Virulent Serovar Research Articles

Comparison of Phenotype Nutritional Profiles and Phosphate Metabolism Genes in Four Serovars of Salmonella enterica from Water Sources.

The surveillance of foods for Salmonella is hindered by bias in common enrichment media where serovars implicated in human illness are outgrown by less virulent serovars. We examined four Salmonella serovars, two common in human illness (Enteritidis and Typhimurium) and two that often dominate enrichments (Give and Kentucky), for factors that might influence culture bias. The four serovars had similar growth kinetics in Tryptic Soy Broth and Buffered Peptone Water. Phenotype microarray analysis with 950 chemical substrates to assess nutrient utilization and stress resistance revealed phenotype differences between serovars. Strains of S. Enteritidis had better utilization of plant-derived sugars such as xylose, mannitol, rhamnose, and fructose, while S. Typhimurium strains were able to metabolize tagatose. Strains of S. Kentucky used more compounds as phosphorus sources and grew better with inorganic phosphate as the sole phosphorus source. The sequences of nine genes involved in phosphate metabolism were compared, and there were differences between serovars in the catalytic ATP-binding domain of the histidine kinase phoR. Analysis of the predicted PhoR amino acid sequences from additional Salmonella genomes indicated a conservation of sequences each within the Typhimurium, Give, and Enteritidis serovars. However, three different PhoR versions were observed in S. Kentucky.

Immune responses following neonatal vaccination with conserved F4 fragment of VtaA proteins from virulent Glaesserella parasuis adjuvanted with CAF®01 or CDA

Glaesserella parasuis is a Gram-negative bacterium that colonizes the upper airways of swine, capable of causing a systemic infection called Glässer’s disease. This disease is more frequent in young post-weaning piglets. Current treatments against G. parasuis infection are based on the use of antimicrobials or inactivated vaccines, which promote limited cross-protection against different serovars. For this reason, there is an interest in developing novel subunit vaccines with the capacity to confer effective protection against different virulent strains. Herein, we characterize the immunogenicity and the potential benefits of neonatal immunization with two different vaccine formulations based on the F4 polypeptide, a conserved immunogenic protein fragment from the virulence-associated trimeric autotransporters of virulent G. parasuis strains. With this purpose, we immunized two groups of piglets with F4 combined with cationic adjuvant CAF®01 or cyclic dinucleotide CDA. Piglets immunized with a commercial bacterin and non-immunized animals served as control groups. The vaccinated piglets received two doses of vaccine, at 14 days old and 21 days later. The immune response induced against the F4 polypeptide varied depending on the adjuvant used. Piglets vaccinated with the F4+CDA vaccine developed specific anti-F4 IgGs, biased towards the induction of IgG1 responses, whereas no anti-F4 IgGs were de novo induced after immunization with the CAF®01 vaccine. Piglets immunized with both formulations displayed balanced memory T-cell responses, evidenced upon in vitro re-stimulation of peripheral blood mononuclear cells with F4. Interestingly, pigs immunized with F4+CAF®01 controlled more efficiently a natural nasal colonization by a virulent serovar 4 G. parasuis that spontaneously occurred during the experimental procedure. According to the results, the immunogenicity and the protection afforded by F4 depend on the adjuvant used. F4 may represent a candidate to consider for a Glässer‘s disease vaccine and could contribute to a better understanding of the mechanisms involved in protection against virulent G. parasuis colonization.

Salmonella in Poultry; An Overview

An essential part of commercial poultry production is safeguarding flocks of birds against contamination by unwanted microorganisms. Currently, a serious issue called salmonellosis poses a threat to the chicken industry globally. The two most virulent serovars in avian species, Salmonella gallinarium and Salmonella pullorum (Fowl Typhoid) cause systemic infection and significant financial losses in the poultry sector. Salmonella nontyphoidal serotypes (Paratyphoid illness) pose a risk to the public's health because of their role in cases of food poisoning and their significance as zoonotic agents. All animals, including humans, can transmit the intestinal bacterium known as salmonella. Out of a total of 2500 serovars, Salmonella pullorum (pullorum illness) and S. gallinarum (bird typhoid) are the two host-adapted serovars that primarily harm poultry. Salmonella can spread horizontally from contaminated environments to birds as well as vertically from parent flocks to offspring. The danger of the introduction and persistence of infections can be substantially reduced by good management of biosecurity. This study is an overview of Salmonella in poultry which tell us some diagnostic and treatment of salmonella. By reading this study people will get sound knowledge regarding salmonella even they will be able to do desirable management in order to save their birds from Salmonella.

