Pasteurella multocida isolates associated with ovine pneumonia are toxigenic

doi:10.1016/j.vetmic.2019.04.006

Veterinary Microbiology

Volume 232, May 2019, Pages 70-73

https://doi.org/10.1016/j.vetmic.2019.04.006 Get rights and content

Highlights

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Most (94.7%) ovine toxA+ P. multocida isolates express the PMT toxin antigen.
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PMT displayed a cytopathic effect identical to that of porcine toxigenic isolates.
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Isolates of P. multocida associated with pneumonia in sheep are toxigenic.

Abstract

The P. multocida toxin (PMT), a dermonecrotic protein encoded by the toxA gene, is the major virulence factor of capsular type D P. multocida strains causing progressive atrophic rhinitis (PAR) in pigs. A high frequency of P. multocida isolates harboring the toxA gene has been found among ovine pneumonic isolates, although the ability of these isolates to express PMT has never been examined. In this study we have investigated the ability of ovine toxA+ P. multocida isolates (n = 57) to express a functional toxin by detection of PMT toxin antigen using an ELISA test and its cytopathic effect in a Vero cell assay. PMT antigen was expressed in the great majority (54/57; 94.7%) of toxA+ isolates. Moreover, the 100% toxA+ ovine isolates analyzed produced a cytopathic effect in Vero cells within 24–48 h post-inoculation, identical to that described for porcine toxigenic P. multocida isolates. These results show for the first time that, in addition to isolates associated with PAR, isolates of P. multocida associated with pneumonia in sheep are also toxigenic. In addition, we found a total agreement (Kappa = 1; C.I. 0.75–1.25) between the detection of the toxA gene and the toxigenic capability of P. multocida isolates, indicating the PCR detection of toxA would be a suitable predictive marker of the toxigenic fitness of P. multocida.

Introduction

Pasteurella multocida is associated with a number of diseases in domestic and wild animals, including progressive atrophic rhinitis in pigs (PAR) and pneumonia in pigs, cattle and sheep (Harper et al., 2006; Wilson and Ho, 2013). The P. multocida toxin (PMT) is the major virulence factor of P. multocida responsible for the turbinate bone degeneration manifested during infection in PAR (Lax and Chanter, 1990). It is a dermonecrotic protein (146-kDa) encoded by the toxA gene on a lysogenic bacteriophage residing only in the genome of toxicogenic strains (Pullinger et al., 2004). Apart from its well-known role in the pathogenesis of PAR, PMT has several other biological functions. Inoculation of purified PMT can induce liver and kidney damage in addition to atrophy of the nasal turbinate bones in pigs (Lax and Chanter, 1990) and pneumonic lesions in rabbits (Chrisp and Foged, 1991). PMT is also a potent mitogen (Rozengurt et al., 1990) and interacts with several signal transduction pathways to disturb cell growth and differentiation, favoring the evasion of the immune system (Kubatzky et al., 2013). It has been suggested that PMT might dampen the immune response by different toxin-related immune evasion strategies, facilitating the multiplication and survival of P. multocida in the host (Kubatzky et al., 2013).

The synthesis of PMT is mainly associated with capsular type D-toxA+ pig isolates causing PAR, although PMT has been occasionally detected in porcine strains of capsular type A (Djordjevic et al., 1998; Davies et al., 2003). Unexpectedly, a high frequency of toxA+ P. multocida isolates has been found among ovine pneumonic isolates of both capsular types A and D in the last few years (Ewers et al., 2006; Sarangi et al., 2015; Vougidou et al., 2015; Einarsdottir et al., 2016; Shirzad and Tabatabaei, 2016; García-Alvarez et al., 2017), although the ability of these isolates to express PMT has not been investigated. Isolates from sheep and pigs belong to genetically different subpopulations of P. multocida (García-Alvarez et al., 2017). This circumstance together with the fact that not all genes are necessarily expressed (Bavananthasivam et al., 2018) led us to investigate the ability of ovine toxA+ P. multocida isolates to express a functional toxin by detection of PMT toxin antigen using an ELISA test and its cytopathic effect in a Vero cell assay.

Section snippets

P. multocida strains

This study included 57 pneumonic ovine P. multocida isolates harboring the toxA gene (toxA+): 43 capsular type A, 13 capsular type D and one nontypeable (NT) as determined by multiplex PCR (García-Alvarez et al., 2017). Detailed information about the 57 toxA+ ovine isolates is shown in Table 1. In addition, three pneumonic toxA− isolates of capsular types A (M172), D (P69) and NT (M478), were also included as negative controls in both the ELISA and Vero cell assays. A toxigenic capsular type D

Results and discussion

PMT antigen was detected in 54 of the 57 toxA+ isolates (94.7%, C.I. 85.4–98.9%; Table 2). No significant differences (P > 0.05; OR 1.7, CI 0.14–20.5) were observed between isolates of capsular type A (95.3%; 41/43) and D (92.3%; 12/13), indicating that the toxA gene is transcribed and expressed in the great majority of the ovine toxA+ P. multocida isolates investigated, regardless of their capsular type. Analysis of the DNA sequences of toxA gene of strains M297 and M135 (capsular types A and

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

The authors thank Paula Marino Rey for her excellent technical assistance. This study was supported by AGL2009-10136 (Ministerio de Ciencia e Innovación, Spain).

