Discovery of novel treponemes associated with pododermatitis in elk (Cervus canadensis).

Pododermatitis, also known as treponeme-associated hoof disease (TAHD), presents a significant challenge to elk (Cervus canadensis) populations in the northwestern USA, with Treponema spp. consistently implicated in the lesion development. However, identifying species-specific Treponema strains from these lesions is hindered by its culture recalcitrance and limited genomic information. This study utilized shotgun sequencing, in silico genome reconstruction, and comparative genomics as a culture-independent approach to identify metagenome-assembled Treponema genomes (MATGs) from skin scraping samples collected from captive elk experimentally challenged with TAHD. The genomic analysis revealed 10 new MATGs, with 6 representing novel genomospecies associated with pododermatitis in elk and 4 corresponding to previously identified species-Treponema pedis and Treponema phagedenis. Importantly, genomic signatures of novel genomospecies identified in this study were consistently detected in biopsy samples of free-ranging elk diagnosed with TAHD, indicating a potential etiologic association. Comparative metabolic profiling of the MATGs against other Treponema genomes showed a distinct metabolic profile, suggesting potential host adaptation or geographic uniqueness of these newly identified genomospecies. The discovery of novel Treponema genomospecies enhances our understanding of the pathogenesis of pododermatitis and lays the foundation for the development of improved molecular surveillance tools to monitor and manage the disease in free-ranging elk.IMPORTANCETreponema spp. play an important role in the development of pododermatitis in free-ranging elk; however, the species-specific detection of Treponema from pododermatitis lesions is challenging due to culture recalcitrance and limited genomic information. The study utilized shotgun sequencing and in silico genome reconstruction to identify novel Treponema genomospecies from elk with pododermatitis. The discovery of the novel Treponema species opens new avenues to develop molecular diagnostic and epidemiologic tools for the surveillance of pododermatitis in elk. These findings significantly enhance our understanding of the genomic landscape of the Treponemataceae consortium while offering valuable insights into the etiology and pathogenesis of emerging pododermatitis in elk populations.

Transmission and lesion progression of treponeme-associated hoof disease in captive elk (Cervus canadensis).

Treponeme-associated hoof disease (TAHD) is a debilitating disease of free-ranging elk (Cervus canadensis) in the northwestern U.S. While treponemes are associated with lesions, the etiology and transmissibility between elk are unknown. Our objective was to determine whether the disease can be environmentally transmitted to captive elk. Four individually housed treatment elk and 2 control elk were challenged with soil mixed with inoculum prepared from free-ranging elk hooves from TAHD-positive elk or autoclaved hooves from normal elk, respectively. The inoculum for each group was applied to the interdigital space and added to pre-existing soil in each pen. Eight challenges were conducted at 1-4-week intervals and lesion development was assessed during a 138-day challenge period that was followed by a 170-day monitoring period to document lesion progression. All treatment elk, but no control elk, developed gross and histologic lesions consistent with TAHD. Treponema phylotypes similar to those in bovine digital dermatitis in cattle were detected using 16S rRNA gene amplicon sequencing from lesions in all treatment elk, but no control elk, during the challenge period. Lesions progressed from ulcerations in the interdigital space to extensive ulceration and underrunning of the hoof capsule by 35 and 173 days following the initial inoculation, respectively. Lameness in treatment elk was correlated with lesion development (R = 0.702, p≤0.001), and activity of infected elk was reduced during the challenge (p≤0.001) and monitoring periods (p = 0.004). Body condition was significantly lower in treatment than control elk 168 days following the initial inoculation (p = 0.05) and at each individual elk's study endpoint (p = 0.006). Three of 4 treatment elk were euthanized when they reached humane endpoints, and one elk recovered. These results provide direct evidence that TAHD is a transmissible infectious disease in elk. As such, actions that reduce transmission risk can support disease management and prevention.

Dimethyl adenosine transferase (KsgA) contributes to cell-envelope fitness in Salmonella Enteritidis.

