Cellular immune response after vaccination with Salmonella Gallinarum 9R in laying hens and addition of aflatoxin and absorbent in the feed.

Salmonella Gallinarum greatly impacts commercial flocks and vaccination with S. Gallinarum 9R (SG9R) is one of the most effective control strategies in some countries. However, mycotoxins can affect immunization success. Herein, we measured the cellular immune response in SG9R-vaccinated hens, quantified the impact of aflatoxins on the immune response, and determined whether the anti-mycotoxin additive (adsorbent) influences immunity after vaccination. One-day-old chicks of commercial laying hens were raised until 49 days of age and were assigned to six groups. T1 (control group): control diet (no detectable concentration of aflatoxin), no vaccine or adsorbent. T2: vaccine SG-9R at day 28, aflatoxins 2.5 ppm from day 1 to day 49, and adsorbent 2.5 Kg/ton. T3: control diet and vaccine. T4: aflatoxins and vaccine. T5: control diet and aflatoxins. T6: aflatoxins and adsorbent. Body weights were evaluated on days 1, 31, and 41. Cellular immune response was evaluated by flow cytometry at 31, 41, and 49 days of age. T lymphocytes, B lymphocytes, monocytes, phagocytic monocytes and heterophils were evaluated. Aflatoxins suppressed peripheral and mucosal helper T lymphocytes, and mucosal cytotoxic T lymphocytes in vaccinated birds (T2 and T4). However, inclusion of the adsorbent in the feed of vaccinated birds neutralized the effects of aflatoxin (T6). The concentration of immune cells may show differences after SG9R vaccination, particularly an increase in the monocyte concentration. The SG9R vaccine reduced the concentration of activated cytotoxic T lymphocytes, making this marker a good parameter to analyze before and three weeks after immunization.

Antimicrobial and antibiofilm activity of silver nanoparticles against Salmonella Enteritidis.

Salmonella enterica serotype Enteritidis is one of the main pathogens associated with foodborne illnesses worldwide. Biofilm formation plays a significant role in the persistence of pathogens in food production environments. Owing to an increase in antimicrobial resistance, there is a growing need to identify alternative methods to control pathogenic microorganisms in poultry environments. Thus, this study aimed to synthesize silver nanoparticles (AgNPs) and evaluate their antibiofilm activity against poultry-origin Salmonella Enteritidis in comparison to a chemical disinfectant. AgNPs were synthesized, characterized, and tested for their minimum inhibitory concentration, minimum bactericidal concentration, and antibiofilm activity against S. Enteritidis strains on polyethylene surfaces. The synthesized AgNPs, dispersed in a liquid medium, were spherical in shape with a mean diameter of 6.2 nm. AgNPs exhibited concentration-dependent bactericidal action. The bacterial reduction was significantly higher with AgNPs (3.91 log10 CFU [Formula: see text] cm-2) than that with sanitizer (2.57 log10 CFU âˆ™ cm-2). Regarding the time of contact, the bacterial count after a contact time of 30 min was significantly lower than that after 10 min. The AgNPs exhibited antimicrobial and antibiofilm activity for the removal of biofilms produced by S. Enteritidis, demonstrating its potential as an alternative antimicrobial agent. The bactericidal mechanisms of AgNPs are complex; hence, the risk of bacterial resistance is minimal, making nanoparticles a potential alternative for microbial control in the poultry chain.

Prevalence and distribution of pathogenic genes in Campylobacter jejuni isolated from poultry and human sources.

INTRODUCTION: Campylobacter jejuni is one of the most common bacterial causes of human gastroenteritis. Despite its public health importance, the virulence factors and mechanisms underlying C. jejuni pathogenesis remain poorly understood and the relationships between these genes and the sources of the strains are not clear. We aimed to determine the virulence profiles of C. jejuni isolated from poultry and human cases of Campylobacteriosis. METHODOLOGY: A total of 50 strains of C. jejuni isolated from poultry and human cases of Campylobacteriosis were screened by polymerase chain reaction (PCR) for the presence of six pathogenic genes (flaA, iam, wlaN, cdtA, cdtB, cdtC). RESULTS: A total of 40% (10/25) of the human isolates presented only one virulence marker. In contrast, 64% (16/25) of the poultry-derived strains showed four or five virulence markers. cdtA and flaA occurred more frequently in poultry-derived strains than in human strains. Ten different virulence profiles were observed among the human isolates and 11 among the poultry strains. Only four profiles were common to both sources: profiles 3 (flaA, cdtA, cdtB, and cdtC), 5 (cdtA and cdtB), 7 (flaA and cdtB), and 10 (iam, flaA, cdtA, cdtB, and cdtC). The human-derived strains had a higher Shannon diversity index (1.9396) and Simpson index (0.8367), indicating a more diversified population than found in poultry (1.7742 and 0.7333, respectively). CONCLUSIONS: We found variations in the genetic profiles of the circulating strains based on the isolation source and genes that are potentially pathogenic to humans were detected in poultry-derived strains.

