Use of live attenuated recombinant Newcastle disease virus carrying avian paramyxovirus 2 HN and F protein genes to enhance immune responses against species A rotavirus VP6 protein.

Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector.

Establishment of an Indirect ELISA Method for the Detection of the Bovine Rotavirus VP6 Protein.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 µg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Introduction: Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. Methods: In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. Results: The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. Discussion: The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

Complete genome constellation of a dominant Bovine rotavirus genotype circulating in Bangladesh reveals NSP4 intragenic recombination with human strains.

Rotavirus A is a leading cause of non-bacterial gastroenteritis in humans and domesticated animals. Despite the vast diversity of bovine Rotavirus A strains documented in South Asian countries, there are very few whole genomes available for phylogenetic study. A cross-sectional study identified a high prevalence of the G6P[11] genotype of bovine Rotavirus A circulating in the commercial cattle population in Bangladesh. Next-generation sequencing and downstream phylogenetic analysis unveiled all 11 complete gene segments of this strain (BD_ROTA_CVASU), classifying it under the genomic constellation G6P[11]-I2-R2-C2-M2-A13-N2-T6-E2-H3, which belongs to a classical DS-1-like genomic backbone. We found strong evidence of intragenic recombination between human and bovine strains in the Non-structural protein 4 (NSP4) gene, which encodes a multifunctional enterotoxin. Our analyses highlight frequent zoonotic transmissions of rotaviruses in diverse human-animal interfaces, which might have contributed to the evolution and pathogenesis of this dominant genotype circulating in the commercial cattle population in Bangladesh.

A multiplex real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses.

Introduction: Calf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high. Methods: To address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes. Results: This method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/µL, 96.0 copies/µL, 12.8 copies/µL, 16.4 copies/µL, 18.2 copies/µL, and 65.3 copies/µL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence. Discussion: Additionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry.

Genetic characterisation of south african and mozambican bovine rotaviruses reveals a typical bovine-like artiodactyl constellation derived through multiple reassortment events

This study presents whole genomes of seven bovine rotavirus strains from South Africa and Mozambique. Double-stranded RNA, extracted from stool samples without prior adaptation to cell culture, was used to synthesise cDNA using a self-annealing anchor primer ligated to dsRNA and random hexamers. The cDNA was subsequently sequenced using an Illumina MiSeq platform without prior genome amplification. All strains exhibited bovine-like artiodactyl genome constellations (G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3). Phylogenetic analysis revealed relatively homogenous strains, which were mostly related to other South African animal strains or to each other. It appears that these study strains represent a specific bo-vine rotavirus population endemic to Southern Africa that was derived through multiple reassortment events. While one Mozambican strain, MPT307, was similar to the South African strains, the second strain, MPT93, was divergent from the other study strains, exhibiting evi-dence of interspecies transmission of the VP1 and NSP2 genes. The data presented in this study not only contribute to the knowledge of circulating African bovine rotavirus strains, but also em-phasise the need for expanded surveillance of animal rotaviruses in African countries in order to improve our understanding of rotavirus strain diversity

