Copper nanoparticles effectively reduce Salmonella Enteritidis in broiler chicken diet and water.

ABSTRACTThe use of copper nanoparticles (CuNP) in the diet of broiler chickens has been studied as a potential alternative to antibiotic growth promoters. This study aimed to analyze the antimicrobial properties of CuNP in the feed and water of broiler chickens against Salmonella Enteritidis and to assess the intestinal integrity and toxicity of CuNP supplementation in their diet. The antimicrobial activity of CuNP against S. Enteritidis was tested in microplates to evaluate three water samples with different mineral compositions and in an in vitro digestibility model that simulated the three primary intestinal compartments of birds to assess feed samples. To evaluate in vivo intestinal integrity and toxicity, the birds were divided into four groups (30 birds per group): (1) basal diet (control); (2) basal diet + CuNP (100 ppm); (3) basal diet + enramycin (10 ppm); and (4) basal diet + CuNP (100 ppm) + enramycin (10 ppm). Intestinal samples were collected for histomorphometric evaluation and lactic acid bacteria count, while chest muscle and whole blood samples were collected to determine copper content. A significant reduction in the S. Enteritidis count was observed in both in vitro treatments (water and feed) with CuNP inclusion, compared to the control group. No significant differences histomorphometric measurements, weight gain, or total lactic acid bacterial counts were found compared to those in the control. These results demonstrate the effectiveness of CuNP in reducing the occurrence of S. Enteritidis and their non-interference with the intestinal integrity of broiler chickens, highlighting the potential of CuNP as an alternative antimicrobial agent in the poultry production chain.

Development of Predictive Modeling for Removal of Multispecies Biofilms of Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni from Poultry Slaughterhouse Surfaces.

Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni are among the most common foodborne pathogens worldwide, and poultry products are strongly associated with foodborne pathogen outbreaks. These pathogens are capable of producing biofilms on several surfaces used in the food processing industry, including polyethylene and stainless steel. However, studies on multi-species biofilms are rare. Therefore, this study aimed to develop predictive mathematical models to simulate the adhesion and removal of multispecies biofilms. All combinations of microorganisms resulted in biofilm formation with differences in bacterial counts. E. coli showed the greatest ability to adhere to both surfaces, followed by S. Enteritidis and C. jejuni. The incubation time and temperature did not influence adhesion. Biofilm removal was effective with citric acid and benzalkonium chloride but not with rhamnolipid. Among the generated models, 46 presented a significant coefficient of determination (R2), with the highest R2 being 0.88. These results provide support for the poultry industry in creating biofilm control and eradication programs to avoid the risk of contamination of poultry meat.

Adhesion capacity of Salmonella Enteritidis, Escherichia coli and Campylobacter jejuni on polystyrene, stainless steel, and polyethylene surfaces.

Poultry products are recognized as the main source of Salmonella and Campylobacter jejuni infections in humans, while avian pathogenic Escherichia coli may have zoonotic potential and can be transmitted from chicken meat to humans. Biofilm formation contributes to their spread through the food chain. This study aimed to compare the adhesion of Salmonella Enteritidis, E. coli, and C. jejuni strains isolated from poultry, food implicated in outbreaks, and poultry slaughterhouses on three surfaces widely used in poultry production (polystyrene, stainless steel, and polyethylene). S. Enteritidis and E. coli adhesion on the three surfaces tested were not significantly different (p > 0.05). Interestingly, the number of C. jejuni cells on stainless steel (4.51-4.67 log10 CFU/cm.-2) was significantly higher (p = 0.0004) than that on polystyrene (3.80-4.25 log10 CFU/cm.-2), but similar (p > 0.05) to that on polyethylene (4.03-4.36 log10 CFU/cm.-2). However, C. jejuni adhesion was significantly lower (p < 0.05) than S. Enteritidis and E. coli adhesion, regardless of the surface evaluated. In addition, scanning electron microscopy analyses have shown an increased irregularity of the stainless steel surface when compared to polyethylene and polystyrene. These irregularities form small spaces ideal for microbial adhesion.

