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Mycoplasmas: Brain invaders?
Research in Veterinary Science 113 (2017) 56–61
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Review
Mycoplasmas: Brain invaders? a
b
MARK b
c
Rubén S. Rosales , Roberto Puleio , Guido R. Loria , Salvatore Catania , Robin A.J. Nicholas
d,⁎
a Instituto Universitario de Sanidad Animal y Seguridad Alimentaria, Universidad de Las Palmas de Gran Canaria, C/Trasmontaña s/n, Arucas, 35416, Gran Canaria, Spain b Istituto Zooprofilattico Sperimentale della Sicilia, Via G. Marinuzzi 3, 90129 Palermo, Italy c Avian Medicine Laboratory, SCT-1, Istituto Zooprofilattico delle Venezie, Viale dell'Università 10, 35020 Legnaro, PD, Italy d The Oaks, Nutshell Lane, Upper Hale, Farnham, Surrey GU9 0HG, UK
A R T I C L E I N F O
A B S T R A C T
Keywords: Mycoplasmas Mollicutes Spiroplasmas Brain Central nervous system TSE
Mycoplasmas of humans and animals are usually associated with respiratory, autoimmune, genital and joint diseases. Human mycoplasmas have also been known to affect the brain. Severe central nervous system (CNS) diseases, such as encephalitis, have been linked to Mycoplasma pneumoniae and ureaplasma infections. Less well known is the sheep and goat pathogen, Mycoplasma agalactiae, which has been found in large quantities in the brain where it may be responsible for non-purulent encephalitis as well as ataxia in young animals. Experimental intra-mammary infections of sheep with this mycoplasma have resulted in histopathological changes 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. There have been no reported detections of two other avian pathogens, M. meleagridis or M. iowae in the CNS. Over the last few decades, mycoplasmas have been isolated from the brains of sea mammals dying in large numbers in the North Sea although it was concluded that their role may be secondary to underlying viral disease. Finally, evidence has been advanced that certain Spiroplasma species may have a role in the development of the transmissible spongiform encephalopathies (TSE). Invasion of the brain by mycoplasmas may be as a result of direct entry following damage to the inner ear as seen with M. bovis or across the blood brain barrier by mechanisms as yet uncertain.
1. Introduction Mycoplasmas, belonging to the class Mollicutes, which contains wallless bacteria and also includes ureaplasmas, spiroplasmas, haemoplasmas and acholeplasmas, are some of the smallest known self-replicating organisms. During their long evolution from Gram-positive bacteria they have lost, in addition to genes for cell wall biosynthesis, those for many biosynthetic processes to a point where they have become totally dependent on the host for survival. Some contain fewer than 500 genes making them close to the concept of the minimal cell i.e. the minimum number of genes required to sustain life. The majority have almost become the “ideal parasites” colonising the epithelial mucosa of the respiratory and urogenital tracts placing very few demands on the host as their energy requirements are very low (Razin, 1992). Consequently due to their extracellular location, mycoplasmas rarely provoke the host immune system. There are however exceptions and those include some of the most important pathogens of man and animals though their pathogenicity may often be a result of a miss-
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functioning immune system rather than the virulence of the mycoplasma itself (Pilo et al., 2007). These pathogenic mycoplasmas are usually associated with respiratory, urogenital and autoimmune diseases in man and, occasionally, arthritis, eye disease and mastitis in animals. They are rarely linked to diseases of the brain and the occasional reported isolations are often considered circumstantial. These organisms were also considered to be extracellular rarely entering the circulation, but increasing evidence has now shown that many mycoplasmas can enter, survive and multiply inside host cells where they may be transported to a range of body sites (Hegde et al., 2014). This review examines selective cases where mycoplasmas have been isolated from the CNS of animals, discusses whether they are more widely prevalent in diseases of the brain and speculates on the potential mechanisms of penetration. 2. Human mycoplasmas
Corresponding author. E-mail address: [email protected] (R.A.J. Nicholas).
http://dx.doi.org/10.1016/j.rvsc.2017.09.006 Received 16 June 2017; Received in revised form 16 August 2017; Accepted 3 September 2017 0034-5288/ © 2017 Elsevier Ltd. All rights reserved.
In addition to causing respiratory disease, human mycoplasmas
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have been known to affect the brain, though, seemingly, infrequently. Severe CNS diseases, such as encephalitis, have been occasionally linked to Mycoplasma pneumoniae infections (Tsiodras et al., 2005). There is evidence that Ureaplasma species may be pathogenic in premature babies causing inflammation of the CNS and abscesses in the brains of adults (Glaser and Speer, 2015). Even the apparently nonpathogenic M. hominis has been linked to brain abscesses although this is usually following cranial trauma or surgery (Whitson et al., 2014). Several species of mycoplasma including M. fermentans, M. penetrans and M. pirum have been isolated from tissues including the brains of HIV positive patients (Wohlman et al., 2001). Although the exact role that the mycoplasmas are playing in AIDS is not clear, it is known that M. fermentans increases the cytopathogenicity and toxicity of HIV. Furthermore this mycoplasma can increase the production of glucocorticoids which bind to brain tissue contributing to the neuropathology in AIDS patients (Wohlman et al., 2001).
Fig. 1. Inner ear of bull showing purulent exudates and necrotic osteitis consisting of dry and grey bone. Mycoplasma bovis was isolated from the ear.
