Resilience of swine nasal microbiota to influenza A virus challenge in a longitudinal study.

Influenza A virus (IAV) is an important contributing pathogen of porcine respiratory disease complex (PRDC) infections. Evidence in humans has shown that IAV can disturb the nasal microbiota and increase host susceptibility to bacterial secondary infections. Few, small-scale studies have examined the impact of IAV infection on the swine nasal microbiota. To better understand the effects of IAV infection on the nasal microbiota and its potential indirect impacts on the respiratory health of the host, a larger, longitudinal study was undertaken to characterize the diversity and community composition of the nasal microbiota of pigs challenged with an H3N2 IAV. The microbiome of challenged pigs was compared with non-challenged animals over a 6-week period using 16S rRNA gene sequencing and analysis workflows to characterize the microbiota. Minimal changes to microbial diversity and community structure were seen between the IAV infected and control animals the first 10 days post-IAV infection. However, on days 14 and 21, the microbial populations were significantly different between the two groups. Compared to the control, there were several genera showing significant increases in abundance in the IAV group during acute infection, such as Actinobacillus and Streptococcus. The results here highlight areas for future investigation, including the implications of these changes post-infection on host susceptibility to secondary bacterial respiratory infections.

Comparison of Two Multilocus Sequence Typing Schemes for Mycoplasma bovis and Revision of the PubMLST Reference Method.

Mycoplasma bovis causes pneumonia, pharyngitis, otitis, arthritis, mastitis, and reproductive disorders in cattle and bison. Two multilocus sequence typing (MLST) schemes have been developed for M. bovis, with one serving as the PubMLST reference method, but no comparison of the schemes has been undertaken. Although the PubMLST scheme has proven to be highly discriminatory and informative, the recent discovery of isolates missing one of the typing loci, adh-1, raises concern about its suitability for continued use. The goal of our study was to compare the performance of the two MLST schemes and identify a new reference scheme capable of fully typing all isolates. We evaluated 448 isolates from diverse geographic and anatomic sites that collectively represent cattle, bison, deer, and a goat. The discrimination indexes (DIs) for the PubMLST and the alternative scheme are 0.909 (91 sequence types [STs]) and 0.842 (77 STs), respectively. Although the PubMLST scheme outperformed the alternative scheme, the adh-1 locus must be retired from the PubMLST scheme if it is to be retained as a reference method. The DI obtained using the six remaining PubMLST loci (0.897, 79 STs) fails to reach the benchmark recommended for a reference method (0.900), mandating the addition of a seventh locus. Comparative analysis of genome sequences from the isolates used here identified the dnaA locus from the alternative scheme as the optimal replacement for adh-1 This revised scheme, which will be implemented as the new PubMLST reference method, has a DI of 0.914 and distinguishes 88 STs from the 448 isolates evaluated.

Treponeme-Associated Hoof Disease of Free-Ranging Elk ( Cervus elaphus) in Southwestern Washington State, USA.

A novel foot disease in free-ranging elk ( Cervus elaphus) in southwestern Washington State emerged in 2008 and spread throughout the region. Initial studies showed adult elk had chronic hoof overgrowth, sole ulcers, and sloughed hoof capsules, but no cause was determined. To identify possible causes and characterize the earliest lesions, 9-, 7-, and 3-month-old elk were collected. Nine-month-old elk had sole ulcers (3/9 elk) and sloughed/overgrown hoof capsules (4/9 elk) similar to adults. Histologically, lesions consisted of coronary, heel bulb, and interdigital ulcers with suppurative inflammation, epithelial hyperplasia, deeply invasive spirochetes, and underrunning of the hoof capsule and heel-sole junction. Spirochetes were identified as Treponema via immunohistochemistry and polymerase chain reaction (PCR). Seven-month-old elk had similar underrunning foot ulcers (6/8 elk) with Treponema identified in all lesions but no chronic overgrowth or sloughed hoof capsules. Three-month-old calves had superficial coronary erosions with no inflammation or identifiable spirochetes (3/5 elk) but were culture/PCR positive for Treponema, suggesting possible early lesions. Lesions from 9- and 7-month-old elk included aerobic and anaerobic bacteria, many of which are associated with infectious foot disease in livestock. Antibody enzyme-linked immunosorbent assay of 7- and 3-month-old elk from the enzootic region showed a trend toward increased Treponema antibody titers compared to normal control elk from outside the region, further supporting the significance of Treponema in the pathogenesis of foot disease. Treponeme-associated hoof disease (TAHD) in elk, a debilitating and progressive condition, shares similarities to bovine digital dermatitis and contagious ovine digital dermatitis.

