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CD8 + T cell tissue resident memory (T RM ) cells are especially suited to control pathogen spread at mucosal sites. However, their maintenance in lung is short-lived. TCR-dependent NFkB signaling is crucial for T cell memory but how and when NFkB signaling modulates tissue resident and circulating T cell memory during the immune response is unknown. Here, we find that enhancing NFkB signaling in T cells once memory to influenza is established, increases pro-survival Bcl-2 and CD122 levels thus boosting lung CD8 + T RM maintenance. By contrast, enhancing NFkB signals during the contraction phase of the response leads to a defect in CD8 + T RM differentiation without impairing recirculating memory subsets. Specifically, inducible activation of NFkB via constitutive active IKK2 or TNF interferes with TGFβ signaling, resulting in defects of lung CD8 + T RM imprinting molecules CD69, CD103, Runx3 and Eomes. Conversely, inhibiting NFkB signals not only recovers but improves the transcriptional signature and generation of lung CD8 + T RM . Thus, NFkB signaling is a critical regulator of tissue resident memory, whose levels can be tuned at specific times during infection to boost lung CD8 + T RM ., (© 2023. The Author(s).)

Several Francisella spp., including Francisella noatunensis, are regarded as important emerging pathogens of wild and farmed fish. However, very few studies have investigated the virulence factors that allow these bacterial species to be pathogenic in fish. The Francisella pathogenicity island (FPI) is a well-described, gene-dense region encoding major virulence factors for the genus Francisella. pdpA is a member of the pathogenicity-determining protein genes carried by the FPI that are implicated in the ability of the mammalian pathogen Francisella tularensis to escape and replicate in infected host cells. Using a sacB suicide approach, we generated pdpA knockouts to address the role of PdpA as a virulence factor for F. noatunensis . Because polarity can be an issue in gene-dense regions, we generated two different marker-based mutants in opposing polarity (the F. noatunensis subsp. orientalis Δ pdpA1 and Δ pdpA2 strains). Both mutants were attenuated ( P < 0.0001) in zebrafish challenges and displayed impaired intracellular replication ( P < 0.05) and cytotoxicity ( P < 0.05), all of which could be restored to wild-type (WT) levels by complementation for the Δ pdpA1 mutant. Importantly, differences were found for bacterial burden and induction of acute-phase and proinflammatory genes for the F. noatunensis subsp. orientalis Δ pdpA1 and Δ pdpA2 mutants compared to the WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with the F. noatunensis subsp. orientalis Δ pdpA1 mutant led to protection ( P < 0.012) against an acute 40% lethal dose (LD 40 ) challenge with WT F. noatunensis in the zebrafish model of infection. Taken together, the results from this study further demonstrate physiological similarities within the genus Francisella relative to their phylogenetic relationships and the utility of zebrafish for addressing virulence factors for the genus.

Staphylococcus agnetis has been previously associated with subclinical or clinically mild cases of mastitis in dairy cattle and is one of several staphylococcal species that have been isolated from the bones and blood of lame broilers. We reported that S. agnetis could be obtained frequently from bacterial chondronecrosis with osteomyelitis (BCO) lesions of lame broilers (A. Al-Rubaye et al., PLoS One 10:e0143336, 2015 [https://doi.org/10.1371/journal.pone.0143336]). A particular isolate, S. agnetis 908, can induce lameness in over 50% of exposed chickens, exceeding normal BCO incidences in broiler operations. We reported the assembly and annotation of the genome of isolate 908. To better understand the relationship between dairy cattle and broiler isolates, we assembled 11 additional genomes for S. agnetis isolates, an additional chicken BCO strain, and ten isolates from cattle milk, mammary gland secretions, or udder skin from the collection at the University of Missouri. To trace phylogenetic relationships, we constructed phylogenetic trees based on multilocus sequence typing and genome-to-genome distance comparisons. Chicken isolate 908 clustered with two of the cattle isolates, along with three isolates from chickens in Denmark and an isolate of S. agnetis we isolated from a BCO lesion on a commercial broiler farm in Arkansas. We used a number of BLAST tools to compare the chicken isolates to those from cattle and identified 98 coding sequences distinguishing isolate 908 from the cattle isolates. None of the identified genes explain the differences in host or tissue tropism. These analyses are critical to understanding how staphylococci colonize and infect different hosts and potentially how they can transition to alternative niches (bone versus dermis). IMPORTANCE Staphylococcus agnetis has been recently recognized as associated with disease in dairy cattle and meat-type chickens. The infections appear to be limited in cattle and systemic in broilers. This report details the molecular relationships between cattle and chicken isolates in order to understand how this recently recognized species infects different hosts with different disease manifestations. The data show that the chicken and cattle isolates are very closely related, but the chicken isolates all cluster together, suggesting a single jump from cattle to chickens., (Copyright © 2020 American Society for Microbiology.)

