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Mycoplasma host specificity: Fact or fiction?
The Veterinary Journal The Veterinary Journal 170 (2005) 300–306 www.elsevier.com/locate/tvjl
Review
Mycoplasma host specificity: Fact or fiction? D.G. Pitcher a, R.A.J. Nicholas
b,*
a
b
Respiratory and Systemic Infection Laboratory, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK Mycoplasma Group, Department of Statutory and Exotic Bacteria, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK Accepted 7 August 2004
Abstract Bacteria of the genus Mycoplasma are the smallest organisms known to be capable of self-replication. They only occur in association with animal host cells on which they are dependant for many pre-formed nutrients since they lack many of the metabolic pathways associated with energy production and the synthesis of cell components found in other species of bacteria. It is generally thought that most species of Mycoplasma are very host specific but there are many reports of mycoplasmas in hosts that are not perceived as their normal habitat. Sometimes these ‘‘crossings’’ may have a pathological impact particularly where there may be predisposing conditions such as immunodeficiency. These are often reported in humans but may also occur in animals whose immune or physiological status is not known. This review brings together some of these reported incidents and speculates on their potential impact for laboratory diagnosis. Crown Copyright Ó 2004 Published by Elsevier Ltd. All rights reserved. Keywords: Mycoplasmas; Host specificity; Cell culture contamination
1. Introduction Mycoplasmas are ubiquitous throughout the animal kingdom and virtually every mammal, bird, reptile, amphibian and fish that has been tested for mycoplasmas has revealed unique species. Although it is often assumed most Mycoplasma species are extremely host specific, it is well known that some species including Mycoplasma bovis, M. agalactiae, M. mycoides subsp. mycoides SC and LC types have a broader host range and can pass easily between sheep, goats and cattle and that M. gateae is found equally in cats and dogs. The host may simply be the species in which the mycoplasma is most frequently detected. It was formerly believed that human mycoplasma flora were restricted to a few recognised human species. *
Corresponding author. Tel.: +44 932 357379; fax: +44 932 357423. E-mail address: [email protected] (R.A.J. Nicholas).
However, over the last decade there have been reports of animal mycoplasmas being isolated from humans who are more often, though not always, immunocompromised. While it is true that such patients are susceptible to a wide range of microbial infections, it is well established that patients with hypogammaglobulinaemia or who are receiving immunosuppressive drugs have a particular susceptibility to mycoplasma infections (Roifman et al., 1986; Baseman and Tully, 1997), often to species which are normally commensal in the human respiratory or urogenital tract such as M. salivarum, M. orale, M. hominis or Ureaplasma urealyticum (Gregory et al., 1978; So et al., 1983; Paessler et al., 2002). In fact, mycoplasmas are the commonest cause of arthritis in patients with primary immunodeficiency (Franz et al., 1997). Mycoplasmas are most likely to be acquired by their host by intimate contact and transfer of material between mucosal surfaces, for example from mother to child or through sexual contact. It is possible that
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D.G. Pitcher, R.A.J. Nicholas / The Veterinary Journal 170 (2005) 300–306
interspecies transfer may also occur on occasion as between man and domestic animals but whether the foreign mycoplasma will survive for long in its new environment is not always known. If there is a deliberate transfer of tissue between species as occurs in xenotransplantation, it is possible that mycoplasmas may be transferred if stringent precautions are not adopted. Infection of C3H mouse embryonic cells with M. fermentans or M. penetrans has been observed to stimulate high levels of H-ras and c-myc oncogenes with permanent transformation of the cells (Zhang et al., 1997) so there may be a potential for malignant transformation in mycoplasma infected cells. Mycoplasmas may also possess potent immunomodulatory properties for example the macrophage activating lipopeptide (MALP) of M. fermentans that can trigger a cascade of cytokine activity (Calcutt et al., 1999). Although there are no proven links between mycoplasmas and cancer in vivo, recent studies on gastric cancer patients using PCR and the sequencing of gastric biopsy samples, identified 23/56 samples as positive for mycoplasmas. Of these M. faucium, a rare human species, was identified in 13 of the samples (Kwon et al., 2004).
