Influence of early use of antimicrobial on the health and performance of Holstein calves in the first month of life / Influência do uso precoce de antimicrobiano na sanidade de bezerras holandesas no primeiro mês de vida

The early use of antimicrobial therapy has been introduced in many farms to prevent diarrhea and respiratory disease in young calves; however, there is controversy about whether this practice has a beneficial effect on the health of these animals. This study evaluated the influence of the early use of antimicrobials on the health and performance of neonatal Holstein calves. Twenty-six Holstein calves were screened and divided into two groups, according to the administration (ATB+), or not (ATB-) of tulathromycin (2.5mg/kg, subcutaneously) within the first 12 hours of life. Calves were evaluated by general clinical examination, fecal score, respiratory score, and external palpation of the umbilical region, besides fecal output of dry matter. Anemia was determined by using an automatic system and, also, using a commercial kit for iron dosage. Diarrhea was diagnosed by a centrifuge-flotation technique using a sugar solution (Cryptosporidium) and multiplex semi-nested RT-PCR (rotavirus/coronavirus). The performance of the calves was estimated by Daily Weight Gain (DWG). The young dairy calves were evaluated within 12 hours of birth (≤12h) and at 3-5th (D3-5), 7-9th (D7-9), 13-15th (D13-15), 20-23rd (D20-23), and 27-30th (D27-30) days of life. No difference was noted between the ATB+ and ATB- groups concerning heart rate, respiratory frequency, and rectal temperature. Erythrogram showed a higher frequency of anemia in ATB- group (P=0.016) at the D3-5 check-up; lower values of serum iron were also observed simultaneously (P=0.051). Thirteen cases of respiratory disease were detected during this study; however, no significant difference was observed between the groups in this regard. The frequency of diarrhea (fecal score 2-3) was high in both groups, peaking at D13-D15. No differences were noted between the groups regarding the frequency of diarrhea when considering the dry fecal matter. The predominant etiological agent for diarrhea was Cryptosporidium spp.. The DWG was similar between groups, with maximum weight reduction on D13-15. The administration of tulathromycin in prophylactic dose (2.5mg/kg) at birth decreased the frequency of anemia but did not influence weight gain or the prevalence of diarrhea.(AU)

O uso precoce de antimicrobianos tem sido adotado em muitas fazendas para profilaxia das diarreias e doença respiratória em bezerras, no entanto existem controvérsias sobre os beneficios desta prática na saúde desses animais. Esta pesquisa avaliou a influência do uso precoce de antimicrobiano na sanidade e desempenho de bezerras holandesas recém-nascidas. Para tanto foram selecionadas 26 bezerras Holandesas distribuídas de acordo com a aplicação (ATB+) ou não (ATB-) de tulatromicina (2,5mg/Kg) por via subcutânea até 12h de vida. As bezerras foram examinadas por meio de exame clínico geral, escore fecal, escore respiratório e palpação externa da região umbilical, além da matéria seca fecal. A presença de anemias foi determinada pelo eritrograma utilizando sistema automático e além da dosagem de ferro utilizando kit comercial. O diagnóstico etiológico das diarreias foi investigado por meio da técnica de flutuação em solução saturada de sacarose (Cryptosporidium) e multiplex semi-nested RT-PCR (rotavírus/coronavírus). O desempenho das bezerras foi estimado pelo ganho de peso. As bezerras foram avaliadas até doze horas após o nascimento (≤12h); 3-5º (D3-5); 7-9º (D7-9); 13-15º (D13-15); 20-23º (D20-23); e 27-30º dias de vida (D27-30). Não foram encontradas diferenças entre os grupos ATB+ e ATB- em relação à frequência cardíaca, frequência respiratória e temperatura retal. O eritrograma revelou maior frequência de anemias no grupo ATB- (P=0,016) no D3-5. Neste momento também foram observados menores valores de ferro sérico (P=0,051). Foram detectados treze casos de doença respiratória durante o estudo, no entanto não foi possível detectar diferença entre os grupos. A frequência de diarreias (escore fecal 2 e 3) foi alta em ambos os grupos, observando-se pico no D13-15 (ATB+=92,3%; ATB-=92,3%). Não observamos diferenças entre os grupos em relação a frequência de diarreia considerando-se a matéria seca fecal. O agente etiológico predominante nas diarreias foi o Cryptosporidium. O ganho de peso diário foi igual entre grupos, com intensa redução no GPD no D13-15. A administração de tulatromicina na dose profilática (2,5mg/Kg) ao nascimento diminuiu a frequência de anemias e não influenciou no ganho de peso e prevalência de diarreias.(AU)

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.

