Accuracy of PCR targeting different markers for Staphylococcus aureus identification: a comparative study using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as the gold standard.

Staphylococcus aureus is considered a major pathogen in veterinary and human medicine, and the emergence of multidrug-resistant strains, such as livestock-associated methicillin-resistant S. aureus, means that reliable, inexpensive, and fast methods are required to identify S. aureus obtained from animal sources. We tested the accuracy of a PCR targeting the genes femA, nuc, and coa in identifying S. aureus from animals. A total of 157 Staphylococcus spp. isolates were examined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; 18 different Staphylococcus species were identified. Of 68 S. aureus isolates, the genes femA, nuc, and coa were found in 61, 53, and 32 isolates, respectively. Considering MALDI-TOF as the gold standard, the PCR assays targeting all 3 genes showed 100% specificity; the sensitivity values were 89.7, 77.9, and 47.0% for femA, nuc, and coa, respectively. Sensitivity was 100% when femA and nuc markers were targeted simultaneously. These results confirm PCR as an accurate method to identify S. aureus species from animal sources and strongly suggest the simultaneous use of primers targeting femA and nuc genes.

Serological and molecular detection of Theileria equi in sport horses of northeastern Brazil.

Theileriosis is a worldwide protozoal tick-borne disease caused by Theileria equi, which may produce a variety of clinical signs and turn infected horses into lifetime carriers. This study has aimed to perform a serological and molecular detection of T. equi and associated factors in sports horses from six areas of northeastern Brazil. In overall, 59.6% horses were positive by indirect immunofluorescence assay and 50.4% by polymerase chain reaction. No significant association was found when presence of ticks, age, gender, anemia or total plasma proteins was analyzed with seropositivity and molecular techniques. Although a significant association of infection was found in two cities. Thus, local risk factors other than presence of ticks, horse age, gender, anemia and total plasmatic proteins may dictate prevalence of T. equi infection in sports horses, even in highly endemic areas with no control of infection prior to horse competitions.

Diarrhea outbreaks in suckling piglets due to rotavirus group C single and mixed (rotavirus groups A and B) infections / Surtos de diarreia em leitões lactentes por rotavírus grupo C em infecções singulares e mistas (rotavirus grupos A e B)

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 (<21-day-old) piglets, with 70 percent to 80 percent and 20 percent to 25 percent of morbidity and lethality rates, respectively, in three pig herds located in the state of Santa Catarina, Brazil. The diagnosis of PoRV in the diarrheic fecal samples was performed using polyacrylamide gel electrophoresis (PAGE) to identify the presence of porcine rotavirus groups A, B (PoRVB), and C, and by RT-PCR (PoRVA and PoRVC) and semi-nested (SN)-PCR (PoRVB) to partially amplify the VP4 (VP8*)-VP7, NSP2, and VP6 genes of PoRVA, PoRVB, and PoRVC, respectively. […] The PoRVB strains (first and second outbreaks) and the PoRVC strains (first, second, and third outbreaks) showed higher nt identity and clustered in the phylogenetic tree with PoRVB and PoRVC strains that belong to the N4 and I1 genotypes, respectively. This is the first description in Brazil of the involvement of PoRVC in the etiology of diarrhea outbreaks in suckling piglets. The results of this study demonstrated that PoRVC, in both single and mixed infections, is an important enteropathogen involved in neonatal diarrhea outbreaks in piglets and that the use of more sensitive diagnostic techniques allows the identification of mixed infections involving two or even three groups of PoRV, which may be more common than previously reported.

O rotavírus suíno grupo A (PoRVA) é uma das principais causas de diarreia neonatal em leitões lactentes e recém-desmamados em todo o mundo. As descrições do envolvimento de rotavírus não-grupo A em quadros de diarreia neonatal em leitões são esporádicas. No Brasil não há relatos do envolvimento do rotavírus suíno grupo C (PoRVC) na etiologia dos surtos de diarreia em leitões. O objetivo deste estudo foi descrever a identificação de rotavírus grupo C em infecções singulares e mistas com os rotavírus grupos A e B em três surtos de diarreia neonatal em leitões lactentes (<21 dias de idade), com taxas de morbidade de 70 por cento a 80 por cento e de letalidade de 20 por cento a 25 por cento, em três rebanhos suínos localizados no estado de Santa Catarina, Brasil. O diagnóstico de PoRV nas amostras de fezes diarreicas foi realizado por eletroforese em gel de poliacrilamida (PAGE) para identificar a presença dos grupos A, B (PoRVB), e C de rotavírus suíno e por RT-PCR (PoRVA e PoRVC) e semi-nested (SN)-PCR (PoRVB) com a amplificação parcial dos genes VP4 (VP8*)-VP7, NSP2 e VP6 de PoRVA, PoRVB e PoRVC, respectivamente. [...] As cepas de PoRVB (primeiro e segundo surtos) e as cepas de PoRVC (primeiro, segundo e terceiro surtos) mostraram maior identidade de nt com cepas de PoRVB e PoRVC que pertencem aos genotipos N4 e I1, respectivamente. Esta é a primeira descrição realizada no Brasil do envolvimento de PoRVC na etiologia de surtos de diarreia em leitões lactentes. Os resultados deste estudo demonstram que o PoRVC, tanto em infecções singulares quanto em infecções mistas, é um importante enteropatógeno envolvido em surtos de diarreia neonatal em leitões e que o uso de técnicas de diagnóstico mais sensíveis permite caracterizar que infecções mistas, com dois ou até mesmo com três grupos de PoRV, podem ser mais comuns do que anteriormente relatado.

