Genomic Features and Phylogenetic Analysis of Antimicrobial-Resistant Salmonella Mbandaka ST413 Strains.

In recent years, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has been increasingly isolated from laying hens and shell eggs around the world. Moreover, this serovar has been identified as the causative agent of several salmonellosis outbreaks in humans. Surprisingly, little is known about the characteristics of this emerging serovar, and therefore, we investigated antimicrobial resistance, virulence, and prophage genes of six selected Brazilian strains of Salmonella Mbandaka using Whole Genome Sequencing (WGS). Multi-locus sequence typing revealed that the tested strains belong to Sequence Type 413 (ST413), which has been linked to recent multi-country salmonellosis outbreaks in Europe. A total of nine resistance genes were detected, and the most frequent ones were aac(6')-Iaa, sul1, qacE, blaOXA-129, tet(B), and aadA1. A point mutation in ParC at the 57th position (threonine → serine) associated with quinolone resistance was present in all investigated genomes. A 112,960 bp IncHI2A plasmid was mapped in 4/6 strains. This plasmid harboured tetracycline (tetACDR) and mercury (mer) resistance genes, genes contributing to conjugative transfer, and genes involved in plasmid maintenance. Most strains (four/six) carried Salmonella genomic island 1 (SGI1). All S. Mbandaka genomes carried seven pathogenicity islands (SPIs) involved in intracellular survival and virulence: SPIs 1-5, 9, and C63PI. The virulence genes csgC, fimY, tcfA, sscA, (two/six), and ssaS (one/six) were absent in some of the genomes; conversely, fimA, prgH, and mgtC were present in all of them. Five Salmonella bacteriophage sequences (with homology to Escherichia phage phiV10, Enterobacteria phage Fels-2, Enterobacteria phage HK542, Enterobacteria phage ST64T, Salmonella phage SW9) were identified, with protein counts between 31 and 54, genome lengths of 24.7 bp and 47.7 bp, and average GC content of 51.25%. In the phylogenetic analysis, the genomes of strains isolated from poultry in Brazil clustered into well-supported clades with a heterogeneous distribution, primarily associated with strains isolated from humans and food. The phylogenetic relationship of Brazilian S. Mbandaka suggests the presence of strains with high epidemiological significance and the potential to be linked to foodborne outbreaks. Overall, our results show that isolated strains of S. Mbandaka are multidrug-resistant and encode a rather conserved virulence machinery, which is an epidemiological hallmark of Salmonella strains that have successfully disseminated both regionally and globally.

Low temperatures do not impair the bacterial plasmid conjugation on poultry meat.

Conjugation plays an important role in the dissemination of antimicrobial resistance genes. Besides, this process is influenced by many biotic and abiotic factors, especially temperature. This study aimed to investigate the effect of different conditions of temperature and storage (time and recipient) of poultry meat, intended for the final consumer, affect the plasmid transfer between pathogenic (harboring the IncB/O-plasmid) and non-pathogenic Escherichia coli organisms. The determination of minimal inhibitory concentrations (MIC) of ampicillin, cephalexin, cefotaxime, and ceftazidime was performed before and after the conjugation assay. It was possible to recover transconjugants in the poultry meat at all the treatments, also these bacteria showed a significant increase of the MIC for all antimicrobials tested. Our results show that a non-pathogenic E. coli can acquire an IncB/O-plasmid through a conjugation process in poultry meat, even stored at low temperatures. Once acquired, the resistance genes endanger public health especially when it is about critically and highly important antimicrobials to human medicine.

Salmonella enterica serovars in absence of ttrA and pduA genes enhance the cell immune response during chick infections.

Salmonella spp. is one of the major foodborne pathogens responsible for causing economic losses to the poultry industry and bringing consequences for public health as well. Both the pathogen survival ability in the intestinal environment during inflammation as well as their relationship with the host immune system, play a key role during infections in poultry. The objective of this study was to quantify the presence of the macrophages and CD4+/CD8+ cells populations using the immunohistochemistry technique, in commercial lineages of chickens experimentally infected by wild-type and mutant strains of Salmonella Enteritidis and Salmonella Typhimurium lacking ttrA and pduA genes. Salmonella Enteritidis ∆ttrA∆pduA triggered a higher percentage of the stained area than the wild-type, with exception of light laying hens. Salmonella Typhimurium wild-type strain and Salmonella Typhimurium ∆ttrA∆pduA infections lead to a similar pattern in which, at 1 and 14 dpi, the caecal tonsils and ileum of birds showed a more expressive stained area compared to 3 and 7 dpi. In all lineages studied, prominent infiltration of macrophages in comparison with CD4+ and CD8+ cells was observed. Overall, animals infected by the mutant strain displayed a positively stained area higher than the wild-type. Deletions in both ttrA and pduA genes resulted in a more intense infiltration of macrophages and CD4+ and CD8+ cells in the host birds, suggesting no pathogen attenuation, even in different strains of Salmonella.

