Carlos Tadeu Pippi Salle

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.

Antibiotic resistance in Salmonella Enteritidis isolated from broiler carcasses

Eighty Salmonella Enteritidis strains isolated from broiler carcasses between May 1995 and April 1996 in the State of Rio Grande do Sul, Brazil, were tested for antibiotic susceptibility using the disk diffusion method. Resistance to colistin, novobiocin, erythromycin and tetracycline was observed in 100% of the isolates. The strains showed intermediate resistance at different levels to kanamycin (1.25%), enrofloxacin (3.75%), neomycin (3.75%), fosfomycin (20%), sulphonamides (86.25%) and nitrofurantoin (90%). Resistance to ciprofloxacin, norfloxacin, gentamicin, polymyxin B, sulphametrim and sulphazotrim was not found. Since resistance to antibiotics especially those introduced in the last decades, was detected, it is recommended that their use must be based on the results of resistance tests or minimum inhibitory concentration tests.

Polymerase chain reaction (PCR) for the detection of Salmonella in artificially inoculated chicken meat

The aim of this study was to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in artificially contaminated chicken meat. Tests were performed with different dilutions of Salmonella Typhimurium or Salmonella Enteritidis cells (10(-7), 10(-8) or 10(-9) CFU/mL) inoculated in chicken meat samples, in order to establish the limits of detection, incubation times (0, 6, 8 and 24 hours of pre-enrichment in PBW 1%) and three DNA extraction protocols (phenol-chloroform, thermal treatment and thermal treatment and Sephaglass). The assay was able to detect until 10(-9) CFU/mL of initial dilution of Salmonella cells inoculated in chicken meat, which allows detection of Salmonella within 48 hours, including 24 hours of pre-enrichment and using the phenol-chloroform DNA extraction protocol. As the results are obtained in a shorter time period than that of microbiological culture, this procedure will be useful in the methodology for detection of Salmonella in chicken.

Inoculation of newly hatched broiler chicks with two Brazilian isolates of Salmonella Heidelberg strains with different virulence gene profiles, antimicrobial resistance, and pulsed field gel electrophoresis patterns to intestinal changes evaluation

Salmonella Heidelberg is one of the 3 most frequently isolated serovars from human Salmonella cases in Canada, and the fourth most commonly reported Salmonella serovar in human foodborne disease cases in the United States. Since 1962, Salmonella Heidelberg has been isolated and reported in poultry and poultry products in Brazil. The poultry industry has focused efforts on reducing salmonellae incidence in live production in an effort to reduce Salmonella in the processing plant. A better understanding of the initial infection in chicks could provide approaches to control Salmonella contamination. The objective of the present study was to evaluate 2 Salmonella Heidelberg strains that differed in the presence of virulence genes invA, agfA, and lpfA; antimicrobial resistance profiles; and epidemiologic profiles on aspects of pathogenicity and intestinal morphology. Newly hatched broiler chicks were inoculated with 2 strains (SH23 and SH35) of Salmonella Heidelberg and cecal morphometry, histopathology, electron microscopy, and bacterial counts in the liver and cecum were assessed. The SH23 and SH35 strains resulted in different changes in villi height and crypt depth and inflammatory cell infiltration in the cecum. The SH35 group had higher liver and cecum bacterial cell counts when compared with SH23 strains.

Occurrence and Characterization of Campylobacter in the Brazilian Production and Processing of Broilers

