Monitoring of Pig Slaughter Stages and Correlation in the Prevalence of Pathogens and Levels of Microorganisms That Indicate Microbiological Quality and Hygiene Using a Predictive Model.

Pig production is relevant to the Brazilian economy. Different stages of the raising and slaughtering process influence the microbiological quality of pig products and by-products. Microbiological analysis and hazard analysis and critical control points (HACCPs) are tools for monitoring microbiological quality indicator microorganisms. The construction of predictive models can assist the process of monitoring the microbiological quality of pig products. This study aimed to map the slaughter stages and develop a model to predict the absence or presence of Salmonella based on the process variables (distance from the farm to the slaughterhouse and aerobic mesophilic) and analyze their influence on contamination indicator microorganisms. A total of 810 samples were collected at nine stages of the slaughter process (bleeding, scalding, dehairing, singeing, washing, evisceration, inspection, final washing, and chilling). The binary class predictive model was used as a microbiological quality predictor at the slaughter stages. Salmonella was identified at all process stages, with lower contamination levels at the scalding and chilling stages, whereas the highest levels were found at the dehairing and bleeding stages. The predictive model revealed an accuracy of about 85% for Salmonella being a tool to monitor the microbiological quality of pig slaughter.

Microbiological Quality of Pig Carcasses in a Slaughterhouse under Risk-Based Inspection System.

Meat product inspection is one of the procedures adopted more than a century ago to guarantee food quality and safety for consumption. Due to technology and regulation advancement for farming and slaughtering pigs, a change in zoonotic profile attributed to pork has been identified. Thus, a global movement began to establish inspection parameters based on epidemiological risk profiles, culminating in the publication of a new regulation in Brazil in 2018. This normative instruction establishes that slaughterhouses under federal inspection must implement risk-based inspection until 2028. Changes in the inspection system can generate questions and objections on the part of customers and consumer markets. In order to assess microbiological contamination when adopting a risk-based inspection system, the occurrence of Salmonella spp. and the quantification of Enterobacteriaceae and mesophilic aerobic counts were compared in pig carcasses slaughtered under traditional and risk-based inspection systems. A statistical significance reduction was identified regarding the quantification of Enterobacteriaceae (log -0.18 to -1.61 CFU/cm2) and mesophilic aerobic counts (log 4.60 to 3.49 CFU/cm2). The occurrence of Salmonella spp. did not show a significant difference (4% to 5.3%). The results allowed us to conclude that adopting risk-based inspection systems improves food safety through Enterobacteriaceae and mesophilic aerobic counts reduction.

Different Swine Production Systems Can Shape Slurry Resistome at Mechanism and Class Levels Based on Swine Manure Evaluation.

Antimicrobial resistance is a major threat to public health. Antimicrobial use in animal husbandry is a major concern since it can favor an increase in antimicrobial resistance among farms. Herein, we aim to better understand and characterize the main resistome profiles in microbial communities found in pig farms. Sampling of swine manure was performed in two different timepoints (October 2019 and January 2020) in each of the 14 different swine farms, located in the mesoregion of Western Santa Catarina state in Brazil, a pole of swine product production of worldwide importance. Samples were divided into three groups: farms with the opened regimen and no usage of antimicrobials (F1; n = 10), farms with the closed regimen and usage of antimicrobials (F2; n = 16), and farms with the closed regimen and no usage of antimicrobials (F3; n = 2). The metagenomic evaluation was performed to obtain and identify genetic elements related to antimicrobial resistance using nanopore sequencing. We used ResistoXplorer software to perform composition, alpha and beta diversity, and clustering analysis. In addition, PCR reactions were performed to confirm the presence or absence of seven different beta-lactamase family genes and five phosphoethanolamine transferase gene variants clinically relevant. Our findings based on the identification of resistance genes at the mechanism level showed a prevalence of alteration of the drug target (72.3%) profile, followed by drug inactivation (17.5%) and drug efflux (10.1%). We identified predominantly aminoglycosides (45.3%), tetracyclines (15.9%), and multiclass (11,2%) resistance genes. PCoA analysis indicates differences between F1 and F2 profiles. F2 samples showed increased diversity when compared to the F1 group. In addition, herein we first report the identification of mcr-4 in a slurry sample (C1F1.1) in Santa Catarina State. In general, our findings reinforce that many factors on the practices of animal husbandry are involved in the resistome profile at the mechanism and class levels. Further studies to better understand microbiome and mobilome aspects of these elements are necessary to elucidate transmission pathways between different bacteria and environments.

Removal or substitution of in feed antimicrobials in swine production.

