Evaluation of protection conferred by a Salmonella Typhimurium inactivated vaccine in Salmonella-infected finishing pig farms

The efficacy of an inactivated S. Typhimurium vaccine administered to pigs at the beginning of the fattening period was evaluated in four clinical trials (trials A, B, C and D). Faecal shedding and the systemic antibody response during fattening, as well as, the cecal contents and mesenteric lymph nodes collected after slaughtering were used to assess the outcome. Salmonella shedders prevalence in the control groups was six times higher than in the treated groups in trials A and D, both herds infected by S. Typhimurium. The risk of positive pens was also four or five times higher for the pens housing control pigs in trials A and C. Lower prevalence of Salmonella was observed in the slaughter samples from the vaccinated pigs in trial D and in the cecal content samples in trial A, when just the S. Typhimurium results were compared. The results suggest the effective homologous protection of the vaccinated pigs; however, the high humoral response elicited in the vaccinated pigs complicates their use in farms under serological surveillance programmes.     

Non-typhoidal Salmonella encephalopathy involving lipopolysaccharide in cattle

This study assessed the involvement of lipopolysaccharide (LPS) in the non-typhoidal Salmonella encephalopathy (NTSE) caused by a unique isolate of Salmonella enterica serovar Saint-paul (SstpNPG). NTSE was prevented by genetic (deletion of murE) or pharmacologic (polymyxin) disruption of LPS on SstpNPG although the disruption of LPS did not deter brain penetration of the strain. This is the first study to demonstrate that LPS is involved in the manifestations of NTSE.     

Rapid testing and quantification of Salmonella in ileocaecal lymph nodes of Austrian pigs slaughtered for consumption

Traditionally, quantitative microbial risk assessment (QMRA) is based on culture-dependent technologies. However, molecular quantification could forge additional, detailed information. A prerequisite of quantitative real-time PCR in animal science is a tissue preparation method where large volumes of tissue material can be reduced and particularly target cells can be concentrated. An easy-to-use sample preparation method for food (Matrix-Lysis) was recently adapted to tissues and now permits quantification of target cells from up to 5 g of organic matrix. The aim of this study was to examine the suitability of Matrix-Lysis for quantification of Salmonella in porcine ileocaecal lymph nodes (ICLNs). After demonstrating constant recovery rates, ICLNs from 540 pigs were examined for Salmonella spp. with Matrix-Lysis. Samples were also analysed using ISO 6579:2002, a combined enrichment/qPCR method and a lateral flow test. It could be shown that qPCR coupled with Matrix-Lysis can contribute to QMRA in food safety by enabling reproducible quantitative data, even at low contamination rates.     

Review of pathogenesis and diagnostic methods of immediate relevance for epidemiology and control of Salmonella Dublin in cattle

Salmonella enterica subsp. enterica serovar Dublin (S. Dublin) receives increasing attention in cattle production. It is host-adapted to cattle, and leads to unacceptable levels of morbidity, mortality and production losses in both newly and persistently infected herds. Cattle health promoting institutions in several countries are currently constructing active surveillance programmes or voluntary certification programmes, and encourage control and eradication of S. Dublin infected cattle herds. There is a need to understand the underlying pathogenesis of the infection at both animal and herd level to design successful programmes. Furthermore, knowledge about and access to diagnostic tests for use in practice including information about test accuracy and interpretation of available diagnostic test methods are requested.     

A rabbit model of non-typhoidal Salmonella bacteremia

Bacteremia is an important cause of morbidity and mortality in humans. In this study, we focused on the development of an animal model of bacteremia induced by non-typhoidal Salmonella. New Zealand White rabbits were inoculated with a human isolate of non-typhoidal Salmonella strain CVD J73 via the intra-peritoneal route. Blood samples were collected at specific time points and at euthanasia from infected rabbits. Additionally, tissue samples from the heart, lungs, spleen, gastrointestinal tract, liver and kidneys were obtained at euthanasia. All experimentally infected rabbits displayed clinical signs of disease (fever, dehydration, weight loss and lethargy). Tissues collected at necropsy from the animals exhibited histopathological changes indicative of bacteremia. Non-typhoidal Salmonella bacteria were detected in the blood and tissue samples of infected rabbits by microbiological culture and real-time PCR assays. The development of this animal model of bacteremia could prove to be a useful tool for studying how non-typhoidal Salmonella infections disseminate and spread in humans.     

