Respiratory disease and otitis were seen on a northern Italian farm in weaned bulls obtained from multiple sources. Mycoplasma bovis was consistently identified in nasal swabs, tonsil, ears, eyes and lung samples from affected animals. Unusually the closely related Mycoplasma agalactiae, the main cause of contagious agalactia (CA) in small ruminants, was also isolated. It was identified by molecular methods in bulls but only from eye, ear and brain samples. Outbreaks were reduced by retaining the bulls on their original farms for weaning, then moving them directly to the fattening areas on the farm. This prevented the spread of mycoplasma from infected to uninfected calves from different sources. As no small ruminants were within close proximity and the area is free of CA, it is possible that M agalactiae may be more prevalent in cattle than previously thought.
- Otitis media
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Otitis in calves can be caused by a variety of bacteria including Histophilus somni, Mannheimia haemolytica, Pasteurella multocida and, occasionally, Mycoplasma bovis (Foster and others 2009). Clinical signs associated with mycoplasma infection may include head tilt and neurological signs but are rarely seen in adult cattle. M bovis is closely related genetically, immunologically and biochemically to the small ruminant pathogen Mycoplasma agalactiae, the major cause of the World Organisation of Animal Health (OIE)-listed disease contagious agalactia (CA), and indeed was once classified as M agalactiae var bovis until more discriminatory tools became available (Askaa and Erno 1976). Molecular typing has shown that M bovis strains can be separated into two distinct groups: one probably arising from the USA and the second from Europe (McAuliffe and others 2004). Interestingly the European group is more closely related to the type strain of M agalactiae than to the type strain of M bovis that was isolated from the USA (Loria and others 2006). Despite this close relationship, there are very few reports of the isolation of M agalactiae from cattle, although, conversely, M bovis has been isolated from goats in the UK (Nicholas and others 2008). The authors report here the isolation of both M bovis and M agalactiae from a herd of local Simmental bulls in northern Italy presenting with severe otitis and pneumonia.
The farm in Friuli (Friuli Venezia Giulia), northern Italy, contained up to 500 Simmental bulls originating from over 90 different companies. Approximately 5–10 cattle arrived on the farm every week where they were weaned over three months before being moved to fattening areas elsewhere. They were fully vaccinated against bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), respiratory syncytial virus and PI3. The housing conditions were unfavourable with poor ventilation, no heating during weaning and cattle were cared for by inexperienced staff with a high turnover rate. The farm had a history of chronic otitis, pneumonia and enteritis, and previous investigations had shown the presence of Mycoplasma species, in particular M bovis. At the time of the most recent investigation, about 20 per cent of cattle were showing signs of respiratory disease and 2–3 per cent presenting ear infections. Two bulls of approximately 11–12 months of age were inspected carefully and shown to be of poor nutritional status, unsteady on their feet, with deviation of the head, mild conjunctivitis and lip ptosis, but had no fever. For welfare reasons, they were euthanased for postmortem examination.
There were no small ruminants on the farm or within about 3 km of the cattle and the region had been CA-free for several years.
Postmortem of bull 1 revealed hepatomegaly associated with congestion and accentuated lobular pattern of the left lobe, enteritis associated with hyperaemia in the vessel wall of the small intestine, thinning of the proximal end, serious kidney congestion and an enlargement of mesenteric lymph nodes. Within the chest cavity, there was severe fibrinopurulent pneumonia, hepatisation of the cranial lobe and severe mediastinal lymphadenopathy. There was also hyperaemia of the trachea. No other macroscopical lesions were visible. Examination of the second bull revealed a diffuse bilateral suppurative pyelonephritis associated with multiple renal calculi. In the thoracic cavity, lungs showed multifocal areas of marked consolidation of the cranial and middle lobes, and multifocal areas of fibrinosuppurative pneumonia (Fig 1). Investigation of the inner ear area showed the presence of brownish exudates in the auditory external duct. A transverse section of pars petrosa revealed purulent exudates with a necrotic osteitis consisting of dry and grey bone (Fig 2). The lumen of the inner ear also contained purulent exudates.
The samples for mycoplasma detection were placed directly into 2 ml of mycoplasma experience (ME) liquid medium (Reigate, UK) following conventional procedures. Briefly, the inoculated cultures were incubated at 37°C with 5 per cent of CO2 for at least seven days. The broths were checked daily in order to detect any change in colour or increased turbidity. Broths that showed any change were immediately inoculated onto a plate containing semi-solid ME agar medium; the broths that did not show any change for seven days were inoculated onto agar medium. If no colonies were seen in these samples after seven days, then they were considered negative. The broths that showed growth and produced colonies were tested by PCR/parainfluenza virus (DGGE) method (McAuliffe and others 2005). The colonies were also examined by immunofluorescence (IF) test (Rosendal and Black 1972).