2. Mycoplasma transfer between animals and man Closely related species of mammals may be able to exchange mycoplasma strains fairly readily. For example, primates and humans may share identical or similar species of commensal mycoplasmas such as M. orale or M. salivarium (Martinez-Lahoz et al., 1970). One species originally isolated from the genital tract of women is M. primatum, but this species is more frequently isolated from non-human primates (Tully, 1993). On occasion, previously uncharacterised species may be detected which may be closely related to human mycoplasmas. For example, a mycoplasma, very similar to M. salivarum, found frequently in the human buccal cavity, was isolated from 5% of pigs, some of which had seroconverted to the mycoplasma (Erickson et al., 1988). No disease was associated with its presence. However, species that are potentially pathogenic for man may also cause disease in primates. M. pneumoniae is the most important mycoplasma pathogen for humans causing atypical pneumonia although experimental infections in chimpanzees can induce a very similar condition (Barile et al., 1990). The related species M. genitalium, a cause of non-gonococcal urethritis in man, can also cause lower genital tract infections when inoculated iatrogenically into chimpanzees (Tully et al., 1986). Stipkovits et al. (1989) reported that the respiratory tracts of 13 monkeys which died of severe pneumonia during transportation were
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colonised with M. pneumoniae. Furthermore the human urogenital species M. hominis or U. urealyticum colonised the urethra of monkeys following intra-urethral inoculations that led to a mild urethritis (Stipkovits et al., 1990). An unusual case where it was not possible to state whether the transfer occurred from human to animal or vice versa occurred in a Budapest school, where 30 students became ill with fever and respiratory tract catarrhal symptoms; two students developed pneumonia that was serologically shown to be due to M. pneumoniae. This organism was also isolated from the lungs of a number of hamsters that were being cared for in the classroom of the students (Balogh et al., 1997). A previous study has shown that hamsters are susceptible to experimental infection with M. pneumoniae (Collier and Clyde, 1974). Reports of infections in man with animal species of mycoplasma are rare, but probably occur more frequently than is generally recognised, the majority of cases remaining undiagnosed because so few laboratories are equipped to detect and identify mycoplasmas, or infection may present mild or no symptoms. In some cases there is no certain evidence that the infecting organism was acquired from contact with another animal species. This is the case with M. fermentans, a frequent contaminant of laboratory cell lines. The species has been isolated from man on rare occasions but is very commonly detected by PCR in the synovial fluid of patients with rheumatoid arthritis (Schaeverbeke et al., 1996) and has been associated with infections in patients with AIDS (Dawson et al., 1993) and Gulf War Syndrome (Nicolson and Nicolson, 1996). Despite the apparent frequency with which M. fermentans is detected by PCR, no evidence of the species natural habitat has been reported and it is far from certain that humans are its only host. Recently, Nicholas et al. (1998) reported the isolation of M. fermentans from genital infections in sheep; since then further isolations have been made from both rams and ewes with genital lesions in three other sheep flocks in England (Nicholas et al., 2000a). No route of transmission from sheep to man or vice versa has yet been identified though it is possible to speculate. Occasionally, a foreign species may colonise human body sites as secondary invaders without causing disease. Madoff et al. (1979) reported a case, where M. bovis a cause of mastitis, and infections of the joints and respiratory tract in cattle, was isolated from the sputum of a woman with bronchopneumonia and central nervous system abnormalities. The only contact the patient had with cattle was exposure to cow manure during gardening activities three weeks before developing the symptoms. There was serological evidence of M. pneumoniae but this species was not isolated. Attempts to identify M. bovis antibodies in the patientÕs blood were