Characterization of avian pathogenic Escherichia coli isolates based on biofilm formation, ESBL production, virulence-associated genes, and phylogenetic groups.

Escherichia coli is a part of both animal and human commensal microbiota. Avian pathogenic E. coli (APEC) is responsible for colibacillosis in poultry, an economically important disease. However, the close similarities among APEC isolates make it difficult to differentiate between pathogenic and commensal bacteria. The aim of this study was to determine phenotypic and molecular characteristics of APEC isolates and to compare them with their in vivo pathogenicity indices. A total of 198 APEC isolates were evaluated for their biofilm-producing ability and extended-spectrum ß-lactamase (ESBL) production phenotypes. In addition, 36 virulence-associated genes were detected, and the isolates were classified into seven phylogenetic groups using polymerase chain reaction. The sources of the isolates were not associated with biofilms, ESBL, genes, or phylogroups. Biofilm and ESBL production were not associated with pathogenicity. Group B2 had the highest pathogenicity index. Groups B2 and E were positively associated with high-pathogenicity isolates and negatively associated with low-pathogenicity isolates. In contrast, groups A and C were positively associated with apathogenic isolates, and group B1 was positively associated with low-pathogenicity isolates. Some virulence-associated genes showed positive or negative associations with specific phylogenetic groups. None of the individual techniques produced results that correlated with the in vivo pathogenicity index. However, the combination of two techniques, namely, detection of virulence-associated genes and the phylogenetic groups, could help the classification of the isolates as pathogenic or commensal.

Antibiofilm activity of the biosurfactant and organic acids against foodborne pathogens at different temperatures, times of contact, and concentrations.

Biofilm formation has been suggested to play a significant role in the survival of pathogens in food production. Interest in evaluating alternative products of natural origin for disinfectant use has increased. However, there is a lack of information regarding the effects of biosurfactants and organic acids on Salmonella enterica serotype Enteritidis, Escherichia coli, and Campylobacter jejuni biofilms, mainly considering temperatures found in environments of poultry processing, as well as simulating the contact times used for disinfection. The aim of this study was to evaluate the antibiofilm activity of rhamnolipid, malic acid, and citric acid on the adhesion of S. Enteritidis, E. coli, and C. jejuni on polystyrene surfaces at different temperatures (4, 12, and 25 °C), compound concentrations, and times of contact (5 and 10 min), and to analyze the potential use of these compounds to disrupt formed biofilms. All three compounds exhibited antibiofilm activity under all analyzed conditions, both in the prevention and removal of formed biofilms. Contact time was less important than temperature and concentration. The antibiofilm activity of the compounds also varied according to the pathogens involved. In the food industry, compound selection must consider the temperature found in each stage of product processing and the target pathogens to be controlled.

Bacterial community identification in poultry carcasses using high-throughput next generation sequencing.

Poultry products are susceptible to contamination by pathogenic and spoilage bacteria during the slaughtering process. Molecular techniques have been used to assist in the identification of microorganisms in various microbiomes. The aim of this study was to identify bacterial components of the microbiome in poultry carcasses during the slaughter process, using high-throughput next generation sequencing (HT-NGS). Samples were collected from three slaughterhouses (A, B, and C) located in southern Brazil and included those taken from three points (initial, middle, and end) in the chiller tanks and two carcass pools (at the entrance to the clean area and after the final carcass packaging) at each establishment. A total of 104 carcasses were collected from each slaughterhouse. For this study, HT-NGS allows for a precise, quantitative and culture-independent microbiome assessment in poultry products. Three phyla (Firmicutes, Bacteroidetes, and Proteobacteria) were found in all establishments, and one phylum (Verrucomicrobia) was found only in Establishment A. Common set of genera (Anaerotruncus, Bacteroides, Campylobacter, Erysipelatoclostridium, Faecalibacterium, Lachnoclostridium, and Subdoligranulum) was identified in processing establishments along with the groups unique to a particular site. Pathogenic and spoilage bacteria, as well as other microorganisms that were not expected in poultry products, were detected by HT-NGS technique. The Shannon diversity index was the highest in Establishment B (2.40), followed by establishments C (1.98) and A (1.43). As we progressed through sample analysis, from the entrance of the clean area to the final carcass packaging area, we found significant reductions (p < 0.05) in the quantities of sequences of all phyla in establishments A and B. Significant differences (p < 0.05) in the quantities of sequences of all phyla were found between different stages in the slaughtering process. More stringent control procedures in establishments A and B were associated with reduced contamination even though all establishments followed the official sanitary standards. Our findings provide new insight into the chicken meat microbiome, and can be used in future studies to help ensure food safety in slaughterhouses.

