Original Research Article

Mycoplasmas in the brains of animals: An overlooked site of infection

Dr Robin A J Nicholas,
Reviewed by:
Dr İmdat Orhan
Dr Mohamed Zakaria

Recent experimental infections of sheep have shown histopathological changes caused by this mycoplasma in the CNS. The cattle pathogen, M. bovis, has been reported occasionally in the brains of calves and adult cattle showing a range of histopathological lesions including abscesses and fibrinous meningitis.

*Corresponding author:

Robin A. J. Nicholas
robin.nicholas@btinternet.com

Keywords:

Mycoplasmas, mollicutes, spiroplasmas, brain, central nervous system, TSE

Unlike mollicutes found in humans such as Mycoplasma pneumoniae and Ureaplasma species, those of animals are not usually associated with diseases of the central nervous system (CNS). Animal mycoplasmas are usually causes of respiratory and joint disease. However Mycoplasma agalactiae, the main cause of the OIE-listed contagious agalactia in small ruminants, is found in large quantities in the brain where it may be responsible for undiagnosed non-purulent encephalitis as well as ataxia in young animals. Recent experimental infections of sheep have shown histopathological changes caused by this mycoplasma in the CNS. The cattle pathogen, M. bovis, has been reported occasionally in the brains of calves and adult cattle showing a range of histopathological lesions including abscesses and fibrinous meningitis. Two avian pathogens, M. gallisepticum and M. synoviae have been isolated from the brains of poultry showing meningeal vasculitis and encephalitis.
Mycoplasmas have been isolated from the brains of sea mammals which died explicably in large numbers but it is thought that the mycoplasma exacerbated the effects of an underlying viral disease. Finally evidence has been advanced that certain Spiroplasma species may have a role in the development of the transmissible spongiform encephalopathies. This review critically examines the role of mycoplasmas in diseases in the brains of animals.

In addition to causing respiratory disease, human mycoplasmas, small wall-less bacteria belonging to the class Mollicutes, have long been known to affect the brain. Severe central nervous system (CNS) diseases, such as encephalitis, have been commonly linked to Mycoplasma pneumoniae infections.There is evidence that Ureaplasma species may be pathogenic in preterm babies causing inflammation of the CNS and abscesses in the brains of adults [1]. Even the apparently non pathogenic M. hominis has been linked to brain abscesses usually following cranial trauma or surgery [2].
Animal mycoplasmas on the other hand are usually associated with respiratory disease, arthritis and keratoconjunctivitis and, additionally, mastitis in ruminants. They generally do not cause neurological signs and so are not considered pathogens of the CNS. However recent evidence suggests that mycoplasmas may play a role in some neurological conditions in animals and should be considered when carrying out differential diagnosis. This review examines instances where mycoplasmas have been isolated from the CNS of livestock and discusses whether they are more widely prevalent in diseases of the brain.

Contagious agalactia (CA), caused predominantly by Mycoplasma agalactiae, is probably one of the least well known of the diseases listed by the World Association for Animal Health (OIE) for its socio-economic impact, mainly because it is a disease most keenly felt by poor farmers whose animals often subsist on marginal land. It is primar- ily a disease of sheep and goats that are kept for milk and dairy products using traditional husbandry, rather than for production on an industrial scale. The disease is ?rst noticed when milk output falls, usually a few days after the introduction of new asymptomatic stock or from mixing with affected herds at markets, pasture or water sources. Milk becomes thickened, discoloured and granular then production ceases in one or both udders, sometimes per- manently. Kerato-conjunctivitis and arthritis are chronic sequelae particularly severe in young animals. The young born from affected females have been reported to be ataxic (figure 1) which may be related to septicaemia and/or the presence of the mycoplasma in the brain [3].

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Figure 1: Lamb with polyarthritis and ataxia born to ewe affected by contagious agalactia.

