Genetic parameters of resistance to pasteurellosis using novel response traits in rabbits.

BACKGROUND: Pasteurellosis (Pasteurella infection) is one of the most common bacterial infections in rabbits on commercial farms and in laboratory facilities. Curative treatments using antibiotics are only partly efficient, with frequent relapses. Breeding rabbits for improved genetic resistance to pasteurellosis is a sustainable alternative approach. In this study, we infected 964 crossbred rabbits from six sire lines experimentally with Pasteurella multocida. After post-mortem examination and bacteriological analyses, abscess, bacteria, and resistance scores were derived for each rabbit based on the extent of lesions and bacterial dissemination in the body. This is the first study to use such an experimental design and response traits to measure resistance to pasteurellosis in a rabbit population. We investigated the genetic variation of these traits in order to identify potential selection criteria. We also estimated genetic correlations of resistance to pasteurellosis in the experimental population with traits that are under selection in the breeding populations (number of kits born alive and weaning weight). RESULTS: Heritability estimates for the novel response traits, abscess, bacteria, and resistance scores, ranged from 0.08 (± 0.05) to 0.16 (± 0.06). The resistance score showed very strong negative genetic correlation estimates with abscess (- 0.99 ± 0.05) and bacteria scores (- 0.98 ± 0.07). A very high positive genetic correlation of 0.99 ± 0.16 was estimated between abscess and bacteria scores. Estimates of genetic correlations of the resistance score with average daily gain traits for the first and second week after inoculation were 0.98 (± 0.06) and 0.70 (± 0.14), respectively. Estimates of genetic correlations of the disease-related traits with average daily gain pre-inoculation were favorable but with high standard errors. Estimates of genetic and phenotypic correlations of the disease-related traits with commercial selection traits were not significantly different from zero. CONCLUSIONS: Disease response traits are heritable and are highly correlated with each other, but do not show any significant genetic correlations with commercial selection traits. Thus, the prevalence of pasteurellosis could be decreased by selecting more resistant rabbits on any one of the disease response traits with a limited impact on the selection traits, which would allow implementation of a breeding program to improve resistance to pasteurellosis in rabbits.

Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms.

BACKGROUND: Pasteurella multocida type A (PmA) is considered a secondary agent of pneumonia in pigs. The role of PmA as a primary pathogen was investigated by challenging pigs with eight field strains isolated from pneumonia and serositis in six Brazilian states. Eight groups of eight pigs each were intranasally inoculated with different strains of PmA (1.5 mL/nostril of 10e7 CFU/mL). The control group (n = 12) received sterile PBS. The pigs were euthanized by electrocution and necropsied by 5 dpi. Macroscopic lesions were recorded, and swabs and fragments of thoracic and abdominal organs were analyzed by bacteriological and pathological assays. The PmA strains were analyzed for four virulence genes (toxA: toxin; pfhA: adhesion; tbpA and hgbB: iron acquisition) by PCR and sequencing and submitted to multilocus sequence typing (MLST). RESULTS: The eight PmA strains were classified as follows: five as highly pathogenic (HP) for causing necrotic bronchopneumonia and diffuse fibrinous pleuritis and pericarditis; one as low pathogenic for causing only focal bronchopneumonia; and two as nonpathogenic because they did not cause injury to any pig. PCR for the gene pfhA was positive for all five HP isolates. Sequencing demonstrated that the pfhA region of the HP strains comprised four genes: tpsB1, pfhA1, tpsB2 and pfhA2. The low and nonpathogenic strains did not contain the genes tpsB2 and pfhA2. A deletion of four bases was observed in the pfhA gene in the low pathogenic strain, and an insertion of 37 kb of phage DNA was observed in the nonpathogenic strains. MLST clustered the HP isolates in one group and the low and nonpathogenic isolates in another. Only the nonpathogenic isolates matched sequence type 10; the other isolates did not match any type available in the MLST database. CONCLUSIONS: The hypothesis that some PmA strains are primary pathogens and cause disease in pigs without any co-factor was confirmed. The pfhA region, comprising the genes tpsB1, tpsB2, pfhA1 and pfhA2, is related to the pathogenicity of PmA. The HP strains can cause necrotic bronchopneumonia, fibrinous pleuritis and pericarditis in pigs and can be identified by PCR amplification of the gene pfhA2.