Bulk soil bacterial community structure and function respond to long-term organic and conventional agricultural management.

Understanding how soil microbiomes respond to management is essential to maximizing soil health. We contrasted microbiomes in bulk soil under long-term organic and conventional management in a grain production setting. Management category significantly impacted the relative abundances of 17% of the most abundant taxa. Both conventional and organic management favored particular taxa, but these effects were not reflected in summary richness and diversity indices. Management systems also lead to differences in soil edaphic properties, including pH and nutrient status; this may have been the mechanism by which change in the prokaryote community was enacted. Community change between years of sampling was less pronounced, with only 6 taxa differentially abundant among years. Management category also impacted the abundance of functional genes related to the production and consumption of greenhouse gases. Particulate methane monooxygenase genes were more frequent in soil under organic management, while soluble methane monooxygenase genes were more frequent in soil under conventional management in 1 of 2 years. Nitrous oxide reductase genes were significantly less abundant in soils under second-year alfalfa than in soils under corn. This work highlights the ability of agricultural management to enact broad rearrangements to the structure of bulk soil bacterial communities.

Experimental Transmission of Bovine Digital Dermatitis to Sheep: Development of an Infection Model.

Digital dermatitis is an infectious cause of lameness primarily affecting cattle but also described in sheep, goats, and wild elk. Digital dermatitis is a polymicrobial infection, involving several Treponema species and other anaerobic bacteria. Although the exact etiology has not been demonstrated, a number of bacterial, host, and environmental factors are thought to contribute to disease development. To study host-bacterial interactions, a reproducible laboratory model of infection is required. The objective of this study was to demonstrate key aspects of bovine digital dermatitis lesions in an easy-to-handle sheep model. Crossbred sheep were obtained from a flock free of hoof disease. Skin between the heel bulb and dewclaw was abraded before wrapping to emulate a moist, anaerobic environment. After 3 days, abraded areas were inoculated with macerated lesion material from active bovine digital dermatitis and remained wrapped. By 2 weeks postinoculation, experimentally inoculated feet developed erosive, erythematous lesions. At 4 weeks postinoculation, microscopic changes in the dermis and epidermis were consistent with those described for bovine digital dermatitis, including erosion, ulceration, hyperkeratosis, ballooning degeneration of keratinocytes, and the presence of neutrophilic infiltrates. Silver staining of lesion biopsy sections confirmed that spirochetes had penetrated the host epidermis. The model was then perpetuated by passaging lesion material from experimentally infected sheep into naïve sheep. This model of bovine digital dermatitis will allow for future novel insights into pathogenic mechanisms of infection, as well as the development of improved diagnostic methods and therapeutics for all affected ruminants.

Short communication: Lymphocyte proliferative responses in cattle naturally infected with digital dermatitis consist of CD8+ and γδ-T cells but lack CD4+ T cells.

Digital dermatitis is an infectious disease of cattle and the leading cause of lameness. This disease is complicated by the reoccurrence of the lesions and the observation of lesions on more than one limb at different time points, indicating infection may not result in a protective immune response. The objective of this study was to characterize the peripheral blood cellular response in naturally infected and naïve cattle to bacterial antigens derived from pathogens associated with digital dermatitis lesions. Peripheral blood mononuclear cells were isolated from dairy cattle identified as having active or chronic lesions during routine hoof-trimming. Following bacterial antigen stimulation, cells were analyzed for proliferation and phenotype by flow cytometry, and culture supernatants were analyzed for IFN-γ secretion. Digital-dermatitis-infected animals had greater serum antibody titers to treponemal antigens, higher percentages of proliferating CD8+, γδ-T cells, and B cells, and increased IFN-γ secretion in vitro when compared with responses of naïve animals. No increase in proliferation of CD4+ T cells was detected in infected or naïve cattle. Although CD8+ and γδ-T cell responses may be antigen specific, the memory nature or long-lived response is yet unknown. The lack of responsiveness of CD4+ memory cells to treponemal antigens could explain the high rate of reoccurrence of digital dermatitis in infected animals.

