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Melhorar o desempenho reprodutivo é importante para aumentar a lucratividade das propriedades. Assim, nós avaliamos a contagem de folículos antrais (CFA), o escore de condição corporal (ECC), o peso vivo e o comportamento animal sobre à taxa de concepção de vacas Brahman submetidas à inseminação artificial em tempo-fixo (IATF). Vacas (n=122) receberam um protocolo convencional de IATF de três manejos (D0, 8 e 10) a base de estrógeno e progesterona, além do monitoramento da expressão de estro antes da inseminação. O ECC e o comportamento animal foram avaliados em todos os dias do protocolo, o peso foi mensurado no D0 e no diagnóstico de gestação (D40) e a CFA no D0. Para análise, estabeleceu-se os grupos de CFA (baixa, intermediária e alta), do comportamento (calma, inquieta e agitada), do peso (ganhando, mantendo e perdendo) e do ECC (ganhando, mantendo e perdendo). A taxa de concepção geral foi de 50%, e não esteve associada (p>0,05) a classificação da CFA (baixa/52,6%, intermediária/50,9% e alta/45,4%), do peso (ganhando/43,0%, mantendo/54,7% e perdendo/47,1%), do ECC (ganhando/44,0%, mantendo/54,3% e perdendo/37,5%) e do comportamento (calma/43,0%, inquieta/54,7% e agitada/47,1%). No entanto, a baixa CFA apresentou maior (p=0,05) proporção de animais com alta intensidade de estro (94,7%) e foi observado maior escore de reatividade (p=0,001) no primeiro dia de manejo da IATF em relação aos outros dias. Vacas com baixa CFA resultaram em maior proporção de expressão de estro e o primeiro dia de manejo da IATF determinou uma maior reatividade animal em relação aos outros dias do protocolo. Palavras-chave: Condição Corporal. Inseminação Artificial. Performance Reprodutiva. Peso Vivo. Abstract Improving reproductive performance is important to increase farm profitability. The relationship of antral follicle count (AFC), body condition score (BCS), weight, and animal behavior on the conception rate in cows submitted to timed artificial insemination (TAI). Cows (n=122) received a conventional TAI protocol of three managements (D0, 8, and 10) based on estrogen and progesterone, in addition to monitoring estrus expression before insemination. The BCS and behavior score were assessed every day of the TAI protocol. Weight was measured at D0 and in the pregnancy diagnosis (D40), and AFC was determined at D0. For analysis, the groups were established according to AFC (low, intermediate, and high), behavior animal (calm, restless, and agitated), weight variation (gaining, maintaining, and losing), and BCS variation (gaining, maintaining, and losing). The overall conception rate was 50%, and it was not associated with (P>0.05) the AFC classification (low/52.6%, intermediate/50.9%, and high/45.4%), weight variations (gaining/43.0%, maintaining/54.7%, and losing/47.1%), BCS variations (gaining/44.0%, maintaining/54.3%, and losing/37.5%) and animal behavior (calm/43.0%, restless/54.7%, and agitated/47.1%). However, the low AFC showed a higher (P=0.05) proportion of animals with high intensity of estrus expression (94.7%). In addition, a higher score for animal reactivity (P=0.001) was noted on the first day of the management of TAI protocol than to the other days of the hormonal protocol. The low AFC resulted in a higher proportion of cows with high estrus expression and the management practices for TAI determined a greater animals reactivity at the beginning of the breeding program. Keywords: Body Condition. Artificial Insemination. Reproductive Performance. Live Weight.

This study is aimed at detecting Feline paramyxovirus (FPaV) and Feline morbillivirus (FeMV) in 35 urine samples from domestic cats, collected in 2019, with or without clinical signs of uropathies using a reverse transcription-polymerase chain reaction (RT-PCR) followed by semi-nested polymerase chain reaction (SN-PCR) assays to amplify a partial paramyxovirus L gene. Eight (22.9%) out of the 35 urine samples were positive for paramyxoviruses. Sequencing and phylogenetic analyses revealed that three samples were positive for FPaV, four samples were positive for FeMV, and it was not possible to determine which virus was present in one RT-SN-PCR positive urine sample. FPaV strains showed 100% nucleotide (nt) identity with each other and 97% nt identity with a Japanese 163 FPaV strain. The FeMV strains showed 85.9% nt identity with each other; three strains were similar to previously described Brazilian FeMV strains, and one strain clustered in a different branch of the phylogenetic tree together with the first described Chinese FeMV strain. This study provides the first description of FPaV strains in cats from Brazil and provides new information about the molecular characteristics of FPaV and FeMV strains circulating in domestic cats in Brazil.

Nos últimos meses, em todo o mundo, tem-se falado muito em COVID-19, uma doença que ocasionou um surto na China e rapidamente espalhou-se pelo mundo, tendo se transformado em uma pandemia. Em meio a esse cenário, dentre diversas questões, foi levantada uma dúvida em relação à possibilidade de infecção e posterior transmissão dessa doença viral dos animais de companhia, mais especificamente, cães e gatos, para indivíduos destas mesmas espécies ou até mesmo para os humanos. Este trabalho tem por objetivo elucidar, de forma clara e objetiva, o que é a COVID-19, sua origem e comportamento do agente viral responsável pela doença, com o intuito de disseminar informações sobre a atual pandemia, além de esclarecer a real participação de cães e gatos no ciclo de transmissão do vírus. Para isso, artigos recentes sobre o assunto foram acessados, em plataformas tais como: PubMed, SciELO e Google Acadêmico, além de livros e documentos eletrônicos, os quais foram utilizados na obtenção das informações aqui descritas, a fim de atingir não apenas a comunidade científica, mas toda a sociedade. Apesar de haver evidências de infecção por SARS-CoV-2 em cães e gatos, a transmissão do vírus por meio desses animais para os seres humanos ainda não foi confirmada. É importante também ressaltar que devido à atual situação e ao caráter dinâmico da doença, novas questões podem ser levantadas, bem como confirmações ou exclusões de hipóteses no decorrer do tempo. Palavras-chave: Coronavírus. SARS-CoV-2. Pandemia. Quarentena. Abstract In the last few months, all over the world, much has been said about COVID-19, a disease that caused an outbreak in China and quickly spread around the world, becoming a pandemic. In this scenario, among several issues, a doubt was raised regarding the possibility of infection and subsequent transmission of this viral disease of pets, more specifically, dogs and cats, to individuals of the same species or even to humans. This study aims to elucidate, in a clear and objective way, what is COVID-19, its origin and the viral agent behavior responsible for the disease, in order to disseminate information about the current pandemic, in addition to clarifying the actual participation of dogs and cats in the virus transmission cycle. Recent articles were accessed, on platforms such as: PubMed, SciELO, and Google Scholar, also books and electronic documents, which were used to obtain the information described herein, in order to reach not only the scientific community, but also the society. Although there is evidence of infection with SARS-CoV-2 in dogs and cats, the virus transmission through these animals to humans has not been confirmed yet. It is also important to emphasize that, due to the current situation and its dynamic character, new questions can be raised, as well as confirmation or exclusion of hypotheses over time. Keywords: Coronavirus. SARS-CoV-2. Pandemic. Quarantine.

Made available in DSpace on 2021-06-25T12:23:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-23 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazilian institute: Financing of Studies and Projects (FINEP) Brazilian institute: Araucaria Foundation (FAP/PR) Funadesp The crab-eating fox (Cerdocyon thous) is a small wild mammal present in all Brazilian biomes and in some countries of South America. This study aimed to verify the involvement of viral infectious agents in the death of a wild crab-eating fox pup (Cerdocyon thous) in Brazil. The Center for Medicine and Research of Wild Animals of the Universidade Estadual Paulista received a free-living crab-eating fox aged approximately 21 days and apparently healthy. After 13 days, the animal presented anorexia, diarrhea, fever, prostration, and neurological signs progressing to death with an inconclusive diagnosis. In a retrospective study, tissue fragments stored at - 80 degrees C were used to identify nucleic acids from major canine viruses, such as canine parvovirus-2 (CPV-2), canine adenovirus A types 1 and 2, canid alphaherpesvirus 1, and canine distemper virus. The amplified product with the expected length for CPV-2 was obtained from the heart fragment. After performing nucleotide (nt) sequencing of the amplicon, it was possible to demonstrate that the crab-eating fox strain exhibited high (99.8%) nt identity with the CPV-2b prototype (CPV-39 strain). Additionally, deduced amino acid (aa) sequence analysis showed the GAT codon for the aa Asp (D) at position 426 of the CPV-2 viral protein VP2, which characterizes the subtype 2b. To the best of the authors' knowledge, this report describes the first detection of CPV-2b DNA in tissue fragments from a crab-eating fox. Univ Estadual Londrina, Lab Anim Virol, Dept Vet Prevent Med, Celso Garcia Cid Rd,PR455 Km 380,POB 10011, BR-86057970 Londrina, Parana, Brazil Univ Estadual Londrina, Multiuser Anim Hlth Lab, Mol Biol Unit, Dept Vet Prevent Med, Celso Garcia Cid Rd,PR455 Km 380,POB 10011, BR-86057970 Londrina, Parana, Brazil Univ Pitagoras Unopar, Post Grad Program Anim Hlth & Prod, Arapongas, Parana, Brazil Univ Estadual Paulista, Vet Hosp, Ctr Med & Res Wild Anim, POB 560, BR-18618000 Botucatu, SP, Brazil Univ Estadual Paulista, Vet Hosp, Anim Pathol, POB 560, BR-18618000 Botucatu, SP, Brazil Univ Estadual Paulista, Vet Hosp, Ctr Med & Res Wild Anim, POB 560, BR-18618000 Botucatu, SP, Brazil Univ Estadual Paulista, Vet Hosp, Anim Pathol, POB 560, BR-18618000 Botucatu, SP, Brazil

Made available in DSpace on 2020-12-11T17:20:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-14 Canine parvovirus type 2 (CPV-2) is classified into three subtypes (CPV-2a, CPV-2b, and CPV-2c) and is the main cause of enteritis and myocarditis in young domestic and wild animals. This study aimed to evaluate the presence of CPV-2 in the feces of asymptomatic free-living coatis from Garden Forest Reserve, Palmital city, SP, Brazil. Fecal samples from 21 coatis (both sexes, different ages, and different aspects of feces) were collected in August 2014 and March 2015. The nucleic acid extracted was submitted to a polymerase chain reaction (PCR) assay to amplify a fragment of the VP2 gene of CPV-2. Eight (38%) fecal samples were positive in the PCR assay and were confirmed by sequencing. The 7 nucleotide (nt) sequences analyzed showed 100% nt identity with the prototype strain of CPV-2b (CPV-39 strain). The analysis of the deduced amino acid (aa) sequence revealed the presence of the GAT codon (aa D-Asp) at position 426 of the VP2 viral protein (subtype 2b). This study describes for the first time the identification of CPV-2b in asymptomatic free-living coatis (Nasua nasua) and suggests that coatis are susceptible to Carnivore protoparvovirus 1 infection and are important as a reservoir and an asymptomatic carrier to other wild and domestic animal species. Univ Estadual Londrina, Dept Vet Prevent Med, Lab Anim Virol, Celso Garcia Cid Rd,PR455 Km 380,POB 10011, BR-86057970 Londrina, Parana, Brazil Univ Estadual Londrina, Dept Vet Prevent Med, Mol Biol Unit, Multiuser Anim Hlth Lab, Celso Garcia Cid Rd,PR455 Km 380,POB 10011, BR-86057970 Londrina, Parana, Brazil Univ Pitagoras Unopar, Post Grad Program Anim Hlth & Prod, Arapongas, Parana, Brazil Univ Estadual Paulista, Vet Hosp, Ctr Med & Res Wild Anim, POB 560, BR-18618000 Botucatu, SP, Brazil Univ Estadual Paulista, Vet Hosp, Ctr Med & Res Wild Anim, POB 560, BR-18618000 Botucatu, SP, Brazil

We describe the molecular analysis of a wild-type field strain of bovine viral diarrhea virus (BVDV) identified in a mummified fetus from a small Brazilian dairy cattle herd. Nucleic acids extracted from samples of the lung, liver, heart, spleen, and kidney were tested by PCR assays for bovine alphaherpesvirus 1, Neospora caninum, Leptospira spp., Histophilus somni, and Brucella abortus, a nested PCR assay for Mycoplasma bovigenitalium and Ureaplasma diversum, and a RT-PCR assay for BVDV. Amplicons were only obtained in the RT-PCR assay for the partial amplification of the BVDV 5′UTR (288 bp) in kidney and spleen samples and the Npro (438 bp) gene in the kidney sample. Nucleotide sequencing of the amplified products and phylogenetic analyses based on the 2 BVDV genomic regions enabled the BVDV strain to be classified as subgenotype 1a.

