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Acquired Resistance to Saliva Anticoagulants by Prey Previously Fed upon by Vampire Bats (Desmodus rotundus): Evidence for Immune Response

Horacio A. Delpietro , Roberto G. Russo
DOI: http://dx.doi.org/10.1644/07-MAMM-A-374.1 1132-1138 First published online: 15 October 2009

Abstract

Ranchers have observed that residual hemorrhage decreases after livestock suffer repeated bites from common vampire bats (Desmodus rotundus). We hypothesized that repeated exposure to the anticoagulants in the bats' saliva generated an immune response. We compared clotting time of blood mixed with Desmodus saliva between individuals of several species of livestock that were regularly exposed to vampire bats (“bat-attacked”) and individuals living where bat predation was not observed (“bat-naïve”). We also compared the bleeding times of domestic sheep following a single defensive bite from a bat, before and after subjecting the animals to a series of feeding bites by Desmodus. Clotting time was significantly shorter in bat-attacked than in bat-naïve livestock. Bleeding time after a provoked defensive bat bite was significantly shorter after sheep were exposed to a series of feeding bites from Desmodus. Bat predation induced increased resistance by the prey against the anticoagulants, suggesting an immune response.

Key words
  • acquired resistance
  • anticoagulants
  • common vampire bat
  • Desmodus rotundus
  • vampire saliva

The common vampire bat (Desmodus rotundus) is a strictly hematophagus species that is abundant in cattle-raising areas from Mexico to northern Argentina. At the present time, common vampire bats feed mainly on domesticated cattle, horses, goats, pigs, and sheep, and to a lesser extent on poultry, wild prey, and humans (Crespo et al. 1961; Dalquest 1955; Greenhall et al. 1983; Lord et al. 1981). The presence of vampire bats is closely related to the availability of prey, roosts, and suitable habitats such as forests or hills or both, resulting in zones in which vampire bat predation is endemic, and other zones in which predation is sporadic or nonexistent (Crespo et al. 1961; Delpietro et al. 1992). Every year, paralytic rabies transmitted by vampire bats kills hundreds of thousands of cattle and dozens of people, as well as causing cases of rabies in wild species (Da Rosa et al. 2006; Delpietro et al. 1997; World Health Organization 1984, 1992, 2004).

Special anatomical and physiological traits of vampire bat digestive and urinary systems facilitate blood feeding. Vampire bats have large, sharp upper incisor teeth that make possible the removal of a circular piece of skin with a single bite, exposing the subcutaneous tissue of the prey from which it licks the blood for about 20–30 min (De Verteuil and Urich 1936; Greenhall 1972; Greenhall et al. 1969, 1983). The saliva of the bat contains anticoagulant and fibrinolytic proteins, which are inhibitors of coagulation, plasminogen activators, and inhibitors of platelet aggregation (Apitz-Castro et al. 1995; Di Santo 1960; Fernandez et al. 1998, 1999; Gardell et al. 1989; Hawkey 1966, 1967; Kratzschmar et al. 1991, 1992; Romana 1939). These proteins help maintain bleeding while the bat is feeding and liquidity of the blood inside the stomach and intestine during digestion (Greenhall et al. 1969, 1983; Lord et al. 1981; McFarland and Wimsatt 1969; Mitchell and Tigner 1970; Rouk and Glass 1970; Wimsatt 1969; Wimsatt and Guerriere 1962). The anticoagulant effect of the bat saliva is intense and persistent and causes in the prey “residual hemorrhaging,” that is, blood continues to flow from the wound sometimes for hours after the bat has finished feeding (Dalquest 1955; Goodwin and Greenhall 1961; Greenhall et al. 1969, 1983; McFarland and Wimsatt 1969). The anticoagulant effect of the bat saliva also can be observed through in vitro mixing of the saliva with blood (Apitz-Castro et al. 1995; Romana 1939). The defensive bites of the bat (i.e., bites without relation to feeding) are swift and produce wounds and hemorrhaging, although wounds are smaller in size than those produced by the feeding bites (Dalquest 1955; Greenhall 1972).