Antimicrobial substitutes are being used in pig production systems, to maintain the health of the animals without compromising their performance. The aim of this study is to evaluate the impact of either the removal of in feed antimicrobials or their substitution for feed additives, at the nursery and growing/finishing stages. At weaning, 1091 piglets were sexed, vaccinated, homogenized by weight and allocated to six treatments during the nursery stage (26-63 d): T1- feed with no antimicrobials nor additives; T2 - feed with antimicrobials; T3 - feed with prebiotic; T4 - feed with probiotic; T5 - feed with essential oils; T6 - feed with organic acids. The same treatments were applied to 840 pigs during the growing/finishing stages (64-167 d). There was no effect of the treatments on feed conversion at the nursery (P = 0.222) and the growing/finishing (P = 0.809) stages. The average daily gain did not differ across treatments in the nursery (P = 0.342) and in growing/finishing (P = 0.050). The cost of the interventions with injectable drugs was not different between the treatments neither at the nursery (P = 0.990) nor at the growing/finishing (P = 0.310). However, the pneumonia and pleurisy index for all treatments was equal or above 1.0, which indicates a respiratory challenge. There was an increase in the cost with antimicrobials or additives per kg of feed produced, which impacts the cost per kg of pig produced. In conclusion, the removal of antimicrobials in pig diets is financially feasible and their substitution by additives did not impact growing performance.

Effect of gaseous ozone application during chilling on microbial and quality attributes of pig carcasses.

Ozone application has been suggested as an additional measure to the slaughter animals under hygiene programs. In this study, we determined the efficacy of gaseous ozone applied to pig carcasses during chilling (16 h at 2-5°C). Forty carcasses were allocated to each treatment: control, without ozone application (T1) and 5 ppm gaseous ozone application (T2), divided in two 4-h periods. The carcasses were sampled before and after chilling. The average counts of total aerobic mesophilic (TAM) bacteria before chilling were not different (p = 0.55) between T1 and T2. In turn, after chilling, the ozone-treated carcasses had significantly reduced about 0.4 colony-forming units (CFU)/cm2 of TAM counts (p < 0.001) than the control carcasses. No significant reduction was observed in the number of carcasses positive for Listeria sp. and Escherichia coli after gaseous ozone treatment; while a tendency (p = 0.08) of lower number of Salmonella positive carcasses in T2 was observed. Common macrorestriction (pulsed-field gel electrophoresis) patterns of S. enterica were observed in the carcasses before and after chilling. Pork samples from treated and untreated carcasses with ozone showed no lipid oxidation or altered color and pH. The results indicate that the gaseous ozone in the tested protocol is effective in reducing TAM populations, but not effective in decreasing the number of carcasses positive for E. coli and Listeria sp. Regarding Salmonella, the tendency of positive carcasses reduction may encourage further studies by testing other protocols of gaseous ozone application inside the chilling chamber.

Phylogenetic relationship and genomic characterization of Salmonella Typhimurium strains isolated from swine in Brazil.

Salmonella Typhimurium has been transmitted between humans and animals. Although, Brazil has been one of the largest pork meat exporters worldwide, there are few studies that characterized epidemiologically S. Typhimurium strains from swine. The aims of this work were to study the phylogenetic relationship of S. Typhimurium genomes isolated from swine in Brazil among themselves and with other genomes isolated from several sources and countries using wgMLST and cgMLST and to perform the search of Salmonella pathogenicity islands (SPIs). In addition, for S. Typhimurium strains from swine to compare the virulence and antimicrobial resistance genes by VFDB and ResFinder, genetic content by BLAST Atlas and orthologous proteins clusters by OrthoVenn. The constructed phylogenetic trees by wgMLST and cgMLST grouped the majority (92.3% and 80.7%, respectively) of the strains isolated from swine in Brazil into the same group. All the isolates contained important SPIs (SPI-1, SPI-2, SPI-3, SPI-5 and SPI-9). A total of 100 and 31 virulence and resistance genes were detected in the S. Typhimurium strains isolated from swine, respectively. The BLAST Atlas and orthologous proteins analysis found regions of phages and differences in metabolic, regulatory and cellular processes among S. Typhimurium LT2 and S. Typhimurium isolates from swine. In conclusion, molecular typing based in the wgMLST and cgMLST suggested that the S. Typhimurium isolates from swine studied were genetically related. The pathogenic potential of the strains studied was corroborated by the presence of important SPIs and virulence genes. The high number of antimicrobial resistance genes detected is worrying and reinforced their potential risk in swine in Brazil. The comparison by BLAST Atlas suggested differences in mobile genetic elements among S. Typhimurium LT2 and S. Typhimurium isolates from swine in Brazil. The orthologous proteins analysis revealed unique genes related to important cellular processes in the strains from swine.

Antimicrobial resistance in commensal Escherichia coli and Enterococcus spp. isolated from pigs subjected to different antimicrobial administration protocols.