Veterinarians, among other health professionals, are considered health professionals at high risk of exposure to and contraction of COVID-19. The main objective of this study is to assess changes in the clinical practices of veterinarians during the COVID-19 pandemic around prophylactic and biosafety measures, as well as to evaluate changes in workload and cost-benefit ratio. An online questionnaire was sent to veterinary professionals from July 2020 to July 2021 using Google Forms. A total of 1134 veterinarians answered the questionnaire on clinical experiences and biosafety practices during the COVID-19 pandemic. Veterinarians changed their routine clinical practices, as there was a reduction in working hours, and applied new patient approaches and advice to their owners, as well as restricting the number of people allowed inside. Biosafety measures were added in their workplaces, with an increase in the use of personal protective equipment. COVID-19 tests were administered at least once in 19.0%, and more than once in 9.5% of the respondents. Flu symptoms were present in 23.8% of the respondents, and 31.0% of the veterinarians attended to COVID-19 positive pet owners. Therefore, most veterinarians altered their routine practices, and some were exposed to sources of COVID-19 infection.Os médicos veterinários, entre outros profissionais de saúde, são considerados profissionais de saúde sob alto risco de exposição e contração do COVID-19. O objetivo principal do presente estudo foi avaliar as mudanças na prática clínica de médicos veterinários durante a pandemia de COVID-19 em torno das medidas profiláticas e de biossegurança, assim como avaliar as mudanças na carga de trabalho e a relação custo-benefício. De julho a dezembro de 2020, um questionário online foi enviado aos profissionais por meio da ferramenta Formulários Google. Um total de 1.134 veterinários responderam ao questionário relacionado às experiências clínicas e práticas de biossegurança durante a pandemia COVID-19. Os médicos veterinários mudaram suas práticas clínicas rotineiras, pois houve redução da jornada de trabalho, novas abordagens dos pacientes e orientações aos proprietários, além da restrição do número de pessoas nos locais. Medidas de biossegurança foram adicionadas aos locais de trabalho, com aumento do uso de equipamentos de proteção individual. Os testes COVID-19 foram realizados pelo menos uma vez em 19,0% e mais de uma vez em 9,5% dos entrevistados. Sintomas de gripe estavam presentes em 23,8% dos profissionais entrevistados e 31,0% dos veterinários atendiam tratadores de animais positivos para COVID-19. Portanto, a maioria dos veterinários alterou suas práticas de rotina e alguns foram expostos a fontes de infecção COVID-19.

The aim of this study was to investigate and compare the frequency of occurrence of avian rotavirus (AvRV) in poultry flocks according to its Performance Efficiency Index (PEI) scores. A total of 256 individual intestinal content samples of small sized-chicks (runts) with clinical signs of Runting Stunting Syndrome (RSS) and 24 clinically healthy chicks (control) were collected from twelve flocks in southern Brazil with different PEI scores: good (n = 4, PEI mean = 365); moderate (n = 4, PEI mean = 342) or poor (n = 4, PEI mean = 319). Silver-stained polyacrylamide gel electrophoresis (ss-PAGE) was used to detect and identify the AvRV species followed by RT-PCR and sequencing of the partial VP6 gene for species confirmation. AvRV was detected in 83% (10/12) of the flocks and 23.4% (60/256) of the chicks. The electrophoretic migration patterns of viral dsRNA segments were compatible with AvRV species A (AvRV- A), D (AvRV-D) and F (AvRV-F) in 9 (15%), 18 (30%), and 33 (55%) of the positive chicks fecal samples, respectively. The AvRV species identified by ss-PAGE were confirmed by RT-PCR and partial sequence analysis of the VP6 gene. The AvRV detection rate was statistically higher (p = 0.007) in chicks from flocks with poor PEI when compared to those with good PEI. The occurrence of AvRV-D and AvRV-F was statistically higher in 7 to 9 days old chicks, while AvRV-A was detected only in 13 to 14 days old animals.

The reports of rotavirus C (RVC) involvement in diarrhea outbreaks in newborn piglets have been increasing in recent years. This longitudinal study, conducted over a 37-day period, aimed to evaluate the frequency of RVC infection in piglets aged up to 7 days obtained from a pig herd with a previous diagnosis of RVC infection in this age group. Piglets from 50 different litters were monitored daily for the occurrence of diarrhea, and all litters were classified into the following categories: sow parity order (PO) 1 to 5; litter size (LS) ≤ 10 piglets and > 10 piglets; and piglet birth weight (BW) 1.2 to 1.3 kg and > 1.3 to 1.4 kg. Two hundred six diarrheic fecal samples were collected and classified according to the fecal consistency score (pasty, semiliquid, liquid). Ten fecal samples were collected from asymptomatic piglets (control group). Fecal samples were screened for rotavirus (RV) by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), and samples with inconclusive and negative-ss-PAGE results were submitted to RVC VP6 gene amplification by RT-PCR. RVC was identified in 71 (34.5%) samples, in 1 (10%) sample of the control group, and in piglets from 33 (66%) litters. The electrophoretic profile of RV species A was identified in only two samples. Of the 72 RVC-positive samples, 51 (70.8%) presented semiliquid or liquid consistency. There was no significant difference in either group regarding the production parameters (PO, LS, BW) evaluated. An analysis of the whole VP6 gene of three RVC field strains collected on the first, fifteenth, and last day of the experiment enabled us to identify genotype I6. This report describes the first longitudinal study examining epidemiological aspects of RVC infection in newborn piglets.

The livestock sector has achieved many technological advances, which have resulted in continued improvements in animal production systems and in the reproductive efficiency of herds. The associated use of reproductive biotechnology and genetic improvements combined with adequate sanitary and nutritional management are essential conditions for sustainable intensified animal production and financial autonomy within farms. Timed artificial insemination (TAI) represents one of the strategies with the greatest impact of expansion in providing genetic improvements and increased reproductive efficiency at a decreased cost. Despite the high proportion of cows receiving TAI, this market still exhibits considerable potential for expansion. After a TAI procedure, approximately 40 to 60% of females become pregnant. This result can vary depending on such factors as the hormonal protocol employed, female category, body condition score, ovarian status, farm management and aspects related to bulls and semen. The fertility and genetic quality of the bull plays an important role in the herd because a single bull can influence the entire production system. Another important strategy is the use of sex-sorted semen associated with TAI, primarily when associated with management practices to improve the pregnancy rate. This paper presents a review of the intensification of TAI, supplying practical information regarding the implementation of TAI commercial programs.

Bovine viral diarrhea virus (BVDV) causes a significant economic impact on the beef and dairy industries worldwide. Fetal infection with a non-cytopathic strain may lead to the birth of persistently infected (PI) offspring, the main event in the epidemiological chain of BVDV infection. This report describes the birth of 10 PI dairy calves from Brazil and the infecting BVDV subgenotype. Serum and blood samples were collected from 10 cows and 10 calves; all 10 calves were previously deemed BVDV positive by the ear notch rapid test. Serum samples were used in the virus neutralization technique to detect anti-BVDV antibody. Blood samples were used to detect BVDV RNA using the reverse transcription polymerase chain reaction (RT-PCR) assay of the 5' UTR and Npro genes. All 10 cows were negative for BVDV RT-PCR, while all 10 calves were RT-PCR positive. Phylogenetic analyses were performed and the strain was classified as BVDV-1d. High titers of BVDV-specific antibodies in the serum of cows indicated recent circulation of BVDV in the dairy herd, whereas calves presented intermediate, low, or no anti-BVDV-1a antibody titers. The monitoring of circulating BVDV subgenotypes and the detection of PI animals is of great importance in disease control, and regular vaccination alone is insufficient to prevent BVDV infection.

This study describes the etiological diversity observed in a severe neonatal diarrhea outbreak with morbidity and mortality rates of 80 and 20%, respectively, with detection of mixed infections with viral, bacterial, and protozoan disease agents in a dairy calf rearing unit. Diarrheic fecal samples were collected from eight 5 to 18 days of age calves and were submitted to the investigation of the presence of rotavirus A (RVA), bovine coronavirus (BCoV), bovine kobuvirus (BKV), bovine viral diarrhea virus 1 and 2 (BVDV-1 and BVDV-2), enteropathogenic Escherichia coli (ETEC), Salmonella sp., and Cryptosporidium spp. Fragments of the small intestine of one calf with diarrhea that spontaneously died were submitted for histopathological analyses. The most frequent infectious agent detected in diarrheic fecal samples was BKV (8/8—100%), followed by RVA (5/8—62.5%), BVDV (5/8—62.5%), Cryptosporidium parvum (5/8—62.5%), ETEC (4/8—50%), and Cryptosporidium ryanae (1/8—12.5%). These etiological agents were found in mixed infections with two or more pathogens per diarrheic fecal sample. The association of viral and protozoan pathogens was the most frequently identified (37.5%) in these samples, followed by viral and bacterial (25%); viral, bacterial, and protozoan (25%); and only viral agents (12.5%). BCoV and Salmonella sp. were not identified in the diarrheic fecal samples analyzed. Additionally, histopathology of the small intestine diagnosed chronic lymphocytic enteritis. In conclusion, in calf rearing units, the adoption and strict monitoring of health management practices are critical to the success of this calf creation system. Supplementary Information The online version contains supplementary material available at 10.1007/s42770-021-00565-5.

Highlights • RVA vaccination program reduces the frequency and intensity of diarrhea in dairy calves. • The vaccination immune pressure can select specific genotypes in RVA field strains. • RVA genotype G10P[11] in fecal samples of calves from G6P[5] vaccinated dairy cattle herds., Worldwide, neonatal diarrhea is one of the most important health issues affecting dairy calves, and rotavirus A (RVA) is one of its primary causes. Among the measures to mitigate the risk of diarrhea outbreaks, cow vaccination stands out as one of the most important. However, the immune pressure resulting from routine vaccination may be able to select specific G and P genotypes in RVA field strains. This study aimed to determine the frequency and intensity of neonatal diarrhea and the incidence of RVA and attempted to monitor the G and P genotypes present in the RVA strains circulating in a high milk yield cattle herd vaccinated with RVA G6P[5] strain. Fecal samples (n = 1220) from 122 Holstein heifer calves between 0–30 days old that were born from RVA-vaccinated cows were collected at 10 different time points, regardless of the presence or absence of diarrhea. The presence of RVA in fecal samples was determined by the polyacrylamide gel electrophoresis (PAGE) technique and confirmed by reverse transcription polymerase chain reaction (RT-PCR). G and P amplicons from 10 RVA-positive fecal samples from calves of different ages and collections were subjected to nucleotide sequencing. The proportion of the calves and fecal samples that were positive for RVA were 62.3% (76/122) and 8.1% (99/1220), respectively. Using sequence analysis, all 10 RVA field strains presented genotype G10P[11]. The protection of G6P[5] vaccination is clear, as this genotype was not detected in this study, and it is known that vaccination against RVA reduces the incidence of diarrhea independent of genotype involved. This result demonstrates the importance of epidemiological monitoring of RVA genotypes circulating in vaccinated dairy cattle herds to the early detection of new potential pathogenic RVA strains.

Fecal samples from 27 pigs were longitudinally analyzed for Teschovirus A (TV-A), Sapelovirus A (SV-A), and Enterovirus G (EV-G) RNA presence. Suckling piglet fecal samples were negative for the three enteric picornaviruses. However, these picornaviruses were detected in 22/27 weaned pig fecal samples. This study provides new data on TV-A, SV-A, and EV-G infection dynamics. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-018-0018-1) contains supplementary material, which is available to authorized users.

Dairy calf rearing unit is a management system that is only recently being implemented by some milk producer’s cooperatives in southern Brazil. However, aspects related to the health profile of the heifer calves that arrive in the rearing unit as well as about biosecurity practices and microbiological challenges have not yet been evaluated in this rearing system in a tropical country. Diarrhea is the main and most frequent consequence of enteric infections in newborn calves. This study, through some etiological and epidemiological characteristics of an outbreak of neonatal diarrhea, has the aim to alert to the possibility of pathogenic microorganism spread in a dairy heifer calf rearing unit. The diarrhea outbreak presented some non-regular characteristics observed in bovine coronavirus (BCoV) enteric infections in dairy calves. The spread of infection was extremely rapid (1 week); the attack rate (> 50%) was much higher than that observed in calves subjected to conventional rearing; and the age range (5 to 90 days) of the affected heifer calves was much broader than that often observed in the BCoV diarrhea worldwide. These unusual epidemiological characteristics observed in this BCoV diarrhea outbreak raise awareness of the health threat present in calf rearing units as well as of the easy and rapid viral spread in a population of young animals from different dairy herds and, therefore, with very distinct immunological status.

Rotavirus (RV) is an important viral pathogen causing diarrhea in piglets and other mammals worldwide. We describe 34 cases from 4 diarrheal outbreaks caused by RV in unvaccinated farrowing units in southern Brazil from 2011 to 2013. We performed autopsy, histologic examinations, bacterial culture, RV immunohistochemistry (IHC), and enteric virus detection through molecular assays for rotavirus A, B, and C, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, sapovirus, norovirus, and kobuvirus. Histologically, villus atrophy (29 of 34) and epithelial vacuolation (27 of 34) occurred in all 4 outbreaks. Cell debris in the lamina propria occurred in 20 cases, mostly from outbreaks A (8 of 11), C (4 of 6), and D (7 of 11). IHC was positive for RV in 21 of 34 samples. RT-PCR was positive for RV in 20 of 30 samples; RV-C was the most frequently detected RV ( n = 17). Kobuvirus was detected in 11 samples, and, in 3 of them, there was single detection of this enteric virus.

Highlights • BALF is a good biological sample for the molecular diagnosis of BRD in dairy calves. • Mixed infections of viruses and bacteria were frequent in dairy calves with respiratory disease. • BVDV 1d in BALF samples of dairy heifer calves in a BRD outbreak was characterized. • The calf rearing unit without prophylactic measures for respiratory infections represent a risk factor for BRD., The etiological agents involved in a bovine respiratory disease (BRD) outbreak were investigated in a dairy heifer calf rearing unit from southern Brazil. A battery of PCR assays was performed to detect the most common viruses and bacteria associated with BRD, such as bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine alphaherpesvirus 1 (BoHV-1), bovine coronavirus (BCoV), bovine parainfluenza virus 3 (BPIV-3), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Bronchoalveolar lavage fluid (BALF) samples were taken from 21 heifer calves (symptomatic n = 15; asymptomatic n = 6) that, during the occurrence of the BDR outbreak, were aged between 6 and 90 days. At least one microorganism was detected in 85.7% (18/21) of the BALF samples. Mixed infections were more frequent (72.2%) than single infections (27.7%). The interactions between viruses and bacteria were the most common in coinfections (55.5%). The frequencies of BRD agents were 38.1% for BRSV, 28.6% for BVDV, 33.3% for BCoV, 42.85% for P. multocida, 33.3% for M. bovis, and 19% for H. somni. BoHV-1, BPIV-3, and M. haemolytica were not identified in any of the 21 BALF samples. Considering that BALF and not nasal swabs were analyzed, these results demonstrate the etiological multiplicity that may be involved in BRD outbreaks in dairy calves.

