The Fur-like regulatory protein MAP3773c modulates key metabolic pathways in Mycobacterium avium subsp. paratuberculosis under in-vitro iron starvation.

Johne's disease (JD) is a chronic enteric infection of dairy cattle worldwide. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of JD, is fastidious often requiring eight to sixteen weeks to produce colonies in culture-a major hurdle in the diagnosis and therefore in implementation of optimal JD control measures. A significant gap in knowledge is the comprehensive understanding of the metabolic networks deployed by MAP to regulate iron both in-vitro and in-vivo. The genome of MAP carries MAP3773c, a putative metal regulator, which is absent in all other mycobacteria. The role of MAP3773c in intracellular iron regulation is poorly understood. In the current study, a field isolate (K-10) and an in-frame MAP3773c deletion mutant (ΔMAP3773c) derived from K-10, were exposed to iron starvation for 5, 30, 60, and 90 min and RNA-Seq was performed. A comparison of transcriptional profiles between K-10 and ΔMAP3773c showed 425 differentially expressed genes (DEGs) at 30 min time post-iron restriction. Functional analysis of DEGs in ΔMAP3773c revealed that pantothenate (Pan) biosynthesis, polysaccharide biosynthesis and sugar metabolism genes were downregulated at 30 min post-iron starvation whereas ATP-binding cassette (ABC) type metal transporters, putative siderophore biosynthesis, PPE and PE family genes were upregulated. Pathway analysis revealed that the MAP3773c knockout has an impairment in Pan and Coenzyme A (CoA) biosynthesis pathways suggesting that the absence of those pathways likely affect overall metabolic processes and cellular functions, which have consequences on MAP survival and pathogenesis.

Apparent prevalence and selected risk factors of methicillin-resistant Staphylococcus aureus and non-aureus staphylococci and mammaliicocci in bulk tank milk of dairy herds in Indiana, Ohio, and Michigan.

The purpose of this study was to determine the apparent prevalence and risk factors of methicillin-resistant Staphylococcus aureus and non-aureus staphylococci and mammaliicocci (NASM) in bulk tank milk (BTM) obtained from 300 dairy farms that belong to a cooperative collecting milk from Indiana, Michigan, and Ohio. Dairy field personnel recorded information about selected farm level risk factors and collected and froze BTM samples (n = 300) that were sent to Michigan State University researchers. Milk samples were thawed at room temperature and pre-enriched by adding 1 to 4 mL of Mueller-Hinton broth supplemented with 6.5% NaCl and incubated at 37°C for 24 h. Subsequently, 10 µL was plated on mannitol salt agar and Mueller-Hinton agar supplemented with 2.5% NaCl containing 2 mg/L oxacillin and 20 mg/L aztreonam. Colonies that grew on the selective media were subcultured on blood agar and identified using MALDI-TOF mass spectrometry. Phenotypic methicillin resistance was tested using cefoxitin disk diffusion. Conventional PCR was used to detect mecA and mecC in phenotypically resistant isolates. Of 550 isolates that were obtained from mannitol salt agar plates and 10 isolates from Mueller-Hinton agar plates, 16 species of NASM accounted for 84% of staphylococci, while S. aureus accounted for the remaining 16%. Among S. aureus, 4 isolates from 4 farms (1.3%) demonstrated phenotypic resistance to methicillin resistance but none carried mecA or mecC genes. Among NASM, 45 isolates from 40 farms (13.3%) demonstrated phenotypic resistance to methicillin. However, only 13 NASM isolates (7 Mammaliicoccus sciuri, 2 Staphylococcus haemolyticus, 1 Mammaliicoccus fleuretti, 1 Staphylococcus epidermidis, 1 Staphylococcus saprophyticus, and 1 Staphylococcus hyicus) from 13 farms were positive for mecA, whereas all were negative for mecC. Thus, the prevalence of mecA-positive NASM in BTM was 4.3%. Based on molecular results, this study demonstrated a low prevalence of methicillin resistance NASM from BTM samples collected from farms in the Upper Midwest. Dairy farms that contained ≤200 lactating cows and had swine located on the farm had a higher prevalence of methicillin-resistant NASM than smaller farms that did not contain swine.