Chitosan and HPMCAS double-coating as protective systems for alginate microparticles loaded with Ctx(Ile21)-Ha antimicrobial peptide to prevent intestinal infections.

The incorrect use of conventional drugs for both prevention and control of intestinal infections has contributed to a significant spread of bacterial resistance. In this way, studies that promote their replacement are a priority. In the last decade, the use of antimicrobial peptides (AMP), especially Ctx(Ile21)-Ha AMP, has gained strength, demonstrating efficient antimicrobial activity (AA) against pathogens, including multidrug-resistant bacteria. However, gastrointestinal degradation does not allow its direct oral application. In this research, double-coating systems using alginate microparticles loaded with Ctx(Ile21)-Ha peptide were designed, and in vitro release assays simulating the gastrointestinal tract were evaluated. Also, the AA against Salmonella spp. and Escherichia coli was examined. The results showed the physicochemical stability of Ctx(Ile21)-Ha peptide in the system and its potent antimicrobial activity. In addition, the combination of HPMCAS and chitosan as a gastric protection system can be promising for peptide carriers or other low pH-sensitive molecules, adequately released in the intestine. In conclusion, the coated systems employed in this study can improve the formulation of new foods or biopharmaceutical products for specific application against intestinal pathogens in animal production or, possibly, in the near future, in human health.

Molecular genotyping reveals inter-regional relatedness among antimicrobial resistant Salmonella Minnesota strains isolated from poultry farm and humans, Brazil.

Genetic profiles of Salmonella Minnesota isolates were analyzed using pulsed-field gel electrophoresis (PFGE). In total, 13 isolates obtained from the broiler industry collected in the states of Minas Gerais (11) and São Paulo (2), as well as five recovered from cases of foodborne infections in humans in the states of Minas Gerais (2), Santa Catarina (1), and Rio Grande do Sul (2), were submitted to PFGE. These 18 S. Minnesota isolates together with other 12 of poultry origin were also subjected to antimicrobial susceptibility testing. The PFGE analysis of 18 strains of S. Minnesota generated a dendrogram that grouped the isolates with 83-90% similarity into four main clusters. Among them, cluster "A" grouped the majority of isolates (13), including two of human origin that showed 90% similarity with a broiler isolate, both recovered in Minas Gerais. The S. Minnesota isolates showed resistance to tetracycline (80%), cefoxitin (80%), ceftazidime (46.7%), nalidixic acid (23.3%), ciprofloxacin (13.3%), and streptomycin (10%). No resistance to gentamicin, chloramphenicol, meropenem, nitrofurantoin, and sulfamethoxazole-trimethoprim was found. Moreover, 23.3% of the evaluated isolates presented multi-resistance profile, all from Minas Gerais. The results highlight the importance of further studies involving S. Minnesota, which is prevalent in the Brazilian broiler flocks and could provoke foodborne infection in humans.

Evaluation of transfer of maternal immunity to the offspring of broiler breeders vaccinated with a candidate recombinant vaccine against Salmonella Enteritidis.