Characterization of Demodex musculi Infestation, Associated Comorbidities, and Topographic Distribution in a Mouse Strain with Defective Adaptive Immunity.

A colony of B6.Cg-Rag1tm1Mom Tyrp1B-w Tg(Tcra,Tcrb)9Rest (TRP1/TCR) mice presented with ocular lesions and ulcerative dermatitis. Histopathology, skin scrapes, and fur plucks confirmed the presence of Demodex spp. in all clinically affected and subclinical TRP1/TCR mice examined (n = 48). Pasteurella pneumotropica and Corynebacterium bovis, both opportunistic pathogens, were cultured from the ocular lesions and skin, respectively, and bacteria were observed microscopically in abscesses at various anatomic locations (including retroorbital sites, tympanic bullae, lymph nodes, and reproductive organs) as well as the affected epidermis. The mites were identified as Demodex musculi using the skin fragment digestion technique. Topographic analysis of the skin revealed mites in almost all areas of densely haired skin, indicating a generalized demodecosis. The percentage of infested follicles in 8- to 10-wk-old mice ranged from 0% to 21%, and the number of mites per millimeter of skin ranged from 0 to 3.7. The head, interscapular region, and middorsum had the highest proportions of infested follicles, ranging from 2.3% to 21.1% (median, 4.9%), 2.0% to 16.6% (8.1%), and 0% to 17% (7.6%), respectively. The pinnae and tail skin had few or no mites, with the proportion of follicles infested ranging from 0% to 3.3% (0%) and 0% to 1.4% (0%), respectively. The number of mites per millimeter was strongly correlated with the percentage of infested follicles. After administration of amoxicillin-impregnated feed (0.12%), suppurative infections were eliminated, and the incidence of ulcerative dermatitis was dramatically reduced. We hypothesize that the Rag1-null component of the genotype makes TRP1/TCR mice susceptible to various opportunistic infestations and infections, including Demodex mites, P. pneumotropica, and C. bovis. Therefore, Rag1-null mice may serve as a useful model to study human and canine demodecosis. D. musculi should be ruled out as a contributing factor in immunocompromised mouse strains with dermatologic manifestations.

Genome wide host gene expression analysis in mice experimentally infected with Pasteurella multocida.

Pasteurella multocida causes acute septicemic and respiratory diseases, including haemorrhagic septicaemia, in cattle and buffalo with case fatality of 100%. In the present study, mice were infected with P. multocida (1.6 × 103 cfu, intraperitoneal) to evaluate host gene expression profile at early and late stages of infection using high throughput microarray transcriptome analyses. Several differentially expressed genes (DEGs) at both the time points were identified in P.multocida infected spleen, liver and lungs. Functional annotation of these DEGs showed enrichment of key pathways such as TLR, NF-κB, MAPK, TNF, JAK-STAT and NOD like receptor signaling pathways. Several DEGs overlapped across different KEGG pathways indicating a crosstalk between them. The predicted protein-protein interaction among these DEGs suggested, that the recognition of P. multocida LPS or outer membrane components by TLR4 and CD14, results in intracellular signaling via MyD88, IRAKs and/or TRAF6 leading to activation of NFκB and MAPK pathways and associated cytokines.

The Role of Necroptosis, Apoptosis, and Inflammation in Fowl Cholera-Associated Liver Injury in a Chicken Model.