Ranchers frequently report observations that when horses and cattle from areas free of vampire bats (“bat-naïve” animals) were introduced to areas of endemic bat predation, residual hemorrhages produced by the 1st bat bites were worse and more prolonged than those of the resident “bat-attacked” animals. Ranchers also observed that, as the bat-naïve animals continue to suffer bites, the residual hemorrhaging begins to diminish until reaching, after about 4–6 months, a level of intensity and persistence similar to that observed in the resident animals. We hypothesized that contacts between the anticoagulants of the bat saliva and the tissues of the prey during repeated feeding bites of bats could generate an immunological response by the prey that would neutralize or destroy part of those anticoagulants. This could be observed in nature by comparing the clotting time of blood mixed with saliva of vampire bats between bat-attacked and bat-naïve animals of the same species. This also could be observed experimentally by comparing the clotting time of blood mixed with bat saliva, and the bleeding time of the wound caused by a single, provoked defensive bite from a vampire bat, in a bat-naïve animal before and after exposure to a series of bat feeding bites.

Materials and Methods

Bats.—We used 37 adult common vampire bats captured in an area where there were no outbreaks of rabies in the previous 5 years. After a quarantine of 90 days during which they remained healthy and no rabies virus was isolated from 2 samplings of saliva made 30 and 60 days following capture, bats were considered to be rabies free and appropriate for use in the study. The bats were divided in 2 groups: 20 bats were used for extraction of saliva and the other 17 to obtain feeding and defensive bites on sheep. This was done to avoid handling and inoculations of pilocarpine to those bats that were to be used as the source of feeding bites and defensive bites on sheep. Each group was placed in a cage 3.8 m long × 3.2 m wide × 2.2 m high, made of wood and wire mesh, and kept inside a building of the Regional Laboratory of Servicio Nacional de Sanidad y Calidad Agroalimentaria, located in a rural area 10 km southwest of Posadas, Misiones Province, Argentina. Bats were fed with defibrinated (stirring with a stick) cattle blood administered in dishes at ambient temperature and ad libitum, or on live sheep during the experiment. Collection and observations of vampire bats were made under provincial and federal permits and followed the guidelines approved by the American Society of Mammalogists for the use of wild mammals in research (Gannon et al. 2007).

Extraction and conservation of bat saliva.—Saliva was extracted from each bat once every 6–9 days. For extraction, the bat was immobilized with a straitjacket (Fig. 1) to diminish the risk of the operator suffering bites, and to avoid harm to the bat due to its struggles. The bat was then inoculated subcutaneously with 0.2 mg of pilocarpine (0.1 ml of pilocarpine diluted 0.2% in Ringer's solution) to stimulate salivation. Maintaining the bat with the head hung down, the secreted saliva was collected for 15–20 min in a petri dish placed on ice under the bat. The quantity of saliva obtained per bat per extraction varied between 0.3 and 0.6 ml. The saliva obtained from 8 to 10 bats was placed in a tube and centrifuged for 15 min at 5,000 rpm for elimination of cells and other impurities. The supernatant was kept in another tube and maintained at −30°C. When sufficient saliva was obtained for the study, the tubes were thawed and placed in a dish on ice. There, saliva was mixed carefully for 10 min and then fractioned to tubes (0.5 ml each) and maintained at −30°C until use. Thus, the same batch of saliva was used throughout the study.

Fig. 1

Common vampire bat (Desmodus rotundus) biting the edge of a sheep ear during a provoked defensive bite.

Observations of clotting time.—To compare resistance against the anticoagulants of saliva of the vampire bat among prey animals, we observed the clotting time of blood of the animals mixed with saliva of the bat (in nature, the bat ingests the blood of the prey mixed with its saliva). The saliva was mixed in the proportion 1:60 (0.1 ml saliva, 5.9 ml blood) for observations in cattle and horses, and in the proportion 1:30 (0.1 ml saliva, 2.9 ml blood) for observations in goats, sheep, and pigs, because in previous assays we observed that cattle and horses were more sensitive to anticoagulants than are goats, sheep, and pigs. The use of different dilutions and volumes in the clotting experiments is valid because comparisons were made between bat-attacked and bat-naïve animals of the same species, using the same volume and dilution for each species. Blood was obtained by syringe from the jugular vein and was placed in a glass tube (13 mm diameter, 90 mm long) containing the recently thawed bat saliva. The tube was inverted and rotated for 15 s to mix the contents and then was placed vertically at a constant temperature of 38°C. To observe coagulation the tube was inclined about 150° every 4–6 min. We considered coagulation to be observation of a fixed clot in the bottom of the inclined tube, or a clot slipping toward the stopper without losing its form. Increased viscosity or formation of soft clots that returned to a liquid state when the tube was inclined was not considered to be coagulation. As a control, we observed the clotting time of 3 ml of blood without saliya.