Identification of several clades of novel single-stranded circular DNA viruses with conserved stem-loop structures in pig feces.

Metagenomic analysis of fecal samples collected from diarrheal swine detected sequences encoding a replication initiator protein (Rep). The genomes of ten novel single-stranded DNA viruses were determined, and they exhibited a similar genome organization. The two putative open reading frames (ORFs) encoding Rep and the capsid protein are bidirectionally transcribed and separated by two intergenic regions. Stem-loop structure(s) typical of genomes that undergo the rolling-circle DNA replication mechanism were observed. Phylogenetically, these ten genomes are in a monophyletic clade with the previously described porcine stool-associated virus (PoSCV) but are divergent enough to be further classified into to six distinct virus clades.

In-feed antibiotic effects on the swine intestinal microbiome.

Antibiotics have been administered to agricultural animals for disease treatment, disease prevention, and growth promotion for over 50 y. The impact of such antibiotic use on the treatment of human diseases is hotly debated. We raised pigs in a highly controlled environment, with one portion of the littermates receiving a diet containing performance-enhancing antibiotics [chlortetracycline, sulfamethazine, and penicillin (known as ASP250)] and the other portion receiving the same diet but without the antibiotics. We used phylogenetic, metagenomic, and quantitative PCR-based approaches to address the impact of antibiotics on the swine gut microbiota. Bacterial phylotypes shifted after 14 d of antibiotic treatment, with the medicated pigs showing an increase in Proteobacteria (1-11%) compared with nonmedicated pigs at the same time point. This shift was driven by an increase in Escherichia coli populations. Analysis of the metagenomes showed that microbial functional genes relating to energy production and conversion were increased in the antibiotic-fed pigs. The results also indicate that antibiotic resistance genes increased in abundance and diversity in the medicated swine microbiome despite a high background of resistance genes in nonmedicated swine. Some enriched genes, such as aminoglycoside O-phosphotransferases, confer resistance to antibiotics that were not administered in this study, demonstrating the potential for indirect selection of resistance to classes of antibiotics not fed. The collateral effects of feeding subtherapeutic doses of antibiotics to agricultural animals are apparent and must be considered in cost-benefit analyses.

Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions.

Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

Transcriptomic profiles of Mannheimia haemolytica planktonic and biofilm associated cells.

Mannheimia haemolytica is the principal agent contributing to bovine respiratory disease and can form biofilms with increased resistance to antibiotic treatment and host immune defenses. To investigate the molecular mechanisms underlying M. haemolytica biofilm formation, transcriptomic analyses were performed with mRNAs sequenced from planktonic and biofilm cultures of pathogenic serotypes 1 (St 1; strain D153) and St 6 (strain D174), and St 2 (strain D35). The three M. haemolytica serotypes were cultured in two different media, Roswell Park Memorial Institute (RPMI) 1640 and brain heart infusion (BHI) to form the biofilms. Transcriptomic analyses revealed that the functions of the differentially expressed genes (DEGs) in biofilm associated cells were not significantly affected by the two media. A total of 476 to 662 DEGs were identified between biofilm associated cells and planktonic cells cultured under BHI medium. Functional analysis of the DEGs indicated that those genes were significantly enriched in translation and many biosynthetic processes. There were 234 DEGs identified in St 1 and 6, but not in St 2. The functions of the DEGs included structural constituents of ribosomes, transmembrane proton transportation, proton channels, and proton-transporting ATP synthase. Potentially, some of the DEGs identified in this study provide insight into the design of new M. haemolytica vaccine candidates.

Correlation of lesion severity with bacterial changes in Treponeme-Associated Hoof Disease from free-roaming wild elk (Cervus canadensis).