Detection and Quantification of Campylobacter in Poultry Slaughterhouses Using Conventional Microbiological Technique, Most Probable Number, and Real-Time PCR.

Campylobacteriosis is one of the most common bacteria causing human gastroenteritis. Poultry is a major reservoir of Campylobacter spp. as well as the main source of transmission. Due to the increased occurrence of campylobacteriosis, poultry slaughterhouses are under pressure to deliver carcasses with low contamination. However, a few studies have been carried out to evaluate Campylobacter contamination of broiler carcasses in Brazilian slaughter lines. Therefore, in this study, we aimed at detecting and quantifying the thermotolerant Campylobacter spp. at different stages of the poultry slaughtering process. The samples were collected from 12 points in three slaughterhouses in southern Brazil, at an interval of 12 months, and were tested for Campylobacter spp. by conventional microbiological technique, the most probable number, and real-time PCR. A total of 432 samples were analyzed. The majority of strains belonged to Campylobacter jejuni (92%), and the flock positivity among the three techniques was similar in most cases. Campylobacter was detected in all slaughtering stages. Although contamination has remained similar (p > 0.05) throughout almost all the slaughter process, evisceration seemed to be an important source of contamination. Our results reinforce the idea that the final carcass quality after the slaughtering process is directly influenced by the level of contamination of the broiler flocks on arrival at the processing plant.

Avian pathogenic Escherichia coli (APEC) and uropathogenic Escherichia coli (UPEC): characterization and comparison.

INTRODUCTION: Avian pathogenic E. coli (APEC) and uropathogenic E. coli (UPEC) are responsible for avian colibacillosis and human urinary tract infections, respectively. There are genetic similarities between the APEC and UPEC pathotypes, suggesting the APEC strains could be a potential reservoir of virulence and antimicrobial-resistance genes for the UPEC strains. This study aimed to characterize and compare APEC and UPEC strains regarding the phylogroup classification, pathogenicity and antimicrobial susceptibility. METHODOLOGY: A total of 238 APEC and 184 UPEC strains were selected and characterized. The strains were assayed for antimicrobial susceptibility and classified into phylogenetic groups using a multiplex-PCR protocol. In addition, the APEC strains had previously been classified according to their in vivo pathogenicity. RESULTS: The results showed that both pathotypes had variation in their susceptibility to most of the antimicrobial agents evaluated, with few strains classified as multidrug resistant. The highest resistance rate for both pathotypes was to amoxicillin. Classifying the APEC and UPEC strains into phylogenetic groups determined that the most frequently frequencies were for groups D and B2, respectively. These results reflect the pathogenic potential of these strains, as all the UPEC strains were isolated from unhealthy patients, and most of the APEC strains were previously classified as pathogenic. CONCLUSIONS: The results indicate that distribution into phylogenetic groups provided, in part, similar classification to those of in vivo pathogenicity index, as it was possible to adequately differentiate most of the pathogenic and commensal or low-pathogenicity bacteria. However, no relationship could be found between the specific antimicrobial agents and pathogenicity or phylogenetic group for either pathotype.

Identification of pathogenic genes in Campylobacter jejuni isolated from broiler carcasses and broiler slaughterhouses.

Campylobacter jejuni is one of the most common causes of foodborne diseases worldwide. There are few reports on Campylobacter strains isolated from Latin-American countries. Here, 140 C. jejuni strains isolated from cloacal and transport boxes swabs, water from chiller tanks, and broiler carcasses of five poultry companies in Southern Brazil were identified using phenotypic and genotypic methods. Polymerase chain reaction (PCR) was used to analyze eight C. jejuni virulence markers: flaA, cadF, and invasion-associated (iam) genes, cdtABC operon (associated with the cytolethal distending toxin), and plasmidial virB11 and wlaN genes were present in 78.5%, 77.8%, 0%, 74.2%, 22.1%, and 10.7% of samples, respectively. There were 25 different virulence profiles: 1 (cdtA, cdtB, cdtC, flaA, and cadF), 2 (cdtA, cdtB, cdtC, flaA, cadF, and virB11), and 3 (cdtA, cdtB, cdtC, flaA, cadF, and wlaN) were the most common (> 60% of strains). We provide insight into factors related to the occurrence of this pathogen and their epidemiology.

Electrochemically-Activated Water Presents Bactericidal Effect Against Salmonella Heidelberg Isolated from Poultry Origin.

Salmonella spp. are among the most important pathogens in poultry farming, and Salmonella Heidelberg (SH) is one of the most frequent serotypes isolated in Brazil. SH has a zoonotic potential and stands out as a pathogen that is difficult to eliminate from the poultry chain due to its resistance to disinfectants. One alternative to traditional disinfectants is the electrochemically-activated water (ECA), a bactericidal compound produced from the electrolysis of salt and water. ECA generators produce a compound that consists of free chlorine, hypochlorous acid, and other free radicals. This alternative control method is safe for human health and reduces environmental contamination. The present study aimed at evaluating the efficacy of ECA against 30 SH isolates from poultry origin in scenarios that simulated the chiller environment (4°C, 5 and 50 parts per million [ppm], 5 and 40 min of exposure) and the cleaning and disinfection process (25°C, 200 ppm, 5 and 10 min of exposure). In the quantitative test, SH was susceptible to ECA. The mean bacterial counts decreased significantly compared to the control group, especially at 200 ppm. At this concentration, ECA inhibited the growth of almost 87% of the Salmonella strains, and the results showed a significant decrease in the mean bacterial counts for both exposure times (5 and 10 min). These findings demonstrate that ECA is effective against SH in vitro and it is a possible alternative to disinfection in the poultry industry for the control of this pathogen. However, in situ tests in the food industry are needed.