Enhanced protective immune responses against Salmonella Enteritidis infection by Salmonella secreting an Escherichia coli heat-labile enterotoxin B subunit protein

Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is a potent mucosal adjuvant. In this study, the effect of an attenuated Salmonella secreting LTB protein as an adjuvant strain (JOL1228) for a live Salmonella Enteritidis (SE) vaccine candidate (JOL919) was evaluated. In a single immunization experiment, chickens immunized with a mixture of JOL919 (5 parts) and JOL1228 (1 part) showed enhanced mucosal and cellular immune responses and efficient protection against salmonellosis as compared to those unimmunized control chickens. In further analysis, chickens were primed at one day of age and were boosted at the fifth week of age to prolong immune responses and to maximize the protection efficacy against salmonellosis. The immunized groups B (prime and booster with JOL919), C (prime with JOL919-JOL1228 mixture and booster with JOL919), and D (prime and booster with JOL919-JOL1228 mixture) showed significantly higher humoral and cellular immune responses as compared to those in the unimmunized control group A. In addition, immunized groups C and D showed fewer gross lesions in the liver and spleen and a lower number of SE-positive organs, with the lowest bacterial counts in the SE challenge strain as compared to the control group. These results indicate that SE vaccination with the LTB strain can have an adjuvant effect on the vaccine candidate by enhancing immune responses, and that a prime-boost strategy with the addition of the adjuvant strain can efficiently protect birds against salmonellosis.     

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 ± 0.09 and 0.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 ± 0.21 and 1.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.     

Salmonella Antimicrobial Activity of Selected Strains of Enterolactobacillus Species Isolated from the Gastrointestinal Tract of the Horse

The gastric mucosa and the mucosa of the right and left dorsal colon were biopsied in each of the 15 horses, and a total of 45 samples were collected. Mucosal samples were cultured using a Lactobacillus enrichment broth. While numerous Lactobacillus strains were identified, Lactobacillus reuteri was the most common organism identified. Sixteen strains of Lactobacillus reuteri were selected for antimicrobial testing. Salmonella antimicrobial activity was identified in six out of 16 strains tested. Organisms with Salmonella antimicrobial activity were cultured from the stratified squamous epithelium of the stomach and the mucosa of the right and left dorsal colon.     

Prevalence and antimicrobial susceptibility of Salmonella isolates in Pekin ducks from South Korea

An investigation was carried out to determine the prevalence and antibiotic resistance of Salmonella serotypes at South Korean duck farms. A total of 7119 samples collected from 72 duck farms in five provinces were examined from 2011 to 2012. The overall prevalence of Salmonella serotypes was 43.4% (69/159) in duck flocks from 65.2% (47/72) of the duck farms. Eighty-five strains were isolated from 69 duck flocks. Three serotypes of Salmonella enterica were identified such as S. Typhimurium (39/85), S. Enteritidis (44/85), and S. London (2/85). The prevalence of Salmonella infection decreased significantly in 3-week-old ducks compared to that in 1-week-old ducks (P < 0.05). All isolates except one were resistant to at least one antimicrobial and 27% of the isolates were resistant to 5–16 antimicrobials. Our findings provide baseline information on the degree of Salmonella infection and distribution of Salmonella serotypes in ducks and indicate that ducks should be considered an important source of foodborne pathogens.     

Prevalence and antimicrobial susceptibility of Salmonella spp. in small Indian mongooses (Herpestes auropunctatus) in Grenada,West Indies

Intestinal samples from 156 small Indian mongooses (Herpestes auropunctatus) collected island-wide in Grenada from April 2011 to March 2013 were examined for the presence of Salmonella enterica spp. Nineteen (12%) mongooses were culture-positive for S. enterica spp. of which five serotypes were identified. Salmonella javiana and S. Montevideo were the most commonly isolated serotypes. The other serotypes isolated were S. Rubislaw, S. Panama and S. Arechavaleta. All isolates were susceptible to amoxicillin–clavulanic acid, ampicillin, cefotaxime, ceftazidime, ciprofloxacin, enrofloxacin, gentamicin, nalidixic acid, imipenem and trimethoprim–sulfamethoxazole. One isolate (S. Montevideo) showed resistance to tetracycline and intermediate resistance to streptomycin. The five isolated Salmonella serotypes are potential human pathogens suggesting that the mongoose may play a role in the epidemiology of human salmonellosis in Grenada.     

