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Anti-Leptospira spp. antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance
Accepted Manuscript Title: Anti-Leptospira spp. antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance Author: T.C.S. Rodrigues A.L.Q. Santos A.M.C. Lima D.O. Gomes V.L.C. Brites PII: DOI: Reference:
S0001-706X(16)30059-6 http://dx.doi.org/doi:10.1016/j.actatropica.2016.02.006 ACTROP 3850
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
13-7-2015 8-2-2016 9-2-2016
Please cite this article as: Rodrigues, T.C.S., Santos, A.L.Q., Lima, A.M.C., Gomes, D.O., Brites, V.L.C., Anti-Leptospira spp.antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Anti-Leptospira spp. antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance
Rodrigues, T.C.S.a; Santos, A.L.Q.a; Lima, A.M.C.b; Gomes, D.O.b; Brites, V.L.C.c
a
Wildlife Teaching and Research Laboratory (LAPAS), College of Veterinary
Medicine (FAMEV), Federal University of Uberlândia (UFU). Avenida Amazonas 2245,
Jardim
Umuarama,
38405-302
Uberlândia,
MG,
Brazil.
[email protected] b
Laboratory of Infectious Diseases, College of Veterinary Medicine (FAMEV),
Federal University of Uberlândia (UFU). Rua Ceará, s/n, Bloco 2D, Sala 33, Campus Umuarama, 38405-315, Uberlândia, MG, Brazil. [email protected] c
Reptile Sector, Institute of Biology, Federal University of Uberlândia (UFU). Rua
Ceará, s/n, Bloco 2D, Campus Umuarama, 38405-315, Uberlândia, MG, Brazil. [email protected]
Corresponding author:
Thaís Carneiro Santos Rodrigues. Postal Address: Rua das
Petúnias, 131 Cidade Jardim, 38412-112 Uberlândia, MG, Brazil. Phone: 55(34)8861-2836. E-mail: [email protected]
2
Graphical Abstract
Highlights
We studied the occurrence of anti-Leptospira spp. antibodies in captive rattlesnakes 90% of the snakes were positive in the microscopic agglutination test Most frequent serovars were Javanica, Andamana and Patoc These snakes can present high titers despite the absence of clinical symptoms We focus on the importance of leptospirosis as an occupational zoonosis
3 ABSTRACT: Leptospirosis is a worldwide spread zoonosis that can affect all groups of vertebrates, including reptiles. Because it has been little studied in snakes, this study focused on determining the occurrence of anti-Leptospira spp. antibodies in 64 Crotalus durissus collilineatus kept in captivity and on identifying the most common serovars in these animals, using the microscopic agglutination test. Of these, almost 90% were positive and there were reactions to the 22 serovars used in the study. The most common serovar in these snakes was Javanica, Andamana and Patoc. Most frequent titers were 25 and 50, although high titers (such as 1600) were also recorded, despite the absence of clinical symptoms. The possibility should be considered of captive snakes serving as a serious source of leptospiral infection in humans, which is why it is essential to study, prevent and control the disease in breeding centers and serpentariums.
Keywords: Leptospirosis; Reptiles; Snakes; Serology; Zoonosis.
4 1. INTRODUCTION Leptospirosis is the world’s most widespread zoonotic disease (Jamshidi et al., 2009) and is therefore extremely important to human and animal health (Faine et al., 1999). It is caused by spirochetes of the genus Leptospira and these bacteria comprise several pathogenic and saprophytic species that are classified into serogroups. Each serogroup is composed of a number of varieties, called serovars (Adler and Moctezuma, 2010). A single animal can be host to various serovars, and one serovar can infect multiple hosts (Quinn et al., 2005). Many species of domestic and wild mammals are known to act as natural or accidental hosts of several Leptospira serovars. However, little is known about the participation of reptiles in the maintenance and dissemination of the pathogen in the environment (Lindtner-Knific et al., 2013). These animals can play important roles in the epidemiological cycle of the disease by keeping the infectious agent in the environment and transmitting it to other animals (Glosser et al.,1974; Lindtner-Knific et al., 2013). According to Hyakutake et al. (1980), snakes may be important natural reservoirs of leptospira, particularly because of their normal diet of rodents, which are the main reservoirs of the pathogen in the environment (Faine et al., 1999). Leptospirosis in snakes is usually not associated with clinical symptoms (Wallach, 1983), but the occurrence of renal changes in an infected snake has been reported in the past (Abdulla and Karstad, 1962). The number of snakes raised in captivity has increased considerably in recent years. In addition to becoming pets, there are numerous snake breeding centers for scientific and commercial purposes. Despite the absence of reports about the transmission of leptospirosis from reptiles to humans (Ebani and Fratini, 2005), it is
5 important to investigate the occurrence of leptospirosis in these animals to prevent the risk of exposure to the causative agent of the disease (Silva et al., 2009). Biologists, veterinarians and snake handlers may also be at risk of infection (Ebani and Fratini, 2005). The incidence of different serovars in human populations is highly dependent on reservoir hosts in the region in question and on the serovars they carry (Bharti et al., 2003). Thus, determining which animal species act as hosts of leptospira in a given area is critical to the control and prevention of leptospirosis (WHO, 2003; Desvars et al., 2010). Therefore, the purpose of this study was to evaluate the occurrence of anti-Leptospira spp. agglutinins in Crotalus durissus collilineatus rattlesnakes living in captivity in a breeding facility, and to identify the serovars appearing most frequently in these animals, using the microscopic agglutination test (MAT).