The objectives of this study were (1) to report the rates of new intramammary infection (IMI) and spontaneous IMI cure over the dry period in 3 dairy goat herds; (2) to evaluate the factors predicting infection dynamics over the dry period; and (3) to define milk quality parameter thresholds that predict infection dynamics over the dry period. Two consecutive udder-half milk samples were collected 10 to 14 d apart before dry-off from 288 goats in 3 herds, and 2 consecutive udder-half samples were collected 7 to 14 d apart in the following lactation, with the first sample being collected ≤10 d in milk, from 200 of the same goats. In 2 of the herds, udder-half milk samples were also collected at the same time points (n = 312 halves; 157 goats) for measurement of milk quality parameters. Standard aerobic culture of milk samples was performed for the detection of mastitis pathogens. To rule out the presence of Mycoplasma spp. IMI, milk samples were also cultured on modified Hayflick medium. Non-Mycoplasma isolates were speciated using MALDI-TOF mass spectrometry. Staphylococcal isolates, when not identified by MALDI-TOF, were speciated using partial gene sequence analysis of rpoB or tuf. When >1 sample from an udder half yielded the same species, available isolates from the first and last positive samples for that species were strain-typed using pulsed-field gel electrophoresis. Incidence of new IMI and cure rate were computed. Generalized linear mixed regression models were built to evaluate the associations between new IMI and pre-dry somatic cell score (SCS), between IMI persistence and half-level SCS, and between IMI persistence and pre-dry IMI species. Thresholds for pre-dry SCS and lactose concentration were computed to predict IMI persistence. Overall, 12.6% (48/380) of halves had a persistent IMI. Cumulative incidence of new IMI over the dry period was 13.2%, and cure rate was 52.0%. Pre-dry SCS was not associated with odds of new IMI or IMI persistence. Pre-dry IMI species was not associated with odds of persistence. Lactose concentration was not associated with odds of persistence. Regardless of culture data, the optimal pre-dry SCS threshold to detect IMI that would persist into the next lactation was 8.7, with sensitivity and specificity of 50 and 73.8%, respectively. Further studies on the effect of control measures on species-specific incidence and cure rates during the dry period are warranted., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mycoplasmosis is a well-known cause of morbidity and mortality in small ruminants. Previously recognized outbreaks have involved arthritis, and pneumonia or pleuropneumonia. Modern bacteriology procedures rely less on isolation techniques that require special media for mollicutes given that these species are notoriously difficult to isolate, and rely more on PCR tests. We report an outbreak of arthritis, pleuropneumonia, and mild meningitis affecting dairy goat kids, spanning a period of 3 y, which had unusual epidemiologic characteristics related to husbandry practices. Lesions were characterized by polyarthritis of the appendicular joints, with copious joint fluid and extension of arthritic exudate beyond the joint itself. The cause remained unknown until serendipitous isolation of a mycoplasma on blood agar. Mycoplasmosis was not detected from synovial samples by a general mycoplasma PCR, despite multiple attempts. Isolated colonies were also negative by this general PCR assay. The isolate was identified as Mycoplasma mycoides subspecies capri, using universal 16S primers and amplicon sequencing. Testing of additional isolates from other diseased goats in the herd confirmed that this was the cause of illness. A failure to recognize the distinct nature of organisms of the M. mycoides group of mycoplasmas meant that a PCR test that cannot detect this group of organisms was utilized at first, and the etiology of the illness was overlooked for a period of time. Veterinary pathologists and microbiologists must be aware of the limitations of some PCR assays when confronted with joint disease and pleuropneumonia in small ruminants.