Clinically relevant multidrug resistant Salmonella enterica in swine and meat handlers at the abattoir

The presence of multidrug resistant (MDR) Salmonella serotypes in slaughtered swine, carcasses, meat and meat handlers is scarcely evaluated. Recently we demonstrated that diverse Salmonella serotypes are frequently present in swine, pork meat and carcasses, and meat handlers at Portuguese abattoirs. Here we have characterized their antibiotic resistance phenotypes and genotypes, helping elucidate the flow of MDR Salmonella in the food chain. Testing 60 Salmonella isolates from different serotypes, the highest frequencies of resistance were observed for tetracycline (T) [70% (n = 42/60), tet(A)/tet(B)/tet(G)], streptomycin (S) [63% (n = 38/60), aadA2/strA/strB], sulfamethoxazole (Sul) [62% (n = 37/60), sul1/sul2/sul3] and ampicillin (A) [57% (n = 34/60), bla_PSE-1/bla_TEM]. Thirty-seven percent (n = 22/60) carried class 1 integrons and multidrug resistance was frequently observed (63% n = 38/60), including those serotypes common to human infections [S. Typhimurium 78% n = 25/32; S. 4,[5],12:i:- 67% n = 2/3; S. Rissen 75% (n = 3/4); S. London 67% n = 2/3; S. Derby 55%; n = 6/11)]. The emergent S. 4,[5],12:i:- isolates were mostly characterized by ASSuT phenotype [bla_TEM/strA-strB/sul2/tet(B)], typical of the European clone, while for the first time the ST phenotype [strA-strB-tet(A)-tet(B)] was also observed. Moreover, we report a first finding of a MDR phenotype in S. London [ANSSuT; bla_TEM-strA-strB-sul2-tet(A)]. Our findings suggest that the abattoir environment and the slaughter operations seem not only to harbor MDR serotypes that originated in the pig reservoir, but also propagate them through cross-contamination processes, involving meat handlers. The present study suggests a probable relationship between swine and human salmonellosis throughout the food chain, which is of interest for epidemiological, animal health and public health purposes.     

Evaluation of efficacy of a new live Salmonella Typhimurium vaccine candidate in a murine model

Efficacy of a new live Salmonella Typhimurium vaccine candidate attenuated by two virulence gene deletions was evaluated in this study. Live form of the vaccine was orally administered and formalin-inactivated form was intramuscularly (IM) administered. 105 female BALB/c mice were used and divided into 5 groups, A to E, containing 21 mice per group. Serum IgG and secretory IgA titers and levels of serum IFN-γ, IL-4, TNF-α, IL-12 of the immunized groups, especially group C (oral prime-oral booster) significantly increased. In addition, all animals in groups C showed no clinical symptoms and survived the virulent challenges, whereas all or some of mice from the other groups died of the challenge. These indicate that the vaccine candidate can be an effective tool for prevention of Salmonella infections by inducing robustly protective immune responses and leading to the production of inflammatory cytokines. Booster immunization, especially via oral administration, is necessary to optimize its protection.     

6-hydroxydopamine-mediated release of norepinephrine increases faecal excretion of Salmonella enterica serovar Typhimurium in pigs

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. In pigs, transport and social stress are associated with reactivation and spread of Salmonella Typhimurium infection. The stress-related catecholamine norepinephrine (NE) has been reported to activate growth and virulence factor expression in Salmonella; however the extent to which NE contributes to stress-associated salmonellosis is unclear. We studied the impact of releasing NE from endogenous stores during Salmonella Typhimurium infection of pigs by administration of 6-hydroxydopamine (6-OHDA), which selectively destroys noradrenergic nerve terminals. Treatment of pigs with 6-OHDA 7 or 16 days post-oral inoculation with Salmonella Typhimurium produced elevated plasma NE levels and transiently, but significantly, increased faecal excretion of the challenge strain. Oral administration of NE to Salmonella Typhimurium-infected pigs also transiently and significantly increased shedding; however pre-culture of the bacteria with NE did not alter the outcome of infection. Salmonella has been proposed to sense and respond to NE via a homologue of the adrenergic sensor kinase QseC. A ΔqseC mutant of Salmonella Typhimurium was consistently excreted in lower numbers than the parent strain post-oral inoculation of pigs, though not significantly so. 6-OHDA treatment of pigs infected with the ΔqseC mutant also increased faecal excretion of the mutant strain, albeit to a lesser extent than observed upon 6-OHDA treatment of pigs infected with the parent strain. Our data support the notion that stress-related catecholamines modulate the interaction of enteric bacterial pathogens with their hosts.     

