Elsevier

Veterinary Microbiology

Volume 174, Issues 1–2, 7 November 2014, Pages 172-179
Veterinary Microbiology

Sequence diversity, cytotoxicity and antigenic similarities of the leukotoxin of isolates of Mannheimia species from mastitis in domestic sheep

https://doi.org/10.1016/j.vetmic.2014.08.009Get rights and content

Highlights

  • The lktA genes of Mannheimia haemolytica causing mastitis in sheep are more diverse than those carried by M. glucosida.

  • The lktA genes in Mannheimia species form ovine mastitis appear to have been transferred horizontally.

  • The leukotoxin of Mannheimia glucosida is more potent those that of Mannheimia haemolytica.

  • LktA of M. glucosida induces more broadly cross-reactive antibody than LktAs of M. haemolytica.

Abstract

Species within the genus Mannheimia are among the most important causes of ovine mastitis. Isolates of these species can express leukotoxin A (LktA), a primary virulence factor of these bacteria. To examine the significance of variation in the LktA, the sequences of the lktA genes in a panel of isolates from cases of ovine mastitis were compared. The cross-neutralising capacities of rat antisera raised against LktA of one Mannheimia glucosida, one haemolytic Mannheimia ruminalis, and two Mannheimia haemolytica isolates were also examined to assess the effect that variation in the lktA gene can have on protective immunity against leukotoxins with differing sequences. The lktA nucleotide distance between the M. haemolytica isolates was greater than between the M. glucosida isolates, with the M. haemolytica isolates divisible into two groups based on their lktA sequences. Comparison of the topology of phylogenetic trees of 16S rDNA and lktA sequences revealed differences in the relationships between some isolates, suggesting horizontal gene transfer. Cross neutralisation data obtained with monospecific anti-LktA rat sera were used to derive antigenic similarity coefficients for LktA from the four Mannheimia species isolates. Similarity coefficients indicated that LktA of the two M. haemolytica isolates were least similar, while LktA from M. glucosida was most similar to those for one of the M. haemolytica isolates and the haemolytic M. ruminalis isolate. The results suggested that vaccination with the M. glucosida leukotoxin would generate the greatest cross-protection against ovine mastitis caused by Mannheimia species with these alleles.

Introduction

Mastitis is an important disease of sheep, with significant effects on animal welfare and flock productivity. In meat and wool production systems, Mannheimia species, including Mannheimia haemolytica and Mannheimia glucosida, can be as important as, and sometimes more significant than, Staphylococcus aureus as a cause of mastitis (Omaleki et al., 2011). Leukotoxin A (LktA), a member of the RTX (repeats in toxin) family of toxins, is a well characterised virulence factor of Mannheimia species. The role of LktA in the pathogenesis of ruminant pneumonia and of anti-LktA antibodies in providing partial protection against this pneumonia has been studied extensively (Pandher et al., 1999, Shewen and Wilkie, 1988). Although lktA-bearing isolates of Mannheimia species have been obtained from ovine mastitis, the role of LktA in mastitis has not been well characterised (Omaleki et al., 2010). Production and secretion of the M. haemolytica leukotoxin is encoded by an operon of four genes, lktC, lktA, lktB and lktD, with lktA encoding the toxin (Lo et al., 1987). Allelic variation in lktA has been reported previously, and is suggestive of host selection for antigenic variants (Davies et al., 2002), as a neutralising monoclonal antibody against LktA of one serotype cannot neutralise LktA of some other serotypes (Gerbig et al., 1992).

The current study aimed to compare lktA sequence diversity in isolates of Mannheimia species obtained from ovine mastitis and to investigate cross-neutralisation by rat anti-lktA antibodies raised against LktA from different isolates of Mannheimia species.

Section snippets

Bacterial isolates

A total of twenty lktA positive Mannheimia isolates, including ten M. haemolytica, nine M. glucosida and one haemolytic Mannheimia ruminalis were included in this study. One non-haemolytic M. ruminalis was used as a control. These isolates were from a panel of isolates obtained from cases of ovine mastitis in nine different Poll Dorset flocks (Table 1), designated A-H and J, and one mixed-breed flock, mostly composed of Merinos and Merino crosses, designated I (Omaleki et al., 2010, Omaleki et

Analysis of nucleotide and amino acid sequences

The complete DNA sequences of lktA in 5 M. haemolytica, 7 M. glucosida and the haemolytic M. ruminalis isolate were determined and compared with those of the European isolates (Davies et al., 2001) listed in Table 1 (the mean nucleotide and amino acid sequence diversity for the lktA genes are in Supplementary Table 2). The M. haemolytica lktA sequences could be divided into two groups. The first group (C1 and I1) had an lktA sequence very similar to those of the European isolates PH284 and

Discussion

The similarity between lktA sequences of M. haemolytica isolates was less than the similarity between those of M. glucosida isolates (89.4% and 99%, respectively), in agreement with previous studies on Mannheimia species from cases of pneumonia (Davies et al., 2001). Phylogenetic analyses of lktA and 16S rDNA of M. haemolytica and M. glucosida suggested horizontal gene transfer, as the locations of some isolates were incongruent in the two phylogenetic trees. Extensive horizontal gene transfer

Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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