Serum concentrations of acute phase proteins and immunoglobulins of calves with rotavirus diarrhea / Concentração sérica de proteínas de fase aguda e imunoglobulinas em bezerros acometidos por diarreia por rotavírus

The aim of the present study was to characterize changes in acute phase protein levels according to the occurrence of rotavirus diarrhea in calves in the first month of life. Blood and fecal samples were taken before colostrum intake and at 1, 2, 7, 15, 21 and 30 days of age from 24 Holstein calves allotted in three experimental groups: calves that did not present diarrhea (group A), calves that presented diarrhea, but tested negative for rotavirus in feces (group B), and calves that presented diarrhea and tested positive for rotavirus in feces (group C) (experiment 1). When the animals presented episodes of diarrhea, blood and fecal samples were taken at 24-hour intervals until the end of clinical signs (experiment 2). Serum proteins were separated by SDS-PAGE technique and rotavirus in feces was detected by PAGE. Data of experiment 1 were analyzed by ANOVA and Tukey's test, considered significant at P<0.05. Data of experiment 2 were subjected to the HSD test. Total protein, globulins, and IgG concentrations were lower in group C than in groups A and B. Ceruloplasmin and transferrin levels were higher in group C than in groups A and B. Serum concentrations of haptoglobin and α1-acid glycoprotein did not differ significantly between groups throughout the experimental period. Calves presented diarrhea between 10.4 and 14.6 days of age in group B, and between 10.3 and 14.6 days of age in group C. In the moments of diarrhea manifestation, least square means of IgA, haptoglobin and α1-acid glycoprotein concentrations did not differ significantly between groups B and C, but ceruloplasmin and transferrin concentrations were higher in group C than in group B, as opposed to what occurred with IgG levels. These findings show that optimizing passive immunity transfer of immunoglobulins decrease the likelihood of calves developing diarrhea caused by rotavirus. In addition, ceruloplasmin presents characteristics of a biomarker of rotavirus infection in calves.(AU)

O objetivo do presente estudo foi avaliar alterações nos teores de proteínas de fase aguda de acordo com a ocorrência de diarreia por rotavírus em bezerros no decorrer do primeiro mês de vida. Amostras de sangue e fezes de 24 bezerros da raça Holandesa foram coletadas antes da ingestão de colostro e com um, dois, sete, quinze, vinte um e trinta dias de idade, sendo os bezerros alocados em três grupos: bezerros que não apresentaram diarreia (grupo A), bezerros que apresentaram diarreia, mas foram negativos para a detecção de rotavírus nas fezes (grupo B) e bezerros que apresentaram diarreia e foram positivos para detecção de rotavírus nas fezes (grupo C) (experimento 1). Sempre que os animais apresentavam episódio de diarreia, amostras de sangue e fezes eram coletadas em intervalos de 24 horas até o término dos sinais clínicos (experimento 2). As proteínas séricas foram separadas por meio da técnica de SDS-PAGE e a pesquisa de rotavírus nas fezes foi realizada por meio da técnica de PAGE. Os resultados do experimento 1 foram analisados por meio de ANOVA e do teste de Tukey, considerado significativo quando P<0,05. Os dados do experimento 2 foram submetidos ao teste HSD. Os teores de proteína total, globulinas e IgG foram menores no grupo C que nos grupos A e B, os teores de ceruloplasmina e transferrina foram maiores no grupo C que nos grupos A e B e as concentrações séricas de haptoglobina e α1-glicoproteína ácida não diferiram significativamente entre grupos. Os bezerros manifestaram diarreia, em média, com 10,4 a 14,6 dias de idade no grupo B e com 10,3 a 14,6 dias de idade no grupo C. Nos momentos de manifestação de diarreia, os teores de IgA, haptoglobina e α1-glicoproteína ácida não diferiram significativamente entre os grupos B e C, mas os teores de ceruloplasmina e transferrina foram maiores no grupo C que no grupo B, oposto ao verificado para o teor de IgG. Esses achados mostram que a otimização da transferência de imunidade passiva de imunoglobulinas reduz a probabilidade de os animais apresentarem diarreia por rotavírus. Adicionalmente, a ceruloplasmina apresenta características de um biomarcador da infecção por rotavírus em bezerros.(AU)

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.