We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis, an electrokinetic tool, in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists.

Genetically distinct lineages of Salmonella Typhimurium ST313 and ST19 are present in Brazil.

In sub-Saharan Africa, two genetically distinct lineages of multi-drug resistant non-typhoidal Salmonella (NTS) serovar Typhimurium sequence type 313 (ST313) are known to cause invasive disease among people. S. Typhimurium ST313 has evolved to become more human-adapted and is commonly isolated from systemic sites (eg., blood) from febrile patients in sub-Saharan Africa. Epidemiological studies indicate that S. Typhimurium is frequently isolated from systemic sites from human patients in Brazil, however, it is currently unknown if this pathogen has also evolved to become more invasive and human-adapted in this country. Here we determined genotypic and phenotypic divergence among clinical S. Typhimurium strains isolated from systemic and non-systemic sites from human patients in Brazil. We report that a subset (8/38, 20%) of epidemiologically diverse human clinical strains of S. Typhimurium recovered from systemic sites in Brazil show significantly higher intra-macrophage survival, indicating that this subset is likely more invasive. Using the whole genome sequencing and phylogenetic approaches, we identified S. Typhimurium ST313-lineage in Brazil that is genetically and phenotypically distinct from the known African ST313-lineages. We also report the identification of S. Typhimurium ST19-lineage in Brazil that is evolving similar to ST313 lineages from Africa but is genetically and phenotypically distinct from ST19-lineage commonly associated with the gastrointestinal disease worldwide. The identification of new S. Typhimurium ST313 and ST19 lineages responsible for human illnesses in Brazil warrants further epidemiological investigations to determine the incidence and spread of a genetically divergent population of this important human pathogen.

Biochemical Reference Intervals for Backyard Hens.

Keeping backyard poultry has become increasingly popular in urban and suburban households. With this rise in popularity comes an increased need for veterinarians who are willing to serve this demographic and a need for lab reference intervals that capture the range of clinically healthy backyard hens. We developed blood chemistry reference intervals for birds in backyard chicken flocks. Between June and August 2016, 133 adult, actively laying hens from 34 different flocks in Western Washington were sampled via medial metatarsal venipuncture. Whole heparinized blood was analyzed using a VetScan VS2 with Avian/Reptilian Profile Plus reagent rotors. Packed cell volume was determined via centrifugation of microhematocrit tubes. Reference intervals were calculated by Reference Value Advisor V2.1 software using the nonparametric method. Seven currently published reference intervals for the Gallus gallus domesticus could not be validated for use in backyard hens, according to the guidelines established by the Clinical and Laboratory Standards Institute. Of flock owners who participated in the study, 47% reported they would consider paying for a blood test if a veterinarian thought it would be beneficial.

Identification of new CpG oligodeoxynucleotide motifs that induce expression of interleukin-1ß and nitric oxide in avian macrophages.

Unmethylated CpG motifs are known to stimulate mammalian toll-like receptor-9 expressing cells such as macrophages. However, the magnitude of immune-stimulation by CpG-motif can be sequence- and host-specific, implying the importance of identifying new immune-stimulatory motifs. This study aimed to determine the frequency distribution of 256 unique hexamers CpG-motifs in the Salmonella genome and to characterize their immune-stimulatory activity in avian host. We synthesized 256 CpG oligodeoxynucleotides (CpG-ODNs) each containing triplicates of a unique hexamer CpG-motif and tested their ability to induce expression of pro-inflammatory cytokine IL-1ß in avian macrophages using q-RT PCR in four rounds of screening assays. CpG-ODNs that induced significantly higher IL-1ß expression were also subjected to Griess assay to determine their ability to induce nitric oxide (NO) production in avian macrophages. This analysis resulted in identification of 7 CpG-ODNs that consistently induced IL-1ß expression and NO production in avian macrophages at a level similar to the expression achieved using commercially available PTO-CpG-ODN 2007 and LPS derived from Salmonella. To the best of our knowledge, this is the first report showing comprehensive screening of all possible unique CpG hexamer (n=256) motifs for their ability to induce IL-1ß expression and NO production in avian macrophages. We also show that the newly identified CpG-motifs with high immune-stimulatory activity are widely distributed in Salmonella genome. The CpG-ODNs identified in this study may serve as promising immunoprophylactics to potentiate innate responses in chickens against Salmonella and other infectious agents.

