The Veterinary Journal 203 (2015) 92–96

Contents lists available at ScienceDirect

The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l

Point prevalence of infection with Mycoplasma bovoculi and Moraxella spp. in cattle at different stages of infectious bovine keratoconjunctivitis Christiane Schnee *, Martin Heller, Evelyn Schubert, Konrad Sachse Institute of Molecular Pathogenesis, Friedrich-Loeffler-lnstitut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany

A R T I C L E

I N F O

Article history: Accepted 13 November 2014 Keywords: Bovine Infectious bovine keratoconjunctivitis Moraxella spp Mycoplasma bovoculi

A B S T R A C T

Infectious bovine keratoconjunctivitis (IBK) has significant economic consequences and a detrimental impact on animal welfare. Although Moraxella (Mor.) bovis is the primary causative agent, the role of other bacteria, such as Mor. ovis, Mor. bovoculi and Mycoplasma (Myc.) bovoculi, is not well understood. To assess the prevalence of infection with these organisms, and to correlate this with outbreaks of IBK, conjunctival samples from four herds of cattle in Germany of differing IBK status were examined. Herds were selected to represent a hypothetical course of IBK ranging from the pre-outbreak stage (herd 1), to the acute disease stage (herd 2), to a stage where treatment had ceased (herd 3). Unaffected animals were also included (herd 4). To facilitate effective, sensitive sample analysis, a new real-time PCR for Myc. bovoculi was developed and used in concert with established real-time PCR protocols for Myc. bovis and Moraxella spp. Herds 1 and 2 showed similarly high rates of detection for Myc. bovoculi (92.5% and 84.0%, respectively), whereas herds 3 and 4 had a lower prevalence (35.5% and 26.2%, respectively). Mor. bovis and Mor. ovis were more prevalent in herd 1 (32.5% and 87.5%, respectively) and herd 2 (38% and 58%, respectively) than herd 3 (10.4% and 1.3%, respectively) and herd 4 (9.8% and 31.1%, respectively). Mor. bovoculi was the only pathogen that correlated with clinical signs of IBK; at 20% prevalence, it was almost exclusively detected in herd 2. The results indicate that herds with high Myc. bovoculi prevalence are more predisposed to outbreaks of IBK, possibly due to a synergistic interaction with Moraxella spp. © 2014 Elsevier Ltd. All rights reserved.

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).

* Corresponding author. Tel.: +49 3641 8042435. E-mail address: christiane.schnee@fli.bund.de (C. Schnee). http://dx.doi.org/10.1016/j.tvjl.2014.11.009 1090-0233/© 2014 Elsevier Ltd. All rights reserved.

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 and Barton, 1987; Alexander, 2010; O’Connor et al., 2012) and is the only pathogen proven to reproduce IBK in challenge models (Henson and 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

C. Schnee et al./The Veterinary Journal 203 (2015) 92–96

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 and 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. Materials and methods 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 discharges, uveitis, corneal opacity, keratitis and/or ulceration of the corneal epithelium. Although affected animals were treated repeatedly by subconjunctival injection of oxytetracycline (OTC; Baxyl), this did not mitigate the clinical signs. Herd 3 comprised 264 animals (beef suckler cows and a few calves) pastured from March to October and housed overwinter. At the time of sampling, most of the cows had already exhibited signs of severe IBK and had been successfully treated with IM injections of long-acting OTC (20 mg/kg body weight; Oxipra); no clinical signs were evident at the time of sampling 1–4 weeks after treatment. The IBKfree control group (herd 4) consisted of 61 untreated, clinically normal dairy calves housed in six groups of 9–11 animals at the Friedrich-Loeffler-Institut, Jena, Germany. These four herds of differing IBK status will be referred to as ‘pre-IBK’ (herd 1), ‘acute IBK’ (herd 2), ‘post-IBK’ (herd 3), and ‘unaffected by IBK’ (herd 4).