Global-scale GWAS associates a subset of SNPs with animal-adapted variants in M. tuberculosis complex.

BACKGROUND: While Mycobacterium tuberculosis complex (MTBC) variants are clonal, variant tuberculosis is a human-adapted pathogen, and variant bovis infects many hosts. Despite nucleotide identity between MTBC variants exceeding 99.95%, it remains unclear what drives these differences. Markers of adaptation into variants were sought by bacterial genome-wide association study of single nucleotide polymorphisms extracted from 6,362 MTBC members from varied hosts and countries. RESULTS: The search identified 120 genetic loci associated with MTBC variant classification and certain hosts. In many cases, these changes are uniformly fixed in certain variants while absent in others in this dataset, providing good discriminatory power in distinguishing variants by polymorphisms. Multiple changes were seen in genes for cholesterol and fatty acid metabolism, pathways previously proposed to be important for host adaptation, including Mce4F (part of the fundamental cholesterol intake Mce4 pathway), 4 FadD and FadE genes (playing roles in cholesterol and fatty acid utilization), and other targets like Rv3548c and PTPB, genes shown essential for growth on cholesterol by transposon studies. CONCLUSIONS: These findings provide a robust set of genetic loci associated with the split of variant bovis and variant tuberculosis, and suggest that adaptation to new hosts could involve adjustments in uptake and catabolism of cholesterol and fatty acids, like the proposed specialization to different populations in MTB lineages by alterations to host lipid composition. Future studies are required to elucidate how the associations between cholesterol profiles and pathogen utilization differences between hosts and MTBC variants, as well as the investigation of uncharacterized genes discovered in this study. This information will likely provide an understanding on the diversification of MBO away from humans and specialization towards a broad host range.

Attenuated but immunostimulatory Mycobacterium tuberculosis variant bovis strain Ravenel shows variation in T cell epitopes.

Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC) organisms, affects a range of humans and animals globally. Mycobacterial pathogenesis involves manipulation of the host immune system, partially through antigen presentation. Epitope sequences across the MTBC are evolutionarily hyperconserved, suggesting their recognition is advantageous for the bacterium. Mycobacterium tuberculosis var. bovis (MBO) strain Ravenel is an isolate known to provoke a robust immune response in cattle, but typically fails to produce lesions and persist. Unlike attenuated MBO BCG strains that lack the critical RD1 genomic region, Ravenel is classic-type MBO structurally, suggesting genetic variation is responsible for defective pathogenesis. This work explores variation in epitope sequences in MBO Ravenel by whole genome sequencing, and contrasts such variation against a fully virulent clinical isolate, MBO strain 10-7428. Validated MTBC epitopes (n = 4818) from the Immune Epitope Database were compared to their sequences in MBO Ravenel and MBO 10-7428. Ravenel yielded 3 modified T cell epitopes, in genes rpfB, argC, and rpoA. These modifications were predicted to have little effect on protein stability. In contrast, T cells epitopes in 10-7428 were all WT. Considering T cell epitope hyperconservation across MTBC variants, these altered MBO Ravenel epitopes support their potential contribution to overall strain attenuation. The affected genes may provide clues on basic pathogenesis, and if so, be feasible targets for reverse vaccinology.

Cold Cas: reevaluating the occurrence of CRISPR/Cas systems in Mycobacteriaceae.

Bacterial CRISPR/Cas systems target foreign genetic elements such as phages and regulate gene expression by some pathogens, even in the host. The system is a marker for evolutionary history and has been used for inferences in Mycobacterium tuberculosis for 30 years. However, knowledge about mycobacterial CRISPR/Cas systems remains limited. It is believed that Type III-A Cas systems are exclusive to Mycobacterium canettii and the M. tuberculosis complex (MTBC) of organisms and that very few of the >200 diverse species of non-tuberculous mycobacteria (NTM) possess any CRISPR/Cas system. This study sought unreported CRISPR/Cas loci across NTM to better understand mycobacterial evolution, particularly in species phylogenetically near the MTBC. An analysis of available mycobacterial genomes revealed that Cas systems are widespread across Mycobacteriaceae and that some species contain multiple types. The phylogeny of Cas loci shows scattered presence in many NTM, with variation even within species, suggesting gains/losses of these loci occur frequently. Cas Type III-A systems were identified in pathogenic Mycobacterium heckeshornense and the geological environmental isolate Mycobacterium SM1. In summary, mycobacterial CRISPR/Cas systems are numerous, Type III-A systems are unreliable as markers for MTBC evolution, and mycobacterial horizontal gene transfer appears to be a frequent source of genetic variation.