Fowl cholera resulting from infection with Pasteurella multocida causes huge economic losses in the poultry industry. Necrotic hepatitis is reported to be a significant lesion associated with fowl cholera in chickens. Clarifying the underlying molecular mechanism of hepatic injury caused by P. multocida infection is needed to develop new strategies to control fowl cholera. Pasteurella multocida Q (the standard reference strain) and P. multocida 1G1 (a clinical strain) were used to infect healthy laying hens. Clinical signs were observed and gross lesions in livers were observed postmortem. Histologic lesions and the localization and expression of protein molecules associated with necroptosis, apoptosis, and inflammation in hepatic tissues were examined by hematoxylin and eosin staining and immunohistochemistry. Western blot analysis was used to determine the expression of liver injury-related genes. Necroptotic molecules such as RIPK1 (receptor interaction protein kinases 1), RIPK3 (receptor interaction protein kinases 3), and MLKL (mixed lineage kinase domain-like protein) were observed by immunostaining primarily in the cytoplasm of hepatocytes within or around necrotic foci, and inflammatory mediators HMGB1 (high-mobility group box 1) and IL-6 (interleukin-6) were found in the cytoplasm of heterophils, monocytes/macrophages, and hepatic sinusoids. In addition, MMP9 (matrix metalloproteinase 9) and TIMP1 (tissue inhibitor of metalloproteinase 1) were observed in hepatic parenchymal cells, inflammatory cells, and interstitial spaces, whereas the apoptotic effector molecule caspase-3 (cysteine-containing aspartic proteolytic enzymes 3) was mainly found in hepatocytes. The expression of RIPK1, RIPK3, and MLKL was significantly higher in the infected chickens than in the controls. HMGB1 and IL-6 protein levels were also increased in infected chickens relative to those in controls. Both MMP9 and TIMP1 were highly expressed in infected chickens. In addition, caspase-3 protein levels were significantly elevated in infected chickens. Necroptosis, apoptosis, and inflammation played a significant role in hepatic injury caused by P. multocida.

Neonatal Pulmonary Macrophage Depletion Coupled to Defective Mucus Clearance Increases Susceptibility to Pneumonia and Alters Pulmonary Immune Responses.

Resident immune cells (e.g., macrophages [MΦs]) and airway mucus clearance both contribute to a healthy lung environment. To investigate interactions between pulmonary MΦ function and defective mucus clearance, a genetic model of lysozyme M (LysM) promoter-mediated MΦ depletion was generated, characterized, and crossed with the sodium channel ß subunit transgenic (Scnn1b-Tg) mouse model of defective mucus clearance. Diphtheria toxin A-mediated depletion of LysM(+) pulmonary MΦs in wild-type mice with normal mucus clearance resulted in lethal pneumonia in 24% of neonates. The pneumonias were dominated by Pasteurella pneumotropica and accompanied by emaciation, neutrophilic inflammation, and elevated Th1 cytokines. The incidence of emaciation and pneumonia reached 51% when LysM(+) MΦ depletion was superimposed on the airway mucus clearance defect of Scnn1b-Tg mice. In LysM(+) MΦ-depleted Scnn1b-Tg mice, pneumonias were associated with a broader spectrum of bacterial species and a significant reduction in airway mucus plugging. Bacterial burden (CFUs) was comparable between Scnn1b-Tg and nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice. However, the nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice exhibited increased airway inflammation, the presence of neutrophilic infiltration, and increased levels of inflammatory cytokines in bronchoalveolar lavage fluid compared with Scnn1b-Tg mice. Collectively, these data identify key MΦ-mucus clearance interactions with respect to both infectious and inflammatory components of muco-obstructive lung disease.

Experimental Study of the Pathogenicity of Pasteurella multocida Capsular Type B in Rabbits.

The increased frequency of isolation of Pasteurella multocida capsular type B from rabbitries in north-western India prompted this investigation into the role of this organism in inducing disease in rabbits. Ten rabbits were divided into two groups of five animals. Group I rabbits were infected intranasally (IN) with 1 ml of inoculum containing 2 × 10(5) colony forming units/ml, while rabbits in group II were given 1 ml phosphate buffered saline IN. The rabbits in group I developed respiratory distress, increased rectal temperature and severe dyspnoea, with death occurring 24-48 h post infection. The main pathological findings were severe congestion and haemorrhage in the trachea, fibrinopurulent pneumonia, bacteraemia and septicaemia. The nasal secretions of all group I animals contained P. multocida. These observations indicate that in addition to P. multocida capsular types A and D, P. multocida capsular type B can also be highly pathogenic for rabbits.

Pasteurella multocida toxin interaction with host cells: entry and cellular effects.