The second case occurred on a beef farm in a newly introduced male calf which developed respiratory disease. After an apparent recovery following antibiotic treatment, it failed to grow and developed clinical signs of lethargy, depression, apparent blindness, severe weight loss and was also grinding its teeth. Post mortem examination revealed multiple areas of necrosis within the cerebral hemispheres of the brain and a fibrinous lesion in the heart; M. bovis was subsequently identified from both organs. Further confirmation of the invasive role of M. bovis this time in the brain of an aborted foetus was provided by Hermeyer et al. (2012) using immunocytochemical methods. In an unusual case of otitis and pneumonia in cattle where both M. bovis and the closely related small ruminant pathogen, M. agalactiae were isolated from the same herd, the latter was detected in the cerebrospinal fluid and optic chiasmas of affected animals (Catania et al., 2016b). Investigation of the inner ear of one of the animals showed the presence of brownish exudates in the auditory external duct. A transverse section of the pars petrosa revealed purulent exudates with a necrotic osteitis consisting of dry and grey bone (Fig. 1). The lumen of the inner ear also contained purulent exudates. Histopathological examination of the brain, from where the mycoplasma was isolated showed the characteristic lymphocytic perivascular cuffing surrounding the meningeal blood vessels (Fig. 2). Thus it is feasible the damage to the middle and inner ear could lead to the invasion of the brain via the cranial nerves leading to meningitis (Lamm et al., 2004). The prevalence of M. bovis is almost certainly underestimated (Nicholas and Ayling, 2003), but these cases where M. bovis was the sole pathogen isolated from bovine brains emphasise the invasive
3. Bovine mycoplasmosis Mycoplasma bovis is an important emerging pathogen of cattle causing pneumonia, arthritis and mastitis (Pfützner and Sachse, 1996) and has also been linked to other clinical diseases including keratoconjunctivitis and otitis media (Maeda et al., 2003). Investigators do not usually associate mycoplasma infections with the brain and therefore attempts to isolate them would not normally be considered. However, there are increasing reports of isolations of M. bovis from the brains of individual calves. Stipkovits et al. (1993) described cases from a farm where 3 to 18-day-old calves were affected by M. bovis causing serofibrinous 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. (1987) 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 cellular fibrinous meningitis in the cerebrum and cerebellum. Maeda et al. (2003) also reported the isolation of M. bovis from the brains of two calves with moderate meningitis in the cerebellum, but failed to show M. bovis antigen in the brain tissue by immunohistochemistry. In the Veneto region of Italy, a bull with a chronic head tilt was examined post mortem revealing a chronic catarrhal bronchopneumonia (Catania, unpublished results). Furthermore, a significant inflammatory lesion involving the inner ear was seen as well as a large abscess at the base of the mesencephalic area of the brain from which M. bovis was isolated. The microbiological examination also showed the presence of various bacteria including Trueperella pyogenes, Staphylococcus spp., and Corynebacterium spp. which were almost certainly secondary invaders to the mycoplasma infection which was responsible for extensive pathognomonic lung lesions. Two separate cases of M. bovis isolation from brain tissue of calves were reported by Ayling et al., 2005. The first case occurred on a dairy farm 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, recumbent and developed CNS signs including convulsions. Post mortem 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 post mortem examination. 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.
Fig. 2. Histopathological examination of Mycoplasma bovis infected brain from bull showing characteristic lymphocytic perivascular cuffing surrounding the meningeal blood vessels. (H & E).
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nature of the organism and the extensive range of diseases it can cause. We speculate from these cases that the pathogen may enter the brain through invasive infections from the affected inner ear or via the blood stream where there is no evidence of otitis. 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 differential diagnoses of similar cases. 4. Sheep and goat mycoplasmas Mycoplasma agalactiae is the main cause of contagious agalactia (CA), which is probably one of the least well known of the diseases listed by the World Organisation for Animal Health (OIE) for its socioeconomic impact, mainly because it is a disease most keenly felt by poor farmers whose animals often subsist on marginal land (Loria and Nicholas, 2013). It is a disease of sheep and goats that are kept for milk and dairy products using traditional husbandry mainly involving manual milking, rather than for industrial scale production (Loria et al., 2007). The disease is first seen when milk output falls, usually a few days after the introduction of carriers 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 permanently. Kerato-conjunctivitis and arthritis are chronic sequelae which are particularly severe in young animals. The young born from affected females have been reported to be ataxic which may be associated with septicaemia and/or the presence of the mycoplasma in the brain (Loria et al., 2007). Until relatively recently effective formalin-inactivated vaccines against CA were produced in Italy from the mammary and brain tissue of sheep using the intramammary infection route pioneered by Zavagli (1951). In 2013, Loria and Nicholas (2013) examined the original research notes of Dr. Zavagli at the IZS library in Palermo and confirmed that the brain of infected sheep contained very high concentrations of mycoplasma antigen, second only to the udder. Unfortunately the production of this vaccine was discontinued following the deaths of many goats caused by the scrapie agent probably present in the brains of some of the experimentally infected sheep (Caramelli et al., 2001). Puleio et al. (2014) reported for the first time histopathological lesions in brains of sheep experimentally infected with M. agalactiae via the mammary gland. All brain tissues from sheep euthanized after 1 and 2 months were positive for mycoplasma DNA by real time PCR. Microscopically the brain tissue showed an accumulation of leukocytes in the adventitia of vessels and in the perivascular Virchow-Robin space; neuronal degeneration, characterised by chromatolysis with swollen cells, pyknotic nuclei and pale pink cytoplasm was evident in stained sections (Fig. 3). The most common inflammatory cells were lymphocytes which accumulated around blood vessels, known as perivascular cuffing, 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, which is a commonly seen lesion at necropsy but not usually associated with mycoplasma. In the same year Hegde et al. (2014) also reported the detection of M. agalactiae in macrophages in the sheep brain by immunocytochemistry following an intramammary infection although there was no description of pathology. The authors concluded that the mycoplasma was capable of invading and partially or fully surviving the phagocytosis after having been disseminated around the body, in particular, to the brain. Mycoplasma mycoides subsp. capri is a member of the important M. mycoides cluster, a group which contains ruminant pathogens including those that cause the OIE-listed contagious bovine and caprine pleuropneumonias. In addition to being another aetiological agent of CA, it can cause respiratory disease in goats. In 2012 Gomez-Martin et al. isolated the mycoplasma from the brains of 3 male goats which had been identified as chronic auricular carriers of M. m. capri over one year before. However, no clinical or histopathological signs were reported.
Fig. 3. Brain of sheep experimentally infected with Mycoplasma agalactiae. Lymphocytes have accumulated around blood vessels “perivascular cuffing” (top). Also showing focal microgliosis and neuronal degeneration; cell shrinkage with condensed hyper-eosinophilic cytoplasm and apoptotic nuclei is evident (bottom). (H & E).
More recently the carcass of a 40-day-old kid was examined at the diagnostic laboratory of the IZS Venezie (Catania, unpublished results). The medical history was scant but the owner reported that the animal had been depressed for several days before death but had shown no specific neurological signs. Post mortem examination did not show any important lesions with the exception of very mild enteritis probably caused by clostridia which were isolated from the gut. No bacteria were found in the brain with the exception of very small haemolytic microcolonies evident after 48 h in culture. These colonies were sub-cultured onto Mycoplasma Experience medium (Reigate, UK) and later identified as M. m. capri by PCR/denaturing gradient electrophoresis (McAuliffe et al., 2005) and confirmed by 16S rDNA sequencing. It was not clear whether the mycoplasma was the cause of the depression and death. Post mortem examination by the IZS Venezie of a 4 month-old goat presenting chronic respiratory disease revealed a severe fibrinous pleuropneumonia and pericarditis. M. ovipneumoniae, a worldwide pathogen, and the ubiquitous M. arginini were not unexpectedly isolated from the lungs and associated tissues. However, only M. arginini was isolated following investigations of the brain although no conspicuous lesions were seen. This raises further questions on the role of this opportunistic mycoplasma in disease processes (Nicholas et al., 2008).
