Aline Fernandes Barry

First detection of kobuvirus in farm animals in Brazil and the Netherlands

Animal kobuviruses have been described in pigs, cattle, sheep and bats in countries in Asia and Europe. The virus can be detected in fecal and serum samples of infected animals with or without diarrhea, but most of the clinical as well as epidemiological features of kobuvirus infection are still unknown. This study reports the first detection of kobuvirus in farm animals from Brazil and the Netherlands and the molecular analysis of the detected strains. In Brazil, 53% (61/115) of the pigs (suckling, weaned and sows) were shedding porcine kobuvirus in feces, while in the Netherlands 16.7% (3/18) of the tested weaned pigs were infected. Kobuviruses detected in fecal samples of pigs in Brazil showed association (p=0.0002) with diarrhea. In pig serum, kobuvirus was detected at different ages (3, 21, 36, 60, 75, and 180days), with an overall rate of 76.7% (23/30). The sequencing of amplicons detected in serum of pigs of different ages suggested reinfection and no persistent infection. Kobuvirus was also detected in sheep and cattle feces from Brazil and the Netherlands, respectively. Phylogenetic analyses of Brazilian and Dutch kobuviruses from pig, cattle and sheep revealed genetic variability, particularly in one strain detected in sheep feces, which was more closely related to human Aichi virus. The molecular and phylogenetic analyses performed with other published kobuvirus strains and the strains presented in this study, showed that, in most of the cases, kobuvirus seems to group according to host species, but not to geographical region of origin. The data presented in this study contribute to the comprehension of kobuvirus epidemiology and also to the molecular identification of kobuvirus strains circulating worldwide.

High genetic diversity in RdRp gene of Brazilian porcine sapovirus strains

Sapovirus is one genus within Caliciviridae family that causes diarrhea in humans and animals. Sapovirus (SaV) has been classified into seven genogroups (GI to GVII). The GIII, GVI, and GVII, which prototype is Cowden, JJ681, and K7/JP strains, respectively, infect pigs. The objective of this study was to characterize wild-type Brazilian SaV strains from piglet stool samples and determine SaV infection frequency, age distribution and association with diarrheic disease. Stool samples from 113 piglets up to 28-days-old were collected from 34 pig farms located in the States of Minas Gerais (MG), Mato Grosso do Sul (MS), Paraná (PR), Santa Catarina (SC), and Rio Grande do Sul (RS), during 2004 and 2005. The specimens were evaluated for enteric calicivirus by RT-PCR assay with primers p289/290, designed to detect the polymerase gene of SaV and norovirus. Thirty four (30.1%) samples were positive for SaV and five amplicons were sequenced. Phylogenetic analyses placed BRA29-MS/04 and BRA52-PR/05 sequences into the GIII of SaV genus. BRA04-SC/04, BRA21-RS/04, and BRA37-MG/05 demonstrated low identity with the Cowden strain but were closely related (up to 86.3%) to the Japanese and Dutch SaV strains, grouping together in a new cluster (GVIII?) in the phylogenetic tree. SaV infection was detected more frequently (p=0.0001) in animals between 22 and 28 days of age, in equal frequencies in piglets with and without diarrhea (p=0.59), and in the five Brazilian States. In this study, such as other unclassified worldwide SaVs, the Brazilian strains showed high genetic variability. Furthermore, the distribution and frequency of SaV infection provides evidence that the virus is circulating in Brazilian pig herds.

Molecular detection and characterization of hepatitis E virus in naturally infected pigs from Brazilian herds.

The aim of this study was to investigate the presence of swine hepatitis E virus (HEV) from pigs of different production categories and from different pig farms in South Brazil. A total of 170 porcine faecal samples from breeder sows, boars, suckling piglets, weaned and growing pigs were collected from 14 pig farms. The faecal samples were screened by nested RT-PCR using primers targeting the ORF2 region of HEV genome. The samples that were positive from this screening were used in a nested RT-PCR targeting the ORF1 region. The screening detected HEV RNA in 62.5% of the pig farms and in 15.3% of the faecal samples. In 15 faecal samples, it was possible to amplify the HEV RNA with both the ORF1 and ORF2 regions. The phylogenetic analyses obtained for both ORFs confirmed that all of the Brazilian swine HEV isolates clustered with genotype 3b, the same genotype described previously in humans in Brazil.

Frequency of BCoV detection by a semi-nested PCR assay in faeces of calves from Brazilian cattle herds.

