Lessons from a Multilaboratorial Task Force for Diagnosis of a Fatal Toxoplasmosis Outbreak in Captive Primates in Brazil.

Toxoplasmosis is an important zoonotic disease caused by the parasite Toxoplasma gondii and is especially fatal for neotropical primates. In Brazil, the Ministry of Health is responsible for national epizootic surveillance, but some diseases are still neglected. Here, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among eleven neotropical primates housed at a primatology center in Brazil. After presenting non-specific clinical signs, all animals died within four days. A wide range of pathogens were evaluated, and we successfully identified T. gondii as the causative agent within four days after necropsies. The liver was the most affected organ, presenting hemorrhage and hepatocellular necrosis. Tachyzoites and bradyzoite cysts were observed in histological examinations and immunohistochemistry in different organs; in addition, parasitic DNA was detected through PCR in blood samples from all specimens evaluated. A high prevalence of Escherichia coli was also observed, indicating sepsis. This case highlights some of the obstacles faced by the current Brazilian surveillance system. A diagnosis was obtained through the integrated action of researchers since investigation for toxoplasmosis is currently absent in national guidelines. An interdisciplinary investigation could be a possible model for future epizootic investigations in animals.

IgM antibody responses against Plasmodium antigens in neotropical primates in the Brazilian Atlantic Forest.

Introduction: Zoonotic transmission is a challenge for the control and elimination of malaria. It has been recorded in the Atlantic Forest, outside the Amazon which is the endemic region in Brazil. However, only very few studies have assessed the antibody response, especially of IgM antibodies, in Neotropical primates (NP). Therefore, in order to contribute to a better understanding of the immune response in different hosts and facilitate the identification of potential reservoirs, in this study, naturally acquired IgM antibody responses against Plasmodium antigens were evaluated, for the first time, in NP from the Atlantic Forest. Methods: The study was carried out using 154 NP samples from three different areas of the Atlantic Forest. IgM antibodies against peptides of the circumsporozoite protein (CSP) from different Plasmodium species and different erythrocytic stage antigens were detected by ELISA. Results: Fifty-nine percent of NP had IgM antibodies against at least one CSP peptide and 87% against at least one Plasmodium vivax erythrocytic stage antigen. Levels of antibodies against PvAMA-1 were the highest compared to the other antigens. All families of NP showed IgM antibodies against CSP peptides, and, most strikingly, against erythrocytic stage antigens. Generalized linear models demonstrated that IgM positivity against PvCSP and PvAMA-1 was associated with PCR-detectable blood-stage malaria infection and the host being free-living. Interestingly, animals with IgM against both PvCSP and PvAMA-1 were 4.7 times more likely to be PCR positive than animals that did not have IgM for these two antigens simultaneously. Discussion: IgM antibodies against different Plasmodium spp. antigens are present in NP from the Atlantic Forest. High seroprevalence and antibody levels against blood-stage antigens were observed, which had a significant association with molecular evidence of infection. IgM antibodies against CSP and AMA-1 may be used as a potential marker for the identification of NP infected with Plasmodium, which are reservoirs of malaria in the Brazilian Atlantic Forest.

Bifidobacteria define gut microbiome profiles of golden lion tamarin (Leontopithecus rosalia) and marmoset (Callithrix sp.) metagenomic shotgun pools.

Gut microbiome disruptions may lead to adverse effects on wildlife fitness and viability, thus maintaining host microbiota biodiversity needs to become an integral part of wildlife conservation. The highly-endangered callitrichid golden lion tamarin (GLT-Leontopithecus rosalia) is a rare conservation success, but allochthonous callitrichid marmosets (Callithrix) serve as principle ecological GLT threats. However, incorporation of microbiome approaches to GLT conservation is impeded by limited gut microbiome studies of Brazilian primates. Here, we carried out analysis of gut metagenomic pools from 114 individuals of wild and captive GLTs and marmosets. More specifically, we analyzed the bacterial component of ultra filtered samples originally collected as part of a virome profiling study. The major findings of this study are consistent with previous studies in showing that Bifidobacterium, a bacterial species important for the metabolism of tree gums consumed by callitrichids, is an important component of the callitrichid gut microbiome - although GTLs and marmosets were enriched for different species of Bifidobacterium. Additionally, the composition of GLT and marmoset gut microbiota is sensitive to host environmental factors. Overall, our data expand baseline gut microbiome data for callitrichids to allow for the development of new tools to improve their management and conservation.