Characterization of adaptive immune responses induced by a new genetically inactivated Salmonella Enteritidis vaccine

The superior conservation of antigenic determinants on the surface of genetically inactivated bacterial ghosts makes them attractive immunogenic inactivated vaccine candidates. The efficacy of Salmonella Enteritidis (SE) ghost vaccination was evaluated in chickens by characterizing the nature of the adaptive immune response. Chickens from the immunized group demonstrated significant increases in SE-specific plasma IgG, intestinal secretory IgA, and lymphocyte proliferative response. The populations of CD4, CD8, and TCR γδ T-cells in immunized chickens were significantly greater than in the controls. Increased levels of IFN-γ, IL-2, IL-6 and IL-10 were observed in peripheral blood mononuclear cells stimulated with SE specific antigen. After virulent SE challenge, the immune system of immunized chickens was rapidly stimulated, as indicated by significantly increased population of CD4 and CD8 T-cells. Furthermore, the immunized group exhibited decreased challenge strain recovery of the internal organs compared to the non-immunized group. Together, these data indicate that the immunization induced humoral and cell-mediated immunity might be responsible for significant reduction of the virulent challenge strain load in the internal organs of immunized chickens.     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul, Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Dynamic of nasal colonization by methicillin-resistant Staphylococcus aureus ST398 and ST1 after mupirocin treatment in a family in close contact with pigs

Nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) was evaluated after a mupirocin treatment in a family previously colonized by MRSA sequence type ST398 and ST1, who lived close to a pig farm. Eight nasal samples were swabbed from each of the four family members on different moments after mupirocin treatment. The efficacy of treatment was low in those family members who worked in the farm, and higher in the remaining two family members with sporadic contact with pigs. In addition, nasal and skin swabs from randomly selected pigs of the farm were taken. MRSA were detected in 33% of pigs tested. All MRSA isolates obtained were characterized by Staphylococcal-Cassette-Chromosome mec (SCCmec) determination, Multilocus-Sequence-Typing (MLST), spa- and agr-typing, Pulsed-field-gel-electrophoresis (PFGE), antimicrobial susceptibility, detection of antimicrobial resistance genes, and toxin gene profiling. Spa-types t011, t1255 and t1197 were detected in humans and animals, with indistinguishable PFGE patterns, suggesting animal to human MRSA transmission. Each spa-type was ascribed to a specific pulsotype. Spa-types t127 and t108 were only detected in MRSA isolates obtained from humans, and t012 only in those from animals. MRSA ST1-t127 isolates and some ST398-t011 and ST398-t1197 isolates presented a multiantimicrobial-resistance phenotype. None of them harbored lukF/lukS, tst, eta and etb virulence genes. This study showed that the efficacy of nasal MRSA decolonization in healthy people with very close contact with pigs is especially low.     

Immunological study of an attenuated Salmonella Typhimurium expressing ApxIA,ApxIIA, ApxIIIA and OmpA of Actinobacillus pleuropneumoniae in a mouse model

Salmonella Typhimurium strain expressing the Actinobacillus pleuropneumoniae antigens, ApxIA, ApxIIA, ApxIIIA and OmpA, was previously constructed as a vaccine candidate for porcine pleuropneumonia. This strain was a live attenuated (∆lon∆cpxR∆asd)Salmonella as a delivery host and contained a vector containing asd. An immunological study of lymphocyte proliferation, T-lymphocyte subsets and cytokines in the splenocytes of a mouse model was carried out after stimulation with the candidate Salmonella Typhimurium by intranasal inoculation. The splenic lymphocyte proliferation and the levels of IL-4, IL-6 and IL-12 of the inoculated mice were significantly increased, and the T- and B-cell populations were also elevated. Collectively, the candidate may efficiently induce the Th1- and Th2-type immune responses.     

Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada

In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8 months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II.All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in wild and domestic ruminants sharing pastures in Galicia (Northwest Spain)

The prevalence of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and cELISA, respectively, in 160 roe deer (Capreolus capreolus), 177 sheep and 178 cattle sharing pastures in Galicia (Northwest Spain). The seroprevalence for T. gondii was 13.7% in roe deer, 57% in sheep and 7.3% in cattle. The seroprevalence for N. canimum was 6.8%, 10.1% and 24.1% in roe deer, sheep and cattle, respectively. Statistically significant differences were observed between sheep and the other species for T. gondii and between cattle and the other ruminants for N. caninum. Only 19/515 animals were positive for both, T. gondii and N. caninum. Statistically significant differences were observed among different geographical areas for T. gondii but not for Neospora, seroprevalence being higher in the coastal area lower than in other areas. This study reveals a widespread exposure to T. gondii in Galician ruminants, and therefore, those species, particularly sheep, should be regarded as a potential source of infection for humans.