Virulence and antimicrobial resistance profile of non-typhoidal Salmonella enterica serovars recovered from poultry processing environments at wet markets in Dhaka, Bangladesh.

The rapid emergence of virulent and multidrug-resistant (MDR) non-typhoidal Salmonella (NTS) enterica serovars is a growing public health concern globally. The present study focused on the assessment of the pathogenicity and antimicrobial resistance (AMR) profiling of NTS enterica serovars isolated from the chicken processing environments at wet markets in Dhaka, Bangladesh. A total of 870 samples consisting of carcass dressing water (CDW), chopping board swabs (CBS), and knife swabs (KS) were collected from 29 wet markets. The prevalence of Salmonella was found to be 20% in CDW, 19.31% in CBS, and 17.58% in KS, respectively. Meanwhile, the MDR Salmonella was found to be 72.41%, 73.21%, and 68.62% in CDW, CBS, and KS, respectively. All isolates were screened by polymerase chain reaction (PCR) for eight virulence genes, namely invA, agfA, IpfA, hilA, sivH, sefA, sopE, and spvC. The S. Enteritidis and untyped Salmonella isolates harbored all virulence genes while S. Typhimurium isolates carried six virulence genes, except sefA and spvC. Phenotypic resistance revealed decreased susceptibility to ciprofloxacin, streptomycin, ampicillin, tetracycline, gentamicin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid, and azithromycin. Genotypic resistance showed a higher prevalence of plasmid-mediated blaTEM followed by tetA, sul1, sul2, sul3, and strA/B genes. The phenotypic and genotypic resistance profiles of the isolates showed a harmonic and symmetrical trend. According to the findings, MDR and virulent NTS enterica serovars predominate in wet market conditions and can easily enter the human food chain. The chi-square analysis showed significantly higher associations among the phenotypic resistance, genotypic resistance and virulence genes in CDW, CBS, and KS respectively (p < 0.05).

Two homologous Salmonella serogroup C1-specific genes are required for flagellar motility and cell invasion

BackgroundSalmonella is a major bacterial pathogen associated with a large number of outbreaks of foodborne diseases. Many highly virulent serovars that cause human illness belong to Salmonella serogroup C1, and Salmonella ser. Choleraesuis is a prominent cause of invasive infections in Asia. Comparative genomic analysis in our previous study showed that two homologous genes, SC0368 and SC0595 in Salmonella ser. Choleraesuis were unique to serogroup C1. In this study, two single-deletion mutants (Δ0368 and Δ0595) and one double-deletion mutant (Δ0368Δ0595) were constructed based on the genome. All these mutants and the wild-type strain were subjected to RNA-Seq analysis to reveal functional relationships of the two serogroup C1-specific genes.ResultsData from RNA-Seq indicated that deletion of SC0368 resulted in defects in motility through repression of σ28 in flagellar regulation Class 3. Consistent with RNA-Seq data, results from transmission electron microcopy (TEM) showed that flagella were not present in △0368 and △0368△0595 mutants resulting in both swimming and swarming defects. Interestingly, the growth rates of two non-motile mutants △0368 and △0368△0595 were significantly greater than the wild-type, which may be associated with up-regulation of genes encoding cytochromes, enhancing bacterial proliferation. Moreover, the △0595 mutant was significantly more invasive in Caco-2 cells as shown by bacterial enumeration assays, and the expression of lipopolysaccharide (LPS) core synthesis-related genes (rfaB, rfaI, rfaQ, rfaY, rfaK, rfaZ) was down-regulated only in the △0368△0595 mutant. In addition, this study also speculated that these two genes might be contributing to serotype conversion for Salmonella C1 serogroup based on their apparent roles in biosynthesis of LPS and the flagella.ConclusionA combination of biological and transcriptomic (RNA-Seq) analyses has shown that the SC0368 and SC0595 genes are involved in biosynthesis of flagella and complete LPS, as well as in bacterial growth and virulence. Such information will aid to revealing the role of these specific genes in bacterial physiology and evolution within the serogroup C1.