For routine bacteriology, samples including lung, inner ear and kidney were inoculated onto blood agar (Biolife, Milan) MacConkey agar (CONDA, Madrid) and brain heart infusion broth (CONDA, Madrid), and cultivated at 37°C in an aerobic, anaerobic and 5 per cent CO2 environment for 48 hours. Moreover, cephalorachidian fluid samples were tested by PCR for H somni (Angen and others 1998).
H somni, M haemolytica and P multocida were not detected in the two bulls. H somni was not detected in the cephalorachidian fluid by PCR.
Numerous treatments of oral antibiotics (e.g. sulphonamides, amoxicillin) and intramuscular antibiotics (enrofloxacin, florfenicol, cephalosporins) were made during the course of the outbreaks. There was very little improvement in the condition of affected animals following treatment.
Outcome and follow-up
Results of PCR and mycoplasma culture showed a mixture of M bovis and, unusually, M agalactiae in both bulls and sometimes in the same sample (Table 1). In bull 1, M agalactiae was isolated then identified by PCR/DGGE in the eye and ear (Fig 3); direct PCR and IF test (Fig 4) for M agalactiae confirmed these results. M bovis was isolated and identified by PCR/DGGE in the tonsillar crypt. Direct PCR was more sensitive with positive results for M bovis in nasal swabs, eye, tonsillar crypt, ear lungs and kidney. Proteus mirabilis, an anaerobic Gram-negative bacteria that can cause nephritis, mastitis and pneumonia in cattle, was detected in the lungs and kidneys. Bull 2 was a richer source of mycoplasmas though M bovis was isolated and identified by DGGE only from the inner ear and lungs mixed with Mycoplasma dispar and Mycoplasma bovirhinis, respectively. These mycoplasmas are of unknown pathogenicity. Many more samples were positive by direct M bovis PCR including nasal swabs, eyes, ears and optic and auditory nerves. Interestingly M agalactiae was isolated and identified by DGGE in pure culture from both eyes, ears, optic chiasmas and brain spinal fluid. All positive detections were confirmed by direct M agalactiae PCR (Subrahamaniam and others 1998); the OIE reference Laboratory for Contagious Agalactia (APHA, UK) confirmed the identification of M agalactiae from both bulls. Real-time PCR for M bovis was negative following testing of two isolates of M agalactiae from each bull (Sachse and others 2010).
It was clear from a study of the present outbreaks that the critical infection point was at the weaning phase where calves from many different sources were mixed, facilitating the spread of the mycoplasmas. To overcome this, calves were retained on their original farms for weaning, then moved directly to the fattening areas on the farm. This led to a major reduction in cases of otitis that are presently being monitored.
The isolation and detection of M agalactiae from numerous samples taken from the two bulls is highly unusual. In view of the close genetic relationship between M bovis and M agalactiae, it is not unreasonable to suspect that the various diagnostic tests may have lacked specificity. However, the PCR/DGGE is quite unambiguous with clear differentiation between the two mycoplasmas (McAuliffe and others 2005). Moreover, the real-time PCR for M bovis was also quite definitively negative for the M agalactiae isolates. It is interesting to note that M agalactiae was not detected in samples from the respiratory systems but only from the eyes, ears and brain of the bulls. This could reflect the tissue tropism of the mycoplasma in its normal small ruminant host where it is rarely found in the lungs. M bovis, on the other hand, was found more widely. The source of the M agalactiae is unknown as there were no small ruminants on the farm or in the vicinity and there have been no reported outbreaks of CA for over five years. It is of course possible that M agalactiae is more prevalent in cattle than previously believed but is misidentified as M bovis because of its close biological similarities.
Further research including molecular typing of isolates, which is planned, should help us further understand the relationship between the isolates found here. These results have important implications because M agalactiae is a notifiable organism in many countries and its isolation from cattle in countries free of CA would be problematic.
Acknowledgement The authors are grateful for the help of the Animal and Plant Health Agency, UK, for their confirmation of the identity of some of the isolates in this work.
Contributors SC is the leader of the project responsible for planning of the work and writing the first drafts of the paper. FG performed animal inspections, supervised the diagnostic tests and analysed results. ES carried out the diagnostic tests and provided advice. and RAJN provided consultancy and completed writing the paper.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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