The antimicrobial resistance (AMR) in human and animal pathogens is a global concern, and antimicrobial use (AMU) is considered the most important driver for its increase. The aim of this study was to assess AMR in Escherichia coli and Enterococcus spp. in faecal samples of pigs subjected to four different AMU protocols from birth to finishing: G1, no in-feed antimicrobials; G2: a total average dose 6018 mg antimicrobials/pig; G3: a total average dose 8127 mg antimicrobials/pig; and G4: a total average dose 15,678 mg antimicrobials/pig. Faecal samples were collected at six time points and AMR was assessed in both bacteria. The microbiota composition was assessed by 16S rRNA sequencing. Minor differences on the microbiota profile was observed among groups, but a lower Firmicutes:Bacteroidetes ratio was noted in G4. Escherichia coli and Enterococcus spp. strains isolated from all groups showed a high level of multi-drug resistance (MDR). The amount of antimicrobials used was significantly positively associated with the probability of MDR in both bacteria. Approximately 43% of the variation in MIC90 for colistin could be explained by AMU, and a one-day increase in administration of colistin increased MIC90 by 0.05 µg mL-1. In conclusion, the results suggest that the higher the use of antimicrobials in farms, the higher the MDR frequency and resistance to the highest priority critically important antimicrobials for humans in commensal gut bacteria of pigs.

Re-Emergence of Salmonellosis in Hog Farms: Outbreak and Bacteriological Characterization.

Clinical salmonellosis has been increasing significantly in Brazil in recent years. A total of 130 outbreaks distributed among 10 swine-producing states were investigated. One representative Salmonella isolate from each outbreak was characterized through serotyping, antimicrobial resistance profiles, PFGE, and WGS. From 130 outbreaks: 50 were enteric, 48 were septicemic, 17 cases were characterized as hepato-biliary invasive, 13 as nodal and two were not classified. The most prevalent serovars were a monophasic variant of S. typhimurium (55/130), Choleraesuis (46/130), and Typhimurium (14/130). Most of the strains (86.92%) demonstrated a high rate of multi-drug resistance. The identification of a major Choleraesuis clonal group in several Brazilian states sharing the same resistance genes suggested that these strains were closely related. Six strains from this clonal group were sequenced, revealing the same ST-145 and 11 to 47 different SNPs. The detected plasmid type showed multiple marker genes as RepA_1_pKPC-CAV1321, the first to be reported in Salmonella. All AMR genes detected in the genomes were likely present on plasmids, and their phenotype was related to genotypic resistance genes. These findings reveal that salmonellosis is endemic in the most important pig-producing states in Brazil, emphasizing the need to make data available to aid in reducing its occurrence.

Synthetic gene as target to assess the sensitivity of PCR to detect Trichinella spp. larvae in meat from a non-endemic region.

Trichinellosis is a zoonotic disease exotic in Brazil but commonly found worldwide including South American countries like Argentina. International trading of swine meat needs an official Trichinella-free diagnosis commonly carried out by pepsin-HCl digestion of diaphragm tissue fragments followed by microscopic examination for the presence or absence of Trichinella larvae. The easiness of this diagnostic method allows it to be performed at slaughtering plants but, in contrast, it lacks sensitivity and does not allow species differentiation, which is fundamental for determining geographical and species distribution of different genotypes. In our study, we aimed to evaluate a highly sensitive diagnostic method based on the polymerase chain reaction (PCR) that would allow us to detect and classify different species of Trichinella. Thus, we designed a synthetic gene and selected five sets of primers targeting specific regions of the Trichinella genome. The synthetic gene was cloned into a plasmid and then used to optimize PCR conditions. Using our PCR, we were able to detect 0.001 pg of the synthetic gene, which corresponded to 0.01 larvae. Then, we collected 175 samples of Suidae (domestic and wild boars) diaphragm fragments that were pooled into groups, digested with pepsin-HCl, and had the DNA extracted for analysis by PCR. The clinical samples evaluated were negative by PCR. Our results indicate that the PCR-based method might be a useful diagnostic method complementary to the pepsin-HCl digestion method currently in use, mostly in non-endemic areas.

Comparison of Meat Juice Serology and Bacteriology for Surveillance of Salmonella in the Brazilian Pork Production Chain.

This study assessed an enzyme-linked immunosorbent assay (ELISA) based assay to detect Salmonella in swine as a potential tool to predict the presence of Salmonella in swine carcasses. The following samples were collected from 10 swine batches: blood (n = 100); environment (barn floor, n = 10, and lairage floor, n = 10); meat juice (n = 100, obtained after defrosting of diaphragm); tonsils (n = 100); mesenteric lymph nodes (MLNs) (n = 100); and carcasses after bleeding (n = 100), after singeing (n = 100), after evisceration (n = 100), and after final rinsing (n = 100). Blood and meat juice were subjected to ELISA to detect antibodies against Salmonella, and other samples were subjected to Salmonella detection by ISO 6579. Salmonella was detected in 3 samples from barn floors, 7 lairage floors, 45 tonsils, 43 MLNs and in 3 carcasses. Based on ELISA, Salmonella positive samples were: 86 and 46 blood serum (20% and 40% cut-offs) and 68 and 46 meat juice (20% and 40% cut-offs). Optical density readings from blood serum and meat juice presented a high and significant correlation (r = 0.93, p < 0.001), and a substantial agreement for Salmonella detection (K = 0.69, ELISA 40% cut-off). The agreement between ELISA and microbiological analysis for Salmonella detection in pig carcasses were absent or poor, with the exception of results obtained by ELISA 40% cut-off from blood serum and meat juice with MLNs (K = 0.49 and 0.50, respectively) and tonsils (K = 0.29 and 0.30, respectively). Based on the obtained results, meat juice can be considered an alternative to blood serum as a matrix for ELISA for preliminary detection of Salmonella, allowing the identification of potential sources of contamination during slaughtering.

