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.

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% das amostras diarréicas e em 70,0% das amostras não-diarréicas. No grupo de bezerros com diarréia, E. coli (69,0%) foi o agente mais freqüentemente isolado, seguido de Cryptosporidium spp. (30,0%), coronavírus (16,0%) e rotavírus (11,0%). No grupo controle, E. coli, Cryptosporidium spp. e coronavírus foram detectados, respectivamente, em 66,7%, 10,0% e 3,3% 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%). 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.

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.

Neonatal diarrhea is an important cause of economic losses for cattle farmers. The main viral etiologies of enteric diseases are group A rotaviruses (GARV) and the bovine coronavirus (BCoV). Although both viruses infect calves of the same age, the occurrence of mixed infections is still under studied. The present study describes the co-infection of BCoV and GARV in stool samples. Forty-four diarrheic fecal samples from calves up to 60 days old that had previously tested positive for GARV by SS-PAGE were analyzed using semi-nested PCR for BCoV. A product with 251 bp of the BCoV nucleoprotein gene was amplified in 15.9% (7/44) of the samples, demonstrating that coinfection is not an unusual event.These results reinforce the need for testing for both GARV and BCoV, even in fecal samples that previously tested positive for one virus.

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% to 80% and 20% to 25% 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. One RT-PCR (PoRVA and PoRVC) and SN-PCR (PoRVB) product of each group of rotavirus of each diarrhea outbreak was submitted to nucleotide (nt) sequence analysis. Based on the PAGE technique, 4 (25%) and 1 (6.25%) of the 16 diarrheic fecal samples evaluated in the first outbreak presented PoRVA and PoRVC electropherotype, respectively, and 11 (68.75%) were negative. In the second outbreak, 3 (42.85%) of the 7 fecal samples evaluated presented PoRVA electropherotype, and in 3 (42.85%) and in 1 (14.3%) fecal samples were detected inconclusive and negative results, respectively. Three (30%) of the 10 fecal samples of the third outbreak presented PoRVC electropherotype; 5 (50%) and 2 (20%) samples showed negative and inconclusive results, respectively. Based on the RT-PCR and SN-PCR assays in the first neonatal diarrhea outbreak, PoRVC was detected in 13 (81.2%) of the 16 diarrheic fecal samples evaluated. PoRVC single infection was identified in 4 (25%) of these samples and mixed infections with PoRVA and PoRVB in 9 (56.2%) fecal samples. All of the seven diarrheic fecal samples evaluated from the second neonatal diarrhea outbreak were positive for PoRVC, whereas its mixed infection with other PoRV groups was detected in 4 (57.2%) samples. In the third outbreak, PoRVC in single infection was detected in all of the 10 diarrheic fecal samples analyzed. In the nt sequence analysis, the PoRVA strains of the first and second outbreaks demonstrated higher nt identity with G4P[6] and G9P [23] genotypes, respectively. The PoRVB strains (first and second outbreaks) and the PoRVC