Renicolids are parasites that inhabit the renal tubules and ureters of molluscivorous and piscivorous birds. Puffinus puffinus is a migratory seabird that was identified as the definitive host of Renicola spp. Studies focusing on the renicolid species and the resulting renal lesions are valuable for their association with causes of stranding in seabirds. The aim of this study was to identify the renicolid trematodes and evaluate the histological findings in two P. puffinus stranded on the coast of Paraná state, Brazil. The parasites were evaluated by histologic, ultrastructural and molecular assays, while tissue changes were analyzed by histologic methods. The morphological and morphometrical characteristics of the parasites, along with polymerase chain reaction and sequencing assays (ribosomal and mitochondrial regions), identified the species as Renicola sloanei. The results also suggest that this helminth can be the adult form of Cercaria pythionike. The dilation of collecting ducts was the main histological finding in the kidneys. In conclusion, R. sloanei was identified, and for the first time, P. puffinus was described as a host of this digenean inducing mild renal changes. Resumo Renicolídeos são parasitos que habitam túbulos renais e ureteres de aves que se alimentam de moluscos e peixes. Puffinus puffinus, ave marinha migratória, foi registrada como hospedeiro definitivo de Renicola spp. Estudos relacionados com as espécies de renicolídeos e as lesões renais resultantes são importantes para o entendimento das causas de óbito de aves marinhas. O objetivo deste estudo foi identificar os trematódeos renicolídeos e avaliar os achados histológicos em dois P. puffinus encalhados no litoral do Estado do Paraná, Brasil. Os parasitos foram avaliados por ensaios histológicos, ultraestruturais e moleculares, enquanto as alterações teciduais foram analisadas por métodos histológicos. As características morfológicas e morfométricas dos parasitos, juntamente com a reação em cadeia da polimerase e sequenciamento (regiões ribossomal e mitocondrial), identificaram a espécie como Renicola sloanei. Os resultados também sugerem que este helminto pode ser a forma adulta de Cercaria pythionike. A dilatação dos ductos coletores foi o principal achado histológico renal. Em conclusão, R. sloanei foi identificado, e pela primeira vez P. puffinus foi descrito como hospedeiro deste digenético induzindo alterações renais discretas.

This study presents the pathological, immunohistochemical, and molecular findings associated with the extra-intestinal detection of canine kobuvirus (CaKV) in a 5-month-old Chihuahua puppy, that had a clinical history of bloody-tinged feces. Principal pathological findings were interstitial pneumonia, necrotizing bronchitis, and parvovirus-induced enteritis. Molecular diagnostic methods identified CaKV within the cerebellum, cerebrum, lung, tonsil, and liver. CaKV and rotavirus were not identified within the feces and intestine. Immunohistochemistry (IHC) assays detected antigens of CDV and CAdV-1 in the lungs. These results confirmed the extra-intestinal detection of CaKV in this puppy and represent the first extra-intestinal detection of CaKV in a dog.

Psychrotrophic microorganisms, especially Pseudomonas spp., are present in the microbiota of refrigerated milk as they can grow at refrigeration temperatures irrespective of their optimal growth temperature. Psychrotrophic counts ranging from 105 to 108 CFU/mL in refrigerated raw milk effect cheese quality, since the synthesized thermoresistant enzymes affect the nutritional value, sensory properties, and texture. Cheese is the dairy product with the highest growth rate in the food industry in Brazil in recent years and meets the current consumption trends of nutritious and practical foods. The objective of this review was to address the importance and influence of the psychrotrophic raw milk microbiota on the quality and sensory properties of cheese. The enzymes produced by psychrotrophic microorganisms lead to taste changes, undesirable clotting times, increased concentrations of free fatty acids and free amino acids, and a reduced shelf-life, in addition to negatively affecting cheese yields. Proteases from psychrotrophs are also associated with slicing problems and progressive loss of the elasticity of cheese, a bitter taste, and increased clotting times of cheese produced with pasteurized milk. On the other hand, their lipases increase the clotting time and have a negative effect on the sensory properties by providing a rancid, soap, metallic, or oxidized smell and taste. The control of the psychrotrophic population found in refrigerated raw milk contributes to better cheese production yields and desirable texture and sensory properties, which extends the shelf-life of cheese and improves consumer acceptance. Los microorganismos psicrotróficos, especialmente el género Pseudomonas spp, conforman el microbiota de la leche refrigerada porque son capaces de multiplicarse a temperaturas de refrigeración, independientemente de su temperatura de crecimiento óptima. La leche cruda refrigerada con recuentos psicrotróficos entre 105 y 108 UFC/mL compromete la producción de queso porque las enzimas termorresistentes sintetizadas deterioran la calidad nutricional, sensorial y de textura. En Brasil, dentro del sector lácteo, el queso representa el producto con mayor tasa de crecimiento en los últimos años, actualmente cumpliendo con las tendencias de alimentos nutritivos y prácticos para el consumo. El objetivo de esta revisión fue abordar la importancia y la influencia del microbiota psicrotrófica de la leche cruda en la calidad y las características sensoriales de los quesos. Las enzimas psicrotróficas promueven cambios en el sabor, tiempo de coagulación indeseable de las proteínas, mayor concentración de ácidos grasos y aminoácidos libres, limitación de la vida útil, así como efectos negativos en los rendimientos. Las proteasas psicrotróficas también se asocian con problemas de corte y pérdida progresiva de elasticidad del queso, sabor amargo y aumento del tiempo de coagulación de los quesos producidos con leche pasteurizada. Las lipasas psicrotróficas, por otro lado, confieren defectos como el sabor y el olor de rancio, jabón, metálico u oxidado a los quesos, además de aumentar el tiempo de coagulación. El control de la población psicrotrófica de la leche cruda refrigerada contribuye a un mejor rendimiento en la producción de quesos, textura y características sensoriales deseables, lo que asegura una mayor vida útil de los quesos y aceptado por el consumidor. Os micro-organismos psicrotróficos, com destaque ao gênero Pseudomonas spp, compõem a microbiota do leite refrigerado pois são capazes de se multiplicar em temperaturas de refrigeração, independentemente de sua temperatura ótima de crescimento. O leite cru refrigerado com contagens de psicrotróficos entre 105 e 108 UFC/mL compromete a produção de queijos pois as enzimas termorresistentes sintetizadas prejudicam a qualidade nutricional, sensorial e a textura. No Brasil, dentro do setor de lácteos, o queijo representa o produto com a maior taxa de crescimento nos últimos anos, atendendo atualmente às tendências de alimentos nutritivos e práticos para o consumo. O objetivo desta revisão foi abordar a importância e a influência da microbiota psicrotrófica do leite cru na qualidade e nas características sensoriais dos queijos. As enzimas psicrotróficas promovem mudanças no sabor, tempo de coagulação indesejável de proteínas, aumento da concentração de ácidos gordurosos e aminoácidos livres, limitação da vida útil, bem como efeitos negativos sobre os rendimentos. As proteases psicrotróficas também estão associadas a problemas de fatiamento e perda progressiva da elasticidade dos queijos, gosto amargo e aumento do tempo de coagulação da massa dos queijos produzidos com leite pasteurizado. Já as lipases psicrotróficas conferem defeitos como sabor e odor de ranço, de sabão, metálico ou oxidado aos queijos, além do aumento do tempo de coagulação. O controle da população psicrotrófica do leite cru refrigerado contribui para um melhor rendimento na produção de queijos, textura e caraterísticas sensoriais desejáveis, o que garante maior vida de prateleira dos queijos e o aceite pelo consumidor.

The present review aims to show the main aspects related to bluetongue virus (BTV) infection in sheep. The bluetongue (BT) is a viral, infectious, and non-contagious disease caused by a virus (BTV) of the Orbivirus genus, transmited by a hematophagous vector of the Culicoides genus, to domestic and wild ruminants, mainly to sheep, the most susceptible species. It is caused by the association of endemic with climate conditions, with high temperatures and humidity. Economic loss is directly linked to death, abortion, weight loss, loss of milk, and meat production, and, indirectly, to the restriction on the export of animals and their by-products. The study concludes that the BTV is worldwidely spread, and probably persists due to the warm and humid climate that leads to the proliferation of Culicoides sp., being necessary to adopt measures that reduce the risk factors associated to the BTV infection. RESUMO A presente revisão objetivou apresentar os principais aspectos relacionados à infecção causada pelo vírus da língua azul em ovinos. A língua azul é uma doença viral, infecciosa e não contagiosa, causada por um vírus (BTV) do gênero Orbivírus, transmitida por meio de vetores hematófagos do gênero Culicoides a ruminantes domésticos e selvagens, principalmente aos ovinos, a espécie mais susceptível. A infecção ocorre de forma endêmica, associada a condições climáticas com elevada temperatura e umidade. As perdas econômicas estão ligadas diretamente à morte, ao abortamento, à perda de peso, à perda na produção de leite e carne, e, indiretamente, devido à restrição na exportação de animais e seus subprodutos. O estudo conclui que a língua azul está disseminada mundialmente e persiste, provavelmente, devido ao clima quente e úmido que propicia a proliferação de Culicoides sp., sendo necessário adotar medidas que diminuam os fatores de risco associados à infecção pelo vírus.

Worldwide diarrhea outbreaks in cattle herds are more frequently detected in calves being that diarrhea outbreaks in adult cattle are not common. Winter dysentery (WD) is a bovine coronavirus (BCoV) enteric infection that is more reported in Northern hemisphere. Seasonal outbreaks of WD in adult cattle occur mainly in dairy cows. WD has not been described in beef cattle herds of tropical countries. This study describes the molecular detection of BCoV in a diarrhea outbreak in beef cattle steers (Nellore) raised on pasture in Parana, southern Brazil. During the outbreak, the farm had about 600 fattening steers. Watery and bloody diarrhea unresponsive to systemic broad-spectrum antibiotic therapy reveals a morbidity rate of approximately 15 %. The BCoV N gene was identified in 42.9 % (6/14) of the diarrheic fecal samples evaluated by semi-nested polymerase chain reaction (SN-PCR) technique. Other enteric microorganisms occasionally identified in adult cattle and evaluated in this study such as bovine groups A, B, and C rotavirus, bovine viral diarrhea virus, bovine torovirus, aichivirus B, and Eimeria sp. were not identified in the fecal samples. To the best knowledge of the authors, this is the first description of the BCoV diagnosis in fecal samples collected in a diarrhea outbreak in adult beef cattle grazing in the grass in a tropical country.

Helicobacter infection has been associated with hepatobiliary diseases in humans and animals. The aims of this study were to identify Helicobacter species in the hepatobiliary tract of dogs and to elucidate the possible association of these bacteria in liver diseases. Twenty-seven gastric and hepatobiliary samples were collected from 33 dogs with hepatic lesions and 17 dogs with no liver histological changes. Warthin-Starry staining, immunohistochemical assay, and PCR were performed to detect the presence of Helicobacter. Helicobacter genus was detected in 21.2% of the samples with hepatic lesions. The main lesion was chronic hepatitis. Immunohistochemistry revealed infection in liver (1/5) and gallbladder (1/3) 32 samples. The sequence analysis of seven amplicons of the 16S rRNA gene of Helicobacter genus from hepatobiliary samples showed 97.8 to 100% of nucleotide identity with gastric helicobacter. One amplicon of the ureA and ureB gene of Helicobacter genus from the stomach showed 89.1 to 90.7% nucleotide identity with H. heilmannii. The presence of Helicobacter genus in liver samples showing hepatic lesions suggests the involvement of these bacteria in the etiology of hepatobiliary disease in dogs. DNA sequences were similar to gastric Helicobacter species, reinforcing the hypothesis of bacterial translocation from the stomach to liver by the biliary pathway.

The aim of this study was to perform the molecular characterization of the eleven genes of a G6P[5] bovine group A rotavirus (RVA) strain detected in a diarrhea outbreak from a vaccinated beef cattle herd. The outbreak affected 80 % of calves between 15-30 days old. RVA was identified by RT-PCR in 12 (70.6 %) out of 17 diarrheic fecal samples evaluated. The rotavirus wild-type strain had the genotype constellation G6(IV)-P[5](IX)-I2c-R2-C2-M2-A3-N2-T6-E2e-H3a. This study confirms the importance of homotypic immunity against the bovine RVA P[5] genotype in neonatal diarrhea in cattle herds that are regularly vaccinated against rotaviruses.