Salmonella Enteritidis (SE) is a major cause of foodborne diseases in humans being frequently related to the consumption of poultry products. Therefore, guaranteeing early immunity to chicks is an important tool to prevent the colonization and infection by this pathogen. The present study evaluated the effectiveness of a candidate recombinant vaccine against SE. Thirty female and five male broiler breeders that were ten weeks-old were divided into 3 groups: unvaccinated (UV), vaccinated with recombinant vaccine candidate (VAC) and vaccinated with commercial bacterin (BAC). Samples of serum and embryonated egg were collected at seven and twelve weeks after the booster dose to quantify the transfer rate of IgY to egg yolks and offspring. Subsequently, forty day-old offspring were divided into two groups (UV and VAC) and challenged on the following day with 107 CFU/chick of SE. Samples of serum, intestine, liver, and cecal content were harvested. Throughout the experiment period, significantly higher levels of IgY were observed in the egg yolk and also in the serum of broiler breeders and offspring of the VAC group in comparison to the UV group. In addition, increased transfer rates of IgY were observed in the VAC group when compared to the BAC group. Furthermore, higher villus-crypt ratios were found out in duodenum, jejunum and ileum at four days post-infection in the offspring from the VAC group. A high challenge dose of SE (107 CFU per chick) was used and despite the stronger humoral immune response provoked by the candidate vaccine, there were no statistical differences in the recovery of viable SE cells from the offspring cecal contents. Therefore, the effect of vaccination to improve intestinal quality may affect the development of the chickens and consequently increase the resistance to lower SE challenge doses.

Heat stress impairs egg production in commercial laying hens infected by fowl typhoid.

Salmonella Gallinarum (SG) is an avian-restricted pathogen that causes fowl typhoid in poultry. Although it has been reported frequently over many decades in poultry flocks worldwide, the microorganism is more commonly associated with poultry in developing countries, particularly those with high ambient temperatures, where the acute form of the disease results in considerable economic losses. A more detailed investigation of environmental factors that affect the course of disease may assist in identifying effective prevention and control measures. Heat stress is known to impair the immunological response to a variety of pathogens and clearly may be an important contributory factor in the prevalence of disease in countries with warm or hot climates. Thus, the objective of the present study was to evaluate the effects of heat stress on chickens infected with SG. For this, light and semi-heavy commercial laying hens were distributed randomly within four groups as follows: infected and non-infected groups in rooms held at ambient temperature, and infected and non-infected groups under heat stress. Clinical signs, egg production, and mortality were recorded daily. Bacteriological counts in liver and spleen samples were estimated at 2, 5, 7, and 14 days post-infection. The results showed that both SG infection and heat stress had similar effects on egg production and a synergistic effect of the two stressors was observed. The data show an interaction between disease and heat stress which could point towards environmental and biosecurity approaches to resolving the possible 30% fall in production observed in such countries.

Emergent multidrug-resistant nontyphoidal Salmonella serovars isolated from poultry in Brazil coharboring blaCTX-M-2 and qnrB or blaCMY-2 in large plasmids.

The number of foodborne gastroenteritis caused by nontyphoidal Salmonella (NTS) worldwide is estimated to be 80.3 million each year. Currently, antimicrobial-resistant NTS disseminated in the animal environment increases the risk of aggravated foodborne outbreaks. Poultry are important source of foodborne NTS infections. This study was conducted to evaluate the phenotypic and genotypic characteristics of 83 NTS isolates from poultry, classified within 36 different serovars. The most prevalent serovar was S. Schwarzengrund (10/83), from which 8/10 were multidrug resistant (MDR). The antimicrobial susceptibility testing showed a total of 18 MDR isolates, from which 8/18 coharbored blaCTX-M-2 and qnrB5. The genes qnrB5, blaCTX-M-2, qnrB2, or blaCMY-2 were also found alone in other MDR isolates. All resistance genes were harbored in large plasmids, ranging from 30 to 270 kb. The pColE replicon was present in 8 MDR isolates; however it was not associated with resistance. ISCR1 and class I integron structures were always associated with blaCTX-M-2.

Inactivation of phoPQ genes attenuates Salmonella Gallinarum biovar Gallinarum to susceptible chickens

Abstract Salmonella Gallinarum is a host-restrict pathogen that causes fowl typhoid, a severe systemic disease that is one of the major concerns to the poultry industry worldwide. When infecting the bird, SG makes use of evasion mechanisms to survive and to replicate within macrophages. In this context, phoPQ genes encode a two-component regulatory system (PhoPQ) that regulates virulence genes responsible for adaptation of Salmonella spp. to antimicrobial factors such as low pH, antimicrobial peptides and deprivation of bivalent cations. The role of the mentioned genes to SG remains to be investigated. In the present study a phoPQ-depleted SG strain (SG ΔphoPQ) was constructed and its virulence assessed in twenty-day-old laying hens susceptible to fowl typhoid. SG ΔphoPQ did cause neither clinical signs nor mortality in birds orally challenged, being non-pathogenic. Furthermore, this strain was not recovered from livers or spleens. On the other hand, chickens challenged subcutaneously with the mutant strain had discreet to moderate pathological changes and also low bacterial counts in liver and spleen tissues. These findings show that SG ΔphoPQ is attenuated to susceptible chickens and suggest that these genes are important during chicken infection by SG.