Detection of Plasmodium simium gametocytes in non-human primates from the Brazilian Atlantic Forest.

BACKGROUND: Plasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium. METHODS: Whole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting 18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte. RESULTS: Overall, 87.5% of the 26 samples, previously diagnosed as P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL. CONCLUSIONS: For the first time, a molecular detection of P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.

Isolation and genetic characterization of Toxoplasma gondii from a captive black-and-gold howler monkey (Alouatta caraya Humboldt, 1812) in Brazil.

Toxoplasma gondii was isolated in mice from different tissues of a captive black-and-gold howler monkey (Alouatta caraya) kept in a colony at the Primatology Center of Rio de Janeiro State, Brazil, and it was genotypically characterized based on using PCR-RFLP and Microsatellite Analysis (MS), later on. T. gondii was successfully isolated from inocula deriving from heart, liver and tissue pool (heart, liver, lungs, axillary lymph nodes and cerebellum) samples. The isolate was named TgBgHmBrRJ1. The high virulence of the aforementioned strain was observed in infected mice. Non-archetypal genotype (ToxoDB PCR-RFLP #206) was obtained through PCR-RFLP. This genotype had been previously described in 12 isolates from different hosts, also in Southeastern Brazil, a fact that indicates likely high circulation of this genotype in this region. The isolate was also classified as non-archetypal, based on MS genotyping, as well as presented genotypic identity close to that of strains isolated from free-range non-symptomatic chickens (TgCkBr244,245,278,279) in Espírito Santo State. It is worth emphasizing that despite the large number of reports about clinical toxoplasmosis in neotropical primates in Brazil, this is just the second isolate of this parasite ever reported in this group of animals.

Neotropical Sylvatic Mosquitoes and Aedes aegypti Are Not Competent to Transmit 17DD Attenuated Yellow Fever Virus from Vaccinated Viremic New World Non-Human Primates.

Beside humans, thousands of non-human primates (NHPs) died during the recent outbreak caused by the yellow fever virus (YFV) in Brazil. Vaccination of NHPs against YFV with the YF 17DD attenuated virus has emerged as a public health strategy, as it would reduce sylvatic transmission while also preserving endangered susceptible species. The hypothesis of establishing an uncontrolled transmission of this attenuated virus in nature was raised. We assessed vector competence of four sylvatic mosquito species, Haemagogus leucocelaenus, Haemagogus janthinomys/capricornii, Sabethes albiprivus, and Sabethes identicus, as well as the urban vector Aedes aegypti for YF 17DD attenuated vaccine virus when fed directly on eleven viremic lion tamarins or artificially challenged with the same virus. No infection was detected in 689 mosquitoes engorged on viremic lion tamarins whose viremia ranged from 1.05 × 103 to 6.61 × 103 FFU/mL, nor in those artificially taking ≤ 1 × 103 PFU/mL. Low viremia presented by YF 17DD-vaccinated New World NHPs combined with the low capacity and null dissemination ability in sylvatic and domestic mosquitoes of this attenuated virus suggest no risk of its transmission in nature. Thus, vaccination of captive and free-living NHPs against YFV is a safe public health strategy.

Subacute multisystemic toxoplasmosis in a captive black-and-gold howler monkey (Alouatta caraya) indicates therapy challenging.

A 10-year-old black howler monkey presented with a 36-day subacute clinicopathological picture of fever, prostration, inappetence, intestinal hypomotility, and emaciation. Therapy was trimethoprim-sulfamethoxazole with streptomycin. The liver, lungs, lymph nodes, and spleen presented lesions. Toxoplasma gondii isolation and PCR determined the diagnosis, and indirect fluorescent antibody tests confirmed an increase in antibody titers.

Metastatic endometrioid carcinoma in a hybrid Leontopithecus sp.

