Identification of third stage larvae of strongyles and molecular diagnosis of Strongylus vulgaris in the feces of Thoroughbred horses kept in training centers in Rio de Janeiro, Brazil.

The aims of the present study were to identify strongyles in the feces of Thoroughbred horses based on larval morphology; to detect Strongylus vulgaris using molecular diagnosis and compare results to those of feces culture; and to determine the association between the presence of S. vulgaris with corresponding animal information (age range, gender, and anthelmintic use). Feces of horses kept in six Training Centers in Rio de Janeiro State, that showed the presence of ≥500 eggs per gram of feces (EPG) were subjected to strongyle identification. Of the 520 fecal samples collected, 35 had an EPG ≥ 500. After fecal culture for L3 larvae identification, DNA was extracted, subjected to PCR to amplify the ITS2 region DNA fragment of S. vulgaris, and sequenced. A total of 3500 larvae were analyzed. Most were classified as small strong (99.7%), with an emphasis on the type A subfamily of Cyathostominae. Forms of S. vulgaris only corresponded to 0.2%. In all, 25 samples showed amplified S. vulgaris DNA products and 11 showed nucleotide sequences with high sequence identity. Fecal culture and PCR results showed poor agreement (kappa = 0.105) for S. vulgaris diagnosis. Age, gender, anthelmintic use, and anthelmintic administration interval were not statistically significant. The present study showed the presence of S. vulgaris in the feces of horses kept in Rio de Janeiro Training Centers, mainly seen via PCR, which has emerged as the most effective tool for diagnosis. This study made it possible to identify strongyles that infect horses in the region, emphasizing upon the necessity for constant monitoring of the animals.

Piroplasmid Infections Among Domestic Dogs in the Mountain City of Rio de Janeiro, Brazil.

PURPOSE: The aim of the present study was to analyze the frequency of the piroplasmids in blood from dogs and ticks recovered from these animals in Teresópolis city, located in the mountain region of Rio de Janeiro state, Brazil. In addition to the clinical and hematological profile. METHODS: A total of 400 dogs attended in a veterinary clinic in this city between 2020 and 2021 were included. The blood was collected from the dogs, along with ticks and information on these dogs was obtained through a questionnaire applied to the owners. Thin-smear analyses and complete blood counts were performed. All forms characteristic of piroplasmids were measured and classified morphologically. The blood was also subjected to PCR assays based on the genes 18S rRNA and hsp70. In addition, the ixodid ticks were classified morphologically and subjected to PCR for piroplasmids research. The amplified products were sent for gene sequencing. RESULTS: Piroplasmids were detected in 2.3% of the dogs. The variables statistically associated with infections in these animals were hemorrhage/bleeding, jaundice, anisocytosis, activated monocytes and macroplatelets (p ≤ 0.05). Piriform, ring-shaped, oval and aberrant structures were viewed in erythrocytes, neutrophils and monocytes, with lengths greater than and less than 2.5 µm. The nine positive samples from these dogs were characterized as due to Rangelia vitalii. However, one sequence from B. vogeli was detected in a single adult specimen of R. sanguineus. CONCLUSION: Although circulation of two species of piroplasmids potentially infective for domestic dogs has been observed in the mountain city of Rio de Janeiro, infection due to R. vitalii was mostly seen in the dogs of the present study.

Diagnosis of endoparasite species and subtypes of Cryptosporidium spp. with one health importance, in feces from captive snakes in Rio de Janeiro, Brazil.

In captivity, snakes may present chronic infections with high mortality, such as those caused by Cryptosporidium serpentis, or they may be pseudoparasitized by species that present zoonotic potential. Thus, the aim of this study was to evaluate the frequency of helminths and protozoa in the feces of captive snakes, characterize the species and subtypes of Cryptosporidium spp. and correlate the parasites detected with other information obtained from these animals. Feces were collected from 189 snakes kept at the Vital Brazil Institute, Rio de Janeiro, including samples from Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops atrox, Bothrops leucurus, Crotalus durissus and Lachesis muta. All the samples were subjected to microscopy techniques and the polymerase chain reaction (PCR) in association with sequencing, to identify Cryptosporidium spp.. Forms of parasites infecting the snakes were identified through microscopy in 50.8% of the samples. Helminths were detected more often than protozoa in the feces of these animals, mainly comprising eggs resembling Kalicephalus sp. and oocysts of Eimeria sp.. Pseudoparasites such as Syphacia sp., Aspiculuris sp. and Hymenolepis nana were also detected. Through correlating the results obtained from parasitological staining techniques and PCR, the total frequency of Cryptosporidium sp. was found to be 19%. The species C. tyzzeri and C. parvum were identified. Characterization using the target gp60 showed subtypes with high potential for zoonotic transmission, especially IIaA15G2R1 and IIaA14G2R1 of C. parvum and IXbA8 of C. tyzzeri. This study highlighted the need for more intensive health management in the Institute's serpentarium and, especially, in its bioterium where rodents are reared as a food source for these snakes.

