Publications by authors named "Robert E Briggs"

32 Publications

Septicemic pasteurellosis causing peracute death and necrotizing myositis in a beef heifer calf in Alberta, Canada.

Can Vet J 2020 12;61(12):1303-1306

Diagnostic Services Unit and Department of Veterinary Clinical and Diagnostic Sciences (Doyle-Baker, Davies), Department of Production Animal Health (Janzen), University of Calgary Faculty of Veterinary Medicine, 11877 85th Street NW, Calgary, Alberta T3R 1J3; Prairie Diagnostic Services and Department of Veterinary Microbiology, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N 5B4 (Ngeleka); United States Department of Agriculture, National Animal Disease Center, 1920 Dayton Avenue, Ames, Iowa, USA 50010 (Briggs).

Septicemic pasteurellosis is an acute and fatal bacterial disease of cattle and wild ungulates caused by certain serotypes of . Here we report a single case of septicemic pasteurellosis in a 6-month-old, Red Angus heifer from a cow-calf operation in Alberta, Canada. Postmortem examination revealed necrotizing and hemorrhagic myositis, fibrinous pericarditis and multisystemic bacterial emboli. was isolated from muscle in pure culture, and the capsular antigen group was identified as serogroup B using polymerase chain reaction. To the best of our knowledge, this is the first reported case of septicemic pasteurellosis in beef cattle in Canada. Key clinical message: Veterinary practitioners and diagnosticians should include septicemic pasteurellosis on their list of differential diagnoses when they encounter similar presentations of peracute death and severe necrotizing myositis in cattle in Canada.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659874PMC
December 2020

Identification of a reliable fixative solution to preserve the complex architecture of bacterial biofilms for scanning electron microscopy evaluation.

PLoS One 2020 29;15(5):e0233973. Epub 2020 May 29.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, Ames, Iowa, United States of America.

Bacterial biofilms are organized sessile communities of bacteria enclosed in extracellular polymeric substances (EPS). To analyze organization of bacteria and EPS in high resolution and high magnification by scanning electron microscopy (SEM), it is important to preserve the complex architecture of biofilms. Therefore, fixation abilities of formalin, glutaraldehyde, and Methacarn (methanol/chloroform/acetic acid-6:3:1) fixatives were evaluated to identify which fixative would best preserve the complex structure of bacterial biofilms. Economically important Gram-negative Mannheimia haemolytica, the major pathogen associated with bovine respiratory disease complex, and Gram-positive Staphylococcus aureus, the major cause of chronic mastitis in cattle, bacteria were selected since both form biofilms on solid-liquid interface. For SEM analysis, round glass coverslips were placed into the wells of 24-well plates and diluted M. haemolytica or S. aureus cultures were added, and incubated at 37°C for 48-72 h under static growth conditions. Culture media were aspirated and biofilms were fixed with an individual fixative for 48 h. SEM examination revealed that all three fixatives were effective preserving the bacterial cell morphology, however only Methacarn fixative could consistently preserve the complex structure of biofilms. EPS layers were clearly visible on the top, in the middle, and in the bottom of the biofilms with Methacarn fixative. Biomass and three-dimensional structure of the biofilms were further confirmed spectrophotometrically following crystal violet staining and by confocal microscopy after viability staining. These findings demonstrate that Methacarn fixative solution is superior to the other fixatives evaluated to preserve the complex architecture of biofilms grown on glass coverslips for SEM evaluation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233973PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259777PMC
August 2020

Synthetic bovine NK-lysin-derived peptide (bNK2A) does not require intra-chain disulfide bonds for bactericidal activity.

PLoS One 2019 19;14(6):e0218507. Epub 2019 Jun 19.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, United Sates Department of Agriculture, Ames, IA, United States of America.

Bovine NK-lysins are cationic antimicrobial proteins found predominantly in the cytosolic granules of T lymphocytes and NK-cells. NK-lysin-derived peptides show antimicrobial activity against both Gram positive and Gram negative bacteria. Mature NK-lysin protein has six well-conserved cysteine residues. This study was performed to assess whether synthetic bovine NK-lysin-derived peptide (bNK2A) forms disulfide bonds and whether disulfide bonds were essential for bNK2A antimicrobial activity. Two 30-mer bNK2A peptides were synthesized: one with two original cysteines and an analog with cysteines substituted with two serines. Mass spectrometry revealed lack of disulfide bonds in original peptide while CD spectrophotometry showed both peptides have similar α-helical structures. Since both peptides were equally inhibitory to Histophilus somni, disulfide bonds appeared dispensable for synthetic bNK2A peptide antibacterial activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218507PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6583974PMC
February 2020

Erratum for Petruzzi et al., "Capsular Polysaccharide Interferes with Biofilm Formation by Serogroup A".

mBio 2018 02 13;9(1). Epub 2018 Feb 13.

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA

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http://dx.doi.org/10.1128/mBio.00176-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821099PMC
February 2018

Capsular Polysaccharide Interferes with Biofilm Formation by Serogroup A.

mBio 2017 11 21;8(6). Epub 2017 Nov 21.