Dimethyl adenosine transferase (KsgA) deficiency in Salmonella enterica Serovar Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens.

Dimethyl adenosine transferase (KsgA) performs diverse roles in bacteria, including ribosomal maturation and DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that a ksgA mutation in Salmonella enterica serovar Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of a ksgA mutant (the ksgA::Tn5 mutant) of S. Enteritidis in orally challenged 1-day-old chickens. The ksgA::Tn5 mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens compared to those of the wild-type (WT) parent. Phenotype microarray (PM) was employed to compare the ksgA::Tn5 mutant and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C, and 42°C. At chicken body temperature (42°C), the ksgA::Tn5 mutant showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur, and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations of ≥6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, the ksgA::Tn5 mutant displayed a severe growth defect in high-osmolarity (6.5% NaCl) conditions in nutrient-rich (LB) and nutrient-limiting (M9 minimum salts) media at 42°C. Moreover, the ksgA::Tn5 mutant showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike Escherichia coli, the ksgA::Tn5 mutant did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity, and altered susceptibility to kasugamycin and chloramphenicol.

Production and evaluation of chicken egg-yolk-derived antibodies against Campylobacter jejuni colonization-associated proteins.

Campylobacter jejuni is one of the most important causes of foodborne gastroenteritis. Chickens are considered a reservoir host of C. jejuni, and epidemiological studies have shown that contaminated chicken meat is a primary source of human infection. The objective of this study was to produce chicken egg-yolk-derived antibody (IgY) against the five C. jejuni colonization-associated proteins or CAPs (CadF, FlaA, MOMP, FlpA, and CmeC). Recombinant C. jejuni CAPs were expressed in Escherichia coli and were purified by affinity chromatography. Specific-pathogen-free laying hens were hyperimmunized with each recombinant CAP to induce production of α-CAP-specific IgY. Egg yolks were collected from immunized and nonimmunized hens and were lyophilized to obtain egg-yolk powder (EYP) with or without α-C. jejuni CAP-specific IgY. IgY was purified from EYP, and the antibody response in serum and egg yolk was tested by indirect enzyme-linked immunosorbent assay. The α-C. jejuni CAP-specific IgY levels were significantly (p<0.05) higher in both serum and EYP obtained from immunized hens as compared with the nonimmunized hens. Each α-C. jejuni CAP-specific IgY reacted with the C. jejuni cells and recombinant CAPs as detected by immunofluorescence microscopy and Western blot assays, respectively. We also show that α-CadF, α-MOMP, and α-CmeC IgY significantly reduced adherence of C. jejuni to the chicken hepatocellular carcinoma (LMH) cells, suggesting that these α-C. jejuni CAP-specific IgY may be useful as a passive immunotherapeutic to reduce C. jejuni colonization in chickens.

Transposon mutagenesis of Salmonella enterica serovar Enteritidis identifies genes that contribute to invasiveness in human and chicken cells and survival in egg albumen.

Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nal(r) strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis.

Discovery of a gene conferring multiple-aminoglycoside resistance in Escherichia coli.

Bovine-origin Escherichia coli isolates were tested for resistance phenotypes using a disk diffusion assay and for resistance genotypes using a DNA microarray. An isolate with gentamicin and amikacin resistance but with no corresponding genes detected yielded a 1,056-bp DNA sequence with the closest homologues for its inferred protein sequence among a family of 16S rRNA methyltransferase enzymes. These enzymes confer high-level aminoglycoside resistance and have only recently been described in Gram-negative bacteria.