2. MATERIAL AND METHODS All the procedures were performed with the approval of the Ethics Committee on Animal Use – CEUA/UFU (Protocol 120/14) and the authorization of the Brazilian Institute of Environment and Renewable Natural Resources – IBAMA (SISBIO Permit No. 46845).
2.1.
Animals and study area
Sixty-four samples of blood serum from clinically healthy adult C. durissus collilineatus belonging to the Reptile Sector (Conservation Breeding Center for Scientific Purposes) of the Federal University of Uberlândia (UFU). All the rattlesnakes were sent to the breeding center by IBAMA, the Environmental Police,
6 the Zoonoses Laboratory (city of Uberlândia), or by members of the community, because they were found in rural and peri-urban areas in the regions of the Minas Triangle and High Paranaíba in the state of Minas Gerais. The snakes were fed every 15 days with mice bred in the vivarium of the Reptile Sector and had free access to water. There is no strict control of synanthropic rodents at the breeding center and the presence of roof rats (Rattus rattus) was discovered on two occasions.
2.2.
Collection of material
The rattlesnakes were restrained by trained handlers, using hooks and a foam mattress, as described by Francisco (1997) and Goulart (2004). After disinfection with 70% alcohol, blood was drawn by puncture of the vertebral venous plexus, as described by Zippel et al. (2001), between the occipital bone and the atlas, using 13x4.5 mm disposable hypodermic needles and 3 ml disposable syringes. The blood was transferred to tubes without anticoagulant and centrifuged at 2,500 rpm for 10 minutes to obtain serum samples, which were placed in individually identified microtubes. The microtubes were then stored at -20ºC until the MAT was performed.
2.3.
Microscopic Agglutination Test (MAT)
The MAT was performed at the Laboratory of Infectious Diseases of UFU, using a panel of 22 serovars (Table 1). Given the absence of a standard test for reptiles, a cutoff dilution of 1:25 was established, which was used by Rossetti et al. (2003) on crocodilians. Flat bottom microwell plates were used for the MAT, placing
7 23 μL of saline 0.9%, 2 μL of serum from each animal, and 25 μL of antigen (each of the 22 serovars) in each well, resulting in a final solution of 50 µL. The final solution was gently stirred by hand and allowed to rest for one hour at room temperature. Reading was then carried out by dark field microscopy directly from the plate wells, using a 10X objective lens and eyepiece. Samples showing agglutination of more than 50% of the field were considered positive, as specified by the Ministry of Health (Ministério da Saúde, 1995). The samples considered positive in the screening test were subjected to antibody titration. This involved subjecting the serum of each sample to consecutive two-fold dilutions (1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200), including the antigen. Reading was performed like in the initial test, after the same resting time at room temperature. The titer of each sample was the highest dilution at which agglutination corresponded to 50% or more of the field. A sample can be considered positive to more than one serovar, due to cross-reactions or exposure to multiple serovars. In this case, the presumptive infecting serovar is based on highest titers (Ministério da Saúde, 1995).
2.4.
Statistical Analysis
Statistical analysis was performed using the binomial test for comparing two proportions, using the BioEstat 5.0 program (Ayres et al., 2007) to determine the most frequent serovars and titers in the snakes under study. Significant differences were inferred at p<0.05.