The episodes of diarrhea caused by neonatal bovine rotavirus group A (BoRVA) constitute one of the major health problems in the calf rearing worldwide. The main G (VP7) and P (VP4) genotypes of BoRVA strains involved in the etiology of diarrhea in calves are G6P[1], G10P[11], G6P[5], and G8P[1]. However, less frequently, other G and P genotypes have been described in BoRVA strains identified in diarrheic fecal samples of calves. This study describes the identification and molecular characterization of an emerging genotype (G6P[11]) in BoRVA strains involved in the etiology of a diarrhea outbreak in beef calves in a cattle herd of high production in extensive management system. The diarrhea outbreak, which showed high morbidity (60%) and lethality (7%) rates, occurred in calves (n= 384) Nelore (Bos indicus) up to 30-day-old from the State of Mato Grosso do Sul, Brazil. BoRVA was identified in 80% (16/20) of the fecal samples analyzed by polyacrylamide gel electrophoresis (PAGE) technique. In all PAGE-positive fecal samples were amplified products with 1,062-bp and 876-bp in the RT-PCR assays for VP7 (G type) and VP4 (VP8*) (P type) of BoRVA, respectively. The nucleotide sequence analysis of VP7 and VP4 genes of four wild-type BoRVA strains showed G6-III P[11]-III genotype/lineage. The G6P[11] genotype has been described in RVA strains of human and animal hosts, however, in calves this genotype was only identified in some cross-sectional studies and not as a single cause of diarrhea outbreaks in calves with high morbidity and lethality rates as described in this study. The monitoring of the G and P genotypes of BoRVA strains involved in diarrhea outbreaks in calves is important for both animal and public health by allowing the identification of the most frequent genotypes, the characterization of novel genotypes and to identify reassortments with genotypes described in animal and human hosts. The results of this study show the importance of the monitoring of the genotypes of BoRVA strains involved in episodes of bovine neonatal diarrhea as for characterization of frequency of occurrence and pathogenic potential of uncommon genotypes as for monitoring of the emergency of different BoRVA genotypes not included in commercial vaccines. Os episódios de diarreia neonatal ocasionados pelo rotavírus bovino grupo A (BoRVA) constituem-se em um dos principais problemas sanitários na criação de bezerros em todo o mundo. Os principais genotipos G (VP7) e P (VP4) de cepas de BoRVA envolvidos na etiologia da diarreia em bezerros são G6P[1], G10P[11], G6P[5] e G8P[1]. No entanto, com menor frequência, outros genotipos G e P têm sido descritos em cepas de BoRVA identificadas em amostras de fezes diarreicas de bezerros. Este estudo descreve a identificação e caracterização molecular de um genotipo emergente (G6P[11]) em cepas de BoRVA envolvidas na etiologia de um surto de diarreia em bezerros de um rebanho bovino de corte de alta produção em sistema de manejo extensivo. O surto, que apresentou altas taxas de morbidade (60%) e de letalidade (7%), ocorreu em bezerros (n=384) da raça Nelore (Bos indicus) com até 30 dias de idade, provenientes do estado do Mato Grosso do Sul, Brasil. O BoRVA foi identificado em 80% (16/20) das amostras fecais analisadas pela técnica de eletroforese em gel de poliacrilamida (PAGE). Em todas as amostras fecais PAGE-positivas foi possível a amplificação por RT-PCR de produtos com 1.062 pb e 876 pb referentes aos genes VP7 (G tipo) e VP4 (VP8*) (P tipo), respectivamente, de BoRVA. A análise da sequência de nucleotídeos dos genes VP7 e VP4 de quatro cepas de BoRVA demonstrou a presença do genotipo/linhagem G6-III P[11]-III. O genotipo G6P[11] tem sido descrito em cepas de RVA de hospedeiros humanos e animais. Contudo, em bezerros, este genotipo foi apenas identificado em alguns estudos transversais e não como a única causa de surtos de diarreia em bezerros com altas taxas de morbidade e letalidade como descrito neste estudo. O monitoramento dos genotipos G e P de cepas de BoRVA envolvidos em surtos de diarreia em bezerros é relevante tanto para a saúde animal quanto para a saúde pública por possibilitar a identificação dos genotipos mais frequentes, a caracterização de novos genotipos e por identificar reassortments com genotipos descritos em hospedeiros humanos e animais. Os resultados deste estudo demonstram a importância do monitoramento dos genotipos de cepas de BoRVA envolvidas em surtos de diarreia neonatal bovina tanto para a caracterização da frequência de ocorrência e potencial patogênico de genotipos incomuns quanto para o monitoriamento da emergência de genotipos distintos daqueles incluídos em vacinas comerciais.

Porcine group A rotavirus (PoRVA) is a major cause of neonatal diarrhea in suckling and recently weaned piglets worldwide. The involvement of non-group A rotavirus in cases of neonatal diarrhea in piglets are sporadic. In Brazil there are no reports of the porcine rotavirus group C (PoRVC) as etiologic agent of the diarrhea outbreaks in piglets. The aim of this study was to describe the identification of rotavirus group C in single and in mixed infection with rotavirus groups A and B in three neonatal diarrhea outbreaks in suckling (

There are few studies involving the detection of Aichivirus B in cattle herds worldwide, and this virus has never been diagnosed in South America. This study evaluated 222 diarrhoeic faecal samples from four Brazilian geographical regions (South, Southeast, Midwest, and North), collected between February 2010 to May 2012. To evaluate the frequency of occurrence in different types of livestock, samples from beef (n = 105) and dairy (n = 117) cattle herds were evaluated. To determine the category of animals more susceptible to infection, the sampling included samples from calves (n = 182) and adults animals (n = 40). The 216 bp fragment of the Aichivirus RdRp gene was amplified by a RT-PCR assay in 18.2 % (40/222) of the samples evaluated in both beef and dairy cattle animals. The highest (P

We determined nucleotide and deduced amino acid sequences of the rotavirus gene encoding viral protein 6 from 3 fecal samples collected from piglets with diarrhea in Brazil, 2012. The analyses showed that the porcine rotavirus strains in Brazil are closely related to the novel species H rotavirus.

A diarreia neonatal é um dos principais problemas sanitários que acomete leitões em todo o mundo. Em sistemas intensivos de produção o rotavírus suíno grupo A (RVA) é uma das principais etiologias de diarreia em leitões lactentes e recém-desmamados. A caracterização dos genotipos das cepas de RVA detectadas em surtos de diarreia é de grande importância para a identificação de novos genotipos virais, para a análise da diversidade genética, identificação de eventos de ressortimento e transmissão interespécies. O objetivo deste estudo foi realizar análises filogenéticas em três cepas de RVA identificadas em fezes diarreicas de leitões. As duas cepas virais do primeiro estudo (BRA381 e BRA382) foram selecionadas durante um estudo retrospectivo de genotipagem realizado em amostras fecais diarreicas positivas para o RVA pela técnica de eletroforese em gel de poliacrilamida, por apresentarem resultados negativos em testes de genotipagem utilizando primers genotipo-específico para os principais protótipos de RVA de origem humana e animal (bovino e suíno). A cepa viral do segundo estudo (BRA843) foi selecionada para a análise de todos os genes por ser uma cepa G4P[6] diferente do protótipo suíno, a cepa Gottfried. No primeiro estudo os produtos da RT-PCR dos genes VP7 (G) e VP4 (P) foram sequenciados e a análise filogenética possibilitou a identificação do genotipo G26P[13] e G26P[X] nas cepas BRA381 e BRA382, respectivamente. G26 é um genotipo raro que, até então, somente havia sido identificado em cepas de RVA suíno da Ásia (Japão e China) e, possivelmente, em uma cepa de origem humana da Tailândia. O genotipo P[13] é o terceiro genotipo mais comum em RVA suíno, porém a associação G26P[13] ainda não havia sido descrita em cepas de RVA em todo o mundo. No segundo estudo, a análise dos 11 genes de uma cepa (BRA843) de RVA suíno G4-IX P[6]-If, anteriormente caracterizada como distinta do protótipo G4P[6] (Gottfried) de origem suína, revelou estreita relação com cepas de RVA detectadas em suínos e também com cepas detectadas em humanos e animais provenientes de ressortimento com cepas suínas. Estas características sugerem que a cepa BRA843 seja de origem suína. A constelação de genotipos (G4-P[6]-I5-R1-C1-M1-A8-C1-T7-E1-H1) identificada nesta cepa brasileira de RVA suíno tem estrutura Wa-like com genotipos comumente detectados em cepas de RVA de origem suína. O genotipo T7 é um genotipo raro que foi primeiramente descrito na cepa de RVA UK (protótipo) de origem bovina. Posteriormente, o genotipo T7 foi relatado em cepas de RVA de humanos provenientes de ressortimento com cepas suínas e bovinas. Recentemente, o genotipo T7 foi também identificado em cepas de RVA suíno reforçando a origem suína deste genotipo. No Brasil, este é o primeiro estudo com análise de todos os genes de uma cepa suína de RVA G4P[6], bem como a primeira descrição de uma cepa G4P[6] suína detectada em um hospedeiro suíno carreando o genotipo T7. Os dois estudos revelaram a complexidade de genotipos de RVA circulantes em rebanhos suínos brasileiros, bem como a importância da análise de todos os genes para a compreensão dos mecanismos de evolução das cepas de rotavírus e o papel dos suínos como reservatórios de uma constelação de genotipos de RVA passíveis de infectar tanto animais quanto humanos. Neonatal diarrhea is a major health problem that affects piglets worldwide. In intensive pig production systems, the porcine group A rotavirus (RVA) is one of the main causes of diarrhea in suckling and recently weaned piglets. The characterization of genotypes of the RVA strains detected in diarrhea outbreaks is important for the identification of new genotypes, for the analysis of genetic diversity, identification of reassortment and interspecies transmission events. The aim of this study was to perform the phylogenetic analysis of three RVA strains identified in diarrheic fecal samples of piglets. The two strains from the first study (BRA381 and BRA382) were selected during a retrospective genotyping study performed in RVA-positive diarrheic fecal samples by polyacrylamide gel electrophoresis technique, by negative results in genotyping assays using genotype-specific primers for the main RVA prototypes of human and animal origin (bovine and porcine). The strain from the second study (BRA843) was chosen for analysis of the all genes for being a G4P[6] strain different to porcine prototype, the Gottfried strain. In the first study, the RT-PCR products of the VP7 (G) and VP4 (P) genes were sequenced and the phylogenetic analysis allow the identification of the genotype G26P[13] and G26P[X] in the BRA381 and BRA382 strains, respectively. G26 is a rare genotype, which until then, had only been identified in porcine RVA strains from Asia (Japan and China) and, probably, in a human origin strain from Thailand. P[13] is the third most common genotype in porcine RVA, but the combination G26P[13] had not still been reported in RVA strains worldwide. In the second study, the analysis of the all genes of a G4-IX P[6]-If porcine RVA strain (BRA843), previously characterized as distinct from the G4P[6] porcine prototype (Gottfried), revealed close relationship with RVA strains detected in pigs and also with porcine-like human and porcine-like animal RVA strains. These characteristics suggest that BRA843 strain is a porcine strain. The genotype constellation (G4-P[6]-I5-R1-C1-M1-A8-C1-T7-E1-H1) identified in this Brazilian porcine RVA field strain has the Wa-like structure with genotypes commonly detected in porcine RVA strains. T7 is a rare genotype that was first described in the UK RVA strain (prototype) of bovine origin. Afterwards, the T7 genotype was identified in human RVA strains derived from reassortment with porcine and bovine RVA strains. Recently, the T7 genotype was also identified in porcine RVA strains reinforcing the porcine origin of this genotype. In Brazil, this is the first study where all genes of a porcine G4P[6] RVA strain were analyzed, as well as, the first description of G4P[6] porcine RVA strain detected in a pig host bearing the T7 genotype. The two studies reveals the complexity of RVA genotypes circulating in Brazilian pig herds, as well as, the importance of analyzing all genes for understanding the mechanisms of evolution of the RVA strains and the role of pigs as reservoirs of a RVA genotype constellation able to infect both animals and humans.