Until relatively recently highly effective vaccines against CA had been produced for over 50 years at the Italian Istituto Zoopro?lattico Sperimentale of Latium and Tuscany from the mammary glands and brain tissue of artificially-affected sheep [4]. Harvested tissues were then diluted and inactivated with formalin before use. In 2013, Loria and Nicholas [5] examined the original research notes of Dr Zavagli at the IZS library in Palermo and con?rmed that the brain of affected sheep contained high concentrations of mycoplasma antigen, second only to the udder. Unfortunately the production of this vaccine was discontinued following severe adverse reactions seen in goats caused by the scrapie agent probably present in the brains of some of the experimentally infected sheep[6].

Puleio et al [7] reported for the first time histopatho- logical lesions in brains of sheep experimentally infected with M.agalactiae via the mammary gland. All brain tissues from sheep euthanized after1 and 2 months were positive for mycoplasma DNA by real time PCR, while culture tests on some of the brains were also positive. Microscopically the brain tissue showed an accumula- tion of leukocytes in the adventitia of vessels and in the perivascular Virchow-Robin space; neuronal degenera- tion, characterised by chromatolysis with swollen cells, pyknotic nuclei and pale pink cytoplasm was evident in stained sections. The most common inflammatory cells were lymphocytes which accumulated around blood ves- sels, known as perivascular cuffing (figure 2), and were spread diffusely in the infected tissue. Microglial cells formed small collections around dead infected neurons. Microscopically non-purulent encephalitis was the major finding (figure 3). Exactly how the mycoplasma colonises the brain, and what effect this has, is presently uncertain, but may well account for a high percentage of undiagnosed inflammatory lesions presently classifed as non-purulent encephalitis. Further studies are required to clarify the clini- cal and epidemiological implications and moreover to inves- tigate the still unknown role of the CNS in the pathogenesis of mycoplasma diseases generally and CA specifically.

Mycoplasma bovis is an important pathogen of cattle and a primary cause of pneumonia, arthritis and mastitis [8] and has also been linked to other clinical disease including kerato-conjunctivitis and otitis media [9]. Investigators do not usually associate mycoplasma infections with the brain and therefore attempts to isolate them would not normally be considered.

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Figure 2: Brain of sheep experimentally infected with Mycoplasma agalactiae lymphocytes which accumulated around blood vessels (perivascular cuffing)20x (H&E).
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Figure 3: Brain of sheep experimentally infected with Mycoplasma agalactiae showing focal microgliosis and neuronal degeneration; cell shrinkage with condensed hyper-eosinophilic cytoplasm and apoptotic nuclei is evident. 20x H&E.

However, there are a few reports of isolation of M.bovis from the brain of individual calves. Stipkovits et al [10] described cases from a Hungarian farm where 3-to 18-day-old calves were affected by M.bovis causing sero-fibrinous arthritis accompanied by pneumonia; fibrinous meningitis was also observed in an 8-day-old animal that had shown nervous signs accompanied by arthritis. Machado et al [11] reported a similar, single case in Portugal where M.bovis was isolated from a calf that had a rapidly evolving illness where nervous signs predominated. Histological examination revealed cellu- lar fibrinous meningitis in the cerebrum and cerebellum. Maeda et al [9] also reported the isolation of M.bovis from the brains of two calves with moderate meningitis in the area of the cerebellum, but failed to show M.bovis antigen in the brain tissue by immunohistochemistry.