Primary female reproductive neoplasms in Platyrrhines species are few reported. We present the gross, histological, and immunohistochemical findings of metastatic endometrioid carcinoma in the uterus, urinary bladder, jejunum, and rectum of a Leontopithecus sp. The neoplastic endometrial cells expressed strong cytoplasmic immunolabeling of cytokeratin 7.

Infection by Prosthernorchis elegans (Diesing, 1851) in captive Callithrix aurita (É. Geoffroy, 1812) and Leontopithecus rosalia (Linnaeus, 1766) from Rio de Janeiro, Brazil.

This study reports on infection by Prosthernorchis elegans of Callithrix aurita and Leonthopithecus rosalia through biometry on adults and by molecular biology. Seventy-eight helminths were recovered from the animals' intestine. This is a detailed morphological description and the first molecular characterization of P. elegans in animals from Brazil.

Spontaneous disseminated T-cell lymphoma in a buff-headed capuchin (Sapajus xanthosternos).

Reports of spontaneous hematopoietic neoplasms in Platyrrhines species are scarce. We present the gross, histological, and immunohistochemical findings of disseminated T-cell lymphoma in a male 25-year-old Sapajus xanthosternos kept in a Brazilian conservation center. No molecular evidence of betaherpesvirus or gammaherpesvirus was associated with the occurrence of this neoplasm.

The genome of the zoonotic malaria parasite Plasmodium simium reveals adaptations to host switching.

BACKGROUND: Plasmodium simium, a malaria parasite of non-human primates (NHP), was recently shown to cause zoonotic infections in humans in Brazil. We sequenced the P. simium genome to investigate its evolutionary history and to identify any genetic adaptions that may underlie the ability of this parasite to switch between host species. RESULTS: Phylogenetic analyses based on whole genome sequences of P. simium from humans and NHPs reveals that P. simium is monophyletic within the broader diversity of South American Plasmodium vivax, suggesting P. simium first infected NHPs as a result of a host switch of P. vivax from humans. The P. simium isolates show the closest relationship to Mexican P. vivax isolates. Analysis of erythrocyte invasion genes reveals differences between P. vivax and P. simium, including large deletions in the Duffy-binding protein 1 (DBP1) and reticulocyte-binding protein 2a genes of P. simium. Analysis of P. simium isolated from NHPs and humans revealed a deletion of 38 amino acids in DBP1 present in all human-derived isolates, whereas NHP isolates were multi-allelic. CONCLUSIONS: Analysis of the P. simium genome confirmed a close phylogenetic relationship between P. simium and P. vivax, and suggests a very recent American origin for P. simium. The presence of the DBP1 deletion in all human-derived isolates tested suggests that this deletion, in combination with other genetic changes in P. simium, may facilitate the invasion of human red blood cells and may explain, at least in part, the basis of the recent zoonotic infections.

Profiling Humoral Immune Response Against Pre-Erythrocytic and Erythrocytic Antigens of Malaria Parasites Among Neotropical Primates in the Brazilian Atlantic Forest.

Human malaria due to zoonotic transmission has been recorded in the Atlantic Forest, an extra-Amazonian area in Brazil, which are a challenge for malaria control. Naturally acquired humoral immune response against pre-erythrocytic and erythrocytic antigens of Neotropical primates (NP) was evaluated here to improve the knowledge about the exposure of those animals to the malaria transmission and support the identification of the potential reservoirs of the disease in the Atlantic Forest. Blood samples of 154 monkeys from three areas of the Atlantic Forest were used to identify IgG antibodies against peptides of the repeat region of the major pre-erythrocytic antigen, the circumsporozoite protein (CSP), of Plasmodium vivax (PvCSP), Plasmodium brasilianum/Plasmodium malariae (Pb/PmCSP), and Plasmodium falciparum (PfCSP) by ELISA. Antibodies against erythrocytic recombinant antigens of P. vivax, Apical membrane antigen 1 (PvAMA-1), Erythrocyte binding protein 2 (PvEBP-2) and domain II of Duffy binding protein (PvDBPII) were also evaluated. Parameters, such as age, sex, PCR positivity, and captivity, potentially associated with humoral immune response were analyzed. Eighty-five percent of NP had antibodies against at least one CSP peptide, and 76% against at least one P. vivax erythrocytic antigen. A high percentage of adults compared to non-adults were seropositive and showed increased antibody levels. Neotropical primates with PCR positive for P. simium had a significantly higher frequency of positivity rate for immune response against PvEBP-2, PvDBPII and also higher antibody levels against PvDBPII, compared to PCR negative NPs for this species. Monkeys with PCR positive for P. brasilianum/P. malariae showed higher frequency of seropositivity and antibody levels against Pb/PmCSP. Levels of antibodies against Pb/PmCSP, PvEBP-2 and PvDBPII were higher in free-living than in captive monkeys from the same area. All Platyrrhine families showed antibodies against CSP peptides, however not all showed IgG against erythrocytic antigens. These findings showed a high prevalence of naturally acquired antibodies against CSP repeats in all studied areas, suggesting an intense exposure to infected-mosquitoes bites of NP from all families. However, mainly monkeys of Atelidae family showed antibodies against P. vivax erythrocytic antigens, suggesting blood infection, which might serve as potential reservoirs of malaria in the Atlantic Forest.

