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Balantioides coli is a ciliated protist that can cause dysentery in humans, pigs and nonhuman primates and may have the potential for zoonotic transmission. Its diagnosis is routinely performed through conventional parasitological techniques, and few studies have used culturing techniques to isolate it, applying molecular tools for the characterization of this protozoan. Thus, the objective of this study was to confirm B. coli diagnosis using molecular tools and to characterize the genetic variants of this parasite isolated from pigs kept on family farms in Brazil using three different culture media that differed in the serum added. Fecal samples from pigs were inoculated in Pavlova medium plus coconut water (PC), fetal bovine serum (PB) and horse serum (PH). Of the 127 samples positive for forms compatible with the phylum Ciliophora, 31 were selected for isolation. The most successful medium for isolation was PB 19/31 (61.3%), followed by PH 18/31 (58.1%) and PC 11/31 (35.5%). Of the nucleotide sequences generated, 20 were classified as genetic variant type B0, two as A1 and 15 as A0. The results indicated that PC, despite having allowed the isolation of B. coli for a short period, was not an adequate medium for the maintenance of this parasite in vitro, therefore requiring improvement.

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Intestinal protozoa, which can be asymptomatic or cause diarrhea, dysentery and even death, are among the main agents that affect nonhuman primates (NHPs) kept under human care. Nevertheless, information on the molecular and morphometric profiles of parabasilids in the Neotropics is still scarce. In this context, the objective of this study was to isolate the Parabasalia protozoa detected in the feces of NHPs and their keepers in Pavlova and TYSGM9 media and to characterize the isolates by molecular biology and morphometry. Fecal samples from NHPs from five Brazilian institutions were analyzed. Direct examination was performed immediately after obtaining the samples. A total of 511 fecal samples from NHPs were collected, and 10.6% contained parabasilids. Regarding the handlers, of the 74 samples analyzed, three were positive. In vitro-generated parabasilid isolates were successfully obtained from all positive samples, as identified via microscopy. Isolates of the parasite were obtained both from New World NHPs, including the genera Leontopithecus, Saguinus, Leontocebus, Aotus, Saimiri, Sapajus, and Alouatta, and from the Old World primate Pan troglodytes. Forty-nine NHP isolates were molecularly identified: Pentatrichomonas hominis (16), Trichomitus batrachorum (14), Tetratrichomonas brumpti (13) and Hypotrichomonas hampli (6). The human isolates were identified as Tetratrichomonas sp. (2) and T. batrachorum (1). Visualization and morphometric analysis revealed trophozoites with piriform or rounded shapes that presented variable measurements. The isolates previously characterized as P. hominis had up to five free flagella, while T. batrachorum and Tetratrichomonas sp. had up to four free flagella, and H. hampli had a maximum of three free flagella. These morphometric characteristics corroborated the molecular identification. In general, a variety of parabasilids were observed to infect NHPs, and T. batrachorum was isolated from biological samples from both NHPs and their keepers, a finding that reinforces the susceptibility of these hosts to infections by parabasilids in Brazil.

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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.

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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.

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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.

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The frequency of gastrointestinal parasites with an emphasis on Strongylus vulgaris was investigated among the Brazilian Pony breed kept on farms in the municipality of Teresópolis, state of Rio de Janeiro. Fecal samples were collected in three stud farms: A (n= 22 animals), B (n= 3), and C (n= 2). Fecal samples were subjected to the quantitative Mini-FLOTAC technique, using three different solutions, and to qualitative techniques. The parasite prevalence was found to be 81.4%. Eggs from strongylids were identified in 74% of the ponies. Eggs of Parascaris spp. were detected in 22.7% of the animals, which were all females of farm A. At this locality, mares were kept with their foals in fenced paddocks all the time. The NaCl solution of d = 1.200 g/ml was generally the one that presented the highest frequency of diagnosis of nematode eggs and the highest mean of fecal eggs per gram. The fecal samples were also subjected to the polymerase chain reaction for amplification of DNA from the ITS2 region for Strongylus vulgaris. Twelve samples presented nucleotide sequences for S. vulgaris. In the end, this study revealed the high frequency (96.3%) of S. vulgaris among ponies on farms in Teresópolis, Rio de Janeiro, Brazil.

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The frequency of gastrointestinal parasites with an emphasis on Strongylus vulgaris was investigated among the Brazilian Pony breed kept on farms in the municipality of Teresópolis, state of Rio de Janeiro. Fecal samples were collected in three stud farms: A (n= 22 animals), B (n= 3), and C (n= 2). Fecal samples were subjected to the quantitative Mini-FLOTAC technique, using three different solutions, and to qualitative techniques. The parasite prevalence was found to be 81.4%. Eggs from strongylids were identified in 74% of the ponies. Eggs of Parascaris spp. were detected in 22.7% of the animals, which were all females of farm A. At this locality, mares were kept with their foals in fenced paddocks all the time. The NaCl solution of d = 1.200 g/ml was generally the one that presented the highest frequency of diagnosis of nematode eggs and the highest mean of fecal eggs per gram. The fecal samples were also subjected to the polymerase chain reaction for amplification of DNA from the ITS2 region for Strongylus vulgaris. Twelve samples presented nucleotide sequences for S. vulgaris. In the end, this study revealed the high frequency (96.3%) of S. vulgaris among ponies on farms in Teresópolis, Rio de Janeiro, Brazil.(AU)