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unsuccessful. The disease subsided following tetracycline therapy. The patientÕs symptoms were consistent with M. pneumoniae as the primary aetiological agent and the presence of M. bovis could not be explained. In another case of M. bovis infection in a human that responded to tetracycline treatment, there was no evidence of M. pneumoniae (HJ Ball, personal communication). Introduction of almost any foreign mycoplasma species through skin trauma may be a potential hazard in apparently healthy individuals even when the organism is not known to be pathogenic in its natural host. A curious case of laboratory-acquired infection occurred in 1971 when a microbiologist accidentally injected a thumb with a viable suspension of M. caviae with FreundÕ s adjuvant while working with guinea pigs. This species is found in guinea pigs but apparently nonpathogenic for rodents. Within four days, the hand became swollen and painful and the subject acquired serum antibodies to the organism but fortunately the condition was cured after oral tetracycline therapy (Hill, 1971). Probably the most documented observations of nonhost mycoplasma infections in humans are those received from seal bites (Hartley and Pitcher, 2002). M. phocicerebrale is a species frequently isolated from the wounds of injured harbour seals in the UK (Ayling et al., 2001). This species has been isolated from the hands of patients with ‘‘seal finger’’, a painful condition common in sealers which results from bites or from skinning or handling sea mammals (Stadtlander and Madoff, 1994). Supporting evidence for the implication of this species was produced when the mycoplasma was isolated both from the swollen finger of a young female trainer and the front teeth and pharynx of a seal which had bitten her (Baker et al., 1998). The condition is treatable with tetracycline but not with penicillin or erythromycin backing the argument that mycoplasmas may be a cause. Mycoplasma infections are particularly common in immuno-compromised patients and cases of human infection with mycoplasmas normally inhabiting domestic animals are probably due to persistent and intimate contact between them in these susceptible patients. Armstrong et al. (1971) demonstrated colonisation of the throat by M. canis in several family members who were in very close contact with their dog. One family member, who was receiving anti-neoplastic chemotherapy, developed a respiratory infection that was treatable by tetracycline. A case of septic arthritis due to M. felis has been reported in a woman with hypo-gammaglobulinaemia. The woman had worked in an animal shelter and had kept cats for 20 years. She had received a cat bite shortly before her illness and the wound had been treated with antibiotics and healed (Bonilla et al., 1997).
An unknown mycoplasma, later shown to be part of the mycoplasma flora of the feline respiratory tract, was the only bacterial species isolated from the infected hand of a veterinarian with soft tissue cellulitis following a cat scratch (McCabe et al., 1987). Treatment with vibramycin was eventually successful after erythromycin had failed to control the initial infection. Mycoplasmas should be considered as potential pathogens in disease following any animal bite and scratch events where other common causes such as bartonellosis can be ruled out. A fatal infection as a result of pneumonia and septicaemia in an abattoir worker with advanced non-HodgkinÕs lymphoma due to Mycoplasma arginini has been reported (Yechouron et al., 1992). The natural habitat of M. arginini is unknown and the species has been isolated from a wide range of domestic animals but most commonly from sheep and goats and is probably one of the least host specific mycoplasmas known. It is also one of the commonest species to infect laboratory cell lines.
3. Mycoplasma transfer between animal species The canine species, M. canis, appears to be accepted as a part of the bovine flora where there is contact between dogs and cattle (ter Laak et al., 1993). Interestingly while it mainly causes reproductive disease in dogs, in cattle, respiratory disease is most frequent. Its widespread occurrence in pneumonic calves in the UK since its first isolation in 1995 indicates its successful colonisation of this host (Nicholas et al., 2000b) M. felis, normally found in cats has several times been reported in outbreaks of respiratory disease in horses (Wood et al., 1997) though firm evidence for its role as a pathogen is lacking. M. gallisepticum is an important respiratory pathogen of poultry. It is found in many birds and may be transmitted by songbirds (Hartup et al., 2000). It is a close relative in evolutionary terms to M. pneumoniae on the basis of 16S rRNA sequence homology. M. pneumoniae is genetically very homogeneous in contrast to M. gallisepticum that is a heterogeneous species as shown by genetic typing (Santha et al., 1988). Comparison of these two mycoplasmas suggests that genetic diversity within a species may be an indication of a wider host range and is independent of its evolutionary relationships with other mycoplasmas. The presence of the goat pathogen, M. mycoides subsp. mycoides LC, in cattle has been described previously but was thought to be avirulent for this species (Nicholas and Bashiruddin, 1995). However, the spread of LC from goats to calves through infected unpasteurised goat milk in New Zealand recently led to severe out-