The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

There is a worldwide problem of disease caused by Mycoplasma (M.) bovis in cattle; it has a significant detrimental economic and animal welfare impact on cattle rearing. Infection can manifest as a plethora of clinical signs including mastitis, pneumonia, arthritis, keratoconjunctivitis, otitis media and genital disorders that may result in infertility and abortion. Current diagnosis and control information are reviewed and analysed to identify gaps in knowledge of the causative organism in respect of the disease pathology, diagnosis and control methods. The main considerations are as follows: no vaccines are commercially available; antimicrobial resistance is increasing; diagnostic and antimicrobial sensitivity testing needs to be improved; and a pen-side test would facilitate more rapid diagnosis and implementation of treatment with antimicrobials. More data on host susceptibility, stress factors, immune response and infectious dose levels are required. The impact of asymptomatic carriers, M. bovis survival in the environment and the role of wildlife in transmitting the disease also needs investigation. To facilitate development of vaccines, further analysis of more M. bovis genomes, its pathogenic mechanisms, including variable surface proteins, is required, along with reproducible disease models., (© 2018 Blackwell Verlag GmbH.)

The purpose of this study was to describe the prevalence and distribution of staphylococcal species on the teat and inguinal skin of dairy heifers across the various stages of the heifer life cycle. The cross-sectional study included 106 Holstein heifers with an age range of 0 d to 27 mo that were selected from 11 different groups, based on housing type and age, on a single dairy operation. A composite swabbing sample including all 4 teats and a second composite sample including both inguinal regions of each heifer were collected using gas-sterilized electrostatic dusters (Swiffers; Procter and Gamble, Cincinnati, OH). Swabbing samples were mixed with 10 mL of sterile saline, agitated, and cultured on mannitol salt agar plates. At 24 h, plates were read and up to 10 staphylococcal colonies were saved for further analysis. Staphylococcal isolates were speciated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or PCR amplification and partial sequencing of rpoB or tuf. The prevalence of staphylococci was compared between the inguinal and teat regions using the chi-squared or Fisher's exact test, as applicable. Logistic regression models were used to investigate the relationship between a heifer's age (treated as a quantitative continuous variable) and the probability of isolating a given staphylococcal species from a given body site (inguinal region or teats). Overall, the most common species identified were Staphylococcus haemolyticus followed by Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus devriesei, and Staphylococcus sciuri. Staphylococcus aureus was more prevalent on the teat than in the inguinal region, whereas Staphylococcus arlettae was more prevalent in the inguinal region than on the teat. All other staphylococcal species were as likely to be found on the teat skin as the inguinal region skin. Isolation from the inguinal and teat skin was associated with age for Staphylococcus agnetis, S. chromogenes, S. devriesei, Staphylococcus equorum, S. haemolyticus, Staphylococcus lentus, S. sciuri, Staphylococcus vitulinus, and S. xylosus. The probability of finding S. chromogenes and S. agnetis on the teat and inguinal region increased with age, whereas the probability of S. devriesei and S. haemolyticus decreased with age. This study provides further insight into the ecology of staphylococcal species involved in heifer mastitis., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Pythiosis is a potentially fatal but non-contagious disease affecting humans and animals living in tropical and subtropical climates, but is also reasonably widespread in temperate climates, throughout the world. The most commonly reported affected animal species with pythiosis are equine and canine, with fewer cases in bovine and feline. Extracutaneous infections caused by Pythium insidiosum have been rarely described in the cat., Case Presentation: Sublingual pythiosis was diagnosed in a 2-year-old, male, Domestic Shorthair cat. The cat had a multilobulated, sublingual mass present for 3 months. Histopathological examination revealed severe multifocal coalescing eosinophilic granulomatous inflammation. Centers of the inflammation contained hyphae that were 3-7 μm-wide, non-parallel, uncommonly septate and rarely branching. The fungal-like organism was identified as P. insidiosum by polymerase chain reaction and subsequent amplicon sequencing., Conclusions: Only a few feline pythiosis cases have been reported and, when encountered, it usually causes granulomatous diseases of the skin or gastrointestinal tract. This case presents an unusual manifestation of feline pythiosis, representing the first involving the oral cavity in cats or dogs.

