Bovine Pathogens Research Articles

Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV) in cattle

BackgroundBovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen.FindingsReplicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection.ConclusionsReplicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

Study on the Prevalence, Incidence, Drug sensitivity and Extra Chromosomal Impact on Bovine Mastitis Pathogens

India is the highest milk producer in the world, but the per capita availability of milk still remains half of the world average. One of the reasons for low productivity is poor animal health, particularly mastitis, which is single largest problem in dairy animal in terms of economical loses in India. Mastitis destroys the milk secreting cells. Scar or connective tissue replaces the milk secreting tissue, resulting in a permanent loss of productive ability. Mastitis is a multietiological disease wherein about 95 % of reported cases are caused by Staphylococcus. In this study plasmid DNA of the strain was isolated and the antibiotic sensitivity was done for strain with plasmid and without plasmid in order to found genetic impact of bovine mastitis.

Host-Mycobacterium avium subsp. paratuberculosis interactome reveals a novel iron assimilation mechanism linked to nitric oxide stress during early infection.

BackgroundThe initial interaction between host cell and pathogen sets the stage for the ensuing infection and ultimately determine the course of disease. However, there is limited knowledge of the transcripts utilized by host and pathogen and how they may impact one another during this critical step. The purpose of this study was to create a host-Mycobacterium avium subsp. paratuberculosis (MAP) interactome for early infection in an epithelium-macrophage co-culture system using RNA-seq.ResultsEstablishment of the host-MAP interactome revealed a novel iron assimilation system for carboxymycobactin. Iron assimilation is linked to nitric oxide synthase-2 production by the host and subsequent nitric oxide buildup. Iron limitation as well as nitric oxide is a prompt for MAP to enter into an iron sequestration program. This new iron sequestration program provides an explanation for mycobactin independence in some MAP strains grown in vitro as well as during infection within the host cell. Utilization of such a pathway is likely to aid MAP establishment and long-term survival within the host.ConclusionsThe host-MAP interactome identified a number of metabolic, DNA repair and virulence genes worthy for consideration as novel drug targets as well as future pathogenesis studies. Reported interactome data may also be utilized to conduct focused, hypothesis-driven research. Co-culture of uninfected bovine epithelial cells (MAC-T) and primary bovine macrophages creates a tolerant genotype as demonstrated by downregulation of inflammatory pathways. This co-culture system may serve as a model to investigate other bovine enteric pathogens.

Specific Molecular Detection and Characterization of <i>Anaplasma marginale</i> in Mongolian Cattle

Anaplasma marginale is an etiologic agent of bovine anaplasmosis. This study aimed to molecularly detect and characterize A. marginale that is prevalent in Mongolian cattle populations. A highly specific and sensitive nested PCR (nPCR) method based on the Msp5 gene was developed to detect A. marginale (Msp5 nPCR). The method detected A. marginale from the positive DNA samples obtained from different countries, while no amplicons were observed from DNA samples of several other bovine blood pathogens tested. The detection limit of Msp5 nPCR was determined to be 2 copies/μl. The method was tested against field blood DNA samples prepared from 300 Mongolian cattle in 2010. Results indicated a prevalence rate of 8.7% (26 of 300). On the other hand, partial DNA fragments of an Anaplasma sp. closely related to A. ovis (with 95.0% identity) were detected using a different nPCR method based on groEL gene. The phylogenetic analyses based on the Msp5, groEL and 16S rRNA genes demonstrated that A. marginale isolates in Mongolia were not divergent from the isolates distributed in other countries. The present study successfully established a new nPCR assay that can detect A. marginale, and reported the first molecular detection and characterization of A. marginale and an Anaplasma sp. closely related to A. ovis in Mongolian cattle populations.