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA

is an important multihost animal and zoonotic pathogen that is capable of causing respiratory and multisystemic diseases, bacteremia, and bite wound infections. The glycosaminoglycan capsule of is an essential virulence factor that protects the bacterium from host defenses. However, chronic infections (such as swine atrophic rhinitis and the carrier state in birds and other animals) may be associated with biofilm formation, which has not been characterized in Biofilm formation by clinical isolates was inversely related to capsule production and was confirmed with capsule-deficient mutants of highly encapsulated strains. Capsule-deficient mutants formed biofilms with a larger biomass that was thicker and smoother than the biofilm of encapsulated strains. Passage of a highly encapsulated, poor-biofilm-forming strain under conditions that favored biofilm formation resulted in the production of less capsular polysaccharide and a more robust biofilm, as did addition of hyaluronidase to the growth medium of all of the strains tested. The matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS), as determined by gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion. However, a putative glycogen synthesis locus was not differentially regulated when the bacteria were grown as a biofilm or planktonically, as determined by quantitative reverse transcriptase PCR. Therefore, the negatively charged capsule may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix from encasing large numbers of bacterial cells. This is the first detailed description of biofilm formation and a glycogen EPS by is an important pathogen responsible for severe infections in food animals, domestic and wild birds, pet animals, and humans. was first isolated by Louis Pasteur in 1880 and has been studied for over 130 years. However, aspects of its lifecycle have remained unknown. Although formation of a biofilm by has been proposed, this report is the first to characterize biofilm formation by Of particular interest is that the biofilm matrix material contained a newly reported amylose-like glycogen as the exopolysaccharide component and that production of capsular polysaccharide (CPS) was inversely related to biofilm formation. However, even highly mucoid, poor-biofilm-forming strains could form abundant biofilms by loss of CPS or following passage under biofilm growth conditions. Therefore, the carrier state or subclinical chronic infections with may result from CPS downregulation with concomitant enhanced biofilm formation.
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http://dx.doi.org/10.1128/mBio.01843-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698555PMC
November 2017

Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni.

PLoS One 2017 21;12(8):e0183610. Epub 2017 Aug 21.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, Ames, Iowa, United States of America.

Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all the important bacterial pathogens that are involved in the bovine respiratory disease complex have been studied. Therefore the objective of the present study was to evaluate the antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni. Four, 30-mer peptides corresponding to the functional region of NK-lysin helices 2 and 3 were synthesized and assessed for antibacterial activity on four bovine pneumonic H. somni isolates. Although there were some differences in the efficiency of bactericidal activity among the NK-lysin peptides at lower concentrations (2-5 μM), all four peptides effectively killed most H. somni isolates at higher concentrations (10-30 μM) as determined by a bacterial killing assay. Confocal microscopic and flow cytometric analysis of Live/Dead Baclight stained H. somni (which were preincubated with NK-lysin peptides) were consistent with the killing assay findings and suggest NK-lysin peptides are bactericidal for H. somni. Among the four peptides, NK2A-derived peptide consistently showed the highest antimicrobial activity against all four H. somni isolates. Electron microscopic examination of H. somni following incubation with NK-lysin revealed extensive cell membrane damage, protrusions of outer membranes, and cytoplasmic content leakage. Taken together, the findings from this study clearly demonstrate the antimicrobial activity of all four bovine NK-lysin-derived peptides against bovine H. somni isolates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183610PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565109PMC
October 2017

A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6.

J Microbiol Methods 2017 08 24;139:155-160. Epub 2017 May 24.

Agriculture and Agri-Food Canada Research Centre, Lethbridge, Canada. Electronic address:

Mannheimia haemolytica is an important respiratory pathogen of ruminants. Of the 12 capsular serovars identified, 1 and 6 are most frequently associated with disease in cattle, while 2 is largely a commensal. Comparative analysis of 24 M. haemolytica genomes was used to identify unique genes associated with capsular polysaccharide synthesis as amplification targets in a multiplex PCR assay to discriminate between serotype 1, 2, and 6 strains. The specificity of serotype specific gene targets was evaluated against 47 reference strains representing 12 known serovars of M. haemolytica and 101 field isolates identified through antisera agglutination as serotypes 1, 2, or 6. The results suggest this simple and cost-effective serotype specific PCR assay can be used as an alternative to agglutination based techniques to serotype the majority of M. haemolytica collected from bovines, thus averting the need to use animals and invest in expensive sera development for agglutination assays. In addition, the gene targets identified in this study can be used in silico to identify serotype 1, 2, and 6 strains in sequenced M. haemolytica isolates without the need for culture based analysis.
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http://dx.doi.org/10.1016/j.mimet.2017.05.010DOI Listing
August 2017

Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica.

PLoS One 2016 4;11(3):e0151083. Epub 2016 Mar 4.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, Ames, Iowa, United States of America.

Human respiratory syncytial virus (HRSV) is a leading cause of severe lower respiratory tract infection in children under five years of age. IL-17 and Th17 responses are increased in children infected with HRSV and have been implicated in both protective and pathogenic roles during infection. Bovine RSV (BRSV) is genetically closely related to HRSV and is a leading cause of severe respiratory infections in young cattle. While BRSV infection in the calf parallels many aspects of human infection with HRSV, IL-17 and Th17 responses have not been studied in the bovine. Here we demonstrate that calves infected with BRSV express significant levels of IL-17, IL-21 and IL-22; and both CD4 T cells and γδ T cells contribute to this response. In addition to causing significant morbidity from uncomplicated infections, BRSV infection also contributes to the development of bovine respiratory disease complex (BRDC), a leading cause of morbidity in both beef and dairy cattle. BRDC is caused by a primary viral infection, followed by secondary bacterial pneumonia by pathogens such as Mannheimia haemolytica. Here, we demonstrate that in vivo infection with M. haemolytica results in increased expression of IL-17, IL-21 and IL-22. We have also developed an in vitro model of BRDC and show that co-infection of PBMC with BRSV followed by M. haemolytica leads to significantly exacerbated IL-17 production, which is primarily mediated by IL-17-producing γδ T cells. Together, our results demonstrate that calves, like humans, mount a robust IL-17 response during RSV infection; and suggest a previously unrecognized role for IL-17 and γδ T cells in the pathogenesis of BRDC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151083PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778910PMC
July 2016

Draft Genome Sequence of Pasteurella multocida Isolate P1062, Isolated from Bovine Respiratory Disease.