93

Table 1 Mollicute reference strains used in specificity testing of the PCR for Mycoplasma bovoculi. Species

Reference strain

Culture medium

Acholeplasma laidlawii Mycoplasma agalactiae Mycoplasma alkalescens Mycoplasma arginini Mycoplasma bovigenitalium Mycoplasma bovirhinis Mycoplasma bovis Mycoplasma bovoculi Mycoplasma californicum Mycoplasma canadense Mycoplasma canis Mycoplasma conjunctivae Mycoplasma dispar Mycoplasma hyopneumoniae Mycoplasma hyorhinis Mycoplasma leachii Mycoplasma mycoides subsp. mycoides Mycoplasma ovipneumoniae

PG8 PG2 PG31 G230 PG11 PG3 PG45 M165/69 St-6 275C PG14 HRC581 462/2 J BTS-7 PG50 PG1

Modified Hayflick Modified Hayflick Modified Hayflick Modified Hayflick MLM (Mycoplasma Experience) Modified Hayflick Modified Hayflick MLM (Mycoplasma Experience) MLM (Mycoplasma Experience) MLM (Mycoplasma Experience) MLM (Mycoplasma Experience) MLM (Mycoplasma Experience) MLM (Mycoplasma Experience) FRIIS Modified Hayflick Modified Hayflick Modified Hayflick

Y98 (b)

Modified Hayflick

Dissociation curves (95 °C for 60 s, 55 °C for 30 s and 95 °C for 30 s) were analysed to assess the specificity of the amplification. In addition, PCR products were separated by standard ethidium bromide-stained agarose gel electrophoresis and visualised using the gel documentation system G:Box (Syngene). The sensitivity and reproducibility of the assay were evaluated by running 10-fold serial dilutions of DNA extracted from each type strain, both in elution buffer and in DNA extracts from conjunctival swabs that were previously tested negative by PCR for Myc. bovoculi, in quadruplicate in six independent runs. Detection of Myc. bovoculi was further verified in 14 and 18 randomly selected samples from herds 2 and 3, respectively, using a Mycoplasma spp. microarray assay (Schnee et al., 2012). Specificity of Mycoplasma bovoculi SYBR Green PCR Reference strains were selected on the basis of close phylogenetic relatedness to Myc. bovoculi or because of their probable occurrence in cattle (Table 1). In addition, several Myc. bovoculi field strains and strains from clinical samples that had previously tested positive for Myc. bovoculi by microarray or sequencing analysis were included. Strains were recovered from lyophilised stocks and propagated in broth cultures of modified Hayflick medium containing 20% horse serum, FRIIS (Freundt, 1983) or commercially available MLM (‘Mycoplasma Experience’) medium for 2–4 days at 37 °C. Cells were harvested by centrifugation and chromosomal DNA was extracted and tested as described earlier. Statistical analysis

Sample collection and preparation Conjunctival swabs were collected from animals randomly selected from herds 1 (n = 50) and 2 (n = 40) on two occasions in September and December 2012. In herd 3, conjunctival swabs were collected from 231 cows randomly selected on two consecutive days in March 2012. Conjunctival swabs were also collected from all 61 animals in herd 4. Only one conjunctiva per animal was swabbed in each herd and DNA was extracted from the swabs using the High Pure PCR Template Preparation Kit (Roche Diagnostics).

Real-time PCR To detect Moraxella spp., a quadruplex real-time PCR with TaqMan probes and quantitative PCR Mastermix No ROX (Eurogentech) was performed (Shen et al., 2011), with the additional inclusion of an internal amplification control (Hoffmann et al., 2006). Myc. bovis DNA was detected using quantitative real-time PCR according to Sachse et al. (2010). A SYBR Green PCR based on the target gene rpoB was designed for specific detection of Myc. bovoculi DNA using primers MbovocF (5′-CACATCAGCAAAATCAAGATCA-3′) and MbovocR (5′-GAATAATGATTTCATTGTGTCTG3′). The 20 μL reaction mix contained Dynamo Flash Sybr Green (Finnzyme) and 500 nM of each primer. Amplification was performed with the Mx3000P (Agilent) thermocycler at 95 °C for 5 min, then 40 cycles of 95 °C for 30 s, 54 °C for 30 s and 72 °C for 45 s. PCR products were sequenced on both strands (Eurofins MWG Operon) and analysed using BLAST.1

1

See: http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed 10 November 2014).