BACKGROUND: Treponeme-Associated Hoof Disease (TAHD) is a polybacterial, multifactorial disease affecting free-ranging wild elk (Cervus canadensis) in the Pacific Northwest. Previous studies have indicated a bacterial etiology similar to digital dermatitis in livestock, including isolation of Treponema species from lesions. The lesions appear to progress rapidly from ulcerative areas in the interdigital space or along the coronary band to severe, ulcerative, necrotic, proliferative lesions under-running the hoof wall, perforating the sole, and contributing to hoof elongation, deformity, and overgrowth. Eventually the lesions undermine the laminal structure leading to sloughing of the hoof horn capsule. The objective of this study was to characterize the bacterial communities associated with hoof lesions, which were categorized into 5 stages or disease grade severities, with 0 being unaffected tissue and 4 being sloughed hoof capsule. We also wanted to determine if the etiology of TAHD through morphological changes was dominated by Treponema, as observed in hoof diseases in livestock. RESULTS: The bacterial 16S rRNA gene was sequenced from 66 hoof skin biopsy samples representing 5 lesion grades from samples collected by Washington Department of Fish and Wildlife as part of a voluntary hunter program. Analysis of the relative abundance of bacterial sequences showed that lesions were dominated by members of the bacterial phyla Proteobacteria, Firmicutes, Spirochaetes, Bacteroidetes and Actinobacteria. In lesion samples, members of the genus Treponema, Porphyromonas, and Mycoplasma increased with lesion severity. Association analysis indicated frequent identification of Treponema with Porphyromonas, Bacteroides and other anaerobic Gram-positive cocci. CONCLUSIONS: The bacterial 16S rRNA gene sequencing confirmed the presence of Treponema species at all stages of TAHD lesions, treponeme specie-specific PCR and histopathology, indicating that the morphological changes are a continual progression of disease severity with similar bacterial communities. Association and abundance of these other pathogenic genera within lesions may mean synergistic role with Treponema in hoof disease pathogenesis. Characterizing bacteria involved in lesion development, and their persistence during disease progression, provides evidence for science-based management decisions in TAHD infected elk populations.

Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion.

BACKGROUND: Bovine papillomatous digital dermatitis (DD) is the leading cause of lameness in dairy cattle and represents a serious welfare and economic burden. Found primarily in high production dairy cattle worldwide, DD is characterized by the development of an often painful red, raw ulcerative or papillomatous lesion frequently located near the interdigital cleft and above the bulbs of the heel. While the exact etiology is unknown, several spirochete species have been isolated from lesion material. Four isolates of Treponema phagedenis-like spirochetes were isolated from dairy cows in Iowa. Given the distinct differences in host, environmental niche, and disease association, a closer analysis of phenotypic characteristics, growth characteristics, and genomic sequences of T. phagedenis, a human genitalia commensal, and the Iowa DD isolates was undertaken. RESULTS: Phenotypically, these isolates range from 8.0 to 9.7 µm in length with 6-8 flagella on each end. These isolates, like T. phagedenis, are strictly anaerobic, require serum and volatile fatty acids for growth, and are capable of fermenting fructose, mannitol, pectin, mannose, ribose, maltose, and glucose. Major glucose fermentation products produced are formate, acetate, and butyrate. Further study was conducted with a single isolate, 4A, showing an optimal growth pH of 7.0 (range of 6-8.5) and an optimal growth temperature of 40 °C (range of 29 °C-43 °C). Comparison of partial genomic contigs of isolate 4A and contigs of T. phagedenis F0421 revealed > 95% amino acid sequence identity with amino acid sequence of 4A. In silico DNA-DNA whole genome hybridization and BLAT analysis indicated a DDH estimate of >80% between isolate 4A and T. phagedenis F0421, and estimates of 52.5% or less when compared to the fully sequenced genomes of other treponeme species. CONCLUSION: Using both physiological, biochemical and genomic analysis, there is a lack of evidence for difference between T. phagedenis and isolate 4A. The description of Treponema phagedenis should be expanded from human genital skin commensal to include being an inhabitant within DD lesions in cattle.

A divergent clade of circular single-stranded DNA viruses from pig feces.

Using metagenomics and molecular cloning methods, we characterized five novel small, circular viral genomes from pig feces that are distantly related to chimpanzee and porcine stool-associated circular viruses, (ChiSCV and PoSCV1). Phylogenetic analysis placed these viruses into a highly divergent clade of this rapidly growing new viral family. This new clade of viruses, provisionally named porcine stool-associated circular virus 2 and 3 (PoSCV2 and PoSCV3), encodes a stem-loop structure (presumably the origin of DNA replication) in the small intergenic region and a replication initiator protein commonly found in other biological systems that replicate their genomes via the rolling-circle mechanism. Furthermore, these viruses also exhibit three additional overlapping open reading frames in the large intergenic region between the capsid and replication initiator protein genes.