Genome Announc 2015 Oct 22;3(5). Epub 2015 Oct 22.

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa, USA

Here, we report the draft genome of Pasteurella multocida isolate P1062 recovered from pneumonic bovine lung in the United States in 1959.
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http://dx.doi.org/10.1128/genomeA.01254-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4616194PMC
October 2015

Genome Sequences of Mannheimia haemolytica Serotype A2 Isolates D171 and D35, Recovered from Bovine Pneumonia.

Genome Announc 2015 Mar 12;3(2). Epub 2015 Mar 12.

USDA, ARS, National Animal Disease Center, Ames, Iowa, USA

Here, we report two genomes, one complete and one draft, from isolates of serotype A2 Mannheimia haemolytica recovered from pneumonic bovine lung.
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http://dx.doi.org/10.1128/genomeA.00093-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357755PMC
March 2015

Genome Sequences of Serotype A6 Mannheimia haemolytica Isolates D174 and D38 Recovered from Bovine Pneumonia.

Genome Announc 2015 Mar 5;3(2). Epub 2015 Mar 5.

USDA, ARS, National Animal Disease Center, Ames, Iowa, USA

Here, we report two genomes, one complete and one draft, from virulent bovine strains of Mannheimia haemolytica serotype A6 recovered prior to the field usage of modern antimicrobial drugs.
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http://dx.doi.org/10.1128/genomeA.00086-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358395PMC
March 2015

Genome Sequences of Mannheimia haemolytica Serotype A1 Strains D153 and D193 from Bovine Pneumonia.

Genome Announc 2013 Oct 17;1(5). Epub 2013 Oct 17.

USDA, ARS, National Animal Disease Center, Ames, Iowa, USA.

Here we report two genome sequences, one complete and one draft, from virulent bovine strains of Mannheimia haemolytica serotype A1 recovered prior to the field usage of modern antimicrobial drugs.
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http://dx.doi.org/10.1128/genomeA.00848-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798457PMC
October 2013

Bivalent vaccination against pneumonic pasteurellosis in domestic sheep and goats with modified-live in-frame lktA deletion mutants of Mannheimia haemolytica.

Microb Pathog 2013 Nov 8;64:43-7. Epub 2013 Sep 8.

US Department of Agriculture, National Animal Disease Center, 1920 Dayton Avenue, Ames, IA 50010, USA. Electronic address:

A temperature-sensitive shuttle vector, pBB80C, was utilized to generate in-frame deletion mutants of the leukotoxin structural gene (lktA) of Mannheimia haemolytica serotypes 1, 2, 5, 6, 7, 8, 9, and 12. Culture supernatants from the mutants contained a truncated protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Sheep and goats vaccinated intramuscularly with a mixture of serotypes 5 and 6 mutants were resistant to virulent challenge with a mixture of the wild-type parent strains. These vaccinates responded serologically to both vaccine serotypes and exhibited markedly-reduced lung lesion volume and pulmonary infectious load compared to control animals. Control animals yielded a mixture of serotypes from lung lobes, but the proportion even within an individual animal varied widely from 95% serotype 5-95% serotype 6. Cultures recovered from liver were homogeneous, but two animals yielded serotype 5 and the other two yielded serotype 6 in pure culture.
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http://dx.doi.org/10.1016/j.micpath.2013.08.004DOI Listing
November 2013

Comparative genome analysis of an avirulent and two virulent strains of avian Pasteurella multocida reveals candidate genes involved in fitness and pathogenicity.

BMC Microbiol 2013 May 14;13:106. Epub 2013 May 14.

Department of Veterinary and Biomedical Sciences, University of Minnesota, St, Paul, MN, USA.

Background: Pasteurella multocida is the etiologic agent of fowl cholera, a highly contagious and severe disease of poultry causing significant mortality and morbidity throughout the world. All types of poultry are susceptible to fowl cholera. Turkeys are most susceptible to the peracute/acute forms of the disease while chickens are most susceptible to the acute and chronic forms of the disease. The whole genome of the Pm70 strain of P. multocida was sequenced and annotated in 2001. The Pm70 strain is not virulent to chickens and turkeys. In contrast, strains X73 and P1059 are highly virulent to turkeys, chickens, and other poultry species. In this study, we sequenced the genomes of P. multocida strains X73 and P1059 and undertook a detailed comparative genome analysis with the avirulent Pm70 strain. The goal of this study was to identify candidate genes in the virulent strains that may be involved in pathogenicity of fowl cholera disease.