5. Avian mycoplasmosis Four mycoplasmas are considered pathogenic for domestic poultry: M. gallisepticum, M. synoviae, M. meleagridis and M. iowae giving a range of clinical signs including respiratory disease, synovitis and joint disease. Mycoplasmas have been isolated sporadically from avian brains although their significance is not known but almost certainly underreported as the CNS is infrequently sampled during routine diagnosis. Meningo-encephalitis was seen for the first time in several turkey flocks between 1998 and 2005 (Wyrzykowski et al., 2013). 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 subacute multifocal parenchymal necrosis, perivascular cuffing, leptomeningitis and vasculitis. Many birds were seropositive for M. gallisepticum and specific antigen was detected in formalin-fixed sections of brain. Much et al. (2002) were able to reproduce brain lesions using aerosol inoculation of low but not high passaged strains of M. gallisepticum. This was linked to the ability of the low passaged strain to invade eukaryotic cells in vitro. 58
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which S. mirum induced clinical neurological damage in rodents very similar to experimental scrapie (Bastian et al., 1984). Furthermore, spiroplasmas isolated from scrapie-affected sheep brains caused spongiform encephalopathies in sheep, goats and deer (Bastian et al., 2007). These conclusions have been challenged by Alexeeva et al. (2006) who were unable to detect mollicute or other bacterial gene sequences in the brains of hamsters with experimental scrapie. While Bastian's work has not yet been reproduced by other groups, an important later finding has been the discovery of the spiroplasma biofilm which would account for the increased resistance of the pathogen and may also be involved indirectly in the formation of prion amyloid (Bastian, 2014). Clearly further work is urgently needed to resolve this important issue.
M. synoviae was isolated from the brains of 22-week-old commercial meat turkeys displaying severe synovitis and occasional CNS signs (Chin et al., 1991). 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, accumulating as perivascular cuffs, damaged the vessel wall and involved surrounding meninges. Some arteries were thrombotic. Detection of M. synoviae from the brains of layer hens following experimental infections via the trachea were recently reported by Catania et al. (2016a); moreover, the presence of M. synoviae in the brains of broiler was detected in an experimental intranasal and conjunctival inoculation (Catania, unpublished report). It is interesting to note that no mycoplasmas could be recovered from the brains of young turkeys experimentally infected with strains of M. iowae of differing virulence during attempts to develop a persistent infection (Jordan et al., 1992). So far there are no reports of M. meleagridis involvement with the CNS.
8. Pathogenesis of animal mycoplasma brain infections Blood-borne infections of the brain are very rare because of the effectiveness of the semi-permeable blood-brain barrier (BBB) which is composed of endothelial cells connected by tight junctions along the relevant capillaries. Bacteria that are able to cross this barrier include those that cause meningitis including Streptococcus pneumoniae, Haemophilus influenzae type B and Neisseria meningitidis. Inflammation of the meninges, the membrane that surrounds the brain and spinal cord, by toxic bacterial products following infection leads to an increase in permeability of the BBB and thus can allows the entry of some pathogens (van Sorge and Doran, 2012). The pathogenesis of brain infection in animals by mycoplasma remains elusive as little evidence of toxins like those of other bacteria has been found. M. pneumoniae, a human pathogen linked to cases of atypical pneumonia and sporadic CNS disease, produces a compound known as community-acquired respiratory distress syndrome (CARDS) toxin (Kannan and Baseman, 2006) which is directly linked to epithelial airway damage and plays a primary role in M. pneumoniae pathogenesis; however, there is no direct evidence of its association with CNS invasion by the mycoplasma. Interestingly, CARDS toxin is homologous to the Bordetella pertussis toxin subunit 1 (PT toxin S1) which has been linked to experimental autoimmune encephalitis by increasing the permeability of the BBB or by modulating immune response (Carbonetti, 2010). Narita (2009) described an evidence-based classification scheme for neurologic manifestations caused by M. pneumoniae. The pathogenesis of neurological infections by M. pneumoniae was divided into three categories: a direct type caused by locally produced cytokines; an indirect type linked to autoimmunity; and a vascular occlusion type associated with vasculitis or thrombosis. Interestingly, vasculitis has been linked to many cases of mycoplasma brain infections in birds and mammals (Chin et al., 1991; Wyrzykowski et al., 2013; Puleio et al., 2014). Autoimmunity has also been suggested as a possible pathogenicity mechanism in a cat with meningo-encephalomyelitis which was refractory to treatment with the fluoroquinolone antibiotic, marbofloxacin. M felis had colonised the bloodstream of the brain and was detected in the cerebrospinal fluid (Beauchamp et al., 2011). Therefore, the scheme proposed by Narita could also be considered relevant for mycoplasma brain infections in animals although more evidence is needed. Another crucial process leading to CNS invasion may be the ability of mycoplasmas to invade blood cells which would provide protection from the host immune system. The colonisation of brain tissue by mycoplasmas would then be linked to haematogenous dissemination or possibly by direct entry to the CNS (Ilha et al., 2010). It has long been known that M. neurolyticum was capable of inducing neurological signs in experimentally infected young rodents though the exact nature of the “toxin” was unknown (Tully, 1981). Later, neurominidase enzymatic activity (NEAC), found in many pathogenic bacteria, was detected in this mycoplasma and suggested as a potential virulence factor for CNS invasion (Berčič et al., 2012). Subsequently NEAC was shown in dogs with granulomatous meningo-