Vacinação pré-parto de vacas Nelore contra diarreia neonatal

A diarreia neonatal dos bezerros (DNB) é um dos eventos de saúde mais importantes durante o período de criação de bezerros de corte, sendo responsável por grande parte da morbidade e mortalidade nesta fase. Entre as medidas para mitigar o risco de surtos de DNB, a vacinação da vaca na última fase da gestação é uma das mais importantes. O objetivo desta pesquisa foi avaliar a eficácia da vacinação pré-parto contra DNB, especialmente Rotavírus Bovino tipo A (BoRVA) e Coronavírus Bovino (BCoV), em animais da raça Nelore. Um total de 117 animais, divididos em vacas (n=81) e novilhas (n=36) foram distribuídos em dois grupos experimentais, sendo estes: grupo de animais vacinados (VAC: vacas=40; novilhas=19) e grupo de animais não vacinados (NVAC: vacas=41; novilhas=17). O tratamento dos animais ocorreu entre 60 a 50 dias antes da data prevista para o parto, sendo aplicado no grupo VAC uma única dose de uma vacina comercial (2mL), administrado por via intramuscular, e no grupo NVAC uma dose de solução salina 0,9%. Amostras de sangue foram coletadas imediatamente antes da vacinação e após 30 dias, para avaliar a resposta de anticorpos específicos (IgG1) contra BoRVA e BCoV. O nascimento dos bezerros foi monitorado e a transferência de imunidade passiva foi avaliada entre o segundo e o terceiro dias de vida. A diarreia foi monitorada nos primeiros 30 dias de idade, e amostras de fezes foram coletadas para identificação do agente etiológico em animais com diarreia. Como controle, amostras de fezes de bezerros saudáveis com idade semelhante à dos animais com diarreia, também foram coletadas. Os anticorpos específicos (IgG1) contra BoRVA e BCoV foram medidos usando a técnica Enzyme Linked Immuno Sorbent Assay (ELISA). A comparação de médias pelo teste T de Student mostrou títulos mais elevados de IgG1 contra BoRVA e BCoV no grupo VAC quando comparado ao grupo NVAC na categoria de vacas (P<0,0001) e no total de animais (P<0,0001). Em relação aos bezerros, não houve diferenças (P>0,05) entre os grupos para a concentração de proteína total sérica (PTS) e taxa (%) de falha da transferência imune passiva (FTIP). O título de IgG1 específico no soro de bezerros refletiu a resposta das mães, observando títulos mais elevados de IgG1 para BoRVA (P<0,0016) e BCoV (P<0,0095) nos bezerros nascidos da categoria de vacas do grupo VAC; e títulos mais elevados de IgG1 para BoRVA (P<0,0171) e BCoV (P<0,0200) nos bezerros nascidos do total de animais do grupo VAC. A incidência geral de diarreia observada foi de 18,6% (11/59) e 29,3% (17/58) nos bezerros nascidos do grupo VAC e NVAC, respectivamente. Apesar da diferença observada, o pequeno tamanho amostral não permitiu detectar diferenças estatísticas entre os grupos experimentais para diarreia (P>0,05). Entre bezerros com diarreia, o agente mais prevalente foi o BoRVA. A vacinação pré-parto com uma única dose da vacina testada aumentou os títulos de IgG1 contra BCoV e BoRVA, podendo ser utilizada como estratégia preventiva para diminuir a ocorrência de DNB em animais da raça Nelore.

Neonatal calf diarrhea (NCD) is one of the most important health events during the rearing period of beef calves, accounting for much of the morbidity and mortality at this stage. Among the measures to mitigate the risk of NCD outbreaks, cow vaccination in the last stage of pregnancy is one of the most important. The objective of this research was to evaluate the effectiveness of prepartum vaccination against NCD, especially Bovine Rotavirus type A (BoRVA) and Bovine Coronavirus (BCoV), in Nelore animals. A total of 117 animals, divided into cows (n=81) and heifers (n=36) were divided into two experimental groups, namely: vaccinated animal group (VAC: cows=40; heifers=19) and animal group unvaccinated (NVAC: cows=41; heifers=17). The treatment of the animals occurred between 60 to 50 days before the expected date of calving, with a single dose of a commercial vaccine (2mL) being applied to the VAC group, administered intramuscularly, and to the NVAC group a dose of saline solution 0, 9%. Blood samples were collected immediately before vaccination and after 30 days to assess the specific antibody response (IgG1) against BoRVA and BCoV. The calf birth was monitored and the transfer of passive immunity was evaluated between the second and third days of life. Diarrhea was monitored in the first 30 days of age, and stool samples were collected to identify the etiologic agent in animals with diarrhea. As a control, fecal samples from healthy calves with age similar to that of animals with diarrhea were also collected. Specific antibodies (IgG1) against BoRVA and BCoV were measured using the Enzyme Linked Immuno Sorbent Assay (ELISA) technique. Comparison of means by Student's t test showed higher IgG1 titers against BoRVA and BCoV in the VAC group when compared to the NVAC group in the cows category (P<0.0001) and in total animals (P<0.0001) . In relation to calves, there were no differences (P>0.05) between groups for serum total protein concentration (STP) and rate (%) of passive immune transfer failure (PITF). The specific IgG1 titer in the serum of calves reflected the response of the dams, observing higher IgG1 titers for BoRVA (P<0.0016) and BCoV (P<0.0095) in calves born from the category of cows in the VAC group; and higher IgG1 titers for BoRVA (P<0.0171) and BCoV (P<0.0200) in calves born from the total number of animals in the VAC group. The overall incidence of diarrhea observed was 18.6% (11/59) and 29.3% (17/58) in calves born in the VAC and NVAC groups, respectively. Despite the observed difference, the small sample size did not allow detecting statistical differences between the experimental groups for diarrhea (P>0.05). Among calves with diarrhea, the most prevalent agent was BoRVA. Prepartum vaccination with a single dose of the tested vaccine increased IgG1 titers against BCoV and BoRVA, and can be used as a preventive strategy to reduce the occurrence of NCD in Nelore animals.