Protection efficacy of the rLTB-R1 chimera against experimental swine mycoplasmal pneumonia

Background: Mycoplasma hyopneumoniae is the etiological agent of the Swine Mycoplasmal Pneumonia (SMP), one of the most economically significant diseases in the swine industry worldwide. Commonly used vaccines for SMP control consist of inactivated whole cells (bacterins). These vaccines are efficacious against M. hyopneumoniae challenge, but do not prevent colonization by the pathogen or completely eliminate pneumonia. P97 adhesin is conserved in the M. pneumoniae virulent strains, therefore it is an attractive target to be used in recombinant vaccines against M. hyopneumoniae. The aim of the present study was to evaluate protection afforded by rLTB-R1, a recombinant chimera composed by LTB fused with the R1 repeat region of P97 adhesin of M. hyopneumoniae, in specific-pathogen-free (SPF) piglets vaccinated by intranasal or intramuscular route and challenged with a pathogenic strain of M. hyopneumoniae. Materials, Methods & Results: PCR products of the LTB and R1 coding sequences were fused, then cloned into pETDEST42™ expression vector. The rLTB-R1 was expressed in Escherichia coli BL21 (DE3) Salt induction (SI). The piglets were divided into three groups: four piglets were intranasally vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IN rLTB-R1 group); four piglets were intramuscularly vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IM rLTB-R1 group); three piglets were intranasally and intramuscularly inoculated with 1 mL of PBS (control group). Two weeks after the last immunization (28 day), piglets were intratracheally challenged with 10 mL of a suspension containing 109 color-changing unit (CCU) of pathogenic M. hyopneumoniae 7448 strain on three consecutive days. Until the challenge (28 days), intranasal and intramuscular vaccination with rLTB-R1 induced seroconversions of antiR1 systemic antibodies of 1.6 and 4.6 ×, respectively. The IN rLTB-R1 group had no pulmonary lesion, rLTB-R1 conferred protection against experimental SMP. On the other hand, IM rLTB-R1 and control groups had on average 7.24% and 8.46% of pulmonary lesion, respectively, showing that intramuscular vaccination with rLTB-R1 did not confer protection. Discussion: The rLTB-R1, when intranasally administrated to mice, elicited production of anti-R1 IgA in trachea and bronchi as well as specific Th1 response, suggesting an adequate stimulation of the mucosal immune system. We believe that rLTB-R1 induced a similar immune response in piglets intranasally vaccinated, conferring protection against experimental SMP. The present study, the rLTB-R1 alone, without any chemical adjuvant, stimulated a significant seroconversion of anti-R1 systemic antibodies in pigs intramuscularly vaccinated, showing the potential of LTB as a parenteral adjuvant in swine vaccination. Previous work has shown that the intramuscular administration route was evaluated in pigs because mice intramuscularly vaccinated with rLTB-R1 presented significant levels of anti-R1 IgA in trachea and bronchi, suggesting that rLTB can stimulate some degree of mucosal immunity even if not delivered by a mucosal route. However, in the present study, piglets intramuscularly vaccinated with rLTB-R1 presented high levels of anti-R1 systemic antibodies, they were not protected. On the other hand, intranasal vaccination of piglets with rLTB-R1 elicited low levels of antiR1 systemic antibodies (1.6 × at 28 days), but it conferred full protection against experimental SMP. The present study demonstrated that intranasal vaccination of piglets with rLTB-R1 conferred protection against experimental SMP. A more detailed analysis of the protective immune response induced by rLTB-R1 in pigs is currently being performed.

Protection efficacy of the rLTB-R1 chimera against experimental swine mycoplasmal pneumonia