Binary data were analysed using the χ2 test in WinEpi2 and 95% confidence intervals were calculated. P values 25% of samples in both herds harboured at least two Moraxella spp. (Fig. 2). The simultaneous occurrence of more than two species was uncommon in herds 3 and 4, which also had lower overall prevalences of infection with these organisms (12.5% and 41%, respectively). In contrast to studies by Kelly et al. (1983) and O’Connor et al. (2012), we found Mor. ovis to be the most prevalent Moraxella spp. Misidentification of this species, to the detriment of the prevalence of Mor. bovoculi, as has been assumed in previous reports (Angelos, 2010; O’Connor et al., 2012), can be ruled out in the current study due to the use of the multiplex real-time PCR (Shen et al., 2011), which provides reliable differentiation of the three Moraxella spp. Similar to Mor. ovis, Mor. bovis was more prevalent in herds affected by IBK than in unaffected herds, but at lower levels (32.5% and 38% in herds 1 and 2, respectively). Mor. bovoculi was the only agent whose presence correlated with the occurrence of clinical cases of IBK, i.e. it was almost exclusively detected in herd 2. However, its prevalence at 20% was relatively low and was not consistent with the large number of animals in this herd with clinical signs. Similarly, the prevalence of the other Moraxella spp. was also relatively low when considered individually and could not be correlated directly with the large number of animals with clinical signs in herd 2. Again, our data suggest a synergistic interaction between Myc. bovoculi and one of the three Moraxella spp. in the induction of IBK in herd 2. We can only speculate as to the effect of systemic treatment with OTC in herd 3 on the prevalence of infection, since no comparative data were available from this herd at the acute stage of disease. Assuming a similar prevalence distribution as herd 2, such treatment would have resulted in almost complete removal of Mor. bovoculi and Mor. ovis, whereas Mor. bovis and Myc. bovoculi were still detectable by real-time PCR. One of the limitations of the present study was the small number of herds investigated and their heterogeneity in terms of management system and age of animals. These factors may have contributed to some of the differences in apparent pathogen prevalences. 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. Affected herds also had higher overall prevalences and a more complex range of Moraxella spp. than herds in which IBK did not occur. Longitudinal cohort studies involving collection of multiple samples during the course of IBK outbreaks are required; this will require PCR-based technology, as used in the current study, to deal with the high sample throughput.

96

C. Schnee et al./The Veterinary Journal 203 (2015) 92–96

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. References Alexander, D., 2010. Infectious bovine keratoconjunctivitis: A review of cases in clinical practice. Veterinary Clinics of North America: Food Animal Practice 26, 487–503. Angelos, J.A., 2010. Moraxella bovoculi and infectious bovine keratoconjunctivitis: Cause or coincidence? Veterinary Clinics of North America: Food Animal Practice 26, 73–77. Angelos, J.A., Spinks, P.Q., Ball, L.M., George, L.W., 2007. Moraxella bovoculi sp. nov., isolated from calves with infectious bovine keratoconjunctivitis. International Journal of Systematics and Evolutionary Microbiology 57, 789–795. Barber, D.M., Jones, G.E., Wood, A., 1986. Microbial flora of the eyes of cattle. Veterinary Record 118, 204–206. Brown, M.H., Brightman, A.H., Fenwick, B.W., Rider, M.A., 1998. Infectious bovine keratoconjunctivitis: A review. Journal of Veterinary Internal Medicine 12, 259–266. Cerny, H.E., Rogers, D.G., Gray, J.T., Smith, D.R., Hinkley, S., 2006. Effects of Moraxella (Branhamella) ovis culture filtrates on bovine erythrocytes, peripheral mononuclear cells, and corneal epithelial cells. Journal of Clinical Microbiology 44, 772–776. Freundt, E.A., 1983. Culture media for classic mycoplasmas. In: Razin, S., Tully, J.G. (Eds.), Methods in Mycoplasmology. Vol. I: Mycoplasma Characterization. Academic Press, London, UK, pp. 127–135. Friis, N.F., Pedersen, K.B., 1979. Isolation of Mycoplasma bovoculi from cases of infectious bovine keratoconjunctivitis. Acta Veterinaria Scandinavica 20, 51–59. Gould, S., Dewell, R., Tofflemire, K., Whitley, R.D., Millman, S.T., Opriessnig, T., Rosenbusch, R., Trujillo, J., O’Connor, A.M., 2013. Randomized blinded challenge study to assess association between Moraxella bovoculi and infectious bovine keratoconjunctivitis in dairy calves. Veterinary Microbiology 164, 108–115. Henson, J.B., Grumbles, L.C., 1960. Infectious bovine keratoconjunctivitis. II. Susceptibility of laboratory animals to Moraxella (Hemophilus) bovis. Cornell Veterinarian 50, 445–458. Hoffmann, B., Depner, K., Schirrmeier, H., Beer, M., 2006. A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses. Journal of Virological Methods 136, 200–209. Kelly, J.I., Jones, G.E., Hunter, A.G., 1983. Isolation of Mycoplasma bovoculi and Acholeplasma oculi from outbreaks of infectious bovine keratoconjunctivitis. Veterinary Record 112, 482.