Abstract Twenty-two commercial broiler flocks and their carcasses, totaling 546 samples (450 collected from a poultry farm and 96 from a slaughterhouse), were surveyed for the presence of Campylobacter. The positive results for Campylobacter among the analyzed samples were homogeneous, yielding 81.8% for cecal droppings, 80.9% for feces, and 80.4% for cloacal swabs. Pre-enrichment and direct plating showed that 77.85% and 81.8% of cloacal swabs, respectively, were positive for Campylobacter compared to 99.0% and 97.9% of carcasses testing positive with the pre-enrichment and direct plating methods. The Campylobacter count averaged 7.0 log10 colony-forming units (CFU)/g in cecal droppings, 5.15 log10 CFU/carcass after defeathering, and 4.24 log10 CFU/carcass after chilling. The samples were identified by the API Campy system as Campylobacter jejuni subsp. jejuni (68.8%), Campylobacter coli (8.3%), Campylobacter jejuni subsp. doylei (6.3%), Campylobacter upsaliensis (4.2%), and Campylobacter fetus subsp. fetus (2.1%). The analyzed broiler flocks were positive for Campylobacter in 81.8% of the cases, thus characterizing the occurrence of this pathogen in a broiler-producing region in southern Brazil. These results highlight the importance of programs targeted at the reduction of Campylobacter in poultry products, in order to minimize the risks for consumers. Abbreviations: CFU = colony-forming units; mCCDA = modified charcoal cefoperazone deoxycholate agar; TTC = triphenyl tetrazolium chloride Nota de Investigación—Ocurrencia y caracterización de Campylobacter en pollos de engorde procesados y en producción. Para evaluar la presencia de Campylobacter, se realizó una encuesta en 22 lotes comerciales de pollos de engorde y sus canales, con un total de 546 muestras de las cuales 450 fueron tomadas de una granja de pollos y 96 de la planta de proceso. Entre las muestras analizadas, los resultados positivos para Campylobacter fueron homogéneos, encontrándose un 81.8% en las heces cecales, 80.9% en las heces y 80.4% en los hisopos cloacales. Los resultados obtenidos mediante pre-enriquecimiento y siembra directa mostraron una positividad para el Campylobacter del 77.85% y 81.8%, respectivamente, para los hisopos cloacales, comparado con el 99.0% y 97.9% de positividad para las canales. Los promedios de los recuentos de Campylobacter fueron 7.0 log10 unidades formadoras de colonia (UFC) por gramo en las heces cecales, 5.15 log10 UFC en las canales después del desplume, y 4.24 log10 UFC para las canales después del enfriamiento. Mediante el sistema API Campy, las muestras fueron identificadas como Campylobacter jejuni subsp. jejuni (68.8%), Campylobacter coli (8.3%), Campylobacter jejuni subsp. doylei (6.3%), Campylobacter upsaliensis (4.2%), y Campylobacter fetus subsp. fetus (2.1%). Los lotes de pollos analizados fueron positivos a Campylobacter en el 81.8% de los casos, caracterizando de esta manera la ocurrencia de este patógeno en la región del sur del Brasil productora de pollos. Estos resultados enfatizan la importancia de establecer programas enfocados a la reducción del Campylobacter en los productos avícolas, con el objeto de disminuir el riesgo para los consumidores.

Diagnosis of skin lesions in condemned or downgraded broiler carcasses — a microscopic and macroscopic study

Eight hundred skin samples from broiler carcasses condemned or downgraded for skin lesions were collected at five processing plants. Histologically, 45.25% were cellulitis, 19.00% were atypical fowlpox, 3.25% were dermal squamous cell carcinoma (DSCC), 17.00% were non-specific dermatitis, 0.5% were focal haemorrhages and 0.25% were haemangiomas. Of the samples, 14.75% were histologically normal. Macroscopically, increased thickening of the skin was a frequent finding occurring in all the skin diseases, as well as skin discoloration, dark nodules in feather follicles and crusts. Skin scratches were associated with all of the diseases, except DSCC. Crater-like ulcers occurred in DSCC and fowlpox. Cellulitis was more frequent on the abdomen, fowlpox on the dorsum, DSCC in thighs, and non-specific dermatitis on the dorsum, thighs, legs and breast. It was concluded that macroscopic changes are not specific and examinations at slaughterhouses do not allow classification of broiler skin diseases. Histological examination appears to be an important tool to provide more accurate data.

Effects of Probiotics and Maternal Vaccination on Salmonella Enteritidis Infection in Broiler Chicks

Abstract The effects of probiotics and maternal vaccination with an inactivated Salmonella Enteritidis (SE) vaccine on day-old chicks challenged with SE were evaluated. A 2 × 3 factorial arrangement was used (with or without probiotics; breeders nonvaccinated, vaccinated intramuscularly, or vaccinated intraperitoneally). Three trials were conducted in isolation cabinets and SE challenge was different between trials. The number of SE organisms per chick and the time interval between housing and introduction of seeder birds (hereafter called challenge) were 1.6 × 108 and 1 hr (Trial I), 1.8 × 106 and 12 hr (Trial II), and 1.2 × 104 and 24 hr (Trial III). SE recovery was assessed in ceca and liver at 3, 5, and 7 days postchallenge, and the number of colony-forming units (CFU) in ceca was evaluated at 5 and 7 days postchallenge. The number of SE (log CFU) in the ceca reduced 0.56 log (from 7.59 to 7.03) and 1.45 log (7.62 to 6.17) because of the treatment with probiotics in Trials II and III, respectively. The greater reduction in Trial III indicates the importance of the early use of probiotics on the prevention of SE infection. Treatment with probiotics resulted in a smaller number of SE-positive livers after 5 days postchallenge on Trial III. Although there was no significant effect of maternal vaccination on the number of SE CFU in the ceca, a significant effect of maternal vaccination on the SE CFU was observed in the liver, but not in the ceca at 5 days after challenge.