Background: Mycoplasma hyopneumoniae is the etiological agent of the Swine Mycoplasmal Pneumonia (SMP), one of the most economically significant diseases in the swine industry worldwide. Commonly used vaccines for SMP control consist of inactivated whole cells (bacterins). These vaccines are efficacious against M. hyopneumoniae challenge, but do not prevent colonization by the pathogen or completely eliminate pneumonia. P97 adhesin is conserved in the M. pneumoniae virulent strains, therefore it is an attractive target to be used in recombinant vaccines against M. hyopneumoniae. The aim of the present study was to evaluate protection afforded by rLTB-R1, a recombinant chimera composed by LTB fused with the R1 repeat region of P97 adhesin of M. hyopneumoniae, in specific-pathogen-free (SPF) piglets vaccinated by intranasal or intramuscular route and challenged with a pathogenic strain of M. hyopneumoniae. Materials, Methods & Results: PCR products of the LTB and R1 coding sequences were fused, then cloned into pETDEST42™ expression vector. The rLTB-R1 was expressed in Escherichia coli BL21 (DE3) Salt induction (SI). The piglets were divided into three groups: four piglets were intranasally vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IN rLTB-R1 group); four piglets were intramuscularly vaccinated with 1 mg of rLTB-R1 solubilized in 1 mL of PBS at 0 and 14 days (IM rLTB-R1 group); three piglets were intranasally and intramuscularly inoculated with 1 mL of PBS (control group). Two weeks after the last immunization (28 day), piglets were intratracheally challenged with 10 mL of a suspension containing 109 color-changing unit (CCU) of pathogenic M. hyopneumoniae 7448 strain on three consecutive days. Until the challenge (28 days), intranasal and intramuscular vaccination with rLTB-R1 induced seroconversions of antiR1 systemic antibodies of 1.6 and 4.6 ×, respectively. The IN rLTB-R1 group had no pulmonary lesion, rLTB-R1 conferred protection against experimental SMP. On the other hand, IM rLTB-R1 and control groups had on average 7.24% and 8.46% of pulmonary lesion, respectively, showing that intramuscular vaccination with rLTB-R1 did not confer protection. Discussion: The rLTB-R1, when intranasally administrated to mice, elicited production of anti-R1 IgA in trachea and bronchi as well as specific Th1 response, suggesting an adequate stimulation of the mucosal immune system. We believe that rLTB-R1 induced a similar immune response in piglets intranasally vaccinated, conferring protection against experimental SMP. The present study, the rLTB-R1 alone, without any chemical adjuvant, stimulated a significant seroconversion of anti-R1 systemic antibodies in pigs intramuscularly vaccinated, showing the potential of LTB as a parenteral adjuvant in swine vaccination. Previous work has shown that the intramuscular administration route was evaluated in pigs because mice intramuscularly vaccinated with rLTB-R1 presented significant levels of anti-R1 IgA in trachea and bronchi, suggesting that rLTB can stimulate some degree of mucosal immunity even if not delivered by a mucosal route. However, in the present study, piglets intramuscularly vaccinated with rLTB-R1 presented high levels of anti-R1 systemic antibodies, they were not protected. On the other hand, intranasal vaccination of piglets with rLTB-R1 elicited low levels of antiR1 systemic antibodies (1.6 × at 28 days), but it conferred full protection against experimental SMP. The present study demonstrated that intranasal vaccination of piglets with rLTB-R1 conferred protection against experimental SMP. A more detailed analysis of the protective immune response induced by rLTB-R1 in pigs is currently being performed.

Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms.

BACKGROUND: Pasteurella multocida type A (PmA) is considered a secondary agent of pneumonia in pigs. The role of PmA as a primary pathogen was investigated by challenging pigs with eight field strains isolated from pneumonia and serositis in six Brazilian states. Eight groups of eight pigs each were intranasally inoculated with different strains of PmA (1.5 mL/nostril of 10e7 CFU/mL). The control group (n = 12) received sterile PBS. The pigs were euthanized by electrocution and necropsied by 5 dpi. Macroscopic lesions were recorded, and swabs and fragments of thoracic and abdominal organs were analyzed by bacteriological and pathological assays. The PmA strains were analyzed for four virulence genes (toxA: toxin; pfhA: adhesion; tbpA and hgbB: iron acquisition) by PCR and sequencing and submitted to multilocus sequence typing (MLST). RESULTS: The eight PmA strains were classified as follows: five as highly pathogenic (HP) for causing necrotic bronchopneumonia and diffuse fibrinous pleuritis and pericarditis; one as low pathogenic for causing only focal bronchopneumonia; and two as nonpathogenic because they did not cause injury to any pig. PCR for the gene pfhA was positive for all five HP isolates. Sequencing demonstrated that the pfhA region of the HP strains comprised four genes: tpsB1, pfhA1, tpsB2 and pfhA2. The low and nonpathogenic strains did not contain the genes tpsB2 and pfhA2. A deletion of four bases was observed in the pfhA gene in the low pathogenic strain, and an insertion of 37 kb of phage DNA was observed in the nonpathogenic strains. MLST clustered the HP isolates in one group and the low and nonpathogenic isolates in another. Only the nonpathogenic isolates matched sequence type 10; the other isolates did not match any type available in the MLST database. CONCLUSIONS: The hypothesis that some PmA strains are primary pathogens and cause disease in pigs without any co-factor was confirmed. The pfhA region, comprising the genes tpsB1, tpsB2, pfhA1 and pfhA2, is related to the pathogenicity of PmA. The HP strains can cause necrotic bronchopneumonia, fibrinous pleuritis and pericarditis in pigs and can be identified by PCR amplification of the gene pfhA2.