Observations of bleeding time.—To compare resistance against the anticoagulants of bat saliva in sheep before and after exposure to feeding by vampire bats, the bleeding time of a single wound caused by a provoked defensive bite of a bat was observed. For that purpose, the bat was immobilized with a straitjacket that left free only the head, which was moved close to the edge of the sheep's ear, which was quickly bitten by the bat (Fig. 1). The bite produced a wound of 4–6 mm with a shape between semicircular and triangular that bled quickly (Fig. 2). To check hemorrhaging, the wound was touched at 15- to 25-s intervals with a strip of cellulose filter paper (grade 1; Whatman, Maidstone, Kent, United Kingdom), and the time was measured with a digital electronic stopwatch. We considered bleeding time to be the time between the bite and the 1st touch of the wound in which the paper was retracted without a blood stain. As control, the bleeding time of an incision by scalpel (5 mm long, 2 mm deep) in the edge of the other ear was observed. Although the incision by the scalpel differed in shape from the wound caused by the defensive bite of the vampire bat, it was similar in size to a defensive bat bite and was free of bat saliva.

Fig. 2

Wound in a sheep ear caused by the provoked defensive bite of a common vampire bat (Desmodus rotundus).

Field observations.—Observations on adult cattle, horses, goats, sheep, and pigs were made in northern Argentina (Table 1). In each species (using animals of the same breed and reared in similar conditions) the clotting time of blood mixed with bat saliva was compared between bat-attacked and bat-naïve animals (Table 2).

View this table:
Table 1

Farms in northern Argentina with predation of the common vampire bat (Desmodus rotundus; +) and without vampire bat predation (−) where the clotting time was compared among bat-attacked and bat-naïve animals, province, coordinates, species observed, and sample size.

FarmProvinceCoordinatesSpecies observedSample size
Granada S.A.+Corrientes27°39′S,55°59′WCattle47
E. Oria −Corrientes29°22′S,58°45′WCattle38
Granada S.A.+Corrientes27°39′S,55°59′WHorses29
E. Oria −Corrientes29°22′S,58°45′WHorses27
J. Soriano+Salta25°55′S,65°52′WGoats38
B. Martorell −Salta25°59′S,65°49′WGoats37
J. Soriano+Salta25°55′S,65°52′WSheep19
SENASA −Misiones27°28′S,55°48′WSheep20
A. Bentancur +Misiones27°36′S,55°58′WPigs13
M. Godina −Misiones27°26′S,55°57′WPigs16
View this table:
Table 2

Comparisons among bat-attacked (+) and bat-naïve animals (−) of the same species (see Table 1) of the clotting time (min) of blood mixed with saliva of the common vampire bat (Desmodus rotundas)a and of blood (as control), sample size (n), mean value ± standard deviation ( ± SD), range, and P-values of the differences in each species from t-tests or Mann–Whitney U-tests depending on whether the data were normally distributed or not.

Clotting time (min)
Blood mixed with bat salivaaBlood (controls)
Species and bat predationnX̄ ±SDRangePX̄ ± SDRangeP
Cattle+4732 ±2110–13011 ± 29–16
Cattle −3842 ± 2419–151< 0.0211 ± 19–140.89
Horses +2938 ± 288–11015 ± 47–26
Horses ℒ27161 ± 10555–380« 0.000116 ± 57–290.48
Goats +3823 ± 87–4311 ± 27–15
Goats −3731 ± 912–49< 0.000211 ±27–160.84
Sheep +1918 ± 97–3311 ± 27–15
Sheep −2030 ± 620–41< 0.000110 ± 27–160.09
Pigs +1339 ± 385–1205 ± 14–8
Pigs−1677 ± 4415–142< 0.035 ± 14–90.91
  • a For observations with cattle and horses saliva was mixed in the proportion 1:30 (1 part saliva, 29 parts blood) and for observations with goats, sheep, and pigs in the proportion 1:60 (1 part saliva, 59 parts blood).