An infection with Histoplasma capsulatum was diagnosed in a farmed reindeer in Missouri, an endemic area for histoplasmosis, localized in the intestine. The intrahistiocytic organisms were identified in tissue sections using histologic methods and confirmed by immunohistochemistry. This is the first report of histoplasmosis in a reindeer or in any deer species.

Staphylococcus hyicus and Staphylococcus agnetis are two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positive S. hyicus based on phenotypic species identification methods and to develop a PCR-based method for differentiating S. hyicus , S. agnetis , and Staphylococcus aureus Isolates ( n = 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiate S. hyicus , S. agnetis , and S. aureus was developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be either S. agnetis ( n = 43) or S. hyicus ( n = 1). Overall, 88% (37/42) of coagulase-positive S. agnetis isolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positive S. agnetis ranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest that S. agnetis is likely more prevalent on dairy farms than S. hyicus Also, some S. agnetis isolates in this study appeared to be contagious and associated with persistent infections., (Copyright © 2017 American Society for Microbiology.)

Presented here is the first completely assembled genome sequence of Kocuria palustris, an actinobacterial species with broad ecological distribution. The single, circular chromosome of K. palustris MU14/1 comprises 2,854,447 bp, has a G+C content of 70.5%, and contains a deduced gene set of 2,521 coding sequences., (Copyright © 2015 Calcutt and Foecking.)

The draft genome sequence of a novel Mycoplasma strain, designated Mycoplasma sp. HU2014, has been determined. The genome comprises 1,084,927 nucleotides and was obtained from a mycoplasma-infected culture of chicken DT40 cells. Phylogenetic analysis places this taxon in a group comprising the closely related species Mycoplasma yeatsii and Mycoplasma cottewii., (Copyright © 2015 Calcutt et al.)

Mycoplasma capricolum subspecies capricolum is both a pathogen of small ruminants and a model recipient organism for gene transplantation and synthetic biology. With the availability of the complete genome of the type strain California kid (released in 2005), a draft genome of strain GM508D was determined to investigate genomic variation in this subspecies. Differences in mobile genetic element location and complement, catabolic pathway genes, contingency loci, surface antigen genes and type II restriction-modification systems highlight the plasticity and diversity within this taxon., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Presented here is the complete and annotated genome sequence of Mycoplasma hominis Sprott (ATCC 33131). The chromosome comprises 695,214 bp, which is approximately 30 kb larger than the syntenic genome of M. hominis PG21(T). Tetracycline resistance of strain Sprott is most probably conferred by the tetM determinant, harbored on a mosaic transposon-like structure., (Copyright © 2015 Calcutt and Foecking.)

Mycoplasma yeatsii is a goat mycoplasma species that, although an obligate parasite, accommodates this lifestyle as an inapparent commensalist. High-frequency transformation has also been reported for this species. The complete 895,051-bp genome sequence of strain GM274B has been determined, enabling an analysis of the features of this potential cloning host., (Copyright © 2015 Calcutt et al.)