Adhesion of Fusobacterium necrophorum to bovine endothelial cells is mediated by outer membrane proteins

Fusobacterium necrophorum, a Gram-negative anaerobe, is frequently associated with suppurative and necrotic infections of animals and humans. The organism is a major bovine pathogen, and in cattle, the common fusobacterial infections are hepatic abscesses, foot rot, and necrotic laryngitis. The species comprises two subspecies: F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme. Bacterial adhesion to the host cell surface is a critical initial step in the pathogenesis, and outer membrane proteins (OMP) play an important role in adhesion and establishment of certain Gram-negative bacterial infections. The means by which F. necrophorum attaches to epithelial or endothelial cells has not been determined. We evaluated whether OMP of F. necrophorum, isolated from a liver abscess, mediated adhesion to bovine endothelial cells (adrenal gland capillary endothelial cell line). The extent of binding of subsp. necrophorum to the endothelial cells was higher than that of F. necrophorum subsp. funduliforme. Trypsin treatment of bacterial cells decreased their binding to endothelial cells indicating the protein nature of adhesins. Preincubation of endothelial cells with OMP extracted from F. necrophorum decreased the binding of bacterial cells. In addition, binding of each subspecies to endothelial cells was inhibited by polyclonal antibodies raised against respective OMP and the antibody-mediated inhibition was subspecies specific. The western blot analysis of OMP bound to endothelial cells with anti-OMP antibodies showed four OMP of 17, 24, 40 and 74kDa. We conclude that OMP of F. necrophorum play a role in adhesion of bacterial cells to the endothelial cells.

In vitro susceptibility testing of ceftiofur against bovine mastitis pathogens isolated as part of an ongoing surveillance program (2002- 2010)

Although many antimicrobial susceptibility reports have been published that include mastitis pathogens, few have compared susceptibility trends over time and geographical areas. In 2001, Pfizer Animal Health (PAH) initiated a pilot susceptibility surveillance program, and requested that six veterinary diagnostic laboratories submit bacterial pathogens isolated from naturally occurring mastitis infections for susceptibility testing at PAR. That year, 354 pathogens were tested. The program has grown and more than 1,200 isolates are tested each year. Results of ceftiofur activity against mastitis pathogens for the years 2002 to 2010 are reported here. SPECTRAMAST® was approved in 2005 for the treatment of clinical mastitis associated with coagulase-negative staphylococci (CNS), Strep. dysgalactiae, and Escherichia coli in lactating dairy cattle and for the treatment of subclinical mastitis associated with Staphylococcus aureus, Strep. dysgalactiae, and Streptococcus uberis in dairy cattle at the time of dry off.

Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and bacteriological culture for 4 common bovine intramammary pathogens

Bacteriological culture (BC) is the traditional method for intramammary infection diagnosis but lacks sensitivity and is time consuming. Multiplex real-time PCR (mr-PCR) enables testing the presence of several bacteria and reduces diagnosis time. Our objective was to estimate bacterial species-specific sensitivity (Se) and specificity of both BC and mr-PCR tests for detecting bacteria in milk samples from clinical mastitis cases and from apparently normal quarters, using a Bayesian latent class model. Milk samples from 1,014 clinical mastitis cases and 1,495 samples from apparently normal quarters were analyzed by BC and mr-PCR. Two positive culture definitions were used: ≥1cfu/0.01mL and ≥10cfu/0.01mL of the specified bacteria. The mr-PCR was designed to simultaneously detect Staphylococcus aureus, Streptococcus uberis, Escherichia coli, and Streptococcus agalactiae. The priors used in our Bayesian model were weakly informative, with BC priors using the best available error data. Results were compared with those obtained using uniform priors for mr-PCR to test robustness. Weak and uniform priors gave about the same posterior distributions except for Strep. uberis from normal quarters and Strep. agalactiae. Multiplex real-time PCR Se on milk from clinical mastitis were lower than mr-PCR Se on milk from normal quarters. Multiplex real-time PCR Se was higher than BC on milk from normal quarters. Multiplex real-time PCR Se was generally lower than BC on milk from clinical mastitis and it varied by clinical severity. The estimate specificities of detection for all pathogens were ≥99%, regardless of sample type. The effect of milk sample preservation before testing was evaluated and may have been a factor that affected our observed results. A significant association was observed between sample age and mr-PCR results leading to reduced detection of E. coli and Strep. agalactiae in nonclinical samples. Differences in sample age between conduct of BC and of mr-PCR did not concur with any apparent differences between Se estimates of the 2 tests. Further work should be done to extend these results to other PCR-based tests for detecting bacterial species in milk samples, for which presented results could be used as prior parameter distributions. Limits of sample handling and storage and the potential existence of substances in clinical case samples that may interfere with PCR reactions also are worth further investigation.

