Pasteurella Multocida Serogroup Research Articles

Pathogenic and genomic characterization of rabbit-sourced Pasteurella multocida serogroup F isolates recovered from dead rabbits with respiratory disease.

The 19 rabbit-sourced Pasteurella multocida serogroup F isolates showing high virulence to rabbits were avirulent to the broilers. Notably, polymorphisms were determined in the lipopolysaccharide outer core biosynthetic genes natC and gatF among all serogroup F strains of different hosts. However, the sequences of natC and gatF from rabbit-sourced strains (except for SD11) were identical, which might be responsible for the host specific of the 19 isolates.

Antimicrobial susceptibility screening test of Mannheimia haemolytica and Pasteurella multocida (serogroup A) Moroccan strains isolated from ruminants

The present study is the first report aimed to determine the antibiotic susceptibility profiles of Mannheimia haemolytica and Pasteurella multocida serogroup A Moroccan isolates. Each isolate was tested for sensitivity to amoxicillin (A), amoxicillin/clavulanic acid (AMC), gentamicin (CN), streptomycin (STR), florfenicol (FFC), doxycycline (DO), erythromycin (E), spiramycin (SP), nalidixic acid (NA), flumequine (UB), enrofloxacin (ENF) and sulfamethoxazole (SXT). All isolates showed resistance to the antibiotics tested at a rate greater than 14%, except for one P. multocida isolate which had no resistance profile against AMC. The highest level of resistance was found against NA for P. multocida (100%) and against UB (82.4%) for M. haemolytica. The sensitivity rates for P. multocida were between 0 (against NA) and 85.7% (against STR, AMC, FFC), whereas sensitivity of M. haemolytica isolates ranged from 17.6% against UB and 79.4% against AMC. For both Pasteurellaceae species, the AMC was the most effective antimicrobial agent, however multi-drug resistance was observed in all isolates raising the interest to monitor the antimicrobial susceptibility of Pasteurellaceae species to determine appropriate antibiotic for treatment of pasteurellosis.

Conservation of ptfA gene encoded Type IV fimbrial protein among circulating Pasteurella multocida serogroup A strains causing pneumonia in sheep

Respiratory infections are most common in small ruminants. Differentiation of homogenous bacterial strains originated from similar clinical forms (acute or chronic) of infections in a particular or diverse host origin and regions posses a greater challenge for rapid epidemiological studies. Pasteurella multocida, being a multi-host pathogen with wide range of infections among small ruminants especially sheep, is of greater economical concern among small and marginal farmers. In our study, we report ptfA gene sequence based analysis of circulating P. multocida strains recovered from clinically ailing sheep either with pneumonia or septicaemia belonging to different geographical regions of Karnataka. All the 29 P. multocida strains were characterized by conventional methods as well as molecular methods which indicated homogeneity as they belonged to serogroup A and possessed highly conserved ptfA gene by phylogenetic analysis. The study highlighted the conservation of ptfA gene/fimbrial protein among P. multocida strains from identical/diverse clinical conditions and could be employed in rapid epidemiological studies in routine surveillance of circulating pathogenic bacterial strains as well as pasteurellosis outbreak investigations among animals and birds.

L-Ascorbic Acid Shapes Bovine Pasteurella multocida Serogroup A Infection.

Bovine Pasteurella multocida serogroup A (bovine PmA) is one of the most important pathogens causing fatal pneumonia in cattle. However, it is largely unknown how nutrition shapes bovine PmA infection. Here, we discovered that the infected lung held the highest bacterial density than other tissues during infection. By screening the different metabolites between high (lung)- and low (liver)-bacterial density tissues, the present work revealed that L-ascorbic acid and L-aspartic acid directly influenced bovine P. multocida growth. Interestingly, L-ascorbic acid, which is expressed at higher levels in the infected livers, inhibited bovine PmA growth as well as virulence factor expression and promoted macrophage bactericidal activity in vitro. In addition, ascorbic acid synthesis was repressed upon bovine PmA infection, and supplementation with exogenous L-ascorbic acid significantly reduced the bacterial burden of the infected lungs and mouse mortality. Collectively, our study has profiled the metabolite difference of the murine lung and liver during bovine PmA infection. The screened L-ascorbic acid showed repression of bovine PmA growth and virulence expression in vitro and supplementation could significantly increase the survival rate of mice and reduce the bacterial load in vivo, which implied that L-ascorbic acid could serve as a potential protective agent for bovine PmA infection in clinic.

