Search

Made available in DSpace on 2018-12-11T17:14:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-01 We describe molecular testing for felid alphaherpesvirus 1 (FHV-1), carnivore protoparvovirus 1 (CPPV-1), feline calicivirus (FCV), alphacoronavirus 1 (feline coronavirus [FCoV]), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and canine distemper virus (CDV) in whole blood samples of 109 free-ranging and 68 captive neotropical felids from Brazil. Samples from 2 jaguars (Panthera onca) and 1 oncilla (Leopardus tigrinus) were positive for FHV-1; 2 jaguars, 1 puma (Puma concolor), and 1 jaguarundi (Herpairulus yagouaroundi) tested positive for CPPV-1; and 1 puma was positive for FIV. Based on comparison of 103 nucleotides of the UL24-UL25 gene, the FHV-1 sequences were 99–100% similar to the FHV-1 strain of domestic cats. Nucleotide sequences of CPPV-1 were closely related to sequences detected in other wild carnivores, comparing 294 nucleotides of the VP1 gene. The FIV nucleotide sequence detected in the free-ranging puma, based on comparison of 444 nucleotides of the pol gene, grouped with other lentiviruses described in pumas, and had 82.4% identity with a free-ranging puma from Yellowstone Park and 79.5% with a captive puma from Brazil. Our data document the circulation of FHV-1, CPPV-1, and FIV in neotropical felids in Brazil. Departments of Preventive Veterinary Medicine and Animal Health School of Veterinary Medicine and Animal Science University of São Paulo Departments of Pathology School of Veterinary Medicine and Animal Science University of São Paulo Jaguar Conservation Fund Instituto Onça-Pintada Institute for the Conservation of Neotropical Carnivores Instituto Pró-Carnívoros Institute for Ecological Research Instituto de Pesquisas Ecológicas (IPE) Department of Microbiology and Immunology Biosciences Institute State University of São Paulo (UNESP) Júlio de Mesquita Filho Brazilian Institute for Conservation Medicine Instituto Brasileiro para Medicina da Conservação (TRÍADE) Chico Mendes Institute for Biodiversity Conservation ICMBio Department of Microbiology and Immunology Biosciences Institute State University of São Paulo (UNESP) Júlio de Mesquita Filho

Highlights • We performed a search for canine coronavirus in fecal samples and organs from dogs. • Pantropic canine coronavirus was found in different organs. • Similar lesions to those found in canine parvovirus were observed. • This study reinforces the need for differential diagnosis between canine parvovirus and canine coronavirus., Characterization of canine coronavirus (CCoV) strains currently in circulation is essential for understanding viral evolution. The aim of this study was to determine the presence of pantropic CCoV type IIa in tissue samples from five puppies that died in Southern Brazil as a result of severe gastroenteritis. Reverse-transcriptase PCR was used to generate amplicons for sequence analysis. Phylogenetic analysis of the CCoV-IIa strains indicated that they were similar to those found in other countries, suggesting a common ancestor of these Brazilian isolates. This is the first report of pantropic CCoV-II in puppies from Latin America and our findings highlight that CCoV should be included as a differential diagnosis when dogs present with clinical signs and lesions typically seen with canine parvovirus infection.

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle.

O coronavírus canino (CCoV) causa gastroenterite em cães jovens, podendo ser letal, sobretudo quando há coinfecção com parvovírus canino (CPV). Os objetivos do presente projeto foram investigar a presença de CCoV e CPV em amostras fecais de cães jovens; estudar a diversidade molecular das amostras de CCoV com base em sequenciamento parcial dos genes M, S, 3b e N, incluindo amostras vacinais, e estudar a evolução in vitro de CCoV em células A72 de fibroma canino. Foram detectados 40,17% (47/117) animais positivos para CCoV e 13,68% (16/117) para CPV. Estudos filogenéticos demonstraram que oito amostras foram classificadas como CCoV-II, vinte e cinco como CCoV-I. Análises para o gene M destacaram alta identidade de CCoV-I com amostras de coronavírus da peritonite infecciosa felina (PIF) e uma possível amostra pantrópica foi demonstrada pela análise do gene S. O gene do nucleocapsídeo de CCoV é altamente conservado entre os tipos I e tipo II, com uma resolução mais baixa em relação a árvores para os genes M e S. Amostras dos tipos I e II apresentam um polimorfismo baixo para o gene 3b, sem marcadores estáveis para diferenciação dos tipos de CCoV. Uma amostra de CCoV-II putativamente pantrópica foi isolada em células A72, resultando em efeito citopático no 5o dia da 5a passagem. No estudo evolutivo, a amostra vacinal CCV 1-71 e nove passagens desta em células A72 foram submetidas a amplificação parcial e clonagem molecular do gene S seguida de sequenciamento de DNA. Os resultados mostraram mutações não silenciosas, silenciosas e três deleções de aminoácidos, mas nenhuma mutação compartilhada entre as diversas passagens. Amostras vacinais de CCoV-II adaptadas em células podem ser altamente geneticamente estáveis após passagens em série em uma mesma linhagem celular, acumulando substituições de nucleotídeos principalmente sinônimas no gene S devido a relação célula - hospedeiro estável. Estes resultados de epidemiologia molecular e processos evolutivos de CCoV podem servir para uma melhor compreensão da virologia básica e ser base de dados para estudos em outros coronavírus Canine coronavirus (CCoV) causes gastroenteritis in young dogs and can be lethal, especially when there is co-infection with canine parvovirus (CPV). The objectives of this project were to investigate the presence of CCoV and CPV in stool samples from young dogs, the molecular diversity of CCoV strains based on partial sequencing of genes M, S, N and 3b, and the in vitro evolution of CCoV in A72 canine fibroma. Of the fecal samples studied, 40.17% animals (47/117) were positive for CCoV and 13.68% (16/117) for CPV. Phylogenetic studies have shown that eight strains were CCoV-II and twenty five CCoV-I. Phylogenetic analysis for the M gene highlighted the high identity of CCoV-I strains with a feline coronavirus strain (FCoV) that causes feline infectious peritonitis (FIP). Further analysis based on the spike gene showed a putative pantropic CCoV strain (CCoV-II/dog50). CCoV nucleocapsid gene is highly conserved among type I and type II, with a lower resolution relative to trees based on M and S genes. CCoV Types I and II had a low polymorphism for 3b gene, without any stable markers to differentiate thee types. Regarding the virus isolation trial, a putative pantropic CCoV-II strain was successfully isolated in A72 cells from, resulting in cytopathic effect on the 5th day of the 5th passage. In the evolutionary study, the vaccine strain CCV 1-71 and nine passages of this strain in A72 were submitted to partial S gene amplification and molecular cloning followed by DNA sequencing. Missense, silent, and three amino acids deletions were found amongst diverse clones of each passage, but no mutation was repeatedly found among passages. Cell culture-adapted CCoV-II vaccine strains can be highly genetically stable upon serial passage in a same cell line, accumulating primarily synonymous nucleotide substitutions in the S gene due to a stable cell-host relationship. In short, all data gathering herein on molecular epidemiology and evolutionary processes of CCoV can serve for a better understanding of basic virology and as a basis for studies on other coronaviruses