Um dos principais problemas sanitarios que acomete as criacoes de leitoes em todo o mundo e a diarreia neonatal. O rotavirus suino sorogrupo A (PoRV-A) e uma etiologia viral frequentemente detectada nos episodios de diarreia em leitoes lactentes e recem-desmamados em sistemas de producao intensivo. Para controlar a infeccao sao preconizadas varias acoes de manejo sanitario, incluindo a vacinacao das matrizes. Entretanto, a grande diversidade antigenica encontrada nas estirpes de PoRV-A podem ser responsaveis por falhas vacinais. As proteinas VP7 e VP4, presentes na camada externa do capsideo viral do RV-A, apresentam importante acao na imunidade induzindo anticorpos neutralizantes. Com base nas caracteristicas moleculares dos genes que codificam as proteinas VP7 e VP4, os RV-A podem ser classificados em genotipos G e P, respectivamente. Dentre os genotipos P identificados em estirpes de PoRV-A, o genotipo P[6] destaca-se pela sua frequencia de ocorrencia e pela grande diversidade. Ate o momento foram descritas cinco linhagens (I-V) e oito sublinhagens (Ia-If; Va-Vb) do genotipo P[6] em estirpes de RV-A de origem humana e suina. Este estudo teve por objetivo a caracterizacao molecular do genotipo P (gene VP4) presente em estirpes de PoRV-A identificadas em um foco de diarreia neonatal em suinos. O foco ocorreu em uma granja altamente tecnificada, localizada no estado de Santa Catarina, com esquema de vacinacao regular contra a rotavirose suina. Foram colhidas 80 amostras de fezes sendo 49 diarreicas e 31 normais (controle). As amostras foram colhidas de leitoes lactentes (n=57) e recem-desmamados (n=23). O diagnostico de rotavirus foi realizado por ss-PAGE e RT-PCR. As amostras positivas foram genotipadas por multiplex-nested-PCR utilizando primers genotipo P-especificos e sequenciadas para a definicao da epidemiologia molecular da infeccao. Foram identificadas 22 (27,5%) amostras fecais positivas para o PoRV-A sendo 16 (72,7%) provenientes de animais com diarreia e 6 (27,3%) de animais assintomaticos. O PoRV-A foi identificado em leitoes de todas as faixas etarias incluidas no estudo. Vinte estirpes de PoRV-A identificadas foram classificadas como genotipo P[6] de origem humana pois foram amplificadas (267 pb) com o primer desenhado para a amplificar estirpes M37-like de RV-A de origem humana. Nenhuma das amostras foi amplificada com o primer desenhado especificamente para amplificacao de estirpes de RV-A genotipo P[6] de origem suína (estirpe Gottfried). A analise da sequencia de nucleotideos e a reconstrucao da arvore filogenetica, realizada em quatro amostras permitiram a classificacao de duas estirpes virais (BRA838/07-Po e BRA844/07-Po) como pertencentes ao genotipo P[6] linhagem I, sublinhagem Ie e outras duas estirpes (BRA843/07-Po e BRA898/07-Po) como sublinhagem If. Os genotipos P[6]-Ie e If foram recentemente descritos em estirpes de RV-A de origem humana. Essa e a primeira descricao de estirpes de PoRV-A genotipos P[6]-Ie e If identificadas em um foco de diarreia neonatal em suinos. Estes resultados sugerem que o foco de diarréia pode ter resultado da transmissão interespecie do RV-A. A vacinação intensiva das matrizes com vacina comercial contendo o genótipo P[6] de origem suína (estirpe Gottfried) pode ter influenciado na seleção desses genótipos, ate então somente descritos em estirpes virais de origem humana. A possibilidade da ocorrência de infecções heterologas, em situação de pressão imunológica, alerta ainda para a necessidade do constante monitoramento das características antigênicas e moleculares de estirpes de PoRV-A identificadas em surtos de diarreia neonatal em rebanhos suínos regularmente vacinados contra a rotavirose. Neonatal diarrhea is the major piglet health problem in swine production systems worldwide.In intensive production the porcine serogroup A rotaviruses (PoRV-A) are common cause of diarrhea in suckling and recently weaned piglets. The VP4 and VP7 proteins of the outer layer viral capsid of RV-A are relevant for immunity against rotavirus infection by inducing neutralizing antibodies. According to the antigenic and molecular characteristics of the genes that encode VP4 and VP7 proteins, the RV-A strains can be classified in genotypes P and G, respectively. In the modern swine production the main health management to control the PoRV-A infections is the sows vaccination. However, the diversity of G (VP7) and P (VP4) genotypes identified in wild-type PoRV-A strains can be responsible for vaccine failures. Despite of several P genotypes identified in PoRV-A strains, the P[6] genotype is considered important for the frequency and for the great antigenic diversity. So far, have been described 5 lineages (I-V) and 8 sublineages (Ia-If; Va-Vb) of P[6] genotype in RV-A strains of human and porcine origin. The present study was developed to detect P genotype (VP4 gene) in PoRV-A strains identified during an outbreak of diarrhea in piglets. The outbreak occurred in a pig farm located in the state of Santa Catarina, South area of Brazil that presented good management practices and regular vaccination against porcine rotaviruses. Were collected 80 fecal samples being 49 diarrheic and 31 with normal consistency (control group). Were evaluated 57 stool samples of suckling (≤ 3-week-old) and 23 of recently-weaned (4-weekold) piglets. PoRV-A diagnosis was carried out by silver-stained polyacrylamide gel electrophoresis and RT-PCR assay. Positive samples were genotyping by multiplex-nested- PCR with genotype-specific primers and four PoRV-A strains were sequenced to define the molecular epidemiology of the infection. During this study were identified 22 (27.5%) PoRVA positive stool samples of which 16 (72.7%) were from symptomatic and 6 (27.3%) from asymptomatic piglets. PoRV-A was detected in fecal samples either from suckling as weaned piglets. In all the 20 wild-type PoRV-A strains included in this study were only amplified a PCR products with 267 bp length that belong to the P[6] genotype present in RV-A strains of human origin (M37-like strains). The sequencing and phylogenetic analysis carried out in a 586 bp length product of the VP4 gene (VP8*) of four Brazilian wild-type PoRV-A strains allowed to classify two P[6] strains (BRA838/07-Po and BRA844/07-Po) as P[6]-Ie genotype and the other two P[6] strains (BRA843/07-Po and BRA898/07-Po) as P[6]-If genotype. The genotype sublineages P[6]-Ie and If were recently described in RV-A strains of human origin.This is the first description of P[6]-Ie and If genotypes in RV-A strains of porcine origin. These findings suggest that the neonatal diarrhea outbreak might have been resulted of interspecies transmission of RV-A. The regularly vaccination of the sows with a rotavirus commercial vaccine containing P[6] genotype of porcine origin (Gottfried strain) might have provide the disappearance of the genotype P[6]-II (Gottfried-like) of porcine origin and the selection of RV-A strains with VP4 gene described only in RV-A strains of human origin such as P[6]-Ie and If. Heterologous infections are described in rotavirus infections in human beings and in animals. Rotavirus vaccination fails can occur in RV-A infections with virus strains carrying P and/or G genotypes different from those presents in the vaccine. The neonatal diarrhea outbreak in a regularly vaccinated pig herd and the characterization of PoRV-A strains with atypical P genotypes described in this study highlight the importance of the epidemiological studies of human and animal rotaviruses.

leptospirose, causada por espiroquetas do gênero Leptospira, é uma zoonose globalmente disseminada, negligenciada e emergente. O isolamento de leptospiras é o primeiro passo para sua caracterização molecular e genotipagem, no entanto, para os sorovares mais fastidiosos, a cultura e isolamento são controversos e laboriosos. Com relação ao controle e tratamento da leptospirose, muitos estudos são realizados, mas ainda não existe uma vacina eficaz contra todas as espécies patogênicas ou variedade de antimicrobianos com atuação sobre o quadro de leptospirúria. Assim o presente estudo visou primeiramente desenvolver estratégias de cultura e isolamento para as leptospiras fastidiosas e utilizar métodos rápidos de diagnóstico molecular para caracterização e tipificação dos sorovares. Posteriormente, a partir do isolamento de novas estirpes, o sequenciamento genômico foi realizado com a finalidade de obtenção de dados de genômica comparativa com outras estirpes disponíveis no GenBank para uma busca in silico de alvos drogáveis e vacinais. No primeiro estudo, três formulações de meios de cultura líquidos básicos foram produzidas para isolar e manter leptospiras. Em cada formulação (A, B e C) foram adicionados diferentes suplementos para ajudar no crescimento de leptospiras fastidiosas: piruvato de sódio, enzima superóxido dismutase e soro fetal bovino. Durante o período aproximado de 55 dias, adotou-se estratégias de troca entre as três formulações de acordo com a observação em microscopia de campo escuro e ao final desse período houve sucesso no isolamento de três estirpes do sorovar Hardjo (dois genotipos, Hardjobovis e Hardjoprajitno) com adaptação total ao meio de cultura. Posteriormente, estas culturas foram avaliadas com uso da microscopia eletrônica e notou-se diferenças na morfologia e viabilidade de acordo com a composição do meio. No segundo estudo, os genomas desses dois genotipos foram sequenciados, montados e depositados no GenBank. A partir desses dados e também de outros genomas de outras espécies de Leptospira foi possível realizar uma abordagem de com base na vacinologia reversa, genômica comparativa e de docking molecular com o uso de ferramentas de bioinformática. Para os alvos vacinais, os resultados com base nas características da probabilidade de adesão, TMHMM e densidade de epítopos, sugerem que cinco alvos são bons candidatos e podem ser testados rapidamente em novas formulações de vacinas e posteriormente testados in vivo. Para a etapa de docking molecular, oito proteínas preditas como citoplasmáticas e identificadas como essenciais para a sobrevivência de leptospiras foram consideradas bons alvos para novos medicamentos. Destacando a proteína de divisão celular FtsZ, que foi o alvo identificado com as melhores características de ligação e, por meio de uma triagem virtual, o ZINC04259719 foi identificado como a melhor molécula para ligar a essa proteína, a fim de inibir sua função eliminando o patógeno. A vacinologia reversa e o docking molecular são poderosas ferramentas de bioinformáticas que possibilitam uma busca rápida e sem procedimentos laboriosos, por alvos proteicos específicos que possam ser candidatos a uma nova vacina ou medicamento no controle da leptospirose. Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonosis. The leptospires isolation is the first step towards its molecular characterization and genotyping, however, for the most fastidious serovars, the culture and isolation are controversial and laborious. Regarding the control and treatment of leptospirosis, many studies have been carried out, but there is not yet an efficient vaccine against all pathogenic species or more variety of antimicrobials acting on leptospiruria. Thus, the present study aimed primarily to develop culture and isolation strategies for fastidious leptospires and using rapid molecular diagnostic methods for characterization and typing of serovars. Subsequently, from the isolation of new strains, genomic sequencing was carried out in order to obtain comparative genomic data with other strains available at GenBank for an in silico search for drug and vaccine targets. In the first study, three formulations of basic liquid culture media were produced to isolate and maintain leptospires. In each formulation (A, B and C) different supplements were added to help the growth of fastidious leptospires: sodium pyruvate, superoxide dismutase enzyme and fetal bovine serum. During the approximate period of 55 days, exchange strategies were adopted between the three formulations according to the observation in dark field microscopy and at the end of this period, there was success in the isolation of three strains of serovar Hardjo (two genotypes, Hardjobovis and Hardjoprajitno) with total adaptation to the culture medium. Subsequently, these culture was evaluated through electron microscopy and differences in morphology and viability were noted according to the composition of the medium. In the second study, the genomes of these two genotypes were sequenced, assembled and deposited on GenBank. From these data and also from other genomes of Leptospira species, it was possible to carry out an approach based on reverse vaccinology, genomic comparative and molecular docking with the use of bioinformatics tools. For vaccine targets, results based on characteristics of probability of adherence, TMHMM and epitope density, suggest that five targets are good candidates and can be tested quickly in new vaccine formulations and subsequently tested in vivo. For the molecular docking step, eight proteins predicted as cytoplasmic and considered essential for the survival of leptospires were considered good targets for new drugs. Highlighting the cell division protein FtsZ, which was the target identified with the best binding characteristics and through a virtual screening the ZINC04259719 was identified as the best molecule for linked to this protein in order to inhibit its function and consequently eliminating the microorganism. Reverse vaccinology and molecular docking are powerful bioinformatics tools that enable a quick discovery, without laborious procedures, for specific protein targets that may be candidates for a new vaccine or therapeutic target to leptospirosis control.

Com o objetivo de avaliar se a contagem de folículos antrais (CFA), o escore de condição corporal (ECC), o peso vivo e o escore de comportamento no tronco exercem influência sobre a taxa de concepção em vacas submetidas ao protocolo de inseminação artificial em tempo fixo (IATF), foram avaliadas 122 fêmeas da raça Brahman (Bos taurus indicus), com ECC variando de 2,5 a 3,5 (escala de 1-5) e peso entre 300 a 560 kg. Em um dia aleatório do ciclo estral (D0), as vacas receberam um dispositivo intravaginal contendo 1,2 g de progesterona (P4) e aplicação de 2,0 mg de benzoato de estradiol (BE) por via intramuscular (IM). Após 8 dias (D8), o dispositivo foi removido e administrou-se 500 μg de cloprostenol sódico (PGF2α), 300 UI de gonadotrofina coriônica equina (eCG) e 1,0 mg de cipionato de estradiol (CE) por via IM. Neste mesmo dia, realizou-se a marcação da base da cauda para avaliar a demonstração de estro e 48 horas depois procedeu-se a IATF. O ECC e o escore de comportamento foram avaliados em todos os dias do protocolo de IATF. O peso foi mensurado no D0 e no diagnóstico de gestação (D40). A CFA (folículos ≥ 3 mm) de cada fêmeas foi determinada no D0. Para análise, estabeleceu-se os grupos de CFA (baixa ≤ 15 folículos, intermediaria ≥ 16 e ≤ 29 folículos e alta contagem ≥ 30 folículos), da classificação do comportamento (calma, média de escore = 1,0; inquieta, média de escore > 1,0 e ≤ 2,0; e agitada, média de escore ≥ 2,1) de variação do peso (ganhando, variação positiva de +10 a +40 kg; mantendo, variação de -9 a +9 kg; e perdendo, variação negativa de -10 a -40 kg) e variação do ECC (ganhando, variação positiva de +0,25 a +0,75 pontos; mantendo, variação de -0,25 a +0,25 pontos; e perdendo, variação negativa de -0,25 a -0,75 pontos). Taxas de concepção foram analisadas pelo modelo de regressão logística binaria. A taxa de concepção geral do estudo foi de 50% (61/122), e não sofreu influência (p > 0,05) dos grupos de CFA, classificação de reatividade, variações de peso e ECC. Animais de baixa, intermediaria e alta CFA apresentaram taxas de concepção de 52,6, 50,9 e 45,4%, respectivamente. No entanto, a baixa CFA apresentou maior (p = 0,050) proporção de animais com alta intensidade de manifestação de estro (94,7%) no momento da IATF. Vacas classificadas como calmas, inquietas e agitadas apresentaram taxas de concepção de 43,0, 54,7 e 47,1%, respectivamente. Animais que ganharam, mantiveram e perderam peso tiveram taxas de concepção de 43,0, 54,7 e 47,1%, respectivamente. Já aquelas consideradas como ganhando, mantendo e perdendo ECC apresentaram taxas de concepção de 44,0, 54,3 e 37,5%, respectivamente. Nas condições do presente estudo, conclui-se que a taxa de concepção de vacas da raça Brahman (Bos taurus indicus) submetidas a IATF não sofrem influência da contagem de folículos antrais, da classificação de reatividade e nem das variações de peso e ECC. No entanto, a baixa CFA resultou em maior proporção de fêmeas bovinas da raça Brahman com manifestação de estro de alta intensidade. In order to evaluate if the antral follicle count (AFC), the body condition score (BCS), the live weight and the behavioral score in the trunk, influence the design rate in cows submitted to timed artificial insemination (TAI), 122 Brahman (Bos taurus indicus) cows, with BCS ranging from 2.5 to 3.5 (range of 1-5) and weight between 300 and 560 kg were evaluated. On a random day of estrous cycle (D0), cows received an intravaginal device containing 1.2 g of progesterone (P4) and 2.0 mg of estradiol benzoate (EB) intramuscularly (IM). After 8 days (D8), the device was removed and 500 μg of sodium cloprostenol (PGF2α), 300 IU of equine chorionic gonadotrophin (eCG) and 1.0 mg estradiol cypionate (EC) were administered IM. On the same day, the base of the tail was marked to evaluate the estrus demonstration and 48 hours later the TAI was performed. The BCS and the behavior score were assessed every day of the TAI protocol. The weight was measured at the D0 and at the gestation diagnosis (D40). The AFC (follicles ≥ 3 mm) of each female was determined at D0. For analysis, the AFC groups (low ≤15 follicles, intermediate ≥16 and ≤29 follicles and high ≥30 follicles), behavioral classification (calm, mean score = 1.0, restlessness, mean score> 1.0 and ≤ 2.0, and agitated, mean score ≥ 2,1) of weight variation (gaining, positive variation of +10 to +40 kg, maintaining, variation from -9 to +9 kg; and losing, negative variation of -10 to -40 kg) and BCS variation (gaining, positive variation of +0.25 to +0.75 points, maintaining a variation of -0.25 to +0.25 points; losing, negative change from -0.25 to -0.75 points). Design rates were analyzed by the binary logistic regression model. The overall design rate of the study was 50% (61/122), and it was not influenced (p> 0.05) by the AFC groups, reactivity classification, weight variations and BCS. Animals of low, intermediate and high AFC presented design rates of 52.6, 50.9 and 45.4%, respectively. However, the low AFC presented a higher (p = 0.050) proportion of animals with high estrus manifestation intensity (94.7%) at the time of TAI. Cows classified as calm, uneasy and restless had design rates of 43.0, 54.7 and 47.1%, respectively. Animals that gained, maintained and lost weight had design rates of 43.0, 54.7 and 47.1%, respectively. Those considered to be gaining, maintaining and losing BCS had conception rates of 44.0, 54.3 and 37.5%, respectively. Under the conditions of the present study, it was concluded that the conception rate of Brahman cows (Bos taurus indicus) submitted to AFC is not influenced by antral follicle count, reactivity classification, or weight and BCS variations. However, low AFC resulted in a higher proportion of Brahman cows with high intensity estrus.