Global dissemination of influenza A virus is driven by wild bird migration through arctic and subarctic zones.

Influenza A viruses (IAV) circulate endemically among many wild aquatic bird populations that seasonally migrate between wintering grounds in southern latitudes to breeding ranges along the perimeter of the circumpolar arctic. Arctic and subarctic zones are hypothesized to serve as ecologic drivers of the intercontinental movement and reassortment of IAVs due to high densities of disparate populations of long distance migratory and native bird species present during breeding seasons. Iceland is a staging ground that connects the East Atlantic and North Atlantic American flyways, providing a unique study system for characterizing viral flow between eastern and western hemispheres. Using Bayesian phylodynamic analyses, we sought to evaluate the viral connectivity of Iceland to proximal regions and how inter-species transmission and reassortment dynamics in this region influence the geographic spread of low and highly pathogenic IAVs. Findings demonstrate that IAV movement in the arctic and subarctic reflects wild bird migration around the perimeter of the circumpolar north, favouring short-distance flights between proximal regions rather than long distance flights over the polar interior. Iceland connects virus movement between mainland Europe and North America, consistent with the westward migration of wild birds from mainland Europe to Northeastern Canada and Greenland. Though virus diffusion rates were similar among avian taxonomic groups in Iceland, gulls play an outsized role as sinks of IAVs from other avian hosts prior to onward migration. These data identify patterns of virus movement in northern latitudes and inform future surveillance strategies related to seasonal and emergent IAVs with potential public health concern.

Mycobacterium bovis Strain Ravenel Is Attenuated in Cattle.

Mycobacterium tuberculosis variant bovis (MBO) has one of the widest known mammalian host ranges, including humans. Despite the characterization of this pathogen in the 1800s and whole genome sequencing of a UK strain (AF2122) nearly two decades ago, the basis of its host specificity and pathogenicity remains poorly understood. Recent experimental calf infection studies show that MBO strain Ravenel (MBO Ravenel) is attenuated in the cattle host compared to other pathogenic strains of MBO. In the present study, experimental infections were performed to define attenuation. Whole genome sequencing was completed to identify regions of differences (RD) and single nucleotide polymorphisms (SNPs) to explain the observed attenuation. Comparative genomic analysis of MBO Ravenel against three pathogenic strains of MBO (strains AF2122-97, 10-7428, and 95-1315) was performed. Experimental infection studies on five calves each, with either MBO Ravenel or 95-1315, revealed no visible lesions in all five animals in the Ravenel group despite robust IFN-γ responses. Out of 486 polymorphisms in the present analysis, 173 were unique to MBO Ravenel among the strains compared. A high-confidence subset of nine unique SNPs were missense mutations in genes with annotated functions impacting two major MBO survival and virulence pathways: (1) Cell wall synthesis & transport [espH (A103T), mmpL8 (V888I), aftB (H484Y), eccC5 (T507M), rpfB (E263G)], and (2) Lipid metabolism & respiration [mycP1(T125I), pks5 (G455S), fadD29 (N231S), fadE29 (V360G)]. These substitutions likely contribute to the observed attenuation. Results from experimental calf infections and the functional attributions of polymorphic loci on the genome of MBO Ravenel provide new insights into the strain's genotype-disease phenotype associations.

Genome Sequences of Two Mycobacterium tuberculosis Isolates from Asian Elephants in Nepal.

This report describes the genome sequences of two Mycobacterium tuberculosis isolates, S1 and S3, recovered from Asian elephants in Nepal. These genome sequences will enhance our understanding of the genomic epidemiology of Mycobacterium tuberculosis in Asian elephants.

Genome Sequences of Mycobacterium Strains Recovered from Captive Elephants with Tuberculosis.