Use of artificial neural networks to estimate production variables of broilers breeders in the production phase

1. Although the poultry industry uses state-of-the-art equipment and up-to-date services, in Brazil it generally makes decisions involving all its production variables based on purely subjective criteria. This paper reports the use of artificial neural networks to estimate performance in production birds belonging to a South Brazilian poultry farm. 2. Recorded data from 22 broiler production breeder flocks were obtained, from April, 1998 to December, 1999, which corresponded to 689 data lines of weekly recordings. 3. These data were processed by artificial neural networks using the software NeuroShell 2® version 4·0TM (Ward Systems Group®). The artificial neural network models generated were compared and selected based on their largest determination coefficient (R 2), lowest Mean Squared Error (MSE), as well as on a uniform scatter in the residual plots. The authors conclude that it is possible to explain the performance variables of production birds, with the use of artificial neural networks. 4. The method allows the decisions made by the technical staff to be based on objective, scientific criteria, allows simulations of the consequences related to these decisions, and reports the contribution of each variable to the variables under study.

Behavior of Salmonella heidelberg and Salmonella enteritidis strains following broiler chick inoculation: evaluation of cecal morphometry, liver and cecum bacterial counts and fecal excretion patterns

Over the years, Salmonella Heidelberg (SH) has gained prominence in North America poultry production and in the poultry production of other countries. Salmonella Heidelberg has been isolated and reported from poultry and poultry products in Brazil since 1962, whereas Salmonella Enteritidis (SE) has only emerged as a serious problem in poultry and public health since 1993. These strains of Salmonella can cause intestinal problems in newly hatched chicks, and infection may persist until adulthood. Upon slaughter of chickens, Salmonella can contaminate carcasses, a condition that poses a threat to human health. The aim of this study was to compare the fecal excretion of Salmonella Enteritidis and Salmonella Heidelberg in newly hatched chicks (orally inoculated with 105ufc/mL each) until 20 days of age. In addition, the ratio of cecal villus height:crypt depth (morphometry) and liver and cecum cell counts was analyzed in chicks ranging from 0 to 3 days of age and infected with these two Salmonella strains. One hundred seventeen chicks were separated into one of three experimental groups: a control group, an SE-infected group and an SH-infected group. Eight chicks per group were euthanized at 6, 12 and 72 hours post-inoculation (pi) to allow for Salmonella isolation from the liver and cecum and for the collection of the cecum for villi and crypt analysis. Other birds were allowed to mature to 20 days of age and cloacal swabs were taken at 2, 6, 13 and 20 days pi to compare the fecal excretion of inoculated strains. The Salmonella Enteritidis group had a higher number of cells excreted during the trial. Both strains were isolated from the liver and cecum by 6h pi. At 12h pi the Salmonella Heidelberg group had high cell counts in the cecum. No difference was found in liver cell counts. Both strains showed lower villus height:crypt depth ratio than the control group post-infection.

Prevalence of antibodies against Ornithobacterium rhinotracheale in broilers and breeders in Southern Brazil.

Abstract In this investigation, we determined the prevalence of the Ornithobacterium rhinotracheale (ORT) infection in broilers and broiler breeders in southern Brazil. We also correlated the presence of antibodies in broilers with performance. Sera from 1550 broilers from 50 flocks were collected during the slaughter time in nine companies with federal veterinary inspection of the state of Rio Grande do Sul. Sera from 480 meat-type breeders of 40 flocks from 14 companies in southern Brazil were also analyzed by enzyme-linked immunosorbent assay, and the prevalence of antibodies was determined. The prevalence of ORT antibodies in broiler flocks was 63.83%, but in each individual flock only 6.52% of the birds were positive. The prevalence in broiler breeder flocks was 100.00%, and in each individual flock 94.62% of the birds were positive. There was a positive correlation between the presence of antibodies to ORT and decreased body weight in broilers. There was no significant correlation between presence of antibodies to ORT and age, lineage, efficiency index, feed conversion, and mortality. There was a positive correlation between the presence of respiratory signs and antibodies to ORT, although the reverse correlation was not significant. These results confirm that ORT is present and widespread in broilers and broiler breeders in southern Brazil.