English

Colistin is an important drug of last resort against lethal infections with multidrugresistant gram-negative bacteria, this antibiotic interacts on phosphate groups of lipopolysaccharides present on the outer membrane of gram-negative bacteria. This first mechanism of transferable colistin resistance involves a gene called mcr-1. Here, we described different tools such as Colistin susceptibility test, Minimal Inhibitory Concentration to find the colistin profiles of Mannheimia haemolytica and Pasteurella multocida serogroup A Moroccan strain isolated from nasal swabs and lung taken from sheep, goat and cattle with respiratory diseases during January 2015 to December 2017 in six different regions, in addition, we investigated either real time PCR to detect mcr-1 gene. Antimicrobial sensitivity test, was achieved for Fortyone isolates, the resistance rates of isolates from Pasteurellaceae species were between 59 and 71%, which respectively correspond to Mannheimia haemolytica and Pasteurella multocida segroup A. The sensitive ratios were between 29 and 41% which respectively correspond to Pasteurella multocida segroup A and Mannheimia haemolytica. Also, the MIC test was done against colistin, the results showed a resistant profile with a MIC >=64 μg/ml except for 3 strains from ruminants which have a value <2 μg/ml. The real time PCR screening test was detected with ct values ranging from 23 to 31 and confirmed the results obtained by MIC test. To our knowledge the present study is the first study which reports the resistance to colistin in Pasteurellaceae species strains isolated from ruminants in Morocco, our results suggest the necessity and the urgency for establishing a national program for monitoring antibacterial resistance against colistin

RESPIRATORY TRACT INFECTIONS IN CATTLE

In the article a literature review of Bovine respiratory diseases (BRD) is presented. Respiratory diseases are considered to be one of the most harmful diseases of cattle, which cause great economic damage for the operators of the cattle industry. The BRD complex is a multifactorial and multi-etiological disease. The BRD complex is a multifactorial and multi-etiological disease. The main factors providing the BRD development are the management status of rearing cattle, the impact of the environment and pathogens. Without neglecting the importance of the first two factors, pathogenic microorganisms remain the major etiological factor of BRD. Respiratory tract infections in cattle are caused by viruses and bacteria, moreover the diseases often develop in an associated form. However, the bacterial factor in the etiology of respiratory diseases plays a main role. Mannheimia haemolytica serotype 1 is the main pathogen of BRD, which can cause disease as a single etiologic agent and as in association with other pathogens (Histophilus somni, Mycoplasma bovis). In most cases, fibrinous pneumonia or fatal acute pneumonia is often associated with Mannheimia haemolytica. Pasteurella multocida is considered to be a less virulent bacteria than Mannheimia haemolytica, and for a higher level of infection need to initiate the inflammatory process in the respiratory tract of animals. Pathogenic strains of Pasteurella multocida serogroup A are a significant etiologic factor of severe enzootic pneumonia in dairy calves. Respiratory diseases caused by mycoplasma remain one of the serious infectious diseases of cattle. Mycoplasma bovis is the most invasive and dangerous mycoplasma for young cattle. This type of mycoplasma is usually present in the upper respiratory tract of clinically healthy calves who are bacterial carriers. When the zootechnical conditions of brieding and feeding the calves are disturbed and for other stress factors there is an active proliferation of mycoplasmas and they successfully colonize the lower respiratory tract of the animals, causing an inflammatory process in the lungs. Other commensal bacteria of the upper respiratory tract, Histophilus somni, can cause pneumonia that usually occurs in subacute or chronic form. The pathogenic forms of this bacteria are often isolated together with Mannheimia hemolytica. Other opportunistic bacteria (Arcanobacterium pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Chlamydiales spp., Fusobacterium necrophorum, Corynebacterium bovis) may be etiological factors for the development of BRD. Depending on the etiologic agent, the clinical symptoms of calf bronchopneumonia have some specificity, herewith the degree of lung damage depends on the duration of the disease and the virulence of the pathogen.