Mycoplasma flocculare is a commensal or low-virulence pathogen of swine. The complete 778,866-bp genome sequence of M. flocculare strain Ms42(T) has been determined, enabling further comparison to genomes of the closely related pathogen Mycoplasma hyopneumoniae. The absence of the p97 and glpD genes may contribute to the attenuated virulence of M. flocculare., (Copyright © 2015 Calcutt et al.)

The complete genome sequence of Mycoplasma hominis LBD-4 has been determined and the gene content ascribed. The 715,165-bp chromosome contains 620 genes, including 14 carried by a strain-variable prophage genome related to Mycoplasma fermentans MFV-1 and Mycoplasma arthritidis MAV-1. Comparative analysis with the genome of M. hominis PG21(T) reveals distinctive arrangements of repeat-containing surface proteins., (Copyright © 2015 Calcutt and Foecking.)

Staphylococcus hyicus is the primary etiological agent of exudative epidermitis in swine. Analysis of the complete genome sequence of the type strain revealed a locus encoding a type VII secretion system and a large chromosomal island harboring the genes encoding exfoliative toxin ExhA and an EDIN toxin homolog., (Copyright © 2015 Calcutt et al.)

Microbiological and histological analysis of a sample from a swollen testicle of a 2-year-old Border Collie dog revealed a mixed infection of the fungus Blastomyces dermatitidis and the Gram-negative bacterium Aureimonas altamirensis. When subjected to an automated microbial identification system, the latter isolate was provisionally identified as Psychrobacter phenylpyruvicus, but the organism shared several biochemical features with Brucella canis and exhibited agglutination, albeit weakly, with anti-B. canis antiserum. Unequivocal identification of the organism was only achieved by 16S ribosomal RNA gene sequencing, ultimately establishing the identity as A. altamirensis. Since its first description in 2006, this organism has been isolated infrequently from human clinical samples, but, to the authors' knowledge, has not been reported from a veterinary clinical sample. While of unknown clinical significance with respect to the pathology observed for the polymicrobial infection described herein, it highlights the critical importance to unambiguously identify the microbe for diagnostic, epidemiological, infection control, and public health purposes., (© 2014 The Author(s).)

The assembled and annotated genome of Actinobacillus suis ATCC 33415(T) is reported here. The 2,501,598-bp genome encodes 2,246 open reading frames (ORFs) with strain variable incursion of an integrative conjugative element into a tRNA locus. Comparative analysis of the deduced gene set should inform our understanding of pathogenesis, genomic plasticity, and serotype variation., (Copyright © 2014 Calcutt et al.)

Presented here is a draft genome sequence for Staphylococcus agnetis CBMRN 20813338, isolated from a lactating dairy cow with subclinical mastitis. The genome is approximately 2,416 kb and has 35.79% G+C content. Analysis of the deduced open reading frame (ORF) set identified candidate virulence attributes in addition to potential molecular targets for species identification., (Copyright © 2014 Calcutt et al.)

Coagulase-negative staphylococcal species are a common cause of subclinical bovine mastitis, with Staphylococcus chromogenes being one of the most frequently identified species in these cases. The draft genome sequence of an S. chromogenes isolate (MU 970) recovered from the milk of a cow with a chronic intramammary infection is reported here., (Copyright © 2014 Fry et al.)

Mycoplasma californicum is one of several mycoplasmal species associated with bovine mastitis. The complete genome sequence of 793,841 bp has been determined and annotated for the M. californicum ST-6 type strain, providing a resource for the identification of surface antigens and putative pathoadaptive features., (Copyright © 2014 Calcutt et al.)

Moraxella bovoculi is a recently identified species, recovered from the bovine eye, which is under investigation as an etiological agent of infectious bovine keratoconjunctivitis. A draft genome sequence of the Moraxella bovoculi type strain 237(T) has been determined to identify features that may be important during host colonization., (Copyright © 2014 Calcutt et al.)

