[Show abstract][Hide abstract] ABSTRACT: Pasteurella multocida serotype B:2 is the causative agent of hemorrhagic septicemia in cattle and buffaloes in Asia. It is an acute fatal disease and is considered one of the most economically important diseases in this region of the world. We present here the draft genome sequences of strains 2213 and 3213 of P. multocida.
[Show abstract][Hide abstract] ABSTRACT: 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 to 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Frontiers in Microbiology 01/2011; 2:163. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ornithobacterium rhinotracheale is a gram-negative bacterium responsible for the sporadic outbreaks of airsacculitis in poultry, accounting for millions of dollars in losses to the poultry industry annually. Although the organism was originally classified as non-β-hemolytic, recent North American field isolates of O. rhinotracheale obtained from pneumonic lungs and air sacs indicated hemolytic activity on blood agar plates upon extended incubation for 48 hr at room temperature in air after initial incubation at 37 C for 48 hr under 7.5% CO2. This report characterizes the β-hemolytic activity of O. rhinotracheale isolates by using in vitro kinetic hemolysis assays with sheep red blood cells, western blotting with leukotoxin-specific monoclonal antibodies, and isobaric tagging and relative and absolute quantitative (iTRAQ) analysis of O. rhinotracheale outer membrane protein digest preparations. The kinetic analyses of the hemolytic activity with red blood cells indicated that the protein is a pore former. iTRAQ analysis with membrane preparations revealed four peptides with homology to Mannheimia haemolytica leukotoxin and two peptides with homology to Actinobacillus actinoacetemcomitans leukotoxin. This is the first report that North American field isolates of O. rhinotracheale may express a hemolysin-like activity.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
103 feedlot calves from New Mexico and 100 from Arkansas.
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.
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.
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.
American Journal of Veterinary Research 09/2006; 67(8):1412-20. · 1.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Canadian journal of veterinary research = Revue canadienne de recherche vétérinaire 05/2006; 70(2):121-7. · 1.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Applied and Environmental Microbiology 12/2005; 71(11):7187-95. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Applied and Environmental Microbiology 12/2005; 71(11):7196-202. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Canadian journal of veterinary research = Revue canadienne de recherche vétérinaire 08/2005; 69(3):161-9. · 1.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.