8 3. RESULTS Of the 64 tested samples, 56 (87.5%) were positive and among them, 51 (91.1%) samples reacted to two or more serovars, while two samples agglutinated all the tested serovars except for the serovar Sentot. There were reactions to all the serovars used in the study, and the most common serovar in these rattlesnakes was Javanica, which occurred in 83.9% of the samples, followed by Andamana (60.7%) and Patoc (51.8%). Figure 1 illustrates the number of samples considered positive for each serovar and the statistical differences in frequency for each of them. The titers varied from 25 to 1600, with titers of 50 (47.8%) and 25 (40.1%) being the most frequent ones (Figure 2). The highest titers were from the serovars Whitcombi (1600), Panama (1600) and Patoc (800).
4. DISCUSSION The high percentage of positive reaction and the occurrence of these serovars in snakes underscores their importance as possible sources of infection for humans. There are no reports of direct transmission of Leptospira spp. from snakes to humans, but the latter may become infected by contact with the environment in which reptiles live (Ebani and Fratini, 2005). That is why professionals who handle wild animals in captivity, such as keepers, veterinarians and biologists in zoos and snake breeding centers, are more susceptible to this type of infection. According to Feuer and Domash-Martinez (2011), nine handlers of crocodilians in the United States contracted leptospirosis after contact with the animals, and some of them had to be hospitalized. The transmission of leptospira may have been direct (from the reptiles to the hands of the handlers) or, more likely, indirect, by exposure to water contaminated with the urine of infected animals. These
9 authors also reported a case of human leptospirosis after contact with a crocodile carcass in the state of Florida. Veen et al. (2012) pointed out the importance of establishing control and prevention protocols for zoonoses such as leptospirosis in zoos, particularly to prevent transmission of the disease to pregnant workers. According to these authors, many handlers of captive wildlife are young women who are more prone to infection during pregnancy because of their weakened immune system. Leptospirosis can increase the rate of miscarriage, placental problems and neonatal jaundice (Puliyath and Singh, 2012). All the rattlesnakes of this study appeared to be healthy, even those that presented high antibody titers of up to 1600. Biscola et al. (2011) reported that both free-living and captive snakes may act as hosts of several of leptospiral serovars without showing clinical symptoms. The absence of clinical signs makes it difficult to identify animals that are hosts to the pathogen, and this may facilitate its transmission to humans and to other snakes. Unlike mammals, reptiles rarely exhibit clinical signs of kidney disease (Miller, 1998). After experimentally infecting snakes with serovar Pomona, Abdulla and Karstad (1962) detected interstitial nephritis in one animal. Biscola et al. (2011), on the other hand, reported reproductive problems in pit vipers (Bothrops jararaca) seropositive for serovar Hardjo. All the rattlesnakes of this study were kept in captivity. Stressful environments, large concentrations of animals, grouping together of different species, and incorrect handling are factors that facilitate the multiplication and transmission of pathogens in captive reptiles. All these factors reduce the animals’ immune response and increase their risk of developing diseases such as leptospirosis. The establishment of
10 sanitation and personal hygiene practices can minimize the risk of transmission (Rataj et al., 2011). Moreover, periodic serological tests are essential to identify the occurrence of the zoonosis in breeding stocks, since the physical examination of snakes may be insufficient. Animals that will be introduced into the breeding stock or taken home as pets must also undergo diagnostic tests. At most snake breeding facilities, some of the animals are taken from the wild while others are the result of crossbreeding wild snakes with snakes born in captivity (Rataj et al., 2011), and the introduction of Leptospira spp. host animals can be an important mode of transmission of leptospirosis in these serpentariums. Snakes can become infected by direct contact with animal hosts of leptospira, or by drinking contaminated water (Hyakutake et al., 1980). The free-living rattlesnakes used in this study lived in rural or peri-urban areas before being confined. Therefore, it is possible that some animals came into contact with leptospira in their natural environment and introduced this agent in the breeding center, contaminating other snakes. Rodents are natural reservoirs of the serovars Javanica, Andamana and Patoc (Shenberg et al., 1975; Priya et al., 2007), which were frequent in the snakes of this study. Therefore, one should also consider the possibility that these rattlesnakes were exposed to Leptospira spp. by eating infected prey, since there is no control of synanthropic rodents at the breeding center. This possibility of transmission to snakes through the food chain was also suggested in the studies of Andrews et al. (1965) and Hyakutake et al. (1980). Brasil (2011) reported that no reptile at the zoo in João Pessoa, Paraíba tested positive by MAT (at a cutoff of 1:100), and the author