Members of the Mycobacterium tuberculosis complex cause tuberculosis, infamous for enormous impacts on human health. As zoonoses, they also threaten endangered species like African/Asian elephants. We report the whole-genome sequences of Mycobacterium tuberculosis bv. tuberculosis and Mycobacterium tuberculosis bv. bovis from two zoo elephants in the United States.

Apoptosis in the late replication phase of Bovine alphaherpesvirus 1 in experimentally infected calves.

Bovine alphaherpesvirus 1 (BoHV-1) is a pathogen causing respiratory and reproductive clinical signs in cattle. Infected animals may develop rhinotracheitis, vulvovaginitis, balanoposthitis, and abortion. Viral latency is generally established in neuronal ganglia simultaneously to a decrease in both genes or genome expression and viral replication. Under stressful conditions, infection is reactivated leading to viral replication and the manifestation of clinical signs. In this study, we evaluated both viral reactivation and apoptosis in trigeminal ganglia cells as BoHV-1 progressed from the latent to the acute phase of infection after dexamethasone administration in experimentally infected calves. To test ganglia cell death as a consequence of BoHV-1 infection, we stained the BoHV-1 samples with TUNEL after the viral shedding by the calves. RT-qPCR of apoptotic genes was also performed, showing the upregulation of the caspase 8 gene in the trigeminal ganglia from cattle experimentally infected with BoHV-1. These results showed the occurrence of apoptosis in ganglion cells of calves infected by BoHV-1.

Genome Sequences of Mycobacterium tuberculosis Biovar bovis Strains Ravenel and 10-7428.

We report the draft genomes of two Mycobacterium tuberculosis biovar bovis strains. Strain Ravenel was isolated in the 1900s and has been shown to be attenuated in cattle. Strain 10-7428 is considered highly pathogenic in cattle and was isolated from a bovine tuberculosis outbreak.

Prevalence, antibiotic resistance, virulence and genetic diversity of Staphylococcus aureus isolated from bulk tank milk samples of U.S. dairy herds.

BACKGROUND: Colonization of dairy cows by Staphylococcus aureus (S. aureus), especially those which are multi-drug resistant and toxin producing, is a concern for animal health and well-being as well as public health. The objective of this study was to investigate the prevalence, antibiotic resistance, gene content and virulence determinants of S. aureus in bulk tank milk samples (BTM) from U.S. dairy herds. RESULTS: BTM samples were collected, once in winter and once in summer, from 189 U.S. dairy herds. Of 365 BTM samples cultured, the sample and herd prevalence of S. aureus in BTM was 46.6% (170 of 365 samples) and 62.4% (118 of 189 herds), respectively. Among a subset of 138 S. aureus isolates that were stored for further analysis, 124 were genome sequenced after being confirmed as S. aureus using phenotypic tests. The most commonly identified antimicrobial resistance-associated gene was norA (99.2%) and mecA gene responsible for methicillin resistance (MRSA) was identified in one isolate (0.8%). The most frequently detected putative virulence genes were aur (100%), hlgB (100%), hlgA, hlgC, hlb (99.2%), lukE (95.9%) and lukD (94.3%). In the 53 staphylococcal enterotoxin positive isolates, sen (37.9%), sem (35.5%), sei (35.5%) and seg (33.1%) were the most frequently detected enterotoxin genes. Among the 14 sequence types (ST) and 18 spa types identified, the most common was ST2187 (20.9%) and t529 (28.2%), respectively. The most predominant clone was CC97 (47.6%) followed by CC unknown (36.3%). The single MRSA isolate belonged to ST72-CC8, spa type t126 and was negative for the tst gene but harbored all the other virulence genes investigated. CONCLUSION: Our findings indicated a high prevalence of S. aureus in BTM of U.S. dairy herds, with isolates showing little evidence of resistance to antibiotics commonly used to treat mastitis. However, isolates often carried genes for the various enterotoxins. This study identified predominant genetic clones. Despite lower prevalence, the presence of MRSA and multi-drug resistant strains in BTM poses a significant risk to animal and public health if their number were to increase in dairy environment. Therefore, it is necessary to continuously monitor the use of antibiotics in dairy cows.

