Point prevalence of infection with Mycoplasma bovoculi and Moraxella spp. in cattle at different stages of infectious bovine keratoconjunctivitis
Introduction
Infectious bovine keratoconjunctivitis (IBK), commonly known as ‘pinkeye’, is highly contagious, and spreads rapidly within a herd through direct contact, nasal or ocular discharges and via insect vectors (Kopecky et al., 1986). The early stages of the disease are characterised by excessive lachrymation and photophobia with blepharospasm. Subsequently, mucopurulent conjunctival exudates and corneal opacity can occur, progressing to corneal ulceration and oedema, along with conjunctivitis of varying severity (Brown et al, 1998, Alexander, 2010).
Although corneal ulceration often heals without therapeutic intervention and cattle may spontaneously recover from IBK, corneal rupture resulting in complete and permanent loss of vision can occur in severe cases, with marked ocular discomfort (Williams, 2010). Apart from these welfare implications, IBK also has a considerable economic impact, particularly due to reduced weight gain in calves at weaning (Slatter et al., 1982) and in the costs of antibiotic treatment (McConnel et al., 2007).
Moraxella (Mor.) bovis is considered to be the major causative agent of IBK, since it is the bacterium most frequently isolated from clinical cases (Lepper, Barton, 1987, Alexander, 2010, O'Connor et al, 2012) and is the only pathogen proven to reproduce IBK in challenge models (Henson, Grumbles, 1960, Vogelweid et al, 1986, Gould et al, 2013). However, Mor. bovis can also colonise conjunctivae opportunistically without causing clinical signs (Pugh and McDonald, 1986). These differences in virulence have been attributed to variability in expression of two virulence factors, capsular pili and production of a β-haemolytic cytotoxin (Brown et al, 1998, Postma et al, 2008). Other Moraxella spp. suspected to be causally associated with IBK include Moraxella ovis (Cerny et al., 2006) and Moraxella bovoculi (Angelos et al., 2007), although clear experimental proof is lacking (O'Connor et al., 2012).
Infection with Mycoplasma (Myc.) spp. also often has a high prevalence in the conjunctivae of cattle with IBK, with Myc. bovoculi being the most prominent (Langford and Leach, 1973). Whether Myc. bovoculi alone can cause clinical IBK, or whether it acts as a predisposing factor enhancing the effects of Moraxella spp. is currently unclear. Friis and Pedersen (1979) and Rosenbusch (1983) demonstrated the importance of prior Myc. bovoculi infection to the pathogenicity of Mor. bovis. An outbreak of IBK has even been attributed to the synergistic action of Myc. bovoculi and Mycoplasma bovis, following the possible predisposing effects of bovine respiratory disease (Levisohn et al., 2004). However, doubts have also been raised over the ability of Myc. bovoculi to cause IBK in the absence of pathogenic Moraxella spp. (Kelly et al, 1983, Schoettker-Wegner et al, 1990).
In the past, classical culture methods were used for epidemiological investigations of Mycoplasma spp. infections (Friis, Pedersen, 1979, Kelly et al, 1983, Barber et al, 1986, Levisohn et al, 2004). However Mycoplasma spp. are difficult to isolate, and culture for Moraxella spp. is not routinely performed in most veterinary laboratories. PCR may circumvent some of these diagnostic challenges, since it is sensitive, rapid, robust and suitable for high-throughput applications.
The present study was conducted to investigate the cause of two outbreaks of IBK using a novel real-time PCR for Myc. bovoculi, Myc. bovis, Mor. bovis, Mor. ovis and Mor. bovoculi. Results from affected herds were compared with those from a herd that subsequently developed IBK, and from an unaffected control herd. A SYBR Green real-time PCR assay targeting Myc. bovoculi was employed in tandem with an established Moraxella spp. real-time PCR protocol (Shen et al., 2011) for the rapid screening of large numbers of swab samples.
Section snippets
Selection of animals
Herds 1 and 2 consisted of 100 and 150 dairy calves, respectively, which were kept separately in pens on the same farm in Eastern Saxony-Anhalt, Germany. The calves were purchased at 14 days old and raised in herd 1 until 4 months of age. At this point, they were transferred to herd 2, where they stayed until 6 months of age. Once calves were transferred as a batch from herd 1 to herd 2, they exhibited the characteristic clinical signs of IBK, including unilateral/bilateral conjunctival
SYBR Green real-time PCR for detection of Mycoplasma bovoculi
A rpoB fragment of Myc. bovoculi was amplified by real-time PCR to produce a 567 base pair amplicon, the identity of which was confirmed by sequencing. Amplicons of Myc. bovoculi type strain M165/69, of two field strains and of selected Myc. bovoculi-containing clinical samples had a melting temperature (Tm) of 81.0 ± 0.5 °C. In contrast, no detectable PCR bands in agarose gel electrophoresis were produced by any other Mycoplasma spp. and dissociation curves exhibited no melting peak or
Discussion
Numerous epidemiological observations and experimental challenge models over the last 50 years have implicated Mor. bovis as the major causal agent of IBK (Henson, Grumbles, 1960, Lepper, Barton, 1987, O'Connor et al, 2012, Gould et al, 2013). Nevertheless, the disease is clearly multifactorial; outbreaks are influenced by co-infections with other bacteria, such as Mor. bovoculi, Myc. bovoculi, and Staphylococcus spp., or by viral infection or abiotic factors (Brown et al, 1998, Angelos, 2010).
Conclusions
Although experimental challenge is the preferred method of elucidating causal relationships between potential pathogens and disease, prevalence studies investigating a naturally occurring disease at different stages help to elucidate interactions between pathogens and their contribution to pathogenesis. Our results indicate that herds with a high prevalence of Myc. bovoculi are more predisposed to acute IBK, possibly due to this pathogen facilitating enhanced infection with Moraxella spp.
Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
Acknowledgements
The authors wish to thank Susann Bahrmann and Simone Bettermann for technical assistance, and Roland Diller and Beate Burkert for help with the statistical analysis. We are also grateful to the attending veterinarians Sibylle Börngen and Helmut Schuster. Preliminary results of the study were presented as a poster at the European Mycoplasma Meeting, Dubrovnik, Croatia, 6–7, June 2013.
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