vapA Gene Research Articles

Commonality of Virulence-Promoting Function in Rhodococcus equi Virulence Associated Proteins (Vaps)

Rhodococcus equi is a Gram-positive facultative intracellular pathogen associated with life-threatening bronchopneumonial disease in foals. Key to R. equi’s intracellular survival in host macrophages is the production of virulence associated proteins (Vaps). Numerous vap genes are found on virulence plasmids isolated from different species, and the Vaps share a high degree of sequence identity. VapA has been extensively studied, and although vapK and vapN genes from other R. equi virulence plasmids have been shown to be essential for R. equi intracellular survival, their mode of action is less characterised. We, therefore, examined whether VapK and VapN worked mechanistically in the same way as VapA. Indeed, like VapA, VapK and VapN neutralised lysosomal pH and reduced lysosomal hydrolase activity. A loss of VapA and R. equi virulence could be regained by the presence of either VapK or VapN. The acid-neutralisation activity was also observed to a lesser extent with VapB. There was a differential activity across these virulence-promoting Vaps with the most “acid-neutralising” activity found with VapN, then VapA and K, and finally VapB. These data suggest that VapA production, which is often found in equine infections, can be substituted by VapK and B (produced by plasmids often found in porcine species) or VapN (produced by plasmids often isolated in bovine and human samples). These data imply that the molecular mechanism(s) that VapA uses to neutralise lysosomal acidity should also be seen in VapN and K which will help guide researchers in identifying their precise mode of action and aid the future development of targeted therapeutics.

Evaluation of serum concentration of acute-phase proteins (haptoglobin and serum amyloid A) in the affected Arabian foals with rhodococcosis.

Early detection of Rhodococcus equi pneumonia in foals is essential for horse health and for veterinarians. This study aimed to demonstrate the usefulness of assessing the serum concentration of acute-phase proteins (APPs) in the early diagnosis of pneumonia. The study evaluated APPs in 19 Arabian foals with R. equi pneumonia and compared them with 18 normal Arabian foals in equestrian clubs in Tabriz, Iran. Affected foals were identified through history, clinical findings and bacterial culture of tracheal washing. Biochemical methods and polymerase chain reaction tests were performed by examining the 16S rRNA and vapA genes to confirm the diagnosis of bacterial isolates. Blood samples were taken from all sick and healthy horses, and their serum was isolated. APPs in the serum were measured in all the samples. Rhodococcosis increased the serum concentration of haptoglobin (Hp) and serum amyloid A (SAA) (p < 0.001). The relationship between SAA and Hp was meaningful in the infected group (r = 0.933) but not in the healthy group. In cases where there are clinical findings of R. equi pneumonia, the concentration of SAA and Hp can help the effectiveness of treatment. Serum concentration analysis of APPs can be helpful in early diagnosis and successfully treating foals with R. equi pneumonia.

Development of Two Recombinase Polymerase Amplification EXO (RPA-EXO) and Lateral Flow Dipstick (RPA-LFD) Techniques for the Rapid Visual Detection of Aeromonas salmonicida.

Aeromonas salmonicida is the pathogen underlying furunculosis, causing a septicemic infection that influences both salmonid and non-salmonid fish. Early diagnosis of these contagions is essential for disease surveillance and prevention, so a rapid and sensitive approach is needed. Herein, a recombinase polymerase amplification EXO (RPA-EXO) assay and RPA with a lateral flow dipstick (RPA-LFD) were produced for testing A. salmonicida. The RPA-EXO and RPA-LFD primer sets were devised based on the conserved fragment sequence of the vapA gene. Then, RPA-EXO and RPA-LFD reaction systems were established, and the reaction temperature and time were optimized. After optimization, the RPA-EXO method was capable of testing A. salmonicida within 10min, and the RPA-LFD method could detect A. salmonicida in only 5min. The RPA-EXO and RPA-LFD methods exhibited high specificity with no cross-reaction with other strains. To assess sensitivity, a partial vapA gene was cloned, and serial plasmid dilutions were created ranging from 1 × 106 to 1 × 10-1 copies/μL. The detection limit of RPA-EXO was 1 × 102 copies/μL, and the detection limit of RPA-LFD was 1 copy/μL. For spiked turbot tissue samples, the sensitivity detection of A. salmonicida was 1.2 × 101CFU/mL and 1.2CFU/mL by RPA-EXO and RPA-LFD, respectively. In comparative analyses of clinical samples, the diagnostic results of RPA-EXO and RPA-LFD were compared with those of the standard conventional PCR test and showed nearly 100% consistency. Therefore, our RPA-EXO and RPA-LFD assays exhibited excellent specificity and sensitivity, which provided two simple, fast and dependable methods to conduct large-scale field investigations of A. salmonicida in resource-limited settings.

