Cláudio Wageck Canal

Detection and identification of salmonellas from poultry-related samples by PCR.

A polymerase chain reaction (PCR) assay was developed for the generic detection of Salmonella sp. and the identification of S. Enteritidis (SE), S. Gallinarum (SG), S. Pullorum (SP) and S. Typhimurium (ST) in material collected in the field from poultry. The specificity and sensitivity of the assay combined with Rappaport-Vassiliadis selective enrichment broth (PCR-RV) were determined, and field samples were analyzed to verify the validity of the method application. Specificity of the assay was tested using 29 SE, 11 SG, 10 ST and 10 SP strains, along with 75 strains of 28 other Salmonella serovars and 21 strains of other bacterial genera. The assay was 100% specific for Salmonella detection and ST identification. The primer pair for SE, SG and SP also detected S. Berta. PCR detection limits for Salmonella at the genus level were 2 ST, 8 SE, 1.1x10(3) SG and 1.8x10(5) SP cells. At the serovar level, detection limits were 7 ST, 1.2x10(3) SE, 4.4x10(7) SG and 1.8x10(6) SP cells. At the genus level, PCR-RV detected approximately 128% more positive field samples than the standard microbiological techniques and results were ready in 48h instead of 7 days. PCR-RV method is diagnostic of Salmonella at the genus level and ST at the serovar level, although other tests are needed to identify SE, SG and SP to serovar level.

Detection of antibodies to bovine viral diarrhoea virus (BVDV) and characterization of genomes of BVDV from brazil

An ELISA for the detection of antibodies to bovine viral diarrhea virus (BVDV) was developed based on antigens derived from a genotype I BVDV strain isolated in Switzerland. Using monoclonal antibodies we showed that this antigen contained the conserved non-structural protein NS3 whereas it essentially lacked the more strain-specific E2 surface glycoprotein. This ELISA has a sensitivity of 97.5% and a specificity of 99.2% as compared to the serum neutralization test (SNT). Preliminary experiments showed that this ELISA reliably detects antibodies to BVDV strains circulating in Brazil. Serum samples obtained from 430 adult cattle on 19 farms of the State of Rio Grande do Sul (Brazil) and one farm from Corrientes (Argentina) were tested for antibodies by means of this ELISA. We found antibodies in 56% +/- 15.1% of the cattle sera tested, which indicates that, in Brazil, the prevalence of infection with BVDV is similar to that found in Europe and the USA. Our sequence analysis of two BVDV isolates showed that BVDV of both genotypes I and II circulate in Brazil.

Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection

Aims: To compare PCR combined with enrichment media with the standard microbiological techniques (SMT) and to determine the most sensitive method for the detection of Salmonella and the identification of Salm. typhimurium (ST), Salm. enteritidis (SE), Salm. gallinarum (SG) and Salm. pullorum (SP).

Serological Characterization and Prevalence of spvR Genes in Salmonella Isolated from Foods Involved in Outbreaks in Brazil

Salmonella strains (n = 75) isolated from foods involved in foodborne outbreaks occurred in Rio Grande do Sul State, Brazil, during 1999 and 2000 were studied. Strains were serotyped and submitted to PCR analysis to verify the prevalence of Salmonella plasmid virulence (spvR) regulatory gene. Among the 75 isolates, 73 (97%) were classified as Salmonella enterica serovar Enteritidis. All of the Salmonella strains isolated in 1999 were classified as serotype Enteritidis, whereas in 2000 two isolates were serotyped as Salmonella Derby and Salmonella Typhimurium. Regarding the prevalence of spvR gene, 62 strains (82.7%) were PCR positive, and a positive correlation (P < 0.05) between the strains of Salmonella Enteritidis and the presence of spvR gene was demonstrated, which suggests that this gene is a characteristic of the Salmonella Enteritidis analyzed.

Antimicrobial resistance and subtyping of Salmonella enterica subspecies enterica serovar Enteritidis isolated from human outbreaks and poultry in southern Brazil

To investigate antimicrobial resistance, 96 Salmonella enterica subspecies enterica serovar Enteritidis strains isolated from salmonellosis outbreaks and poultry-related products obtained in southern Brazil were analyzed. Macrorestriction patterns, obtained by pulsed-field gel electrophoresis and phage types, were assessed. Although 43.75% of samples were sensitive to all drugs tested, resistance to sulfonamide (34.37%), trimethoprim-sulfamethoxazole (25.00%), nalidixic acid (14.58%), streptomycin (2.08%), gentamicin, and tetracycline (1.04%) was identified. Furthermore, 89.60% of strains belonged to phage type 4, and a predominant pulsed-field gel electrophoresis genotype represented by 82.29% of the strains was identified, suggesting that a clonal group was distributed in poultry, food, and human isolates. Although it was not possible to associate strains from different sources, the occurrence of antimicrobial-resistant Salmonella Enteritidis strains supports the need to establish monitoring programs to identify the emergence of potential resistance patterns and to direct policies for use of these drugs in food-producing animals.