11 attributed this finding to the type of food fed to the animals, which consisted of mice supplied by a vivarium with strict sanitary control. There are few studies on the occurrence of leptospiral antibodies in snakes, and no research was found about the disease in C. durissus collilineatus. To the best of our knowledge, reactions to serovars Djasiman, Cynopteri, Sentot and Whitcombi have not yet been described in snakes. However, the others that were seroreactive in this study have already been reported in these animals (Andrews et al., 1965; Hyakutake et al., 1980; Stanchi et al., 1986; Calle et al., 2001; Biscola et al., 2011; Lindtner-Knific et al., 2013). The most frequent serovars in the animals of this study could be due to cross-agglutinations. Serovar Javanica is common in Southeast Asia (WHO, 2011), while serovars Andamana and Patoc are considered saprophytic varieties (L. biflexa). On the other hand, more than 50% of the rattlesnakes in this study were seroreactive to Andamana and Hyakutake et al. (1980) suggested that snakes may act as serovar reservoirs, in view of the high frequency of animals positive to this variety of leptospire. These authors also isolated Andamana from the kidney of a snake. As for titers, there are reports of snakes that showed high titers of up to 3200 (Biscola et al., 2011) and 6400 (Hyakutake et al., 1980) in the MAT. On the other hand, Calle et al. (2001) found lower titers of 100 and 200 in green anacondas (Eunectes murinus) in Venezuela. Lower titers, such as those that occurred more frequently in this study, may indicate recent infections, residual antibodies from past infections, antibody response to leptospira present in preys, or cross-reactions against serovars that were not been tested in the MAT (Calle et al., 2001). Conversely, high titers such as 800 and 1600 may indicate acute disease, but there are no studies about the cut-off point to be used in reptile serology. The
12 mechanisms of immune system response of snakes have yet to be fully elucidated, but it is known that these animals can keep active pathogenic leptospira in their bodies even without showing detectable titers of antibodies in serological tests (Minette, 1983).
5. CONCLUSIONS Approximately 90% of the C. durissus collilineatus rattlesnakes of this study come into contact with leptospira and the most frequent serovars in these snakes were Javanica, Andamana and Patoc. Some of them presented high titers (such as 1600), despite the absence of clinical symptoms. Attention should focus on the importance of leptospirosis as an occupational zoonosis and the possibility of captive snakes acting as important sources of leptospiral infection in humans. It is essential that the pathogen be controlled in breeding centers and serpentariums, particularly via periodic serological tests on the breeding stock, to prevent the introduction of infected individuals and to establish sanitation and control practices of synanthropic rodents.
Conflicts of interest: none
Acknowledgments: The authors acknowledge the support offered by the Federal University of Uberlândia (UFU), Conselho Nacional de Pesquisa (CNPq), Fundação de
Amparo
à
Pesquisa
em
Minas
Gerais
(Fapemig),
Coordenação
de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Reptile Sector staff member Graciele Freitas Cardoso to which we are grateful for.
13 6. REFERENCES
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17
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19 Figure 1 – Number of serum samples from Crotalus durissus collilineatus rattlesnakes positive for at least one of the 22 serovars used in the microscopic agglutination test.
Different letters (a, b and c) indicate a statistical difference at a level of 0.05 by the Binomial Test for Two Proportions.
Figure 2 – Number of serum samples from Crotalus durissus collilineatus rattlesnakes positive for each titer in the microscopic agglutination test.
20
Different letters (a, b and c) indicate a statistical difference at a level of 0.05 by the Binomial Test for Two Proportions.
21 Table 1 – Leptospira spp. species, serogroups and serovars used in the microscopic agglutination test Species
Serogroup
Serovar
L. biflexa
Andamana
Andamana
L. interrogans
Autumnalis
Autumnalis
L. interrogans
Australis
Australis
L. interrogans
Bataviae
Bataviae
L. interrogans
Australis
Bratislava
L. interrogans
Canicola
Canicola
L. kirschneri
Cynopteri
Cynopteri
L. interrogans
Icterohaemorrhagiae
Copenhageni
L. interrogans
Djasiman
Djasiman
L. kirschneri
Grippotyphosa
Grippotyphosa
L. interrogans
Sejroe
Hardjo
L. interrogans
Hebdomadis
Hebdomadis
L. interrogans
Icterohaemorrhagiae
Icterohaemorrhagiae
L. borgpetersenii
Javanica
Javanica
L. noguchii
Panama
Panama
L. biflexa
Semarang
Patoc
L. interrogans
Pomona
Pomona
L. interrogans
Pyrogenes
Pyrogenes
L. interrogans
Djasiman
Sentot
L. borgpetersenii
Tarassovi
Tarassovi
L. borgpetersenii
Celledoni
Whitcombi
L. interrogans
Sejroe
Wolffi
Source: Baranton, 2006
S0001-706X(16)30059-6 http://dx.doi.org/doi:10.1016/j.actatropica.2016.02.006 ACTROP 3850
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
13-7-2015 8-2-2016 9-2-2016
Please cite this article as: Rodrigues, T.C.S., Santos, A.L.Q., Lima, A.M.C., Gomes, D.O., Brites, V.L.C., Anti-Leptospira spp.antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Anti-Leptospira spp. antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance
Rodrigues, T.C.S.a; Santos, A.L.Q.a; Lima, A.M.C.b; Gomes, D.O.b; Brites, V.L.C.c
a
Wildlife Teaching and Research Laboratory (LAPAS), College of Veterinary
Medicine (FAMEV), Federal University of Uberlândia (UFU). Avenida Amazonas 2245,
Jardim
Umuarama,
38405-302
Uberlândia,
MG,
Brazil.