Draft Genome Sequences of 20 Salmonella enterica subsp. enterica Serovar Typhimurium Strains Isolated from Swine in Santa Catarina, Brazil.

Salmonellosis is a disease with a high incidence worldwide, and Salmonella enterica subsp. enterica serovar Typhimurium is one of the most clinically important serovars. We report here the draft genome sequences of 20 S. Typhimurium strains isolated from swine in Santa Catarina, Brazil. These draft genomes will improve our understanding of S. Typhimurium in Brazil.

Genotyping and antimicrobial resistance in Escherichia coli from pig carcasses / Genotipagem e resistência antimicrobiana de Escherichia coli em carcaças suínas

The increasing antimicrobial resistance observed worldwide in bacteria isolated from human and animals is a matter of extreme concern and has led to the monitoring of antimicrobial resistance in pathogenic and commensal bacteria. The aim of this study was to evaluate the antimicrobial resistance profile of Escherichia coli isolated from pig carcasses and to assess the occurrence of relevant resistance genes. A total of 319 E. coli isolates were tested for antimicrobial susceptibility against different antimicrobial agents. Moreover, the presence of extended-spectrum β-lactamase (ESBL) and inducible ampC-β-lactamase producers was investigated. Eighteen multi-resistant strains were chosen for resistance gene detection and PFGE characterization. The study showed that resistance to antimicrobials is widespread in E. coli isolated from pig carcasses, since 86.2% of the strains were resistant to at least one antimicrobial and 71.5% displayed multi-resistance profiles. No ampC-producing isolates were detected and only one ESBL-producing E. coli was identified. Genes strA (n=15), floR (n=14), aac(3)IVa (n=13), tetB (n=13), sul2 (n=12), tetA (n=11), aph(3)Ia (n=8) and sul3 (n=5) were detected by PCR. PFGE analysis of these multi-resistant E. coli strains showed less than 80% similarity among them. We conclude that antimicrobial multi-resistant E. coli strains are common on pig carcasses and present highly diverse genotypes and resistance phenotypes and genotypes.(AU)

O incremento de resistência frente aos antimicrobianos, observado em bactérias isoladas de humanos e animais, tem sido motivo de preocupação mundial e levado ao monitoramento dos perfis de resistência em bactérias patogênicas e comensais. O objetivo desse estudo foi avaliar o perfil de resistência em Escherichia coli isolada de carcaças suínas e descrever a ocorrência de alguns genes de resistência relevantes. Um total de 319 isolados de E. coli foi testado quanto à suscetibilidade frente a diversos antimicrobianos. A presença de isolados produtores de β-lactamases (ESBL) de espectro estendido e β-lactamase induzível do tipo ampC foi também investigada. Dezoito cepas multirresistentes foram escolhidas para investigação de genes de resistência e caracterização por macro-restrição (PFGE). Os resultados demonstraram que a resistência a antimicrobianos está disseminada, pois 86,2% dos isolados de E. coli foram resistentes ao menos a um antimicrobiano e 71,5% apresentaram perfil de multirresistência. Uma cepa de E. coli produtora de ESBL e nenhuma produtora de ampC induzível foram identificadas. Os genes strA (n=15); floR (n=14);aac(3)-IVa (n=13); tetB (n=13); sul2 (n=12); tetA (n=11); aph(3')Ia (n=8); sul3 (n=5) foram detectados por PCR. A análise de PFGE demonstrou que cepas de E. coli multirresistentes apresentaram similaridade inferior a 80% entre si. Concluiu-se que cepas multirresistentes de E. coli são frequentes em carcaças de suínos e apresentam uma alta diversidade genotípica, bem como de fenótipos e genes de resistência.(AU)

Effect of slaughterhouse and day of sample on the probability of a pig carcass being Salmonella-positive according to the Enterobacteriaceae count in the largest Brazilian pork production region.

Sources of contamination of carcasses during slaughter include infected pigs as well as environmentally related sources. There are many microbial indicators that can be used in the processing of food to assess food hygiene and the safety of food processing. The presence of some microbial indicators can be viewed as a result of direct or indirect contamination of a food with fecal material. The presence of Enterobacteriaceae is often used as a hygiene indicator, as they are found both in the environment and in the intestine of warm-blooded animals. An association between Salmonella isolation and Enterobacteriaceae count (EC) on pre-chill carcasses has been described, however the impact of slaughterhouse and the day of sampling on the occurrence of Salmonella has not been previously investigated. To this end, mixed logistic regressions (MLRs) with random effects and fixed slopes were performed to assess the change in EC and its correlation with Salmonella occurrence using two data sets. The first describes the EC and Salmonella isolation in 60 pork carcasses in one slaughterhouse sampled at 11 different slaughter steps, including the carcass as a random effect. The second describes the EC and Salmonella isolation on 1150 pre-chill carcasses sampled in 13 slaughterhouses over 230 sampling days, and the model combined two random intercepts, slaughterhouse and date of sampling nested with slaughterhouse (day/slaughterhouse). Statistically significant associations (p<0.0001) between the log of the EC and Salmonella occurrence were found in all models. Nevertheless, although a strong association was found between Enterobacteriaceae and Salmonella contamination in pork carcasses, this association was not constant, given that there was a high variation in the probability of a carcass being positive for Salmonella according to the EC mainly between days of samples. The effect of the day of sampling on Salmonella prevalence was so large that the predictive value of the EC count for Salmonella isolation on a daily basis was compromised. It is possible that on some days batches with a high prevalence of Salmonella carriers shedding a high number of Salmonella were slaughtered. On these days, the potential for contamination/cross-contamination of carcasses will be so large that even hygienic slaughter, confirmed by the low EC on carcasses, will not be able to prevent the presence of Salmonella on some carcasses. The results of this study demonstrate that, despite the statistically significant association found, it may be difficult to predict when hygiene failure measured via EC actually indicates Salmonella contamination, and neither the inverse.

