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    In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella
    (Oxford University Press on behalf of Genetics Society of America, 2021-01) Paulson AR; O'Callaghan M; Zhang X-X; Rainey PB; Hurst MRH; Oliver B
    The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.
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    Elastic Light Scatter Pattern Analysis for the Expedited Detection of Yersinia Species in Pork Mince: Proof of Concept.
    (Frontiers Media S.A., 2021-02-17) On SLW; Zhang Y; Gehring A; Patsekin V; Chelikani V; Flint S; Wang H; Billington C; Fletcher GC; Lindsay J; Robinson JP; Fusco V
    Isolation of the pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis from foods typically rely on slow (10-21 day) "cold enrichment" protocols before confirmed results are obtained. We describe an approach that yields results in 39 h that combines an alternative enrichment method with culture on a non-selective medium, and subsequent identification of suspect colonies using elastic light scatter (ELS) analysis. A prototype database of ELS profiles from five Yersinia species and six other bacterial genera found in pork mince was established, and used to compare similar profiles of colonies obtained from enrichment cultures from pork mince samples seeded with representative strains of Y. enterocolitica and Y. pseudotuberculosis. The presumptive identification by ELS using computerised or visual analyses of 83/90 colonies in these experiments as the target species was confirmed by partial 16S rDNA sequencing. In addition to seeded cultures, our method recovered two naturally occurring Yersinia strains. Our results indicate that modified enrichment combined with ELS is a promising new approach for expedited detection of foodborne pathogenic yersiniae.
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    Temperature- and host-dependent transcriptional responses in the entomopathogenic bacterium, Yersinia entomophaga MH96 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany Campus, New Zealand
    (Massey University, 2020) Paulson, Amber Rose
    Yersinia entomophaga MH96 is a virulent pathogenic bacterium that is infective towards a broad range of insects and is under development as a biopesticide. MH96 produces insecticidal toxin complex called Yen-TC that is secreted at temperatures of 25 °C and below and has been shown to be the primary virulence factor (VF) during per os challenge against the New Zealand grass grub, Costelytra giveni and other agricultural pests (Hurst et al., 2011a, 2019). New insights into the pathobiology of MH96 during insect infection were gained from the in vivo transcriptome, including identification of a core secreted weaponry of co-expressed/co-secreted VFs, including Yen-TC and other exoenzymes; however, many other diverse types of VFs, including toxins, effectors, fimbriae, secretion systems, efflux pumps, iron acquisition, stress response and metabolic adaptation were also identified as highly expressed under in vivo conditions. A small DNA-binding protein, Yen6, was shown to be under thermoregulation at the transcriptional level and host-dependent-regulation at the post-transcriptional level and contributed to virulence during intrahemocoelic infection of Galleria mellonella at 37 °C. The in vivo transcriptome of Δyen6 and in vitro DNA-binding specificity analysis provided evidence that Yen6 is a novel LytTR-containing regulator that activates a ribose uptake/metabolism gene cluster, rbsD-xylG-rbsC-xylF-rbsK-ccpA, and represses a fructose uptake/metabolism gene cluster, IIA-fruK-IIB and a gene for RNA-binding protein yhbY during infection at 37 °C. Another small DNA-binding protein, Yen7, was also implicated as a potential temperature-dependent activator of Yen-TC component genes and over-expression of yen7 resulted in restored secretion by MH96 at 37 °C; however, deletion of yen7 did not abrogate Yen-TC production. Experimental investigations into potential regulatory linkages between Yen6 and yen7 were undertaken, and evidence to date does not support Yen6 as transcriptional repressor of yen7. A 17.5 Kb unstable element within the genome of MH96 with linkages to Yen-TC and toxin secretion, motility and cell shape was identified. Overall the findings presented in this thesis represent the most detailed investigation of MH96 pathogenesis to date, reinforcing MH96 as one of the most highly entomopathogenic bacteria known to humankind; yet suggesting MH96 has possibly maintained at least one core thermoregulatory mechanism more typical of an opportunistic pathogen.
