Carla Rosane Rodenbusch

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).

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.

DETECTION OF SALMONELLA SP. FROM PORCINE ORIGIN: A COMPARISON BETWEEN A PCR METHOD AND STANDARD MICROBIOLOGICAL TECHNIQUES

The aim of this study was to compare a polymerase chain reaction (PCR) method combined with selective enrichment in Rappaport-Vassiliadis broth (PCR-RVB) with standard microbiological techniques (SMT) for the generic detection of Salmonella in samples of porcine origin. Two hundred sixty eight field samples consisting of 42 sets of pooled porcine mandibular lymph nodes and tonsils, 44 samples of intestinal content, 38 pork sausage meat samples and 144 samples of feed collected from swine farms were submitted to the PCR-RVB and SMT protocols. Salmonella was detected in 54 samples using the PCR-RVB assay and in 42 samples by SMT, three of the SMT Salmonella-positive samples (one each of S. Derby, S. Panama and S. Typhimurium) being Salmonella-negative by PCR-RVB. For the PCR-RVB method 15 Salmonella-positive samples were negative by SMT, a significant difference according to the Mac Nemar’s chi-squared test (p=0.0153). Subsequent serological typing of the SMT isolates showed the following Salmonella serovars, the number of positive samples being given in parentheses: Typhimurium (12); Bredeney (10); Panama (5); Saint-paul (5); Minnesota (3); Mbandaka (2); Derby (1); Enteritidis (1); Orion (1) and Salmonella sp. (2). We concluded that, although the use of both PCR-RVB and SMT increased the number of positive samples, the PCR-RVB, due to its higher sensitivity and greater speed in giving results, can be implemented to detect Salmonella in samples of porcine origin.

Molecular diagnosis of Salmonella species in captive psittacine birds

Cloacal swabs were collected from 280 captive psittacine birds belonging to 13 species. Samples of dna were tested by pcr using a pair of primers that amplify a 284 base pair fragment of the Salmonella genus invA gene, and the pcr-positive samples were tested by standard microbiological techniques. Thirteen per cent of the samples were positive by pcr, but negative by microbiological techniques. The infection rates were significantly different among the 13 species, the most commonly infected being Amazona amazonica (28 per cent) and Amazona pretrei (20 per cent). Specific tests for Salmonella Typhimurium Salmonella Enteritidis, Salmonella Pullorum and Salmonella Gallinarum did not produce positive results.

Fibropapillomatosis in green turtles Chelonia mydas in Brazil: characteristics of tumors and virus

Fibropapillomatosis (FP) is a benign neoplasia that affects physiological functions of sea turtles and may lead to death. High prevalence of FP in sea turtle populations has prompted several research groups to study the disease and the associated herpesvirus, chelonid herpesvirus 5 (ChHV5). The present study detected and quantified ChHV5 in 153 fibropapilloma samples collected from green turtles Chelonia mydas on the Brazilian coast between 2009 and 2010 to characterize the relationship between viral load and tumor characteristics. Of the tumor samples collected, 73 and 87% were positive for ChHV5 in conventional PCR and real-time PCR, respectively, and viral loads ranged between 1 and 118.62 copies cell⁻¹. Thirty-three percent of turtles were mildly, 28% were moderately and 39% were severely affected with FP. Skin samples were used as negative control. High viral loads correlated positively with increasing FP severity in turtles sampled on the Brazilian coast and with samples from turtles found dead in the states of São Paulo and Bahia. Six viral variants were detected in tumor samples, 4 of which were similar to the Atlantic phylogenetic group. Two variants were similar to the western Atlantic/eastern Caribbean phylogenetic group. Co-infection in turtles with more than one variant was observed in the states of São Paulo and Bahia.

Detection and characterization of fibropapilloma associated herpesvirus of marine turtles in Rio Grande do Sul, Brazil

Fibropapillomatosis (FP) is a benign tumoral disease that affects sea turtles, hampering movement, sight and feeding, ultimately leading to death. In Brazil, the disease was described for the first time in 1986. Research suggests the involvement of a herpesvirus in association with environmental and genetic factors as causal agents of FP. The objective of the present study was to detect and characterize this herpesvirus in sea turtles living in the coast of state Rio Grande do Sul (RS), Brazil. From October 2008 to July 2010, 14 turtles were observed between the beaches of Torres and Tavares, of which 11 were green turtles (Chelonia mydas) and 3 were loggerhead turtles (Caretta caretta). All turtles were young and mean curved carapace length was 37.71±7.82cm, and varied from 31 to 55cm. Only one green turtle presented a 1cm, papillary, pigmented fibropapilloma. Skin and fibropapilloma samples were analyzed by conventional and real time PCR assays to detect and quantify herpesvirus. All skin samples were negative, though the fibropapilloma specimen was positive in both tests. Viral load was 9,917.04 copies of viral genome per milligram of tissue. The DNA fragment amplified from the fibropapilloma sample was sequenced and allocated in the Atlantic phylogeographic group. This study reports the first molecular characterization of herpesvirus associated with fibropapilloma in turtles from the coast of RS.