Evaluation of Real-Time Quaking-Induced Conversion, ELISA, and Immunohistochemistry for Chronic Wasting Disease Diagnosis.

Chronic wasting disease (CWD) is a transmissible prion disorder, primarily affecting free-ranging and captive cervids in North America (United States and Canada), South Korea, and Europe (Finland, Norway, and Sweden). Current diagnostic methods used in the United States for detection of CWD in hunter harvested deer involve demonstration of the causal misfolded prion protein (PrPCWD) in the obex or retropharyngeal lymph nodes (RLNs) using an antigen detection ELISA as a screening tool, followed by a confirmation by the gold standard method, immunohistochemistry (IHC). Real-time quaking-induced conversion (RT-QuIC) assay is a newer approach that amplifies misfolded CWD prions in vitro and has facilitated CWD prion detection in a variety of tissues, body fluids, and excreta. The current study was undertaken to compare ELISA, IHC, and RT-QuIC on RLNs (n = 1,300 animals) from white-tailed deer (WTD) in Michigan. In addition, prescapular, prefemoral and popliteal lymph nodes collected from a small subset (n = 7) of animals were tested. Lastly, the location of the positive samples within Michigan was documented and the percentage of CWD positive RLNs was calculated by sex and age. ELISA and RT-QuIC detected PrPCWD in 184 and 178 out of 1,300 RLNs, respectively. Of the 184 ELISA positive samples, 176 were also IHC positive for CWD. There were seven discordant results when comparing IHC and ELISA. RT-QuIC revealed that six of the seven samples matched the IHC outcomes. One RLN was negative by IHC, but positive by ELISA and RT-QuIC. RT-QuIC, IHC, and ELISA also detected PrPCWD in prescapular, prefemoral and popliteal lymph nodes. CWD infection heterogeneities were observed in different age and sex groups, with young males having higher CWD prevalence. All, except one, CWD positive RLNs analyzed were from ten Counties geographically located in the West Michigan region of the Lower Peninsula. Taken together, we show evidence that the RT-QuIC assay is comparable to ELISA and IHC and could be helpful for routine CWD detection in surveillance programs. RT-QuIC also demonstrated that CWD prions are distributed across lymph nodes in a variety of anatomic locations. A multi-laboratory validation on blinded sample panels is underway and is likely to help to provide insight into the variability (lab-to-lab), analytical sensitivity, and specificity of gold standard diagnostics vs. RT-QuIC assay.

Genetic variability of influenza A virus in pigs at weaning in Midwestern United States swine farms.

Suckling piglets play an important role at maintaining influenza A virus (IAV) infections in breeding herds and disseminating them to other farms at weaning. However, the role they play at weaning to support and promote genetic variability of IAV is not fully understood. The objective here was to evaluate the genetic diversity of IAV in pigs at weaning in farms located in the Midwestern USA. Nasal swabs (n = 9,090) collected from piglets in breed-to-wean farms (n = 52) over a six-month period across seasons were evaluated for the presence of IAV. Nasal swabs (n = 391) from 23 IAV-positive farms were whole-genome sequenced. Multiple lineages of HA (n = 7) and NA (n = 3) were identified in 96% (22/23) and 61% (237/391) of the investigated farms and individual piglets, respectively. Co-circulation of multiple types of functional HA and NA was identified in most (83%) farms. Whole IAV genomes were completed for 126 individual piglet samples and 25 distinct and 23 mixed genotypes were identified, highlighting significant genetic variability of IAV in piglets. Co-circulation of IAV in the farms and co-infection of individual piglets at weaning was observed at multiple time points over the investigation period and appears to be common in the investigated farms. Statistically significant genetic variability was estimated within and between farms by AMOVA, and varying levels of diversity between farms were detected using the Shannon-Weiner Index. Results reported here demonstrate previously unreported levels of molecular complexity and genetic variability among IAV at the farm and piglet levels at weaning. Movement of such piglets infected at weaning may result in emergence of new strains and maintenance of endemic IAV infection in the US swine herds. Results presented here highlight the need for developing and implementing novel, effective strategies to prevent or control the introduction and transmission of IAV within and between farms in the country.