The mitogenic dermonecrotic toxin from Pasteurella multocida (PMT) is a 1285-residue multipartite protein that belongs to the A-B family of bacterial protein toxins. Through its G-protein-deamidating activity on the α subunits of heterotrimeric G(q)-, G(i)- and G(12/13)-proteins, PMT potently stimulates downstream mitogenic, calcium, and cytoskeletal signaling pathways. These activities lead to pleiotropic effects in different cell types, which ultimately result in cellular proliferation, while inhibiting cellular differentiation, and account for the myriad of physiological outcomes observed during infection with toxinogenic strains of P. multocida.

Molecular biology of Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT) is the causative agent of progressive atrophic rhinitis in swine. The 146 kDa single-chain toxin harbours discrete domains important for receptor binding, internalisation and biological activity. The molecular basis of the toxin's activity is the deamidation of a specific glutamine residue in the α-subunit of heterotrimeric G proteins. This results in an inhibition of the inherent GTPase activity leading to a constitutively active phenotype of the G protein. Due to the ability of the toxin to act on various families of heterotrimeric G proteins, a large subset of signal transduction pathways is stimulated.

Pasteurella multocida lipopolysaccharide: the long and the short of it.

Pasteurella multocida is a capsulated, gram-negative cocco-bacillus that can cause serious disease in a wide range of mammals and birds. P. multocida strains are classified into 16 serovars based on lipopolysaccharide (LPS) antigens. LPS is an essential virulence factor of P. multocida; mutants expressing severely truncated LPS are completely attenuated in chickens. LPS is also a major immunogen of P. multocida and protection against infections caused by P. multocida is generally considered to be serovar specific. In this review we summarize current knowledge of the structure and genetics of LPS assembly of P. multocida strains belonging to five different serovars. These include strains belonging to serovars 1 and 3, the most common serovars found in the poultry industry, and strains belonging serovars 2 and 5, the serovars associated with bovine haemorrhagic septicaemia outbreaks. A number of the serovars are genetically related; serovars 1 and 14 share the same LPS outer core biosynthesis locus, but due to a mutation within the phosphocholine biosynthesis gene, pcgA, the serovar 14 strain produces a truncated LPS structure. Similarly serovars 2 and 5 share an identical LPS outer core locus and express near-identical LPS structures. However, due to a single point mutation in the phosphoethanolamine (PEtn) transferase gene, lpt_3, the serovar 2 strain does not elaborate a PEtn residue on heptose II. Knowledge of the genetic basis for the LPS structures expressed by P. multocida will facilitate the development of rapid molecular methods for typing and diagnosis and will be essential for a rational approach to vaccine formulation.

Molecular epidemiology of Japanese avian Pasteurella multocida strains by the single-enzyme amplified fragment length polymorphism and pulsed-field gel electrophoresis.

Molecular epidemiology analyses of the 36 clinical isolates of Pasteurella multocida from various avian hosts in Japan between 1976 to 2007 including 5 reference strains from the U.S.A., Taiwan and Indonesia were performed by employing the single-enzyme amplified fragment length polymorphism (SE-AFLP) comparison with the classical ApaI-based pulsed-field gel electrophoresis (PFGE). As the results, SE-AFLP gave 21 profiles while PFGE gave 20 profiles. The Simpson's index of diversity analysis indicated that SE-AFLP gave a high discrimination power than PFGE. This concluded that SE-AFLP is a higher discrimination power than PFGE to differentiate avian P. multocida isolates in Japan. In addition, the genetical profiles suggested that there is the evolution of somatic serotype 3 strain in the indigenous host of Japan.

Identification of Riemerella anatipestifer genes differentially expressed in infected duck livers by the selective capture of transcribed sequences technique.