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    Assessment of the sensitivity of current standard procedures for the isolation of Yersinia enterocolitica from pork mince : a dissertation presented in partial fulfilment (25%) of the requirements for the degree of Master of Veterinary Studies in Veterinary Public Health at Massey University
    (Massey University, 1999) Subramaniam, Durai
    Y. enterocolitica and related species have been isolated from many types of food. The majority of isolates differ in biochemical and serological characteristics from typical pathogenic strains and are termed non-pathogenic or environmental strains. Usually the number of Y. enterocolitica organisms present in food products is low compared with the dominant background flora. The ability of current enrichment procedures to recover pathogenic strains of Y. enterocolitica from different foods is often inadequate probably because different strains require different conditions for optimum growth (De Boer 1992). An efficient enrichment procedure should confer some selective advantage to the desired type of microorganism by promoting its growth relative to the competing microflora. At present, there is no single ideal isolation procedure available for the recovery of pathogenic strains of Y. enterocolitica from foods. The aim of this study was to determine the recovery rate of Y. enterocolitica biotype 4/serotype 0:3 from samples of pork mince inoculated with known numbers of the microorganism using different enrichment parameters (Time, temperature and pH) and Cefsulodin-Irgasan-Novobiocin (CIN) agar as the selective medium. The experiment was conducted in two trials using different bacterial dilutions. Three pork mince samples in duplicate were inoculated with known quantities of Y. enterocolitica biotype 4/serotype 0:3 organisms and subjected to cold enrichment in phosphate buffered saline (PBS) with a pH of 7.6, 6.6 and 5.5 at 25°C for 2 days, 10°C for 7 days and 4°C for 21 days. CIN agar was used as the selective medium. Pre-inoculation control samples were selected and plated in CIN on day O and on day 21 after PBS enrichment at 4°C. In Trial one Y. enterocolitica organisms were recovered from all 3 samples incubated at 25°C for 2 days and from 1 out of 3 inoculated samples incubated at 4°C for 21 days. There were no organisms recovered from other inoculated samples. The control sample did not show any environmental contamination with Yersinia species. In Trial two, Y. enterocolitica was recovered from 1 out of 3 duplicate samples enriched in PBS with pH 6.6 and incubated at 25°C for two days. Y. enterocolitica was not recovered from other inoculated samples. Y. intermedia was isolated from all pH, temperature and time combinations and also from control samples. The following conclusions can be drawn from this experiment. Incubation at high temperature (25°C) and short duration (48 hours) can be used as an efficient method for isolating Y. enterocolitica from pork samples. The standard incubation period of 21 days required for cold enrichment at 4°C is too long for the isolation of pathogenic strains, because of possible growth of environmental microorganisms. A pH of 6.6 is less efficient than 7.6 for enrichment although occasional isolation can be made using this pH. Enrichment in PBS with a pH of 5.5 with any time as well as temperature combinations and incubation at 10°C for 7 days are not ideal for isolation of pathogenic Yersinia enterocolitica strains. Of the three enrichments (PBS 7.6, 6.6, 5.5) used in this experiment, PBS with pH 7.6 was found to be most efficient to others.
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    The tonsillar carriage of Yersinia species by pigs: a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Veterinary Science at Massey University
    (Massey University, 1994) De Allie, Claude Adrian
    The impetus for this study arose due to the increasing isolation of species of Yersinia from people, with pigs being suspected as major reservoirs of human pathogenic strains of the organism in New Zealand. The general aims of the study, conducted in two phases, among pigs from several herds sent for slaughter at an abattoir in Palmerston North were: (i) to determine the presence of human pathogenic strains of Yersinia in the tonsils of slaughtered pigs and their distribution among selected herds. (ii) to determine the seasonal effect on prevalence of isolation and type of organism isolated, and (iii) to determine the in vitro virulence characteristics of strains of the organism isolated from the tonsils of slaughter pigs, and their potential public health implications. The first phase involved a cross-sectional study, conducted between August and September, 1993. Tonsils were collected from 124 pigs from eight farms and were examined for the presence of species of Yersinia. A total of 77 (62.1%) strains of Yersinia were isolated, this consisted of 42 (33.9%), 27 (21.8%), 7 (5.6%) and 1 (0.8%) strains of Y. enterocolitica. Y. pseudotuberculosis, Y. frederiksenii and Y. kristensenii respectively. Yersinia enterocolitica serotypes 0:3, 0:5, 27 and Y. pseudotuberculosis comprised 26 (33.8%), 12(15.6%) and 27 (35.1%) of the total number of isolates respectively. Yersiniae were isolated from all eight farms with individual farm prevalences ranging from 20% to 100%, while the number of species per farm ranged from 1 to 3. The pyrazinamidase activity test correctly identified 48 of the isolates as pathogenic or non-pathogenic yersiniae, (a specificity of 96%). The second phase, a longitudinal study, was conducted over a period of twelve months (February 1993 – January 1994), among pigs from four farms, selected according to the particular strain of Yersinia prevailing in the herd. A total of 705 pigs were examined for the carriage of species of Yersinia in their tonsils. A total of 264 isolates were obtained, consisting of 198 (75%), 55 (20.8%), 5 (1.9%), and 1 (0.4%) strains of Y. enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii and Y. kristensenii respectively. Yersinia enterocolitica serotypes 0:5,27 and 0:3 comprised 105 (39.8%) and 78 (29.5%) of the total number of isolates respectively. Yersinia pseudotuberculosis comprised 55 (20.9%) with serotype III, 39 (14.8%) the most consistently isolated serotype. Yersiniae were isolated throughout the year particularly in the colder months. Yersinia enterocolitica serotypes 0:3 and 0:5,27 were found throughout the year with the lowest prevalence in the warmer months. However, a seasonal variation existed among serotypes of Y. pseudotuberculosis, with serotypes I and II found only in the winter and spring. Serotype III was found throughout the year, except for February. During phase two of the study, 150 isolates of Yersinia were tested for in vitro virulence-associated characteristics. The autoagglutination test. CR-MOX agar, and the pyrazinamidase assay, coupled with salicin and aesculin tests, were highly successful in separating pathogenic from non-pathogenic strains of Y. enterocolitica. Likewise, the three assays successfully identified virulence activity in the majority of strains of Y. pseudotuberculosis with specificity among the three assays ranging between 90-100% for both Y. pseudotuberculosis and Y. enterocolitica. The study also revealed marked variation in prevalence and type of Yersinia species isolated from pigs from different farms. The fact that particular serotypes predominate and persist on specific farms strongly suggest that there are factors such as source of pigs, management practices or contact with other animals which determine their status. Identification of these determinates could lead to control or eradication of important yersiniae from pig farms. The overall prevalence of 41.1% ranks New Zealand among countries with reported high isolation rates of the organism and further emphasises the fact that pigs constitute major reservoirs for human pathogenic strains of Yersinia worldwide. The infection among slaughter pigs in New Zealand may be of human health concern and thus warrants further investigation particularly to determine whether the strains isolated from pigs are identical to those involved in human disease.
