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Equine influenza is one of the major respiratory infectious diseases in horses. An equine influenza virus outbreak was identified in vaccinated and unvaccinated horses in a veterinary school hospital in São Paulo, SP, Brazil, in September 2015. The twelve equine influenza viruses isolated belonged to Florida Clade 1. The hemagglutinin and neuraminidase amino acid sequences were compared with the recent isolates from North and South America and the World Organisation for Animal Health recommended Florida Clade 1 vaccine strain. The hemagglutinin amino acid sequences had nine substitutions, compared with the vaccine strain. Two of them were in antigenic site A (A138S and G142R), one in antigenic site E (R62K) and another not in antigenic site (K304E). The four substitutions changed the hydrophobicity of hemagglutinin. Three distinct genetic variants were identified during the outbreak. Eleven variants were found in four quasispecies, which suggests the equine influenza virus evolved during the outbreak. The use of an out of date vaccine strain or updated vaccines without the production of protective antibody titers might be the major contributing factors on virus dissemination during this outbreak.

ABSTRACT:Peccaries and pigs, Tayassuidae and Suidae respectively, diverged approximately one million years ago from a common ancestor. Because these families share some pathogens, peccaries can act as reservoirs of infectious pathogens for domestic and wild swine. We evaluated the presence of swine infectious agents in the spleen and lung tissues of white-lipped peccaries (WLP; Tayassu pecari) and collared peccaries (CP; Pecari tajacu) in Brazil. Samples from 10 adult CP and three WLP, which had been hunted by locals or hit by motor vehicles, were obtained from two free-ranging Brazilian populations. The samples were tested by PCR for Mycoplasma hyopneumoniae, Bordetella bronchiseptica, Pasteurella multocida, porcine circovirus 2 (PCV2), Suid herpesvirus 1 (SuHV-1), and porcine parvovirus (PPV). Positive samples were sequenced. Both species were negative for PPV and B. bronchiseptica and positive for PCV2 and SuHV-1. The lungs of two animals were positive for M. hyopneumoniae and P. multocida. This report is the first demonstration of PCV2 and SuHV-1 swine viruses and of M. hyopneumoniae and P. multocida bacteria in peccaries. One factor contributing to this detection was access to tissue samples, which is uncommon. The role of these infectious agents in peccaries is unknown and further epidemiologic studies should be performed. This study identified several infectious agents in peccaries and highlighted the importance of the tissue type used to detect pathogens.

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.

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.

Introduction: Staphylococci are the most important agents associated with bovine mastitis. This study aimed at characterizing resistance factors to antimicrobials in Staphylococcus spp. isolated from the milk of cows with subclinical mastitis. Methodology: In vitro resistance of 243 Staphylococcus spp. isolates to antimicrobials commonly used in clinical practice was evaluated. The detection and expression of genes encoding resistance mecA (gene encoding penicillin binding protein 2a) mecALGA251 (mecA homologue), blaZ (gene encoding penicillin resistance), femA and femB (genes encoding essential factors - A and B - for the expression of methicillin resistance) and aacA-aphD (gene encoding for a bifunctional enzyme that confers resistance to gentamicin) using PCR and RT-PCR was investigated. Results: One or more genes encoding resistance to different antimicrobials were detected in 184 Staphylococcus spp. samples. The femA and femB genes were the most frequent. Regarding the variables’ detection (N = number of strains) and expression (% of strains), the following results were obtained: blaZ (N = 40 – 82.5%), femA (N = 147 – 47.6%), aacAaphD (N = 30 – 43.3%), femB (N = 138 – 29.7%), mecA (N = 33 – 27.3%), mecALGA251 (N = 01 – 0.0%). There was a higher occurrence of phenotypic resistant strains for amoxicillin, ampicillin and penicillin in isolates positive for detection and/or expression of blaZ gene when compared with the other genes. Conclusions: The present study provides new information on genotypic traits of Staphylococcus isolates from bovine subclinical mastitis especially regarding the evaluation of expression of genes associated with antimicrobial resistance in Staphylococcus spp. using molecular tools.

RESUMO.-[Detecção rápida do Coronavírus Bovino (BCoV) por meio de uma semi-nested RT-PCR.]O Coronavírus bovino (BCoV) pertence ao grupo 2 do gêne-ro Coronavirus (Nidovirales: Coronaviridae) e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doença respiratória em bezerros. O presente estudo teve por objetivo desenvolver uma seminested RT-PCR para a detecção do BCoV com base em seqüências representativas e recentes do gene do nucleocapsídeo, região conservada do genoma dos coronavírus. Três primers foram desenhados, a primeira amplificação com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analítica foi determinada pela diluição do BCoV cepa Kakegawa (título HA: 256) na base de 10 em água ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensão fecal negativa para o BCoV, onde foram encontrados resultados positivos até a diluição de 10 -2 , 10 -3 e 10 -7 , respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitação dos pellets pelo método de extração utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10 -2 , 10 -3 and 10 -7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Lyssavirus rabies (RABV) is an RNA virus and, therefore, is subject to mutations due to low RNA polymerase replication fidelity, forming a population structure known as a viral quasispecies, which is the core of RNA viruses’ adaptive strategy. Under new microenvironmental conditions, the fittest populations are selected, and the study of this process on the molecular level can help determine molecular signatures related to virulence. Our aim was to survey gene signatures on nucleoprotein and glycoprotein genes that might be involved in virulence modulation during the in vitro evolution of RABV lineages after serial passages in a neuronal cell system with or without the presence of neutralizing antibodies based on replicative fitness, in vivo neurotropism and protein structure and dynamics. The experiments revealed that amino acids at positions 186 and 188 of the glycoprotein are virulence factors of Lyssavirus rabies, and site 186 specifically might allow the attachment to heparan as a secondary cell receptor, while polymorphism at position 333 might allow the selection of escape mutants under suboptimal neutralizing antibodies titers.