Immunogenicity of recombinant outer membrane protein (OmpW) of Pasteurella multocida serogroup B:2 in mouse model

Haemorrhagic septicaemia (HS) caused by Pasteurella multocida serogroup B:2, in cattle and buffalo especially in tropical regions of Asian and African countries, is known to possess several outer membrane proteins (OMPs) as virulent factors which are being targeted to evaluate their immunogenicity and protective efficacy as candidate antigens for vaccine. In the present study, ompW gene encoding for OmpW protein of P. multocida serogroup B:2 strain P52, an Indian HS vaccine strain, has been cloned and over-expressed in recombinant Escherichia coli. The recombinant OmpW fusion protein (~37 kDa) including histidine tag was purified by affinity chromatography under denaturing condition and confirmed by Western blotting. Further, mice immunized with rOmpW (50μg/ dose) along with FCA/FIA resulted in antigen specific IgG antibodies as well as subtypes (IgG1 and IgG2a). The study indicated the potential possibilities to use the rOmpW antigen in developing subunit vaccine for HS as well as other diseases caused by members of Pasteurellaceae.

Molecular Identification and Serogrouping of Pasteurella mutocida Field Isolats

Haemorrhagic septicaemia (HS) is a serious disease affecting buffalo and cattle worldwide mainly caused by Pasteurella multocida (PM) serogroup B and E. In Indonesia cases of HS have been reported every year from 2005 to 2017. The bacterial identification was done primarily based on morphological and biochemical characteristics, while the serogroup has never been determined. This research was aimed to implement molecular biological techniques for identification and serogrouping of three archival PM isolates; two isolates were originated from HS cases in South Sulawesi (SulSel) and one isolate from East Nusa Tenggara (NTT). Polymerase chain reaction (PCR) amplifications of PM family-specific gene 16SrRNA, species-specific gene kmt and serotype-specific gene bcbD identified all of the isolates were PM, which was substantiated by inoculation of VITEK® 2 compact. The three PM isolates were belong to capsular serogroup B. Sequence analysis of 690 nucleotides of the capsular gene and the respective hypothetical amino acid was further confirmed their identity. Two isolates of SulSel and NTT were 100% identical to M1404 B: 2 (AF169324.1), meanwhile one silent mutation was noted in the other SulSel isolate. These finding substantiate the application of PCR as a rapid, sensitive and specific method for PM identification and characterization.

Host responses and bacterial colonization following inoculation of Pasteurella multocida P52 strain into unvaccinated and aluminum hydroxide gel hemorrhagic septicemia vaccinated mice

Hemorrhagic septicemia is a highly infectious and contagious disease caused by Pasteurella multocida serogroup B:2 in tropical Asian and African countries. The acute inflammatory responses induced by Pasteurella multocida are the main cause of death in hemorrhagic septicemia. Therefore, present study was undertaken to examine the blood cytokine expression profiles (TNF-α, IL-1β, and IL-6), bacterial colonization and histopathological changes of intraperitoneally and subcutaneously challenged vaccinated and unvaccinated mice with 102 CFU of P. multocida P52. The observations were made at 6, 12, 18, 24 h and 48 h intervals. Real-time PCR based blood cytokine profiles (TNF-α, IL-1β, and IL-6) measurement revealed a significantly higher amount of pro-inflammatory cytokines expression in the unvaccinated challenged group of mice than the vaccinated challenged group. There was heavy bacterial load in all organs of mice viz. trachea, lung, spleen, within 6 h of challenge in both vaccinated and unvaccinated group of mice, but bacterial load increased in the unvaccinated challenged group of mice with respect to time whereas the load were constant in the vaccinated challenged group. Histopathological changes were mild in the vaccinated challenged group of mice in comparison to the unvaccinated challenged group. There was no significant difference in the bacterial load, histopathological changes and cytokines expression when challenged through different routes.