Virulence plasmids in clinical isolates of Rhodococcus equi from sick foals in the Netherlands.

Clinical samples from 123 foals with suspected rhodococcosis submitted to the Veterinary Microbiological Diagnostic Centre of the Faculty of Veterinary Medicine between 1993 and 2006 were tested for the presence of the virulence gene vapA. Of the 123 samples, 120 were vapA-positive and 3 vapA-negative Rhodococcus equi were isolated. The 120 vapA-positive R. equi were isolated from 70 tracheal wash, 19 lung tissues, 7 lymph nodes, 6 synovial fluids, 13 abscesses or pus and single isolates from the uterus, gut, cerebrospinal fluid, abdomen fluid and faeces. Of the 120 isolates, 46 were from Dutch warmblood horses, 23 from Friesian horses, 14 from Trotters, 4 from Holsteiners, 3 from Arab breed, 2 from ponies, 1 from a Welsh pony and 27 from undefined breed horses. Using plasmid profile analysis of the 120 isolates, 117 isolates contained the 85-kb type I plasmid, 2 contained the 87-kb type I plasmid and 1 contained the novel 52-kb non-mobilizable virulence plasmid reported recently. These results showed that the virulent R. equi strains harbouring a virulence plasmid of 85-kb type I or 87-kb type I, which have been detected in clinical isolates from five European countries, are widespread in the Netherlands. This is the first report of plasmid types of clinical R. equi isolates in the Netherlands.

Fatal Infection in an Alpaca (Vicugna pacos) Caused by Pathogenic Rhodococcus equi

Simple SummarySerious consequences of septicemic bacterial infections include the formation of purulent and pyogranulomatous inflammation resulting in abscesses in inner organs. Different bacteria are known to cause these infections in livestock. In this study, we report in detail on a case of a fatal Rhodococcus (R.) equi infection in an alpaca (Vicugna pacos), to our knowledge, for the first time. R. equi is a member of the actinomycetes, a bacterial group known to contain several pathogenic bacteria. R. equi primarily affects equine foals and other domestic animals, but also humans, which renders this bacterium a zoonotic agent. The rhodococcal infection of the alpaca reported herein caused septicemia, resulting in emaciation and severe lesions in the lungs and heart. The onset of infection was presumably caused by aspiration pneumonia, resulting in abscesses exclusively in the lungs. The R. equi isolate proved to be pathogenic, based on the virulence gene vapA encoding the virulence-associated protein A. Antibiotic susceptibility testing revealed a susceptibility to doxycycline, erythromycin, gentamycin, neomycin, rifampicin, trimethoprim/sulfamethoxazole, tetracycline and vancomycin. This report of an R. equi infection in an alpaca makes clear that we still have knowledge gaps about bacterial infectious diseases in alpacas and potential zoonotic impacts. Therefore, the determination of pathogenic, zoonotic bacteria in alpacas is essential for treatment and preventive measures with respect to sustaining the health, welfare and productivity of this camelid species.Rhodococcus (R.) equi is a pathogen primarily known for infections in equine foals, but is also present in numerous livestock species including New World camelids. Moreover, R. equi is considered an emerging zoonotic pathogen. In this report, we describe in detail a fatal rhodococcal infection in an alpaca (Vicugna pacos), to our best knowledge, for the first time. The alpaca died due to a septicemic course of an R. equi infection resulting in emaciation and severe lesions including pyogranulomas in the lungs and pericardial effusion. The onset of the infection was presumably caused by aspiration pneumonia. R. equi could be isolated from the pyogranulomas in the lung and unequivocally identified by MALDI-TOF MS analysis and partial sequencing of the 16S rRNA gene, the 16S-23S internal transcribed spacer (ITS) region and the rpoB gene. The isolate proved to possess the vapA gene in accordance with tested isolates originating from the lungs of infected horses. The R. equi isolates revealed low minimal inhibitory concentrations (MIC values) for doxycycline, erythromycin, gentamycin, neomycin, rifampicin, trimethoprim/sulfamethoxazole, tetracycline and vancomycin in antibiotic susceptibility testing. Investigations on the cause of bacterial, especially fatal, septicemic infections in alpacas are essential for adequately addressing the requirements for health and welfare issues of this New World camelid species. Furthermore, the zoonotic potential of R. equi has to be considered with regard to the One Health approach.