Kopecky, K.E., Pugh, G.W., Jr., McDonald, T.J., 1986. Infectious bovine keratoconjunctivitis: Contact transmission. American Journal of Veterinary Research 47, 622–624. Langford, E.V., Leach, R.H., 1973. Characterization of a mycoplasma isolated from infectious bovine keratoconjunctivitis: M. bovoculi sp. nov. Canadian Journal of Microbiology 19, 1435–1444. Lepper, A.W., Barton, I.J., 1987. Infectious bovine keratoconjunctivitis: Seasonal variation in cultural, biochemical and immunoreactive properties of Moraxella bovis isolated from the eyes of cattle. Australian Veterinary Journal 64, 33–39. Levisohn, S., Garazi, S., Gerchman, I., Brenner, J., 2004. Diagnosis of a mixed mycoplasma infection associated with a severe outbreak of bovine pinkeye in young calves. Journal of Veterinary Diagnostic Investigation 16, 579–581. McConnel, C.S., Shum, L., House, J.K., 2007. Infectious bovine keratoconjunctivitis antimicrobial therapy. Australian Veterinary Journal 85, 65–69. Naglic, T., Sankovic, F., Madic, J., Hajsig, D., Seol, B., Busch, K., 1996. Mycoplasmas associated with bovine conjunctivitis and keratoconjunctivitis. Acta Veterinaria Hungarica 44, 21–24. O’Connor, A.M., Shen, H.G., Wang, C., Opriessnig, T., 2012. Descriptive epidemiology of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in beef calves with naturally occurring infectious bovine keratoconjunctivitis (pinkeye). Veterinary Microbiology 155, 374–380. Pfutzner, H., Sachse, K., 1996. Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Revue Scientifique et Technique (International Office of Epizootics) 15, 1477–1494. Postma, G.C., Carfagnini, J.C., Minatel, L., 2008. Moraxella bovis pathogenicity: An update. Comparative Immunology, Microbiology and Infectious Diseases 31, 449–458. Pugh, G.W., Jr., McDonald, T.J., 1986. Identification of bovine carriers of Moraxella bovis by comparative cultural examinations of ocular and nasal secretions. American Journal of Veterinary Research 47, 2343–2345. Rosenbusch, R.F., 1983. Influence of mycoplasma preinfection on the expression of Moraxella bovis pathogenicity. American Journal of Veterinary Research 44, 1621–1624. Sachse, K., Salam, H.S.H., Diller, R., Schubert, E., Hoffmann, B., Hotzel, H., 2010. Use of a novel real-time PCR technique to monitor and quantitate Mycoplasma bovis infection in cattle herds with mastitis and respiratory disease. The Veterinary Journal 186, 299–303. Schnee, C., Schulsse, S., Hotzel, H., Ayling, R.D., Nicholas, R.A., Schubert, E., Heller, M., Ehricht, R., Sachse, K., 2012. A novel rapid DNA microarray assay enables identification of 37 Mycoplasma species and highlights multiple Mycoplasma infections. PLoS ONE 7, e33237. Schoettker-Wegner, H.H., Binder, A., Kirchhoff, H., 1990. Nachweis von Mykoplasmen in Augentupferproben. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health 37, 436–441. Shen, H.G., Gould, S., Kinyon, J., Opriessnig, T., O’Connor, A.M., 2011. Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle. Journal of Applied Microbiology 111, 1037–1043. Slatter, D.H., Edwards, M.E., Hawkins, C.D., Wilcox, G.E., 1982. A national survey of the occurrence of infectious bovine keratoconjunctivitis. Australian Veterinary Journal 59, 65–68. Vogelweid, C.M., Miller, R.B., Berg, J.N., Kinden, D.A., 1986. Scanning electron microscopy of bovine corneas irradiated with sun lamps and challenge exposed with Moraxella bovis. American Journal of Veterinary Research 47, 378–384. Williams, D.L., 2010. Welfare issues in farm animal ophthalmology. Veterinary Clinics of North America: Food Animal Practice 26, 427–435.