Passive immunity to control Bovine coronavirus diarrhea in a dairy herd in Argentina / Inmunidad pasiva en el control de la diarrea por coronavirus bovino en un rodeo lechero de Argentina

Bovine coronavirus (BCoV) is a viral enteric pathogen associated with calf diarrhea worldwide being, in Argentina, mostly detected in dairy husbandry systems. The aim of the present work was to study if maternal IgG1 antibodies (Abs) to BCoV acquired by colostrum intake modulate the development of BCoV infection in calves reared in a dairy farm in Argentina. Thirty Holstein calves were monitored during their first 60 days of age. Animals were classified into two groups depending on their initial BCoV IgG1 Ab titers. The "failure of passive transfer" (FPT) group had significantly lower IgG1 Abs to BCoV than the "acceptable passive transfer" (APT) group of calves (log10 1.98 vs. 3.38 respectively) (p<0.0001). These differences were also observed when the total protein levels in both groups were compared (p = 0.0081). Moreover, 71% (5/7) of calves from the FPT group showed IgG1 seroconversion to BCoV compared to 29.4% (5/17) of animals from the APT group. Regarding viral circulation, BCoV was detected in 10% (3/30) of all calves and BCoV IgG1 Ab seroconversion was detected in 42% of the total animals showing that almost half of the calves were infected with BCoV. In conclusion, calves with high titers of specific BCoV IgG1 (≥1024) were mostly protected against viral infection, while animals with low titers of IgG1 (<1024) were mostly infected with BCoV. IgG1 Abs from colostrum origin are critical for prevention of BCoV infection.

El coronavirus bovino (Bovine coronavirus, BCoV) es un enteropatógeno viral asociado a la diarrea neonatal del ternero. El objetivo del presente trabajo fue estudiar si los anticuerpos IgG1 anti-BCoV adquiridos pasivamente mediante el calostro modulan la infección por BCoV en terneros de un rodeo lechero de Argentina. Se monitorearon 30 terneros raza Holstein durante los primeros 60 días de vida. Estos animales fueron clasificados en dos grupos según sus niveles de IgG1 anti-BCoV maternales: grupo con transferencia de inmunidad pasiva aceptable (APT) y grupo con fallas en la transferencia pasiva (FPT). Este último grupo tenía un título de IgG1 significativamente menor comparado con el primer grupo (log10 1,98 vs. 3,38, respectivamente; p< 0,0001). La misma diferencia se observó cuando se compararon los niveles de proteínas séricas totales (p = 0,0081). Además, el 71% (5/7) de los terneros del grupo FPT mostró seroconversión de IgG1, mientras que el 29,4% (5/17) de los terneros del grupo APT la mostró. Con respecto a la circulación viral, se detectó BCoV en el 10% (3/30) de los terneros así como también seroconversión de IgG1 en el 42% del total de los animales, lo que evidencia que aproximadamente la mitad de los terneros se infectaron con BCoV. Este estudio mostró que los terneros con altos títulos de IgG1 específica (≥ 1.024) estuvieron mayormente protegidos contra la infección con BCoV, mientras que los animales con títulos bajos de IgG1 (< 1.024) estuvieron predispuestos a la infección. Esto confirma que los anticuerpos IgG1 calostrales son críticos para la prevención de la infección por este agente viral.

Identification of pathogens and virulence profile of Rhodococcus equi and Escherichia coli strains obtained from sand of parks

The identification of pathogens of viral (Rotavirus, Coronavirus), parasitic (Toxocara spp.) and bacterial (Escherichia coli, Salmonella spp., Rhodococcus equi) origin shed in feces, and the virulence profile of R. equi and E. coli isolates were investigated in 200 samples of sand obtained from 40 parks, located in central region of state of Sao Paulo, Brazil, using different diagnostic methods. From 200 samples analyzed, 23 (11.5%) strains of R. equi were isolated. None of the R. equi isolates showed a virulent (vapA gene) or intermediately virulent (vapB gene) profiles. Sixty-three (31.5%) strains of E. coli were identified. The following genes encoding virulence factors were identified in E. coli: eae, bfp, saa, iucD, papGI, sfa and hly. Phylogenetic classification showed that 63 E. coli isolates belonged to groups B1 (52.4%), A (25.4%) and B2 (22.2%). No E. coli serotype O157:H7 was identified. Eggs of Toxocara sp. were found in three parks and genetic material of bovine Coronavirus was identified in one sample of one park. No Salmonella spp. and Rotavirus isolates were identified in the samples of sand. The presence of R. equi, Toxocara sp, bovine Coronavirus and virulent E. coli isolates in the environment of parks indicates that the sanitary conditions of the sand should be improved in order to reduce the risks of fecal transmission of pathogens of zoonotic potential to humans in these places.

Differentiation of Bovine coronavirus (BCoV) genotypes by a restriction enzyme assay

This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.