Isolation of Rhodococcus equi from the gastrointestinal contents of earthworms (family Megascolecidae).

Rhodococcus equi was isolated from the gastrointestinal contents of earthworms (family Megascolecidae) and their surrounding soil collected from pastures of two horse-breeding farms in Aomori Prefecture, outdoor pig pens, forest in Towada campus, orange groves and forest where wild boars (Sus scrofa) are established in Tanabe, Wakayama Prefecture. The number of R. equi in the lower gastrointestinal contents of 23 earthworms collected from our campus was significantly larger than that of the upper gastrointestinal content. The mean numbers of R. equi from the gastrointestinal contents of earthworms collected from the various places were 2·3-fold to 39·7-fold more than those of the surrounding soil samples. In all, 1771 isolates from the earthworms and 489 isolates from the soil samples were tested for the presence of vapA and vapB genes using polymerase chain reaction. At the horse-breeding farm N, 9 of the 109 isolates (8·3%) from the earthworms and 7 of the 106 isolates (6·6%) from the soil samples were positive for the vapA gene. At the University's forest, one of the 250 isolates (0·4%) from the gastrointestinal contents of the earthworm was positive for the vapB gene. These results revealed that R. equi can be found in significant quantities in the gastrointestinal contents of earthworms, suggesting that they act as an accumulator of R. equi in the soil environment and as a source or reservoir of animal infection.

Rapid visual detection of Aeromonas salmonicida by loop‐mediated isothermal amplification with hydroxynaphthol blue dye

To make crucial prevention, reduce fish losses and minimize the economic damage of diseases on the fish farm owners, a rapid detection of fish pathogens is mandatory. In this study, a loop-mediated isothermal amplification assay combined with hydroxynaphthol blue dye (LAMP-HNB) was developed and used for the rapid detection of Aeromonas salmonicida that caused significant economic losses in fish farming. Firstly, a pair of outer and inner primers specific for conserved fragment of vapA gene in A. salmonicida were designed and synthesized. Secondly, by optimizing the reaction conditions including reaction temperature, time, Mg2+ concentration, dNTP concentration and primer ratio, a LAMP-HNB assay was successfully established for the detection of A. salmoncida. Thirdly, the assay showed good specificity with no false-positive and false-negative results, and good sensitivity with the detection limit of 3.077×10-6 ng/μl, which was 102 times more sensitive than the conventional PCR. Finally, the LAMP-HNB assay was validated by the fish samples inoculated with different concentrations of A. salmoncida. This is the first development of rapid visual detection of A. salmonicida based on LAMP-HNB assay, which has great application prospect and market for diagnostic testing, health certification and active surveillance programmers.