A anemia infecciosa equina (AIE) é uma doença viral e potencialmente fatal que acomete equídeos de diferentes raças, sexo e idades. Com o objetivo de conhecer a ocorrência desta enfermidade no estado do Paraná, o estudo foi realizado em duas etapas distintas. Na primeira etapa foi realizado um estudo retrospectivo da ocorrência da AIE no estado do Paraná, com a utilização dos registros dos casos diagnosticados pelo serviço oficial em equídeos entre os anos de 2006 a 2017. Foi realizada análise de 126 Formulários de Investigação Inicial (Form-In) de casos positivos de AIE entre os anos de 2012 e 2017. Na segunda etapa, foi realizado inquérito soroepidemiológico entre os meses de março e abril de 2018, com colheita de amostras em 2683 animais e 890 propriedades distribuídas pelo estado, utilizando o Imunodifusão em Ágar gel (IDGA) como teste diagnóstico. A prevalência foi calculada e realizou-se análise descritiva dos dados. Na análise retrospectiva dos registros de 2006 a 2017, foram analisadas 502.091 amostras de soro de equídeos e a AIE foi confirmada em 371 animais (0,073%), com variações do número de casos positivos ao longo dos anos, porém com maior frequência na região sul do Estado. Já no inquérito soroepidemiológico foi detectada a ocorrência da AIE em 14 propriedades (1,57%) e 14 animais (0,52%). Foi possível concluir que a AIE esteve presente nos equídeos do estado do Paraná durante o período avaliado, porém com prevalência reduzida. Com o conhecimento destes dados, o serviço oficial de defesa agropecuária pode atuar com maior eficiência nos municípios mais afetados, bem como realizar alterações na legislação e implantar programas de educação sanitária. Equine infectious anemia (EIA) is a life-threatening viral disease that affects equids of different races, gender and ages. With the purpose of knowing Paraná´s State EIA occurrence, the study was carried out in two distinct stages. At first, a retrospective study was carried out, analysing the records of cases in equids between 2006 and 2017. An analysis of 126 Forms of Initial Investigation (Form-In) of positive cases of EIA between the years of 2012 and 2017 was carried out. In the second stage, a seroepidemiological survey was carried out between March and April 2018, with samples collected in 2683 animals and 890 properties distributed through the state, using Agar Gel Immunodiffusion (AGID) as diagnostic test. Prevalence was calculated and a descriptive analysis of the data was performed. In the retrospective analysis of the records from 2006 to 2017, 502,091 equine serum samples were analyzed and the EIA was confirmed in 371 animals (0.073%), with variations in the number of positive cases over the years, but more frequently in the southern region of State. However, in the seroepidemiological survey the occurrence of EIA was detected in 14 properties (1.57%) and 14 animals (0.52%). It was possible to conclude that EIA was present in the equines of the State of Paraná during the period evaluated, but with a reduced prevalence. With the knowledge of these data, the official animal health service can act with greater efficiency at the most affected municipalities, as well as make legislation improvements and implement animal health education programs.

Pestivírus suíno atípico (APPV) tem sido associado ao tremor congênito (TC) tipo A-II em leitões neonatos. Apesar do número de estudos sobre o APPV ter aumentado, as alterações histopatológicas, bem como outros aspectos relativos à caracterização antigênica e molecular do vírus e aspectos epidemiológicos da infecção em leitões ainda são pouco descritos. O objetivo do primeiro estudo foi descrever os achados patológicos e moleculares associados à infecção simultânea de APPV e teschovírus suíno (PTV) em leitões com manifestações clínicas de TC provenientes de um rebanho do estado do Paraná com altas taxas de letalidade em animais recém-nascidos. Em 2017, foram relatados casos de TC em leitegadas com alta letalidade neonatal em um rebanho paranaense e três leitões da mesma leitegada que morreram espontaneamente foram enviados para diagnóstico. As principais alterações patológicas encontradas em todos os leitões foram desmielinização secundária e hipomielinização no cerebelo, tronco encefálico e medula espinhal confirmadas por histopatologia e coloração Luxol Fast Blue. Alterações histopatológicas adicionais incluíram necrose neuronal multifocal, neuronofagia e gliose encontrados no córtex cerebral e medula espinhal e enterite atrófica de todos os leitões e edema no mesocólon de um deles. RNA de APPV e de PTV foi detectado por nested-RT-PCR no sistema nervoso central (SNC) dos leitões afetados, sendo que PTV também foi detectado no intestino e nas fezes. As alterações patológicas e os resultados moleculares demonstram que os leitões avaliados apresentavam infecção por APPV e PTV, o que pode ter ocasionado a alta letalidade (100%) de leitões recém-nascidos identificada nesse caso. No segundo estudo foram descritas as características histopatológicas da infecção espontânea singular por APPV em quatro leitões com sinais clínicos de TC. Os quatro leitões de dois dias de idade com TC foram avaliados por histopatologia, imunohistoquímica (IHQ) e análise molecular. As principais alterações histopatológicas no encéfalo e medula espinhal incluíram necrose neuronal, gliose, neuronofagia, satelitose, desmielinização, degeneração walleriana e necrose de células de purkinje. Na IHQ, realizada com o objetivo de detectar a proliferação da proteína ácida fibrilar glial (GFAP) em áreas afetadas do encéfalo e medula espinhal, demonstrou que a proliferação de células GFAP positivas e fibras era predominante em leitões com TC infectados com APPV em relação a leitões assintomáticos não infectados da mesma idade. As reações de nested-RT-PCR identificaram RNA de APPV no cérebro, cerebelo e tronco cerebral de todos os leitões, enquanto outros vírus (teschovírus suíno, sapelovírus suíno e vírus Seneca Valley) conhecidos por induzir sinais clínicos semelhantes não foram identificados. Estes resultados sugerem que as alterações histopatológicas induzidas pela infecção singular por APPV nos leitões avaliados são predominantemente degenerativas e necróticas e correlacionadas com as alterações identificadas no primeiro estudo. Com estes resultados podemos sugerir que a necrose neuronal, a gliose, a neuronofagia e a satelitose possam ser consideradas características histológicas importantes da infecção induzida por APPV em leitões recém-nascidos com sinais clínicos de TC. Atypical porcine pestivirus (APPV) has been associated with congenital tremor (CT) type A-II in neonatal piglets. Although the number of APP-based studies is increasing, the histopathological alterations and other aspects related to the antigenic and molecular characterization of the virus and epidemiological aspects of the infection in piglets are still underreported. The objective of the first study was to describe the pathological and molecular findings associated with simultaneous infection of APPV and porcine teschovirus (PTV) in piglets with clinical manifestations of CT from a herd of Paraná State with high rates of lethality in newborn piglets. In 2017, CT cases were reported in litters with high neonatal mortality in a pig herd from Paraná and three piglets from the same litter that died spontaneously were sent for diagnosis. The main pathological alterations in all piglets were secondary demyelination and hypomyelination at the cerebellum, brainstem and spinal cord confirmed by histopathology and luxol fast blue?cresyl violet stain. Additional histopathological alterations included multifocal neuronal necrosis, neuronophagia and gliosis found in the cerebral cortex and spinal cord of all piglets, and atrophic enteritis in all piglets and mesocolonic oedema in one piglet. APPV and PTV RNA was detected by nested-RT-PCR in the central nervous system of affected piglets, and PTV was also detected in the intestine and faeces. The pathological alterations and molecular results together suggested that the piglets evaluated had a dual infection by APPV and PTV, which probably was the cause of the high lethality (100%) of newborn piglets identified in this case. In the second study the histopathologic features of singular spontaneous APPV infection in four piglets with clinical signs of CT were described. Four two-day-old piglets with CT were evaluated by histopathology, immunohistochemistry (IHC), and molecular assay. The main histopathologic alterations at the brain and spinal cord included neuronal necrosis, gliosis, neuronophagia, satellitosis, demyelination, Wallerian degeneration, and Purkinje cell necrosis. In the IHC assay, performed to detect the proliferation of glial fibrillary acidic protein (GFAP) in affected areas of the brain and spinal cord revealed that the proliferation of GFAP+ cells and fibers was predominant in APPV-infected piglets relative to asymptomatic piglets of the same age group. The nested-RT-PCR assays identified APPV RNA in the cerebrum, cerebellum, and brainstem of all piglets, while other viruses (PTV, PSV, SVV) known to induce similar clinical signs were not identified. These results suggested that the histopathological alterations induced by singular APPV infection are predominantly degenerative and necrotic and correlate with alterations identified in the first study. With these results we can suggest that neuronal necrosis, gliosis, neuronophagia, and satellitosis should be considered as important histologic features of APPV-induced infection in newborn piglets with clinical signs of CT.

A diarreia neonatal bovina (DNB) é uma enfermidade multifatorial que, com maior frequência, acomete bezerros com até 30 dias de idade. A DNB é o principal evento sanitário em bezerras leiteiras lactentes e devido às taxas de morbidade e mortalidade a infecção ocasiona perdas econômicas consideráveis à cadeia produtiva do leite. O principal agente infeccioso viral envolvido na etiologia dessa enfermidade é o rotavírus A (RVA). Entre as várias medidas de controle e profilaxia a serem adotadas para a redução da frequência de DNB em rebanhos bovinos leiteiros destaca-se a vacinação das vacas no período pré-parto. O objetivo desse estudo foi monitorar os genotipos G (VP7) e P (VP4) de cepas de RVA identificadas em bezerras leiteiras em rebanhos regularmente vacinados com vacina comercial contendo RVA genotipo G6P[5]. Para isso, foram conduzidos dois experimentos sendo o primeiro de caráter longitudinal e o segundo transversal. No primeiro experimento, 122 bezerras provenientes de um rebanho da raça Holandesa foram avaliadas até os 30 dias de idade. Em todas as bezerras, independentemente da presença ou ausência de DNB, nos dias 1, 4, 7, 10, 14, 17, 21, 24, 28 e 30 de idade foram realizadas colheitas de amostras fecais totalizando 1.220 amostras. O segundo experimento foi realizado em 14 rebanhos bovinos leiteiros regularmente vacinados contra a rotavirose, e em cada rebanho foi realizada apenas uma colheita de amostras fecais diarreicas (n=87) em bezerras de até 30 dias de idade. Como técnica de triagem, a presença de RVA nas amostras fecais foi avaliada pela técnica de eletroforese em gel de poliacrilamida (EGPA). Todas as amostras positivas para RVA por EGPA foram submetidas à amplificação dos genes VP7 e VP4 por RT-PCR e os genotipos G e P, respectivamente foram determinados por sequenciamento dos produtos amplificados e análise das sequencias obtidas. No experimento longitudinal foram identificados 76 (62,3%) e 99 (8,1%) animais e amostras fecais positivas para RVA, respectivamente. Todas as amostras RVA-positivas (n=10) selecionadas para o sequenciamento foram caracterizadas como genotipo G10P[11]. No experimento transversal somente 42,86% (6/14) das propriedades foram analisadas. Oito rebanhos foram excluídos do estudo, por não apresentarem bezerras com diarreia (n=2) ou por não apresentarem amostras fecais positivas para RVA (n=6). Nos seis rebanhos restantes, 17 (25,4%) amostras de fezes foram positivas para RVA. O sequenciamento e análise filogenética dos produtos amplificados dos genes VP7 e VP4 possibilitaram identificar os genotipos G6P[11] em cinco rebanhos e G10P[11] em um. As cepas G6 pertencem a linhagem III enquanto a cepa G6 vacinal pertence a linhagem IV, caracterizando a ocorrência nos rebanhos vacinados de RVA genotipo G6 distinto daquele presente na cepa vacinal. Adicionalmente, nestes rebanhos vacinados com RVA genotipo G6P[5] foi observada a emergência dos genotipos G10 e P[11] caracterizando possível falha na proteção heteróloga. Os resultados, tanto do experimento longitudinal quanto do transversal, demonstraram que nos rebanhos bovinos regularmente vacinados com a cepa G6P[5] de RVA houve redução na frequência de diarreia em bezerros com idade inferior a 30 dias. Contudo, houve circulação de RVA com genotipos G e P distintos daqueles presentes na cepa vacinal. Com isso, evidencia-se a importância do constante monitoramento das cepas de RVA circulantes em rebanhos bovinos regularmente vacinados e também da realização de estudos de vigilância epidemiológica para a identificação de genotipos emergentes e para a caracterização molecular de cepas de RVA com maior virulência e/ou com potencial zoonótico. Neonatal calf diarrhea (NCD) is a multifactorial disease that most frequently affects calves up to 30 days old. The NCD is the main health event in suckling calves and due to the morbidity and mortality rates, the infection causes considerable economic losses to the milk production chain worldwide. The main viral infectious agent involved in the etiology of this syndrome is rotavirus A (RVA). Among the several measures of control and prophylaxis to be adopted to reduce the frequency of NCD in dairy cattle herds stands out the vaccination of cows in the dry period. The aim of this study was to monitor the genotypes G (VP7) and P (VP4) of RVA strains identified in heifer calves in dairy cattle herds regularly vaccinated with commercial vaccine containing RVA genotype G6P[5]. For this, two experiments were conducted, the first being of longitudinal design and the second cross-sectional. In the first experiment, 122 heifer calves up to 30 days old from a Holstein breed were evaluated. In all calves, regardless of the presence or absence of NCD, fecal samples were collected on days 1, 4, 7, 10, 14, 17, 21, 24, 28, and 30, totaling 1,220 samples. The second experiment was performed on 14 dairy cattle herds regularly vaccinated against RVA, and in each herd, only one diarrheic fecal sample (n=87) was collected in heifer calves with NCD. As screening test, the presence of RVA in fecal samples was evaluated by the polyacrylamide gel electrophoresis technique (PAGE). All the positive diarrheic fecal samples for RVA in the screening test were submitted to the amplification of the VP7 and VP4 genes by RT-PCR and the genotypes G and P, respectively were determined by sequencing of the amplified products and analyzes of nucleotide sequence obtained. In the longitudinal experiment were identified 76 (62.3%) and 99 (8.1%) calves and fecal samples positives for RVA, respectively. All RVA-positive samples (n=10) selected for sequencing were characterized as genotype G10P[11]. In the cross-sectional experiment, only 42.9% (6/14) of herd were analyzed. Eight herds were excluded from the study because they did not present heifer calves with diarrhea (n=2) or because they did not present RVA positive fecal samples (n=6). In the remaining six herds, 17 (25.4%) diarrheic fecal samples were positive for RVA. Sequencing and phylogenetic analysis of the VP7 and VP4 genes amplified products allowed to identify the genotype G6P[11] in five herds and the genotype G10P[11] in one. The genotype G6 of RVA field strains migrated into a cluster (lineage III) distinct from the G6 vaccine strain (lineage IV), characterizing the occurrence of G6 genotype phylogenetic distant in vaccinated herds. Additionally, in these herds vaccinated with RVA genotype G6P[5] was observed the emergence of the genotypes G10 and P[11], characterizing a possible failure in heterologous protection. The results of both the longitudinal and cross-sectional experiments showed that in dairy cattle herds regularly vaccinated with the RVA genotype G6P[5] there was a reduction in the frequency of diarrhea in calves up to 30-day-old. However, there were circulations of RVA field strains with different G and P genotypes than those present in the vaccine strain. Thus, it highlights the importance of constant monitoring of RVA strains circulating in regularly vaccinated cattle herds, as well as epidemiological surveillance studies for the identification of emergent genotypes and for the molecular characterization of highly virulent RVA strains and/or strains with zoonotic potential.