6. Sea mammal mycoplasmosis About 30 years ago large numbers of harbor seals died in the North Sea. Clinical signs included pneumonia, skin lesions, diarrhoea, polyarthritis, nervous signs, and abortions in pregnant females (Giebel et al., 1991). In addition 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 investigated. 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 species of mycoplasma which were subsequently named M. phocicerebrale and M. phocirhinis. The role of these mycoplasmas in this and later outbreaks in sea mammals has never been clarified but it is well known that respiratory disease and polyarthritis, seen in many of the affected seals, are highly characteristic of mycoplasma infections in other animals. If not primary causes then their large numbers and widespread distribution throughout the seals would suggest that they must have played some role in the disease process. Mycoplasmas have been subsequently isolated from many different species of sea mammals but few studies examined the CNS for evidence of their presence. 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. 7. Transmissible spongiform encephalopathies Spiroplasmas, which are fastidious mollicutes with spiral morphology, are most commonly associated with vector-borne plant diseases and some, like Spiroplasma mirum, are highly resistant to heat and chemical treatment including formalin. Following the discovery of “spiroplasma-like fibrils” in the brain of a patient with CreutzfeldtJakob disease, Bastian and his team in the USA proposed a role for this mollicute in the aetiology of transmissible spongiform encephalopathies (TSE), most important of which for livestock are bovine spongiform encephalopathy and scrapie (Bastian et al., 1984). The present consensus is that there is a miss-folding of a self-replicating host protein, the prion, which is the sole cause of these untreatable brain diseases. Bastian raised doubts about prion theory and postulated that the normal protein is merely the receptor for a conventional but previously unidentified bacterium. Research revealed that the pathogen was extremely resistant, ultra-filterable and unusually sensitive to tetracycline, strongly suggesting that the pathogen was bacterial. The team showed that the scrapie-associated fibrils seen in the brains of TSE-affected humans and animals were morphologically identical and immunologically similar to the internal fibril proteins of spiroplasmas (Bastian, 1991). These results were supported by animal studies in 59
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encephalomyelitis and necrotizing meningo-encephalitis caused by M. canis (Barber et al., 2012). Furthermore, neuraminidase activity has also been found in avian mycoplasmas involved in CNS infection such as M. synoviae, M. gallisepticum and one serovar of M. iowae (Berčič et al., 2008). Neuraminidase has been proven to facilitate pathogen colonisation, invasion and damage of host tissue (Corfield, 1992) and it is linked to CNS colonisation by other pathogens such as Streptococcus pneumoniae (Uchiyama et al., 2009). Other mycoplasmas carrying the neuraminidase gene include M. alligatoris, a mycoplasma involved in invasive lethal diseases and cases of pyogranulomatous meningitis in alligators and caimans (Brown and Zacher, 2004), M. cynos which is associated with canine respiratory disease (Walker et al., 2013) and the ubiquitous M. arginini (Genbank accession number: NZ_AUAH01000010.1). 9. Conclusions In view of the examples above it is possible to conclude that many pathogenic mycoplasmas are able to cross the BBB and cause neuropathological effects in both man and animals. How they cross this barrier is presently uncertain. They do not appear to possess toxins like other bacteria which are capable of causing inflammation that leads to the increase in its semi-permeability thus allowing micro-organisms across. Several candidate molecules have been identified in mycoplasmas of cats and birds such as neuraminidase. In the case of M. bovis, the damage caused to the inner ear may lead to direct entry to the brain as otitis media often with associated tissue destruction is a common early clinical sign. 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 investigate these findings and to elucidate whether mycoplasmas may be the cause of a number of undiagnosed neuropathological conditions such as the nonpurulent encephalitis which is frequently seen at necropsy. Finally if the scrapie agent is identified as postulated by Bastian then the contamination of the CA vaccine that killed many goats mentioned earlier may be another mycoplasma. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Alexeeva, I., Elliot, E.J., Rollins, S., Gasparich, G.E., Lazar, J., Rohwer, R.G., 2006. Absence of spiroplasma or other bacterial 16S rRNA genes in brain tissue of hamsters with scrapie. J. Clin. Microbiol. 44, 91–97. Ayling, R.D., Nicholas, R.A.J., Wessells, J., Hogg, R., Byrne, W., 2005. Isolation of Mycoplasma bovis from the brain tissue of calves. Vet. Rec. 156, 391–392. Barber, R.M., Porter, B.F., Li, Q., May, M., Claiborne, M.K., Allison, A.B., Howerth, E.W., Butler, A., Wei, S., Levine, J.M., Levine, G.J., Brown, D.R., Schatzberg, S.J., 2012. Broadly reactive polymerase chain reaction for pathogen detection in canine granulomatous meningoencephalomyelitis and necrotizing meningoencephalitis. J. Vet. Intern. Med. 26, 962–968. Bastian, F.O., 1991. Creutzfeldt-Jakob and other transmissible spongiform encephalopathies. Mosby, New York. Bastian, F.O., 2014. The case for involvement of spiroplasma in the pathogenesis of transmissible spongiform encephalopathies. J. Neuropathol. Exp. Neurol. 73, 104–114. Bastian, F.O., Purnell, D.M., Tully, J.G., 1984. Neuropathology of spiroplasma infection in the rat brain. Am. J. Pathol. 114, 496–514. Bastian, F.O., Sanders, D.E., Forbes, W.A., Hagius, S.D., Walker, J.V., Henk, W.G., Enright, F.M., Elzer, P.H., 2007. Spiroplasma spp from transmissible spongiform encephalopathy brains or ticks induce spongiform encephalopathy in ruminants. J. Med. Microbiol. 56, 1235–1242. Beauchamp, D.J., da Costa, R.C., Premanandan, C., Burns, C.G., Cui, J., Daniels, J.B., 2011. Mycoplasma felis-associated meningoencephalomyelitis in a cat. J. Feline Med. Surg. 13, 139–143.
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Whitson, W.J., Ball, P.A., Lollis, S.S., Balkman, J.D., Bauer, D.F., 2014. Postoperative Mycoplasma hominis infections after neurosurgical intervention. J. Neurosurg. Pediatr. 14, 212–218. Wohlman, A., Yirmiya, R., Gallily, R., Weidenfeld, J., 2001. Neuroimmunomodulation 9, 141–147. Wyrzykowski, B., Albaric, O., Moreau, S., Nguyen, F., Fleurance, R., Belluco, S., Wyers, M., Colle, M.A., 2013. Retrospective study of Mycoplasma gallisepticum meningoencephalitis in six turkey flocks in western France. J. Comp. Pathol. 148, 173–177. Zavagli, V., 1951. L'agalaxie contagieuse des brebis et des chèvres. Bull. Off. Int. Epiz. 36, 336–361.