Results: Comparison of virulent versus avirulent avian P. multocida genomes revealed 336 unique genes among the P1059 and/or X73 genomes compared to strain Pm70. Genes of interest within this subset included those encoding an L-fucose transport and utilization system, several novel sugar transport systems, and several novel hemagglutinins including one designated PfhB4. Additionally, substantial amino acid variation was observed in many core outer membrane proteins and single nucleotide polymorphism analysis confirmed a higher dN/dS ratio within proteins localized to the outer membrane.

Conclusions: Comparative analyses of highly virulent versus avirulent avian P. multocida identified a number of genomic differences that may shed light on the ability of highly virulent strains to cause disease in the avian host, including those that could be associated with enhanced virulence or fitness.
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http://dx.doi.org/10.1186/1471-2180-13-106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660278PMC
May 2013

Draft Genome Sequences of Two Virulent Serotypes of Avian Pasteurella multocida.

Genome Announc 2013 Jan 31;1(1). Epub 2013 Jan 31.

Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, USA.

Here we report the draft genome sequences of two virulent avian strains of Pasteurella multocida. Comparative analyses of these genomes were done with the published genome sequence of avirulent P. multocida strain Pm70.
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http://dx.doi.org/10.1128/genomeA.00058-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569333PMC
January 2013

Cross-protection against fowl cholera disease with the use of recombinant Pasteurella multocida FHAB2 peptides vaccine.

Avian Dis 2012 Sep;56(3):589-91

United States Department of Agriculture, Infectious Bacterial Diseases Research Unit, 1920 Dayton Avenue, Ames, IA 50010, USA.

It has been demonstrated that fhaB2 (filamentous hemagglutinin) is an important virulence factor for Pasteurella multocida in development of fowl cholera disease and that vaccination with recombinant FHAB2 peptides derived from P. multocida, P-1059 (serotype A:3) protects turkeys against P-1059 challenge. Here the hypothesis that vaccination with the same rFHAB2 peptides could cross-protect turkeys against challenge with P. multocida chi73 (serotype A:1) was examined. Three rFHAB2 peptides were purified and pooled, and two doses, consisting of equal amounts of each, were administered subcutaneously to turkeys at 2-wk intervals. Simultaneously, control birds were administered sham inoculations. One week later, vaccinates and controls were challenged intranasally with P-1059 or chi73. The results showed vaccination with rFHAB2 peptides significantly protected turkeys against lethal challenge from both P. multocida serotypes (P < 0.01). The high degree of FHAB2 conservation across serotypes likely allow the observed cross-protection.
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http://dx.doi.org/10.1637/9991-111611-ResNote.1DOI Listing
September 2012

Mucosal and parenteral vaccination against pneumonic pasteurellosis in cattle with a modified-live in-frame lktA deletion mutant of Mannheimia haemolytica.

Microb Pathog 2012 May 2;52(5):302-9. Epub 2012 Mar 2.

National Animal Disease Center, US Department of Agriculture, 1920 Dayton Avenue, Ames, IA 50010, USA.

A new temperature-conditional shuttle vector, pBB80C, was constructed and utilized to generate an in-frame deletion in the leukotoxin structural gene of Mannheimia haemolytica serotype 1. Culture supernatants from the mutant contained no detectable cytotoxicity to BL-3 lymphocyte targets, and contained a new protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Calves vaccinated mucosally by top-dressing the live mutant onto feed, or parenterally by subcutaneous injection, were resistant to virulent challenge with the parent strain. Serologic antibody response, reduction in lung lesion, and reduction in pulmonary infectious load were greater among calves mucosally vaccinated than those which were vaccinated by injection.
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http://dx.doi.org/10.1016/j.micpath.2012.02.008DOI Listing
May 2012

Monoclonal Antibodies Bind A SNP-Sensitive Epitope that is Present Uniquely in Mycobacterium avium Subspecies Paratuberculosis.

Front Microbiol 2011 26;2:163. Epub 2011 Jul 26.

Agricultural Research Service, United States Department of Agriculture, National Animal Disease Center Ames, IA, USA.

Due to a close genetic relatedness, there is no known antibody that detects Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne's disease in cattle and sheep, and does not cross-react with other M. avium subspecies. In the present study, a monoclonal antibody (MAb; 17A12) was identified from mice immunized with a cell membrane fraction of MAP strain K-10. This antibody is 100% specific as it detected a 25-kDa protein in all 29 MAP whole cell lysates, but did not bind to any of the 29 non-paratuberculosis strains tested in immunoblot assays. However, the antibody revealed variable reactivity levels in MAP strains as it detected higher levels in bovine isolates but comparably lower levels in ovine isolates of MAP. In order to identify the target binding protein for 17A12, a lambda phage expression library of MAP genomic fragments was screened with the MAb. Four reactive clones were identified, sequenced and all shown to be overlapping. Further analysis revealed all four clones expressed an unknown protein encoded by a sequence that is not annotated in the K-10 genome and overlapped with MAP3422c on the opposing DNA strand. The epitope of 17A12 was precisely defined to seven amino acids and was used to query the K-10 genome. Similarity searches revealed another protein, encoded by MAP1025, possessed a similar epitope (one-amino acid mismatch) that also reacted strongly to the antibody. A single nucleotide polymorphism (SNP) in MAP1025 was then identified by comparative sequence analysis, which results in a Pro28His change at residue 28, the first amino acid within the 17A12 epitope. This SNP is present in all MAP strains but absent in all non-MAP strains and accounts for the specificity of the 17A12 antibody. This new antibody is the first ever isolated that binds only to the paratuberculosis subspecies of M. avium and opens new possibilities for the specific detection of this significant ruminant pathogen.
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http://dx.doi.org/10.3389/fmicb.2011.00163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145258PMC
November 2011

Protection against fowl cholera conferred by vaccination with recombinant Pasteurella multocida filamentous hemagglutinin peptides.