Two separate cases of M.bovis isolation from brain tissue of calves in the British Isles were reported several years ago [12]. The first case occurred on a dairy farm in the north-west of England where four calves, seropositive for M.bovis, displayed head tilt. One of the affected heifer calves had previously shown clinical signs of head tilt at two months of age and was unresponsive to treatment with antibiotics. The animal then became ataxic, recum- bent and developed CNS signs including convulsions. Postmortem findings included cloudiness and scarring of the left cornea, purulent meningitis affecting the left side of the cerebellum, the area adjacent to the left inner/ middle ear (petrous temporal bone) and the ventral part of the brain near the pituitary. M.bovis was isolated from the affected part of the brain close to the left inner/middle ear. No other organs appeared to be affected and no other pathogens were isolated following postmortem examina- tion. Histopathological examination of the brain revealed a unilateral cerebellopontine abscessation consistent with spread to the cranial vault from a focus of otitis media/ interna. The remaining three affected calves, while showing varying degrees of head tilt had histologically normal brains.
The second case occurred in Ireland on a beef farm in a newly introduced male calf which developed respiratory disease. After an apparent recovery following antibiotic treatment, it failed to thrive and developed clinical signs of lethargy, depression, apparent blindness, severe weight loss and was also grinding its teeth. Postmortem exami- nation revealed multiple areas of necrosis within the cer- ebral hemispheres of the brain (figure 4) and a fibrinous lesion in the heart. M.bovis was subsequently identified from the brain and heart lesions. Further confirmation of the invasive role of M.bovis this time in the brain of an aborted foetus was provided by Hermeyer et al [13] using immune-cytochemical methods.

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Figure 4: Brain from calf naturally infected with Mycoplasma bovis shows multiple areas of necrosis within the cerebral hemispheres of the brain (Dr W. Byrne).
The prevalence of M.bovis is almost certainly under- estimated [14], but these cases where M.bovis was the sole pathogen isolated from bovine brains emphasise the invasive nature of the organism and the extensive range of diseases it can cause. The presence of M.bovis in the brain should be brought to the attention of veterinary diagnosticians in order that they may include it for dif- ferential diagnoses of similar cases.

Four mycoplasmas are considered pathogenic for do- mestic poultry: M.gallisepticum, M.synoviae, M.meleagridis and M.iowae giving a range of clinical signs including respiratory disease, synovitis and joint disease. Myco- plasmas have been isolated sporadically from avian brains although their significance is not known but almost cer- tainly under-reported as the CNS is rarely sampled during routine diagnosis.

Meningo-encephalitis was seen for the first time in several turkey flocks in western France between 1998 and 2005 [15]. Affected birds were about 2 months old and showed neurological signs, especially torticollis, with more than half also having respiratory signs. Examination of brain samples showed similar lesions of acute to suba- cute multifocal parenchymal necrosis, perivascular cuffing, leptomeningitis and vasculitis. Many birds were seroposi- tive for M.gallisepticum and specific antigen was detected in formalin-fixed sections of brain. Much et al [16] were able to reproduce brain lesions using aerosol inoculation of a low but not high passage strain of M.gallisepticum. This was linked to the ability of the low passage strain to invade eukaryotic cells in vitro.

M. synoviae was isolated from the brains of 22-week-old commercial meat turkeys displaying severe synovitis and infrequent CNS signs [17]. Histological examination of the brains revealed mild-to-severe meningeal vasculitis which ranged from fibrinoid necrosis with little inflammation to a marked infiltration of lymphocytes and plasma cells. This infiltration disrupted the architecture of the vessel wall, accumulating as perivascular cuffs, and involving surrounding meninges. Some arteries were thrombotic. This was the first known field case of M.synoviae isolation from the brains of turkeys.

It is interesting to note that no mycoplasmas could recovered from the brains of young turkeys experimentally infected with strains of M.iowae of differing virulence dur- ing attempts to develop a persistent infection [18]. There are no reports of M.meleagridis involvement with the CNS.