Acta Vet. Hungarica 41, 73–88. Tsiodras, S., Kelesidis, I., Kelesidis, T., Stamboulis, E., Giamarellou, H., 2005. Central nervous system manifestations of Mycoplasma pneumoniae infections. J. Inf. Secur. 51, 343–344. Tully, J.G., 1981. Mycoplasma toxins. Isr. J. Med. Sci. 17, 604–607. Uchiyama, S., Carlin, A.F., Khosravi, A., Weiman, S., Banerjee, A., Quach, D., Hightower, G., Mitchell, T.J., Doran, K.S., Nizet, V., 2009. The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion. J. Exp. Med. 206, 1845–1852. Walker, C.A., Mannering, S.A., Shields, S., Blake, D.P., Brownlie, J., 2013. Complete genome sequence of Mycoplasma cynos strain C142. 1(1):e00196-12. doi:http://dx. doi.org/10.1128/genomeA.00196-12.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Review
Mycoplasmas: Brain invaders? a
b
MARK b
c
Rubén S. Rosales , Roberto Puleio , Guido R. Loria , Salvatore Catania , Robin A.J. Nicholas
d,⁎
a Instituto Universitario de Sanidad Animal y Seguridad Alimentaria, Universidad de Las Palmas de Gran Canaria, C/Trasmontaña s/n, Arucas, 35416, Gran Canaria, Spain b Istituto Zooprofilattico Sperimentale della Sicilia, Via G. Marinuzzi 3, 90129 Palermo, Italy c Avian Medicine Laboratory, SCT-1, Istituto Zooprofilattico delle Venezie, Viale dell'Università 10, 35020 Legnaro, PD, Italy d The Oaks, Nutshell Lane, Upper Hale, Farnham, Surrey GU9 0HG, UK
A R T I C L E I N F O
A B S T R A C T
Keywords: Mycoplasmas Mollicutes Spiroplasmas Brain Central nervous system TSE
Mycoplasmas of humans and animals are usually associated with respiratory, autoimmune, genital and joint diseases. Human mycoplasmas have also been known to affect the brain. Severe central nervous system (CNS) diseases, such as encephalitis, have been linked to Mycoplasma pneumoniae and ureaplasma infections. Less well known is the sheep and goat pathogen, Mycoplasma agalactiae, which has been found in large quantities in the brain where it may be responsible for non-purulent encephalitis as well as ataxia in young animals. Experimental intra-mammary infections of sheep with this mycoplasma have resulted in histopathological changes 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. There have been no reported detections of two other avian pathogens, M. meleagridis or M. iowae in the CNS. Over the last few decades, mycoplasmas have been isolated from the brains of sea mammals dying in large numbers in the North Sea although it was concluded that their role may be secondary to underlying viral disease. Finally, evidence has been advanced that certain Spiroplasma species may have a role in the development of the transmissible spongiform encephalopathies (TSE). Invasion of the brain by mycoplasmas may be as a result of direct entry following damage to the inner ear as seen with M. bovis or across the blood brain barrier by mechanisms as yet uncertain.
1. Introduction Mycoplasmas, belonging to the class Mollicutes, which contains wallless bacteria and also includes ureaplasmas, spiroplasmas, haemoplasmas and acholeplasmas, are some of the smallest known self-replicating organisms. During their long evolution from Gram-positive bacteria they have lost, in addition to genes for cell wall biosynthesis, those for many biosynthetic processes to a point where they have become totally dependent on the host for survival. Some contain fewer than 500 genes making them close to the concept of the minimal cell i.e. the minimum number of genes required to sustain life. The majority have almost become the “ideal parasites” colonising the epithelial mucosa of the respiratory and urogenital tracts placing very few demands on the host as their energy requirements are very low (Razin, 1992). Consequently due to their extracellular location, mycoplasmas rarely provoke the host immune system. There are however exceptions and those include some of the most important pathogens of man and animals though their pathogenicity may often be a result of a miss-
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functioning immune system rather than the virulence of the mycoplasma itself (Pilo et al., 2007). These pathogenic mycoplasmas are usually associated with respiratory, urogenital and autoimmune diseases in man and, occasionally, arthritis, eye disease and mastitis in animals. They are rarely linked to diseases of the brain and the occasional reported isolations are often considered circumstantial. These organisms were also considered to be extracellular rarely entering the circulation, but increasing evidence has now shown that many mycoplasmas can enter, survive and multiply inside host cells where they may be transported to a range of body sites (Hegde et al., 2014). This review examines selective cases where mycoplasmas have been isolated from the CNS of animals, discusses whether they are more widely prevalent in diseases of the brain and speculates on the potential mechanisms of penetration. 2. Human mycoplasmas
Corresponding author. E-mail address: [email protected] (R.A.J. Nicholas).
http://dx.doi.org/10.1016/j.rvsc.2017.09.006 Received 16 June 2017; Received in revised form 16 August 2017; Accepted 3 September 2017 0034-5288/ © 2017 Elsevier Ltd. All rights reserved.
In addition to causing respiratory disease, human mycoplasmas
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have been known to affect the brain, though, seemingly, infrequently. Severe CNS diseases, such as encephalitis, have been occasionally linked to Mycoplasma pneumoniae infections (Tsiodras et al., 2005). There is evidence that Ureaplasma species may be pathogenic in premature babies causing inflammation of the CNS and abscesses in the brains of adults (Glaser and Speer, 2015). Even the apparently nonpathogenic M. hominis has been linked to brain abscesses although this is usually following cranial trauma or surgery (Whitson et al., 2014). Several species of mycoplasma including M. fermentans, M. penetrans and M. pirum have been isolated from tissues including the brains of HIV positive patients (Wohlman et al., 2001). Although the exact role that the mycoplasmas are playing in AIDS is not clear, it is known that M. fermentans increases the cytopathogenicity and toxicity of HIV. Furthermore this mycoplasma can increase the production of glucocorticoids which bind to brain tissue contributing to the neuropathology in AIDS patients (Wohlman et al., 2001).
Fig. 1. Inner ear of bull showing purulent exudates and necrotic osteitis consisting of dry and grey bone. Mycoplasma bovis was isolated from the ear.
The second case occurred on a beef farm in a newly introduced male calf which developed respiratory disease. After an apparent recovery following antibiotic treatment, it failed to grow and developed clinical signs of lethargy, depression, apparent blindness, severe weight loss and was also grinding its teeth. Post mortem examination revealed multiple areas of necrosis within the cerebral hemispheres of the brain and a fibrinous lesion in the heart; M. bovis was subsequently identified from both organs. Further confirmation of the invasive role of M. bovis this time in the brain of an aborted foetus was provided by Hermeyer et al. (2012) using immunocytochemical methods. In an unusual case of otitis and pneumonia in cattle where both M. bovis and the closely related small ruminant pathogen, M. agalactiae were isolated from the same herd, the latter was detected in the cerebrospinal fluid and optic chiasmas of affected animals (Catania et al., 2016b). Investigation of the inner ear of one of the animals showed the presence of brownish exudates in the auditory external duct. A transverse section of the pars petrosa revealed purulent exudates with a necrotic osteitis consisting of dry and grey bone (Fig. 1). The lumen of the inner ear also contained purulent exudates. Histopathological examination of the brain, from where the mycoplasma was isolated showed the characteristic lymphocytic perivascular cuffing surrounding the meningeal blood vessels (Fig. 2). Thus it is feasible the damage to the middle and inner ear could lead to the invasion of the brain via the cranial nerves leading to meningitis (Lamm et al., 2004). The prevalence of M. bovis is almost certainly underestimated (Nicholas and Ayling, 2003), but these cases where M. bovis was the sole pathogen isolated from bovine brains emphasise the invasive
3. Bovine mycoplasmosis Mycoplasma bovis is an important emerging pathogen of cattle causing pneumonia, arthritis and mastitis (Pfützner and Sachse, 1996) and has also been linked to other clinical diseases including keratoconjunctivitis and otitis media (Maeda et al., 2003). Investigators do not usually associate mycoplasma infections with the brain and therefore attempts to isolate them would not normally be considered. However, there are increasing reports of isolations of M. bovis from the brains of individual calves. Stipkovits et al. (1993) described cases from a farm where 3 to 18-day-old calves were affected by M. bovis causing serofibrinous 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. (1987) 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 cellular fibrinous meningitis in the cerebrum and cerebellum. Maeda et al. (2003) also reported the isolation of M. bovis from the brains of two calves with moderate meningitis in the cerebellum, but failed to show M. bovis antigen in the brain tissue by immunohistochemistry. In the Veneto region of Italy, a bull with a chronic head tilt was examined post mortem revealing a chronic catarrhal bronchopneumonia (Catania, unpublished results). Furthermore, a significant inflammatory lesion involving the inner ear was seen as well as a large abscess at the base of the mesencephalic area of the brain from which M. bovis was isolated. The microbiological examination also showed the presence of various bacteria including Trueperella pyogenes, Staphylococcus spp., and Corynebacterium spp. which were almost certainly secondary invaders to the mycoplasma infection which was responsible for extensive pathognomonic lung lesions. Two separate cases of M. bovis isolation from brain tissue of calves were reported by Ayling et al., 2005. The first case occurred on a dairy farm 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, recumbent and developed CNS signs including convulsions. Post mortem 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 post mortem examination. 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.