[email protected] b
Laboratory of Infectious Diseases, College of Veterinary Medicine (FAMEV),
Federal University of Uberlândia (UFU). Rua Ceará, s/n, Bloco 2D, Sala 33, Campus Umuarama, 38405-315, Uberlândia, MG, Brazil. [email protected] c
Reptile Sector, Institute of Biology, Federal University of Uberlândia (UFU). Rua
Ceará, s/n, Bloco 2D, Campus Umuarama, 38405-315, Uberlândia, MG, Brazil. [email protected]
Corresponding author:
Thaís Carneiro Santos Rodrigues. Postal Address: Rua das
Petúnias, 131 Cidade Jardim, 38412-112 Uberlândia, MG, Brazil. Phone: 55(34)8861-2836. E-mail: [email protected]
2
Graphical Abstract
Highlights
We studied the occurrence of anti-Leptospira spp. antibodies in captive rattlesnakes 90% of the snakes were positive in the microscopic agglutination test Most frequent serovars were Javanica, Andamana and Patoc These snakes can present high titers despite the absence of clinical symptoms We focus on the importance of leptospirosis as an occupational zoonosis
3 ABSTRACT: Leptospirosis is a worldwide spread zoonosis that can affect all groups of vertebrates, including reptiles. Because it has been little studied in snakes, this study focused on determining the occurrence of anti-Leptospira spp. antibodies in 64 Crotalus durissus collilineatus kept in captivity and on identifying the most common serovars in these animals, using the microscopic agglutination test. Of these, almost 90% were positive and there were reactions to the 22 serovars used in the study. The most common serovar in these snakes was Javanica, Andamana and Patoc. Most frequent titers were 25 and 50, although high titers (such as 1600) were also recorded, despite the absence of clinical symptoms. The possibility should be considered of captive snakes serving as a serious source of leptospiral infection in humans, which is why it is essential to study, prevent and control the disease in breeding centers and serpentariums.
Keywords: Leptospirosis; Reptiles; Snakes; Serology; Zoonosis.
4 1. INTRODUCTION Leptospirosis is the world’s most widespread zoonotic disease (Jamshidi et al., 2009) and is therefore extremely important to human and animal health (Faine et al., 1999). It is caused by spirochetes of the genus Leptospira and these bacteria comprise several pathogenic and saprophytic species that are classified into serogroups. Each serogroup is composed of a number of varieties, called serovars (Adler and Moctezuma, 2010). A single animal can be host to various serovars, and one serovar can infect multiple hosts (Quinn et al., 2005). Many species of domestic and wild mammals are known to act as natural or accidental hosts of several Leptospira serovars. However, little is known about the participation of reptiles in the maintenance and dissemination of the pathogen in the environment (Lindtner-Knific et al., 2013). These animals can play important roles in the epidemiological cycle of the disease by keeping the infectious agent in the environment and transmitting it to other animals (Glosser et al.,1974; Lindtner-Knific et al., 2013). According to Hyakutake et al. (1980), snakes may be important natural reservoirs of leptospira, particularly because of their normal diet of rodents, which are the main reservoirs of the pathogen in the environment (Faine et al., 1999). Leptospirosis in snakes is usually not associated with clinical symptoms (Wallach, 1983), but the occurrence of renal changes in an infected snake has been reported in the past (Abdulla and Karstad, 1962). The number of snakes raised in captivity has increased considerably in recent years. In addition to becoming pets, there are numerous snake breeding centers for scientific and commercial purposes. Despite the absence of reports about the transmission of leptospirosis from reptiles to humans (Ebani and Fratini, 2005), it is
5 important to investigate the occurrence of leptospirosis in these animals to prevent the risk of exposure to the causative agent of the disease (Silva et al., 2009). Biologists, veterinarians and snake handlers may also be at risk of infection (Ebani and Fratini, 2005). The incidence of different serovars in human populations is highly dependent on reservoir hosts in the region in question and on the serovars they carry (Bharti et al., 2003). Thus, determining which animal species act as hosts of leptospira in a given area is critical to the control and prevention of leptospirosis (WHO, 2003; Desvars et al., 2010). Therefore, the purpose of this study was to evaluate the occurrence of anti-Leptospira spp. agglutinins in Crotalus durissus collilineatus rattlesnakes living in captivity in a breeding facility, and to identify the serovars appearing most frequently in these animals, using the microscopic agglutination test (MAT).