Experimental observations of sheep.—We observed the clotting time of blood mixed with bat saliva and the bleeding time after bat bites in bat-naïve sheep before and after subjecting them to feeding bites of vampire bats. We used 7 Romney ewes of 20–25 months of age with an average weight of 51 kg, which were maintained in a pasture in an area where vampire bats were not known to occur. To identify individual sheep, we took advantage of the mandatory ear tags implanted during vaccination against foot and mouth disease (Figs. 1 and 2). Procedures used in the experiments with sheep were approved by the Ethics Committee of the Consejo Profesional de Médicos Veterinarios de la Provincia de Misiones, Argentina (license 02, 15 February 2006).

For each exposure to bats, 2 sheep were placed into a cage with 4 vampire bats and remained there for 2 h. After exposure, any fresh bites suffered by each sheep were counted, the wool around the wounds was washed to eliminate residual hemorrhage, and a topical insecticide was applied to avoid myasis. Each sheep received 1–4 bites per exposure. Sheep were exposed to bats 14–16 times from 1 October 2006 to 23 January 2007, until each received 41–43 feeding bites. This procedure did not seem to cause psychological or physical damage to sheep, because they willingly entered into the bat cage to eat corn placed there, and their body weight had increased an average of 1.6 kg after the bouts of exposures compared an average weight gain of 1.2 kg for sheep in the same cohort maintained in the same pasture but not used in the experiment. The clotting time of blood mixed with bat saliva and of blood without bat saliva was examined in the experimental sheep on 27 September 2006 and on 8 February 2007, and the bleeding time after a bat bite and by incision was examined on 29 September 2006 and on 10 February 2007.

Statistics.—We used 2-sample t-tests or Mann–Whitney U-tests to compare the clotting time of blood mixed with bat saliva between bat-attacked and bat-naïve animals in our field observations, depending on whether the data were normally distributed or not (Table 2). In the experimental observations on sheep, we used paired t-tests to compare the clotting time of blood mixed with bat saliva and the bleeding time after a bat bite before and after exposure of the sheep to bat feeding bites (Table 3).

View this table:
Table 3

Comparisons in sheep of the clotting time of blood mixed with bat salivaa (blood + saliva) and of the bleeding time of a provoked defensive bite of the common vampire bat (Desmodus rotundas) on the edge of a sheep ear (see Fig. 2), before and after the sheep received a series of 41–43 feeding bites from a vampire bat. The clotting time of blood and the bleeding time of a scalpel incision were observed as control.

Clotting time (min)Bleeding time (min)
Blood + salivaBlood alone (controls)By bat biteBy incision (controls)
SheepBeforeAfterBeforeAfterBeforeAfterBeforeAfter
468332610119555
46932229108645
470282011119654
472363315139745
473352511107746
4744723111112855
475362612119644
Average352511119645
t = 4.15t = 0.69t = 4.87t = 1.16
P < 0.006P = 0.52P < 0.003P = 0.29
  • a Bat saliva was mixed with blood in proportion 1:30 (1 part saliva, 29 parts blood).

Results

Field observations.—Clotting time of blood mixed with bat saliva was significantly shorter in bat-attacked than in bat-naïve animals in all species observed (Table 2), indicating that resistance to the anticoagulants of bat saliva is stronger in bat-attacked than in bat-naïve animals. Lack of significant differences in the clotting time of blood (controls) between bat-attacked and bat-naïve animals in all species observed (Table 2) indicates that bat predation did not influence the physiological mechanism of blood coagulation of the prey.

Experimental observations.—Clotting time of blood mixed with bat saliva and bleeding time after a bat bite observed 16 and 18 days, respectively, after the last exposure of a sheep to a series of feeding bites by vampire bats were significantly shorter than those observed before the exposure (Table 3). Thus, vampire feeding bites induced a lasting increase in resistance by sheep to the anticoagulants of bat saliva. Lack of significant differences in the clotting time of blood without bat saliva, and in the bleeding time after incisions by a scalpel (Table 3), indicate that exposure to vampire feeding bites did not affect the physiological mechanism of blood clotting of the sheep.