TLR4 single nucleotide polymorphisms (SNPs) associated with Salmonella shedding in pigs.

Toll-like receptor 4 (TLR4) is a key factor in the innate immune recognition of lipopolysaccharide (LPS) from Gram-negative bacteria. Previous studies from our group identified differences in the expression profile of TLR4 and genes affected by the TLR4 signaling pathway among pigs that shed varying levels of Salmonella, a Gram-negative bacterium. Therefore, genetic variation in this gene may be involved with the host's immune response to bacterial infections. The current study screened for single nucleotide polymorphisms (SNPs) in the TLR4 gene and tested their association with Salmonella fecal shedding. Pigs (n = 117) were intranasally challenged at 7 weeks of age with 1 × 10(9) CFU of S. Typhimurium χ4232 and were classified as low or persistent Salmonella shedders based on the levels of Salmonella being excreted in fecal material. Salmonella fecal shedding was determined by quantitative bacteriology on days 2, 7, 14, and 20/21 post exposure, and the cumulative levels of Salmonella were calculated to identify the low (n = 20) and persistent (n = 20) Salmonella shedder pigs. From those 40 animals, the TLR4 region was sequenced, and 18 single nucleotide polymorphisms (SNPs) in TLR4 were identified. Twelve SNPs have been previously described and six are novel SNPs of which five are in the 5' untranslated region and one is in intron 2. Single marker association test identified 13 SNPs associated with the qualitative trait of Salmonella fecal shedding, and seven of those SNPs were also associated with a quantitative measurement of fecal shedding (P < 0.05). Using a stepwise regression process, a haplotype composed of SNPs rs80787918 and rs80907449 (P ≤ 4.0 × 10(-3)) spanning a region of 4.9 Kb was identified, thereby providing additional information of the influence of those SNPs on Salmonella fecal shedding in pigs.

Longitudinal dissemination of Salmonella enterica clonal groups through the slaughter process of Salmonella-positive pig batches.

This study was conducted to assess the dissemination of Salmonella clonal groups in slaughterhouses that received batches of Salmonella -positive pigs and used different routine processing procedures. Eight serial sampling sessions were conducted in three slaughterhouses (A, B, and C). Blood was collected randomly (n = 25) from each batch of pigs and processed for serology. Carcasses (n = 12) were identified and sampled after dehairing, after singeing, after evisceration, and before chilling. A section of cecum also was collected. Salmonella isolates were submitted to pulsed-field gel electrophoresis. The overall seroprevalence of Salmonella was 80.6% (316 of 392 samples), and cecal contents were positive for Salmonella in 23.8% (26 of 109) of the pigs sampled. Carcasses after dehairing had a significantly higher prevalence of Salmonella (P = 0.004) and the highest Salmonella levels (median = 0.26 log CFU/300 cm(2)). The singeing step significantly affected the Salmonella status of the carcasses (P = 0.001); however, the efficacy of singeing differed among slaughterhouses. In the prechilling step, 14.7% (16 of 109) of the carcasses were positive for Salmonella. Salmonella pulsotypes found on the prechill carcasses were also found in the lairage, in the cecal contents, and on carcasses after dehairing, suggesting that the main source of contamination was the slaughter process before singeing. Slaughterhouse C was the most likely (odds ration [OR] = 6.51) to have pigs carrying Salmonella in the gut, and slaughterhouse B was the most likely (OR = 14.66) to have contaminated carcasses at the prechilling step. These findings indicate that the procedures adopted in slaughterhouse B contributed to the spread of Salmonella strains. In contrast, in slaughterhouse C the Salmonella strains carried by the pigs or found in the lairage were not recovered from prechilled carcasses, validating the effectiveness of the slaughterhouse interventions. These results indicate that an effective slaughter process can help decrease the number of Salmonella-positive carcasses in slaughterhouses that receive Salmonella-positive pig batches.