About 30 years ago large numbers of harbour seals died inexplicably in the North Sea. Clinical signs included pneumonia, skin lesions, diarrhoea, polyarthritis, nerv- ous signs, and abortions in pregnant females [19]. In ad- dition to detecting several viruses including one similar to canine distemper virus, over 100 mycoplasma strains were isolated from about a third of the 265 seals investi- gated. Mycoplasmas were found mainly in the respiratory tracts but were also isolated frequently from eyes, hearts, and brains. Indeed mycoplasmas were isolated from the brains of a third of the tested seals. The strains comprised two new mycoplasmas which were subsequently named M.phocicerebrale and M.phocirhinis. The role of these mycoplasmas in this and later outbreaks has never been clarified but respiratory disease and polyarthritis are characteristic of mycoplasma infections in animals. If not primary causes then their large numbers and wide distribution throughout the seals would suggest that they would have played some role in the disease process. Mycoplasmas have been subsequently isolated from many different species of sea mammals but few workers exam- ined the CNS for evidence of these organisms. Finally it is tempting to speculate that brain infections, caused possibly by mycoplasmas, may lead to disorientation and the increasingly frequent strandings of sea mammals on the coast. This theory awaits further study.

A controversial example of where mycoplasmas may play a role is in the aetiology of transmissible spongiform encephalopathies (TSE), most important of which for livestock are bovine spongiform encephalopathy and scrapie. The present consensus is that a miss-folded host protein, the prion, is the sole cause of these untreatable brain diseases. Over the last 30 years Bastian and his team in the USA have raised doubts and postulated that the normal protein is merely the receptor for a conventional but previously unidentified bacterium [20]. Evidence includes the unusual sensitivity of the pathogen to tetracy- cline, extreme resistance and the filterability of the 35 nm pathogen. These results are supported by animal studies in which a mollicute, Spiroplasma mirum, induced clinical neurological damage in rodents very similar to experi- mental scrapie [20]. Furthermore spiroplasmas isolated from scrapie-affected sheep brains caused spongiform encephalopathies in sheep, goats and deer [21]. Clearly further work is urgently needed to clarify this important issue; however there seems a real reluctance of prion workers to engage in the possibility that an infectious agent rather than a protein that does not require nucleic acid to replicate is the cause of TSEs.

In view of these examples it is possible to speculate that many pathogenic mycoplasmas are able to cross the blood-brain barrier and cause neuropathological effects in both man and animals. It is well known that mycoplasma infections are chronic and the organism attempts to escape the host immune system by locating in sites such as the joints and eyes. It is highly likely that the CNS may be another site where the mycoplasma resides between periodic disease eruptions within the host. Further work is necessary to confirm these findings and to investigate whether mycoplasmas may be the cause of a number of undiagnosed neuropathological conditions such as the non-purulent encephalitis described above which are frequently seen at post-mortem examinations. Their potential role in the development of TSE is also of great importance.