A semi-nested RT-PCR assay targeted to hemagglutinin-esterase gene of Bovine Coronavirus / Reação de hemi-nested RT-PCR dirigida ao gene da hemaglutinina-esterase do Coronavírus Bovino

Bovine coronavirus (BCoV) is a non-segmented positive-sense single-stranded RNA virus whose envelope is constituted by a lipid bilayer with four structural proteins (HE, S, E and M) giving its characteristic crown-like virions appearance. Hemagglutinin-esterase (HE), is a polymorphic protein with a function of secondary receptor binder, and studies on the diversity of HE gene allow insights on BCoV evolution and host-parasite interactions. A semi-nested RT-PCR was developed for the amplification of a 441bp-long product of the HE gene of BCoV (nt 543 to 562). Optimal annealing temperatures were tested in a gradient thermocycler for the semi-nested assay and employed in the final protocol. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titer: 256) in a BCoV-free fecal suspension, with positive results up to 10-6 dilution, a high analytical sensitivity without PCR inhibition. The final semi-nested RT-PCR protocol was applied to 21 fecal samples of cows previously positive to BCoV and DNA sequencing of the 441bp amplicons of 14 of these resulted in highly-scored BCoV HE gene sequences after BLAST/n analysis. This semi-nested RT-PCR is a powerful tool for surveys of phylogenetic diversity in field strains of BCoV and for comparative studies among different genes of Coronavirus.

O coronavírus bovino (BCoV) é um vírus RNA simples fita, de sentido positivo, não segmentado com envelope constituído de uma camada dupla de lipídios com quatro proteínas (HE, S, E e M) que resultam no aspecto de coroa dos vírions. Como a HE (hemaglutinina-esterase) é uma proteína polimórfica com uma função de receptor aglutinante secundária, estudos sobre a diversidade do gene HE podem possibilitar maiores informações sobre a evolução e interação hospedeiro-parasita do BCoV. Uma reação de hemi-nested RT-PCR foi desenvolvida para a amplificação de um produto de 441pb do gene HE do BCoV (nt 543 ao 562). Temperaturas ótimas de hibridização foram testadas em um termociclador com gradiente para a reação de hemi-nested e utilizada no protocolo final. A sensibilidade analítica foi determinada por meio da diluição serial na base 10 do BCoV amostra Kakegawa (título HA: 256) em uma suspensão fecal negativa para BCoV, resultando positiva até a diluição de 10-6, mostrando uma alta sensibilidade analítica sem inibição na PCR. O protocolo final da hemi-nested RT-PCR foi aplicado a 21 amostras fecais de vacas previamente positivas para BCoV e o sequenciamento de DNA do produto de 441pb de 14 amostras resultaram em sequências com elevado escore do gene HE do BCoV após a análise no BLAST/n. Essa hemi-nested RT-PCR é uma ferramenta poderosa para estudos de diversidade filogenética de linhagens de campo de BCoV e para estudos comparativos entre os diferentes genes dos Coronavírus.

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.

Cross-reactivity of Vaccine and Fields Strains of Bovine Coronaviruses in Korea

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, and is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. Although the Korean BCoV vaccine strain, BC94, was isolated in 1995, there has still been no report of a molecular characterization of the vaccine strain. To characterize the vaccine strain, relationships between BC94 and field strains were investigated, based on sequence analysis and cross-immunity. We determined the complete sequences of the HE, N, and S genes from BC94 and four NVRQS isolates (SUN5, A3, 0501, 0502). Due to its major role in antigenicity, the spike proteins of the BCoVs were analyzed. BC94 showed distinctive genetic divergence from field isolates collected from 2002 to 2005. BC94, SUN5, and A3 had no virulence-specific sequence and there was a single amino acid change, from asparagine to lysine at residue 175, in the polymorphic region. Strains 0501 and 0502 had virulence-specific sequences at all seven sites. Although the recently isolated Korean BCoVs and BC94 were genetically different, the cleavage site of spike genes at 763~768 (KRRSRR) and the antigenic domain II of the spike protein, amino acid position 528, were conserved in all NVRQS isolates. The antigenic relatedness of KCD9, representative of recent Korean BCoVs, was compared with the Korean vaccine strain BC94. KCD9 showed cross-reactivity against BC94 by virus neutralization (VN) test. These results suggest that BC94 is antigenically closely related to field isolates and is still effective as a vaccine strain.