Fig. 2. Histopathological examination of Mycoplasma bovis infected brain from bull showing characteristic lymphocytic perivascular cuffing surrounding the meningeal blood vessels. (H & E).
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nature of the organism and the extensive range of diseases it can cause. We speculate from these cases that the pathogen may enter the brain through invasive infections from the affected inner ear or via the blood stream where there is no evidence of otitis. 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 differential diagnoses of similar cases. 4. Sheep and goat mycoplasmas Mycoplasma agalactiae is the main cause of contagious agalactia (CA), which is probably one of the least well known of the diseases listed by the World Organisation for Animal Health (OIE) for its socioeconomic impact, mainly because it is a disease most keenly felt by poor farmers whose animals often subsist on marginal land (Loria and Nicholas, 2013). It is a disease of sheep and goats that are kept for milk and dairy products using traditional husbandry mainly involving manual milking, rather than for industrial scale production (Loria et al., 2007). The disease is first seen when milk output falls, usually a few days after the introduction of carriers 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 permanently. Kerato-conjunctivitis and arthritis are chronic sequelae which are particularly severe in young animals. The young born from affected females have been reported to be ataxic which may be associated with septicaemia and/or the presence of the mycoplasma in the brain (Loria et al., 2007). Until relatively recently effective formalin-inactivated vaccines against CA were produced in Italy from the mammary and brain tissue of sheep using the intramammary infection route pioneered by Zavagli (1951). In 2013, Loria and Nicholas (2013) examined the original research notes of Dr. Zavagli at the IZS library in Palermo and confirmed that the brain of infected sheep contained very high concentrations of mycoplasma antigen, second only to the udder. Unfortunately the production of this vaccine was discontinued following the deaths of many goats caused by the scrapie agent probably present in the brains of some of the experimentally infected sheep (Caramelli et al., 2001). Puleio et al. (2014) reported for the first time histopathological lesions in brains of sheep experimentally infected with M. agalactiae via the mammary gland. All brain tissues from sheep euthanized after 1 and 2 months were positive for mycoplasma DNA by real time PCR. Microscopically the brain tissue showed an accumulation of leukocytes in the adventitia of vessels and in the perivascular Virchow-Robin space; neuronal degeneration, characterised by chromatolysis with swollen cells, pyknotic nuclei and pale pink cytoplasm was evident in stained sections (Fig. 3). The most common inflammatory cells were lymphocytes which accumulated around blood vessels, known as perivascular cuffing, 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, which is a commonly seen lesion at necropsy but not usually associated with mycoplasma. In the same year Hegde et al. (2014) also reported the detection of M. agalactiae in macrophages in the sheep brain by immunocytochemistry following an intramammary infection although there was no description of pathology. The authors concluded that the mycoplasma was capable of invading and partially or fully surviving the phagocytosis after having been disseminated around the body, in particular, to the brain. Mycoplasma mycoides subsp. capri is a member of the important M. mycoides cluster, a group which contains ruminant pathogens including those that cause the OIE-listed contagious bovine and caprine pleuropneumonias. In addition to being another aetiological agent of CA, it can cause respiratory disease in goats. In 2012 Gomez-Martin et al. isolated the mycoplasma from the brains of 3 male goats which had been identified as chronic auricular carriers of M. m. capri over one year before. However, no clinical or histopathological signs were reported.
Fig. 3. Brain of sheep experimentally infected with Mycoplasma agalactiae. Lymphocytes have accumulated around blood vessels “perivascular cuffing” (top). Also showing focal microgliosis and neuronal degeneration; cell shrinkage with condensed hyper-eosinophilic cytoplasm and apoptotic nuclei is evident (bottom). (H & E).
More recently the carcass of a 40-day-old kid was examined at the diagnostic laboratory of the IZS Venezie (Catania, unpublished results). The medical history was scant but the owner reported that the animal had been depressed for several days before death but had shown no specific neurological signs. Post mortem examination did not show any important lesions with the exception of very mild enteritis probably caused by clostridia which were isolated from the gut. No bacteria were found in the brain with the exception of very small haemolytic microcolonies evident after 48 h in culture. These colonies were sub-cultured onto Mycoplasma Experience medium (Reigate, UK) and later identified as M. m. capri by PCR/denaturing gradient electrophoresis (McAuliffe et al., 2005) and confirmed by 16S rDNA sequencing. It was not clear whether the mycoplasma was the cause of the depression and death. Post mortem examination by the IZS Venezie of a 4 month-old goat presenting chronic respiratory disease revealed a severe fibrinous pleuropneumonia and pericarditis. M. ovipneumoniae, a worldwide pathogen, and the ubiquitous M. arginini were not unexpectedly isolated from the lungs and associated tissues. However, only M. arginini was isolated following investigations of the brain although no conspicuous lesions were seen. This raises further questions on the role of this opportunistic mycoplasma in disease processes (Nicholas et al., 2008).