The Role of Antibodies Against the Crude Capsular Extract in the Immune Response of Porcine Alveolar Macrophages to In Vitro Infection of Various Serovars of Glaesserella (Haemophilus) parasuis

In Glässer’s disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.

Quantitative Real-Time PCR Assays for the Detection of Pathogenic Leptospira Species in Urine and Blood Samples in Canine Vaccine Clinical Studies: a Rapid Alternative to Classical Culture Methods.

ABSTRACTLeptospirosis is a vaccine-preventable bacterial zoonotic disease caused by pathogenic Leptospira species. The efficacy of Leptospira canine vaccines is assessed by challenging vaccinated and control dogs with virulent serovars of Leptospira, followed by detection of Leptospira in blood and urine. We assessed the consistency between results obtained for urine and blood samples from clinical studies with species-specific real-time quantitative PCR (qPCR) targeting the lipL32 gene and those obtained with the reference culture method. The specificity of the qPCR assay was confirmed by negative results for nonpathogenic Leptospira and for several canine viruses, bacteria, and parasites. The results from the two methods were compared using McNemar’s test, kappa coefficient (κ), and percentage of agreement analyses. The results for numbers of positive and negative dogs were similar, with no false-negative results with the qPCR assay. For both blood and urine, there was strong agreement between the culture method and qPCR results (κ = 0.68 [95% confidence interval (CI), 0.62 to 0.74] and κ = 0.65 [95% CI, 0.59 to 0.71], respectively). However, there was a statistically significant difference between blood samples (P < 0.001) and urine samples (P = 0.028). The negative percentage agreements were 97% and 84% and the positive percentage agreements were 68% and 83% for blood and urine samples, respectively. Although the cell culture method is the recommended gold standard, our results show that qPCR assay is a valid alternative method for the rapid and specific detection of pathogenic Leptospira spp. in urine and blood samples during vaccine efficacy studies, without loss of sensitivity.

Characterization of Salmonella spp. Isolates from Swine: Virulence and Antimicrobial Resistance.