Detection of Rhodococcus equi by PCR from foals and determination of antimicrobial susceptibility

The scope of this research was determination of Rhodococcus equi isolates and antimicrobial susceptibilities in nasal swab specimen taken from animals between 0 and 6 months of age period. R. equi was identified from 10 isolates (11.1%) by automatic identification device. The verification of isolates were conducted by 16S ribosomal RNA PCR. VapA gene presence was detected in two (20%) isolates and multidrug resistance was identified against 11 antimicrobials. The antimicrobial resistance to macrolides and rifampin is arising worldwide since the excessive usage of the chemotherapeutics in various breeding farms just by evaluating monitoring outcomes. These results exhibit the presence of resistant strains among equine populations. The development of fast, economical and highly specific diagnostic technique against the threat of R. equi is essensial for better understanding of gene expressions related to resistance and development of effective immunization.

Development of diagnostic assays for differentiation of atypical Aeromonas salmonicida vapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius).

Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains' host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time-consuming. This paper describes the development of a PCR and a restriction enzyme assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V-specific primers were designed on conserved regions of the vapA gene, and the restriction enzyme assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254bp) and restriction bands (368 and 254bp) for type VI isolates only. In addition, the assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The assays are fast, specific and cost-effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development.

Pathogenic characterization of Aeromonas salmonicida subsp. masoucida turbot isolate from China.

Aeromonas salmonicida is a gram-negative bacterium that is the causative agent of furunculosis. An A.salmonicida strain was isolated from diseased turbot (Scophthalmus maximus) with the sign of furunculosis from North China. Based on vapA gene, the strain was further classified as A.salmonicida subsp. masoucida RZ6S-1. Culturing RZ6S-1 strain at high temperature (28°C) obtained the virulence attenuated strain RZ6S. Genome sequence comparison between the two strains revealed the loss of the type IV secretion system (T4SS) and type III secretion system (T3SS) from the native plasmid pAsmB-1 and pAsmC-1 of wild-type strain RZ6S-1, respectively. Further study demonstrated that the wild-type strain RZ6S-1, but not its derivative mutant RZ6S, can stimulate apoptosis. Elevated protein level of cleaved caspase-3 was detected from epithelioma papulosum cyprinid (EPC) cells infected with wild-type strain RZ6S-1 as compared with that infected with RZ6S strain. Meanwhile, the invasion of the mutant strain RZ6S was about 17-fold higher than the wild-type strain RZ6S-1, suggesting that some protein(s) from A.salmonicida subsp. masoucida RZ6S-1 suppress its invasion. The RZ6S mutant strain was attenuated, since its LD50 is over 10,000 times higher compared to the wild-type strain as revealed in the turbot infection model.

Pinpointing the role of Aeromonas salmonicida in the development of skin ulcerations in common dab (Limanda limanda).

Aeromonas salmonicida was isolated from ulcerations in common dab (Limanda limanda). An experiment was performed to pinpoint its role in ulceration development, considering the importance of the skin barrier and the pigmented and non-pigmented sides. The skin of dab was treated in three zones, one where scales and epidermis were removed, one where mucus was discarded and one non-treated zone. Fish were tagged to allow individual identification and challenged with A.salmonicida. Mortality and severity of the developing lesions were recorded for 21days post-inoculation. Starting 12days post-inoculation, mortality occurred gradually in challenged fish; however, no direct cause could be established. Both control fish and challenged fish developed ulcerations containing A.salmonicida. Sequencing of vapA gene revealed that isolates retrieved from both groups were distinct, suggesting the presence of A.salmonicida prior to the trial. Most ulcerations developed in zones where skin was removed, suggesting that abrasion might be a predisposing factor in ulceration development. Ulcerations were also observed at the insertion site of the tag, where exposed muscle tissue might have favoured the development of ulcerations. In conclusion, A.salmonicida seems to be involved in the development of skin ulcerations in dab, although the exact pathogenesis needs to be elucidated.