Fusobacterium necrophorum is a Gram-negative anaerobic bacterium that causes foot rot and liver abscesses in cattle. F. necrophorum subsp. necrophorum and the less virulent organism F. necrophorum subsp. funduliforme are recognized. We present here a draft genome sequence of the bovine liver abscess isolate F. necrophorum subsp. funduliforme strain B35, which affords a genomic perspective of virulence and bovine adaptation.

Bovine ocular infections compromise animal health and result in significant economic losses. Mycoplasma bovoculi is an etiological agent of conjunctivitis. Presented here is the 760,240-bp complete genome sequence of the M. bovoculi type strain M165/69(T). An analysis of the deduced proteome provides insights into the adherence and antigenic variation mechanisms of the strain.

Coagulase-negative staphylococci are frequently isolated from cases of subclinical bovine mastitis. Reported here is a draft genome sequence of Staphylococcus simulans UMC-CNS-990, an isolate recovered from a chronic intramammary infection of a Holstein cow. Unexpectedly, a cluster of genes encoding gas vesicle proteins was found within the 2,755-kb genome.

Intramammary infections in dairy cattle are frequently caused by staphylococci, resulting in mastitis and associated economic losses. A draft genome sequence was determined for Staphylococcus equorum UMC-CNS-924, isolated from the milk of a Holstein cow, to better understand the genetic basis of its pathogenesis and adaptation to the bovine mammary gland.

A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIH(T). Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (10(4)-10(5) per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host-plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A 3-year-old Texas Longhorn steer had a long history of progressive swelling of the soft tissues of the jaw and neck. At necropsy, multifocal to coalescing dermal and subcutaneous pyogranulomas were surrounded by fibrous tissue. Microscopically, the pyogranulomas contained aggregates of gram-negative coccobacilli surrounded by Splendore-Hoeppli material and were separated by bands of fibrovascular tissue (botryomycosis). Phylogenetic analysis of multilocus sequence-typing data revealed that the bacteria recovered in pure culture from swabs of submandibular tissue were most closely related to Bibersteinia [Pasteurella] trehalosi. The bacterial colonies were immunohistochemically reactive with a rabbit polyclonal anti-Pasteurella class C acid phosphatase antibody. Botryomycosis is a pyogranulomatous inflammation caused by a variety of nonbranching, nonfilamentous bacteria that elicit the formation of Splendore-Hoeppli material. This case of botryomycosis is unique for its association with Bibersteinia trehalosi.

Mycoplasma mycoides subsp. mycoides small colony biotype (SC) is the high-consequence animal pathogen causing contagious bovine pleuropneumonia. We report the complete genome sequences of the pathogenic strain M. mycoides subsp. mycoides SC Gladysdale and a close phylogenetic relative, Mycoplasma leachii PG50(T), another bovine pathogen of the M. mycoides phylogenetic clade.

Mycoplasma hyorhinis impacts swine health and production in many countries, either as a primary pathogen or as a component of a polymicrobial infection. Isolates of this species are also common contaminants of tissue culture lines. The genome sequence of the cell culture isolate M. hyorhinis GDL-1 is presented herein.

Mycoplasma putrefaciens is a causative agent of contagious agalactia in goats. Reported herein is the complete genome sequence of the M. putrefaciens type strain KS1.

Class C acid phosphatases (CCAPs) are 25-30 kDa bacterial surface proteins that are thought to function as broad-specificity 5',3'-nucleotidases. Analysis of the newly published complete genome sequence of Mycoplasma bovis PG45 revealed a putative CCAP with a molecular weight of 49.9 kDa. The expression, purification and crystallization of this new family member are described here. Standard purification procedures involving immobilized metal-ion affinity chromatography and ion-exchange chromatography yielded highly pure and crystallizable protein. Crystals were grown in sitting drops at room temperature in the presence of PEG 3350 and HEPES buffer pH 7.5 and diffracted to 2.3 Å resolution. Analysis of diffraction data suggested a primitive monoclinic space group, with unit-cell parameters a = 78, b = 101, c = 180 Å, β = 92°. The asymmetric unit is predicted to contain six molecules, which are likely to be arranged as three dimers., (© 2011 International Union of Crystallography. All rights reserved.)