Simple SummarySalmonella is a pathogenic bacterium able to infect both humans and animals. It is diffused worldwide and, generally, animals are a source of infection for humans. Among domestic animals, swine represents an important reservoir and a frequent source of human infection, especially in some countries like Italy. To acquire information on Salmonella, in particular about epidemiology, but also virulence, pathogenesis and antimicrobial resistance, is the basis for a cohesive control program. This manuscript describes an investigation conducted on Salmonella isolates from swine, where two important characteristics were evaluated: the pathogenicity and the antimicrobial resistance. A great variability was observed among investigated strains. Salmonella serovar Typhimurium was confirmed as one of the most virulent serovars; indeed, most isolates belonging to this serovar presented many of the searched virulence factors. A high level of antimicrobial resistance was observed for some compounds (sulfonamide, tetracycline, streptomycin and ampicillin), but not for the so-called “last line antibiotics”, such as, for example, ciprofloxacin. The constant monitoring on circulating strains in reservoir animals is important to acquire information and set up adequate prophylaxis measures.Salmonella is one of the most important zoonotic pathogens worldwide. Swine represent typical reservoirs of this bacterium and a frequent source of human infection. Some intrinsic traits make some serovars or strains more virulent than others. Twenty-nine Salmonella spp. isolated from pigs belonging to 16 different serovars were analyzed for gastric acid environment resistance, presence of virulence genes (mgtC, rhuM, pipB, sopB, spvRBC, gipA, sodCI, sopE), antimicrobial resistance and presence of antimicrobial resistance genes (blaTEM, blaPSE-1, aadA1, aadA2, aphA1-lab, strA-strB, tetA, tetB, tetC, tetG, sul1, sul2, sul3). A percentage of 44.83% of strains showed constitutive and inducible gastric acid resistance, whereas 37.93% of strains became resistant only after induction. The genes sopB, pipB and mgtC were the most often detected, with 79.31%, 48.28% and 37.93% of positive strains, respectively. Salmonella virulence plasmid genes were detected in a S. enterica sup. houtenae ser. 40:z4,z23:-strain. Fifteen different virulence profiles were identified: one isolate (ser. Typhimurium) was positive for 6 genes, and 6 isolates (3 ser. Typhimurium, 2 ser. Typhimurium monophasic variant and 1 ser. Choleraesuis) scored positive for 5 genes. None of the isolates resulted resistant to cefotaxime and ciprofloxacin, while all isolates were susceptible to ceftazidime, colistin and gentamycin. Many strains were resistant to sulfonamide (75.86%), tetracycline (51.72%), streptomycin (48.28%) and ampicillin (31.03%). Twenty different resisto-types were identified. Six strains (4 ser. Typhimurium, 1 ser. Derby and 1 ser. Typhimurium monophasic variant) showed the ASSuT profile. Most detected resistance genes sul2 (34.48%), tetA (27.58%) and strA-strB (27.58%). Great variability was observed in analyzed strains. S. ser. Typhimurium was confirmed as one of the most virulent serovars. This study underlines that swine could be a reservoir and source of pathogenic Salmonella strains.

Characterization of novel antigenic vaccine candidates for nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae infection

Streptococcus agalactiae is one of the important pathogens responsible for high mortality and economic losses of the tilapia industry worldwide. Based on ten serovars of S. agalactiae infection, subunit vaccine with conserved antigens is promising strategy corresponding stimulated long-term immunity and provides protection for animals against different serotypes of S. agalactiae. In the present study, eight proteins (AP, AL, LivK, ESAT6, essA, essB, essC and esaA) were selected from the S. agalactiae serotype Ia genome as immunogenic antigens with bioinformation and immune experiment assays. These recombinant proteins were successfully obtained through expression in Escherichia coli and the immunogenicity was assessed in tilapia challenge model. The results showed that the recombinant proteins caused high-level-specific antibodies production and high lysozyme activities, suggesting that the recombinant proteins induced specific humoral immune response and innate immune response of tilapia. The signficant increase were observed in the cytokines levels of TNF-α, IL-1β, IFN-γ, cc1, cc2 and immune-related genes levels of CD8α and MHC factors in the spleen and head kidney tissues, suggesting that the recombinant proteins induced immune response of tilapia through cytokines signal pathway and activated high cytotoxic T-lymphocyte (CTL) activity of tilapia. Furthermore, vaccinated tilapia conferred high levels of protection against challenge with a lethal dose of highly virulent serovar Ⅰa (highest RPS was 91.60% in AL and essC protein groups). Our results indicated that the eight recombinant proteins induced high level of immune responses and offered protection against S. agalactiae infection, could be potential subunit vaccine candidates.

New strategies for Leptospira vaccine development based on LPS removal.

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity.

Galactosylated wall teichoic acid, but not lipoteichoic acid, retains InlB on the surface of serovar 4b Listeria monocytogenes.

Listeria monocytogenes is a Gram‐positive, intracellular pathogen harboring the surface‐associated virulence factor InlB, which enables entry into certain host cells. Structurally diverse wall teichoic acids (WTAs), which can also be differentially glycosylated, determine the antigenic basis of the various Listeria serovars. WTAs have many physiological functions; they can serve as receptors for bacteriophages, and provide a substrate for binding of surface proteins such as InlB. In contrast, the membrane‐anchored lipoteichoic acids (LTAs) do not show significant variation and do not contribute to serovar determination. It was previously demonstrated that surface‐associated InlB non‐covalently adheres to both WTA and LTA, mediating its retention on the cell wall. Here, we demonstrate that in a highly virulent serovar 4b strain, two genes gtlB and gttB are responsible for galactosylation of LTA and WTA respectively. We evaluated the InlB surface retention in mutants lacking each of these two genes, and found that only galactosylated WTA is required for InlB surface presentation and function, cellular invasiveness and phage adsorption, while galactosylated LTA plays no role thereof. Our findings demonstrate that a simple pathogen‐defining serovar antigen, that mediates bacteriophage susceptibility, is necessary and sufficient to sustain the function of an important virulence factor.