Experimental pathogenicity and complete genome characterization of a pig origin Pasteurella multocida serogroup F isolate HN07

Pasteurella multocida serotype F isolates are predominately prevalent in avian hosts, but rarely seen in pigs. However, we isolated several strains of P. multocida serotype F from clinical samples of pigs in China. To understand the pathogenicity of these strains, one of the serotype F isolates designated HN07, was used to challenge experimental chickens, as P. multocida of this serotype is predominately prevalent in avian hosts. However, strain HN07 could not resulted in significant clinical signs in experimental chickens even at an infective dose of ∼109 CFU, suggesting the isolate was avirulent to chickens and therefore raising the possibility that the porcine serotype F isolate is not transmitted by chickens. We then used HN07 to challenge experimental pigs, as this strain was isolated from pigs. As expected, the strain led to the clinical signs and the pathological lesions in experimental pigs that are similar to the pasteurellosis disease. We then determined the complete genome sequence of the pig origin serogroup F isolate HN07 for the first time. Genome comparison between HN07 and the avian serotype F P. multocida Pm70 identified a novel integrative conjugative element (ICE) ICEpmcn07 which was likely to harbor a series of genes responsible for a putative type IV secretion system (T4SS) in HN07. This is the first time that we determined an ICE carrying a T4SS in P. multocida. Besides, comparative analysis also defined a number of virulence-associated genes in HN07 but absent in Pm70 which may have a contribution to the pathogenicity of the strain. This is the first report of the pathogenicity and genome characterization of a pig origin Pasteurella multocida serogroup F isolate. The pathogenic and genomic definition of the pig origin P. multocida serogroup F in our study would have significance on the pathogenesis and genetic diversity and virulence variability of P. multocida.

Recombinant transferrin binding protein A (rTbpA) fragments of Pasteurella multocida serogroup B:2 provide variable protection following homologous challenge in mouse model

Transferrin binding protein A (TbpA), an iron acquisition surface protein that also acts as virulence factor, is widely distributed among strains of Pasteurella multocida. In the present study, a total of seven clones of TbpA fragments (39D to F777; 39D to Q697; 188V to F777; 188V to Q697; 39D to P377; 188V to P377 and 39D to F187) belonging to P. multocida B:2 were constructed, over-expressed and purified as recombinant fusion proteins from Escherichia coli using affinity chromatography. Immunization of mice with rTbpA fragments resulted in a significant (p < 0.05) rise in antigen specific serum total IgG and subtypes (IgG1 and IgG2a) tires. All immunized mice challenged with 8 LD50 of P. multocida B:2 resulted in a variable protective efficacy up to 50%. The study indicated the potential possibilities to incorporate full length TbpA in subunit vaccine formulation composed of synergistic subunit antigens against haemorrhagic septicaemia (HS) in cattle and buffalo.

Structural analysis and cross-protective efficacy of recombinant 87 kDa outer membrane protein (Omp87) of Pasteurella multocida serogroup B:2

Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic septicaemia (HS) in cattle and buffalo especially in tropical regions of Asian and African countries, is known to possess several outer membrane proteins (OMPs) as immunogenic antigens. In the present study, omp87 gene encoding for 87 kDa OMP (Omp87) protein of P. multocida serogroup B:2 strain P52, has been amplified (∼2304 bp), cloned in to pET32a vector and over-expressed in recombinant Escherichia coli as fusion protein. The recombinant Omp87 protein (∼102 kDa) including N-terminus hexa-histidine tag was purified under denaturing condition. Immunization of mice with rOmp87 resulted in increased antigen specific IgG titres in serum and provided protection of 66.6 and 83.3% following homologous (B:2) and heterologous (A:1) challenge, respectively. A homology model of Omp87 revealed the presence of two distinct domains; N-terminal domain with four POTRA repeats in the periplasmic space and a pore forming C-terminal β-barrel domain (β1- β16) in the outer membrane of P. multocida, which belong to Omp85-TpsB transporter superfamily of OMPs. The study indicated the potential possibilities to use rOmp87 protein along with suitable adjuvant in developing subunit vaccine for haemorrhagic septicaemia and pasteurellosis in livestock.

Expression and purification of recombinant type IV fimbrial subunit protein of Pasteurella multocida serogroup B:2 in Escherichia coli

Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic secpticaemia (HS) in cattle and buffalo especially in tropical regions of Asia and African countries, is known to possess a type IV fimbriae (pili) as one of the virulent factors. In the present study, ptfA gene encoding for type IV fimbrial subunit of P. multocida serogroup B:2 (strain p52), an Indian HS vaccine strain, has been cloned and over-expressed in recombinant Escherichia coli. The recombinant type IV fimbrial subunit protein (∼31kDa) including N-terminus histidine tag was purified under denaturing condition and confirmed by western blotting. A homology model of HS causing P. multocida serogroup B:2 fimbrial subunit has also been discussed. The study indicated the potential possibilities to use the recombinant fimbrial protein in developing HS subunit vaccine along with suitable adjuvant.

Antigenicity of partial fragments of recombinant Pasteurella multocida toxin.

Pasteurella multocida serogroup D strain, which produces P. multocida toxin (PMT), is a widespread and harmful pathogen of respiratory diseases such as pneumonia and progressive atrophic rhinitis (PAR) in swine. Vaccination has been considered the most desirable and effective approach for controlling the diseases caused by toxigenic P. multocida. To investigate the antigenicity and immunogenicity of partial fragments of recombinant PMT, recombinant proteins of the N-terminal (PMT-A), middle (PMT-B), Cterminal (PMT-C), and middle-C-terminal (PMT2.3) regions of PMT were successfully produced in an Escherichia coli expression system. The molecular masses of PMT-A, PMT-B, PMT-C, and PMT2.3 were ca. 53, 55, 35, and 84 kDa, respectively, purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. All the recombinant proteins except for PMT-A showed immune responses to antisera obtained from a swine showing symptoms of PAR. Moreover, high titers of PMT-specific antibodies were raised from mice immunized with each of the recombinant proteins; however, the immunoreactivities of the antibodies to authentic PMT and heat-inactivated whole bacteria were different, respectively. In the protection study, the highest protection against homologous challenge was shown in the case of PMT2.3; relatively poor protections occurred for the other PMT fragments.

Experimental study of pathogenicity of Pasteurella multocida serogroup F in rabbits

The role of Pasteurella multocida serogroup F in inducing disease in rabbits was investigated in this study. Three groups of 12 Pasteurella-free rabbits each were intranasally (i.n.), subcutaneously (s.c.), and perorally (p.o.) challenged, respectively. Six rabbits of each group were immunosuppressed using dexamethasone. Eight rabbits (four of them immunosuppressed) inoculated i.n. showed symptoms of respiratory distress resulting in respiratory failure and died or were euthanized in the terminal stage of the disease 3–6 days post-infection (p.i.). The main pathological findings were fibrinopurulent pleuropneumonia (immunocompetent rabbits) or diffuse haemorrhagic pneumonia (immunosuppressed rabbits). Septicemic syndrome ending with shock occurred in 11 rabbits (6 of them immunosuppressed) inoculated s.c., which died or were euthanized in the terminal stage of the disease 2–3 days p.i. The most significant pathological findings were extensive cutaneous and subcutaneous lesions. All of the p.o. inoculated rabbits survived the challenge showing no clinical signs of the disease and no macroscopic lesions. The observations in this study indicate that in addition to serogroups A and D of P. multocida, serogroup F also can be highly pathogenic for rabbits and therefore might be a cause of considerable economic loss in commercial rabbit production.