This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.

Cryptosporidium spp. are cosmopolitan protozoa that infect fishes, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are a group of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for humans and livestock. The aim of this study was to design specific primers for the gene encoding 18S rRNA, potentially capable of amplifying any species or genotype of Cryptosporidium spp. and evaluate the diagnostic attributes of the nested-PCR based on such probes. The primers were designed to amplify the shortest segment as possible to maximize the sensitivity of the test, but preserving the discriminatory potential of the amplified sequences for phylogenetic inferences. The nested-PCR standardized in this study (nPCR-SH) was compared in terms of sensitivity with another similar assay (nPCR-XIAO) that has been largely used for the detection and identification of Cryptosporidium spp. worldwide. We also aimed to molecularly characterize samples of Cryptosporidum spp. isolated from synanthropic rodents using these probes. Forty-five rodents were captured in urban areas of the municipality of Umuarama, Parana State, Brazil. Fecal samples were submitted to three molecular tests (nested-PCRs), two of them targeted to the 18S rDNA gene (nPCR-SH and nPCR-XIAO) and the third targeted to the gene encoding actin (nPCR-actin). The nPCR-SH was tested positive on samples of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis, Cryptosporidum canis, and Cryptosporidum serpentis. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR-actin. Sequencing of amplified fragments allowed the identification of Cryptosporidum muris in three samples of Rattus rattus, and two genotypes of Cryptosporidium, the genotypes mouse II and III. Cryptosporidium genotype mouse II was found in one sample of Mus musculus and genotype mouse III, in twelve samples, being five from R. rattus and seven from M. musculus. The results of this study demonstrated that the primers designed for detection of Cryptosporidium spp. were more efficient than those used in the nPCR-XIAO. Genotypes or species of Cryptosporidium that can be usually transmitted for human beings and livestock were not found in synanthropic rodents, suggesting that the importance of these animals in zoonotic transmission of cryptosporidiosis should be revisited.

Gastroenteritis is one of the leading causes of morbidity and mortality among young and newborn animals and is often caused by multiple intestinal infections, with rotavirus and bovine coronavirus (BCoV) being the main viral causes in cattle. Given that BCoV is better studied than equine coronaviruses and given the possibility of interspecies transmission of these viruses, this research was designed to compare the partial sequences of the spike glycoprotein (S), hemagglutinin-esterase protein (HE), and nucleoprotein (N) genes from coronaviruses from adult cattle with winter dysentery, calves with neonatal diarrhea, and horses. To achieve this, eleven fecal samples from dairy cows with winter dysentery, three from calves, and two from horses, all from Brazil, were analysed. It could be concluded that the enteric BCoV genealogy from newborn and adult cattle is directly associated with geographic distribution patterns, when S and HE genes are taken into account. A less-resolved genealogy exists for the HE and N genes in cattle, with a trend for an age-related segregation pattern. The coronavirus strains from horses revealedBetacoronavirussequences indistinguishable from those found in cattle, a fact previously unknown.

The Visna/Maedi virus is a Lentivirus that causes amultisystem, progressive, slow-developing disease that can affect sheepand goats. After long periods of subclinical infection, the disease canslowly progress to cause the degeneration of various organs, cachexiaand death. The diagnosis is made by the observation of clinical signs andconfirmed by serological tests and isolation and/or identification of thevirus. There is no treatment for the disease. The control is based onsegregated breeding, handling and slaughtering of the positives. As Visna/Maedi is on the list of the World Organization for Animal Health (OIE),reporting of the disease is obligatory and it has been found in some Statesin Brazil, causing negative economic impacts with its spread. Given thelimited seroepidemiological data available in the Brazilian State of Bahia,this study aimed to carry out a seroepidemiological survey to investigatethe occurrence of anti-Visna/Maedi virus antibodies in sheep fromRecôncavo Baiano. Two hundred serum samples from sheep were subjectedto the agar gel immunodiffusion test (AGID). None of the serum sampleswere found to be positive. To confirm the absence of Visna/Maedi in sheepherds in Bahia, there is a need for further studies with significant samplingincluding a more sensitive assay.