José Antonio Jerez

Rotavirus detection and isolation from chickens with or without symptoms

Rotaviruses have been identified as one of the main etiological agents of diarrhea and enteritis in mammals, including humans, and in avian species. Few studies have been published about enteric viruses in Brazilian poultry, including those related to rotavirus infection. Such studies demonstrate significant occurrence and the importance of enteric viruses in poultry presenting intestinal problems. Enteric viruses are the primary cause of injuries to the gut, allowing other agents, especially bacteria, to attach, to penetrate, and to replicate in the enteric tissue, leading to further damage. The aim of the present study was to detect rotavirus in the intestinal contents of layers and broilers by polyacrylamide gel electrophoresis (PAGE) and virus isolation in MA-104 cell culture. A total of 45.3% of all samples were positive to rotavirus; rotavirus frequencies were 48.7% in samples from flocks with diarrhea, 46.4% in flocks with delayed growth, and 30% in asymptomatic flocks. It was possible to isolate rotavirus in MA-104 cells from the nine rotavirus-positive randomly chosen samples. These results indicate that rotavirus may have an important role in pathogenesis of enteric disease.

Molecular analysis of Brazilian strains of bovine coronavirus (BCoV) reveals a deletion within the hypervariable region of the S1 subunit of the spike glycoprotein also found in human coronavirus OC43

Summary.Bovine coronavirus (BCoV) causes enteric and respiratory dis- orders in calves and dysentery in cows. In this study, 51 stool samples of calves from 10 Brazilian dairy farms were analysed by an RT-PCR that amplifies a 488-bp fragment of the hypervariable region of the spike glycoprotein gene. Maximum parsimony genealogy with a heuristic algorithm using sequences from 15 field strains studied here and 10 sequences from GenBank and bredavirus as an outgroup virus showed the existence of two major clusters (1 and 2) in this viral species, the Brazilian strains segregating in both of them. The mean nucleotide identity between the 15 Brazilian strains was 98.34%, with a mean amino acid similarity of 98%. Strains from cluster 2 showed a deletion of 6 amino acids inside domain II of the spike protein that was also found in human coronavirus strain OC43, supporting the recent proposal of a zoonotic spill- over of BCoV. These results contribute to the molecular characterization of BCoV, to the prediction of the efficiency of immunogens, and to the definition of molecular markers useful for epidemiologic surveys on coronavirus-caused diseases.

Prevalência de rotavírus do grupo A em fezes diarréicas de bezerros de corte em sistema semi-intensivo de produção

During three successive seasons of calving (August to November 1999, January to April and August to November 2000) 69 fecal samples were collected from calves between 30 and 60 days of age presenting diarrhea, to account for the detection and prevalence of rotavirus. Samples were submited to an immunoenzymatic assay for rotavirus and adenovirus (kit EIARA-Fiocruz) and polyacrylamide gel electrophoresis (PAGE). Of the 69 samples tested, 63.8% (44/69) were positive by EIARA. At the first calving season rotavirus was detected in feces from 14 of 17 calves (82.4%) with clinical signals of diarrhea. For the year 2000 seasons, rotavirus was detected in 5 of 12 (41.7%) and 25 of 40 (62.5%) of samples, respectively. The analysis of the eletrophoretic pattern of the positive samples showed four distinct group A rotavirus genome eletropherotypes.

Identification of enteropathogens from buffalo calves with and without diarrhoea in the Ribeira Valley, State of São Paulo, Brazil