Analysis of bovine rotavirus strains circulating in diarrheic dairy calves in Uberaba, Minas Gerais, Brazil, during 2008-2009 / Análise de cepas de rotavírus bovino circulantes em bezerros leiteiros com diarreia em Uberaba, Minas Gerais, Brasil, durante 2008-2009

O objetivo deste estudo foi detectar rotavírus em fezes de bezerros com diarreia em Uberaba, MG, e caracterizar os genes VP7 e VP4 por meio da genotipagem e da análise filogenética. Setenta e quatro amostras foram coletadas entre novembro de 2008 e setembro de 2009. A detecção do vírus foi feita por teste de aglutinação e as amostras positivas foram submetidas à transcrição reversa, seguida de reação em cadeia da polimerase (RT-PCR), tipagem por PCR e sequenciamento. A taxa de detecção de rotavírus foi de 6,8% e todas as amostras apresentaram o genótipo G6P[5]. A análise filogenética mostrou que as amostras do genótipo G6 pertencem à linhagem IV e que, para ambos os genes (VP7 e VP4), as amostras deste estudo compõem um sub-cluster à parte daquele das cepas referências e das amostras campo mais similares. O alinhamento das sequências de aminoácidos deduzidas mostrou substituições em regiões antigênicas quando comparadas com as sequências das cepas bovinas UK e NCDV, presentes nas vacinas disponíveis no Brasil. Uma nova sublinhagem genética de G6P[5] foi evidenciada neste estudo. Substituições de aminoácidos nas regiões antigênicas dos rotavírus e a circulação de novas variantes podem representar desafios para as vacinas utilizadas atualmente. O presente estudo contribui para a compreensão da epidemiologia dos rotavírus bovinos no Brasil.(AU)

Analysis of bovine rotavirus strains circulating in diarrheic dairy calves in Uberaba, Minas Gerais, Brazil, during 2008-2009 / Análise de cepas de rotavírus bovino circulantes em bezerros leiteiros com diarreia em Uberaba, Minas Gerais, Brasil, durante 2008-2009

O objetivo deste estudo foi detectar rotavírus em fezes de bezerros com diarreia em Uberaba, MG, e caracterizar os genes VP7 e VP4 por meio da genotipagem e da análise filogenética. Setenta e quatro amostras foram coletadas entre novembro de 2008 e setembro de 2009. A detecção do vírus foi feita por teste de aglutinação e as amostras positivas foram submetidas à transcrição reversa, seguida de reação em cadeia da polimerase (RT-PCR), tipagem por PCR e sequenciamento. A taxa de detecção de rotavírus foi de 6,8% e todas as amostras apresentaram o genótipo G6P[5]. A análise filogenética mostrou que as amostras do genótipo G6 pertencem à linhagem IV e que, para ambos os genes (VP7 e VP4), as amostras deste estudo compõem um sub-cluster à parte daquele das cepas referências e das amostras campo mais similares. O alinhamento das sequências de aminoácidos deduzidas mostrou substituições em regiões antigênicas quando comparadas com as sequências das cepas bovinas UK e NCDV, presentes nas vacinas disponíveis no Brasil. Uma nova sublinhagem genética de G6P[5] foi evidenciada neste estudo. Substituições de aminoácidos nas regiões antigênicas dos rotavírus e a circulação de novas variantes podem representar desafios para as vacinas utilizadas atualmente. O presente estudo contribui para a compreensão da epidemiologia dos rotavírus bovinos no Brasil.(AU)

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 1-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 1-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 1-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 1-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)

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)

Análisis molecular del gen de la VP4 de la cepa de rotavirus bovino tipo P[5] aislada de becerros en el Estado Yaracuy / Molecular analysis of the VP4 genes of bovine rotavirus strain type P[5] isolated from calves in Yaracuy State