Transcriptome profile of a bovine respiratory disease pathogen: Mannheimia haemolytica PHL213

BackgroundComputational methods for structural gene annotation have propelled gene discovery but face certain drawbacks with regards to prokaryotic genome annotation. Identification of transcriptional start sites, demarcating overlapping gene boundaries, and identifying regulatory elements such as small RNA are not accurate using these approaches. In this study, we re-visit the structural annotation of Mannheimia haemolytica PHL213, a bovine respiratory disease pathogen. M. haemolytica is one of the causative agents of bovine respiratory disease that results in about $3 billion annual losses to the cattle industry. We used RNA-Seq and analyzed the data using freely-available computational methods and resources. The aim was to identify previously unannotated regions of the genome using RNA-Seq based expression profile to complement the existing annotation of this pathogen.ResultsUsing the Illumina Genome Analyzer, we generated 9,055,826 reads (average length ~76 bp) and aligned them to the reference genome using Bowtie. The transcribed regions were analyzed using SAMTOOLS and custom Perl scripts in conjunction with BLAST searches and available gene annotation information. The single nucleotide resolution map enabled the identification of 14 novel protein coding regions as well as 44 potential novel sRNA. The basal transcription profile revealed that 2,506 of the 2,837 annotated regions were expressed in vitro, at 95.25% coverage, representing all broad functional gene categories in the genome. The expression profile also helped identify 518 potential operon structures involving 1,086 co-expressed pairs. We also identified 11 proteins with mutated/alternate start codons.ConclusionsThe application of RNA-Seq based transcriptome profiling to structural gene annotation helped correct existing annotation errors and identify potential novel protein coding regions and sRNA. We used computational tools to predict regulatory elements such as promoters and terminators associated with the novel expressed regions for further characterization of these novel functional elements. Our study complements the existing structural annotation of Mannheimia haemolytica PHL213 based on experimental evidence. Given the role of sRNA in virulence gene regulation and stress response, potential novel sRNA described in this study can form the framework for future studies to determine the role of sRNA, if any, in M. haemolytica pathogenesis.

Complete Genome Sequences of Mycoplasma leachii Strain PG50 T and the Pathogenic Mycoplasma mycoides subsp. mycoides Small Colony Biotype Strain Gladysdale

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.

Antimicrobial resistance profiles of common mastitis pathogens on Canadian dairy farms

Monitoring of antimicrobial resistance (AMR) in bacteria has clinical and public health significance. The present study determined prevalence of AMR in common mastitis pathogens Staphylococcus aureus, including methicillin-resistant Staph. aureus (MRSA; n=1,810), Escherichia coli (n=394), and Klebsiella species (n=139), including extended-spectrum β-lactamase (ESBL)-producing E. coli and Klebsiella species, isolated from milk samples on 89 dairy farms in 6 Canadian provinces. Minimum inhibitory concentrations (MIC) were determined using the Sensititer bovine mastitis plate (Trek Diagnostic Systems Inc., Cleveland, OH) and a National Antimicrobial Resistance Monitoring System gram-negative panel containing antimicrobials commonly used for mastitis treatment and control. Denim blue chromogenic agar and real-time PCR were used to screen and confirm MRSA, respectively. Resistance proportion estimates ranged from 0% for cephalothin and oxacillin to 8.8% for penicillin in Staph. aureus isolates, and 15% of the resistant Staph. aureus isolates were multidrug resistant. One MRSA isolate was confirmed (prevalence: 0.05%). Resistance proportion estimates ranged from 0% for ceftriaxone and ciprofloxacin to 14.8% for tetracycline in E. coli, and 0% for amikacin, ceftiofur, ciprofloxacin, and nalidixic acid to 18.6% for tetracycline in Klebsiella species isolates. Further, 62.8 and 55% of the resistant E. coli and Klebsiella species isolates were multidrug resistant, respectively. Resistance to >5 and >2 antimicrobials was most common in E. coli and Klebsiella species isolates, respectively, and no ESBL producers were found. Prevalence of AMR in bovine mastitis pathogens was low. Most gram-negative udder pathogens were multidrug resistant; MRSA was rarely found, and ESBL E. coli and Klebsiella species isolates were absent in Canadian milk samples.