A frequência de parasitos gastrointestinais, com ênfase na pesquisa de Strongylus vulgaris, foi investigada entre os Pôneis Brasileiros criados em haras na cidade de Teresópolis, no estado do Rio de Janeiro. Amostras fecais foram coletadas em três haras: A (n=22 animais), B (n=3) e C (n=2). Amostras fecais foram submetidas à técnica quantitativa de Mini-FLOTAC utilizando três diferentes soluções e técnicas qualitativas. A prevalência de parasitos foi de 81,4%. Ovos de estrôngilos foram identificados em 74% dos pôneis. Ovos de Parascaris spp. foram detectados em 22,7% dos animais, sendo todos fêmeas do haras A. Nesta propriedade, as éguas eram mantidas com os pôneis em piquetes cercados durante todo o tempo. A solução de NaCl, com densidade de 1.200 g/ml, foi a que apresentou a maior frequência diagnóstica de ovos de nematoides e a maior contagem de ovos por grama de fezes. As amostras também foram submetidas à reação de polimerase em cadeia para amplificar DNA da região ITS2 de Strongylus vulgaris. Doze amostras fecais apresentaram sequências nucleotídicas de S. vulgaris. Ao final, este estudo demonstrou a alta frequência (96.3%) de S. vulgaris em pôneis mantidos em haras na cidade de Teresópolis, Rio de Janeiro, Brazil.(AU)

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An analysis was made of the frequency of Cryptosporidium spp. in fecal samples from horses raised on farms in the Teresópolis city, state of Rio de Janeiro, Brazil, and the risk factors that favored this infection. Between 2019 and 2020, 314 samples of equine feces were collected, 287 of which came from English Thoroughbred horses and 27 from ponies. Information on the horses and their management were retrieved from a stud book and forms filled out by trainers. The fecal samples were subjected to macroscopic analysis, modified Sheather's and Lutz parasitological techniques, safranin staining, and to enzyme-linked immunosorbent assay (ELISA) for the detection of coproantigens. All the samples that tested positive by these techniques underwent partial sequence analysis of the 18S rRNA gene to characterize the protozoan species. Cryptosporidium spp. was identified in 35 (11.1%) of the samples, 34 from English Thoroughbred horses and one from a pony. Based on a logistic regression model, it was found that the presence of dogs and small ruminants on the farms, and drinking water from a spring, were significantly associated with the animals' infection by the protozoan (p < 0.05). Eight of the English Thoroughbred horse samples underwent molecular characterization, which revealed the presence of Cryptosporidium felis in one sample and Cryptosporidium parvum in seven. The seven samples containing C. parvum were subjected to gp60 gene analysis, based on which nucleotide sequences typical of the IIa family were identified, which are usually transmitted from animals to humans. In addition, the genotype IIaA15G2R1, which is considered to have the highest profile of zoonotic transmissibility, was identified in one Thoroughbred horse. This is the first study conducted in the state of Rio de Janeiro that molecularly characterized Cryptosporidium spp. in horses, and the first on the American continent to detect C. felis in the feces of these animals.

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Polymyxin resistance is an emerging health issue aggravated by mcr dissemination among Enterobacterales recovered from various sources. Commensal Escherichia coli plays a key role in the spread of antimicrobial resistance in community settings and is likely to spread silently. It may transfer resistance genes to pathogenic bacteria in the gastrointestinal tract and the environment, and may cause difficult-to-treat infections, especially in immunocompromised patients. Unraveling actors disseminating resistance to last-resort antimicrobials might support the future development of control measures. Here we report the occurrence of a commensal ST683/CC155 colistin-resistant mcr-1.1-harboring E. coli (JP24) obtained from touristic coastal water. JP24's genome was sequenced and comparatively analyzed with other genomes from ST683/CC155 isolated worldwide and with mcr-carrying isolates recovered from various sources in Brazil. Besides mcr-1, JP24 carried blaCTX-M-8, tet(A), tet(34), dfrA12, sul2, sul3, aph(3')-Ia, aph(3')-IIa, aadA1, aadA2, cmlA1, Inu(G), mef(B) and mdf(a). mcr-1 and blaCTX-M-8 were transferable by IncX4 and IncI1/Iγ plasmids, respectively. Tree-based phylogeny of the ST683/CC155 isolates core genome revealed two larger clades. E. coli JP24 was grouped into a subclade together with an isolate from Thailand (ERR4221036), both carrying mcr-1. The core genome-based tree of the isolates carrying mcr-1 from Brazil revealed proximity with E. coli ECEST9 recovered from a mangrove also located in Northeastern Brazil. Accessory genome-based tree clustered most environmental isolates apart from the clinical ones and remained JP24 closer to ECEST9. High sequence conservation was observed between mcr-1-harboring plasmids detected in different species and reservoirs in Brazil and other countries. In addition to recreational coastal waters being potential sources for community exposure to antimicrobial-resistant bacteria, our findings reinforce a more prominent role of horizontal gene transfer, other than clonal expansion, in mcr dissemination in the community.

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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.

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