Identification and characterization of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae isolated from healthy poultry in Brazil.

The expression of plasmid-mediated quinolone resistance (PMQR) genes confers low-level quinolone and fluoroquinolones resistance alone. However, the association to chromosomal resistance mechanisms determines an expressively higher resistance in Enterobacteriaceae. These mechanisms are horizontally disseminated within plasmids and have contributed to the emergence of bacteria with reduced susceptibility or resistant to therapies worldwide. The epidemiological characterization of PMQR dissemination is highly relevant in the scientific and medical context, to investigate the dissemination within enterobacteria, from different populations, including humans and food-producing animals. In the present study, 200 Enterobacteriaceae isolates were harvested from poultry with cloacal swabs and identified as Escherichia coli (90.5%), Escherichia fergusonii (5.5%), Klebsiella oxytoca (2.5%) and Klebsiella pneumoniae (1.5%). Among isolates evaluated, 46 (23%) harboured PMQR genes including qnrB (43/200), qnrS (2/200) and aac(6')-Ib-cr (1/200). All isolates carrying PMQR genes showed multidrug-resistance phenotype. The 36 E. coli isolates showed 18 different PFGE types. All E. fergusonii isolates showed the same PFGE type. The two Klebsiella oxytoca belonged to two different PFGE types. The phylogenetic groups A, B1, and D were found among the E. coli harboring PMQR genes. Based on the phylogenetic analysis and PFGE, the population structure of E. coli isolates was diverse, even within the same farm. All isolates carrying qnrB and qnrS genes also harboured ColE-like plasmids. The Southern blot hybridization using the S1-PFGE revealed that the qnrB genes were located on low molecular weight plasmids, smaller than 10Kb. Resistance plasmids were sequenced and showed 100% identity with plasmid pPAB19-3. The association of PMQR genes with mobile genetic elements, such as transferable plasmids, favours the selection and dissemination of (fluoro) quinolones resistant bacteria among food-producing animals, and may play an important role in the current increased prevalence of resistant bacteria in different environments reported worldwide.

Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken

ABSTRACT Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE ΔmotB) or non-flagellated (SE ΔfliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE ΔmotB and SE ΔfliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5 dpi). The SE ΔmotB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE ΔfliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken.

Diversity of plasmids harboring blaCMY-2 in multidrug-resistant Escherichia coli isolated from poultry in Brazil.

Multidrug-resistance (MDR) has been increasingly reported in Gram-negative bacteria from the intestinal microbiota, environment and food-producing animals. Resistance plasmids able to harbor different transposable elements are capable to mobilize antimicrobial resistance genes and transfer to other bacterial hosts. Plasmids carrying blaCMY are frequently associated with MDR. The present study assessed the presence of plasmid-encoded ampC genes (blacmy, blamox, blafox, blalat, blaact, blamir, bladha, blamor) in commensal E. coli isolated from apparently healthy broiler chickens. Furthermore, we characterized the plasmids and identified those harboring the resistance genes. We isolated 144/200 (72%) of E. coli isolates with resistance to cefotaxime and the resistance gene identified was blaCMY-2. The pulsed-field gel electrophoresis (PFGE) analysis showed high diversity of the genetic profiles. The phylogenetic groups A, B1, B2, and D were identified among E. coli isolates and group D was the most prevalent. The PCR-based replicon typing (PBRT) analysis identified four distinct plasmid incompatibility groups (Inc) in MDR isolates. Moreover, plasmids harboring blaCMY-2, ranged in size from 50kb to 150kb and 51/144 (35%) belonged to IncK, 21/144 (14.5%) to IncB/O, 8/144 (5.5%) to IncA/C, 1/144 (0.5%) to IncI, while 63/144 (44.5%) were not typeable by PBRT. Overall, a high prevalence of blaCMY-2 genes was found in a diverse population of commensal MDR E. coli from poultry in Brazil, harbored into different plasmids.

Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken.

Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE ΔmotB) or non-flagellated (SE ΔfliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE ΔmotB and SE ΔfliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5dpi). The SE ΔmotB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE ΔfliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken.