Safety and immunogenicity of 17DD attenuated yellow fever vaccine in howler monkeys (Alouatta spp.).

BACKGROUND: Alouatta spp. are highly susceptible to yellow fever (YF) infection and develop an often fatal disease. The threat posed by an outbreak started in 2016 leads us to investigate vaccination as a potential tool in preventing YF in non-human primates (NHP). METHODS: Susceptible howler monkeys were immunized with three different concentrations of the human Brazilian commercial YF17DD vaccine. Post-vaccination viremia/RNAemia, immunogenicity, and safety were characterized. RESULTS: The vaccine did not produce YF clinical manifestations in any of the NHPs. After immunization, all animals seroconverted demonstrating the ability of the YF vaccine to induce humoral response in Alouatta species. CONCLUSIONS: The present work has demonstrated the safe and immunogenic profile of the existing YF 17DD vaccine in howler monkeys. This knowledge may support further studies with other susceptible monkey species and provide a possible solution for controlling epizootics and preventing the devastation of endangered species.

An Introduction to the Callithrix Genus and Overview of Recent Advances in Marmoset Research.

We provide here a current overview of marmoset (Callithrix) evolution, hybridization, species biology, basic/biomedical research, and conservation initiatives. Composed of 2 subgroups, the aurita group (C aurita and C flaviceps) and the jacchus group (C geoffroyi, C jacchus, C kuhlii, and C penicillata), this relatively young primate radiation is endemic to the Brazilian Cerrado, Caatinga, and Atlantic Forest biomes. Significant impacts on Callithrix within these biomes resulting from anthropogenic activity include (1) population declines, particularly for the aurita group; (2) widespread geographic displacement, biological invasions, and range expansions of C jacchus and C penicillata; (3) anthropogenic hybridization; and (4) epizootic Yellow Fever and Zika viral outbreaks. A number of Brazilian legal and conservation initiatives are now in place to protect the threatened aurita group and increase research about them. Due to their small size and rapid life history, marmosets are prized biomedical models. As a result, there are increasingly sophisticated genomic Callithrix resources available and burgeoning marmoset functional, immuno-, and epigenomic research. In both the laboratory and the wild, marmosets have given us insight into cognition, social group dynamics, human disease, and pregnancy. Callithrix jacchus and C penicillata are emerging neotropical primate models for arbovirus disease, including Dengue and Zika. Wild marmoset populations are helping us understand sylvatic transmission and human spillover of Zika and Yellow Fever viruses. All of these factors are positioning marmosets as preeminent models to facilitate understanding of facets of evolution, hybridization, conservation, human disease, and emerging infectious diseases.

Author Correction: An assay for the identification of Plasmodium simium infection for diagnosis of zoonotic malaria in the Brazilian Atlantic Forest.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An assay for the identification of Plasmodium simium infection for diagnosis of zoonotic malaria in the Brazilian Atlantic Forest.