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.

Non-invasive genetic sampling reveals a habitat use extension of Chrysocyon brachyurus and Leopardus guttulus inside a protected area of Southeastern Brazil / Amostragem genética não-invasiva revela uma extensão do uso de habitat por Chrysocyon brachyurus e Leopardus guttulus em uma área protegida do Sudeste do Brasil

Abstract Anthropogenic activities have modified landscapes leading to environmental damages and to a threatened biodiversity. As a result, protected areas have become the last refuge for many species. Protected areas surrounded by a highly modified landscape may accumulate species, especially large mammals, which may alter their habitual habitat use. Here we used non-invasive DNA analysis, molecular species identification, and landscape analyses to assess the habitat use of carnivore species in an isolated protected area, Itatiaia National Park (PNI). Two species were by far the most sampled within PNI, Chrysocyon brachyurus and Leopardus guttulus. The spatial distribution of each species was assessed to the following landscape variables: altitude; land use/land cover; slope and Euclidean distances from water and from urban buildings. The habitat use of C. brachyurus and L. guttulus was related to altitude and land use/land cover. We tested whether there were differences in the environmental indicators considering both species, which showed that higher altitudes, forest and/or grassland formation were indeed associated with them. We highlighted the unprecedented presence of both species at altitudes up to 2,631 meters. Our results suggest a habitat use extension for both species inside the PNI that could be a consequence of the highly modified landscape where PNI is inserted. Therefore, the results can be helpful for better understanding the species dynamics and their conservation in the face of landscape changes. Further, this study may be of help for management and conservation policies of this emblematic protected area.

Resumo As atividades antropogênicas modificaram as paisagens levando a danos ambientais e a uma biodiversidade ameaçada. Como resultado, as áreas protegidas se tornaram o último refúgio para muitas espécies. Áreas protegidas cercadas por uma paisagem altamente modificada podem acumular espécies, especialmente grandes mamíferos, o que pode alterar o uso de seu habitat habitual. Neste trabalho, usamos análise de DNA não-invasivo, identificação molecular de espécies e análises de paisagem para avaliar o uso de habitat de carnívoros em uma área protegida isolada, o Parque Nacional de Itatiaia (PNI). Duas espécies foram de longe as mais amostradas dentro do PNI, Chrysocyon brachyurus e Leopardus guttulus. A distribuição espacial de cada espécie foi avaliada com as seguintes variáveis paisagísticas: altitude; uso e cobertura do solo; declividade e distâncias euclidianas da água e de construções urbanas. O uso de C. brachyurus e L. guttulus no habitat estava relacionado à altitude e ao uso e cobertura do solo. Testamos se existiam diferenças nos indicadores ambientais considerando ambas as espécies, o que mostrou que altitudes mais elevadas, formação de florestas e/ou campestres estavam de fato associadas a elas. Destacamos a presença incomum de ambas as espécies em altitudes de até 2.631 metros. Nossos resultados sugerem uma extensão do uso do habitat conhecido para ambas as espécies como possível consequência da paisagem altamente modificada onde o PNI está inserido. Portanto, os resultados podem ser úteis para entender melhor a dinâmica das espécies e sua conservação diante das mudanças na paisagem. Além disso, este estudo pode ser útil para as políticas de manejo e conservação desta emblemática área protegida.

Microscopic Detection, Hematological Evaluation and Molecular Characterization of Piroplasms from Naturally Infected Dogs in Rio de Janeiro, Brazil.

PURPOSE: This study aimed to analyze the frequency of piroplasmids in the blood of dogs in Rio de Janeiro, compare the performance of microscopic techniques, assess the risk factors associated with infections and also molecularly and morphologically characterize the piroplasmids identified. METHODS: In all, 407 blood samples were collected from dogs between 2018 and 2019. These were subjected to microscopic parasitological techniques for thin and thick smears, stained with Giemsa and using a rapid staining kit. The slides were read under an optical microscope and the protozoa were characterized morphometrically. In addition, the blood samples were subjected to molecular characterization for diagnosing piroplasmid species using primers that amplified the gene 18S rRNA. RESULTS: Piroplasmids were detected in 38 (9.3%) samples. Of these, 33 samples presented nucleotide sequences compatible with Babesia vogeli. Most of the positive samples were young, male, defined breeds dogs that had been attended in clinics in São Gonçalo city. Thrombocytopenia and leukopenia were the hematological alterations more observed in positive samples, but positive samples without alterations were also detected. The sex was the only variable that showed statistical differences. Males dogs being more often infected than females (p < 0.05). The microscope slides mostly showed piriform and oval merozoites measuring greater than 2.5 µm in length, which were compatible with B. vogeli. However, smaller forms were also identified, thus demonstrating the polymorphic nature of this parasite. CONCLUSION: Babesia vogeli was detected in blood samples from dogs in the metropolitan cities of Rio de Janeiro by molecular techniques in different parasite morphotypes.