Decoration of Histophilus somni lipooligosaccharide with N-acetyl-5-neuraminic acid enhances bacterial binding of complement factor H and resistance to killing by serum and polymorphonuclear leukocytes

The incorporation of N-acetyl-5-neuraminic acid (Neu5Ac), or sialic acid, onto surface components of some bacterial species may enhance their virulence. We have previously shown that Neu5Ac can be incorporated onto the lipooligosaccharide (LOS) of the bovine pathogen Histophilus somni, resulting in diminished antibody binding and enhanced serum resistance (Inzana et al., 2002. Infect. Immun. 70, 4870). In the present study, we assessed the effect of sialylation of H. somni LOS on the interaction with bovine innate host defenses. Incubation of non-sialylated H. somni with pre-colostral calf serum (PCS) resulted in dose-dependent, complement-mediated killing of the bacteria by the alternative pathway. However, sialylated H. somni was significantly more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated and consumed less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized red blood cells, and by Western blotting of C3 activation products. Sialylated H. somni bound more factor H and iC3b and less C3 than non-sialylated bacteria, as determined by enzyme-linked immunosorbent assay, supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni LOS inhibited both polymorphonuclear leukocyte phagocytosis of 3H-thymidine-labeled bacteria and intracellular killing of the bacteria, compared to non-sialylated bacteria. Furthermore, sialylated H. somni bound less non-specific antibodies in normal bovine sera than non-sialylated bacteria. Therefore, sialylation of H. somni LOS had profound effects on resistance of the bacteria to innate bovine host defenses, which should be taken into consideration during in vitro studies of H. somni.

Human Platelet Lysate Gel Provides a Novel Three Dimensional-Matrix for Enhanced Culture Expansion of Mesenchymal Stromal Cells

Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that-apart from heparin-consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix-this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion of MSCs.

Dynamics of bovine herpesvirus type 1 infection in Estonian dairy herds with and without a control programme

Bovine herpesvirus type 1 (BHV-1) is an important bovine pathogen, exacerbating poor health and the productivity of cattle. The aims of this study were to detect the efficacy of vaccination...

Evaluation of envelope glycoprotein Erns of an atypical bovine pestivirus as antigen in a microsphere immunoassay for the detection of antibodies against bovine viral diarrhea virus 1 and atypical bovine pestivirus

Atypical bovine pestiviruses are related antigenically and phylogenetically to bovine viral diarrhea viruses (BVDV-1 and BVDV-2), and may cause the same clinical manifestations in animals. Glycoprotein E(rns) of an atypical bovine pestivirus Th/04_KhonKaen was produced in a baculovirus expression system and was purified by affinity chromatography. The recombinant E(rns) protein was used as an antigen in a microsphere immunoassay for the detection of antibodies against BVDV-1 and atypical bovine pestivirus. The diagnostic performance of the new method was evaluated by testing a total of 596 serum samples, and the assay was compared with enzyme-linked immunosorbent assay (ELISA). Based on the negative/positive cut-off median fluorescence intensity (MFI) value of 2800, the microsphere immunoassay had a sensitivity of 100% and specificity of 100% compared to ELISA. The immunoassay was able to detect antibodies against both BVDV-1 and the atypical pestivirus. This novel microsphere immunoassay has the potential to be multiplexed for simultaneous detection of antibodies against different bovine pathogens in a high-throughput and economical way.