This complete and fully assembled genome sequence of Mycoplasma bovis type strain PG45 is the first available for this species and offers a framework for comparison with additional pathogenic isolates. The single circular chromosome of 1,003,404 bp reveals multiple gene sets and mechanisms involved in variable expression of surface antigens and the incursion of numerous and assorted mobile elements, despite its reduced size.

The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, Mollicutes. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins., Results: Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the Mollicutes. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by HGT and intra-genomic shuffling., Conclusions: We describe novel features of PARCELs (Palindromic Amphipathic Repeat Coding ELements), a set of widely distributed repeat protein domains and coding sequences that were likely acquired through HGT by diverse unicellular microbes, further mobilized and diversified within genomes, and co-opted for expression in the membrane proteome of some taxa. Disseminated by multiple gene-centric vehicles, ORFs harboring these elements enhance accessory gene pools as part of the "mobilome" connecting genomes of various clades, in taxa sharing common niches.

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Class C nonspecific acid phosphatases are bacterial enzymes that are secreted across the cytoplasmic membrane and hydrolyze a variety of phosphomonoesters at acidic pH. These enzymes are of interest for the development of improved vaccines and clinical diagnostic methods. In one case, the category A pathogen Francisella tularensis, the class C phosphatase plays a role in bacterial fitness. Here, the cloning, expression, purification and crystallization methods for the class C acid phosphatases from F. tularensis and Pasteurella multocida are reported. Crystals of the F. tularensis enzyme diffracted to 2.0 A resolution and belonged to space group C222(1), with one enzyme molecule in the asymmetric unit. Crystals of the P. multocida enzyme diffracted to 1.85 A resolution and belonged to space group C2, with three molecules in the asymmetric unit. Diffraction patterns from crystals of the P. multocida enzyme exhibited multiple interpenetrating reciprocal-space lattices, indicating epitaxial twinning. Despite this aberrance, autoindexing was robust and the data could be satisfactorily processed to 1.85 A resolution using MOSFLM and SCALA.

The generation of surface variation among many divergent species of Mollicutes (mycoplasmas) occurs through stochastic expression patterns of diverse lipoprotein genes. The size and wide distribution of such variable gene sets in minimal (approximately 0.6- to 1.4-Mb) mycoplasmal genomes suggest their key role in the adaptation and survival of these wall-less monoderms. Diversity through variable genes is less clearly established among phylogenetically similar mycoplasmas, such as the Mycoplasma mycoides cluster of ruminant pathogens, which vary widely in host range and pathobiology. Using (i) genome sequences from two members of this clade, Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides small colony biotype (SC), (ii) antibodies to specific peptide determinants of predicted M. capricolum subsp. capricolum gene products, and (iii) analysis of the membrane-associated proteome of M. capricolum subsp. capricolum, a novel set of six genes (vmcA to vmcF) expressing distinct Vmc (variable M. capricolum subsp. capricolum) lipoproteins is demonstrated. These occur at two separate loci in the M. capricolum subsp. capricolum genome, which shares striking overall similarity and gene synteny with the M. mycoides subsp. mycoides SC genome. Collectively, Vmc expression is noncoordinate and combinatorial, subject to a single-unit insertion/deletion in a 5' flanking dinucleotide repeat that governs expression of each vmc gene. All vmc genes share modular regions affecting expression and membrane translocation. In contrast, vmcA to vmcD genes at one locus express surface proteins with highly structured size-variable repeating domains, whereas vmcE to vmcF genes express products with short repeats devoid of predicted structure. These genes confer a distinctive, dynamic surface architecture that may represent adaptive differences within this important group of pathogens as well as exploitable diagnostic targets.