Tuberculosis in elephants: Origins and evidence of interspecies transmission.

Tuberculosis (TB) is a devastating disease in elephants caused by either Mycobacterium tuberculosis or M. bovis. It is an ancient disease, and TB in elephants was first reported over two millennia ago in Sri Lanka. Outbreaks of TB worldwide, in captive and free-ranging elephant populations, have been recorded. Interspecies transmission of TB among elephants and humans has been confirmed in several geographic localities using spoligotyping, MIRU-VNTR analysis, and/or comparative genomics. Active surveillance of TB in wild and captive elephants and their handlers is necessary to prevent TB transmission at the elephant-human interface and to aid in the conservation of Asian and African elephants. In this review, we present an overview of diagnosis, reports of TB outbreaks in the past 25 years, TB in wild elephants, its transmission, and possible prevention and control strategies that can be applied at the elephant-human interface.

Elucidating the Regulon of a Fur-like Protein in Mycobacterium avium subsp. paratuberculosis (MAP).

Intracellular iron concentration is tightly regulated to maintain cell viability. Iron plays important roles in electron transport, nucleic acid synthesis, and oxidative stress. A Mycobacterium avium subsp. paratuberculosis (MAP)-specific genomic island carries a putative metal transport operon that includes MAP3773c, which encodes a Fur-like protein. Although well characterized as a global regulator of iron homeostasis in multiple bacteria, the function of Fur (ferric uptake regulator) in MAP is unknown as this organism also carries IdeR (iron dependent regulator), a native iron regulatory protein specific to mycobacteria. Computational analysis using PRODORIC identified 23 different pathways involved in respiration, metabolism, and virulence that were likely regulated by MAP3773c. Thus, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was performed to confirm the putative regulon of MAP3773c (Fur-like protein) in MAP. ChIP-Seq revealed enriched binding to 58 regions by Fur under iron-replete and -deplete conditions, located mostly within open reading frames (ORFs). Three ChIP peaks were identified in genes that are directly related to iron regulation: MAP3638c (hemophore-like protein), MAP3736c (Fur box), and MAP3776c (ABC transporter). Fur box consensus sequence was identified, and binding specificity and dependence on Mn2+ availability was confirmed by a chemiluminescent electrophoresis mobility shift assay (EMSA). The results confirmed that MAP3773c is a Fur ortholog that recognizes a 19 bp DNA sequence motif (Fur box) and it is involved in metal homeostasis. This work provides a regulatory network of MAP Fur binding sites during iron-replete and -deplete conditions, highlighting unique properties of Fur regulon in MAP.

Phylogeography and Antigenic Diversity of Low-Pathogenic Avian Influenza H13 and H16 Viruses.

Low-pathogenic avian influenza viruses (LPAIVs) are genetically highly variable and have diversified into multiple evolutionary lineages that are primarily associated with wild-bird reservoirs. Antigenic variation has been described for mammalian influenza viruses and for highly pathogenic avian influenza viruses that circulate in poultry, but much less is known about antigenic variation of LPAIVs. In this study, we focused on H13 and H16 LPAIVs that circulate globally in gulls. We investigated the evolutionary history and intercontinental gene flow based on the hemagglutinin (HA) gene and used representative viruses from genetically distinct lineages to determine their antigenic properties by hemagglutination inhibition assays. For H13, at least three distinct genetic clades were evident, while for H16, at least two distinct genetic clades were evident. Twenty and ten events of intercontinental gene flow were identified for H13 and H16 viruses, respectively. At least two antigenic variants of H13 and at least one antigenic variant of H16 were identified. Amino acid positions in the HA protein that may be involved in the antigenic variation were inferred, and some of the positions were located near the receptor binding site of the HA protein, as they are in the HA protein of mammalian influenza A viruses. These findings suggest independent circulation of H13 and H16 subtypes in gull populations, as antigenic patterns do not overlap, and they contribute to the understanding of the genetic and antigenic variation of LPAIVs naturally circulating in wild birds.IMPORTANCE Wild birds play a major role in the epidemiology of low-pathogenic avian influenza viruses (LPAIVs), which are occasionally transmitted-directly or indirectly-from them to other species, including domestic animals, wild mammals, and humans, where they can cause subclinical to fatal disease. Despite a multitude of genetic studies, the antigenic variation of LPAIVs in wild birds is poorly understood. Here, we investigated the evolutionary history, intercontinental gene flow, and antigenic variation among H13 and H16 LPAIVs. The circulation of subtypes H13 and H16 seems to be maintained by a narrower host range, in particular gulls, than the majority of LPAIV subtypes and may therefore serve as a model for evolution and epidemiology of H1 to H12 LPAIVs in wild birds. The findings suggest that H13 and H16 LPAIVs circulate independently of each other and emphasize the need to investigate within-clade antigenic variation of LPAIVs in wild birds.