Avian Dis 2009 Jun;53(2):169-74

U.S. Department of Agriculture, Respiratory Diseases of Livestock Unit, National Animal Disease Unit, Agricultural Research Services, 2300 Dayton Road, Ames, IA 50010, USA.

Three gene fragments, derived from Pasteurella multocida strain P-1059 (serotype A:3), encoding approximately the 5' one-third of fhaB2 were overexpressed individually in Escherichia coli. The recombinant peptides were purified, pooled, and administered to turkey poults to evaluate immunity. The results showed that turkeys immunized twice with the recombinant peptides were significantly protected against intranasal challenge with P. multocida strain P-1059. Vaccination elicited antibody responses, based on Western blotting, that were reactive with a wild-type P-1059 cellular product approximately 170 kDa in size and multiple high molecular weight products in culture supernatant. These antibodies did not react with cell or supernatant blots of a P-1059 fhaB2 isogenic mutant. Pasteurella multocida fhaB2 genes of a bovine strain (A:3) and an avian strain (F:3) are highly conserved as is the portion of P-1059 fhaB2 examined here (>99% identities). These findings suggest that broad cross-protection against this heterogeneous pathogen may be achievable through immunization with specific recombinant FHAB2 peptides.
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http://dx.doi.org/10.1637/8471-092308-Reg.1DOI Listing
June 2009

Sialic acid uptake is necessary for virulence of Pasteurella multocida in turkeys.

Microb Pathog 2009 Jun 12;46(6):337-44. Epub 2009 Apr 12.

US Department of Agriculture, Respiratory Diseases of Livestock Unit, National Animal Disease Center, Agricultural Research Service, Ames, IA 50010, USA.

Many pathogenic bacteria employ systems to incorporate sialic acid into their membranes as a means of protection against host defense mechanisms. In Pasteurella multocida, an opportunistic pathogen which causes diseases of economic importance in a wide range of animal species, sialic acid uptake plays a role in a mouse model of systemic pasteurellosis. To further investigate the importance of sialic acid uptake in pathogenesis, sialic acid uptake mutants of an avian strain of P. multocida P-1059 (A:3) were constructed, characterized, and an in-frame sialic acid uptake deletion mutant was assessed for virulence in turkeys. Inactivation of sialic acid uptake resulted in a high degree of attenuation when turkeys were challenged either intranasally or intravenously. Resistance of the sialic acid uptake mutant to killing by turkey serum complement was similar to that of the parent, suggesting other mechanisms are responsible for attenuation of virulence in turkeys.
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http://dx.doi.org/10.1016/j.micpath.2009.04.003DOI Listing
June 2009

Transmission of bovine coronavirus and serologic responses in feedlot calves under field conditions.

Am J Vet Res 2006 Aug;67(8):1412-20

Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, 44691, USA.

Objective: To compare shedding patterns and serologic responses to bovine coronavirus (BCV) in feedlot calves shipped from a single ranch in New Mexico (NM calves) versus calves assembled from local sale barns in Arkansas (AR calves) and to evaluate the role of BCV on disease and performance.

Animals: 103 feedlot calves from New Mexico and 100 from Arkansas.

Procedures: Calves were studied from before shipping to 35 days after arrival at the feedlot. Nasal swab specimens, fecal samples, and serum samples were obtained before shipping, at arrival, and periodically thereafter. Bovine coronavirus antigen and antibodies were detected by use of an ELISA.

Results: NM calves had a high geometric mean titer for BCV antibody at arrival (GMT, 1,928); only 2% shed BCV in nasal secretions and 1% in feces. In contrast, AR calves had low antibody titers against BCV at arrival (GMT, 102) and 64% shed BCV in nasal secretions and 65% in feces. Detection of BCV in nasal secretions preceded detection in feces before shipping AR calves, but at arrival, 73% of AR calves were shedding BCV in nasal secretions and feces. Bovine coronavirus infection was significantly associated with respiratory tract disease and decreased growth performance in AR calves.

Conclusions And Clinical Relevance: Replication and shedding of BCV may start in the upper respiratory tract and spread to the gastrointestinal tract. Vaccination of calves against BCV before shipping to feedlots may provide protection against BCV infection and its effects with other pathogens in the induction of respiratory tract disease.
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http://dx.doi.org/10.2460/ajvr.67.8.1412DOI Listing
August 2006

Challenge with Bovine viral diarrhea virus by exposure to persistently infected calves: protection by vaccination and negative results of antigen testing in nonvaccinated acutely infected calves.

Can J Vet Res 2006 Apr;70(2):121-7

Department of Veterinary Pathobiology, Room 250, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, USA.

Calves persistently infected (PI) with Bovine viral diarrhea virus (BVDV) represent an important source of infection for susceptible cattle. We evaluated vaccine efficacy using calves PI with noncytopathic BVDV2a for the challenge and compared tests to detect BVDV in acutely or transiently infected calves versus PI calves. Vaccination with 2 doses of modified live virus vaccine containing BVDV1a and BVDV2a protected the calves exposed to the PI calves: neither viremia nor nasal shedding occurred. An immunohistochemistry test on formalin-fixed ear notches and an antigen-capture enzyme-linked immunosorbent assay on fresh notches in phosphate-buffered saline did not detect BVDV antigen in any of the acutely or transiently infected calves, whereas both tests had positive results in all the PI calves.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1410726PMC
April 2006

Construction of in-frame aroA deletion mutants of Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus by using a new temperature-sensitive plasmid.

