First report of infectious spleen and kidney necrosis virus (ISKNV) in two native cichlids cultured in Brazil

Peacock bass (syn.: tucunaré, Cichla ocellaris) and the pearl cichlids (syn.: acará, Geophagus brasiliensis) are South American cichlids that are highly valued in both the ornamental and sport fish industries. Since 2017, a number of outbreaks of infectious spleen and kidney necrosis virus (ISKNV) have been reported on Brazilian food and ornamental fish farms. In this study, we detected ISKNV in farmed peacock bass and pearl cichlid by PCR and sequence analysis of the partial major capsid protein (MCP) gene. Moribund peacock bass (n=10) and pearl cichlids (2) from a farm experiencing elevated mortality among juveniles and adults of these species, were submitted for bacteriological and molecular diagnostics. Spleen, liver, brain, and kidney tissues were cultured on 5% sheep blood agar and cystine heart agar with 1% glucose and bovine haemoglobin. No bacteria were isolated from the 12 fish. Additionally, DNA extracts from the liver and spleen of all animals were tested for ISKNV using two conventional polymerase chain reaction (cPCR) assays and two nested PCR (nPCR) assays. ISKNV DNA was amplified in all 12 fish DNA extracts tested, in two or more of the PCR assays. Selected ISKNV amplicons were confirmed by Sanger sequencing. The nucleotide sequences derived from these animals were identical to ISKNV strains previously detected in food (e.g., tilapia and carp) and ornamental species, including strains previously detected in fish from Brazil. To the authors’ knowledge, this is the first report of ISKNV in these native Brazilian cichlids

Comparison between bacterial isolation and PCR for detection of Streptococcus suis type 2 in swine tonsils at slaughter in Santa Catarina

Streptococcus suis is a important pathogen associated to many production and economical losses in the swine industry, in addition to its zoonotic importance. The aims of this study was evaluate the occurrence of Streptococcus suis type 2 in tonsil samples of healthy pigs at slaughter, comparing the bacterial isolation with PCR assay and to determine the presence of the EF factor (extracellular protein factor). Tonsil samples from 302 slaughtered pigs were collected from three abattoir. Isolation of S. suis was made in Columbia Blood Agar Base, and then bacterial phenotypic characterization was made using biochemical assays. The typification genus and species of the isolates was performed with the PCR technique for confirmation, and the presence of the capsular gene (type 2). In the comparison between the techniques, PCR was made directly from the tonsil samples for the 16SrRNA gene (S. suis), capsular genes (type 2) and also the ef gene. The pathogen was detected in 100% of the samples with the PCR, whereas only 84.1% were positive on the isolation technique. Upon confirmation of the S. suis isolates, four of them were negative to the 16S rRNA gene. PCR demonstrated higher sensibility on detecting the type 2 S. Suis, having 89.7% of positive samples against 88% from the isolates. The ef gene was detected in 28.4% of the samples, three of them being detected as the ef variant. Significant difference was found between the PCR and bacterial isolation for S. suis, detection demons that the molecular assay has a higher detection capability than isolationStreptococcus suis é um patógeno responsável por muitas perdas produtivas e econômicas à suinocultura, além da importância zoonótica. O objetivo deste trabalho foi avaliar a ocorrência de Streptococcus suis tipo 2 a partir de tonsilas de suínos sadios no abate, comparando a técnica de isolamento bacteriano com a PCR e determinar a presença do fator protéico extracelular (EF). Foram coletadas tonsilas de 302 suínos provenientes de três frigoríficos, o isolamento foi realizado em Agar Sangue Columbia, após foi realizada a caracterização fenotípica. Esses isolados foram tipificados através da PCR para confirmação do gênero e da espécie, além da presença do gene capsular (tipo 2). Na comparação das técnicas foi realizado PCR diretamente das tonsilas para o gene 16S rRNA (S. suis), capsular (tipo 2) e também foi realizada pesquisa do gene ef (EF). Utilizando PCR diretamente das tonsilas, S. suis foi detectado em 100% das amostras, diferindo do isolamento em que 84,1% das amostras foram positivas. Na confirmação dos isolados sugestivos de S. suis, quatro deles foram negativos na PCR para o gene 16S rRNA. Na comparação para o tipo 2 a PCR demonstrou maior sensibilidade, detectando em 89,7% das tonsilas superando o isolamento, que detectou em 88% dos isolados. O gene ef foi detectado em 28,4% das amostras, sendo que em três amostras foi detectado o ef variante. Houve diferença significativa entre a PCR e o isolamento bacteriano para S. suis, demonstrando que a técnica molecular tem uma maior capacidade de detecção do que o isolament

The Role of <i>Mycoplasma bovirhinis</i> in the Development of Singular and Concomitant Respiratory Infections in Dairy Calves from Southern Brazil