Genetic diversity of influenza A viruses circulating in pigs between winter and summer in a Minnesota live animal market.

There has been little surveillance of influenza A viruses (IAVs) circulating in swine at live animal markets, particularly in the United States. To address this gap, we conducted active surveillance of IAVs in pigs, the air, and the environment during a summer and winter season in a live animal market in St. Paul, Minnesota, that had been epidemiologically associated with swine-origin influenza cases in humans previously. High rates of IAV were detected by PCR in swine lungs and oral fluids during both summer and winter seasons. Rates of IAV detection by PCR in the air were similar during summer and winter, although rates of successful virus isolation in the air were lower during summer than in winter (26% and 67%, respectively). H3N2 was the most prevalent subtype in both seasons, followed by H1N2. Genetically diverse viruses with multiple gene constellations were isolated from both winter and summer, with a total of 19 distinct genotypes identified. Comparative phylogenetic analysis of all eight segments of 40 virus isolates from summer and 122 isolates from winter revealed that the summer and winter isolates were genetically distinct, indicating IAVs are not maintained in the market, but rather are re-introduced, likely from commercial swine. These findings highlight the extent of IAV genetic diversity circulating in swine in live animal markets, even during summer months, and the ongoing risk to humans.

MAC-INMV-SSR: a web application dedicated to genotyping members of Mycobacterium avium complex (MAC) including Mycobacterium avium subsp. paratuberculosis strains.

Genotyping of Mycobacterium avium subsp. paratuberculosis (Map) is an indispensable tool for surveillance of this significant veterinary pathogen. For Map, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRUs) and other variable number variable-number tandem repeats (VNTRs) was established using 8 markers. In the recent past this standard, portable, reproducible and discriminatory typing method has been frequently applied alone or in combinations with multi-locus short-sequence-repeat (MLSSR) sequencing. With the widespread use of these genotyping methods, standardization between laboratories needs to be managed, and knowledge of existing profiles and newly defined genotypes should be indexed and shared. To meet this need, a web application called "MAC-INMV-SSR database" was developed. This freely accessible service allows users to compare MLVA and MLSSR subtype data of their strains with those of existing reference strains analyzed with the same genotyping methods.

Molecular epidemiology of Mycoplasma hyorhinis porcine field isolates in the United States.

Mycoplasma hyorhinis is one of the causative agents of polyserositis and arthritis in post-weaning pigs. Here we describe the development of a multi-locus sequence typing (MLST) protocol for the characterization of M. hyorhinis field isolates. A total of 104 field isolates from different geographical locations, swine production systems, and clinical backgrounds, were analyzed. Twenty-seven genes, including housekeeping and those encoding surface proteins, were evaluated to index diversity. Genes encoding surface proteins were included to increase the discriminatory power of the MLST. Four target gene fragments were selected to be included in the final MLST-s (surface) protocol: pdhB, p95, mtlD and ung. Within each locus the nucleotide variation ranged from 1.4% to 20%. The 104 field isolates were classified into 39 distinct sequence types (STs). Multiple STs were found within the same production system and within the same pig. The majority of STs grouped strains from the same production system; however, cases existed where multiple systems shared a ST, indicating potential relationships between pig flows. The majority of the nucleotide changes observed in these genes generated synonymous changes, while non-synonymous changes were exclusively in the mtlD gene fragment, suggesting that this protein is undergoing selection. Molecular typing of M. hyorhinis will primarily aid swine practitioners with pig flow management and identifying sources of infection during outbreaks.