Appl Environ Microbiol 2005 Nov;71(11):7196-202

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, IA 50010, USA.

A temperature-sensitive (TS) plasmid was generated from the endogenous streptomycin resistance plasmid of Mannheimia hemolytica and used to engineer in-frame aroA deletion mutants of Mannheimia hemolytica, Pasteurella multocida, and Haemophilus somnus. TS replacement plasmids carrying in-frame aroA deletions were constructed for each target species and introduced into host cells by electroporation. After recovery in broth, cells were spread onto plates containing antibiotic and incubated at 30 degrees C, the permissive temperature for autonomous plasmid replication. Transfer of transformants to selective plates cultured at a nonpermissive temperature for plasmid replication selected for single-crossover mutants consisting of replacement plasmids that had integrated into host chromosomes by homologous recombination. Transfer of the single-crossover mutants back to a permissive temperature without antibiotic selection drove plasmid resolution, and, depending on where plasmid excision occurred, either deletion mutants or wild-type cells were generated. The system used here represents a broadly applicable means for generating unmarked mutants of Pasteurellaceae species.
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http://dx.doi.org/10.1128/AEM.71.11.7196-7202.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1287724PMC
November 2005

Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae.

Appl Environ Microbiol 2005 Nov;71(11):7187-95

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, IA 50010, USA.

Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42 degrees C in M. hemolytica but which were fully functional below 31 degrees C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5'-GCGC-3') characterized herein.
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http://dx.doi.org/10.1128/AEM.71.11.7187-7195.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1287723PMC
November 2005

Transmission of bovine viral diarrhea virus 1b to susceptible and vaccinated calves by exposure to persistently infected calves.

Can J Vet Res 2005 Jul;69(3):161-9

Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.

Bovine viral diarrhea virus (BVDV) persistently infected (PI) calves represent significant sources of infection to susceptible cattle. The objectives of this study were to determine if PI calves transmitted infection to vaccinated and unvaccinated calves, to determine if BVDV vaccine strains could be differentiated from the PI field strains by subtyping molecular techniques, and if there were different rates of recovery from peripheral blood leukocytes (PBL) versus serums for acutely infected calves. Calves PI with BVDV1b were placed in pens with nonvaccinated and vaccinated calves for 35 d. Peripheral blood leukocytes, serums, and nasal swabs were collected for viral isolation and serology. In addition, transmission of Bovine herpes virus 1 (BHV-1), Parainfluenza-3 virus (PI-3V), and Bovine respiratory syncytial virus (BRSV) was monitored during the 35 d observation period. Bovine viral diarrhea virus subtype 1b was transmitted to both vaccinated and nonvaccinated calves, including BVDV1b seronegative and seropositive calves, after exposure to PI calves. There was evidence of transmission by viral isolation from PBL, nasal swabs, or both, and seroconversions to BVDV1b. For the unvaccinated calves, 83.2% seroconverted to BVDV1b. The high level of transmission by PI calves is illustrated by seroconversion rates of nonvaccinated calves in individual pens: 70% to 100% seroconversion to the BVDV1b. Bovine viral diarrhea virus was isolated from 45 out of 202 calves in this study. These included BVDV1b in ranch and order buyer (OB) calves, plus BVDV strains identified as vaccinal strains that were in modified live virus (MLV) vaccines given to half the OB calves 3 d prior to the study. The BVDV1b isolates in exposed calves were detected between collection days 7 and 21 after exposure to PI calves. Bovine viral diarrhea virus was recovered more frequently from PBL than serum in acutely infected calves. Bovine viral diarrhea virus was also isolated from the lungs of 2 of 7 calves that were dying with pulmonary lesions. Two of the calves dying with pneumonic lesions in the study had been BVDV1b viremic prior to death. Bovine viral diarrhea virus 1b was isolated from both calves that received the killed or MLV vaccines. There were cytopathic (CP) strains isolated from MLV vaccinated calves during the same time frame as the BVDV1b isolations. These viruses were typed by polymerase chain reaction (PCR) and genetic sequencing, and most CP were confirmed as vaccinal origin. A BVDV2 NCP strain was found in only 1 OB calf, on multiple collections, and the calf seroconverted to BVDV2. This virus was not identical to the BVDV2 CP 296 vaccine strain. The use of subtyping is required to differentiate vaccinal strains from the field strains. This study detected 2 different vaccine strains, the BVDV1b in PI calves and infected contact calves, and a heterologous BVDV2 subtype brought in as an acutely infected calf. The MLV vaccination, with BVDV1a and BVDV2 components, administered 3 d prior to exposure to PI calves did not protect 100% against BVDV1b viremias or nasal shedding. There were other agents associated with the bovine respiratory disease signs and lesions in this study including Mannheimia haemolytica, Mycoplasma spp., PI-3V, BRSV, and BHV-1.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176294PMC
July 2005

Construction and virulence of a Pasteurella multocida fhaB2 mutant in turkeys.

Microb Pathog 2005 Jul-Aug;39(1-2):9-17

US Department of Agriculture, Respiratory Diseases of Livestock Unit, National Animal Disease Centre, Agricultural Research Service, IA 50010, USA.