Efeito de probiótico na infecção e excreção fecal de Salmonella em suínos / Effect of probiotic on the Salmonella infection and fecal excretion in pigs

A transmissão de Salmonella na cadeia produtiva de suínos é um problema de difícil controle. O objetivo do estudo foi avaliar o efeito da administração oral de probiótico sobre a ocorrência de infecção e excreção fecal de Salmonella em suínos em fase de crescimento. Os tratamentos consistiram de ração basal sem aditivos (controle) ou adicionada de probiótico (10(7)ufc g-1 de células viáveis dos gêneros Bifidobacterium, Enterococcus, Lactobacillus e Saccharomyces). Foram alocados seis leitões de 50 dias em cada tratamento, com duas repetições por tratamento. Todos os animais foram inoculados com Salmonella Typhimurium (10(6)ufc mL-1) após 14 dias do alojamento. Semanalmente, foram coletadas amostras de sangue e fezes e no dia 35 pós-inoculação os animais foram sacrificados e necropsiados. Os animais de ambos os tratamentos foram infectados por Salmonella e soroconverteram. Não houve diferença (P>0,05) entre os grupos nas médias de Salmonella, Enterococcus, Lactobacillus e coliformes totais nas fezes, porém a administração de probiótico resultou em menor frequência de isolamento de Salmonella a partir de fígado (P=0,04), linfonodos mesentéricos (P=0,04), pulmão (P=0,03) e baço (P=0,01). Conclui-se que os microrganismos probióticos testados não foram capazes de impedir a infecção ou a excreção fecal de Salmonella em suínos de crescimento, mas diminuíram o número de portadores em linfonodos mesentéricos.

Control of Salmonella transmission has been a challenge for the pork production companies. The aim of this study was to evaluate the effect of oral administration of probiotics on the occurrence of infection and fecal excretion of Salmonella in growers. The treatments consisted of basal diet without additives (control) or added of probiotic (10(7)cfu g-1 of viable cells of the genera Bifidobacterium, Enterococcus, Lactobacillus and Saccharomyces). Six 50 days-old pigs were allocated into each treatment, with two replicates per treatment. All animals were inoculated with Salmonella Typhimurium (10(6)cfu mL-1) after 14 days of housing. Afterwards, blood and feces samples were taken weekly and on day 35 post-inoculation the animals were euthanized and necropsied. The animals in both treatment groups were infected by Salmonella and seroconverted. There was no difference (P>0.05) between groups in mean counts of Salmonella, Enterococcus, Lactobacillus and coliforms in the feces samples, but the probiotic administration resulted in a lower frequency of isolation of Salmonella from liver (P=0.04), mesenteric lymph nodes (P=0.04), lung (P=0.03) and spleen (P=0.01). It was concluded that the probiotic microorganisms tested in this study were not able to protect against the infection or to decrease the fecal excretion of Salmonella in growing pigs, but were able to decrease the number of carriers in the mesenteric lymph nodes.

Efeito do manejo pré-abate sobre alguns parâmetros fisiológicos em fêmeas suínas pesadas / Effect of pre-slaughter management on physiological parameters of heavy-weight female pigs

O objetivo neste trabalho foi avaliar o efeito do período de descanso (3, 5, 7 e 9 horas) dos suínos no frigorífico (PDF) e da localização dos suínos na carroceria do caminhão (PBO), quando transportados, no inverno ou verão, sobre alguns parâmetros fisiológicos avaliados em 64 fêmeas, com peso médio de 130kg para abate, durante o manejo pré-abate. Para a análise estatística, foram considerados, no modelo de análise da variância, os efeitos de bloco, PDF, PBO e da interação (bloco x PDF), entre outros. O PDF influenciou, significativamente, as concentrações de lactato no sangue e cortisol na saliva. Suínos que descansaram 5 e 7 horas apresentaram maior concentração de lactato em relação aos animais que descansaram 3 e 9 horas. No transporte, a freqüência cardíaca foi muito maior em relação aos demais locais avaliados. Concluiu-se que o incremento do PDF não promove mudanças na freqüência cardíaca, nas concentrações de glicose e CPK no sangue e cortisol na saliva, mas interfere na concentração de lactato no sangue dos suínos.

The aim of the research was to evaluate the effect of pig lairage time (PDF=3, 5, 7 and 9 hours) and evaluate the effect of pig position into the truck (PBO) during transportation to slaughterhouse, in winter or summer conditions, on some physiologic parameters evaluated on 64 heavyweight females with mean liveweight of 130kg during pre-slaughter events. The following effects were considered in the statistical analysis of variance model: block (BL=summer farm or winter farm), PDF, PBO and interaction (Block x PDF), under other factors. The PDF influenced significativelly blood lactate and saliva cortisol levels. Pig submitted to 5 and 7 hours of lairage had higher levels of lactate when compared to pigs submitted to 3 and 9 hours of lairage. During transport the heart rate were higher than in other pre-slaughter events. It is concluded that increasing PDF above 3 hours had no effects on heart frequency, glucose and CPK levels and salivary cortisol levels but affects the blood lactate levels.