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breaks of polyarthritis, septicaemia and deaths (Jackson and King, 2002). LC and M. capricolum subsp. capricolum have also been detected serologically in South American camelids (Hung et al., 1991; Nicholas, 1998) but isolates of these mycoplasmas from these animals remain elusive. M. bovis, a major cause of calf pneumonia and mastitis, was originally classified as a subspecies of the small ruminant pathogen M. agalactiae because of its close immunological and biochemical relationship. However, distinct genetic differences particularly in the uvr C gene have been exploited to develop a PCR capable of distinguishing the two species (Subramaniam et al., 1998). This PCR was used to identify M. bovis in goats with mastitis in the UK (Ayling et al., in press); without this test there would have been real concerns that the UK was experiencing its first case of contagious agalactia. Since its first isolation in 1972 in Australia (Carmichael et al., 1972) the unclassified mycoplasma, M. ovine/caprine serogroup 11, has been found sporadically in sheep and goats with a variety of clinical conditions including infertility and vulvovaginitis (Nicholas et al., 2002). The biochemically similar M. bovigenitalium causes similar conditions in cattle. In a comparison of strains from these two species, Nicholas et al. (2002) demonstrated very close genetic, biochemical and immunological relationships between the two species and recommended that the M. serogroup 11 should be reclassified as M. bovigenitalium. One of the recommended sampling sites for pathogenic mycoplasmas in sheep and goats is the ear canal and is believed to be due the presence of ear mites (Psoroptes cuniculi or Railleria caprae) from which mycoplasmas have been isolated (Da Massa and Brooks, 1991). Goat fleas (order Siphonaptera) too have been implicated in disease transmission; Nayak and Bhowmik (1990) showed that the blood taken from polyarthritic goat kids contained up to 105 viable cells per mL of M. mycoides LC organisms and when the fleas were placed on unaffected kids led to polyarthritis with septicaemia. What is not clear from either study is whether this represents just the passive carriage of mycoplasmas from the blood of acutely affected animals or whether mites and fleas act as reservoirs and in which mycoplasmas replicate.
4. Uncultured agents Recently, it has been established through 16S rDNA sequencing that the haemotrophic, unculturable bacteria previously known as Haemobartonella and Eperythrozoon spp., are in fact species of Mycoplasma and several species have been renamed as such (Neimark et al., 2002a). These organisms, commonly referred to
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by the trivial name ‘‘haemoplasmas’’, have been detected within the erythrocytes of cats, dogs, pigs, rodents and cattle where they may cause anaemia. There have been sporadic reports of similar infections in man, but these have been poorly characterised (Kallick et al., 1972; Archer et al., 1979; Dooley, 1980; Puntaric et al., 1986; Duarte et al., 1992; Yang et al., 2000) and to date, they have not been subjected to similar analysis. It has been shown that haemoplasmas can infect primates since asymptomatic carriage of a haemotrophic mycoplasma has been confirmed in squirrel monkeys (Neimark et al., 2002b). There is some evidence to suggest that Eperythrozoon-like species can be experimentally transmitted by arthropods (ticks) (Berkenkamp and Wescott, 1988) but further studies are needed to confirm this. Haemoplasmas appear to show the same host specificity as other mycoplasmas, as judged by their identification through 16S rDNA sequencing (Tasker et al., 2003) but few strains have been studied in detail. In view of the apparent wide distribution of these organisms among host mammals and the current lack of knowledge of their biology or how they are acquired, it is possible that they may transfer between mammals and should be considered potential hazards in the interspecies transfer of biological material.
5. Conclusions Well over 100 Mycoplasma species have been described to date, yet very few laboratories are equipped to test for these organisms and for those that can, the gradual increase in new species means that identification becomes more laborious, as traditionally it is necessary to test each species against new antisera. The task becomes even more difficult if laboratories have to test for mycoplasma species outside their area of general interest because of species crossing between hosts. The advent of PCR has to some extent improved the possibility of detecting them, but where many PCR studies have been carried out there have not been any concurrent attempts to culture mycoplasmas that should be the ‘‘gold standard’’ for the detection of unusual species. Furthermore it should be noted that the PCR can only detect mycoplasmas whose partial or complete sequences are known and the appropriate primers have been designed. For unknown mycoplasmas, 16S rDNA sequencing remains the only technique capable of identification but is presently time consuming and expensive and can sometimes fail to differentiate related species as occurs with M. gallisepticum from poultry and Mycoplasma imitans from ducks which though clearly different on other criteria, their 16S rRNA sequences differ by only two nucleotides (Harasawa et al., 2004).