5. Avian mycoplasmosis Four mycoplasmas are considered pathogenic for domestic poultry: M. gallisepticum, M. synoviae, M. meleagridis and M. iowae giving a range of clinical signs including respiratory disease, synovitis and joint disease. Mycoplasmas have been isolated sporadically from avian brains although their significance is not known but almost certainly underreported as the CNS is infrequently sampled during routine diagnosis. Meningo-encephalitis was seen for the first time in several turkey flocks between 1998 and 2005 (Wyrzykowski et al., 2013). 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 subacute multifocal parenchymal necrosis, perivascular cuffing, leptomeningitis and vasculitis. Many birds were seropositive for M. gallisepticum and specific antigen was detected in formalin-fixed sections of brain. Much et al. (2002) were able to reproduce brain lesions using aerosol inoculation of low but not high passaged strains of M. gallisepticum. This was linked to the ability of the low passaged strain to invade eukaryotic cells in vitro. 58
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which S. mirum induced clinical neurological damage in rodents very similar to experimental scrapie (Bastian et al., 1984). Furthermore, spiroplasmas isolated from scrapie-affected sheep brains caused spongiform encephalopathies in sheep, goats and deer (Bastian et al., 2007). These conclusions have been challenged by Alexeeva et al. (2006) who were unable to detect mollicute or other bacterial gene sequences in the brains of hamsters with experimental scrapie. While Bastian's work has not yet been reproduced by other groups, an important later finding has been the discovery of the spiroplasma biofilm which would account for the increased resistance of the pathogen and may also be involved indirectly in the formation of prion amyloid (Bastian, 2014). Clearly further work is urgently needed to resolve this important issue.
M. synoviae was isolated from the brains of 22-week-old commercial meat turkeys displaying severe synovitis and occasional CNS signs (Chin et al., 1991). 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, accumulating as perivascular cuffs, damaged the vessel wall and involved surrounding meninges. Some arteries were thrombotic. Detection of M. synoviae from the brains of layer hens following experimental infections via the trachea were recently reported by Catania et al. (2016a); moreover, the presence of M. synoviae in the brains of broiler was detected in an experimental intranasal and conjunctival inoculation (Catania, unpublished report). It is interesting to note that no mycoplasmas could be recovered from the brains of young turkeys experimentally infected with strains of M. iowae of differing virulence during attempts to develop a persistent infection (Jordan et al., 1992). So far there are no reports of M. meleagridis involvement with the CNS.
8. Pathogenesis of animal mycoplasma brain infections Blood-borne infections of the brain are very rare because of the effectiveness of the semi-permeable blood-brain barrier (BBB) which is composed of endothelial cells connected by tight junctions along the relevant capillaries. Bacteria that are able to cross this barrier include those that cause meningitis including Streptococcus pneumoniae, Haemophilus influenzae type B and Neisseria meningitidis. Inflammation of the meninges, the membrane that surrounds the brain and spinal cord, by toxic bacterial products following infection leads to an increase in permeability of the BBB and thus can allows the entry of some pathogens (van Sorge and Doran, 2012). The pathogenesis of brain infection in animals by mycoplasma remains elusive as little evidence of toxins like those of other bacteria has been found. M. pneumoniae, a human pathogen linked to cases of atypical pneumonia and sporadic CNS disease, produces a compound known as community-acquired respiratory distress syndrome (CARDS) toxin (Kannan and Baseman, 2006) which is directly linked to epithelial airway damage and plays a primary role in M. pneumoniae pathogenesis; however, there is no direct evidence of its association with CNS invasion by the mycoplasma. Interestingly, CARDS toxin is homologous to the Bordetella pertussis toxin subunit 1 (PT toxin S1) which has been linked to experimental autoimmune encephalitis by increasing the permeability of the BBB or by modulating immune response (Carbonetti, 2010). Narita (2009) described an evidence-based classification scheme for neurologic manifestations caused by M. pneumoniae. The pathogenesis of neurological infections by M. pneumoniae was divided into three categories: a direct type caused by locally produced cytokines; an indirect type linked to autoimmunity; and a vascular occlusion type associated with vasculitis or thrombosis. Interestingly, vasculitis has been linked to many cases of mycoplasma brain infections in birds and mammals (Chin et al., 1991; Wyrzykowski et al., 2013; Puleio et al., 2014). Autoimmunity has also been suggested as a possible pathogenicity mechanism in a cat with meningo-encephalomyelitis which was refractory to treatment with the fluoroquinolone antibiotic, marbofloxacin. M felis had colonised the bloodstream of the brain and was detected in the cerebrospinal fluid (Beauchamp et al., 2011). Therefore, the scheme proposed by Narita could also be considered relevant for mycoplasma brain infections in animals although more evidence is needed. Another crucial process leading to CNS invasion may be the ability of mycoplasmas to invade blood cells which would provide protection from the host immune system. The colonisation of brain tissue by mycoplasmas would then be linked to haematogenous dissemination or possibly by direct entry to the CNS (Ilha et al., 2010). It has long been known that M. neurolyticum was capable of inducing neurological signs in experimentally infected young rodents though the exact nature of the “toxin” was unknown (Tully, 1981). Later, neurominidase enzymatic activity (NEAC), found in many pathogenic bacteria, was detected in this mycoplasma and suggested as a potential virulence factor for CNS invasion (Berčič et al., 2012). Subsequently NEAC was shown in dogs with granulomatous meningo-