Discussion

The fact that bat predation induced an increase in resistance against the anticoagulants of bat saliva without affecting the normal parameters of coagulation of the prey (i.e., the clotting time of blood and the bleeding time of wounds free of vampire saliva) suggests the existence of an immune response of the prey against the anticoagulants. This is not surprising, keeping in mind that at least 3 aspects of bat predation can favor an immune response of the prey against the anticoagulants. First, the feeding behavior of vampire bats is similar to that of parasites because the prey is not killed. Repeated feeding on the same animal therefore may occur, either by the same bat on successive nights or by other bats (Greenhall 1972; Greenhall et al. 1983). Second, the abundant secretion of saliva and the persistent licking of the wound for about 30 min during each bat feeding facilitates a lingering contact between the bat saliva and the subcutaneous or muscular tissues, or both, of the prey (Greenhall 1972; Greenhall et al. 1983). Third, anticoagulants are large molecules (proteins) that can behave as antigens (Apitz-Castro et al. 1995; Fernandez et al. 1998, 1999; Gardell et al. 1989; Hawkey 1966, 1967; Kratzschmar et al. 1991, 1992). Acquired immunity against saliva, tissues, and organs of multicellular parasites has been reported in mammals, a response that facilitates damaging and killing ticks and other parasites (Boag et al. 2003; Dalton and Mulcahy 2001; Momim et al. 1991; Pruett 1999; Trimnell et al. 2002; Viney 2002; Wikel and Alarcon-Chaidez 2001; Willadsen 1999, 2001).

The decrease in residual hemorrhage in the bitten animals, as observed by us and the ranchers, suggests an induced mechanism that targets anticoagulant proteins in the saliva of the bats. On the other hand, these observations suggest that the marked preference of vampire bats to feed on younger animals (Arellano et al. 1971; Dalquest 1955) could be related to the possibility of finding less resistance against their anticoagulants in those animals. Young animals have a shorter period of potential exposures to the antigenic challenges of bat saliva than do adults, so their possibility of generating an immune response is less. Immune responses positively associated with the quantity of antigenic challenges were reported in host-parasite systems between mammals and ticks (Dizij and Kurtenbach 1995; Gebbia et al. 1995; Momin et al. 1991; Need and Butler 1991). Also, the immune system of young animals is still not fully developed and their immune response is weaker than that of adults (Pihlgren et al. 2004; Pilorz et al. 2005).

The possibility of diminishing the hemorrhage caused by bat bites constitutes an advantage for the prey by saving energy and also by diminishing the risk of myasis, which is more frequent in bites with much hemorrhaging (Delpietro et al. 1994, 1999; Kverno and Mitchell 1976; Thompson et al. 1977). Conversely, increasing resistance of the prey against the anticoagulants could be unfavorable for the bats. Less hemorrhaging by the prey implies a larger energy expense and a greater time required to complete feeding for the bats. This also may be hazardous for the bats because of the risk of attacks by their own predators increase while they are feeding (Delpietro et al. 1994). Likewise, the blood of prey with a high level of resistance against the anticoagulants could coagulate inside the stomach of the bat, obstructing or retarding digestion, absorption, and the elimination of excess water, because bats need to maintain the liquidity of the ingested blood for those functions (Greenhall et al. 1983; Lord et al. 1981; McFarland and Wimsatt 1969; Mitchell and Tigner 1970; Rouk and Glass 1970; Wimsatt and Guerriere 1962). Difficulties in ingestion and digestion caused by premature coagulation of blood could be dangerous for vampire bats, due to their inability to withstand prolonged deficiencies in nourishment (McFarland and Wimsatt 1969; Wimsatt and Guerriere 1962).

To our knowledge, this is the 1st report of acquired resistance of mammals against anticoagulants of saliva of common vampire bats. The results of this study encourage future research on immunologic alternatives for control of vampire bats, particularly those that aim to induce a strong immune response by the prey to the anticoagulants. Development of nonchemical methods for controlling vampires is necessary, because current measures of control based on the use of coumarin-derived poisons (Flores Crespo et al. 1976; Greenhall et al. 1993; Linhart et al. 1972; Schmidt et al. 1978) constitute a threat for several species of nonhematophagous bats and other mammals sharing caves with the vampires (Brass 1994; Chiroptera Specialist Group 1996; Greenhall et al. 1993; Mayen 2003; Walker et al. 2001).

Acknowledgments

We thank S. Alvarez de Toledo, G. Cevey, L. Giovannoni, M. Godina, R. Guillen, B. Martorell, L. Oria, P. Pohl, J. Soriano, and J. Zach for assistance in the field. We thank R. D. Lord for assistance in preparation of the manuscript, and for reviewing the several early drafts.

Footnotes

  • Associate Editor was Fritz Geiser.

Literature Cited

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