New Pathological Lesions Developed in Pigs by a "Non-virulent" Strain of Glaesserella parasuis.

Glaesserella parasuis is a Gram-negative bacterium that causes Glässer's disease, a common pathology found in young pigs characterized by polyarthritis, polyserositis, and meningitis. The bacterium has 15 known serovars that have been classified by virulence. Serovars 1, 4, 5, and 12 are considered highly virulent and used in most studies. Serovars 3, 6, 7, 9, and 11 are considered avirulent. Recent reports that serovar 7 is an emerging problem in the pig industry indicate that the association of virulence and serovar may not always be reliable. This led us to infect colostrum-deprived piglets with the reference serovar 7 strain (SV7 strain 174) that had been passaged through pigs and characterize the clinical and pathological signs. We observed that SV7 strain 174 caused clinical signs consistent with Glässer's disease in all infected piglets that succumbed to infection for up to day 5 post-infection. Macroscopic and microscopic lesions were consistent with those found in piglets infected with conventional virulent serovars. In addition, we describe novel microscopic lesions associated with Glässer's disease such as endophthalmitis and thymic depletion. Thus, our findings indicate that SV7 strain 174 causes classical signs of Glässer's disease in colostrum-deprived piglets and some caution should be used in employing vaccine strategies based on association between capsular serovar and virulence.

A crude capsular polysaccharide extract as a potential novel subunit vaccine with cross-protection against the most prevalent serovars of Glaesserella (Haemophilus) parasuis in the Czech Republic

Glaesserella (Haemophilus) parasuis is a part of the normal flora of the respiratory tract of pigs. However, under certain conditions it can also induce severe systemic disease with high morbidity and mortality leading to gross economic losses in the pig industry. The most prevalent serovars in pig herds in the Czech Republic are the virulent serovars 1, 4, 5 and 13. The currently available commercial vaccines are inactivated vaccines with certain limitations, such as no or poor cross-serovar protection. Therefore, the aim of the present study was to construct a subunit vaccine with a crude capsular polysaccharide extract (cCPS) isolated from G. parasuis CAPM 6475 (serovar 5) and evaluate its immunogenicity in a mouse model. Mice were immunised subcutaneously with two doses of the constructed vaccine in a 14-day interval and challenged intraperitoneally with various G. parasuis strains (serovars 1, 4, 5, 13) at 21 days after the second immunisation. The results of the ELISA test showed that the boost dose of the vaccine induced the production of IgG antibodies in high levels. On the basis of the death cases, the pathological findings and the bacterial isolation, the mice immunised with the cCPS were partially protected against the challenge with the homologous serovar 5 as well as with heterologous serovars 1, 4 and 13 of G. parasuis. The cross-reaction of the mixed serum from the immunised mice with the tested serovars was seen in the western-blotting also. Moreover, the most abundant protein found in the cCPS by mass spectrometry was catalase, a protein of molecular weight 55 kDa that may correspond to the strongest reaction seen in the western-blotting. Our findings indicated that the crude capsular polysaccharide extract may provide an effective immunogenicity in preventing a G. parasuis infection caused by the most prevalent serovars in the Czech Republic. However, the evaluation of the efficacy needs to be performed in pigs before any conclusions can be drawn.