Estudamos a morfologia dos gânglios celiaco, celiacomesenterico e mesenterico cranial no bufalo. Estes sao responsaveis pela motilidade gastrointestinal, sendo a sua sistematizacao essencial a clinica e a cirurgia, alem de contribuir para o estabelecimento de uma anatomia funcional comparativa. Para a realizacao desta pesquisa, utilizamos 30 fetos de bufalos, sem raca definida, com idades que variaram entre 4 e 10 meses. Injetamos Neoprene Latex 650 na aorta toracica e, a seguir, fixamos os animais em solucao aquosa de formol a 10% por um periodo minimo de 72 horas. Macroscopicamente, evidenciamos 9 gânglios celiacos, localizados a direita (5) e a esquerda (4) da arteria celiaca. No tocante ao formato desses gânglios, constatamos predominância do tipo retangular (44,4% ± 16,6). O gânglio mesenterico cranial mostrou-se em 2 casos (6,7%), oportunidades em que as suas porcoes direita e esquerda envolviam a arteria mesenterica cranial. Os gânglios celiacomesentericos foram em numero de 30, apresentando diversos arranjos: um gânglio com porcoes direita e esquerda (70%); gânglios independentes (13,33%); gânglio celiacomesenterico direito com uma porcao mesenterica esquerda (6,7%); gânglio celiacomesenterico esquerdo com uma porcao mesenterica direita (10%). O formato do gânglio celiacomesenterico mostrou-se frequentemente semilunar (86,7%). As estruturas de conexao do gânglio celiacomesenterico eram: o nervo esplâncnico maior, menor e nervos esplâncnicos lombares. Observamos ramos celiacos oriundos do tronco vagal dorsal e, em 21 casos (70% ± 8,4), eles eram tambem provenientes do tronco vagal ventral. Microscopicamente detectamos a presenca de massas ganglionares, envolvidas por capsula delicada de tecido conjuntivo contendo fibras elasticas, colagenas e reticulares.Faecal samples of 106 buffalo calves aged 3 to 45 days were collected sequentially, once a week, during six weeks for parasitologic examination. For bacteriologic and virologic exams, faecal samples were collected from all diarrhoeic and the same amount on non-diarrhoeic calves. Blood samples from calves were collected at 3 to 10 days of age for serum IgG determination. Eimeria spp, Strongyloides papillosus and Toxocara vitulorum were the most common parasites. E. coli, Enterobacter cloacae and Klebsiella pneumoniae were the enteropathogens most frequently found (alone or in association with other microrganisms). Heat-stable (STa) enterotoxin was detected in 5 out of 34 samples of E. coli isolated from diarrhoeic calves. Among the antimicrobials tested susceptibilities to norphloxacin, chloramphenicol and gentamycin were the most common. None of the samples had characteristic rotavirus RNA bands in PAGE-electrophoresis. No association was observed between low levels of IgG and diarrhoea.We studied the morphology of the celiac, celiacmesenteric and cranial mesenteric ganglia. These ganglia are responsible by the gastrointestinal motility, being so its anatomotopographical characterization necessary for comparative anatomofunctional purposes. Thirty mixed breed buffalo fetuses aged 4 to 10 months were injected with Neoprene Latex 650 into the thoracic aorta and shortly after fixed in 10% aqueous formalin solution for at least 72 hours. The plexus exhibited 9 celiac ganglionar nodes, 5 of which on the right and 4 on the left side. The cranial mesenteric node occurred in 2 samples, where appeared both in a right and a left portion, in a semilunar disposition. On the other hand, 30 cranial celiac mesenteric ganglionar nodes were observed. They showed different morphological arrangements: single node with right and left portions occurred in 21 samples (70%), independent nodes (13.3%), left celiac mesenteric ganglionar node with a right mesenteric portion (10%), a right celiac mesenteric ganglionar node with a left mesenteric portion (6.7%). In regard to the shape of the cranial celiac mesenteric ganglionar node, we noticed the prevalence of the semilunar type (86.7%). The larger and smaller splanchnic nerves as well as the lumbar splanchnic nerves are considered connection structures of these ganglia. Celiac branches stemmed from the dorsal vagal trunk and in 21 samples they stemmed from the ventral vagal trunk. Microscopic evaluation allowed us the presence of nodular ganglionar tissue, which showed differences characteristics. This tissue was involved by a capsule, made up elastic, reticular and collagen fibers.

Emergence of Enteric Viruses in Production Chickens Is a Concern for Avian Health

Several viruses have been identified in recent years in the intestinal contents of chickens and turkeys with enteric problems, which have been observed in commercial farms worldwide, including Brazil. Molecular detection of these viruses in Brazil can transform to a big threat for poultry production due to risk for intestinal integrity. This disease is characterized by severely delayed growth, low uniformity, lethargy, watery diarrhea, delayed feed consumption, and a decreased conversion rate. Chicken astrovirus (CAstV), rotavirus, reovirus, chicken parvovirus (ChPV), fowl adenovirus of subgroup I (FAdV-1), and avian nephritis virus (ANV) were investigated using the conventional polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR). In addition, the infectious bronchitis virus (IBV), which may play a role in enteric disease, was included. The viruses most frequently detected, either alone or in concomitance with other viruses, were IBV, ANV, rotavirus, and CAstV followed by parvovirus, reovirus, and adenovirus. This study demonstrates the diversity of viruses in Brazilian chicken flocks presenting enteric problems characterized by diarrhea, growth retard, loss weight, and mortality, which reflects the multicausal etiology of this disease.