Molecular characterization of Avian strains of Pasteurella multocida serogroup-A:1 based on amplification of repetitive regions by PCR

Repetitive extragenic palindromic (REP)-PCR (polymerase chain reaction), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and single primer PCR assays were employed to characterize 66 strains of Pasteurella multocida serogroup A:1 isolated from avian species belonging to different regions of India. REP-PCR resulted in amplification of REP sequences from the genome which were in the range of approximately 200 to approximately 3000 bp and accounted for a total of 54 distinguishing profiles (D=0.99). ERIC-PCR analysis also generated amplified products in the range of approximately 200 to approximately 3200 bp categorizing strains into a total of 50 different profiles (D=0.98). Amplification of repetitive regions using a microsatellite primer (GTG)(5), resulted in clear distinctive bands ranging from approximately 200 to approximately 2400 bp. Strains were assigned to 43 profiles (D=0.96). No correlation could be drawn between genotypic profiles and avian hosts with their geographical area of origin. Avian strains of P. multocida serogroup A:1 were found to be highly heterogeneous with diverse profiles. REP-PCR was found to be highly discriminatory and simple method for differentiation of phenotypically similar strains. The present study also indicated that PCR based amplification of repetitive regions of P. multocida is a rapid technique with good discrimination and could be employed directly for routine typing of field isolates from fowl cholera outbreaks.

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468 bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P 52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).

Vaccination against fowl cholera with acapsular Pasteurella multocida A:1

We have previously constructed an acapsular Pasteurella multocida X-73 (serogroup A) mutant strain which was attenuated in virulence for chickens (Chung JY, Wilkie IW, Boyce JD, Townsend KM, Frost AJ, Ghodussi M, Adler B. Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A. Infect. Immun. 2001;69:2487–2492). In this study, we have assessed the ability of this acapsular strain (PBA930) to induce protection against wild-type challenge in mice and the natural host chickens. Intramuscular administration of PBA930 to mice stimulated significant protection against X-73 and the heterologous strain P-1059 (A:3), but not against challenge with P-1662 (A:4). No protection was observed when PBA930 was introduced by the intraperitoneal or subcutaneous routes in mice. Significantly, the acapsular strain PBA930 was able to induce protection against challenge with wild type X-73 in chickens.

Molecular variability among strains of Pasteurella multocida isolated from an outbreak of haemorrhagic septicaemia in India.

The applicability of conventional and molecular methods for rapid detection and differentiation of Pasteurella multocida serogroup B isolates involved in an outbreak of haemorrhagic septicaemia affecting Indian buffaloes, was studied. Five isolates were obtained and were subjected to phenotypic and genotypic characterization. None of the five isolates could be differentiated on the basis of cultural, biochemical, pathogenicity and antimicrobial sensitivity patterns. Polymerase chain reaction (PCR)-based techniques were found to be specific and sensitive for rapid detection and differentiation of isolates. Repetitive extragenic palindromic (REP-) PCR, enterobacterial repetitive intergenic consensus (ERIC-) PCR and single-primer PCR differentiated all the five isolates into different profiles. All the isolates involved in the outbreak were found to have a genetic profile different from standard P. multocida strain (P52). However, three isolates had similar profiles, whereas each of the remaining two had a different profile. The study indicates the involvement of multiple strains of P. multocida in a single outbreak of haemorrhagic septicaemia in buffaloes. The results also indicate that molecular methods of detection and typing are superior to conventional methods for rapid epidemiological investigations of haemorrhagic septicaemia.

PCR assay for rapid detection of Pasteurella multocida serogroup A in morbid tissue materials from chickens with fowl cholera

PCR assay for rapid detection of Pasteurella multocida serogroup A in morbid tissue materials from chickens with fowl cholera