Zoonotic malaria poses a unique problem for malaria control. Autochthonous cases of human malaria in the Atlantic Forest have recently been attributed to Plasmodium simium, a parasite that commonly infects non-human primates in this Brazilian biome. However, due to its close similarity at both the morphological and molecular level to Plasmodium vivax, the diagnosis of P. simium in this region remains problematic. Therefore, a diagnostic assay able to accurately identify P. simium is important for malaria surveillance. Based on mitochondrial genome sequences, primers were designed to amplify a region containing a SNP specific to P. simium. This region can then be digested with the restriction enzyme HpyCH4III, which results in digestion of P. simium sequences, but not of any other malaria parasite. Fifty-two human and monkey blood samples from different regions and infected with different Plasmodium species were used to validate this protocol. This easy and inexpensive tool can be used for the diagnosis of P. simium in non-human primates and human infections from the Atlantic Forest region to monitor zoonotic malaria transmission in Brazil.

Bioquímica plasmática de cascavéis (Caudisona durissa LINNAEUS, 1758) em cativeiro / Blood biochemical parameters from captivity rattlesnake (Caudisona durissa LINNAEUS, 1758)

Acidentes com seres humanos envolvendo a espécie Caudisona durissa possuem a mais alta taxa de letalidade dentre os viperídeos brasileiros. Ressalta-se então a importância para a saúde pública da criação dessa espécie em cativeiro para produção de soro antiofídico. No entanto, essa atividade ainda hoje encontra alguns desafios como a instalação de doenças no plantel, evidenciando a importância de estudos sobre a fisiologia de serpentes. Dessa forma, foram realizadas análises de bioquímica plasmática em 53 serpentes da espécie Caudisona durissa, mantidas em cativeiro. Foram utilizadas amostras de plasma com heparina e as dosagens bioquímicas realizadas em aparelho automatizado (Ciba Corning - Express Plus®). Os resultados e seus respectivos desvios-padrões foram: uréia -1,32mg dL-1 (±1,1); ácido úrico - 2,08mg dL-1 (±1,4); creatinina - 0,52mg dL-1 (±0,2); proteína total - 3,7g dL-1 (±0,7); albumina - 1,62g dL-1 (±0,4); globulinas - 2,08g dL-1 (±0,5); cálcio - 15,25mg dL-1 (±2,8); fósforo - 4,61mg dL-1 (±1,9); colesterol - 171,58mg dL-1 (±52,7); triglicerídeos - 19,29mg dL-1 (±14,3); fosfatase alcalina - 31,04U L-1 (±12,4); aspartato aminotransferase (AST) - 22,25U L-1 (±11,4); alanina aminotransferase (ALT) - 7,11U L-1 (±5,4) e Amilase - 1385,23U L-1 (±568,7). Foram calculados os seguintes índices: relação uréia/creatinina - 2,5 e relação cálcio/fósforo - 3,3. O conjunto de resultados das análises bioquímicas do sangue das cascavéis em cativeiro pode servir como referência para apoio diagnóstico na espécie estudada e para outros trabalhos com o mesmo caráter de observação.

Human accidents involving rattlesnake Caudisona durissa have the highest fatality rate among the Brazilian Viperidae family. Breeding this specie in captivity in order to produce antivenoms is very important to public health. Nevertheless, there are some challenges that this activity must face, like the onset of diseases on the breeding stock, justifying the importance of studying these snakes' physiology. Blood biochemical analysis of 53 Caudisona durissa was performed using plasma samples in an automated equipment (Ciba Corning - Express Plus®). Mean values and its respective Standard deviations are: blood urea nitrogen (BUN) -1.32mg dL-1 (±1.1); uric acid - 2.08mg dL-1 (±1.4); creatinine - 0.52mg dL-1 (±0.2); total protein - 3.7g dL-1 (±0.7); albumin - 1.62g dL-1 (±0.4); globulins - 2.08g dL-1 (±0.5); calcium - 15.25mg dL-1 (±2.8); phosphorus - 4.61mg dL-1 (±1.9); cholesterol - 171.58mg dL-1 (±52.7); triglycerides - 19.29mg dL-1 (±14.3); alkaline phosfatase - 31.04U L-1 (±12.4); aspartate aminotransferase (AST) - 22.25U L-1 (±11.4); alanin aminotransferase (ALT) 7.11U L-1 (±5.4); amylases - 1385.23U L-1 (±568.7). The following indexes were calculated: BUN/creatinine ratio - 2.5 and calcium/phosphorus ratio -3.3. Rattlesnake blood biochemical results above could be used as reference in clinical evaluation for captivity C. durissa.