Achados clínico patológicos durante um surto de disenteria de inverno em bovinos no Rio Grande do Sul / Clinic pathological features during a winter dysentery outbreak in cattle in Rio Grande do Sul

Descreve-se um surto de disenteria de inverno que afetou 10 vacas leiteiras de uma propriedade localizada em Viamão, Rio Grande do Sul. O quadro clínico caracterizou-se por uma diarréia inicialmente líquida esverdeada com estrias de sangue e muco, evoluindo, em alguns animais, para uma diarréia de coloração marrom escura à sanguinolenta, que persistiu, em média, cinco dias. Drástica diminuição na produção de leite e no consumo de alimentos, além de graus variados de depressão também foram observados. Apenas um dos 10 animais afetados morreu. Durante a necropsia, observaram-se mucosas pálidas, conteúdo sanguinolento com presença de grande quantidade de coágulos, principalmente no cólon espiral e petéquias na mucosa do cólon. Os principais achados histológicos foram encontradas no cólon espiral, onde havia criptas dilatadas, sem epitélio de revestimento ou revestidas por epitélio pavimentoso e/ou cuboidal, por vezes com núcleos grandes e nucléolos proeminentes. Algumas criptas eram preenchidas por debris necróticos e polimorfonucleares. Na imuno-histoquímica com anticorpo monoclonal para coronavírus bovino (8F2) em cortes do cólon espiral, havia marcações positivas no citoplasma de enterócitos das criptas, nos debris necróticos destas e em macrófagos na lâmina própria.(AU)

The report describes a winter dysentery outbreak that affected 10 dairy cows from a ranch located in the county of Viamão, Rio Grande do Sul. The most significant clinical sign was profuse and watery diarrhea, which ranged from a greenish to a brownish coloration and from occasional blood streaks with mucus to a bloody diarrhea. Most cases persisted for 5 days and also included depression, drastic decrease in milk production and in food consumption. Only one of the affected animals died. The necropsy revealed pale mucosa and sanguineous content with high quantity of blood clots, particularly within the spiral colon, and petechiae on the colonic mucosa. Histopathological lesions were predominant in the spiral colon, and consisted of a high number of dilated crypts without epithelium or with the replacement of a pavement epithelium with occasional immature cuboidal cells, which sometimes showed enlarged nucleus and proeminent nucleolus. Some crypts were filled with epithelial desquamation and polymorphnuclear cells. Bovine coronavirus (8F2) monoclonal antibody was used for immunohistochemistry on sections of the spiral colon that showed positive reactions in the cytoplasm of the infected crypt epithelium, sloughed necrotic cells, and within macrophages in the lamina propria of both.(AU)

Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8 percent identity for nucleotides and 96.8 percent identity for amino acids) and more similar to the BCoV-ENT strain (98.7 percent for nucleotides and 98.7 percent for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

On the etiology of an outbreak of winter dysentery in dairy cows in Brazil

Winter dysentery (WD) is a seasonal infectious disease described worldwide that causes a marked decrease in milk production in dairy cows. In the Northern hemisphere, where the disease is classically recognized, bovine coronavirus (BCoV) has been assigned as a major etiologic agent of the disease. Nonetheless, in the Southern hemisphere, an in-deep etiological survey on WD cases had not been carried out. This study aimed to survey for BCoV by nested-RT-PCR, rotavirus by polyacrylamide gel electrophoresis (PAGE) and ELISA, bacteria by classical bacteriological methods and PCR for virulence factors and parasites by sugar flotation test on fecal samples of 21 cows from a farm during an outbreak of WD in São Paulo state, Southeastern Brazil. BCoV was detected in all 21 samples, while rotavirus was detected in two symptomatic cows. Escherichia coli, Yersinia intermedia, Providencia rustigianii Proteus penneri, Klebsiella terrigena and Enterobacter aglomerans were detected in samples from both asymptomatic and healthy cows in different associations. The study of E. coli virulence factors revealed that the strains isolated were all apathogenic. Cysts of Eimeria sp. and eggs of Strongyloidea were detected at low numbers in four of the symptomatic cows, with one co-infestation. These results suggest BCoV as the main etiologic agent of the cases of WD in Brazil, a conclusion that, with the clinical and epidemiological patterns of the disease studied herein, match those already described elsewhere. These findings give basis to the development of preventive measures and contribute to the understanding of the etiology of WD.

Em vacas leiteiras, a disenteria de inverno (DI) é uma doença infecciosa sazonal mundialmente relatada que ocasiona uma marcada queda na produção de leite; no hemisfério Norte, onde a doença é classicamente reconhecida, o coronavirus bovino (BCoV) tem um importante papel como agente etiológico. Entretanto, no hemisfério Sul, pesquisas etiológicas aprofundadas em casos de DI nunca forma realizadas. Este estudo objetivou a pesquisa de BCoV utilizando nested-RT-PCR, rotavírus utilizando eletroforese em gel de poliacrilamida (PAGE) e ELISA, bactérias com métodos bacteriológicos clássicos e PCR para fatores de virulência e parasitas pela técnica de flutuação em açúcar em 21 amostras fecais de vacas de uma fazenda durante um surto de DI no estado de São Paulo, Sudeste do Brasil. BCoV foi encontrado em todas as 21 amostras, enquanto que rotavírus foi encontrado em duas vacas sintomáticas. Escherichia coli, Yersinia intermedia, Providencia rustigiani, Proteus penneri, Klebsiella terrigena e Enterobacter aglomerans foram encontradas tanto em amostras de vacas sintomáticas quanto assintomáticas. O estudo de fatores de virulência para E. coli revelou que as amostras isoladas eram todas apatogênicas. Cistos de Eimeria sp. e ovos de Strongyloidea foram encontrados em baixos números em quatro animais sintomáticos, com uma co-infestação. Tais resultados sugerem o BCoV como o principal agente etiológico em casos de DI no Brasil, uma conclusão que, somada aos padrões clínicos e epidemiológicos da doença aqui estudada, concordam com aqueles descritos em outras regiões. Estes achados fornecem base o desenvolvimento de medidas preventivas e também contribuem para o entendimento sobre a etiologia da DI.