La diarrea neonatal bovina indiferenciada es una enfermedad multifactorial que afecta a una alta proporción de becerros (50% o más) causando hasta un 40% de mortalidad en los primeros 30 d de vida. Entre los agentes causales se incluyen muchos enteropatógenos (bacterias, virus, protozoarios), con predominio de infecciones combinadas. Entre los virus, los rotavirus constituyen los principales agentes causales de gastroenteritis aguda en diferentes especies animales. Su genoma, de 11 segmentos de ARN de doble cadena, puede rearreglarse genéticamente en coinfecciones de diferentes cepas, produciendo una progenie viral con un nuevo fenotipo. En la ganadería bovina venezolana, se han descrito los rotavirus con una prevalencia de 20-30% en animales con cuadros de diarrea entre 2-6 sem de edad. Los serotipos G6 y el genotipo P1 se han reportado con mayor frecuencia en becerros de la región central de Venezuela. Cepas de rotavirus bovino del grupo A fueron detectados por inmunoensayo enzimático (ELISA) y electroforesis en gel de poliacrilamida (PAGE), en 32 de 280 (11,43%) muestras de heces de becerros de 15 fincas del estado Yaracuy, Venezuela. La secuencia nucleotídica se logró para una de las variantes detectadas (A610). En el análisis comparativo de las secuencias de la VP8* (fragmento de la VP4) mostró corresponder al genotipo P[5]. Estos datos constituyen el primer reporte de una cepa de rotavirus bovino genotipo P[5], que circula en las fincas del estado Yaracuy, Venezuela, pudiendo servir de apoyo para la incorporación de este grupo viral en las vacunas que se utilizan en el país.

The undifferentiated neonatal bovine diarrhea is a multifactorial disease that affects a high proportion of calves (50% or more) causing up to 40% of mortality in the first 30 d of life. Among the infectious agents known to cause diarrhea in calves are the rotavirus and coronavirus. Rotaviruses are the major causative agents of acute gastroenteritis in different animal species. Their 11-segment genome of double ARN chain can genetically re-arrange different strains, producing a viral progeny with new or atypical phenotype. Rotaviruses have been described in the bovine Venezuelan cattle, with a prevalence of 20-30% in animals between 2-6 weeks of age, with episodes of diarrhea. The serotypes G6 and the genotype P1 have been reported with higher frequency in calves from the central region of Venezuela. In this investigation, rotavirus strains of cattle from Group A were detected by the enzyme-linked immunosorbent assay (ELISA) and the polyacrylamide gel electrophoresis (PAGE) in 32 of 280 (11.43%) stool samples of calves in 15 farms in the State of Yaracuy, Venezuela. The nucleotide sequence was obtained for one of the variants detected (A610) and the comparative analysis of the sequences of the VP8* (the VP4 fragment) corresponded to the P [5] genotype. These data constitute the first report of a strain of bovine rotavirus genotype P[5], which circulates in the States of Yaracuy, Venezuela, and can serve as a support for the inclusion of this viral group to the vaccines that are being used in the country.

DINÂMICA DE INFECÇÃO DA CRIPTOSPORIDIOSE EM BEZERROS NEONATOS COM INFECÇÃO NATURAL E AVALIAÇÃO DA METAFILAXIA COM TOLTRAZURIL

Cryptosporidium spp. é um coccídeo pertencente ao filo Apicomplexa responsável por infectar e causar doença em ampla variedade de hospedeiros, incluindo o homem. Em bovinos, a maior prevalência é registrada nos animais com até um mês de idade que apresentam intensa diarreia aquosa, desidratação e apatia, apesar de se tratar de uma doença auto limitante. O objetivo deste trabalho foi estudar a dinâmica da criptosporidiose em bezerros neonatos e avaliar a eficácia do toltrazuril na metafilaxia da doença. Em uma propriedade rural leiteira localizada no estado de Minas Gerais, foram acompanhados 36 bezerras do nascimento aos 28 dias de idade e criados em sistema de casinha individual. As bezerras foram aleatoriamente divididas em três grupos, sendo o G1 o grupo controle, o G2 o grupo de animais que receberam via oral tratamento de 20 mg/Kg de p.v. de toltrazuril ao nascimento e o G3 o grupo de bezerros que receberam via oral dois tratamentos de 20 mg/Kg de p.v. da droga ao nascimento e ao quinto dia de idade. Amostras de fezes foram coletadas nos dias 1, 3, 5, 7, 9, 11, 13, 15, 21 e 28 e processadas pela técnica de Ziehl-Neelsen modificada a fim de determinar a ocorrência e a intensidade da infecção por Cryptosporidium spp. Além disso, todas as amostras de fezes foram avaliadas quanto à presença de rotavírus bovino em associação com o parasita. A frequência de criptosporiodiose nos bezerros do experimento foi de 91,66% e os animais do grupo controle excretaram oocistos a partir do dia 7, o pico de eliminação ocorreu nos dias 9 a 15 e no dia 28 não foram identificados animais com fezes positivas para o Cryptosporidium spp. O rotavírus bovino foi identificado em 11,12% dos animais em momentos em que não havia excreção de oocistos nas fezes e, portanto, não foi observada associação entre os dois enteropatógenos, o que foi reforçado pela alta correlação entre ocorrência de diarreia e a infecção por Cryptosporidium spp. O toltrazuril administrado tanto em um como em dois momentos não foi capaz de inibir ou reduzir a excreção de oocistos e a intensidade da diarreia. Os animais tratados dos grupos G2 e G3 apresentaram o mesmo padrão de eliminação de oocisto que os bezerros do grupo controle. Conclui-se que o toltrazuril não foi eficaz na metafilaxia da criptosporidiose em bezerros neonatos quando administrado na dose de 20 mg/Kg de p. v. ao nascimento e aos cinco dias de idade.