Avaliação da resposta inflamatória hematológica em cascavéis (Crotalus durissus Linnaeus, 1758) inoculadas com BCG / Assessment of blood inflammatory response in BCG stimulated rattlesnakes (Crotalus durissus Linnaeus, 1758)

A criação de serpentes peçonhentas em cativeiro para produção de soros antipeçonhas possui crescente importância para a saúde pública devido ao aumento do número de notificações de acidentes ofídicos a cada ano no Brasil. Iniciado no século XX, ainda hoje essa atividade apresenta alguns desafios como a instalação de doenças no plantel. O hemograma é um exame de triagem clínica que auxilia no diagnóstico de diversas moléstias que acometem diferentes espécies de animais, no entanto ainda pouco estudado em serpentes. A caracterização das alterações hematológicas em cascavéis inoculadas experimentalmente com BCG pode servir de base na utilização deste exame no auxílio ao diagnóstico de infecções bacterianas na espécie. Dessa forma, foram realizados exames hematológicos em 10 serpentes da espécie Crotalus durissus pertencentes ao plantel da Divisão de Herpetologia do Instituto Vital Brazil. Os animais foram divididos em dois grupos (Grupos 1 e 2), homogêneos entre si em relação ao peso e proporção sexual. Os dois grupos foram inoculados com BCG e submetidos à coleta de sangue antes da inoculação e em três momentos pós-inoculação (3º, 5º, e 7º dias para o Grupo 1 e 11º, 17º e 21º dias para o Grupo 2). O hemograma foi realizado por método semidireto pela utilização de líquido de Natt e Herrick e as lâminas foram coradas pelo Giemsa. Observou-se anemia discreta, com redução dos valores de concentração de hemoglobina corpuscular média e da hemoglobina globular média no Grupo 1 que foi relacionada à doença inflamatória. A trombocitopenia observada no Grupo 2 sugeriu a atuação deste tipo celular em processos inflamatórios. Um único animal do Grupo 1 apresentou granulocitose e alguns animais apresentaram discreta azurofilia. Observaram-se alterações morfológicas nos leucócitos. Os granulócitos apresentaram granulações grosseiras e os azurófilos apresentaram aumento de tamanho e grandes vacúolos. De forma geral, a inoculação de BCG em cascavéis desencadeia ...

The high demand for anti-venom production in response to the increased number of cases of snakebite envenomation highlights the importance of raising and breeding venomous snakes in captivity. Knowledge of types of venoms and anti-venoms is of great interest to public health. The maintenance of venomous serpents in captivity started in the early twentieth century, but still nowadays it is a challenge to manage and prevent diseases in captive fauna. Hematology is commonly used for general health assessment and illness detection. However, data on serpent blood analysis are still scarce. Alterations in hematological parameters were experimentally induced in rattlesnakes by the inoculation of BCG. Based on this, hemograms can be used as a health auxiliary diagnosis method for bacterial diseases in this species. In this study, blood samples were taken from 10 healthy specimens of rattlesnakes (Crotalus durissus) born and bred in captivity in the Herpetological Division of Vital Brazil Institute. Animals were divided into two groups (group 1 and 2) with similar live weight and sex proportion, and were then inoculated subcutaneously with BCG (Bacillus Calmette-Guérin). Blood samples were taken before and after inoculation at three experimental times (days 3, 5 and 7 for group 1 and days 11, 17 and 21 for group 2). Hematological analysis was performed through semi-direct technique, blood samples were diluted in Natt and Herrick solution and smears were stained by Giemsa. Serpents from group 1 developed discrete anemia due to the inflammatory syndrome, and showed significant decrease of MCH and MCHC. Granulocytes were characterized by the presence of rough granules. The azurophils varied in shape and size and showed large amount of cytoplasmic vacuoles. The thrombocytopenia observed in group 2 suggests that these cells participate in the inflammatory process. A single individual from group 1 showed granulocytosis and a few animals showed a discrete ...