New insights about functional and cross-reactive properties of antibodies generated against recombinant TbpBs of Haemophilus parasuis

Vaccines have become fundamental in the control and elimination of Glässer Disease, a systemic disease of pigs caused by Haemophilus parasuis. The classic vaccines available for prevention of this infection were developed without a robust knowledge about host immunological mechanisms. In this study, we demonstrated the presence of cross-reactive epitopes on both the N-lobe and C-lobe of variants of transferrin binding protein B (TbpBs) expressed on the surface of 6 virulent serovars of H. parasuis. Antibodies against TbpB-derived antigens were capable of increasing the phagocytic capacity of neutrophils and were also capable of blocking porcine transferrin from binding to TbpB. Surprisingly, none of the pig or mice antisera from animals immunized with TbpB-derived antigens mixed with Montanide IMS 2215 VG PR adjuvant were able to activate the classical complement pathway (CCP). In contrast, antisera from mice immunized with TbpB-derived antigens adjuvanted with Freund’s adjuvants or Montanide Gel 01 were able to activate the CCP and kill H. parasuis. Our results demonstrate that the type of adjuvant can modulate the functional response induced by TbpB-derived antigens. Based on these results, we propose that a properly formulated TbpB-based vaccine may elicit a functional protective antibody response with broad cross-reactivity against heterologous strains of H. parasuis.

Protective efficacy of cationic-PLGA microspheres loaded with DNA vaccine encoding the sip gene of Streptococcus agalactiae in tilapia

Streptococcus agalactiae (S. agalactiae) is an important fish pathogen, which has received more attention in the past decade due to the increasing economic losses in the tilapia industry worldwide. As existing effective vaccines of S. agalactiae in fish have obvious disadvantage, to select immunoprotective antigens and package materials would undoubtedly contribute to the development of novel oral vaccines. In the present study, surface immunogenic protein (sip) was selected from the S. agalactiae serovar I a genomes as immunogenic protein in DNA vaccine form with cationic chitosan and biodegradable and biocompatible PLGA. The pcSip plasmid in cationic-PLGA was successfully expressed in tissues of immunized tilapia and the immunogenicity was assessed in tilapia challenge model. A significant increase was observed in the cytokine levels of IL-1β, TNF-α, CC1, CC2 in spleen and kidney tissues. Furthermore, immunized tilapia conferred different levels of protection against challenge with a lethal dose of highly virulent serovar I a S. agalactiae. Our results indicated that the pcSip plasmid in cationic-PLGA induced high level of antibodies and protection against S. agalactiae infection, could be effective oral DNA vaccine candidates.

Clinical management of a Labrador retriever dog concurrently infected with Leptospira interrogans, Babesia gibsoni and Dirofilaria repens

Concurrent infection of Leptospira interrogans, Babesia gibsoni, and Dirofilaria repens in a properly vaccinated and dewormed Labrador retriever dog and its successful clinical management are described. The dog was presented with a history of anorexia, vomiting, lethargy, brownish yellow urine, frequent shivering, and weakness. Clinical and laboratory investigations revealed pyrexia, spleenomegaly, icterus, lymphadenopathy, dehydration, hemoglobinuria, anemia, neutrophilia, thrombocytopenia, and hyperbilirubinemia. Concurrent infection of Leptospira interrogans var pyrogenes and autumnalis, Babesia gibsoni and Dirofilaria repens, was confirmed by dark field microscopy of urine sample and blood smear examination, respectively. The animal was stabilized and undergone a combination therapy using Metronidazole (20 mg/kg bid i/v) and Clindamycin (12 mg/kg bid i/v) for 7 days, Doxycycline (5 mg/kg bid po) for 21 days, single dose of Ivermectin was given at 100 μg/kg orally on day 7. Supportive therapy using plasma volume expander, polyionic electrolytes, antiemetics, hepatotonics, vitamin supplements, and hematinics was provided during the course of treatment. The animal made a dramatic recovery by 3 months of care. The case point towards the need of selection of appropriate clinical materials for differential diagnosis, the control of mosquito borne diseases based on ecological characteristics of vector, and the need of incorporating emerging virulent serovars of leptospira in vaccines.