Cryptosporidium spp. is a coccidian of the phylum Apicomplexa responsible for infect and cause disease in a wide variety of hosts, including man. In cattle, the highest prevalence is recorded in animals up to one month of age demonstrating severe watery diarrhea, dehydration and apathy, even though it is a self-limiting disease. The objective of this work was to study the dynamics of cryptosporidiosis in newborn calves and evaluate the efficacy of toltrazuril in diseases metaphylaxis. On a dairy farm in the state of Minas Gerais, were followed 36 calves from birth to 28 days of age and reared in individual house system. The calves were randomly divided into three groups: the control group G1, G2 the group of animals that received oral treatment with 20 mg/kg b.w. toltrazuril birth and G3 group of calves received two treatments orally 20 mg / kg P.V. drug the birth and five days old. Fecal samples were collected on days 1, 3, 5, 7, 9, 11, 13, 15, 21 and 28 and processed by the Ziehl-Neelsen stain modified to determine the occurrence and intensity of infection by Cryptosporidium spp. Moreover, all stool specimens were evaluated for the presence of bovine rotavirus in association with the parasite. The frequency criptosporiodiose the experimental calves was 91.66% and animals excreted oocysts control group from day 7, the peak deletion occurred on days 9 to 15 and on day 28 animals with positive stool were identified for Cryptosporidium spp. The bovine rotavirus was identified in 11-12% of animals in times when there was no excretion of oocysts in the feces and therefore, no association was observed between the two enteropathogens, which was reinforced by the high correlation between the occurrence of diarrhea and infection Cryptosporidium spp. Toltrazuril administered both in one as in two times was not able to inhibit or reduce the excretion of oocysts and intensity of diarrhea. Treated animals of G2 and G3 showed the same pattern oocyst elimination of calves in the control group. It is concluded that toltrazuril was not effective in metafilaxia of cryptosporidiosis in neonatal calves when administered at a dose of 20 mg / kg p. v. at birth and at five days of age.

DINÂMICA DE INFECÇÃO DA CRIPTOSPORIDIOSE EM BEZERROS NEONATOS COM INFECÇÃO NATURAL E AVALIAÇÃO DA METAFILAXIA COM TOLTRAZURIL