A presença de enzimas microbianas deteriorantes termoestáveis proteolíticas e lipolíticas pode comprometer a qualidade e vida útil do leite processado e derivados, mesmo que o leite cru apresente baixas contagens totais de micro-organismos. Além disso, as formas microbianas vegetativas ou esporuladas podem resistir à pasteurização e também atuarem como deteriorantes do leite pasteurizado. Como passo preliminar para redução dos micro-organismos deteriorantes no leite cru refrigerado, aumento da vida útil e do potencial tecnológico do leite pasteurizado, o presente trabalho teve como objetivo identificar a diversidade genética de micro-organismos psicrotróficos e termodúricos com potencial deteriorante proteolítico e/ou lipolítico no leite cru refrigerado de alta qualidade microbiológica. Foram avaliadas 20 amostras de leite cru refrigerado produzido na bacia leiteira da região de Castro, Paraná, nas quais foram utilizados métodos oficiais para a contagem e isolamento de psicrotróficos e termodúricos e técnicas biomoleculares de agrupamento genético hierárquico (Restriction Fragment Length Polymorphism) e caracterização (sequenciamento parcial do gene 16S rRNA e análise filogenética) dos isolados. As contagens médias de psicrotróficos e termodúricos foram 1,1 x 104 e 3,2 x 102 UFC/mL, respectivamente. Entre os psicrotróficos, 47,8% do total de isolados (n = 295) apresentaram atividade deteriorante, dos quais 30,5% foram proteolíticos e lipolíticos simultaneamente, 31,9% exclusivamente proteolíticos e 37,6% lipolíticos. Pelos dendrogramas foram determinados 36 clusters de psicrotróficos proteolíticos e 47 de lipolíticos com 70% de similaridade hierárquica, cuja identificação filogenética determinou o predomínio das espécies com os mesmos perfis polimórficos de Lactococcus lactis (24,5%), Enterobacter kobei (15,4%), Aerococcus urinaeequi (8,4%) e Acinetobacter lwoffii (6,3%) entre os psicrotróficos deteriorantes. Serratia ureilytica, Enterobacter kobei, Pseudomonas spp. e Yersinia enterocolitica foram os psicrotróficos identificados como potencialmente produtores de metaloprotease alcalina (aprX). Entre os termodúricos, 42,6% do total de isolados (n = 310) foram deteriorantes, dos quais 32,6% apresentaram atividade proteolítica e lipolítica simultaneamente, 31% exclusivamente proteolítica e 36,4% apenas lipolítica. Esses isolados proteolíticos e lipolíticos foram agrupados em 41 e 38 clusters, respectivamente, considerando 50% de similaridade. A análise filogenética das sequências parciais do gene 16S rRNA revelou que predominam entre os termodúricos deteriorantes as espécies Bacillus licheniformis (34,1%), Brachybacterium nesterenkovii (14,4%), Enterococcus faecalis (9,1%) e Streptococcus agalactiae (8,3%). Foi observado que 50% das cepas de termodúricos pertencem aos gêneros Bacillus e Paenibacillus, frequentemente descritos como esporulados que resistem a tratamentos térmicos mais intensos que a pasteurização e que comprometem a vida útil do leite pasteurizado dos Estados Unidos. Os resultados do presente trabalho demonstram que a composição da microbiota é tão importante quanto a sua quantificação. Apesar de baixas contagens, psicrotróficos e termodúricos podem comprometer a qualidade do leite e derivados pela sua atividade deteriorante, sendo um fator limitante na manutenção da qualidade do leite fluido e derivados. Portanto, é necessário desenvolver ferramentas sensíveis e eficientes para monitorar e controlar a presença desses micro-organismos no leite cru. The presence of heat-stable spoilage enzymes can compromise the milk and dairy products quality and shelf life, even if raw milk with low total microorganisms counts. In addition, the vegetative or sporulated microbial forms may resist pasteurization and also deteriorate pasteurized milk. The present work aimed to identify the genetic diversity of psychrotrophic and thermoduric microorganisms with proteolytic and/or lipolytic potential in high microbiological quality refrigerated raw milk. Twenty samples of refrigerated raw milk produced in the dairy region of Castro, Paraná State, Brazil, were evaluated per official methods for the counting and isolation of psychrotrophic and thermoduric. Biomolecular techniques of hierarchical genetic grouping (Restriction Fragment Length Polymorphism) and identification by partial sequencing of the 16S rRNA gene and phylogenetic analysis. The mean of psychrotrophic and thermoduric counts were 1.1 x 104 and 3.2 x 102 CFU/mL, respectively. Among the psychrotrophs, 47.8% of the total isolates (n = 295) presented spoilage potential, of which 30.5% were proteolytic and lipolytic simultaneously, 31.9% exclusively proteolytic and 37.6% lipolytic. The dendrograms determined 36 clusters of proteolytic and 47 lipolytic psychrotrophs with 70% genetic hierarchical similarity, whose phylogenetic identification shows the predominance of the species with the same polymorphic profiles of Lactococcus lactis (24.5%), Enterobacter kobei (15.4%), Aerococcus urinaeequi (8.4%) and Acinetobacter lwoffii (6.3%) among the deteriorating psychrotrophs. Serratia ureilytica, Enterobacter kobei, Pseudomonas spp. and Yersinia enterocolitica were the psychrotrophs identified as potentially producing of alkaline metalloprotease (aprX gene positive). Among the thermoduric, 42.6% of the total isolates (n = 310) shows spoilage potential, of which 32.6% presented proteolytic and lipolytic activity simultaneously, 31% were exclusively proteolytic and 36.4% only lipolytic. These proteolytic and lipolytic isolates were grouped in 41 and 38 clusters, respectively, considering 50% of genetic similarity. Phylogenetic analysis of partial sequences of 16S rRNA gene revealed that Bacillus licheniformis (34.1%), Brachybacterium nesterenkovii (14.4%), Enterococcus faecalis (9.1%) and Streptococcus agalactiae (8.3%). It was observed that 50% of the thermoduric strains belong to the genus Bacillus and Paenibacillus, often described as sporulated that resist heat treatments that are more intense at pasteurization and that compromise the shelf life of the pasteurized milk in the United States. The results of the present study demonstrate that the microbiota composition of raw milk as so important that your quantification. Although low counts, psychrotrophic and thermoduric can compromise the milk quality and dairy produtcs by their deteriorating activity, being a limiting factor for the maintenance of the quality. Therefore, it is necessary to development of a sensitive and efficient tools to monitor and control the presence of these microorganisms in raw milk.

Papilomavírus (PV) são vírus oncogênicos que acometem diversas espécies de mamíferos, incluindo o homem. São capazes de induzir tumores benignos (papilomas ou verrugas) e malignos (condilomas) em epitélio cutâneo e mucoso. O genoma viral é constituído por até 10 fases abertas de leitura (ORFs), sendo que a ORF L1, sequência de nucleotídeos mais conservada entre os PVs, é utilizada para a identificação de novos tipos virais. Este estudo teve como objetivo identificar os tipos de papilomavírus bovino (BPV) presentes em cinco amostras de papilomas provenientes de um rebanho bovino leiteiro da região do norte pioneiro do estado do Paraná e determinar a sua classificação taxonômica por meio de comparação dessas sequências com as sequências de nucleotídeos de outros PVs previamente identificados e depositados em base pública de dados (GenBank). O par de primers degenerados FAP54/59 foi utilizado em reação de PCR para a triagem das amostras de papilomas. Os fragmentos de 480 pb obtidos das cinco amostras analisadas, permitiram a identificação de cinco diferentes tipos de BPV no mesmo rebanho, sendo estes BPV10, BPV6 e dois subtipos mais similares a BPV11 e BPV3. A quinta amostra, denominada BPV/BRUEL8, foi identificada no terceiro animal analisado, a partir de uma amostra de papiloma cutâneo. A análise da sequência gerada com o emprego do primeiro par de primers indicou que a cepa BPV/BR-UEL8 apresentava apenas 77% de identidade com o isolado 7Z (KT315748). Adicionalmente, foram empregados primers específicos do gene L1 de BPV6 para a obtenção da sequência completa do gene L1 do provável novo tipo. O produto da PCR foi submetido à clonagem e sequenciamento. A análise filogenética envolvendo a sequência completa da ORF L1 com 1.521 pb possibilitou identificar que a cepa BPV/BR-UEL8 pertence ao gênero Xipapillomavirus com maior identidade (apenas 75,1% identidade de nt) com BPV15. Este resultado possibilitou caracterizar a cepa BPV/BR-UEL8 como um provável novo tipo viral. Nos últimos anos, estudos que avaliaram características moleculares de BPV revelaram grande diversidade de tipos virais envolvidos em infecções singulares e, principalmente, em infecções mistas. A identificação de novos tipos virais pode auxiliar na compreensão da patogenia da doença e na avaliação de novos tratamentos, assim como no desenvolvimento de vacinas eficazes contra a papilomatose bovina. Papillomaviruses (PV) are oncogenic viruses that affect various mammalian species, including man. They can induce benign tumors (papillomas or warts) and malignant tumors (condylomas) in cutaneous and mucosal epithelium. The viral genome consists of up to 10 open reading frames (ORFs), and the L1 ORF, the most conserved nucleotide sequence among the PVs, is used to identify new viral types. The aim of this study was to identify the bovine papillomavirus (BPV) types present in five samples of papillomas from a dairy cattle herd at pioneer northern region of the state of Paraná and to determine their taxonomic classification by comparing these sequences with the sequences of nucleotides from other PVs previously identified and deposited in a public database (GenBank). The degenerate primer pair FAP54/59 was used in PCR reaction for the screening of papillomavirus samples. The 480 bp fragments obtained from the five analyzed samples allowed the identification of five different types of BPV in the same herd, being these BPV10, BPV6 and two subtypes more similar to BPV11 and BPV3. The fifth sample, named BPV/BR-UEL8, was identified in the third animal analyzed from a cutaneous papilloma sample. Analysis of the sequence generated using the first pair of primers indicated that the BPV/BR-UEL8 strain showed only 77% identity with the 7Z isolate (KT315748). In addition, specific primers of BPV6 L1 gene were used to obtain the complete L1 gene sequence of the putative new type. The PCR product was subjected to cloning and sequencing. The phylogenetic analysis involving the complete sequence of the L1 ORF with 1.521 bp allowed to identify that the BPV / BR-UEL8 strain belongs to the genus Xipapillomavirus with identity (only 75.1% identity of nt) with BPV15. This result made it possible to characterize the BPV/BR-UEL8 strain as a putative new viral type. In recent years, studies that have evaluated molecular characteristics of BPV revealed a wide variety of viral types involved in unique infections and, especially, mixed infections. The identification of new viral types can help to understand the pathogenesis of the disease and the evaluation of new treatments, as well as in the development of effective vaccines against bovine papillomatosis.