Increased efficacy of inactivated vaccine candidates prepared with Salmonella enterica serovar Typhimurium strains of predominant genotypes in ducks

Salmonella enterica serovar Typhimurium has been a major causative agent of food-borne human disease, mainly due to consumption of contaminated food animal products. In particular, ducks serve as a reservoir of serovar Typhimurium, and are one of the common sources of human infection. To prevent infection of ducks, and therefore minimize human infection, it is critical to control the persistent epidemic strains in ducks. Here, we analyzed the genetic diversity and virulence of serovar Typhimurium isolates from ducks in Korea to identify the predominant strains that might be used as efficient vaccine candidates for ducks. Among the isolates, 2 representative isolates (ST26 and ST76) of predominant genotypes were selected as vaccine strains on the basis of genotypic analysis by pulsed-field gel electrophoresis and DNA microarrays. Two-week-old ducks were then injected intramuscularly with inactivated vaccine candidates prepared using ST26 or ST76 (10(8) cfu/0.5 mL/duck or 10(9) cfu/0.5 mL/duck), and oral challenge with a highly virulent serovar Typhimurium strain (10(9) cfu/0.5 mL/duck) was carried out 2 wk later. Shedding of the challenge strain was significantly decreased in group 2 after vaccination. The antibody levels by enzyme-linked immunosorbent assay in all vaccinated groups were enhanced significantly (P < 0.05) compared to the unvaccinated control group. Overall, vaccination with ST26 or ST76 reduced bacterial shedding and colonization in internal organs, and induced elevated antibody response. In particular, serovar Typhimurium ST26 (10(8) cfu/0.5 mL/duck) was the most effective vaccine candidate, which can provide efficient protection against serovar Typhimurium in ducks with higher effectiveness compared to a commercial vaccine currently used worldwide.

Identification of secreted proteins as novel antigenic vaccine candidates of Haemophilus parasuis serovar 5

Haemophilus parasuis (H. parasuis) is a swine pathogen responsible for the Glässer's disease, which has received more attention in the past decade due to the increasing economic losses in the pig industry worldwide. As traditional inactive vaccine of H. parasuis has obvious disadvantage, to identify efficient immunoprotective antigens would undoubtedly contribute to the development of novel subunit vaccines. The putative secreted proteins of H. parasuis are potentially essential components of more potent vaccines. In the present study, six secreted proteins (PflA, Gcp, Ndk, HsdS, RnfC and HAPS_0017) were selected from the annotated H. parasuis serovar 5 genome as immunogenic protein with bioinformatic and experimental approaches. These proteins were successfully expressed in Escherichia coli and their immunogenicity was assessed in a mouse challenge model. The results showed that subcutaneous injection with the recombinant proteins resulted in the production of antibodies with high levels. Antigen-specific lymphoproliferative responses were detected in the splenocytes of the immunized animals. CD4+ T-cell populations were higher in the vaccinated animals 3 weeks after the booster immunization than those of the control animals. A significant increase was observed in the cytokine levels of IL-2, IL-4 and IFN-γ in the culture supernatants of splenocytes. Furthermore, immunized mice conferred different levels of protection against challenge with a lethal dose of highly virulent serovar 5 strain (H46). Our results indicate that these six secreted proteins induced a good Th1 response and protection against H. parasuis infection, could be potential subunit vaccine candidates.