Antimicrobial activity of copper surfaces against biofilm formation by Salmonella Enteritidis and its potential application in the poultry industry.

As a consequence of developing antimicrobial resistance to disinfectants, copper, which exhibits antimicrobial activity, has been studied as a possible alternative to the use of stainless steel surfaces. The aim was to evaluate the antimicrobial activity of copper surfaces in preventing biofilm formation by Salmonella Enteritidis and to determine their corrosive capacity. Strains of S. Enteritidis were incubated at 4 °C, 12 °C, and 25 °C with 1 cm2 coupons of electrolytic copper (99.9% Cu), brass (70% Cu), copper coated with tin, and stainless steel (control). A planktonic cell-suspension assay was used, followed by serial dilutions and bacterial counts. The corrosion test was performed with two disinfectants: benzalkonium chloride and sodium hypochlorite (100, 200, and 400 ppm). There was a significant reduction in biofilm production (log10 CFU cm-2) on the copper (2.64 at 4 °C, 4.20 at 12 °C, 4.56 at 25 °C) and brass (2.79 at 4 °C, 3.49 at 12 °C, 4.55 at 25 °C) surfaces compared to the control (5.68 at 4 °C, 5.89 at 12 °C, 6.01 at 25 °C). The antimicrobial surfaces showed uniform corrosion similar to that of surfaces generally used. These results demonstrated the effectiveness of copper surfaces in reducing S. Enteritidis and suggest they can be used as a complementary antimicrobial to control for this pathogen.

Evaluation of thymic lymphocyte loss of broiler using Digital Analysis of the Lymphoid Depletion System (ADDL) / Avaliação de perda de linfócitos do timo em frangos usando análise digital do sistema de depleção linfóide (ADDL)

The thymus is a lymphoid organ and usually evaluated for the degree of lymphocyte loss with subjective histological techniques. This study aimed to adapt and to apply of the digital analysis of the lymphoid depletion system (ADDL) in the thymus in order to obtain a more accurate analysis. Glucocorticoid was used to induce immunosuppression in 55 broilers at 21 days of age; other 15 broilers were the control group. After euthanasia of the broilers, postmortem examination was made. Both thymic chains were collected and six lobes were selected for histological examination of the degree of lymphocyte depletion (scores 1 to 5) and for submission to all stages of processing by the ADDL system. The artificial constructed neural networks (ANN) obtained 94.03% of correct classifications. In conclusion, it was possible to adopt objective criteria to evaluate thymic lymphoid depletion with the ADDL system.(AU)

O timo é um órgão linfóide, que é normalmente avaliado para o grau de perda de linfócitos a partir de técnicas histológicas subjetivas. Este trabalho teve como objetivo a adaptação e aplicação do sistema de análise digital de depleção linfóide (ADDL) para o timo, a fim de tornar sua análise mais acurada. Glicocorticóides foram utilizados a fim de induzir imunossupressão em 55 aves de 21 dias de idade. Outras 15 aves formaram o grupo controle. Posteriormente, para cada um dos aves, realizou-se a eutanásia e necropsia. Ambas as cadeias do timo foram coletadas e foram selecionadas seis lóbulos para processamento histológico, análise quanto ao grau de depleção linfocitária (escores de 1-5) e submissão a todas as fases do processamento pelo sistema ADDL. Observou-se que a rede neural artificial (RNA) construída obteve 94,03% de classificações corretas. Em conclusão, foi possível adotar critérios objetivos para avaliar a depleção linfóide tímica utilizando o sistema ADDL.(AU)

Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

Abstract Pasteurella multocida causes atrophic rhinitis in swine and fowl cholera in birds, and is a secondary agent in respiratory syndromes. Pathogenesis and virulence factors involved are still poorly understood. The aim of this study was to detect 22 virulence-associated genes by PCR, including capsular serogroups A, B and D genes and to evaluate the antimicrobial susceptibility of P. multocida strains from poultry and swine. ompH, oma87, plpB, psl, exbD-tonB, fur, hgbA, nanB, sodA, sodC, ptfA were detected in more than 90% of the strains of both hosts. 91% and 92% of avian and swine strains, respectively, were classified in serogroup A. toxA and hsf-1 showed a significant association to serogroup D; pmHAS and pfhA to serogroup A. Gentamicin and amoxicillin were the most effective drugs with susceptibility higher than 97%; however, 76.79% of poultry strains and 85% of swine strains were resistant to sulphonamides. Furthermore, 19.64% and 36.58% of avian and swine strains, respectively, were multi-resistant. Virulence genes studied were not specific to a host and may be the result of horizontal transmission throughout evolution. High multidrug resistance demonstrates the need for responsible use of antimicrobials in animals intended for human consumption, in addition to antimicrobial susceptibility testing to P. multocida.

Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine.

Pasteurella multocida causes atrophic rhinitis in swine and fowl cholera in birds, and is a secondary agent in respiratory syndromes. Pathogenesis and virulence factors involved are still poorly understood. The aim of this study was to detect 22 virulence-associated genes by PCR, including capsular serogroups A, B and D genes and to evaluate the antimicrobial susceptibility of P. multocida strains from poultry and swine. ompH, oma87, plpB, psl, exbD-tonB, fur, hgbA, nanB, sodA, sodC, ptfA were detected in more than 90% of the strains of both hosts. 91% and 92% of avian and swine strains, respectively, were classified in serogroup A. toxA and hsf-1 showed a significant association to serogroup D; pmHAS and pfhA to serogroup A. Gentamicin and amoxicillin were the most effective drugs with susceptibility higher than 97%; however, 76.79% of poultry strains and 85% of swine strains were resistant to sulphonamides. Furthermore, 19.64% and 36.58% of avian and swine strains, respectively, were multi-resistant. Virulence genes studied were not specific to a host and may be the result of horizontal transmission throughout evolution. High multidrug resistance demonstrates the need for responsible use of antimicrobials in animals intended for human consumption, in addition to antimicrobial susceptibility testing to P. multocida.

Influenza aviária

O documento aborda a influenza aviária, abordando sua história, seu ciclo epidemiológico, evolução, formas de transmissão, diagnóstico e tratamento, prevenção e controle e vacinas.

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.

Campylobacter em humanos e sua relação com animais de produção / Campylobacter in humans and its relationship with farm animals

Bactérias do gênero Campylobacter são patógenos entérios de origem alimentar, sendo o Campylobacter jejuni freqüentemente relatado nas ocorrências de gastroenterite em seres humanos. A associação entre Campylobacter em aves e enterites no homem decorre da persistência do agente no habitat do frango de corte, que proporciona a colonização intestinal assintomática na ave, sendo esta a origem mais importante de contaminação das carcaças.O produto brasileiro é altamente competitivo no mercado mundial, razão pela qual o Campylobacter pode ser o próximo alvo para a imposição de barreiras sanitárias que, por sua vez, dificultarão as exportações do frango brasileiro para diversos mercados. Considerando a necessidade de controlar e minimizar Campylobacter em produtos de origem animal, este trabalho teve por objetivos apresentar dados sobre campilobacterioses em humanos e sua relação com animais de produção, tomando-se como premissa que, se estes tiverem menor nível de colonização por Campylobacter durante a criação, provavelmente o nível de contaminação das carcaças será diminuído, reduzindo as infecções em humanos por Campylobacter.

Virulence factors of avian pathogenic Escherichia coli isolated from broilers from the south of Brazil.