O gênero Kobuvirus, membro da família Picornaviridae, é dividido em seis espécies Aichivirus A a F. O segmento 3D do genoma do Kobuvirus é uma região conservada, que codifica a enzima RNA polimerase dependente de RNA (RdRP), que é amplamente utilizada para o diagnóstico do vírus. A região genômica que codifica a proteína VP1 do capsídeo viral, região mais variável, é utilizada para estudos filogenéticos em cepas de campo de kobuvírus. Kobuvírus tem sido relacionado à diarreia, principalmente em animais jovens e a infecção por kobuvírus ainda é pouco descrita em animais no Brasil. O objetivo deste estudo foi avaliar características da infecção em duas espécies animais sendo um animal de produção e outro de estimação. O primeiro estudo descreve o envolvimento do kobuvírus (Aichivirus B) em um surto de diarreia em bezerros de um rebanho leiteiro de alta produção. Vinte e quatro amostras fecais diarreicas de bezerros com até 30 dias de idade foram coletadas para análise virológica. Cinco amostras fecais de bezerros assintomáticos foram incluídas nas análises como grupo controle. Utilizando ensaios de RT-PCR, o aichivírus B foi detectado em 54,2% (13/24) e rotavírus A G10P[11] em 25% (6/24) das amostras fecais diarreicas avaliadas. Os bezerros assintomáticos foram negativos para ambos os vírus. Aichivírus B foi identificado apenas em bezerros com até duas semanas de idade com 83,3% (10/12) das amostras positivas. A análise filogenética baseada no gene RdRP agrupou as cepas brasileiras em um novo ramo no cluster Aichivirus B. A análise das sequências de nucleotídeos do gene VP1 das cepas brasileiras e chinesas de Aichivirus B permitiu classificar as cepas brasileiras avaliadas na recém proposta, linhagem 1 (genotipo A). Este é o primeiro relato de alta taxa de detecção de aichivírus B em um surto de diarreia em bezerros leiteiros e também o primeiro estudo filogenético com base no gene VP1 realizado com cepas de campo de Aichivirus B identificadas na América do Sul. O segundo estudo descreve a detecção extra-intestinal de kobuvírus canino (CaKV) em um filhote de Chihuahua de 5 meses de idade com histórico clínico de fezes com sangue de morte natural. Fragmentos de tecido foram submetidos à técnica de histopatologia e imuno-histoquímica (IHC) para identificar antígenos do vírus da cinomose (CDV), parvovírus canino tipo 2 (CPV-2) e adenovírus canino tipos 1 e 2 (CAdV-1 e 2). As técnicas de PCR e RT-PCR foram utilizadas para amplificar genes específicos de CaKV, CDV, CPV-2, CAdV-1 e -2 e herpesvírus canino tipo 1 (CaHV-1). Fragmentos de intestino e amostras de fezes foram avaliados para identificar a presença de rotavírus. As principais alterações patológicas encontradas foram peneumonia intersticial, bronquite necrosante, miocardite, enterite e depleção linfóide. A RT-PCR detectou CaKV no cerebelo, cérebro, pulmão, tonsila e fígado. Neste estudo, também foram identificadas infecções mistas de CaKV com CDV e CPV-2 com CaHV-1. CaKV e rotavírus não foram identificados nas fezes e intestino. Os ensaios IHC detectaram antígenos de CDV e CAdV-1 em regiões distintas dos pulmões. A análise filogenética da cepa de CaKV identificada neste estudo revelou semelhança com cepas detectadas em outros países. Os resultados evidenciam a primeira detecção extra-intestinal de CaKV em tecidos em associação com infecções mistas com outros vírus comumente encontrados em cães e a primeira detecção extra-intestinal de CaKV em um cão. Os resultados obtidos nesse estudo adicionaram novos conhecimentos sobre aspectos clínicos e moleculares da infecção por kobuvírus em animais. The Kobuvirus genus is a member of the Picornaviridae family and subdivided into six species Aichivirus A to F. The 3D segment of the Kobuvirus genome is a conserved genomic region which encodes the enzyme RNA-dependent RNA polymerase (RdRP) and widely used for the virus diagnosis. The region encoding the protein VP1 of the viral capsid, region more variable, is used to perform phylogenetic studies in kobuvirus field strains. Kobuvirus has been related with diarrhea mainly in young animals and the kobuvirus infection is still little described in animals in Brazil. This study aimed to evaluate characteristics of the infection in two animal species being one of production and another pet. The first study describes the involvement of kobuvirus (Aichivirus B) in a diarrhea outbreak in calves of a high-producing dairy cattle herd. Twenty-four diarrheic fecal samples from calves with up to 30 days old were collected for virological analysis. As control group were collected five fecal samples from asymptomatic calves. Using RT-PCR assays aichivirus B was detected in 54.2% (13/24) and rotavirus A G10P[11] genotypes in 25% (6/24) of diarrheic fecal samples evaluated. Asymptomatic calves were negative for both viruses. Aichivirus B was only identified in calves with up to two weeks old with 83.3% (10/12) of the positive samples. Phylogenetic analysis based on the RdRP gene the Brazilian strains grouped in a new branch within the Aichivirus B cluster. The analysis of the nucleotide sequences of the VP1 gene of the Brazilian and Chinese Aichivirus B strains allowed classified the Brazilian field strains in the putative lineage 1 (genotype A). This is the first report of the high detection rate of aichivirus B in a diarrhea outbreak in dairy calves and also the first phylogenetic study based in VP1 gene of Aichivirus B wild-type strains in South America. The second study describes the extra-intestinal detection of kobuvirus canino (CaKV) in a Chihuahua 5-month-old puppy, with clinical history of bloody-tinged feces of natural death. Tissue fragments were submitted to technique histopathology, and immunohistochemical (IHC) assays to identify the antigens of canine distemper virus (CDV), canine parvovirus-2 (CPV-2), and canine adenovirus types 1 and 2 (CAdV-1and -2). PCR and RT-PCR assays targeted specific genes of CaKV, CDV, CPV-2, CAdV-1 and -2, and canine herpesvirus-1 (CaHV-1). Intestinal fragments and fecal samples were evaluated to identify the presence of rotavirus. The main pathological findings were interstitial pneumonia, necrotizing bronchitis, and myocarditis, enteritis, and lymphoid depletion. The RT-PCR detected CaKV within the cerebellum, cerebrum, lung, tonsil, and liver. Mixed infections of CaKV with CDV, CPV-2, and CaHV-1 were identified by molecular assays. CaKV and RV were not identified within the feces and intestine. IHC assays detected antigens of CDV and CAdV-1 within distinct regions of the lungs. Phylogenetic analysis of the CaKV strains identified in this study was similar to those strains from other geographical regions. The results confirmed the first extra-intestinal detection of CaKV in tissues in association with mixed infections with others viruses commonly found in dogs and the first extra-intestinal detection of CaKV in a dog. The results obtained in this study added new knowledge of clinical and molecular aspects of kobuvirus infection in animals.

Os picornavírus entéricos suínos (PEVs), Teschovirus A (TV-A), Sapelovirus A (SV-A) e Enterovirus G (EV-G) são agentes infecciosos específicos dos suínos que se encontram disseminados pelos rebanhos suinícolas em todo o mundo. Na maioria dos casos se apresentam de forma assintomática, porém têm sido associados a uma variedade de enfermidades, dentre elas doenças entéricas, reprodutivas, respiratórias e neurológicas. Este estudo visou avaliar a infecção natural pelos PEVs em rebanhos de três importantes regiões geográficas produtoras de suínos do Brasil. Para isso, foram realizados dois estudos, utilizando a reação em cadeia da polimerase precedida por transcrição reversa (RT-PCR) e nested-PCR que tiveram como alvo a região conservada 5´ não traduzida (5’NTR) do genoma destes vírus. O primeiro estudo teve como objetivo avaliar a frequência de infecção natural por TV-A, SV-A e EV-G em criações de suínos localizadas nas regiões Sul, Sudeste e Centro-Oeste do país. Foram selecionadas aleatoriamente 40 amostras de fezes com consistência normal (n=17) e diarreica (n=23) de leitões lactentes (n=22) e desmamados (n=18). Foi possível demonstrar que tanto a infecção pelo TV-A quanto pelo EV-G foi comum entre os leitões lactentes (1 a 3 semanas de idade), com frequência de 40,9% (9/22) em infecções singulares e mistas. O SV-A não foi detectado nesta categoria animal. Nos leitões desmamados (4 a 8 semanas de idade) foi observada maior frequência destes agentes (77,8%, 14/18) nas amostras fecais, sendo mais comum a observação de infecções mistas pelo TV-A e EV-G (4/14). Somente foram detectadas infecções singulares por TV-A e EV-G. O SV-A foi identificado em infecção mista com os outros agentes em 38,8% (7/18) das amostras avaliadas. Para confirmar a especificidade dos fragmentos amplificados neste estudo, duas amostras positivas para cada vírus na nested-PCR foram selecionadas para sequenciamento. As cepas de TV-A, SV-A e EV-G detectadas no Brasil revelaram alta identidade nas sequências de nucleotídeos com as disponíveis em bases públicas de dados. Este estudo concluiu que as regiões geográficas brasileiras pesquisadas apresentam ao menos dois dos vírus estudados circulando entre leitões lactentes e desmamados. O segundo estudo teve como objetivo avaliar a distribuição da infecção pelos PEVs entre javalis do estado do Paraná e avaliar catetos e queixadas como portadores para estes três vírus. Foram analisadas 36 amostras de fezes de animais assintomáticos. Vinte e dois javalis com idade entre 2 e 7 meses (n=14) e entre 2 e 4 anos (n=8) e 14 catetos e queixadas com idade de 6 a 8 meses foram avaliados. Os PEVs foram detectados em 54,5% (12/22) das amostras de javalis. O vírus mais frequente foi EV-G (50%, 11/22), seguido por TV-A (45,5%, 10/22) e SV-A (18,2%, 4/22). De acordo com a idade, animais jovens foram mais frequentemente infectados que os adultos. Um produto amplificado na nested-PCR de cada um dos vírus foi submetido ao sequenciamento. A sequência de nucleotídeos das cepas deste estudo mostrou alta identidade com as sequências de PEVs disponíveis em bases públicas de dados, incluindo as relatadas no Brasil no primeiro estudo. Catetos e queixadas foram negativos para os três vírus. Neste estudo, foi possível comprovar pela primeira vez a circulação de TV-A, SV-A e EV-G em javalis do Brasil e demonstrar que catetos e queixadas não foram positivos para estes patógenos exclusivos dos suínos. Desta forma, foi possível demonstrar que a infecção por TV-A, SV-A e EV-G está presente nas diferentes regiões geográficas produtoras de suínos do Brasil, tanto em fezes com consistência normal quanto em diarreicas, e também em javalis no Brasil. Porcine enteric picornaviruses (PEVs), Teschovirus A (TV-A), Sapelovirus A (SV-A) and Enterovirus G (EV-G) are specific infectious agents of swine spread worldwide. In most cases are asymptomatic, but also associated with a variety of diseases such as enteric, reproductive, respiratory, and neurological disorders. This study aimed to investigate the natural infection with PEVs in herds of three important pig-producing geographical regions of Brazil. Two studies were performed by using the reverse transcription-polymerase chain reaction (RT-PCR) assay and nested-PCR targeting the conserved 5’nontranslated region (5’NTR) of the genome of the viruses. The first study aimed to evaluate the frequency of natural infection by TV-A, SV-A, and EV-G in pig farms located in Southern, Southeast and Midwest regions of the country. Forty diarrheic (n=23) and normal (n=17) consistency fecal samples from suckling (n=22) and nursery (n=18) pigs were randomly selected. The results revealed that TV-A and EV-G infections were common between suckling pigs (1 to 3 weeks old), with frequency of 40.9% (9/22) in single or mixed infections. SV-A was not detected in this age group. For nursery pigs (4 to 8 weeks old), higher detection rate (77.8%, 14/18) of these agents were observed in fecal samples, with mixed infections with TV-A and EV-G more commonly observed (4/14). Only single infection with TV-A and EV-G were observed in these animals. SV-A was detected only in mixed infection with the other agents in 38.8% (7/18) of the samples analyzed. To confirm the specificity of the amplified products in this study, two nested-PCR positive samples from each virus were selected for sequencing analyses. Strains of TV-A, SV-A and EV-G detected in Brazil showed high nucleotides sequences identity with others available in public database. This study concluded that the Brazilian regions evaluated showed at least two of the tested viruses in suckling and nursery pigs. The second study aimed to investigate the PEVs infection distribution in wild boars of Paraná state and to evaluate peccaries as carrier’s species for these three viruses. Fecal samples from 36 asymptomatic wild boars were analyzed. Twenty-two wild boars with ages ranging from two to seven months old (n=14) and two to four years old (n=8) and 14 peccaries aged 6 to 8 months old were evaluated. PEVs were detected in 54.5% (12/22) wild boar samples. EV-G was the most frequently virus detected (50%, 11/22), followed by TV-A (45.5%, 10/22) and SV-A (18.2%, 4/22). Regarding the age groups, young wild boars were more frequently infected than adult animals. One nested-PCR amplified product for each of the viruses was submitted to sequencing analysis. The nucleotide sequences showed high identity with the PEVs sequences described available in public database, including those reported in the first study in Brazil. Peccaries were negative for the three viruses. In this study, it was possible to demonstrate for the first time TV-A, SV-A and EV-G circulating in wild boars in Brazil and demonstrate that peccaries were not positive to these pathogens that are exclusive of swine. In conclusion, it was possible to demonstrate that infection with TV-A, SV-A and EV-G is present in the different pig-producing geographical regions of Brazil, in normal and diarrheic consistency fecal samples and also in wild boars in Brazil.