Diversidade genotípica de rotavírus suínos no Estado de São Paulo

Rotavirus is one the most common causes of diarrhea both in humans and different animal species. It was carried out a transversal study with 144 diarrheic fecal samples of piglets, from 16 commercial swine-producing units distributed among 10 municipalities of Sao Paulo State, Brazil, aiming at the detection of rotavirus occurrence and its molecular characterization according to G and P genotypes. A total of 43 samples (29.86%) were positive for rotavirus by Polyacrylamide Gel Electrophoresis (PAGE) and ELISA, in a parallel screening scheme. The nested-multiplex RT-PCR characterization revealed that, separately, the P[6] genotype was the most frequent, detected in 25.58% of the samples, followed by P[1] (11.63%) and P[7] (9.3%). Concomitant infection of the genotypes P[6]+P[7] (9.3%), P[1]+P[6] (4.65%), P[1]+P[6]+P[7] (2.33%) were also found. Similarly, the G[5] genotype was detected on 30.23% of the samples, followed by G[10] (20.93%), G[6] (4.65%) and G[5]+G[10] (18.6%). The genotype G[5]P[6] was the most frequent (11.63%), but other combinations and untypeable samples were also observed. Considering the diversity porcine rotavirus found in the surveyed population, specific prophylactic measures should take in charge, for its effectiveness, the cross-protection degree between the genotypes present on vaccine formulations and those that really circulates on a region.

Sobre a etiologia de um surto de disenteria de inverno em vacas leiteiras no Brasil

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 Sao 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.

Isolation, propagation, genome analysis and epidemiology of HKU1 betacoronaviruses

From 1 January 2009 to 31 May 2013, 15 287 respiratory specimens submitted to the Clinical Virology Laboratory at the Childrens Hospital Colorado were tested for human coronavirus RNA by reverse transcription-PCR. Human coronaviruses HKU1, OC43, 229E and NL63 co-circulated during each of the respiratory seasons but with significant year-to-year variability, and cumulatively accounted for 7.4-15.6 % of all samples tested during the months of peak activity. A total of 79 (0.5 % prevalence) specimens were positive for human betacoronavirus HKU1 RNA. Genotypes HKU1 A and B were both isolated from clinical specimens and propagated on primary human tracheal-bronchial epithelial cells cultured at the air-liquid interface and were neutralized in vitro by human intravenous immunoglobulin and by polyclonal rabbit antibodies to the spike glycoprotein of HKU1. Phylogenetic analysis of the deduced amino acid sequences of seven full-length genomes of Colorado HKU1 viruses and the spike glycoproteins from four additional HKU1 viruses from Colorado and three from Brazil demonstrated remarkable conservation of these sequences with genotypes circulating in Hong Kong and France. Within genotype A, all but one of the Colorado HKU1 sequences formed a unique subclade defined by three amino acid substitutions (W197F, F613Y and S752F) in the spike glycoprotein and exhibited a unique signature in the acidic tandem repeat in the N-terminal region of the nsp3 subdomain. Elucidating the function of and mechanisms responsible for the formation of these varying tandem repeats will increase our understanding of the replication process and pathogenicity of HKU1 and potentially of other coronaviruses.

First description of group A rotavirus from fecal samples of ostriches (Struthio camelus)

This study investigated the occurrence of rotavirus infections in ostriches (Struthio camelus) reared in Northern Paraná, Brazil. Fecal (n=66) and serum (n=182) samples from nine farms located in four different cities were analyzed by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), RT-PCR assay, virus isolation, and counterimmunoelectroosmophoresis (CIE). Rotavirus group A seropositivity occurred in 5.49% (10/182) of serum samples of ostriches originated from two farms. Only 9.09% (6/66) of fecal samples from ostriches with diarrhea maintained in one farm were positive by ss-PAGE, RT-PCR, and virus isolation. The G (VP7) and P (VP4) genotypes of rotavirus wild strains isolated in cell culture were determined by multiplex-nested PCR. The genotyping identified two rotavirus strains: G6P[1] and G10P[1]. In three rotavirus strains it was only possible to identify the P type; one strain being P[1] and two strains that presented the combination of P[1]+P[7]. These findings might represent the first characterization of rotavirus in ostriches, and the finding of porcine and bovine-like rotavirus genotypes in ostriches might suggest virus reassortment and possible interspecies transmission.

Isolation of rotavirus from asymptomatic dogs in Brazil

is subdivided both serologically, based on the inner capsid VP6 protein, and electropherotypically, based on the arrangement of the 11 dsRNA segments, into seven groups (A to G). Groups A, B, and C are found in both humans and animals, whereas groups D, E, F, and G have been found only in animals to date. In most cases, the electrophoretic pattern of the genome of group A rotaviruses is composed of four high-molecular-weight dsRNA segments (numbered 1 to 4), two middle-sized segments (5 and 6), a distinctive triplet of segments (7 to 9), and two smaller segments (10 and 11) (Estes and Kapikian, 2007). Canine rotavirus most often causes mild enteritis, especially in pups younger than two weeks, but the virus is also found in health animals (Schwers et al., 1983; Mochizuki et al., 1986). To date, only 10 isolates of canine rotavirus have been reported, including three isolates from the USA, two from France, two from Japan, two from Italy, and one from Korea (England and Poston, 1980; Fulton et al., 1981; Hoshino et al., 1982; Hoyois et al., 1982; Mochizuki and Hsuan,