GAPDH, rhbC, and vapA gene expression in Rhodococcus equi cultured under different iron concentrations

The ability of Rhodococcus equi to survive in macrophages and cause pneumonia in foals depends on vapA and rhbC genes, which produce the virulence-associated protein A (VapA) and the rhequichelin siderophore, respectively. Virulent R. equi acquires Fe from transferrin by unknown mechanisms. Our objectives were to determine the role of GAPDH in Fe homeostasis, to further characterize GAPDH, rhbC, and vapA expression under iron homeostasis, and to document the occurrence of rhbC gene in R. equi isolates. Therefore, vapA + R. equi was cultured under excessive, physiologic, and restricted iron concentrations, and quantitative culture and gene expression were performed. The relative expression of GAPDH, rhbC, and vapA after 48 h of culture were analyzed by qPCR. To determine the rhbC occurrence, total DNA was extracted from R. equi isolated from foals with clinical rhodococcosis (n = 22), healthy horses (feces, n = 16; nasal swab, n = 9), soil (n = 6), and 2 ATCC reference strains. Conventional PCR was performed to identify genus/species, vapA, and rhbC genes. Iron restriction proportionally decreased R. equi growth rates, and induced high expression of both GAPDH and vapA. The putative role of GAPDH in R. equi iron homeostasis should be further investigated. rhbC was significantly up-regulated under both Fe excess and critical starvation. The rhbC gene was identified in all clinical isolates and soil, but it was absent in 2 isolates from healthy horses, suggesting that rhequichelin is not required for R. equi nasal and intestinal colonization.

A recombinant adenovirus targeting typical Aeromonas salmonicida induces an antibody-mediated adaptive immune response after immunization of rainbow trout

Aeromonas salmonicida, the oldest known fish pathogen and currently endemic throughout most of the world in both fresh and marine waters, causes severe economic losses to the salmon farming industry. Although there have been many studies on the prevention of furunculosis over the past few decades, it is still prevalent in many fisheries. In this study, a recombinant adenovirus vaccine candidate harboring the highly immunogenic Vapa gene (pAd-easy-cmv-Vapa) was successfully constructed and tested. The immune protection rate and specific antibody levels in the peripheral blood were then determined after immunizing rainbow trout. In addition, relative levels of IgM and IgT in the head kidney and hindgut before and after immunization were measured by quantitative reverse transcription PCR. Western blotting results indicated that the recombinant adenovirus could infect HEK-293 cells and express the A layer protein (encoded by Vapa). Further, survival analysis of fish 28 days after challenge showed that immunization significantly lowered the mortality rate (40%) compared to that in the control group (76.6%) and empty vector group (73.6%). This also led to an increase in specific antibodies in peripheral serum. In addition, levels of IgM and IgT in the head kidney and hindgut were increased to varying degrees. In conclusion, our research provides a candidate vaccine for the prevention of Aeromonas salmonicida A450 infection in rainbow trout and lays the foundation for future research on adaptive immune mechanisms associated with rainbow trout antibodies.

Inhibitory Mechanisms of DHA/CQ on pH and Iron Homeostasis of Erythrocytic Stage Growth of Plasmodium falciparum

Malaria is an infectious disease caused by Plasmodium group. The mechanisms of antimalarial drugs DHA/CQ are still unclear today. The inhibitory effects (IC50) of single treatments with DHA/CQ or V-ATPase inhibitor Baf-A1 or combination treatments by DHA/CQ combined with Baf-A1 on the growth of Plasmodium falciparum strain 3D7 was investigated. Intracellular cytoplasmic pH and labile iron pool (LIP) were labeled by pH probe BCECF, AM and iron probe calcein, AM, the fluorescence of the probes was measured by FCM. The effects of low doses of DHA (0.2 nM, 0.4 nM, 0.8 nM) on gene expression of V-ATPases (vapE, vapA, vapG) located in the membrane of DV were tested by RT-qPCR. DHA combined with Baf-A1 showed a synergism effect (CI = 0.524) on the parasite growth in the concentration of IC50. Intracellular pH and irons were effected significantly by different doses of DHA/Baf-A1. Intracellular pH was decreased by CQ combined with Baf-A1 in the concentration of IC50. Intracellular LIP was increased by DHA combined with Baf-A1 in the concentration of 20 IC50. The expression of gene vapA was down-regulated by all low doses of DHA (0.2/0.4/0.8 nM) significantly (p < 0.001) and the expression of vapG/vapE were up-regulated by 0.8 nM DHA significantly (p < 0.001). Interacting with ferrous irons, affecting the DV membrane proton pumping and acidic pH or cytoplasmic irons homeostasis may be the antimalarial mechanism of DHA while CQ showed an effect on cytoplasmic pH of parasite in vitro. Lastly, this article provides us preliminary results and a new idea for antimalarial drugs combination and new potential antimalarial combination therapies.

Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping.

ABSTRACTA recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15–23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.

Identification of macrolide- and rifampicin-resistant Rhodococcus equi in environmental samples from equine breeding farms in central Kentucky during 2018

Rhodococcus equi causes severe pneumonia in foals and is most often recognized in people as an opportunistic pathogen. Longitudinal studies examining antimicrobial-resistant R. equi from environmental samples are lacking. We hypothesized that antimicrobial-resistant R. equi would be detectable in the ground (pasture soil or stall bedding) and air at breeding farms with previous documentation of foals infected with resistant isolates, and that concentrations of resistant isolates would increase over time during the foaling season. In this prospective cohort study, ground and air samples were collected from stalls and paddocks in January, March, May and July of 2018 at 10 horse-breeding farms with history of foal pneumonia attributed to macrolide- or Rifampicin-resistant R. equi. Environmental samples were cultured in the presence and absence of macrolides and Rifampicin to select for resistant organisms. Data were analyzed with linear mixed-effects and Hurdle models. Concentrations of total R. equi in bedding or air of stalls were significantly (P < 0.05) higher in January than other months. The proportion of resistant R. equi in soil samples from paddocks was significantly (P < 0.05) higher than stall bedding during all months. For each month, air samples from paddocks had a significantly (P < 0.05) higher proportion of resistant isolates than those from stalls. Fifty-five percent of resistant soil isolates and 34% of resistant air isolates were considered virulent by identification of the vapA gene. Concentrations of resistant R. equi isolates did not increase over time during the foaling season. Antimicrobial-resistant R. equi can persist in the environment at farms with a history of pneumonia caused by resistant R. equi infections, and exposure to resistant isolates in paddocks and stalls appears stable during the foaling season. Resistant isolates in the environment not only pose a risk for disease but also can serve as a repository for dissemination of resistance genes.

Molecular analysis of the chromosomal 16S rRNA gene and vapA plasmid gene of Polish field strains of R. equi.

Rhodococcus equi (R. hoagii) is an opportunistic pathogen commonly found in foals up to 6 months old and animal environment. The R. equi genome contains genetically stable chromosomal DNA and an 80–90 kb plasmid containing vapA gene, responsible for virulence. Most reports from around the world focus on the determination of R. equi plasmid profiles. Few studies have attempted to determine differences in nucleotide sequences between virulent strains of R. equi isolated from foals and breeding environment. The aim of the study was to perform a molecular analysis of a fragment of the chromosomal gene encoding the 16S rRNA subunit and the vapA plasmid gene of virulent R. equi strains isolated on Polish studs from foals and from the breeding environment of horses. The sequencing method was used to compare the primary structure of fragments of the chromosomal and plasmid DNA of the virulent R. equi strains. The sequences of 22 clinical and 18 environmental R. equi isolates were compared with the sequences of the gene fragments of reference strains available in the NCBI GenBank database. All sequenced 16S rRNA amplicons of Polish field strains were identical and showed 99.5% similarity to the four randomly selected sequences of this gene fragment in the GenBank database. The results confirm that fragments of the 16S rRNA gene of R. equi strains are highly conserved and do not undergo variation in field conditions. Analysis of the sequencing results for the vapA gene fragment of the strains used in our study revealed two polymorphic variants and clear differences between the sequences of strains isolated from foals and from soil samples. Presumably, R. equi strains present in the breeding environment are more exposed than clinical strains to adverse external factors. This may result in changes in the DNA sequence due to natural selection.