Cross-reactivity of Vaccine and Fields Strains of Bovine Coronaviruses in Korea

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, and is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. Although the Korean BCoV vaccine strain, BC94, was isolated in 1995, there has still been no report of a molecular characterization of the vaccine strain. To characterize the vaccine strain, relationships between BC94 and field strains were investigated, based on sequence analysis and cross-immunity. We determined the complete sequences of the HE, N, and S genes from BC94 and four NVRQS isolates (SUN5, A3, 0501, 0502). Due to its major role in antigenicity, the spike proteins of the BCoVs were analyzed. BC94 showed distinctive genetic divergence from field isolates collected from 2002 to 2005. BC94, SUN5, and A3 had no virulence-specific sequence and there was a single amino acid change, from asparagine to lysine at residue 175, in the polymorphic region. Strains 0501 and 0502 had virulence-specific sequences at all seven sites. Although the recently isolated Korean BCoVs and BC94 were genetically different, the cleavage site of spike genes at 763~768 (KRRSRR) and the antigenic domain II of the spike protein, amino acid position 528, were conserved in all NVRQS isolates. The antigenic relatedness of KCD9, representative of recent Korean BCoVs, was compared with the Korean vaccine strain BC94. KCD9 showed cross-reactivity against BC94 by virus neutralization (VN) test. These results suggest that BC94 is antigenically closely related to field isolates and is still effective as a vaccine strain.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR / Detecção rápida do Coronavírus Bovino (BCoV) por meio de uma semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

O Coronavírus bovino (BCoV) pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae) e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doença respiratória em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a detecção do BCoV com base em seqüências representativas e recentes do gene do nucleocapsídeo, região conservada do genoma dos coronavírus. Três primers foram desenhados, a primeira amplificação com um fragmento esperado de 463pb e a segunda (semi-nested) com um fragmento esperado de 306pb. A sensibilidade analítica foi determinada pela diluição do BCoV cepa Kakegawa (título HA: 256) na base de 10 em água ultra-pura tratada com DEPC, em soro fetal bovino (SFB) e em uma suspensão fecal negativa para o BCoV, onde foram encontrados resultados positivos até a diluição de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitação dos pellets pelo método de extração utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analítica e ausência de possíveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referência, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnóstico apresentaram concordância substancial (kappa: 0,694). A elevada sensibilidade e especificidade do novo método proposto e o fato de que os primers foram desenhados baseados em sequências atuais do BCoV, oferecem bases para o diagnóstico mais acurado de infecções causadas pelo BCoV, assim como para novas perspectivas em protocolos de detecção de outros Coronavírus de importância tanto em saninade animal ...

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.

Winter dysentery in cows associated with Bovine Coronavirus (BCoV)

Descreve-se a pesquisa de BCoV e rotavírus em 13 mostras fecais de vacas de surtos de disenteria utilizando uma nested PCR dirigida ao gene RdRp e PAGE, respectivamente. Todas as amostras fecais foram positivas para BCoV e nenhuma delas apresentou-se positiva para rotavírus em PAGE. O encontro de coronavírus bovino em amostras fecais de vacas com disenteria sugere que este vírus possa ser o agente primário envolvido na etiologia dos casos aqui relatados

Serological Survey of Bovine Coronavirus in Korea

Bovine coronavirus (BCoV) is a causative agent of entero-pathogenic diarrhea in young calves and winter dysentery (WD) in adult cattle. In this study, we conducted a nationwide sero-epidemiological survey of BCoV infection in Korea. In total, 3,029 bovine sera collected between October and December 2005 were screened for the presence of antibodies against BCoV using a hemagglutination inhibition (HI) test. Half (50.0%) of individual cattle tested were positive for BCoV. The regional distribution of the seroprevalence of positive HI antibodies was 55.7% (234/420) in Gyeonggi, 53.0% (316/596) in Jeonra, 51.9% (374/720) in Chungcheong, 48.5% (401/827) in Gyeongsang, 43.9% (79/180) in Jeju, and 38.1% (109/286) in Gangwon Province. Analyzing the distribution of HI titer according to the age of the cattle showed the highest BCoV seropositive rate in 5-year-old cattle, and the incidence of cattle with an HI antibody titer of 1:160 or above was 12.1%.