Influence of the norepinephrine and medium acidification in the growth and adhesion of Salmonella Heidelberg isolated from poultry.

Salmonella spp. are among the leading pathogens responsible for foodborne illnesses worldwide. Bacterial communities use a quorum sensing (QS) system to control biofilm formation. QS is a cell-to-cell signaling mechanism involving compounds called auto-inducers (AI). Norepinephrine utilizes the same bacterial signaling of AI-3 and serves as a signal of QS. Acid stress is a challenge encountered by microorganisms in food processing environments and in the gastrointestinal tracts of hosts. Thus, adaptation to acidic environments may increase the pathogenicity of the strain. The aim of this study was to evaluate the influence of two concentrations of norepinephrine (100 µM and 250 µM) and acidification (pH 3.0) of the medium on the growth and adhesion of Salmonella Heidelberg strains isolated from poultry sources at 12 °C and 25 °C. Furthermore, three genes associated with the biofilm formation process were detected (adrA, csgD, and sidA). Norepinephrine stimulation did not influence the growth or adhesion of Salmonella Heidelberg strains, regardless of the catecholamine concentration and temperature. On the other hand, the use of acidified medium (pH 3.0) resulted in a significant reduction of growth and a significant increase of S. Heidelberg adhesion at both temperatures, indicating that the acidified medium favors the biofilm formation process. The adrA and sidA genes showed higher detection frequencies than csgD. Experiments analyzing the biofilm production process by S. Heidelberg strains are not common, and further studies are necessary to understand this complex process.

Fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli from poultry and human samples assessed by PCR-restriction fragment length polymorphism assay.

The objective of this study was to determine fluoroquinolone resistance in Campylobacter spp from poultry and human isolates. Forty-one Campylobacter jejuni isolates (30 of poultry origin and 11 of human origin) and 11 Campylobacter coli isolates (10 of human origin and 1 of poultry origin) were examined for ciprofloxacin, norfloxacin, and nalidixic acid resistance using the minimal inhibitory concentration (MIC) method. Thereafter, the isolates were analyzed by PCR-Restriction Fragment Length Polymorphism (RFLP) assay for detection of Thr-86 mutation. Finally, DNA sequencing was performed for confirmation of gyrA gene mutation. A complete correlation was observed between MICs, PCR-RFLP assay, and sequencing. The results revealed high quinolone resistance rates for C. jejuni (100%) and C. coli (100%) isolates obtained from poultry and moderate resistance for C. jejuni (9.1%) and C. coli (40%) samples of human origin. A mutation in codon 86 of the gyrA gene with a Thr-to-Ile substitution is reported to be the main cause of high resistance to quinolones. This mutation can be analyzed by PCR-RFLP assay, which has been proven to be a simple and fast method for the detection of fluoroquinolone resistance in Campylobacter spp.

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 10(5)ufc/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.

Análise comparativa entre microbiologia convencional, ELISA e PCR para detecção de Salmonella enteritidis, S. typhimurium, S. gallinarum e S. pullorum em carne de frango contaminada artificialmente

O presente trabalho teve como objetivo realizar uma análise comparativa das técnicas de Reação em Cadeia pela Polimerase(PCR) e Ensaio Imunoenzimático (ELISA SALVIA®) com o método microbiológico convencional para detecção e SalmonellaEnteritidis (SE), S. typhimurium (ST), S. gallinarum (SG) e S. pullorum (SP) em carne de frango. As amostras foram contaminadasartificialmente com diluições de 10-7, 10-8 e 10-9 para SE e ST e de 10-4, 10-5 e 10-6 para SG e SP, com cinco repetições decada diluição, totalizando 300 análises. Os testes foram realizados em cinco diferentes laboratórios para a validação dastécnicas. Na avaliação geral dos dados obtidos, a microbiologia convencional obteve 56,67% (170/300) de recuperação dasamostras contaminadas artificialmente, enquanto as técnicas de ELISA e PCR representaram 71% (213/300) e 75% (225/300), respectivamente. A análise dos resultados de detecção de Salmonella através dos testes ELISA e PCR, em relação aomicrobiológico convencional, apresentaram diferença estatística (p=0,0001, teste de MacNemar). Não houve diferença significativaentre os resultados da PCR e do ELISA. Os resultados alcançados demonstraram que, comparado ao microbiológicoconvencional, tanto o ELISA quanto a PCR foram eficazes para detecção dos sorovares de Salmonella nas amostras de carnede frango avaliadas.