Marine leech Ozobranchus margoi parasitizing loggerhead turtle (Caretta caretta) in Rio Grande do Sul, Brazil

This paper reports the finding of several Ozobranchus margoi (Annelida: Hirudinea) parasitizing a loggerhead turtle (Caretta caretta) that was found in the municipality of Tavares, state of Rio Grande do Sul, southern Brazil. Since this parasite is considered to be a vector of chelonid herpesvirus 5 (ChHV-5), the leeches collected were tested for the presence of this virus. All the specimens were negative on PCR analysis. Although O. margoi is considered to be a common sea turtle parasite, this is the first official record describing collection of this parasite from a loggerhead turtle in southern Brazil, within the countrys subtropical zone. This finding draws attention to the presence of this parasite and to the risk of leech-borne infectious diseases among turtles found along the coast of southern Brazil.

Spatial and temporal description of laboratory diagnosis of bovine rabies in the state of Rio Grande do Sul, Brazil

Background: Rabies remains one of the most important zoonosis worldwide and in Brazil. In the Brazilian state of Rio Grande do Sul (RS) the disease is one of the main causes of death in bovine herds. The confirmation of suspected rabies cases upon clinical examination depends on specific laboratory diagnosis. The Instituto de Pesquisas Veterinarias Desiderio Finamor (IPVDF) is the reference laboratory in RS to diagnose rabies. The objective of the present study was to present a spatial-temporal description of diagnostic procedures of rabies in cattle in the years 2011 to 2015 in RS. Materials, Methods & Results: The results of diagnosis procedures of rabies in cattle carried out in the Laboratory of Virology, IPVDF, between January 2011 and December 2015 were listed. Direct immunofluorescence (DIF) and biological assays for confirming rabies cases were used. Cattle population density per area of a municipality, as well as date, species involved, location of suspected case, and diagnosis were obtained from exams and from official rabies record, and input to a spreadsheet file. Spatial distribution maps of cattle rabies confirmed in laboratory and cattle population density per municipality were constructed using the software TerraView 4.2.2 (INPE). In the study period, 998 samples of brains of cattle presenting neurological symptoms compatible with rabies, of which 582 (58%) were confirmed in the laboratory. The frequency of positive cases on a yearly basis varied between 41 and 65%, with statistically lower number of positives in 2011 (P > 0.05). The annual incidence of rabies for a population of 10,000,000 bovines was 33, 88, 112, 108, and 79, respectively, for the years 2011 to 2015, in that order. A positive correlation was observed between the number of samples analyzed and the incidence of positive cases in the study period, and statistically significant difference in incidence between years (P > 0.05). Samples were from 175 municipalities, of which 114 (65%) had at least one confirmed rabies case and 61 did not have any. The five municipalities that sent most samples for analysis were Viamao (n = 66), Montenegro (n = 40), Camaqua (n = 39), Gravatai (n = 38), and Sao Lourenco do Sul (n = 37). The spatial distribution of rabies in cattle in a year did not follow a defined pattern, though most cases confirmed in the laboratory occurred in the greater Porto Alegre region and in southeast RS. Discussion: High incidence of rabies was observed in 2012 and 2013, and in the two following years the number of cases confirmed in laboratory remained high, compared with 2011. The high incidence of rabies cases confirmed in laboratory correlated with the number of samples analyzed. The fall in vaccine coverage during the study period may also have prompted the increase in suspected cases and in the number of samples examined in the laboratory. A large number of the municipalities presenting rabies cases in cattle was in the greater Porto Alegre region and in southeast RS, which are areas of high and intermediate risk for the disease, according to a previous epidemiological modeling study. The spatial distribution of positive cases was not associated with the cattle population density. The large number of rabies in cattle in RS underscores the importance of the disease and the high risk of exposure, both to humans and animals. The results presented herein may help define control and prevention actions against rabies in RS.