Molecular characterization of Salmonella serovars of zoonotic importance*

Isolates (50) of Salmonella were recovered from 1,132 samples from foods of animal origin and fecal samples from animals and human beings; belonging to 10 different serovars with most prevalent serovar S. Typhimurium (21) followed by S. Weltevreden (12), S. Ughelli (5), S. Essen (3), S. Elisabethville (2), S. Lagos (2), S. Drogana (2), S. Enteritidis (1), S. London (1) and un-typable Salmonella (1). Emerging Salmonella serovars, viz S. Elisabethville, S. Essen, S. Lagos, S. Ughelli and S. Drogana were first time recovered from Pantnagar and its vicinity. S. Drogana was recovered possibly for the first time from human source in India. Multiple Salmonella serovars (up to 3 serovars comprising S. Typhimurium, S. Weltevreden and S. Essen) were recovered from single cattle dung sample while, multiple serovars (up to 2 serovars) were also recorded in many single samples, viz cattle dung (S. Weltevreden and S. Ughelli), poultry droppings (S. Essen and S. Ughelli), pig faeces (S. Weltevreden and S. London), sheep faeces (S. Typhimurium and S. Drogana) and pig faeces (S. Weltevreden and S. Ughelli). Different virulence genes, viz. invA, sipA, sefA, fliC, stn and sopB were detected in Salmonella isolates using PCR-based molecular technique. Among these virulence genes, invA gene was the most prevalent one as is present in 98% Salmonella isolates followed by sopB, stn, sipA, fliC and sefA genes in 96, 86, 78, 32 and 10% Salmonella isolates, respectively. Salmonella serovars of zoonotic importance recovered from varied sources exhibited different virulence enes that may cause serious infections in animals as well as in human beings. Ultimately these virulent serovars may pose great risks to the health and production in the animals and serious health hazards in human.

Map-based comparative genomic analysis of virulent haemophilus parasuis serovars 4 and 5.

Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. However, in conjunction with viral infections in immunocompromised animals H. parasuis can transform into a pathogen that is responsible for causing Glasser's disease which is typically characterized by fibrinous polyserositis, polyarthritis, meningitis and sometimes acute pneumonia and septicemia in pigs. Haemophilus parasuis serovar 5 is highly virulent and more frequently isolated from respiratory and systemic infection in pigs. Recently a highly virulent H. parasuis serovar 4 was isolated from the tissues of diseased pigs. To understand the differences in virulence and virulence-associated genes between H. parasuis serovar 5 and highly virulent H. parasuis serovar 4 strains, a genomic library was generated by TruSeq preparation and sequenced on Illumina HiSeq 2000 obtaining 50 bp PE reads. A three-way comparative genomic analysis was conducted between two highly virulent H. parasuis serovar 4 strains and H. parasuis serovar 5. Haemophilus parasuis serovar 5 GenBank isolate SH0165 (GenBank accession number CP001321.1) was used as reference strain for assembly.Results of these analysis revealed the highly virulent H. parasuis serovar 4 lacks genes encoding for, glycosyl transferases, polysaccharide biosynthesis protein capD, spore coat polysaccharide biosynthesis protein C, polysaccharide export protein and sialyltransferase which can modify the lipopolysaccharide forming a short-chain LPS lacking O-specific polysaccharide chains often referred to as lipooligosaccharide (LOS). In addition, it can modify the outer membrane protein (OMP) structure. The lack of sialyltransferase significantly reduced the amount of sialic acid incorporated into LOS, a major and essential component of the cell wall and an important virulence determinant. These molecules may be involved in various stages of pathogenesis through molecular mimicry and by causing host cell cytotoxicity, reduced inflammatory and immunological response to infection with this organism. The mechanism by which sialyation of LPS contributes to virulence is a key to understanding the pathogenesis of this highly virulent H. parasuis serovar 4. This analysis also revealed the presence of virulence associated genes similar to the MerR family transcriptional regulators, macrophage infectivity potentiator protein, hemolysin, opacity associated protein, toxin antitoxin system, and virulence associated protein D and colicins. Haemophilus parasuis serovar 4 variants also possess extensive metal ion uptake and regulation mechanism which controls various virulence and virulence associated genes. A combination of virulence associated factors and/or genes and proteins with overlapping functions may be responsible for the apparent enhanced virulence of this organism.The extensive structural modification of LOS and OMP of variant H. parasuis serovar 4 strains appear to aid in nasal colonization, are associated with the organisms' ability to evade the host immune response and provide serum-resistance. In addition, the combination of capsule modification and phase variation due to LOS substitutions could help variant H. parasuis serovar 4 transform into a highly virulent pathogen. Based on these results, the variant H. parasuis serovar 4 strains harbor a diverse repertoire of virulence associated genes which have not been previously reported.