Expansion of the in vitro assay for Leptospira potency testing to other serovars: case study with Leptospira Hardjo.

Evaluation of leptospiral vaccines for potency against Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Canicola, and Grippotyphosa is accomplished using the hamster potency test method described in 9 CFR 113.101-104. Applicability of this method to evaluation of bacterins developed for immunization against infection with L. interrogans serovar Hardjo or Leptospira borgpetersenii serovar Hardjo is complicated by several issues. Information from research on target host animal efficacy studies and evaluation of the immune response elicited using effective whole-cell bacterin formulations have revealed problems in relating these studies to either hamster-based or other potency testing methods. Future work on serovar Hardjo vaccines employing recombinant proteins will require preliminary testing methods in models other than the host animal. These models may also prove applicable to evaluation of potency for protein-based vaccines. Both an acute lethal infection model and a chronic infection model have been developed using two different strains of serovar Hardjo and will be described.

Shifts in the swine nasal microbiota following Bordetella bronchiseptica challenge in a longitudinal study.

Bordetella bronchiseptica is a widespread, highly infectious bacterial pathogen that causes respiratory disease in swine and increases the severity of respiratory infections caused by other viral or bacterial pathogens. However, the impact of B. bronchiseptica infection on the swine respiratory microbiota has not been thoroughly investigated. Here, we aim to assess the influence of B. bronchiseptica infection on the community structure and abundance of members of the swine nasal microbiota. To do so, the nasal microbiota of a non-infected control group and a group infected with B. bronchiseptica (BB group) were characterized prior to B. bronchiseptica strain KM22 challenge (day 0) and on selected days in the weeks following B. bronchiseptica challenge (days 1, 3, 7, 10, 14, 21, 36, and 42). Bordetella bronchiseptica was cultured from nasal samples of the BB group to assess nasal colonization. The results showed that B. bronchiseptica colonization did not persistently affect the nasal bacterial diversity of either of the treatment groups (alpha diversity). However, the bacterial community structures (beta diversity) of the two treatment groups significantly diverged on day 7 when peak colonization levels of B. bronchiseptica were detected. This divergence continued through the last sampling time point. In addition, Pasteurella, Pasteurellaceae (unclassified), Mycoplasma, Actinobacillus, Streptococcus, Escherichia-Shigella, and Prevotellaceae (unclassified) showed increased abundances in the BB group relative to the control group at various time points. This study revealed that B. bronchiseptica colonization can disturb the upper respiratory tract microbiota, and further research is warranted to assess how these disturbances can impact susceptibility to secondary infections by other respiratory pathogens.

Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association.

BACKGROUND: The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. RESULTS: Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. CONCLUSIONS: Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences.

Complete Genome Sequence of Four Strains of Leptospira borgpetersenii serovar Hardjo isolated from Cattle in the Central United States.

Pathogenic species of Leptospira cause leptospirosis, a global zoonotic disease affecting humans and all major livestock species. Cattle act as a reservoir host for L. borgpetersenii serovar Hardjo which colonize the kidneys and reproductive tract from which they are excreted and transmitted to other cattle via urine, semen or uterine discharges. Bovine leptospirosis results in reproductive failure, abortion, stillbirth and loss of milk production, and is an occupational risk for those working with infected animals. A recent study determined that 7.2% of cattle from an abattoir in the central United States were actively shedding pathogenic Leptospira. Here, we report and compare the complete genome sequence of four recent isolates of L. borgpetersenii serovar Hardjo designated strain TC112, TC147, TC129, and TC273.

Some like it hot, some like it cold; proteome comparison of Leptospira borgpetersenii serovar Hardjo strains propagated at different temperatures.