Group B Streptococcal Infections

1. Laura Sass, MD* 1. *Division of Infectious Diseases, Children's Specialty Group, Children's Hospital of The King's Daughters, Norfolk, VA; Pediatrics, Eastern Virginia Medical School, Norfolk, VA. * Abbreviations: CDC: : Centers for Disease Control and Prevention CPS: : capsular polysaccharides CSF: : cerebrospinal fluid EOD: : early onset disease GBS: : group B Streptococcus IAP: : intrapartum antibiotic prophylaxis NAAT: : nucleic acid amplification test UTI: : urinary tract infection In 2010, revised guidelines were published with updated algorithms for group B Streptococcus screening, intrapartum prophylaxis, antibiotic doses, and revised management for newborns. After completing this article, readers should be able to: 1. Know the mode of transmission of group B Streptococcus (GBS) and the peripartum risk factors associated with a high risk of infection. 2. Know the major clinical manifestations of GBS and be able to differentiate the epidemiology and clinical presentation of early onset GBS disease from that of late-onset disease. 3. Know the laboratory testing for GBS: isolation, antigen detection, and susceptibility tests. 4. Know the treatment of GBS infections: drugs of choice, alternative drugs, and ineffective drugs. 5. Review the most recent guidelines for the prevention of perinatal GBS disease. 1. Understand the importance of maternal screening tests for GBS. 2. Know the criteria for the treatment of mothers known to be GBS carriers at the time of delivery. 3. Differentiate the prevention of early onset disease from that of late onset. Group B Streptococcus (GBS), or Streptococcus agalactiae , is an encapsulated gram-positive diplococcus that produces a narrow zone of β-hemolysis on blood agar and generally is resistant to bacitracin. GBS can be further differentiated by type-specific capsular polysaccharides (CPS) and protein antigens. Current circulating serotypes include Ia, Ib, Ia/c, II, III, IV, V, VI, VII, VIII, and IX. GBS initially was believed to be a bovine mastitis pathogen, but cases of puerperal sepsis in humans were described in the 1930s. The organism then became more prominent in the 1970s as a cause of maternal and neonatal disease. The most common maternal manifestations are asymptomatic bacteriuria, urinary tract infection (UTI), bacteremia, chorioamnionitis, and endometritis. In …

Evaluation of a commercial rapid test kit for detecting bovine enteric pathogens in feces

Recently a commercial antigen-capture enzyme-linked immunosorbent assay kit in the form of a dipstick (Bovine Enterichek, Biovet Inc.) was made available to bovine practitioners and producers for the rapid detection of Betacoronavirus 1 (BCV-1), Rotavirus A (RV-A), Escherichia coli K99(+), and Cryptosporidium parvum in feces from diarrheic calves. The diagnostic performance of Bovine Enterichek was evaluated in comparison with a multiplex real-time polymerase chain reaction assay (mrtPCR). One hundred fecal samples were procured from diagnostic submissions to Iowa State University Veterinary Diagnostic Laboratory and were used for the assessment. The agreement quotient (kappa) in results for each pathogen between Bovine Enterichek and mrtPCR were 0.095 (BCV-1), 0.521 (RV-A), 0.823 (E. coli K99( + )), and 0.840 (C. parvum). In comparison to mrtPCR, the diagnostic sensitivity of Bovine Enterichek was 60.0%, 42.3%, 71.4%, and 81.5%, and the diagnostic specificity was 51.4%, 100%, 100%, and 98.6% for BCV-1, RV-A, E. coli K99( + ), and C. parvum, respectively. The current study suggested that Bovine Enterichek can be a rapid test tool in the field for detection of RV-A, C. parvum, or E. coli K99(+) in feces from calves at acute stage of clinical disease. However, test results for BCV-1 by the kit should be interpreted with caution due to low specificity and sensitivity of the kit.

Herd-level association between antimicrobial use and antimicrobial resistance in bovine mastitis Staphylococcus aureus isolates on Canadian dairy farms