Protection of chickens against fowl typhoid using field vaccine programs formulated with the live attenuated strain Salmonella Gallinarum ΔcobSΔcbiA / Proteção de aves contra o tifo aviário utilizando a cepa atenuada Salmonella Gallinarum ΔcobSΔcbiA em diferentes programas vacinais de campo

Salmonella enterica serovar Gallinarum biovar Gallinarum (SG) is a host-specific bacteria that causes the fowl typhoid (FT). This disease is highly pathogenic to commercial chickens, specially brown layers and breeders, causing acute septicemia followed by high morbidity and mortality. Vaccination is extensively adopted in the fields as a biosafety tool for prevention of isolated infections and outbreaks in commercial poultry flocks. The present study evaluated the use of an attenuated SG with deletions on genes cobS and cbiA (SGΔcobSΔcbiA) as a live vaccine, using vaccination schemes adjusted for field conditions. To this end, brown layers were used in two different experiments, to evaluate the long-term protection, necessary in the fields. The vaccination scheme on the first experiment consisted of two doses, the first at 4 th week-of-age and the booster dose at 8 th week-of-age with challenge at 16 th week-of-age with wild SG strain. On the second experiment, the vaccination was carried out by different routes using three doses of the live vaccine, at 4 th , 8 th and 12 th weeks-of-age, and the challenge was done at 20 th weeks-of-age. After the challenge, the mortality was recorded during 28 days, and the egg production (experiment 2) was evaluated and compared with the group of unvaccinated layers. In both experiments, the mortality was significantly reduced, and the egg production was not affected in vaccinated layer-hens. In summary, this study shows the efficacy and the protection of different vaccination schemes against FT that can be applied under field conditions in commercial poultry farms.(AU)

Salmonella enterica sorovar Gallinarum biovar Gallinarum (SG) é uma bactéria hospedeira específica que causa o tifo aviário (TA). Essa doença é altamente patogênica em aves comerciais, especialmente galinhas poedeiras de linhagem vermelha e aves reprodutoras pesadas, causando septicemia aguda, e consequentemente, alta morbidade e mortalidade. A vacinação é amplamente utilizada no campo como uma ferramenta de biossegurança para a prevenção de infecções isoladas e surtos nas granjas avícolas comerciais. O atual estudo avaliou o potencial vacinal de uma cepa viva atenuada de SG com deleções nos genes cobS e cbiA (SGΔcobSΔcbiA), utilizando esquemas de vacinação formulados para utilização em campo. Para isso, as galinhas poedeiras de linhagem vermelha foram utilizadas em dois experimentos diferentes, para avaliar a proteção a longo prazo, necessária no campo. O esquema de vacinação no primeiro experimento consistiu em duas doses, a primeira na quarta semana de vida e a dose de reforço na oitava, e o desafio na 16ª semana com a estirpe selvagem SG. No segundo experimento, a vacinação foi realizada por diferentes rotas usando três doses da vacina viva, na quarta, na oitava e na décima segunda semana de vida, e o desafio foi feito na 20ª semana de vida. Após o desafio, a mortalidade foi acompanhada por 28 dias, e no experimento 2 a produção de ovos também foi avaliada e comparada com o grupo de galinhas não vacinadas. Em ambos os experimentos, a mortalidade foi significativamente reduzida, e a produção de ovos não foi afetada nos grupos de galinhas poedeiras vacinadas. Este estudo mostra a eficácia da proteção dos diferentes programas de vacinação contra o TA, que podem ser aplicados em granjas comerciais em condições de campo.(AU)

Evaluation and characterization of plasmids carrying CTX-M genes in a non-clonal population of multidrug-resistant Enterobacteriaceae isolated from poultry in Brazil.

The increasing presence of ESBL-producing bacteria in food-producing animals might impact on public health. In this study, ESBL-producing enterobacteria were investigated in the microbiota of chickens produced in Brazil. We detected blaCTX-M-2, blaCTX-M-8 and blaCTX-M-15 in 13 Escherichia coli isolates, within 9 different PFGE-types. Escherichia fergusonii and Klebsiella pneumoniae were found carrying blaCTX-M-2. Plasmid Inc groups found included repF, FIB, FIC, I1, Y, B/O, A/C, K and HI1. F plasmids were present in 87.5% of the isolates, however, no resistance gene was harbored in this replicon. The pMLST for IncI1 showed ST113 and the novel ST130, ST131 and ST132 harboring blaCTX-M-8. IncK plasmids carried blaCTX-M-2 in one E. coli isolate. Non-typeable plasmids carrying blaCTX-M-2 or blaCTX-M-15 had up to 260kb. blaCTX-M-2 was also associated with class I integron and ISCR1 and blaCTX-M-8 with IS10. Overall, similar resistance elements were disseminated among a diverse population of ESBL-producing enterobacteria.

