The present study focuses on Cryptosporidium infections of foals in Brazil. A total of 92 animals of different breeds from 11 farms in the vicinity of Araçatuba in the state of São Paulo, were examined. According to PCR targeting the 18S rRNA gene, Cryptosporidium sp. DNA was detected in 21.7% (20/92) of foals. Good quality 18S rRNA, actin, HSP70 and gp60 genes nPCR amplicons were obtained from five fecal samples. PCR amplification and sequencing of a fragment of the GP60 sporozoite surface glycoprotein gene revealed C. parvum genotypes IIaA18G3R1, IIaA15G2R1. Interestingly, we also detected in two foals a GP60 genotype related to the human parasite C. hominis.

Since the early 20 th century, the detection of intestinal parasites has improved with the development of several techniques for parasitic structures recovery and identification, which differ in sensitivity, specificity, practicality, cost, and infrastructure demand. This study aims to review, in chronological order, the stool examination techniques and discuss their advantages, limitations, and perspectives, and to provide professionals and specialists in this field with data that lays a foundation for critical analysis on the use of such procedures. The concentration procedures that constitute the main techniques applied in routine research and in parasitological kits are a) spontaneous sedimentation; b) centrifugation-sedimentation with formalin-ethyl acetate; and c) flotation with zinc sulfate solution. While selecting a technique, one should consider the purpose of its application and the technical-operational, biological, and physicochemical factors inherent in the procedures used in stool processing, which may restrict its use. These intrinsic limitations may have undergone procedural changes driven by scientific and technological development and by development of alternative methods, which now contribute to the improvement of diagnostic accuracy.

Due to its important zoonotic potential, cryptosporidiosis arouses strong interest in the scientific community, because, it was initially considered a rare and opportunistic disease. The parasitological diagnosis of the causative agent of this disease, the protozoan Cryptosporidium spp., requires the use of specific techniques of concentration and permanent staining, which are laborious and costly, and are difficult to use in routine laboratory tests. In view of the above, we conducted the feasibility, development, evaluation and intralaboratory validation of a new parasitological technique for analysis in optical microscopy of Cryptosporidium spp. oocysts, called TF-Test Coccidia, using fecal samples from calves from the city of Araçatuba, São Paulo. To confirm the aforementioned parasite and prove the diagnostic efficiency of the new technique, we used two established methodologies in the scientific literature: parasite concentration by centrifugal sedimentation and negative staining with malachite green (CSN-Malachite) and Nested-PCR. We observed good effectiveness of the TF-Test Coccidia technique, being statistically equivalent to CSN-Malachite. Thus, we verified the effectiveness of the TF-Test Coccidia parasitological technique for the detection of Cryptosporidium spp. oocysts and observed good concentration and morphology of the parasite, with a low amount of debris in the fecal smear.

Considering the proximity of sheep farmers to animals that are possibly diseased or releasing fecal oocysts into the environment and the marked pathogenicity in lambs, the aim of this study was to determine the occurrence and to molecularly characterize the infection by Cryptosporidium spp. in lambs in the South Central region of the state of São Paulo, Brazil. A total of 193 fecal samples were collected from sheep of several breeds, males and females, aged up to one year. Polymerase chain reaction (nested-PCR) was used to amplify DNA fragments from the subunit 18S rRNA gene and indicated 15% positivity; sequencing of amplified fragments was possible for 19 samples. Analysis of the obtained sequences showed that the identified species were Cryptosporidium xiaoi for 15 samples, constituting thus the first molecular characterization study of this Cryptosporidium species in Brazil. Cryptosporidium ubiquitum was identified for three samples and Cryptosporidium meleagridis for one sample; the latter two are considered zoonotic species.

With the aim of determining the occurrence of Cryptosporidium spp., 222 fecal samples were collected from Murrah buffalo calves aged up to 6 mo. Fecal DNA was genotyped with a nested polymerase chain reaction targeting the 18S rRNA gene and sequencing of the amplified fragment. Nested 18S PCR was positive for 48.2% of the samples. Sequence analysis showed that the most frequent species in these animals was Cryptosporidium ryanae, which was present in buffalo calves as young as 5 d. The zoonotic species Cryptosporidium parvum was detected in one animal. An uncommon Cryptosporidium 18S genotype was found in buffaloes.