2. MATERIAL AND METHODS All the procedures were performed with the approval of the Ethics Committee on Animal Use – CEUA/UFU (Protocol 120/14) and the authorization of the Brazilian Institute of Environment and Renewable Natural Resources – IBAMA (SISBIO Permit No. 46845).
2.1.
Animals and study area
Sixty-four samples of blood serum from clinically healthy adult C. durissus collilineatus belonging to the Reptile Sector (Conservation Breeding Center for Scientific Purposes) of the Federal University of Uberlândia (UFU). All the rattlesnakes were sent to the breeding center by IBAMA, the Environmental Police,
6 the Zoonoses Laboratory (city of Uberlândia), or by members of the community, because they were found in rural and peri-urban areas in the regions of the Minas Triangle and High Paranaíba in the state of Minas Gerais. The snakes were fed every 15 days with mice bred in the vivarium of the Reptile Sector and had free access to water. There is no strict control of synanthropic rodents at the breeding center and the presence of roof rats (Rattus rattus) was discovered on two occasions.
2.2.
Collection of material
The rattlesnakes were restrained by trained handlers, using hooks and a foam mattress, as described by Francisco (1997) and Goulart (2004). After disinfection with 70% alcohol, blood was drawn by puncture of the vertebral venous plexus, as described by Zippel et al. (2001), between the occipital bone and the atlas, using 13x4.5 mm disposable hypodermic needles and 3 ml disposable syringes. The blood was transferred to tubes without anticoagulant and centrifuged at 2,500 rpm for 10 minutes to obtain serum samples, which were placed in individually identified microtubes. The microtubes were then stored at -20ºC until the MAT was performed.
2.3.
Microscopic Agglutination Test (MAT)
The MAT was performed at the Laboratory of Infectious Diseases of UFU, using a panel of 22 serovars (Table 1). Given the absence of a standard test for reptiles, a cutoff dilution of 1:25 was established, which was used by Rossetti et al. (2003) on crocodilians. Flat bottom microwell plates were used for the MAT, placing
7 23 μL of saline 0.9%, 2 μL of serum from each animal, and 25 μL of antigen (each of the 22 serovars) in each well, resulting in a final solution of 50 µL. The final solution was gently stirred by hand and allowed to rest for one hour at room temperature. Reading was then carried out by dark field microscopy directly from the plate wells, using a 10X objective lens and eyepiece. Samples showing agglutination of more than 50% of the field were considered positive, as specified by the Ministry of Health (Ministério da Saúde, 1995). The samples considered positive in the screening test were subjected to antibody titration. This involved subjecting the serum of each sample to consecutive two-fold dilutions (1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200), including the antigen. Reading was performed like in the initial test, after the same resting time at room temperature. The titer of each sample was the highest dilution at which agglutination corresponded to 50% or more of the field. A sample can be considered positive to more than one serovar, due to cross-reactions or exposure to multiple serovars. In this case, the presumptive infecting serovar is based on highest titers (Ministério da Saúde, 1995).
2.4.
Statistical Analysis
Statistical analysis was performed using the binomial test for comparing two proportions, using the BioEstat 5.0 program (Ayres et al., 2007) to determine the most frequent serovars and titers in the snakes under study. Significant differences were inferred at p<0.05.