Frequency of BCoV detection by a semi-nested PCR assay in faeces of calves from Brazilian cattle herds.

Bovine coronavirus (BCoV) is one of the main causes of neonatal calf diarrhoea. Several diagnostic assays have been employed to detect the presence of the virus in stool samples from calves. Despite this, the frequency of BCoV infection among Brazilian and even South American cattle herds has yet to be well characterised. This study describes the occurrence of BCoV infection among calves from dairy and beef herds in four Brazilian states. A total of 282 stool samples from 1 to 60-day-old calves were evaluated for the presence of BCoV by a semi-nested (SN) PCR assay. The animals were from herds (n = 23) located in three geographical regions in Brazil (south, southeast, and center-west). The specific BCoV amplicon was detected in 15.6% (44/282) of the faecal specimens examined, of which 95.4% (42/44) were from diarrhoeic and 4.6% (2/44) from asymptomatic calves. The specificity of the SN-PCR amplicons was evaluated by restriction fragment length polymorphism (RFLP) analysis. The results show that the BCoV is widespread, mainly among calves from 16 to 30-days-old (p = 0.0023), and verify the association between BCoV infection and clinical signs of diarrhoea (p = 0.005). These findings emphasise the importance of this virus in enteric infections of Brazilian cattle herds.

Diarréia em bezerros da raça Nelore criados extensivamente: estudo clínico e etiológico / Diarrhea in Nelore calves: Clinical and etiologic study

Diarrhea is considered as one of the main causes of morbidity and mortality in neonates calves. Fecal samples from 100 diarrheic and 30 non-diarrheic (control group) Nelore calves less than 9 weeks old were collected for Salmonella spp., Escherichia coli, rotavirus, coronavirus, Cryptosporidium spp., and for helminth eggs investigation. Enteropathogens were detected in 79.0 percent diarrheic samples and 70.0 percent non-diarrheic samples. Among diarrheic calves, Escherichia coli (69.0 percent) was the most common agent found, following by Cryptosporidium spp. (30.0 percent), coronavirus (16.0 percent), and rotavirus (11.0 percent). In the control group, E. coli, Cryptosporidium spp. and coronavirus were detected in 66.7 percent, 10.0 percent and 3.3 percent of the samples, respectively. Salmonella spp. and strongylids were not found in any of the calves from either group. The K99 fimbrial only was detected in E. coli strains from diarrheic calves (5.8 percent). Enrofloxacin, norfloxacin, and gentamicin were the most effective among the antimicrobials tested. The weight of 210-day-old calves did not show statistic differences between diarrheic and non-diarrheic calves.

A diarréia é considerada uma das principais causas de morbidade e mortalidade de bezerros neonatos. Foram colhidas 100 amostras fecais diarréicas e 30 amostras não diarréicas (grupo controle), de bezerros Nelore com até nove semanas de idade com o objetivo de detectar os enteropatógenos Salmonella spp., Escherichia coli, rotavírus, coronavírus, Cryptosporidium spp. e ovos de helmintos. Enteropatógenos foram detectados em 79,0 por cento das amostras diarréicas e em 70,0 por cento das amostras não-diarréicas. No grupo de bezerros com diarréia, E. coli (69,0 por cento) foi o agente mais freqüentemente isolado, seguido de Cryptosporidium spp. (30,0 por cento), coronavírus (16,0 por cento) e rotavírus (11,0 por cento). No grupo controle, E. coli, Cryptosporidium spp. e coronavírus foram detectados, respectivamente, em 66,7 por cento, 10,0 por cento e 3,3 por cento das amostras. Salmonella spp. e ovos de estrongilídeos não foram encontrados nos dois grupos avaliados. A fímbria K99 foi identificada exclusivamente nas linhagens de E. coli isoladas de bezerros com diarréia (5,8 por cento). Entre os antimicrobianos avaliados "in vitro" a enrofloxacina, a norfloxacina e a gentamicina foram os mais efetivos. O peso dos bezerros aos 210 dias de idade não apresentou diferença significativa entre os animais com e sem diarréia.

Improved detection of bovine coronavirus N gene in faeces of calves infected naturally by a semi-nested PCR assay and an internal control.

Bovine coronavirus (BCoV), a positive sense single-stranded RNA virus, is an important causative agent of neonatal diarrhoea in calves from beef and dairy cattle worldwide. The routine detection and diagnosis of BCoV have been mainly dependent on assays with low sensitivity. The aim of the present study was to develop and evaluate a semi-nested PCR (SN-PCR) to amplify a 251bp fragment of BCoV N gene from fresh (n=25) and frozen (n=25) diarrhoeic faecal samples of naturally infected calves. To improve detection of BCoV in faecal samples by the SN-PCR an internal control was developed, and the results were compared with a conventional RT-PCR assay. The rates of positive samples by SN-PCR and RT-PCR were 24% (12/50) and 8% (4/50), respectively (K=0.43). Only fresh samples were positive in RT-PCR while the SN-PCR detected BCoV in both fresh and frozen faecal samples. The sensitivity of SN-PCR was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) that was detected until 10(-7) dilution. The specificity of the amplicons was assessed by restriction fragment length polymorphism and sequence analysis. The inclusion of an internal control provides a way to detect assay inhibition in faecal samples and failure of nucleic acid extraction that allow reduction of the number of false-negative results.