The role of Mycoplasma bovirhinis in the development of pulmonary disease in cattle is controversial and was never evaluated in cattle from Latin America. This study investigated the respiratory infection dynamics associated with M. bovirhinis in suckling calves from 15 dairy cattle herds in Southern Brazil. Nasal swabs were obtained from asymptomatic (n = 102) and calves with clinical manifestations (n = 103) of bovine respiratory disease (BRD) and used in molecular assays to identify the specific genes of viral and bacterial disease pathogens of BRD. Only M. bovirhinis, bovine coronavirus (BCoV), ovine gammaherpesvirus 2 (OvGHV2), Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica were detected. M. bovirhinis was the most frequently diagnosed pathogen in diseased (57.8%; 59/102) and asymptomatic (55.3%; 57/103) calves at all farms. BCoV-related infections were diagnosed in diseased (52%; 53/102) and asymptomatic (51.4%; 53/103) calves and occurred in 93.3% (14/15) of all farms. Similarly, infectious due to OvGHV2 occurred in diseased (37.2%; 38/102) and asymptomatic (27.2%; /28/103) calves and were diagnosed in 80% (12/15) of all farms investigated. Significant statistical differences were not identified when the two groups of calves were compared at most farms, except for infections due to OvGHV2 that affected five calves at one farm. These results demonstrated that the respiratory infection dynamics of M. bovirhinis identified in Southern Brazil are similar to those observed worldwide, suggesting that there is not enough sufficient collected data to consider M. bovirhinis as a pathogen of respiratory infections in cattle. Additionally, the possible roles of BCoV and OvGHV2 in the development of BRD are discussed

Phenotypic and molecular characterization of isolates of Staphylococcus spp. obtained from sheep milk Chapecó-SC<br>Caracterização fenotípica e molecular de isolados de Staphylococcus spp. obtidos de leite de ovelhas do Município de Chapecó-SC

This study aimed to determine the antimicrobial susceptibility profile and to assess the presence of mechanisms of antimicrobial resistance in Staphylococcus spp. (n=36) isolated from mastitis in sheep’s Chapecó-SC. The potential for biofilm production was determined by phenotypic tests of Congo Red Agar, DAPI and Gentian Violet and by PCR for the detection of icaD gene. To evaluate the antimicrobial resistance testing was performed disk diffusion and detection of resistance genes blaZ, mecA, ermA, ermB and ermC and msrA also performed by PCR.The pump test was conducted by efuxo crop growth Muller Hinton agar containing ethidium bromide. The results showed that 1 (2,78%), 36 (100%) and 10 (27,78%) isolates were considered to produce a biofilm on Congo Red Agar test, Gentian Violet and DAPI, respectively, while the gene icaD was observed in only 2 (5.55%) isolates. The lowest percentage of sensitivity was observed for ampicillin (58.33%) and penicillin (58.33%). All strains tested were negative for the mecA, ermA, ermB and ermC genes. However, the isolates were positive for other resistance genes, being the blaZ and the msrA, with percentages of positivity of 58.33% and 11.11% respectively. Only one sample was positive for efflux pump test.O presente trabalho teve como objetivos determinar o perfil de sensibilidade aos antimicrobianos e avaliar a presença de mecanismos de resistência antimicrobiana em Staphylococcus spp. (n=36) isolados de mastite em ovelhas do município de Chapecó-SC. O potencial para produção de biofilme foi determinado pelos testes fenotípicos de Agar Vermelho Congo, DAPI e Violeta de Genciana e por teste molecular pela técnica de PCR para a detecção do gene icaD. Para determinar o perfil de resistência aos antimicrobianos, foi realizado o teste de difusão em disco e detecção dos genes de resistência blaZ, mecA, erm (A, B e C) e msrA. O teste da bomba de efuxo foi realizado através do crescimento das culturas em Agar Muller Hinton contendo brometo de etídio. Os resultados mostraram que 1 (2,78%), 36 (100%) e 10 (27,78%) isolados foram considerados produtores de biofilme pelos testes de Agar Vermelho Congo, DAPI e Violeta de Genciana, respectivamente, enquanto que o gene icaD foi observado em apenas 2 (5,55%) isolados. O menor percentual de sensibilidade foi observado para ampicilina (58,33%) e penicilina (58,33%). Os isolados avaliados foram positivos para os genes de resistência blaZ (58,33%) e msrA (11,11%). Nenhum isolado apresentou os genes de resistência erm (A, B, C) e apenas uma amostra foi positiva para o teste da bomba de efluxo

Clinical and Histopathological Evolution of Acute Intraperitoneal Infection by <i>Streptococcus agalactiae</i> Serotypes Ib and III in Nile Tilapia

Streptococcus agalactiae is a highly invasive bacterium that causes significant economic losses in tilapia aquaculture around the world. Furthermore, it is a pathogen for mammals, including humans, emphasizing its importance in One Health. The aim of this work was to evaluate the evolution of clinical and histopathological lesions caused by acute infection with two serotypes of S. agalactiae. For this, two strains isolated from natural outbreaks in Brazilian aquaculture farms (S13, serotype Ib; S73, serotype III) were used to challenge juvenile Nile tilapia (Oreochromis niloticus) intraperitoneally. Target organ samples were collected ten times, between 1 and 96 h post-infection, for microbiological and histopathological analyses. Anorexia was the first clinical sign and the first death occurred at 24 and 30 h in the fish infected with strains S13 and S73, respectively. Serotype Ib initially caused more pronounced lesions in the nervous system; however, serotype III lesions progressed more aggressively, reaching the same severity as those of serotype Ib. This trend was repeated in the mortality curve after 32 h. These results elucidated the important stages in the pathogenesis of S. agalactiae serotypes Ib and III in tilapia and suggest “tips and tricks” to improve the positive culture rate in the clinical diagnosis of infections in some tissues