance within the cage while demonstrating that the cage provides good conditions for bats in captivity. Acknowledgements The authors are grateful to Carlos Campanerii at the Zoology Museum of São Paulo University and the Entomology Department at CCZ-SP for identification of the Coleoptera. The following people provided volunteer field assistance for bat location and capture: Carlos Alberto Venésio Gomes of the Vigilância Sanitária of Paraisópolis-Minas Gerais; Alberto Dangeri of the Prefeitura Municipal de Itatiba; and Luis Carlos Ismerin and José Carlos Pinto of the Escritório de Defesa Agropecuária of Sorocaba, São Paulo. The authors are also grateful to Vilma A Duarte Sanches, Valtair Santana and Ruberval da Silva of the University of São Paulo School of Medicine. 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