Riemerella anatipestifer is the causative agent of duck septicaemia. Determination of R. anatipestifer virulence mechanisms will help us to effectively control this contagious agent. The differentially expressed gene profile of R. anatipestifer in infected duck livers was therefore identified and compared with in vitro cultures by selective capture of transcribed sequences analysis. A total of 48 genes were identified, of which 43 were genes that encode enzymes for amino acid biosynthesis and metabolism, intermediary metabolism, and energy metabolism, or proteins for regulatory adaptive responses, general microbial stress response, transport proteins and secreted proteinases. Five were unknown, novel genes. Eight genes representing the categories were randomly chosen and verified by real-time reverse transcriptase-polymerase chain reaction analysis. All were upregulated by R. anatipestifer in infected duck livers, with changes ranging from 1.44-fold to 4.62-fold compared with in vitro cultures. The results from the present study revealed a gene expression profile of R. anatipestifer in infected duck livers. The unknown but novel genes may be potential novel virulence factors for R. anatipestifer. In conclusion, the data from this study will provide a molecular basis for further study of R. anatipestifer pathogenesis.

Direct identification of Photobacterium damselae subspecies piscicida by PCR-RFLP analysis.

Fish pasteurellosis is an infectious disease that affects several teleost species living in temperate marine waters. The pathogen responsible, Photobacterium damselae subspecies piscicida, shows high genetic similarity with P. damselae subsp. damselae, making subspecies discrimination extremely laborious. Here we report for the first time a PCR-RFLP method for the identification of P. damselae subsp. piscicida without prior isolation in pure culture. Genomic sequence information was obtained through cloning and sequencing of RAPD products. Two P. damselae-specific primer pairs were developed and tested on 17 strains of P. damselae subsp. piscicida, 10 strains of P. damselae subsp. damselae, and 6 closely related control species. High sensitivity was achieved in PCR amplification on serially diluted samples (<180 fg of pure bacterial DNA or <10 fg, depending on the amplified fragment). Restriction analysis of PCR products showed a unique digestion profile for all P. damselae subsp. piscicida strains. The same PCR-RFLP method was implemented on total DNA samples extracted from experimentally infected sea bream and sea bass. Positive results were obtained on fish with clear signs of the disease as well as on challenged, but asymptomatic, fish. The method presented here might provide a useful tool for both prevention and rapid diagnosis of fish pasteurellosis.

Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian, ovine and porcine origin.

One hundred and fifty-three bovine Pasteurella multocida strains recovered primarily from cases of pneumonia and mastitis in England and Wales over an 11-year period were characterised by capsular PCR typing, comparison of outer membrane protein (OMP) profiles, and multilocus sequence analysis. All of the strains were of capsular type A with the exception of a single capsular type F isolate. Thirteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. However, 85% of the isolates were represented by just five OMP-types and 39% of the strains were of a single OMP-type. Multilocus sequence analysis revealed a limited degree of genetic diversity among bovine P. multocida isolates; strains of the same OMP-type have identical genetic backgrounds and represent distinct clones. Analysis of OMP variation was more discriminating than multilocus sequence analysis because strains of different OMP-types had the same, or similar, genetic backgrounds. The association of a small number of clones with the majority of cases of bovine pneumonia suggests that these clones have an increased capacity to cause disease compared to less frequently recovered clones. Molecular mass heterogeneity of OmpA and OmpH, in strains of the same or similar genetic background, suggests that these proteins are subject to diversifying selection within the host and might play important roles in host-pathogen interactions. Comparison of the OMP profiles of bovine isolates with those of avian, ovine and porcine strains showed that a high proportion of the respiratory tract infections in each of these species are caused by different strains of P. multocida. However, the presence of small numbers of closely related strains in more than one host species suggests that transmission of bacteria between different host species is also a factor in the population biology of P. multocida.

Functional characterization of HgbB, a new hemoglobin binding protein of Pasteurella multocida.

The biological function and role in pathogenesis of a Pasteurella multocida A:1 strain hemoglobin binding protein was investigated. The hgbB gene from the P. multocida A:1 strain, VP161, was cloned and characterized. hgbB was 2991 bp in length and encoded a mature length protein of 111 kDa. HgbB was predicted to be an outer membrane protein and shared 68 and 69% similarity to the hemoglobin/hemoglobin-haptoglobin binding protein, HI0712 from Haemophilus influenzae Rd and HgpC, from H. influenzae b, respectively. HgbB exhibited features typical of TonB dependent receptors, including seven conserved regions typical of these proteins, and conserved invariant residues. Escherichia coli expressing recombinant HgbB was found to bind hemoglobin in a solid phase dot blot binding assay. However, when a truncated form of the protein was expressed in E. coli, cells could no longer bind hemoglobin. Insertional inactivation of hgbB did not affect the ability of P. multocida to bind hemoglobin, nor its ability to produce disease in a mouse model. In addition, recombinant HgbB did not confer any protection against homologous or heterologous challenge.