Surveillance of antimicrobial use and resistance is needed to manage antimicrobial resistance in bacteria. In this study, data were collected on antimicrobial use and resistance in Staphylococcus aureus (n=562), isolated from intramammary infections and (sub)clinical mastitis cases on 89 dairy farms in 4 regions of Canada [Alberta, Ontario, Québec, and the Maritime Provinces (Prince Edward Island, Nova Scotia, and New Brunswick)]. Dairy producers were asked to deposit empty drug containers into specially provided receptacles, and antimicrobial drug use rate was calculated to quantify antimicrobial use. Minimum inhibitory concentrations were determined using the Sensititer bovine mastitis plate system (TREK Diagnostic Systems Inc., Cleveland, OH), containing antimicrobials commonly used for mastitis treatment and control. Multivariable logistic regression models were built to determine herd-level risk factors of penicillin, ampicillin, pirlimycin, penicillin-novobiocin combination, tetracycline and sulfadimethoxine resistance in Staph. aureus isolates. Intramammary administration of the penicillin-novobiocin combination for dry cow therapy was associated with penicillin and ampicillin resistance [odds ratio (OR): 2.17 and 3.10, respectively]. Systemic administration of penicillin was associated with penicillin resistance (OR: 1.63). Intramammary administration of pirlimycin for lactating cow mastitis treatment was associated with pirlimycin resistance as well (OR: 2.07). Average herd parity was associated with ampicillin and tetracycline resistance (OR: 3.88 and 0.02, respectively). Average herd size was also associated with tetracycline resistance (OR: 1.02). Dairy herds in the Maritime region had higher odds of penicillin and lower odds of ampicillin resistance than dairy herds in Québec (OR: 2.18 and 0.19, respectively). Alberta dairy herds had lower odds of ampicillin and sulfadimethoxine resistance than dairy herds in Québec (OR: 0.04 and 0.08, respectively). Ontario dairy herds had lower odds of tetracycline and sulfadimethoxine resistance than dairy herds in Québec (OR: 0.05 and 0.33, respectively). Herd-level use of certain antimicrobials administered for mastitis treatment and control, such as intramammary penicillin and pirlimycin as well as systemically administered penicillin and florfenicol, was positively associated with antimicrobial resistance in bovine mastitis pathogens in the field conditions. Differences in antimicrobial resistance outcomes across 4 regions of Canada were observed.

Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis1 1This paper is an invited article as a result of a presentation at the International Lactoferrin Conference held in Mazatlan, Mexico (May 2011), and has undergone the Journal’s usual peer review process.

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.

Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Bovine Rotavirus

BackgroundBovine rotavirus (BRV) infection is common in young calves. This viral infection causes acute diarrhea leading to death. Rapid identification of infected calves is essential to control BRV successfully. Therefore development of simple, highly specific, and sensitive detection method for BRV is needed.ResultsA reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimized for rapid detection of BRV. Specific primer sets were designed to target the sequences of the VP6 gene of the neonatal calf diarrhea virus (NCDV) strain of BRV. The RT-LAMP assay was performed in a water bath for 60 minutes at 63°C, and the amplification products were visualized either directly or under ultraviolet light. This BRV specific RT-LAMP assay could detect 3.32 copies of subtype A BRV. No cross-reactions were detected with other bovine pathogens. The ability of RT-LAMP to detect bovine rotavirus was further evaluated with 88 bovine rectal swab samples. Twenty-nine of these samples were found to be positive for BRV using RT-LAMP. The BRV-specific-RT-LAMP results were also confirmed by real-time RT-PCR assay.ConclusionsThe bovine rotavirus-specific RT-LAMP assay was highly sensitive and holds promise as a prompt and simple diagnostic method for the detection of group A bovine rotavirus infection in young calves.

A proposal of interpretive criteria for cefoperazone applicable to bovine mastitis pathogens

The correct assessment of mastitis pathogens for their susceptibility/resistance to cefoperazone is currently hampered by the lack of harmonized test conditions and interpretive criteria. The aim of this study was to provide a proposal for clinical breakpoints of cefoperazone which are applicable to Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis from cases of bovine mastitis and better reflect the situation in the bovine udder than breakpoints adopted from human medicine. For this, pharmacological data and clinical efficacy data of the documents submitted for approval of cefoperazone have been revisited. In addition, 1086 bacterial pathogens of the aforementioned six species/groups collected in Germany and in the USA during recent years were tested in parallel for their cefoperazone MICs and the zone diameters using a 75μg disk. Subsequently, MICs were plotted against zone diameters. Based on the pharmacological data, the clinical efficacy and the microbiological data, a proposal was made for veterinary-specific breakpoints which classify members of the aforementioned species/groups as (a) susceptible to cefoperazone when their MIC is ≤2μg/ml and their zone diameters are ≥27mm (staphylococci or E. coli) or ≥21mm (streptococci), (b) intermediate when their MIC is 4μg/ml and their zone diameters are 22–26mm (staphylococci or E. coli) or 16-20mm (streptococci), and (c) resistant when their MIC is ≥8μg/ml and their zone diameters are ≤21mm (staphylococci or E. coli) or ≤15mm (streptococci).