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Pasteurella
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The genus Pasteurella sensu stricto only includes the type species of the genus, Pasteurella multocida, and the species Pasteurella canis, Pasteurella stomatis, Pasteurella dagmatis, and Pasteurella oralis. These species are closely related according to 16S rRNA gene and conserved housekeeping protein sequence phylogeny. Other species are unrelated to P. sensu stricto even though they are validly named Pasteurella. The species excluded from the P. sensu stricto and labeled with the genus name in brackets are only described briefly: [Pasteurella] aerogenes, [Pasteurella] bettyae, [Pasteurella] caballi, [Pasteurella] caecimuris, [Pasteurella] langaaensis, [Pasteurella] mairii, [Pasteurella] skyensis, and [Pasteurella] testudinis. Members of P. sensu stricto are coccobacilli or rods, 0.3–1.0 µm in diameter and 1.0–2.0 µm in length, they are gram-negative, nonmotile, nonacid fast, and endospores are not formed. Growth is aerobic to microaerophilic or facultatively anaerobic. The metabolism is chemoheterotrophic with both oxidative and fermentative types of metabolism. The electron transport system is cytochrome-based with oxygen, nitrate, or fumarate as the terminal electron acceptor. Nitrate reductase is produced. Members of the genus are normally oxidase-positive, and the alkaline phosphatase and the catalase tests are positive. The optimum growth temperature of members of Pasteurella is 35°C–37°C. Members of the genus produce acid from (+)-d-glucose, (+)-d-galactose, (-)-d-fructose, (+)-d-mannose, and sucrose. There is no acid production from adonitol, meso-inositol, (+)-l-rhamnose, and (-)-l-sorbose. Members of the genus have a DNA GC content (mol%) of 36.8–43.5. In human beings, members of Pasteurella are mainly isolated from wound infections inflicted by animals. In this respect, members of the genus are zoonotic, since the main habitat of the genus Pasteurella is the oral cavity and respiratory tract of vertebrates including both mammals and birds. In animals, typical infections associated with P. multocida are hemorrhagic septicemia in cattle and buffaloes, fowl cholera in poultry, atrophic rhinitis in pigs, and snuffles in rabbits.
Toxigenic and non-toxigenic Pasteurella multocida genotypes, based on capsular, LPS, and virulence profile typing, associated with pneumonic pasteurellosis in Iran
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A high prevalence of this gene has been demonstrated in sheep and goat isolates in epidemiological studies (Ewers et al., 2006; Sarangi et al., 2015; García-Alvarez et al., 2017). Although the presence of the toxA gene correlates well with the ability of ovine isolates to produce a functional PMT toxin and therefore these isolates can be classified as toxigenic (Cid et al., 2019), the possible role of this toxin in ovine or caprine isolates remains unknown. Based on the presence or absence of the toxA gene, VP could be classified as toxigenic or non-toxigenic.
Pasteurella multocida is an important cause of pneumonic pasteurellosis in small ruminants. Its prevalence was investigated in 349 pneumonic lungs from sheep (n = 197) and goats (n = 152), and genotypes of isolates were determined by capsular and lipopolysaccharide (LPS) typing as well as by virulotyping based on the detection of 12 virulence-associated genes. P. multocida was isolated from 29.4 % of sheep lungs and 13.8 % of goat lungs. A (78.5 %) and D (21.5 %) capsular types, as well as L3 (41.8 %) and L6 (57.0 %) LPS genotypes, were detected, with the A:L6 genotype being the most prevalent in both sheep (59.6 %) and goat (52.4 %) isolates. A total of 19 virulence profiles (VP) were detected, seven non-toxigenic and 12 toxigenic, which correlated with the capsular–LPS genotype. All isolates of each VP belonged to the same LPS and capsular genotype, except for one isolate of VP1. The diversity in VP was higher among toxigenic (0.29) than non-toxigenic (0.18) isolates. Moreover, the toxigenic VPs showed more diversity in their capsular-LPS genotypes, with the two main toxigenic VPs belonging to genotypes D:L3 (VP2) and A:L3 (VP3). Therefore, the abundance of toxigenic isolates among sheep and goat isolates does not seem to correspond to the expansion of a more virulent lineage associated with pneumonic pasteurellosis in small ruminants. The most prevalent genotypes among sheep isolates were the non-toxigenic VP1:A:L6 (41.4 %) and the toxigenic VP3:A:L3 (17.2 %) genotypes, whereas the most prevalent among goat isolates were the toxigenic VP2:D:L3 (33.3 %) and the non-toxigenic VP1:A:L6 (14.3 %) and VP4:A:L6 (14.3 %) genotypes. These prevalent toxigenic and non-toxigenic genotypes seem to be epidemiologically relevant in pneumonic pasteurellosis of small ruminants.
Comparative genetic diversity analysis based on virulence and repetitive genes profiling of circulating Pasteurella multocida isolates from animal hosts
2020, Infection, Genetics and Evolution
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It is noteworthy that toxA was the least frequently detected gene among all the isolates. Since toxA gene was present in isolates recovered from animals having nasal discharges, strongly indicated its potential role in pneumonia also (Einarsdottir et al., 2016; Cid et al., 2019). The omp87 and ompH genes were detected in >90% of the analyzed samples which encode porins proteins of P. multocida.
Virulence associated and/or housekeeping/repetitive genes either in single or multiple copies are being extensively targeted for bacterial pathogen detection and differentiation in epidemiological studies. In the present study, isolation of Pasteurella multocida from different animals and their genetic profiling based on the capsular types, virulence and repetitive elements (ERIC/REP) were carried out. A total 345 clinical samples from apparently healthy and diseased (pneumonic, septicaemia) animals (sheep, goat, pig, cattle, buffalo and rabbits) from different geographical regions of Karnataka, Uttar Pradesh, Mizoram and Assam states of India were screened. A total of 32% of the samples were found positive, of which 41 P. multocida isolates recovered. Virulence profiling of isolates indicated that omp87, ompA, ptfA, sodA, sodC, nanB, fur and exbB were present in 100% of isolates. Whereas, prevalence of other genes were; nanH (90%), ompH (71%), pfhA (63%), plpB (80%), hsf-1 (12%), hsf-2 (37%), pmHAS (78%), toxA (73%), hgbA (37%), hgbB (81%), tbpA (78%) and fimA (98%), among isolates. There was no influence of host or place on prevalence of virulence genes when assessed by fitting a Hierarchial Bayesian ordinal regression model. There was correlation (positive and negative) between broad groups of virulence genes. Both repetitive gene profiles (ERIC and REP) generated multiple amplicons (~200 to ~4000 bp). Cluster analysis with ERIC profiles revealed 5 clusters and 3 non- typable isolates with higher discriminatory power (D = 0.7991) than the REP-PCR profiles (D = 00.734) which revealed 4 clusters and 6 non- typable isolates. The results showed that a considerable level of genetic diversity exists among circulating P. multocida isolates despite belonging to the same geographical origin. The genetic diversity or clustering based on either virulence or repetitive elements among isolates could be largely driven by multiple factors acting together which lead to manifestations of particular disease symptoms.
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Cluster analysis grouped the lungs into four clusters (Table 2) based on the isolation of M. haemolytica or P. multocida alone (clusters 1 and 4), coisolation of both microorganisms (cluster 3), and isolation of neither (cluster 2), and their characteristics. Most strains of P. multocida were toxigenic, capsular type A (Table 2), even if they were isolated alone (cluster 4) or coisolated with M. haemolytica (cluster 3), in line with previous studies that have demonstrated that the great majority of P. multocidaisolates associated with pneumonia in sheep are capsular type A toxigenic (Garcia-Alvarez et al., 2017; Cid et al., 2019). The diversity of M. haemolytica capsular types was very similar in lungs of clusters 1 and 3 (eight and seven capsular types, respectively; Table 2).
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Short communicationPasteurella multocida isolates associated with ovine pneumonia are toxigenic