Molecular identification of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum by a duplex PCR assay.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S Gallinarum) and biovar Pullorum (S Pullorum) are 2 poultry pathogens that cause major economic losses to the poultry industry worldwide. Control of both diseases mainly relies on the adoption of biosecurity programs, and success is dependent on accurate and fast detection. Based on this concept, we developed a duplex PCR assay, targeting 2 chromosomal sequences, which allowed us to precisely identify and differentiate S Gallinarum and S Pullorum field strains. This assay was validated by testing genomic DNA from 40 S Gallinarum and 29 S Pullorum field strains, 87 other Salmonella serovars, and 7 non-Salmonella strains. The serovar identifier region (SIR) primers produced a fragment only in S Gallinarum and S Pullorum strains, whereas the fragment from the ratA coding sequence, which was previously demonstrated to differentiate the 2 biovars, was also amplified from other Salmonella serovars. Our results showed that the combination of both SIR and ratA amplifications could be used to identify as well as to differentiate colonies of S Gallinarum and S Pullorum reliably. Thus, we believe this methodology can be a useful ancillary tool for routine veterinary diagnostic laboratories by providing rapid, accurate results.

Antimicrobial susceptibility of Salmonella Gallinarum and Salmonella Pullorum isolated from ill poultry in Brazil / Suscetibilidade a antimicrobianos de Salmonella Gallinarum e Salmonella Pullorum isolados de aves doentes no Brasil

ABSTRACT: Salmonella Gallinarum (S. Gallinarum) and Salmonella Pullorum (S. Pullorum) are poultry host-specific, agents of fowl typhoid and pullorum disease, respectively. These biovars cause septicemic infections, resulting in high mortality. Outbreaks are frequently reported worldwide, causing losses due to the elimination of infected flocks and treatments. The use of antimicrobial agents is frequent in poultry farms to prevent or treat gastrointestinal infections. In the present research it was evaluated the antimicrobial susceptibility of 50 S. Gallinarum and S. Pullorum isolates, from outbreaks that occurred between 1987 to 1991 and 2006 to 2013. The comparison of the susceptibility profiles showed that all isolates were susceptible to β-lactams. All isolates from 1987-1991 were susceptible to all antibiotics tested except NAL and CIP (78%). The susceptibility profile of S. Gallinarum (2006 - 2013 period) was the following NAL (58%), CIP (63%), ENR (67%), TET (92%), FFC (96%) and SXT (96%). S. Pullorum isolates (2006 - 2013 period) showed the following susceptibility rates to NAL (65%), CIP (71%), ENR (94%) and TET (94%). All isolates were susceptible to β-lactams tested, however, resistance to quinolones and fluoroquinolones increased over time. Furthermore, low levels of resistance to other antibiotics were found in recent isolates, such as tetracyclines.

RESUMO: Salmonella Gallinarum (S. Gallinarum) e Salmonella Pullorum (S. Pullorum) são patógenos hospedeiro-específico de aves, agentes do tifo aviário e pulorose, respectivamente. Estes biovares causam infecções septicêmicas, resultando em alta mortalidade. Surtos são frequentemente relatados em diversos países, causando prejuízos devido à eliminação de lotes infectados e tratamentos. Agentes antimicrobianos são utilizados frequentemente em granjas avícolas para prevenir ou tratar infecções gastrointestinais. No presente trabalho, foi avaliada a susceptibilidade antimicrobiana de 50 isolados de S. Gallinarum e S. Pullorum, obtidos durante surtos que ocorreram entre 1987 a 1991 e entre 2006 a 2013. A comparação dos perfis de sensibilidade mostrou que todas as amostras são sensíveis aos β-lactâmicos. Todos os isolados de 1987-1991 foram sensíveis a todos os antibióticos testados, exceto NAL e CIP (78%). O perfil de susceptibilidade de S. Gallinarum (surtos de 2006 a 2013) foi NAL (58%), CIP (63%), ENR (67%), TET (92%), FFC (96%) e SXT (96%). Isolados de S. Pullorum (surtos de 2006 a 2013) apresentaram as seguintes taxas de sensibilidade: NAL (65%), CIP (71%), ENR (94%) e TET (94%). Todas as amostras foram sensíveis ao β-lactâmicos testados, no entanto, a resistência às quinolonas e fluoroquinolonas aumentou ao longo do tempo. Além disso, baixos níveis de resistência a outros antibióticos foram encontrados em isolados recentes, tais como as tetraciclinas.