Leptospirosis is a global zoonotic disease affecting humans and livestock species. Bacterin vaccines lack cross protection between serogroups, and include multiple serovars propagated at 29 °C. Recent work demonstrated substantial variation in the transcriptome of identical species and serovars of Leptospira. Here, substantial differences in protein abundance profiles were identified in Leptospira borgpetersenii serovar Hardjo; strain HB203, which was isolated in the 1980s, compared to newer strains TC129 and TC273 isolated in 2016, and whether they were propagated at the routine temperature of 29 °C, compared to 37 °C which more closely emulates host infection. While 388 and 385 significantly differentially expressed (DE) proteins (FDR of 0.01) were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 29 °C respectively, only 66 and 4 DE proteins were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 37 °C respectively. Within each strain comparing temperatures, HB203 had 524 significantly DE proteins, TC129 had 347 DE proteins, and TC273 had 569 DE proteins. Data are available via ProteomeXchange with identifier PXD032831. Results highlight significant differential protein expression among identical serovars of L. borgpetersenii suggesting that bacterin vaccine design can benefit from consideration of strains employed and effects of temperature on growth. SIGNIFICANCE: Leptospirosis is a zoonotic disease caused by spirochete bacteria of the genus Leptospira. While leptospirosis affects over one million people per year, symptoms range vastly in severity from completely asymptomatic, to flu-like, to multi-organ failure and death in severe cases. Incidental hosts become infected after encountering pathogens directly from contact with another host, including domestic or wildlife animals, or indirectly from contaminated environments. Though animal vaccines exist, they lack cross protection across serogroups, and instead rely on inclusion of multiple carefully selected serovars from laboratory strains prepared at ~29 °C. Recent interest in gene expression at the Leptospira strain level, along with a newly achieved culture temperature of 37 °C (which more closely resembles host body temperature), led us to investigate the proteomic profiles of an older, established challenge strain HB203 in comparison to TC129 and TC273, two strains isolated in 2016 from abattoir cattle in the central United States. Herein, we identify substantial proteomic differences not only between strains of the same species and serovar, but notably between growth temperatures, collectively suggesting that bacterin vaccine composition may benefit from investigating strain selection and the temperature employed for growth of the bacteria used in bacterin preparation.

Assessing rodents as carriers of pathogenic Leptospira species in the U.S. Virgin Islands and their risk to animal and public health.

Leptospirosis is a global zoonotic disease caused by pathogenic bacteria of the genus Leptospira. We sought to determine if rodents in U.S. Virgin Islands (USVI) are carriers of Leptospira. In total, 140 rodents were sampled, including 112 Mus musculus and 28 Rattus rattus. A positive carrier status was identified for 64/140 (45.7%); 49 (35.0%) were positive by dark-field microscopy, 60 (42.9%) by culture, 63 (45.0%) by fluorescent antibody testing, and 61 (43.6%) by real-time polymerase chain reaction (rtPCR). Molecular typing indicated that 48 isolates were L. borgpetersenii and 3 were L. kirschneri; the remaining nine comprised mixed species. In the single culture-negative sample that was rtPCR positive, genotyping directly from the kidney identified L. interrogans. Serotyping of L. borgpetersenii isolates identified serogroup Ballum and L. kirschneri isolates as serogroup Icterohaemorrhagiae. These results demonstrate that rodents are significant Leptospira carriers and adds to understanding the ecoepidemiology of leptospirosis in USVI.

Antigen-Specific Urinary Immunoglobulin in Reservoir Hosts of Leptospirosis.

Domestic and wildlife animal species act as reservoir hosts of leptospirosis, a global zoonotic disease affecting more than 1 million people annually and causing significant morbidity and mortality in domestic animals. In contrast to incidental hosts which present with an array of clinical manifestations, reservoir hosts are typically asymptomatic and can shed leptospires from chronically infected kidneys via urine for extended periods of time. Renal excretion of leptospires occurs despite evidence of a humoral and cellular immune response and is reflective of the unique biological equilibrium that exists between certain animal species and specific serovars of Leptospira. Here, we demonstrate that urinary excretion of leptospires is accompanied by the presence of antigen-specific urinary immunoglobulin. In rats experimentally infected with L. interrogans serovar Copenhageni using the intraperitoneal or conjunctival route of inoculation, urinary immunoglobulin (Ig) G specific for protein antigens was detectable within 1 week. Rat urinary IgG was not bound to urinary-derived leptospires. In cattle that were naturally exposed to, and infected with, L. borgpetersenii serovar Hardjo, urinary IgA specific for protein antigens was detected. Collectively, these results demonstrate that urinary excretion of immunoglobulin specific for leptospires is a hallmark of reservoir hosts of infection.