Pasteurella multocida is the causative agent of fowl cholera. The organism can occur as a commensally in the naso-pharyngeal region of apparently healthy animals and it can be a primary or secondary pathogen in the disease process of birds. The complete genome of an avian strain of P. multocida has been sequenced and was shown to possess two filamentous hemagglutinin genes designated fhaB1 and fhaB2. Filamentous hemagglutinin transposon mutants of a bovine strain of P. multocida are attenuated in mice. Here, we report the construction of an fhaB2 P. multocida mutant in an avian strain P-1059 (A:3). The fhaB2 mutant and the parent were assessed for virulence in turkeys by intranasal and intravenous challenge. Inactivation of fhaB2 resulted in a high degree of attenuation when turkeys were challenged intranasally and to a lesser degree when intravenously administered. Resistance of the fhaB2 mutant and parent strain to killing by serum complement was similar.
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http://dx.doi.org/10.1016/j.micpath.2005.05.003DOI Listing
September 2005

Maternally derived humoral immunity to bovine viral diarrhea virus (BVDV) 1a, BVDV1b, BVDV2, bovine herpesvirus-1, parainfluenza-3 virus bovine respiratory syncytial virus, Mannheimia haemolytica and Pasteurella multocida in beef calves, antibody decline by half-life studies and effect on response to vaccination.

Vaccine 2004 Jan;22(5-6):643-9

Department of Veterinary Pathobiology, Oklahoma State University, College of Veterinary Medicine, Stillwater, OK 74078, USA.

The passive immunity transferred to calves from their dams was investigated in a beef herd to determine half-life of antibody, estimated time to seronegative status and effect on immunization. One hundred two beef calves in a commercial ranch under standard management conditions were utilized. Samples were collected at branding (day 0). This was the first possible date to collect samples postcalving. This was approximately 2 months postcalving, and days 95 and 116. The calves were divided into two groups: vaccinates (51) and nonvaccinates (51). The calves were vaccinated with a commercial inactivated viral vaccine containing bovine viral diarrhea virus (BVDV)1a, BVDV2, bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV) on days 0 and 95. Half of the vaccinated and unvaccinated calves also received one dose of an experimental Mannheimia haemolytica and Pasteurella multocida vaccine at day 95. Serums were tested for neutralizing antibody titers to BVDV1a, BVDV1b, BVDV2, BHV-1, PI-3V, and BRSV. Antibodies were detected by ELISA to M. haemolytica whole cell, M. haemolytica leukotoxin, and P. multocida outer membrane protein (OMP). The mean half-life of viral antibodies in nonvaccinated calves to each virus was: BVDV1a, 23.1 days (d); BVDV1b, 22.8 d; BVDV2, 22.9 d; BHV-1, 21.2 d; PI-3V, 30.3 d; and BRSV, 35.9 d. The mean half-life of viral antibodies was greater for vaccinates than for nonvaccinates for all viruses except BRSV. The calculated mean time to seronegative status for nonvaccinates based on titers at day 0 was: BVDV1a, 192.2 d; BVDV1b, 179.1 d; BVDV2, 157.8 d; BHV-1, 122.9 d; PI-3V, 190.6 d; and BRSV, 186.7 d. There was an active immune response after vaccination with two doses to all the viruses, except BRSV. Mean antibody titers of vaccinates at day 116 were statistically higher than nonvaccinates for all viruses except BRSV. However on an individual calf basis there were few seroconversions (four-fold rise or greater to BVDV1a, BVDV1b, BVDV2, PI-3V, or BRSV; or two-fold rise for BHV-1) in the presence of viral antibodies. The predicted time of seronegative status for a group of calves for vaccination programs may not be appropriate as there may be a range of titers for all calves at day 0. In this study the range for BVDV1a was 16-16,384; BVDV1b, 8-8192; BVDV2, 0-8192; BHV-1, 0-935; PI-3V, 8-2048; and BRSV, 8-4096. Using the half-life of 23 d for BVDV1a, the time thereafter for seronegative status would be 46 and 299 d compared to the calculated date of 192.2 d using the mean of estimated time to seronegative status for all the calves. There was an active humoral response in the vaccinated calves to M. haemolytica and P. multocida. Cowherd humoral immunity based on serum antibodies should be monitored as it may relate to transfer of maternal antibodies to calves. Exceptionally high levels of viral antibodies transferred to calves could interfere with the antibody response to vaccination.
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http://dx.doi.org/10.1016/j.vaccine.2003.08.033DOI Listing
January 2004

Response of calves persistently infected with noncytopathic bovine viral diarrhea virus (BVDV) subtype 1b after vaccination with heterologous BVDV strains in modified live virus vaccines and Mannheimia haemolytica bacterin-toxoid.

Vaccine 2003 Jun;21(21-22):2980-5

Room 250, McElroy Hall, Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA.