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A promising new technique, which requires only a single PCR using mycoplasma-specific primers, is denaturing gradient gel electrophoresis (DGGE). This method proved useful in a recent study in which 32 animal mycoplasmas were tested and shown to produce 27 distinct profiles; only members of the M. mycoides cluster, with the exception of M. capricolum subsp. capricolum, and M. mycoides subsp. mycoides SC, showed identical profiles though this may be overcome in future by designing primers from other genes (McAuliffe et al., 2003). Since this time, the DGGE has become a routine technique at the Veterinary Laboratory Agency. It has the distinct advantage over other identification methods in being able to detect and identify mycoplasmas including mixed species directly from clinical material within 2–3 days. In view of their small genome, limited biosynthetic capability and parasitic life style, it is perhaps not surprising that mycoplasmas are often restricted to a single host species. In contrast, another Mollicute genus, Acholeplasma, have relatively large genomes (>1500 kbp), exhibit more metabolic activity than mycoplasmas, are less fastidious and have a wider range of hosts. A. laidlawii, for example, has been isolated from cattle, sheep, horses, cats, pigs, and several avian species (Ernø, 1994). M. genitalium, on the other hand, the smallest mycoplasma known (genome size 580 kbp), has only ever been detected in man. However, the numerous reports of mycoplasmas crossing the species barriers may reflect a greater than previously thought adaptability to different hosts possibly brought out about by their high mutation rates. Furthermore, humans or animals that have deficient immune systems or, perhaps, have undergone physiological change such as stress may be more susceptible to acquiring foreign mycoplasmas with potentially pathogenic consequences. Improved detection methods, like PCR and gene sequencing as well as better mycoplasma medium enabling the growth of more species, are providing a greater insight into host colonisation.
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ology and Molecular Genetics, 4. European Commission, Brussels, pp. 223–226. Nicholas, R.A.J., Baker, S.E., Ayling, R.D., Stipkovits, L., 2000b. Mycoplasma infections in growing cattle. Cattle Practice 8, 115– 118. Nicholas, R.A.J., Khan, L., Houshaymi, B., Miles, R.J., Ayling, R.D., Hotzel, H., Sachse, K., 2002. Close genetic and phenotypic relatedness between Mycoplasma ovine/caprine serogroup 11 and M. bovigenitalium. Systematic and Applied Microbiology 25, 396– 402. Nicolson, G.L., Nicolson, N.L., 1996. Diagnosis and treatment of mycoplasmal infection in Persian Gulf War illness-CFIDS patients. International Journal of Occupational Medicine Immunology and Toxicology 5, 69–78. Paessler, M., Levinson, A., Patel, J.B., Schuster, M., Minda, M., Nachmkin, I., 2002. Disseminated Mycoplasma orale infection in a patient with common variable immunodeficiency syndrome. Diagnostic Microbiology and Infectious Disease 44, 201–204. Puntaric, V., Borcic, D., Vukelic, D., et al., 1986. Eperythrozoonosis in man. Lancet 11 (October), 868–869. Roifman, C.M., Rao, C.P., Lederman, H.M., Lavi, S., Quinn, P., Gelfand, E.W., 1986. Increased susceptibility to mycoplasma