Because canine intestinal parasites are considered cosmopolitan, they carry significant zoonotic potential to public health. These etiological agents are routinely diagnosed using microscopic examination commonly used because of its low cost, simple execution, and direct evidence. However, there are reports in the literature on the poor performance of this test due to low to moderate sensitivity resulting from frequent errors, procedures and interpretation. Therefore, to improve the diagnostic efficiency of microscopic examination in veterinary medicine, we developed and evaluated a unique new protocol. This system was tested in a study involving four genera of highly prevalent canine intestinal parasites in an endemic region in São Paulo state, Brazil. Fecal samples from 104 animals were collected for this research. The new protocol had a significantly higher (p < 0.0001) number of positive cases on image data, including parasites and impurities, and was elaborate to test them with the TF-GII/Dog technique, with a moderate agreement and Kappa index of 0.7636. We concluded that the new Prototic Coproparasitological Test for Dogs (PC-Test Dog) allowed a better visualization of the parasitic structures and showed a favorable result for the diagnosis of intestinal parasites in dogs.

ResumoEstudos referentes á criptosporidiose são essenciais, devido sua relevância em saúde pública e por sua patogenicidade em animais de produção e de companhia. Nos últimos 20 anos, houve uma rápida expansão das pesquisas envolvendo o gênero Cryptosporidium, em grande parte relacionada a estudos moleculares, propiciando a descrição de várias espécies, genótipos e subtipos do parasito. A caracterização molecular de isolados de diferentes origens (animal, humana e ambiental) tem sido amplamente usada com o intuito de investigar o potencial zoonótico deste protozoário. As formas de transmissão documentadas são de animais para o ser humano, de pessoa para pessoa, por meio de ingestão ou do uso de água destinada a lazer contaminada direta ou indiretamente com oocistos esporulados. A elevada taxa de animais naturalmente infectados e a susceptibilidade ao protozoário justificam a importância de se atentar para a ocorrência desta enfermidade. Assim, são demonstrados aspectos epidemiológicos desta zoonose em animais domésticos. Palavras-chave: Cryptosporidium spp., ruminantes, equinos, caninos, felinos AbstractStudies related cryptosporidiosis will be essential, due to its relevance in public health and pathogenicity in pets and production animals. Over the past 20 years, there has been a rapid expansion of research involving the Cryptosporidium genus, largely related to molecular studies, providing a description of

Pesq. Vet. Bras. 34(8):728-732, agosto 2014 728 RESUMO.-O presente estudo teve como objetivo determinar a ocorrência da infecção por Cryptosporidium spp. em cabritos de Quixadá, Ceará, Brasil. Participaram do estudo 400 cabritos, com idade entre três e 360 dias, de ambos os sexos, com e sem padrão racial definido, procedentes de 25 estabelecimentos rurais distribuídos em três circuitos. As fezes foram cadastradas de acordo com o aspecto e cor, distribuídas em tubos tipo "eppendorf ®" e congeladas in natura a -20°C, até o momento das extrações de DNA genô-mico do parasito com auxílio de kit comercial. Para amplificação de fragmentos da subunidade 18S do RNA ribossô-mico (rRNA) foi utilizada a "Nested"-PCR. A ocorrência de Cryptosporidium spp em cabritos de Quixadá foi de 7,50% (30/400). A frequência no período seco e no chuvoso foi de 9,55% (19/199) e 5,47% (11/201), respectivamente (χ 2 =2,39 e P>0,05). Amostras positivas foram identificadas em 64,00% (16/25) das propriedades estudadas e dessas amostras 50,00% (15/30) e 70,00% (21/30) tinham as fezes com aspecto e cor normais, respectivamente, sugerindo que cabritos assintomáticos estão eliminando oocistos. Não foi observada positividade para Cryptosporidium spp.