8 3. RESULTS Of the 64 tested samples, 56 (87.5%) were positive and among them, 51 (91.1%) samples reacted to two or more serovars, while two samples agglutinated all the tested serovars except for the serovar Sentot. There were reactions to all the serovars used in the study, and the most common serovar in these rattlesnakes was Javanica, which occurred in 83.9% of the samples, followed by Andamana (60.7%) and Patoc (51.8%). Figure 1 illustrates the number of samples considered positive for each serovar and the statistical differences in frequency for each of them. The titers varied from 25 to 1600, with titers of 50 (47.8%) and 25 (40.1%) being the most frequent ones (Figure 2). The highest titers were from the serovars Whitcombi (1600), Panama (1600) and Patoc (800).
4. DISCUSSION The high percentage of positive reaction and the occurrence of these serovars in snakes underscores their importance as possible sources of infection for humans. There are no reports of direct transmission of Leptospira spp. from snakes to humans, but the latter may become infected by contact with the environment in which reptiles live (Ebani and Fratini, 2005). That is why professionals who handle wild animals in captivity, such as keepers, veterinarians and biologists in zoos and snake breeding centers, are more susceptible to this type of infection. According to Feuer and Domash-Martinez (2011), nine handlers of crocodilians in the United States contracted leptospirosis after contact with the animals, and some of them had to be hospitalized. The transmission of leptospira may have been direct (from the reptiles to the hands of the handlers) or, more likely, indirect, by exposure to water contaminated with the urine of infected animals. These
9 authors also reported a case of human leptospirosis after contact with a crocodile carcass in the state of Florida. Veen et al. (2012) pointed out the importance of establishing control and prevention protocols for zoonoses such as leptospirosis in zoos, particularly to prevent transmission of the disease to pregnant workers. According to these authors, many handlers of captive wildlife are young women who are more prone to infection during pregnancy because of their weakened immune system. Leptospirosis can increase the rate of miscarriage, placental problems and neonatal jaundice (Puliyath and Singh, 2012). All the rattlesnakes of this study appeared to be healthy, even those that presented high antibody titers of up to 1600. Biscola et al. (2011) reported that both free-living and captive snakes may act as hosts of several of leptospiral serovars without showing clinical symptoms. The absence of clinical signs makes it difficult to identify animals that are hosts to the pathogen, and this may facilitate its transmission to humans and to other snakes. Unlike mammals, reptiles rarely exhibit clinical signs of kidney disease (Miller, 1998). After experimentally infecting snakes with serovar Pomona, Abdulla and Karstad (1962) detected interstitial nephritis in one animal. Biscola et al. (2011), on the other hand, reported reproductive problems in pit vipers (Bothrops jararaca) seropositive for serovar Hardjo. All the rattlesnakes of this study were kept in captivity. Stressful environments, large concentrations of animals, grouping together of different species, and incorrect handling are factors that facilitate the multiplication and transmission of pathogens in captive reptiles. All these factors reduce the animals’ immune response and increase their risk of developing diseases such as leptospirosis. The establishment of
10 sanitation and personal hygiene practices can minimize the risk of transmission (Rataj et al., 2011). Moreover, periodic serological tests are essential to identify the occurrence of the zoonosis in breeding stocks, since the physical examination of snakes may be insufficient. Animals that will be introduced into the breeding stock or taken home as pets must also undergo diagnostic tests. At most snake breeding facilities, some of the animals are taken from the wild while others are the result of crossbreeding wild snakes with snakes born in captivity (Rataj et al., 2011), and the introduction of Leptospira spp. host animals can be an important mode of transmission of leptospirosis in these serpentariums. Snakes can become infected by direct contact with animal hosts of leptospira, or by drinking contaminated water (Hyakutake et al., 1980). The free-living rattlesnakes used in this study lived in rural or peri-urban areas before being confined. Therefore, it is possible that some animals came into contact with leptospira in their natural environment and introduced this agent in the breeding center, contaminating other snakes. Rodents are natural reservoirs of the serovars Javanica, Andamana and Patoc (Shenberg et al., 1975; Priya et al., 2007), which were frequent in the snakes of this study. Therefore, one should also consider the possibility that these rattlesnakes were exposed to Leptospira spp. by eating infected prey, since there is no control of synanthropic rodents at the breeding center. This possibility of transmission to snakes through the food chain was also suggested in the studies of Andrews et al. (1965) and Hyakutake et al. (1980). Brasil (2011) reported that no reptile at the zoo in João Pessoa, Paraíba tested positive by MAT (at a cutoff of 1:100), and the author