Typing of canine parvovirus strains circulating in Brazil between 2008 and 2010.

Abstract Detection and characterisation of the canine parvovirus (CPV-2) strains that are currently circulating are essential for the understanding of viral evolution and the development of measures to control its spread. In the present study, stool samples from 144 dogs were analysed by polymerase chain reaction (PCR) for CPV-2, and 29.2% (42/144) of them were positive. From the 42 positive strains, 71.4% (30) of the dogs had signs of haemorrhagic gastroenteritis. The sequencing of the 583bp fragment of the VP2 gene from the positive strains identified 78.6% (33/42) of them as type 2c, 19% (8/42) as type 2b and 2.4% (1/42) as type 2a. A phylogenetic analysis of the variants circulating in the canine population of Brazil showed that they are very similar to those found in other countries and type 2c has become the predominant type circulating in Brazil.

High rate of viral evolution in the capsid protein of porcine parvovirus

In recent years, it has been shown that some parvoviruses exhibit high substitution rates, close to those of RNA viruses. In order to monitor and determine new mutations in porcine parvovirus (PPV), recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analysed. These samples, together with sequences retrieved from GenBank, were included in three datasets, consisting of the complete NS1 and VP1 genes and a partial VP1 gene. For each dataset, the nucleotide substitution rate and the molecular clock were determined. Analysis of the PPV field isolates revealed that a recently described amino acid substitution, S436T, appeared to be common in the VP2 protein in the Austrian, Brazilian and German virus populations. Furthermore, new amino acid substitutions were identified, located mainly in the viral capsid loops. By inferring the evolutionary dynamics of the PPV sequences, nucleotide substitution rates of approximately 10(-5) substitutions per site per year for the non-structural protein gene and 10(-4) substitutions per site per year for the capsid protein gene (for both viral protein datasets) were found. The latter rate is similar to those commonly found in RNA viruses. An association of the phylogenetic tree with the molecular clock analysis revealed that the mutations on which the divergence for both capsid proteins was based occurred in the past 30 years. Based on these findings, it was concluded that PPV variants are continuously evolving and that vaccines, which are based mainly on strains isolated about 30 years ago, should perhaps be updated.

Detection of virulence genes in Salmonella Enteritidis isolated from different sources

The presence of three virulence genes, invA, spvR, and spvC, was determined in Salmonella Enteritidis isolated from poultry, pigs, humans and food. All isolates were positive for the invA gene, with 91.2% being positive for spvR and 90.2% for spvC. There was no significant difference in the prevalence of the virulence genes between isolates from different sources. The results indicate that there is a putative high virulence potential for the S. Enteritidis isolates characterized.

First detection of canine parvovirus type 2c in Brazil

The presence of canine parvovirus type 2 (CPV-2), 2a and 2b has been described in Brazil, however, the type 2c had not been reported until now. In the current study, seven out of nine samples from dogs with diarrhea were characterized as CPV-2c, indicating that this virus is already circulating in the Brazilian canine population.

Effects of prebiotics and probiotics on the colonization and immune response of broiler chickens challenged with Salmonella Enteritidis

The present study aimed at evaluating the effect of the prebiotic Bio Mos™ (2 kg/ton up to 10 days; 1 kg/ton from 10 to 21 days; and 0.5 kg/ ton from 21 days to slaughter), of the probiotic Lac XCL 5x™ (by spraymixing), of a combination of the two supplements (prebiotic + probiotic), and of one growth promoter antimicrobial agent (Avilamycin at 15 ppm). Birds were orally challenged with inoculated Salmonella Enteritidis (SE) 10 6 CFU at 3 days of age. Four hundred and eighty male Ross chicks were used. The experiment lasted 28 days, and the analyses were conducted at 15 and 28 days of age. Cecum and liver bacterial colonization of production of anti-SE antibiodies, intestinal micrometry and bird performance were assessed. Neither the prebiotic, nor the probiotic influenced performance or production of anti-SE antibodies in SE-challenged birds. Intestinal micrometry and bird mortality were not influenced by prebiotic or probiotic supplementation, or by the antimicrobial agent. The use of an antimicrobial agent produced higher SE CFUs in cecum bacterial counts, while prebiotic and probiotic yielded lower counts. The combination prebiotic+probiotic did not produce significantly different results from the individual use of the additives.