Isolation of bovine coronavirus (BCoV) in monolayers of HmLu-1 cells

A partir de 20 amostras fecais de bezerros, com quadro clínico de diarréia, positivas para BCoV por hemaglutinacão/inibicão da hemaglutinacão (HA/HI), procedeu-se o isolamento viral em monocamadas de células da linhagem HmLu-1. Até a quinta passagem seriada, 13 apresentaram efeito citopático do tipo sincicial, semelhante à amostra padrão Kakegawa de BCoV. Ao serem submetidas a uma reacão de soroneutralizacão com gamaglobulina anti-coronavírus bovino, 8 delas foram consideradas positivas, uma vez que o efeito citopático foi neutralizado. Ao serem tituladas e submetidas à reacão de soroneutralizacão em microplacas, apenas 3 delas puderam ser confirmadas como positivas. As células da linhagem HmLu-1 mostraram-se permissivas para o isolamento de BCoV, todavia a baixa intensidade na replicacão viral demonstrou ser necessário o desenvolvimento de novas metodologias para se poder alcancar esse intuito e a confirmacão do isolamento por soroneutralizacão.

Targeted RNA recombination of the membrane and nucleocapsid protein genes between mouse hepatitis virus and bovine coronavirus

The targeted RNA recombination was attempted to substitute the membrane (M) protein gene and part of the nucleocapsid (N) protein gene of mouse hepatitis virus with the corresponding sequences from bovine coronavirus. Using a defective interfering (DI) RNA-like cDNA construct derived from pMH54, 690 nucleotides representing the entire M gene and the 5' most 915 nucleotides of the N gene of the mouse hepatitis virus Albany 4 mutant were attempted to be replaced. Upon infection of cells with Albany 4 followed by transfection with synthetic RNA transcribed from the DI-like cDNA construct, recombinant mouse hepatitis viruses as the large plaque forming phenotype were isolated by plaque assays at the non-permissive temperature of 391 degrees C. By RT-PCR and sequencing, those large plaque phenotypes were confirmed to have contained the thermostable phenotype marker derived from the transfected RNA, demonstrating that recombination occurred between the Albany 4 genomic RNA and the in vitro RNA transcripts. Further analysis of the recombinant viruses indicated that there combination had taken place within the region of 222 nucleotides between positions 916 and 1,137 of the N gene. This is the region immediately downstream of the replacement sequence and the start of the temperature resistant phenotype marker. The results suggest that the M and part of the N genes of bovine coronavirus may not be able to complement the function of those of mouse hepatitis virus. This study redirects our current approach of utilizing the MHV targeted RNA recombination as a means to study bovine coronavirus genetics towards the construction of an infectious cDNA clone.

Evaluation of serum neutralizing antibodies to bovine coronavirus in cows & their calves using Hmlu-1 cells.

Between 1992 and 1993, 75 paired serum samples from Holstein dairy cows and their calves were collected from Aomori, Tochigi and Okinawa Prefectures, and the neutralizing antibody titres to bovine coronavirus (BCV) were determined using hamster lung (Hmlu)-1 cells. The anti-BCV antibody positive rate in the maternal serum samples was significantly lower (P < 0.001) in Okinawa (72%) than in Aomori (100%) or Tochigi (100%). The geometric mean tire (GMT) of anti-BCV neutralizing antibody was also significantly lower (P < 0.05) in maternal sera from Okinawa (89) than that of Aomori (229) or Tochigi (264). The anti-BCV neutralizing antibody titres in the sera of calves which had ingested the colostrum, significantly correlated with the antibody concentration of the maternal serum samples (P < 0.05). These results suggest an extensive BCV infection among the dairy cattle in these prefectures, with a varied pattern of distribution between the prefectures. Anti-BCV neutralizing antibody in the sera of newborn calves appeared to be transferred from their dams through colostrum.