Highlights

Abstract

Introduction

Section snippets

P. multocida strains

Results and discussion

Conflict of interest

Acknowledgements

Vet. Microbiol.

Vet. Microbiol.

Vet. Microbiol.

Comp. Immunol. Microbiol. Infect. Dis.

Microbiol. Res.

Vaccine

Evaluation of techniques for the detection of toxigenic Pasteurella multocida strains from pigs

J. Vet. Diagn. Invest.

β-Hemolysis may not be a reliable indicator of leukotoxicity of Mannheimia haemolytica isolates

Toxins

Induction of pneumonia in rabbits by use of a purified protein toxin from Pasteurella multocida

Am. J. Vet. Res.

Characterization and comparison of Pasteurella multocida strains associated with porcine pneumonia and atrophic rhinitis

J. Med. Microbiol.

Demonstration that Australian Pasteurella multocida isolates from sporadic outbreaks of porcine pneumonia are non-toxigenic (toxA-) and display heterogeneous DNA restriction endonuclease profiles compared with toxigenic isolates from herds with progressive atrophic rhinitis

J. Med. Microbiol.

Characterisation of dermonecrotic toxin-producing strains ofPasteurella multocida subsp. multocida isolated from man and swine

J. Med. Microbiol.

Variability of Pasteurella multocida isolated from Icelandic sheep and detection of the toxA gene

J. Med. Microbiol.

Short communication
Pasteurella multocida isolates associated with ovine pneumonia are toxigenic