Non-invasive sampling in Itatiaia National Park, Brazil: wild mammal parasite detection.

BACKGROUND: Non-invasive sampling through faecal collection is one of the most cost-effective alternatives for monitoring of free-living wild mammals, as it provides information on animal taxonomy as well as the dynamics of the gastrointestinal parasites that potentially infect these animals. In this context, this study aimed to perform an epidemiological survey of gastrointestinal parasites using non-invasive faecal samples from carnivores and artiodactyls identified by stool macroscopy, guard hair morphology and DNA sequencing in Itatiaia National Park. Between 2017 and 2018, faeces from carnivores and artiodactyls were collected along trails in the park. The host species were identified through macroscopic and trichological examinations and molecular biology. To investigate the parasites, the Faust, Lutz and modified Ritchie and Sheather techniques and enzyme immunoassays to detect Cryptosporidium sp. antigens were used. RESULTS: A total of 244 stool samples were collected. The species identified were Chrysocyon brachyurus, Leopardus guttulus, Canis familiaris, Cerdocyon thous, Puma yagouaroundi, Leopardus pardalis, Puma concolor and Sus scrofa. There were 81.1% samples that were positive for parasites distributed mainly in the high part of the park. Helminths, especially eggs of the family Ascarididae, were more frequently detected in carnivore faeces (70.9%). Protozoa, especially Cryptosporidium sp., represented the highest frequency of infection in artiodactyl faeces (87.1%). This zoonotic protozoon was detected in eight mammalian species, including in a wild boar. High values of structural richness and Shannon and Simpson diversity indices were observed for the parasites, especially in the faeces of C. brachyurus. Significant differences in parasite diversity were observed between wild and domestic animals, such as C. brachyurus and C. familiaris, respectively, and between taxonomically distant species, such as C. brachyurus and S. scrofa. The highest values for parasite similarity were found among the species that frequented similar areas of the park, such as C. brachyurus and L. guttulus. CONCLUSIONS: The animals and parasite infections were identified through the combination of three techniques. High frequency parasite structures were diagnosed. Zoonotic protozoa were found and mainly occurred in samples from introduced species.

First molecular characterization of Balantioides coli (Malmsten, 1857) isolates maintained in vitro culture and from feces of captive animals, Rio de Janeiro, Brazil.

Ciliate protozoa of the genus Balantioides can parasitize a variety of animals. The morphology of the evolutionary forms of the parasite and the host species affected have long been the only characteristics used to taxonomically identify the species of these protozoa, but these variables are not very precise. To confirm species identity, molecular biology tools are currently used. In this context, this study aimed to analyze protozoan isolates maintained in culture medium and from fecal samples from captive animals in Rio de Janeiro, Brazil, by means of molecular tools. Forty isolates maintained in Pavlova modified medium (30 were isolated from feces of pigs and 10 from feces of cynomolgus macaques) were analyzed. In addition, 34 fecal samples (8 from pigs, 8 from cynomolgus macaques and 18 from rhesus macaques) containing Balantioides coli-like cysts were analyzed. All samples were subjected to DNA extraction and the polymerase chain reaction (PCR) to amplify the fragment ITS1 - 5.8s rRNA - ITS2, and the PCR products were purified and sequenced. All samples (100%) presented sequences that were grouped in the Balantioides coli cluster. The type A0 variant predominated. These sequences were 96% to 99% identical to those deposited in GenBank, including a B. coli sequence that had been obtained from human fecal material in Bolivia. It seems that the culturing system did not select variants, because this variant was also seen in the amplified sequences of fecal samples containing cysts. The isolate sequences in the cultures showed few ambiguities and substitutions, thus generating reliable chromatograms. This was the first study to identify B. coli in captive animals in Brazil, through molecular biology. In addition, it was the first to evaluate a large panel of isolates of the parasite through culturing.

Balantidium coli and other gastrointestinal parasites in captives non-human primates of the Rio de Janeiro, Brazil.

BACKGROUND: Parasites are agents of diarrhea in captive non-human primates (NHP). To broaden information about those etiological agents in captive animals in Brazil, gastrointestinal parasites in captive NHP were researched in nurturing Centers of Rio de Janeiro State. METHODS: Fecal samples were collected from primates, of which 960 came from the Research Center (Cecal/Fiocruz) and 115 from the Primate Center (CPRJ/Inea). The study involved species of the New World (NW) primates and of the Old World (OW). RESULTS: The estimated prevalence was 56.7%, of which 91.3% presented protozoans and 7.4% presented helminths. Statistical difference between the nurturing centers occurred in the overall value of parasitosis and in the isolated frequency of Balantidium coli and Entamoeba sp., especially in the samples of OW primates living in Cecal. CONCLUSIONS: These results demonstrated the need for implements of sanitation programs in the sites for captive primates nurturing.