Cloning, expression, purification and crystallization studies of a recombinant class C acid phosphatase from the Category A pathogen Bacillus anthracis are reported. Large diffraction-quality crystals were grown in the presence of HEPES and Jeffamine ED-2001 at pH 7.0. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.4, b = 90.1, c = 104.2 angstroms. The asymmetric unit is predicted to contain two protein molecules with a solvent content of 38%. Two native data sets were collected from the same crystal before and after flash-annealing. The first data set had a mosaicity of 1.6 degrees and a high-resolution limit of 1.8 angstroms. After flash-annealing, the apparent mosaicity decreased to 0.9 degrees and the high-resolution limit of usable data increased to 1.6 angstroms. This crystal form is currently being used to determine the structure of B. anthracis class C acid phosphatase with experimental phasing techniques.

Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4(1)2(1)2, with unit-cell parameters a = 61.96, c = 210.78 A. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 A resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage lambda ZAP Express vector which contains both prokaryotic (P(lac)) and eukaryotic (P(CMV)) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which P(CMV)-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into lambda ZAP Express, and two strongly immunodominant clones, lambda-A8 and lambda-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone lambda-A8 expressed an isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC&#95;0397). Clone lambda-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones lambda-A8 and lambda-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone lambda-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone lambda-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC&#95;0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Francisella tularensis is the etiologic agent of the potentially fatal human disease tularemia and is capable of survival and multiplication within professional phagocytes of the host. While the mechanisms that allow intracellular survival of the bacterium are only now beginning to be elucidated at the molecular level, previous work demonstrated that F. tularensis produces copious levels of an acid phosphatase which in crude and purified form affected the dose-dependent abrogation of the respiratory burst of stimulated neutrophils. The work presented here was undertaken to provide a source of recombinant F. tularensis acid phosphatase for detailed biochemical, biological, and structural studies. Results from this work are consistent with the ability to generate milligram amounts of recombinant enzyme whose attributes are demonstrably equivalent to those of the native enzyme. Such properties include molecular mass, broad substrate specificity, sensitivity and resistance to various inhibitors, pH optimum, and reactivity with rabbit polyclonal antibody to the native enzyme.

The approximately 16 kb genome of the Mycoplasma fermentans phiMFV1 prophage is described, and its mobility, replication and effect on the mycoplasma surface phenotype are demonstrated. In various M. fermentans strains, phiMFV1 was either absent or integrated at diverse (and sometimes multiple) chromosomal sites, each marked by a conserved TTTTTA target sequence that is duplicated upon integration. Precise excision, replication of an extrachromosomal form and loss of phiMFV1 from the mycoplasmal genome were documented in a series of clonal derivatives of M. fermentans propagated in culture. Of 18 open reading frames (ORFs) encoded by phiMFV1, most can be ascribed functions related to phage biology, whereas one encodes a unique coiled-coil membrane surface protein, Mem, that was confirmed to be expressed in propagating populations of M. fermentans. With the exception of Mem and other minor ORFs, the striking similarity between the deduced proteomes of phiMFV1 and the recently described phiMAV1 of arthritogenic strains of Mycoplasma arthritidis, along with the prominent gene synteny between these elements, provides the taxonomic basis for a new family of prophage. Their coding features are consistent with long-term residence in mycoplasma genomes and the divergence of species within a phylogenetic clade of mycoplasmas. The unique Mem protein expressed from phiMFV1 and the unique hypothetical surface lipoproteins encoded by phiMAV1 and phiMFV1 also suggest that prophage-associated genes may provide specific, selectable phenotypic traits during co-evolution of mycoplasma species with their respective mammalian hosts. Retention of these labile prophage elements in organisms with such drastically reduced genome sizes implies a significant role in adaptation and survival.