11 attributed this finding to the type of food fed to the animals, which consisted of mice supplied by a vivarium with strict sanitary control. There are few studies on the occurrence of leptospiral antibodies in snakes, and no research was found about the disease in C. durissus collilineatus. To the best of our knowledge, reactions to serovars Djasiman, Cynopteri, Sentot and Whitcombi have not yet been described in snakes. However, the others that were seroreactive in this study have already been reported in these animals (Andrews et al., 1965; Hyakutake et al., 1980; Stanchi et al., 1986; Calle et al., 2001; Biscola et al., 2011; Lindtner-Knific et al., 2013). The most frequent serovars in the animals of this study could be due to cross-agglutinations. Serovar Javanica is common in Southeast Asia (WHO, 2011), while serovars Andamana and Patoc are considered saprophytic varieties (L. biflexa). On the other hand, more than 50% of the rattlesnakes in this study were seroreactive to Andamana and Hyakutake et al. (1980) suggested that snakes may act as serovar reservoirs, in view of the high frequency of animals positive to this variety of leptospire. These authors also isolated Andamana from the kidney of a snake. As for titers, there are reports of snakes that showed high titers of up to 3200 (Biscola et al., 2011) and 6400 (Hyakutake et al., 1980) in the MAT. On the other hand, Calle et al. (2001) found lower titers of 100 and 200 in green anacondas (Eunectes murinus) in Venezuela. Lower titers, such as those that occurred more frequently in this study, may indicate recent infections, residual antibodies from past infections, antibody response to leptospira present in preys, or cross-reactions against serovars that were not been tested in the MAT (Calle et al., 2001). Conversely, high titers such as 800 and 1600 may indicate acute disease, but there are no studies about the cut-off point to be used in reptile serology. The
12 mechanisms of immune system response of snakes have yet to be fully elucidated, but it is known that these animals can keep active pathogenic leptospira in their bodies even without showing detectable titers of antibodies in serological tests (Minette, 1983).
5. CONCLUSIONS Approximately 90% of the C. durissus collilineatus rattlesnakes of this study come into contact with leptospira and the most frequent serovars in these snakes were Javanica, Andamana and Patoc. Some of them presented high titers (such as 1600), despite the absence of clinical symptoms. Attention should focus on the importance of leptospirosis as an occupational zoonosis and the possibility of captive snakes acting as important sources of leptospiral infection in humans. It is essential that the pathogen be controlled in breeding centers and serpentariums, particularly via periodic serological tests on the breeding stock, to prevent the introduction of infected individuals and to establish sanitation and control practices of synanthropic rodents.
Conflicts of interest: none
Acknowledgments: The authors acknowledge the support offered by the Federal University of Uberlândia (UFU), Conselho Nacional de Pesquisa (CNPq), Fundação de
Amparo
à
Pesquisa
em
Minas
Gerais
(Fapemig),
Coordenação
de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Reptile Sector staff member Graciele Freitas Cardoso to which we are grateful for.
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19 Figure 1 – Number of serum samples from Crotalus durissus collilineatus rattlesnakes positive for at least one of the 22 serovars used in the microscopic agglutination test.
Different letters (a, b and c) indicate a statistical difference at a level of 0.05 by the Binomial Test for Two Proportions.
Figure 2 – Number of serum samples from Crotalus durissus collilineatus rattlesnakes positive for each titer in the microscopic agglutination test.
20
Different letters (a, b and c) indicate a statistical difference at a level of 0.05 by the Binomial Test for Two Proportions.
21 Table 1 – Leptospira spp. species, serogroups and serovars used in the microscopic agglutination test Species
Serogroup
Serovar
L. biflexa
Andamana
Andamana
L. interrogans
Autumnalis
Autumnalis
L. interrogans
Australis
Australis
L. interrogans
Bataviae
Bataviae
L. interrogans
Australis
Bratislava
L. interrogans
Canicola
Canicola
L. kirschneri
Cynopteri
Cynopteri
L. interrogans
Icterohaemorrhagiae
Copenhageni
L. interrogans
Djasiman
Djasiman
L. kirschneri
Grippotyphosa
Grippotyphosa
L. interrogans
Sejroe
Hardjo
L. interrogans
Hebdomadis
Hebdomadis
L. interrogans
Icterohaemorrhagiae
Icterohaemorrhagiae
L. borgpetersenii
Javanica
Javanica
L. noguchii
Panama
Panama
L. biflexa
Semarang
Patoc
L. interrogans
Pomona
Pomona
L. interrogans
Pyrogenes
Pyrogenes
L. interrogans
Djasiman
Sentot
L. borgpetersenii
Tarassovi
Tarassovi
L. borgpetersenii
Celledoni
Whitcombi
L. interrogans
Sejroe
Wolffi
Source: Baranton, 2006