infection in patients with hypogammaglobulinaemia. American Journal of Medicine 95, 477–487. Santha, M., Lukacs, K., Burg, K., Bernath, S., Rasko, I., Stipkovits, L., 1988. Intraspecies genotypic heterogeneity among Mycoplasma gallisepticum strains. Applied and Environmental Microbiology 54, 607–609. Schaeverbeke, T., Gilroy, C.B., Be´be´ar, C., Dehais, J., TaylorRobinson, D., 1996. Mycoplasma fermentans in joints of patients with rheumatoid arthritis and other joint disorders. Lancet 347, 1418. So, A.K.L., Furr, P.M., Taylor-Robinson, D., Webster, A.D.B., 1983. Arthritis caused by Mycoplasma salivary in hypogammaglobulinaemia. British Medical Journal 286, 762–763. Stadtlander, K.-H., Madoff, S., 1994. Characterisation of cytopathogenicity of aquarium sea mycoplasmas and seal finger mycoplasmas by light and scanning electron microscope. Zentrablatt fur Bakteriologie. 280, 458–467. Stipkovits, L., Marantidi, A.N., Dzikidze, E., Krylova, R.I., Vulvovich, J.V., 1989. Isolation of Mycoplasma pneumoniae from monkeys. Zentralblatt fur Veterina¨rmedizin. B 36, 134–138. Stipkovits, L., Marantidi, A.N., Dzikidze, E.K., Krylova, R.I., Csutortoki, V., 1990. Urethral infection of male monkeys by Mycoplasma hominis and Ureaplasma urealyticum. Zentralblatt fur Veterina¨rmedizin. B 37, 125–134. Subramaniam, S., Bergonier, D., Poumerat, F., Capaul, S., Schlatter, J., Nicolet, J., Frey, J., 1998. Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on uvrC genes by PCR. Molecular and Cellular Probes 12, 161–169. Tasker, S., Helps, C.R., Day, M.J., Harbour, D.A., Shaw, S.E., Harrus, S., Baneth, G., Lobetti, R.G., Malik, R., Beaufils, J.P., Belford, C.R., Gruffydd-Jones, T.J., 2003. Phylogenetic analysis of hemoplasma species: an international study. Journal of Clinical Microbiology 41, 3877–3880. ter Laak, E.A., Tully, J.G., Noordergraaf, H.H., Rose, D.L., Carle, P., Bove´, J.M., Smits, M.A., 1993. Recognition of Mycoplasma canis part of the mycoplasma flora of the bovine respiratory tract. Veterinary Microbiology 34, 175–181. Tully, J.G., Taylor-Robinson, D., Rose, D.L., Furr, P.M., Graham, M.F., Barile, M.F., 1986. Urogenital challenge of primate species with Mycoplasma genitalium and characteristics of infection induced in chimpanzees. Journal of Infectious Disease 153, 1046– 1054. Tully, J.G., 1993. Current status of Mollicute flora of humans. Clinical Infectious Diseases 17, S2–S29.
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Wood, J.L., Chanter, N., Newton, J.R., Burrell, M.H., Dugdale, D., Windsor, H.M., Windsor, G.D., Rosendal, S., Townsend, H.G., 1997. An outbreak of respiratory disease in horses associated with Mycoplasma felis infection. Veterinary Record 140, 388–391. Yang, D., Tai, X., Qiu, Y., Yun, S., 2000. Prevalence of Eperythrozoon spp. infection and congenital eperythrozoonosis in humans in Inner Mongolia, China. Epidemiology and Infection 125, 421–426.
Yechouron, A., Lefebvre, J., Robson, H.G., Rose, D.L., Tully, J.G., 1992. Fatal septicaemia due to Mycoplasma arginini : a new human zoonosis. Clinical Infectious Disease 15, 434–438. Zhang, B., Shih, J.W.K., Wear, D.J., Lo, S-C., 1997. High - level expression of H- ras and c-myc oncogenes in mycoplasma mediated malignant cell transformation. Proceedings of the Society for Experimental Biological Medicine 214, 359–366.