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    Faecal and tonsillar carriage of Yersinia spp. by pigs : a dissertation presented in partial fulfilment (25%) of the requirements for the degree of Master of Veterinary Studies in Veterinary Public Health at Massey University
    (Massey University, 1997) Bungay, Alice Alma C
    Pigs are considered important reservoirs for pathogenic Yersinia spp. which cause disease in humans. Results of many surveys have demonstrated the common occurrence of Y. enterocolitica and related species in the tonsils and intestinal tract of healthy slaughter-age pigs. However, the source of infection remains unclear. There is a dearth of information as to when pigs become infected hence, there is a need to conduct further studies not only among slaughter pigs but also among pigs belonging to different age groups on the farm. This study was conducted to determine the faecal and tonsillar carriage of different Yersinia spp. by pigs belonging to different age groups, characterise the species isolated by biochemical and serological methods, and determine the in vitro virulence characteristics of the isolates by simple assays. The study was conducted in two phases: first on a known or suspect Y. enterocolitica-positive farm within Massey University and second in a slaughterhouse where pigs from the suspect farm are sent for slaughter. A total of 54 faecal samples were collected from pigs belonging to six different age groups including 3-4 days, 2 weeks, 3 weeks, 4 weeks, 8 weeks and 12 weeks together with samples from the sow and one pooled sample from the floor. All of the 54 faecal samples collected were found negative for Yersinia spp. after the 21-day cold enrichment, subsequent plating onto CIN agar and use of primary screening tests. In the second phase, a total of 50 samples representing 25 faecal and 25 palatine tonsils were collected from slaughter pigs with ages ranging from 20-24 weeks. Out of the 25 faecal samples, there were two isolates of Yersinia frederiksenii and one isolate of the unusual or new biotype of Yersinia enterocolitica. Of the 25 samples of palatine tonsils collected, three of the isolates were unusual strains of Y. enterocolitica and two were Y. pseudotuberculosis Group I or serotype I. All isolates were confirmed by biochemical tests. The two isolates of Y. pseudotuberculosis were confirmed as Group I by serological tests. In this study, sample collections were done in the months of August and October when Group I was isolated conforming to the results of De Allie (1994) when serotype I was found only in winter and spring. The pathogenic potential of the eight isolates of Yersinia spp. were tested for in vitro virulence associated characteristics using the four simple tests namely salicin fermentation/aesculin hydrolysis, D-xylose fermentation, pyrazinamidase (PYZ) test and Congo Red-magnesium oxalate (CR-MOX). In this study, the unusual strains of Y. enterocolitica isolated from the tonsils and faeces were salicin-aesculin positive, xylose-positive, CR-MOX negative and PYZ negative. Except for PYZ, all three tests suggest the non-pathogenic potential of this unusual strain of Y. enterocolitica. The pathogenic potential of Y. pseudotuberculosis Group I isolated from the pig tonsils was shown by CR-MOX and PYZ tests. Both isolates were CR-MOX positive and PYZ negative. Y. pseudotuberculosis is considered a potential pathogen as shown by in vitro virulence tests and should not be overlooked as a causative of human disease. It was shown in this study that not all pigs are infected with pathogenic and non-pathogenic species of Yersinia even if they have been established to harbour the organisms either in their tonsils or faeces. However, pigs sent for slaughter may carry Yersinia spp. in their tonsils or faeces suggesting the role of other factors such as management and husbandry practices or contact with other animals during lairage, or from possible cross-contamination during processing at the slaughterhouse. Slaughter pigs are important source of Y. enterocolitica infections in humans, hence it is important to prevent or reduce the risk of possible contamination in the slaughterhouse by these organisms. Further investigations are needed to determine the pathogenic potential of the unusual strains of Y. enterocolitica and its possible role as a causative agent in human disease. Since Yersinia spp. were not recovered from faecal samples of pigs in the farm belonging to different age groups in this study, there is a need to conduct further studies using tonsillar samples or swabs instead of faeces. As shown in this study and other published reports, tonsils yield more isolates of Yersinia spp. compared to faecal samples.