Identification of Mycobacterium species and Rhodococcus equi in peccary lymph nodes.

Mycobacterium species and the virulence-associated proteins (vapA, vapB, and vapN genes) of Rhodococcus equi isolated from 330 lymph nodes of collared peccaries (Tayassu tajacu) and white-lipped peccaries (Tayassu pecari) intended for human consumption were investigated. Thirty-six (10.9%) R. equi strains were isolated; 3.3% (n = 11/330) were from white-lipped peccary lymph nodes, and 7.6% (25/330) were from collared peccary lymph nodes. Among the 11 isolates of R. equi from the white-lipped peccaries, 90.9% (n = 10/11) were obtained from the mesenteric lymph nodes, and only 9.1% (n = 1/10) were obtained from the mediastinal lymph nodes. In the 25 isolates of R. equi obtained from the collared peccaries, 40.0% (n = 10/25) were recovered from the mesenteric lymph nodes, 36% (n = 9/25) from the submandibular lymph nodes, and 24.0% (n = 6/25) from the mediastinal lymph nodes. No vapA, vapB, or vapN genes (plasmidless) or three host-associated types (pVAPA, pVAPB, and pVAPN) were identified among the R. equi isolates. Mycobacterium species were isolated in 3.03% (n = 10/330) of all the lymph nodes analyzed. Among the 10 mycobacterial isolates, 60% (n = 6/10) were from the white-lipped peccary lymph nodes, and 40% (n = 4/10) were from the collared peccary lymph nodes. Ten Mycobacterium species were detected by PCR-PRA with a predominance of M. avium type 1. Sequencing of the hsp65 and rpob genes revealed mycobacteria that were saprophytic (M. sinense and M. kumamotonense) and potentially pathogenic (M. colombiense and M. intracellulare) to humans and animals. To our knowledge, this is the first description of R. equi and/or mycobacterial species identified in the lymph nodes of peccary specimens. R. equi (plasmidless) and the mycobacterial species described here have been reported as causes of pulmonary and extrapulmonary infections in both immunocompetent and immunocompromised humans.

Sazlıdere Baraj Gölünde Yaşayan Balık Türlerinde Aeromonas salmonicida Varlığının Araştırılması

Objective: The aim of this study was, to examine the presence of Aeromonas salmonicida in lake water and fish species living in Lake Sazlıdere, a region where fish hunting was also conducted, in Istanbul. Material and Methods: In this study, between April 2014 and March 2015, samples from Lake Sazlıdere were collected for once a month. Samples were divided into three groups including, swabs materials from fish surface (n=48), viscera (n=46) and lake water (n=60). The samples inoculated on to the Furunculosis Agar and Tryptic Soy Agar including Coomassie Brilliant Blue (C-TSA) and incubated at 25°C for 7 days. Presumptive colonies were examined with biochemical tests for the identification of A. salmonicida. Concurrently with bacteriological examination, all of the samples were examined for the presence of vapA gene with Polymerase Chain Reaction (PCR). Results: According to bacteriological examination results, A. salmonicida were not isolated from any of the samples. As a result of PCR, of 60 water samples 3 (5.0%) were positive for vapA gene, while A. salmonicida specific DNA was not detected in the viscere and swab samples. Conclusion: It has been determined that infections caused by A. salmonicida, especially furunculosis disease, under this light do not constitute a risk in Lake Sazlıdere at the time of the study. However, the information obtained from fishermen and the results of PCR, when evaluated together with chemical contamination in general, indicate that continual investigations should be continued in this area, not within a certain period of time.

VapA of Rhodococcus equi binds phosphatidic acid.

Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi-containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA32-189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host-derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA32-189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.