1. K. Glaser, C.P. Speer: Neonatal CNS infection and inflammation caused by Ureaplasma species: rare or relevant? Expert. Rev. Anti. Infect. Ther.(2015) 13, 233-248.
2. W.J.Whitson, P.A. Ball, S.S. Lollis, J.D. Balkman, D.F. Bauer: Postoperative Mycoplasma hominis infections after neurosurgical intervention. J. Neurosurg. Pediatr. (2014) 14, 212-218. Nicholas et al. (2017) Journal of Veterinary and Dairy Sciences 5
3. G. R. Loria, C. Casalone, L. Sparacino, A. Guercio, B. Iulini, F. Oliveri, A. Tamburello, V. Monteverde, S. Caracappa, R. A. J. Nicholas: Mycoplasma agalactiae in sheep brains: a new site of infection? Large AnimalReview (2007) 13, 65-68.
4. V. Zavagli: L’agalaxie contagieuse des brebis et des chèvres. Bull. Off. int. Epiz., (1951) 36, 336-361.
5. G.R. Loria, R.A.J. Nicholas: Contagious agalactia: the shepherd’s nightmare. Vet J. (2013) 198, 5-6.
6. M. Caramelli, G. Ru, C.Casalone, E.Bozzetta, P.L. Acutis, A. Calella,. G.Forloni: Evidence for the transmission of scrapie to sheep and goats from a vaccine against Mycoplasma agalactiae. Vet. Rec. (2001)148, 531-536.
7. R. Puleio, A.Tamburello, A. Cusimano, A. Console, R.A.J. Nicholas, G.R Loria:Isolation of M. agalactiae from the brains of experimentally infected sheep. Proceeding of the 2014 International Organisation of Mycoplasmology, Blumenau, Brazil Poster 49, pp 43-44.
8. H. Pfutzner, K. Sachse: Mycoplasma bovis as an agent of mastitis,pneumonia,arthritis and genital disorders. Rev. Sci. Tech. OIE. (1996) 15, 1477-1494.
9. T.Maeda, T. Shibahara, K. Kimura, Y.Wada, K. Sato,Y. Imada, Y. Ishikawa, K. Kadota: Mycoplasma bovis associated suppurative otitis media and pneumonia in bull calves. J. Comp. Pathol. (2003) 129, 100-110.
10.L. Stipkovits, M. Rády, R. Glávits: Mycoplasmal arthritis and meningitis in calves. Acta Vet. Hungarica. (1993) 41, 73-88.
11.M. Machado, M.M. Monteiro, V. Atalaia: Infection by Mycoplasma bovis in calves. Its isolation from a case of meningitis. Reposit. Trab. Lab. Nac. Invest. Vet. (1987) 19, 67-74.
12.R. D. Ayling, R.A.J. Nicholas, J. Wessells, R. Hogg, W. Byrne: Isolation of Mycoplasma bovis from the brain tissue of calves. Vet. Rec.(2005) 156, 391-392.
13.K. Hermeyer, M. Peters, M. Brügmann, B.J acobsen, M.J. Hewicker-Trautwein: Demonstration of Mycoplasma bovis by immunohistochemistry and in situ hybridization in an aborted bovine fetus and neonatal calf. Vet. Diagn. Invest. (2012) 24, 364-369.
14.R. A. J. Nicholas, R. D. Ayling: Mycoplasma bovis: disease, diagnosis, and control.Res. Vet. Sci.(2003) 74, 105-112.
15.B. Wyrzykowski, O. Albaric, S. Moreau, F. Nguyen , R. Fleurance, S. Belluco, M. Wyers, M.A. Colle: Retrospective study of Mycoplasma gallisepticum meningoencephalitis in six turkey flocks in western France. J. Comp. Pathol. (2013) 148, 173-177.
16.P. Much, F. Winner, L. Stipkovitz, R. Rosengarten, C. Citti:Mycoplasma gallisepticum: influence of cell invasiveness on the outcome of experimental infection in chickens. FEMS Immunol. Med. Microbiol. (2002) 34, 181-186.
17.R. P. Chin, C. U. Meteyer, R.Yamamoto, H. L. Shivaprasad, P. N.Klein: Isolation of Mycoplasma synoviae from the brains of commercial meat turkeys with meningeal vasculitis Avian Dis. (1991) 35, 631-637.
18.F.T. Jordan, B.K. Horrocks, S.K. Jones, C.M.Clee: The production of Mycoplasma iowae infection of turkey poults suitable for monitoring antimicrobials. Avian Pathol. 1992, 307- 313.
19.J. Giebel, J. Meier, A. Binder, J. Flossdorf, J. B. Poveda, R. Schmidt, H. Kirchhoff: Mycoplasma phocarhinis sp. nov.and Mycoplasma phocacerebrale sp. nov., Two New Species from Harbor Seals (Phocavitulina L.) Int.J.System. Bacteriol. (1991) 41, 39-44.
20.F.O. Bastian: Creutzfeldts-Jakob and other transmissible spongiform encephalopathies. Mosby-Year Book, New York (1991).
21.F.O. Bastian, D.E. Sanders, W.A. Forbes, S.D. Hagius, J.V. Walker, W.G. Henk, F.M. Enright, P.H. Elzer: Spiroplasma spp from transmissible spongiform encepathology brains or ticks induce spongiform encephalopathy in ruminants. J. Med. Microbiol.(2007) 56, 1235-12

Received: 16 March 2017

Published: 22 March 2017

Copyright:

Copyright © 2017 Robin A. J. Nicholas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.