Cryptosporidium spp. é um coccídeo pertencente ao filo Apicomplexa responsável por infectar e causar doença em ampla variedade de hospedeiros, incluindo o homem. Em bovinos, a maior prevalência é registrada nos animais com até um mês de idade que apresentam intensa diarreia aquosa, desidratação e apatia, apesar de se tratar de uma doença auto limitante. O objetivo deste trabalho foi estudar a dinâmica da criptosporidiose em bezerros neonatos e avaliar a eficácia do toltrazuril na metafilaxia da doença. Em uma propriedade rural leiteira localizada no estado de Minas Gerais, foram acompanhados 36 bezerras do nascimento aos 28 dias de idade e criados em sistema de casinha individual. As bezerras foram aleatoriamente divididas em três grupos, sendo o G1 o grupo controle, o G2 o grupo de animais que receberam via oral tratamento de 20 mg/Kg de p.v. de toltrazuril ao nascimento e o G3 o grupo de bezerros que receberam via oral dois tratamentos de 20 mg/Kg de p.v. da droga ao nascimento e ao quinto dia de idade. Amostras de fezes foram coletadas nos dias 1, 3, 5, 7, 9, 11, 13, 15, 21 e 28 e processadas pela técnica de Ziehl-Neelsen modificada a fim de determinar a ocorrência e a intensidade da infecção por Cryptosporidium spp. Além disso, todas as amostras de fezes foram avaliadas quanto à presença de rotavírus bovino em associação com o parasita. A frequência de criptosporiodiose nos bezerros do experimento foi de 91,66% e os animais do grupo controle excretaram oocistos a partir do dia 7, o pico de eliminação ocorreu nos dias 9 a 15 e no dia 28 não foram identificados animais com fezes positivas para o Cryptosporidium spp. O rotavírus bovino foi identificado em 11,12% dos animais em momentos em que não havia excreção de oocistos nas fezes e, portanto, não foi observada associação entre os dois enteropatógenos, o que foi reforçado pela alta correlação entre ocorrência de diarreia e a infecção por Cryptosporidium spp. O toltrazuril administrado tanto em um como em dois momentos não foi capaz de inibir ou reduzir a excreção de oocistos e a intensidade da diarreia. Os animais tratados dos grupos G2 e G3 apresentaram o mesmo padrão de eliminação de oocisto que os bezerros do grupo controle. Conclui-se que o toltrazuril não foi eficaz na metafilaxia da criptosporidiose em bezerros neonatos quando administrado na dose de 20 mg/Kg de p. v. ao nascimento e aos cinco dias de idade

Cryptosporidium spp. is a coccidian of the phylum Apicomplexa responsible for infect and cause disease in a wide variety of hosts, including man. In cattle, the highest prevalence is recorded in animals up to one month of age demonstrating severe watery diarrhea, dehydration and apathy, even though it is a self-limiting disease. The objective of this work was to study the dynamics of cryptosporidiosis in newborn calves and evaluate the efficacy of toltrazuril in diseases metaphylaxis. On a dairy farm in the state of Minas Gerais, were followed 36 calves from birth to 28 days of age and reared in individual house system. The calves were randomly divided into three groups: the control group G1, G2 the group of animals that received oral treatment with 20 mg/kg b.w. toltrazuril birth and G3 group of calves received two treatments orally 20 mg / kg P.V. drug the birth and five days old. Fecal samples were collected on days 1, 3, 5, 7, 9, 11, 13, 15, 21 and 28 and processed by the Ziehl-Neelsen stain modified to determine the occurrence and intensity of infection by Cryptosporidium spp. Moreover, all stool specimens were evaluated for the presence of bovine rotavirus in association with the parasite. The frequency criptosporiodiose the experimental calves was 91.66% and animals excreted oocysts control group from day 7, the peak deletion occurred on days 9 to 15 and on day 28 animals with positive stool were identified for Cryptosporidium spp. The bovine rotavirus was identified in 11-12% of animals in times when there was no excretion of oocysts in the feces and therefore, no association was observed between the two enteropathogens, which was reinforced by the high correlation between the occurrence of diarrhea and infection Cryptosporidium spp. Toltrazuril administered both in one as in two times was not able to inhibit or reduce the excretion of oocysts and intensity of diarrhea. Treated animals of G2 and G3 showed the same pattern oocyst elimination of calves in the control group. It is concluded that toltrazuril was not effective in metafilaxia of cryptosporidiosis in neonatal calves when administered at a dose of 20 mg / kg p. v. at birth and at five days of age

Molecular characterization of G and P-types bovine rotavirus strains from Goiás, Brazil: high frequency of mixed P-type infections

In this study, 331 samples from calves less than one month old from a dairy herd in the district of Piracanjuba, state of Goiás, Brazil were tested for rotavirus. Thirty-three samples (9.9 percent) tested positive for rotavirus. Out of those, 31 were submitted to G and P characterization by reverse transcription followed by semi-nested polymerase chain reaction. Two samples were characterized as G6P[1], three as G10P[11] and five as G6P[11]. The majority of the samples (51.6 percent) displayed multiple P genotypes (P-genotype mixtures), including typical human genotypes P[4] and P[6M], suggesting the occurrence of co-infections and genetic reassortment. Also, the detection of human genotypes in bovine samples may be considered evidence of the zoonotic potential of rotaviruses. To our knowledge, this is the first report of such a high frequency of P genotype mixtures in bovine rotavirus samples. It also increases data on G and P rotavirus genotypes circulating in dairy herds in Brazil and can help in the development of more efficient immunization approaches, thereby controlling infection and reducing economical losses.