6. Sea mammal mycoplasmosis About 30 years ago large numbers of harbor seals died in the North Sea. Clinical signs included pneumonia, skin lesions, diarrhoea, polyarthritis, nervous signs, and abortions in pregnant females (Giebel et al., 1991). In addition 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 investigated. 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 species of mycoplasma which were subsequently named M. phocicerebrale and M. phocirhinis. The role of these mycoplasmas in this and later outbreaks in sea mammals has never been clarified but it is well known that respiratory disease and polyarthritis, seen in many of the affected seals, are highly characteristic of mycoplasma infections in other animals. If not primary causes then their large numbers and widespread distribution throughout the seals would suggest that they must have played some role in the disease process. Mycoplasmas have been subsequently isolated from many different species of sea mammals but few studies examined the CNS for evidence of their presence. 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. 7. Transmissible spongiform encephalopathies Spiroplasmas, which are fastidious mollicutes with spiral morphology, are most commonly associated with vector-borne plant diseases and some, like Spiroplasma mirum, are highly resistant to heat and chemical treatment including formalin. Following the discovery of “spiroplasma-like fibrils” in the brain of a patient with CreutzfeldtJakob disease, Bastian and his team in the USA proposed a role for this mollicute in the aetiology of transmissible spongiform encephalopathies (TSE), most important of which for livestock are bovine spongiform encephalopathy and scrapie (Bastian et al., 1984). The present consensus is that there is a miss-folding of a self-replicating host protein, the prion, which is the sole cause of these untreatable brain diseases. Bastian raised doubts about prion theory and postulated that the normal protein is merely the receptor for a conventional but previously unidentified bacterium. Research revealed that the pathogen was extremely resistant, ultra-filterable and unusually sensitive to tetracycline, strongly suggesting that the pathogen was bacterial. The team showed that the scrapie-associated fibrils seen in the brains of TSE-affected humans and animals were morphologically identical and immunologically similar to the internal fibril proteins of spiroplasmas (Bastian, 1991). These results were supported by animal studies in 59
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Future Microbiol 5, 455–469. Catania, S., Gobbo, F., Bilato, D., Gagliazzo, L., Moronato, M.L., Terregino, C., Bradbury, J.M., Ramírez, A.S., 2016a. Two strains of Mycoplasma synoviae from chicken flocks on the same layer farm differ in their ability to produce eggshell apex abnormality. Vet. Microbiol. 193, 60–66. Catania, S., Gobbo, F., Schiavon, E., Nicholas, R.A.J., 2016b. Severe otitis and pneumonia in adult cattle with mixed infection of Mycoplasma bovis and Mycoplasma agalactiae. Vet. Rec. Case Rep. http://dx.doi.org/10.1136/vetreccr-2016-000366. Chin, R.P., Meteyer, C.U., Yamamoto, R., Shivaprasad, H.L., Klein, P.N., 1991. Isolation of Mycoplasma synoviae from the brains of commercial meat turkeys with meningeal vasculitis. Avian Dis. 35, 631–637. Corfield, T., 1992. Bacterial sialidases- roles in pathogenicity and nutrition. Glycobiology 2, 509–521. Giebel, J., Meier, J., Binder, A., Flossdorf, J., Poveda, J.B., Schmidt, R., Kirchhoff, H., 1991. Mycoplasma phocarhinis sp. nov. and Mycoplasma phocacerebrale sp. nov., two new species from harbor seals (Phoca vitulina L.). Int. J. Syst. Bacteriol. 41, 39–44. Glaser, K., Speer, C.P., 2015. Neonatal CNS infection and inflammation caused by Ureaplasma species: rare or relevant? Expert Rev. Anti-Infect. Ther. 13, 233–248. Gomez-Martin, A., De la Fe, C., Amores, J., Sánchez, A., Contreras, A., Paterna, A., Buendía, A.J., Corrales, J.C., 2012. Anatomic location of Mycoplasma mycoides subsp. capri and Mycoplasma agalactiae in naturally infected goat male auricular carriers. Vet. Microbiol. 157, 355–362. Hegde, S., Hegde, S., Spergser, J., Bruthaler, R., Rosengarten, R., Chopra-Dewasthaly, R., 2014. In vitro and in vivo invasion spreading of Mycoplasma agalactiae in the sheep infection model. Int. J. Med. Microbiol. 304, 1024–1031. Hermeyer, K., Peters, M., Brügmann, M., Jacobsen, B., Hewicker-Trautwein, M.J., 2012. 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encephalomyelitis and necrotizing meningo-encephalitis caused by M. canis (Barber et al., 2012). Furthermore, neuraminidase activity has also been found in avian mycoplasmas involved in CNS infection such as M. synoviae, M. gallisepticum and one serovar of M. iowae (Berčič et al., 2008). Neuraminidase has been proven to facilitate pathogen colonisation, invasion and damage of host tissue (Corfield, 1992) and it is linked to CNS colonisation by other pathogens such as Streptococcus pneumoniae (Uchiyama et al., 2009). Other mycoplasmas carrying the neuraminidase gene include M. alligatoris, a mycoplasma involved in invasive lethal diseases and cases of pyogranulomatous meningitis in alligators and caimans (Brown and Zacher, 2004), M. cynos which is associated with canine respiratory disease (Walker et al., 2013) and the ubiquitous M. arginini (Genbank accession number: NZ_AUAH01000010.1). 9. Conclusions In view of the examples above it is possible to conclude that many pathogenic mycoplasmas are able to cross the BBB and cause neuropathological effects in both man and animals. How they cross this barrier is presently uncertain. They do not appear to possess toxins like other bacteria which are capable of causing inflammation that leads to the increase in its semi-permeability thus allowing micro-organisms across. Several candidate molecules have been identified in mycoplasmas of cats and birds such as neuraminidase. In the case of M. bovis, the damage caused to the inner ear may lead to direct entry to the brain as otitis media often with associated tissue destruction is a common early clinical sign. 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 investigate these findings and to elucidate whether mycoplasmas may be the cause of a number of undiagnosed neuropathological conditions such as the nonpurulent encephalitis which is frequently seen at necropsy. Finally if the scrapie agent is identified as postulated by Bastian then the contamination of the CA vaccine that killed many goats mentioned earlier may be another mycoplasma. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Alexeeva, I., Elliot, E.J., Rollins, S., Gasparich, G.E., Lazar, J., Rohwer, R.G., 2006. Absence of spiroplasma or other bacterial 16S rRNA genes in brain tissue of hamsters with scrapie. J. Clin. Microbiol. 44, 91–97. Ayling, R.D., Nicholas, R.A.J., Wessells, J., Hogg, R., Byrne, W., 2005. Isolation of Mycoplasma bovis from the brain tissue of calves. Vet. Rec. 156, 391–392. Barber, R.M., Porter, B.F., Li, Q., May, M., Claiborne, M.K., Allison, A.B., Howerth, E.W., Butler, A., Wei, S., Levine, J.M., Levine, G.J., Brown, D.R., Schatzberg, S.J., 2012. Broadly reactive polymerase chain reaction for pathogen detection in canine granulomatous meningoencephalomyelitis and necrotizing meningoencephalitis. J. Vet. Intern. Med. 26, 962–968. Bastian, F.O., 1991. Creutzfeldt-Jakob and other transmissible spongiform encephalopathies. Mosby, New York. Bastian, F.O., 2014. The case for involvement of spiroplasma in the pathogenesis of transmissible spongiform encephalopathies. J. Neuropathol. Exp. Neurol. 73, 104–114. Bastian, F.O., Purnell, D.M., Tully, J.G., 1984. Neuropathology of spiroplasma infection in the rat brain. Am. J. Pathol. 114, 496–514. Bastian, F.O., Sanders, D.E., Forbes, W.A., Hagius, S.D., Walker, J.V., Henk, W.G., Enright, F.M., Elzer, P.H., 2007. Spiroplasma spp from transmissible spongiform encephalopathy brains or ticks induce spongiform encephalopathy in ruminants. J. Med. Microbiol. 56, 1235–1242. Beauchamp, D.J., da Costa, R.C., Premanandan, C., Burns, C.G., Cui, J., Daniels, J.B., 2011. Mycoplasma felis-associated meningoencephalomyelitis in a cat. J. Feline Med. Surg. 13, 139–143.
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