Sixty-three Escherichia coli strains isolated from broilers with respiratory problems were examined for virulence factors, hemolysin synthesis ability, motility, hemagglutination capacity, operon pap presence, colicin production, and serum resistance. The capacity to hemagglutinate guinea pig erythrocytes was found in 53 (84.1%) of the samples, but only 30 (47.6%) agglutinated chicken erythrocytes. D-mannose-sensitive hemagglutination against guinea pig erythrocytes was found in 19 (30.2%) samples and against chicken erythrocytes, in 15 (23.8%) samples, whereas the D-mannose-resistant hemagglutination with guinea pig erythrocytes was found in 34 (54%) samples, and 13 of these (20.6%) showed this characteristic against chicken erythrocytes. Operon pap, P fimbria codifier, was detected in 26 samples in a total of 34 D-mannose-resistant samples. Colicin production was observed in 55 (87.3%) of the strains, and 41.8% presented V colicin production. Of the samples analyzed, 56 (88.9%) presented serum resistance, six (9.5%) were intermediate, and only one (1.6%) was sensitive to the action of the complement. The diversity of virulence profiles detected in the samples in this study explains in part the multifactorial characteristics of avian colibacillosis.

Isolation of Arcobacter spp from poultry carcasses, in Brazil

Foram isoladas 48 amostras de Arcobacter spp de 37 carcaças de frangos colhidas em frigorífico, prontas para consumo, entre 80 carcaças examinadas. Foram feitas tentativas de cultivo a partir de pele e de músculo, sendo obtidas 25 cultivos positivos de músculo e 23 de pele. As bactérias foram classificadas pelas características fenotípicas e pelo teste de PCR e PCR múltiplo, obtendo-se 41 amostras classificadas como Arcobacter butzleri e 07 com classificaçäo a nível de gênero Arcobacter sp. Estes säo os primeiros relatos sobre a ocorrência das bactérias em carcaças de animais no Brasil.

Problemas locomotores em frangos de três linhagens do tipo corte: I. Aspectos clínicos, ocorrência de discondroplasia tibial e encurvamento do tibiotarso / Locomotor problems in broiler chickens of three commercial strains: I. Clinical aspects, occurrence of tibial dyschondroplasia and bowing of tibiotarsus

Estudou-se a ocorrência de problemas locomotores, discondroplasia tibial e encurvamento do tibiotarso em 18250 frangos do sexo masculino pertencentes a três linhagens do tipo corte (linhagem A: 6000 aves; linhagem B: 6250 aves e linhagem C: 6000 aves) criados nas mesmas condiçöes de manejo e alimentaçäo. Perturbaçöes locomotoras acorreram em 277 (1,5//) aves, näo se constatando diferenças significativas entre linhagens quanto ao número de indivíduos afetados: linhagem A, 90 (1,5//) casos; linhagem B, 103 (1,6//) casos e linhagem C, 84(1,4//) casos. Deformaçöes do tipo "valgus-varus" atingiram 220 (79,4//) animais após os 21 dias de idade. Artrite supurada ocorreu em 11 (4,0//) aves com problemas locomotores, isolando-se, na maioria dos casos, E. coli e Staphylococcus sp. Provas sorológicas foram negativas para Mycoplasma synoviae, Mycoplasma gallisepticum e reovírus (S-1133). Em 46 (16,6//) das 277 aves com problemas da locomoçäo, näo foram constatadas alteraçöes macroscópicas nos membros locomotores. Discondroplasia tibial foi diagnosticada a partir dos 28 dias de idade, em 12 (11,4//) de 105 espécimes clinicamente sadios e em 24 (30,0//) de 80 acometidos de distúrbiosda locomoçäo, näo havendo diferenças estatisticamente significativa entre linhagens quanto à suscetibilidade: linhagem A, 11 casos; linhagem B, oito casos e linhagem C, 17 casos. Encurvamento do tibiotarso afetou 29 (36,0//) de 80 aves com problemas locomotores, após a quinta semana de vida, nem sempre em associaçäo com discondroplasia tibial