Campylobacter jejuni invade chicken LMH cells inefficiently and stimulate differential expression of the chicken CXCLi1 and CXCLi2 cytokines.

Campylobacter jejuni is a major food-borne bacterial pathogen, which is capable of causing diarrhoea containing blood and leukocytes. C. jejuni invasion of the intestinal epithelial cells and the release of proinflammatory molecules contribute to the pathophysiology of campylobacteriosis. Given the commensal relationship of C. jejuni with chickens, we hypothesized that C. jejuni invasion of chicken cells and the release of host cell cytokines would be significantly less than with human cells. To test our hypothesis, we examined the interactions of C. jejuni with chicken LMH cells, and performed in vivo experiments with chickens. The binding and internalization assays revealed that C. jejuni was significantly less invasive of LMH cells relative to human INT 407 cells, even though the bacteria bound to each host cell species equally. We also assessed interleukin-8 (IL-8) transcript, IL-8 secretion, and the release of chemoattractant molecules from the inoculated cells. Inoculation of LMH cells with C. jejuni stimulated expression of both chicken IL-8 orthologues, chCXCLi2 and chCXCLi1, but at levels significantly less than human IL-8 (huCXCL8) expressed from human INT 407 cells inoculated with C. jejuni. Moreover, the supernatant fluids of the C. jejuni-inoculated LMH cells resulted in little heterophil migration. In vivo, C. jejuni were observed bound to the cells lining the glandular crypts, but overt signs of cell invasion or pathology were not observed. These results indicate that cytokine expression in chicken LMH cells in response to C. jejuni is distinct from that of Salmonella typhimurium.

Effect of metC mutation on Salmonella Gallinarum virulence and invasiveness in 1-day-old White Leghorn chickens.

Salmonella enterica serotype Gallinarum (S. Gallinarum) is the causative agent of fowl typhoid (FT) in chickens. FT is a severe systemic disease of chickens causing heavy economic losses to the poultry industry through mortality, reduced egg production and culling of precious breeding stocks. In this study, a metC (encoding cystathionine beta lyase) mutant was produced from a virulent strain of S. Gallinarum by Mini-Tn5 insertional inactivation. The mutant was significantly attenuated in virulence for 1-day-old White Leghorn chickens. Inactivation of metC resulted in 10(4)-fold increase in the LD50 when compared with the wild type parent. The metC mutant showed an in vivo competitiveness defect in the challenged chickens and significantly lower (P < 0.01) bacterial burden in the reticuloendothelial organs when compared with the wild-type parent. These results indicate that metC gene is important for virulence of S. Gallinarum in chickens.

Cloning and expression of 51-kDa antigenic protein of Neorickettsia risticii NR-JA1.

Neorickettsia (Ehrlichia) risticii is a causative agent of acute diarrheal syndrome in horses, commonly known as Potomac horse fever. Korean isolate of N. risticii NR-JA1 was cultivated in mouse macrophage cell line P388D1. A complete ORF of p51 antigenic protein gene was amplified and cloned into pQE32 and pcDNA3.1 vectors and the resultant clones were named as pQE32/Nr-51 and pcDNA3.1/Nr-51, respectively. Recombinant p51 (rp51) protein antigen was expressed in E. coli (pQE32/Nr-51) and cos-7 cell line (pcDNA3.1/Nr-51). The rp51 protein showed immunoreactivity with anti- mouse p51 antibodies. BALB/c mice were inoculated with recombinant plasmid DNA (pcDNA3.1/Nr-51). The serum samples collected from these BALB/c mice showed IgG ELISA titers of 1:128. In a Western immunoblot assay, these serum samples showed a strong reactivity to rp51 expressed in cos-7 cell line transfected with pcDNA3.1/Nr-51. The results of this preliminary indicate that N. risticii p51 protein is an immmuno-dominant antigen and may be a good target for the development of serological or a molecular diagnostic test and possibly an improved recombinant DNA based vaccine against Potomac horse fever.