Bovine coronavirus (BCoV) is one of the main causes of neonatal calf diarrhoea. Several diagnostic assays have been employed to detect the presence of the virus in stool samples from calves. Despite this, the frequency of BCoV infection among Brazilian and even South American cattle herds has yet to be well characterised. This study describes the occurrence of BCoV infection among calves from dairy and beef herds in four Brazilian states. A total of 282 stool samples from 1 to 60-day-old calves were evaluated for the presence of BCoV by a semi-nested (SN) PCR assay. The animals were from herds (n = 23) located in three geographical regions in Brazil (south, southeast, and center-west). The specific BCoV amplicon was detected in 15.6% (44/282) of the faecal specimens examined, of which 95.4% (42/44) were from diarrhoeic and 4.6% (2/44) from asymptomatic calves. The specificity of the SN-PCR amplicons was evaluated by restriction fragment length polymorphism (RFLP) analysis. The results show that the BCoV is widespread, mainly among calves from 16 to 30-days-old (p = 0.0023), and verify the association between BCoV infection and clinical signs of diarrhoea (p = 0.005). These findings emphasise the importance of this virus in enteric infections of Brazilian cattle herds.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

Frequency of group a rotavirus in piglet stool samples from non-vaccinated Brazilian pig herds

ABSTRACT Neonatal diarrhea is one of the main causes of morbidity and mortality in piglets, and it leads to significant economic losses for pig farmers worldwide. The aim of this study was to determine the frequency of diagnosis, age group, and association of group A rotavirus (GARV) infection with diarrhea in piglets from pig herds in two (south and center-west)Brazilian geographical regions. The frequency of GARV diagnosis was evaluated between 2004 and 2007, using SS-PAGE on 681 fecal samples (428 diarrheic and 253 with normal consistency) from 1-4 week-old piglets. The animals were selected from 130 pig herds and 80 counties in the states of Rio Grande do Sul, Santa Catarina, Parana, Mato Grosso do Sul, and Mato Grosso, Brazil. None of the herds were vaccinated against porcine GARV. Rotaviruses with the typical GARV electrophoretic pattern was identified in 193 (28.3%) fecal samples, and of these, 157 (81.3%) were diarrheic (p=0.001).Porcine GARV infection was identified in animals from all age groups evaluated, and the highest infection rate (54.7%; p=0.001) occurred in diarrheic piglets between 21 and 28 days of age. Diarrheic feces from 1-7 day-old piglets also had a high rate of rotavirus presence (32.3%), suggesting a failure in passive immunity. The high rate of porcine GARV infection in all geographical regions studied demonstrates the involvement of rotavirus in the etiology of neonatal diarrhea in Brazilian pig herds. This study highlights the importance of GARV infection for pig raising and the need of control and prophylactic measures for porcine rotavirus infection, including vaccination in the main areas of pork production in Brazil. Key words: piglet, neonatal diarrhea, group A rotavirus, SS-PAGE

Detection and phylogenetic analysis of porcine enteric calicivirus, genetically related to the Cowden strain of sapovirus genogroup III, in Brazilian swine herds

Sapovirus of the Caliciviridae family is an important agent of acute gastroenteritis in children and piglets. The Sapovirus genus is divided into seven genogroups (G), and strains from the GIII, GVI and GVII are associated with infections in swine. Despite the high prevalence in some countries, there are no studies related to the presence of porcine enteric sapovirus infections in piglets in Brazil. In the present study, 18 fecal specimens from piglets up to 28 days were examined to determine the presence of sapovirus genome by RT-PCR assay, using primers designed to amplify a 331 bp segment of the RNA polymerase gene. In 44.4% (8/18) of fecal samples, an amplified DNA fragment was obtained. One of these fragments was sequenced and submitted to molecular and phylogenetic analysis. This analysis revealed high similarity, with nucleotides (87%) and amino acids (97.8%), to the Cowden strain, the GIII prototype of porcine enteric calicivirus. This is the first description of sapovirus in Brazilian swine herds.

VP6 gene diversity in Brazilian strains of porcine group C rotavirus.