Seronegative persistently infected (PI) calves with bovine viral diarrhea virus (BVDV) subtype 1b were vaccinated with each of four modified live virus (MLV) BVDV vaccines and a Mannheimia haemolytica bacterin-toxoid. Nasal swabs and peripheral blood leukocytes (PBL) were collected for virus isolation and serums were collected after vaccination and tested for BVDV1a, BVDV1b, BVDV2, bovine herpesvirus-1 (BHV-1), bovine parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV) antibodies. M. haemolytica and Pasteurella multocida antibodies were detected using ELISA procedures. None of the PI calves developed mucosal disease (MD) after MLV vaccination. None of the BVDV PI calves seroconverted to BVDV1b after MLV vaccination. Calves receiving MLV vaccines seroconverted to the respective type/subtype in the vaccine. Calves receiving a MLV vaccine with noncytopathic (NCP) BVDV1 (subtype not designated) did not seroconvert to BVDV1a, BVDV1b, or BVDV2. The PI calves were positive for BVDV subtype 1b, in the PBL and nasal swabs throughout the study. Calves receiving each of three vaccines with known BVDV1a strains had BVDV1a positive samples after vaccination, in some but not all calves, up to Day 28. The PI BVDV1b calves did not respond with increased M. haemolytica antibodies after vaccination compared to BVDV negative calves receiving the same M. haemolytica vaccine.
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http://dx.doi.org/10.1016/s0264-410x(03)00118-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173202PMC
June 2003

Effect of intranasal exposure to leukotoxin-deficient Mannheimia haemolytica at the time of arrival at the feedyard on subsequent isolation of M haemolytica from nasal secretions of calves.

Am J Vet Res 2003 May;64(5):580-5

USDA, Agriculture Research Service, National Animal Disease Center, PO Box 70, Ames, IA 50010, USA.

Objective: To determine the effect of intranasal exposure to live leukotoxin (LktA)-deficient Mannheimia haemolytica (MH) at the time of feedyard arrival on nasopharyngeal colonization by wild-type MH in calves.

Animals: 200 calves.

Procedure: Calves from Arkansas (AR calves; n = 100; mean body weight, 205 kg) were purchased from an order buyer barn. Calves from New Mexico (NM calves; n = 100; mean body weight, 188 kg) were obtained from a single ranch. Calves were transported to a feedyard, where half of each group was exposed intranasally with LktA-deficient MH at the time of arrival. Calves were observed daily for respiratory tract disease (RTD), and nasal swab specimens were collected periodically to determine nasopharyngeal colonization status with MH. Serum samples were assayed for antibodies to MH.

Results: 15 AR calves had nasopharyngeal colonization by wild-type MH at the order buyer barn, whereas none of the NM calves had nasopharyngeal colonization. Intranasal exposure to LktA-deficient MH elicited an increase in serum antibody titers against MH in NM calves, but titers were less in NM calves treated for RTD. Exposure of NM calves to LktA-deficient MH offered protection from nasopharyngeal colonization by wild-type MH.

Conclusions And Clinical Relevance: Exposure of calves to LktA-deficient MH elicited an increase in serum antibody titers against MH and decreased colonization of the nasopharynx by wild-type MH. Earlier exposure would likely allow an immune response to develop before transportation and offer protection from nasopharyngeal colonization and pneumonia caused by wild-type MH.
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http://dx.doi.org/10.2460/ajvr.2003.64.580DOI Listing
May 2003

Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease.

Can J Vet Res 2002 Jul;66(3):181-90

Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078, USA.

The prevalence of bovine viral diarrhea virus (BVDV) infections was determined in 2 groups of stocker calves with acute respiratory disease. Both studies used calves assembled after purchase from auction markets by an order buyer and transported to feedyards, where they were held for approximately 30 d. In 1 study, the calves were mixed with fresh ranch calves from a single ranch. During the studies, at day 0 and at weekly intervals, blood was collected for viral antibody testing and virus isolation from peripheral blood leukocytes (PBLs), and nasal swabs were taken for virus isolation. Samples from sick calves were also collected. Serum was tested for antibodies to bovine herpesvirus-1 (BHV-1), BVDV1a, 1b, and 2, parainfluenza 3 virus (PI3V), and bovine respiratory syncytial virus (BRSV). The lungs from the calves that died during the studies were examined histopathologically, and viral and bacterial isolation was performed on lung homogenates. BVDV was isolated from calves in both studies; the predominant biotype was noncytopathic (NCP). Differential polymerase chain reaction (PCR) and nucleic acid sequencing showed the predominant subtype to be BVDV1b in both studies. In 1999, NCP BVDV1b was detected in numerous samples over time from 1 persistently infected calf; the calf did not seroconvert to BVDV1a or BVDV2. In both studies, BVDV was isolated from the serum, PBLs, and nasal swabs of the calves, and in the 1999 study, it was isolated from lung tissue at necropsy. BVDV was demonstrated serologically and by virus isolation to be a contributing factor in respiratory disease. It was isolated more frequently from sick calves than healthy calves, by both pen and total number of calves. BVDV1a and BVDV2 seroconversions were related to sickness in selected pens and total number of calves. In the 1999 study, BVDV-infected calves were treated longer than noninfected calves (5.643 vs 4.639 d; P = 0.0902). There was a limited number of BVDV1a isolates and, with BVDV1b used in the virus neutralization test for antibodies in seroconverting calves' serum, BVDV1b titers were higher than BVDV1a titers. This study indicates that BVDV1 strains are involved in acute respiratory disease of calves with pneumonic Mannheimia haemolytica and Pasteurella multocida disease. The BVDV2 antibodies may be due to cross-reactions, as typing of the BVDV strains revealed BVDV1b or la but not BVDV2. The BVDV1b subtype has considerable implications, as, with 1 exception, all vaccines licensed in the United States contain BVDV1a, a strain with different antigenic properties. BVDV1b potentially could infect BVDV1a-vaccinated calves.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC227002PMC
July 2002
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