Characterization of Pasteurella multocida isolates from wetland ecosystems during 1996 to 1999.

We cultured 126 Pasteurella multocida isolates, 92 from water and 34 from sediment samples collected from wetlands in the Pacific and Central flyways of the United States between 1996 and 1999. Most (121) of the isolates were P. multocida serotype 1, but serotypes 3, 3/4, 10, and 11 were also found. Many (82) of the isolates were further characterized by DNA fingerprinting procedures and tested in Pekin ducks for virulence. Almost all the serotype 1 isolates we tested caused mortality in Pekin ducks. Serotype 1 isolates varied in virulence, but the most consistent pattern was higher mortality in male ducks than in females. We found no evidence that isolates found in sediment vs. water, between Pacific and Central flyways, or during El Niño years had consistently different virulence. We also found a number of non-serotype 1 isolates that were avirulent in Pekin ducks. Isolates had DNA fingerprint profiles similar to those found in birds that died during avian cholera outbreaks.

Tetracycline resistance genes in isolates of Pasteurella multocida, Mannheimia haemolytica, Mannheimia glucosida and Mannheimia varigena from bovine and swine respiratory disease: intergeneric spread of the tet(H) plasmid pMHT1.

Tetracycline-resistant isolates of Pasteurella multocida and Mannheimia spp. from respiratory diseases in cattle and swine were investigated for the classes of tet gene and their chromosomal or plasmid location. The 34 isolates comprised eight P. multocida, 23 Mannheimia haemolytica, two Mannheimia varigena and a single Mannheimia glucosida isolate. Identification of the tet genes was achieved by PCR analysis and hybridization with specific probes. Transformation and hybridization experiments served to confirm the plasmid location of tet genes. Selected tet genes and their adjacent regions were sequenced. The tet genes tet(B), tet(G) and tet(H) were detected. The gene tet(H) was present in 26 isolates. The 4.4 kb tet(H)-carrying plasmid pMHT1 was detected in six isolates representing all four species. In the remaining 28 isolates, copies of tet(B), tet(G) and tet(H) were identified as chromosomal. No correlation between the tet gene type and the MIC of tetracycline, or between the number of tet gene copies and the MIC of tetracycline was observed. Tetracycline resistance in P. multocida and Mannheimia spp. is mediated by at least three different tet genes. A new type of tet(H)- carrying plasmid, pMHT1, was identified. The detection of pMHT1 in M. glucosida and M. varigena is the first report of resistance plasmids in isolates of these two species. For the first time, tet(G) genes were detected in members of the family Pasteurellaceae.

MHCII, Tlr4 and Nramp1 genes control host pulmonary resistance against the opportunistic bacterium Pasteurella pneumotropica.

MHCII, Tlr4, and Nramp1 genes are each independently important in pulmonary immunity. To determine the effect of these genes on host resistance, mice carrying various combinations of functional alleles for these three genes were experimentally challenged with the opportunistic bacterium, Pasteurella pneumotropica. MHCII-/-, Tlr4d/d, and Nramp1s/s mice were significantly more susceptible to experimental infections by P. pneumotropica after intranasal challenge compared to mice carrying functional alleles at only one of those genes. P. pneumotropica were cultured from the lungs of challenged mice, and the severity of the pneumonia strongly correlated with the number of isolated bacteria. Mice with the genotype MHCII-/- Tlr4n/n genotype were less susceptible to pneumonia than MHCII+/+, Tlr4d/d mice. It is interesting that the Nramp1 gene contribution to host resistance was apparent only in the absence of functional MHCII or Tlr4 genes. These data suggest that MHCII, Tlr4, and Nramp1 genes are important to pulmonary bacterial resistance.