Immunomodulatory activity and control of Salmonella Enteritidis colonization in the intestinal tract of chickens by Lactobacillus based probiotic.

Lactobacillus-based probiotics (LBP) are used as competitive exclusion to control pathogenic enterobacterial infections and improve the weight gain in broiler chickens. This study assessed the inhibition of Salmonella Enteritidis (SE) infection in one-week-old broiler chicks, using an experimental LBP containing four Lactobacillus strains isolated from chickens (L. acidophilus, L. fermentum, L. reuteri, L. salivarius). The immunomodulatory effects of this treatment were evaluated, through the analysis of cytokines and influx of macrophages, γδ, CD4(+) and CD8(+) T cells in the gut. The intestinal colonization by SE was reduced by 1.8 CFU/g (log10) in chicks treated with LBP (p<0.05). The levels of pro-inflammatory cytokines (IL-1ß, LITAF) were significantly reduced in treated chicks (p<0.05), whilst untreated chicks showed elevated inflammatory stimulus and an increased population of CD8(+) T cells in the intestinal mucosa after challenge (p<0.05). Additionally, the LBP stimulated TLR2 expression in caecal tonsils. The adjuvant property of the Lactobacillus cell wall (LCW) was evaluated, demonstrating good capability to stimulate T helper 2 (Th2) cell proliferation. Pretreatment of chicks with LBP decreased the intestinal colonization by SE, minimizing the tissue lesions and inflammation after challenge and showed a potential use as adjuvant with injectable killed vaccines.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Gallinarum Biovar Pullorum Strain FCAV198, a Brazilian Chicken Pathogen.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum is a bird-restricted pathogen which causes pullorum disease. The strain FCAV198 was isolated from a pool of chicken ovaries in Brazil, and its genome may be helpful for studies involving molecular mechanisms related to pathogenesis and other related applications.

Humoral and cellular immune response generated by different vaccine programs before and after Salmonella Enteritidis challenge in chickens.

The poultry industry has a high demand for Salmonella vaccines in order to generate safer Salmonella-free food for consumers around the world. Vaccination against S. Enteritidis (SE) is vastly undertaken in many countries, although the criteria for the use of live vaccine (LV) or killed vaccine (KV) should also depend on the immune mechanisms triggered by each. In this study, a commercial bacterin (KV) and an attenuated SG mutant (LV) were used in four different vaccine programs (LV; LV+LV; KV; LV+KV). At 1 day before (dbi) and 1, 6 and 9 days after SE challenge (dpi), humoral (IgM, IgG and secretory IgA) and cellular (CD8(+) T cells) immune responses were evaluated along with the production of IL-10, IL-12 and IFN-γ. Although after challenge, all birds from each group had an influx of CD8(+) T cells, birds which received KV had lower levels of these cells in organs and significantly higher levels of immunoglobulins. The expression of the cytokines was up-regulated in all groups post-vaccination, although, after challenge, cytokine expression decreased in the vaccinated groups, and increased in the unvaccinated group A. IL-10 levels were significantly higher at 1 day post-infection in the group that received KV, which may be involved in the weak cellular immune response observed within this group. In caecal tonsils, IFN-γ expression at 1 dbi was higher in birds which received two vaccine doses, and after challenge, the population of CD8(+) T cells constantly increased in birds that were only vaccinated with the LV. This study demonstrated that the development of a mature immune response by CD8(+) T cells, provided by the use of the LV, had better efficacy in comparison to the high antibody levels in the serum stimulated by the KV. However, high secretory IgA levels in the intestinal lumen associated with influx CD8(+) T cells may be indicative of protection as noticed in group E (LV+KV).