Group C rotavirus (RV-C) has been found in Brazilian pig herds; however, wild-type strains have not yet been characterized. We made a molecular analysis of a region of gene 5 in Brazilian RV-C strains. Stool samples from 11 piglets (diarrheic and with normal consistency) positive for the RV-C VP6 gene in an RT-PCR assay were sequenced. A 270-bp amplicon of nine sequences was analyzed. All sequences showed high identity to the Cowden strain of the porcine RV-C prototype and 81.3 to 94.3% to each other (230 nucleotide fragment). Three Brazilian strains were classified in the Cowden group, while the other six showed higher heterogeneity (84.3 to 87.3%) with the prototype strain. Four clusters were formed in the dendrogram, including one human, one bovine, and two porcine clusters; one of these was formed by the six Brazilian strains described in this study. The Brazilian RV-C strains described here did not show any association with the year of collection, the presence of diarrhea, the age of the pig, or the geographical region of herd origin. This strongly suggests that these heterogeneous strains are widely spread in Brazilian pig herds. We conclude that there is genetic polymorphism in the VP6 gene of porcine RV-C strains in Brazil.

Intestinal lesions in pigs affected with postweaning multisystemic wasting syndrome.

Samples of mesenteric lymph nodes and intestines from 79 unthrifty 3- to 5-month-old postweaning pigs, confirmed as naturally affected with postweaning multisystemic wasting syndrome (PMWS), were studied. Pigs originated from 12 farms in southern Brazil and were selected on the basis of clinical signs and/or gross lesions suggestive of enteric disorder. Lymphohistiocytic infiltrates of varying intensity were associated with anti-porcine circovirus type 2 (anti-PCV2) immunostaining (IS) in samples of intestines and mesenteric lymph nodes from all pigs. Although most findings were similar to those described in PCV2-associated enteritis, anti-PCV2 IS in association with depletion of the goblet cell mucin stores (24 pigs), diffuse ileal villous atrophy and fusion (18 pigs), and dilatation of the lymphatic vessels (11 pigs) combined or not with lymphangitis were also observed. PCV2 antigen was immunohistochemically demonstrated in the cytoplasm and nuclei from intralesional epithelial cells, histiocytes, and endothelial-like cells in intestinal tissues. Together these findings imply an association with PCV2. The presence of co-infections by Lawsonia intracellularis, Brachyspira spp., Mycobacterium spp., Salmonella spp., rotavirus, parvovirus, coronavirus and enteric calicivirus with PCV2 in the intestinal lesions was investigated.

Bovine coronavirus detection in a collection of diarrheic stool samples positive for group a bovine rotavirus

ABSTRACT Neonatal diarrhea is an important cause of economic losses for cattle farmers. The main viral etiologies of enteric diseases are group A rotaviruses (GARV) and the bovine coronavirus (BCoV). Although both viruses infect calves of the same age, the occurrence of mixed infections is still under studied. The present study describes the co-infection of BCoV and GARV in stool samples. Forty-four diarrheic fecal samples from calves up to 60 days old that had previously tested positive for GARV by SS-PAGE were analyzed using semi-nested PCR for BCoV. A product with 251 bp of the BCoV nucleoprotein gene was amplified in 15.9% (7/44) of the samples, demonstrating that co-infection is not an unusual event. These results reinforce the need for testing for both GARV and BCoV, even in fecal samples that previously tested positive for one virus. Key words: calf, diarrhea, BCoV, rotavirus, SN-PCR, co-infection * Author for correspondence: alfieri@uel.br INTRODUCTION Neonatal diarrhea is one of the main causes of calf mortality worldwide. The disease results in major economic losses in many dairy and beef cattle herds that result from treatment costs and calf deaths. Calf diarrhea is considered to be a multi-factorial disease. Several environmental, management-related, nutritional, and physiological factors may occur either alone or in synergy with different infectious agents such as protozoans, bacteria, and/or viruses. In calves, bovine group A rotavirus (GARV) and bovine coronavirus (BCoV) are the viruses most commonly associated with neonatal diarrhea and it is not unusual that both viruses can concomitantly infect calves (Snodgrass et al ., 1986; Holland, 1990; Alfieri et al ., 2006; Oliveira Filho et al ., 2007). Rotaviruses belong to the Reoviridae family. They have icosahedral symmetry and a non-enveloped capsid formed by three concentric layers of protein that is 70-90 nm in diameter. The rotavirus genome is composed of 11 double-stranded RNA segments (dsRNA). Based on the common group antigen (VP6 protein), rotaviruses are classified antigenically into seven serogroups (A-G). The co-migration of genomic segments 7, 8, and 9 in polyacrylamide gel electrophoresis (PAGE) is an important characteristic of group A rotaviruses (Estes, 1996). Several techniques have been developed to diagnose bovine GARV by detecting the viral