Review
Mycoplasma host specificity: Fact or fiction? D.G. Pitcher a, R.A.J. Nicholas
b,*
a
b
Respiratory and Systemic Infection Laboratory, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK Mycoplasma Group, Department of Statutory and Exotic Bacteria, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK Accepted 7 August 2004
Abstract Bacteria of the genus Mycoplasma are the smallest organisms known to be capable of self-replication. They only occur in association with animal host cells on which they are dependant for many pre-formed nutrients since they lack many of the metabolic pathways associated with energy production and the synthesis of cell components found in other species of bacteria. It is generally thought that most species of Mycoplasma are very host specific but there are many reports of mycoplasmas in hosts that are not perceived as their normal habitat. Sometimes these ‘‘crossings’’ may have a pathological impact particularly where there may be predisposing conditions such as immunodeficiency. These are often reported in humans but may also occur in animals whose immune or physiological status is not known. This review brings together some of these reported incidents and speculates on their potential impact for laboratory diagnosis. Crown Copyright Ó 2004 Published by Elsevier Ltd. All rights reserved. Keywords: Mycoplasmas; Host specificity; Cell culture contamination
1. Introduction Mycoplasmas are ubiquitous throughout the animal kingdom and virtually every mammal, bird, reptile, amphibian and fish that has been tested for mycoplasmas has revealed unique species. Although it is often assumed most Mycoplasma species are extremely host specific, it is well known that some species including Mycoplasma bovis, M. agalactiae, M. mycoides subsp. mycoides SC and LC types have a broader host range and can pass easily between sheep, goats and cattle and that M. gateae is found equally in cats and dogs. The host may simply be the species in which the mycoplasma is most frequently detected. It was formerly believed that human mycoplasma flora were restricted to a few recognised human species. *
Corresponding author. Tel.: +44 932 357379; fax: +44 932 357423. E-mail address: [email protected] (R.A.J. Nicholas).
However, over the last decade there have been reports of animal mycoplasmas being isolated from humans who are more often, though not always, immunocompromised. While it is true that such patients are susceptible to a wide range of microbial infections, it is well established that patients with hypogammaglobulinaemia or who are receiving immunosuppressive drugs have a particular susceptibility to mycoplasma infections (Roifman et al., 1986; Baseman and Tully, 1997), often to species which are normally commensal in the human respiratory or urogenital tract such as M. salivarum, M. orale, M. hominis or Ureaplasma urealyticum (Gregory et al., 1978; So et al., 1983; Paessler et al., 2002). In fact, mycoplasmas are the commonest cause of arthritis in patients with primary immunodeficiency (Franz et al., 1997). Mycoplasmas are most likely to be acquired by their host by intimate contact and transfer of material between mucosal surfaces, for example from mother to child or through sexual contact. It is possible that
1090-0233/$ - see front matter. Crown Copyright Ó 2004 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2004.08.011
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interspecies transfer may also occur on occasion as between man and domestic animals but whether the foreign mycoplasma will survive for long in its new environment is not always known. If there is a deliberate transfer of tissue between species as occurs in xenotransplantation, it is possible that mycoplasmas may be transferred if stringent precautions are not adopted. Infection of C3H mouse embryonic cells with M. fermentans or M. penetrans has been observed to stimulate high levels of H-ras and c-myc oncogenes with permanent transformation of the cells (Zhang et al., 1997) so there may be a potential for malignant transformation in mycoplasma infected cells. Mycoplasmas may also possess potent immunomodulatory properties for example the macrophage activating lipopeptide (MALP) of M. fermentans that can trigger a cascade of cytokine activity (Calcutt et al., 1999). Although there are no proven links between mycoplasmas and cancer in vivo, recent studies on gastric cancer patients using PCR and the sequencing of gastric biopsy samples, identified 23/56 samples as positive for mycoplasmas. Of these M. faucium, a rare human species, was identified in 13 of the samples (Kwon et al., 2004).
2. Mycoplasma transfer between animals and man Closely related species of mammals may be able to exchange mycoplasma strains fairly readily. For example, primates and humans may share identical or similar species of commensal mycoplasmas such as M. orale or M. salivarium (Martinez-Lahoz et al., 1970). One species originally isolated from the genital tract of women is M. primatum, but this species is more frequently isolated from non-human primates (Tully, 1993). On occasion, previously uncharacterised species may be detected which may be closely related to human mycoplasmas. For example, a mycoplasma, very similar to M. salivarum, found frequently in the human buccal cavity, was isolated from 5% of pigs, some of which had seroconverted to the mycoplasma (Erickson et al., 1988). No disease was associated with its presence. However, species that are potentially pathogenic for man may also cause disease in primates. M. pneumoniae is the most important mycoplasma pathogen for humans causing atypical pneumonia although experimental infections in chimpanzees can induce a very similar condition (Barile et al., 1990). The related species M. genitalium, a cause of non-gonococcal urethritis in man, can also cause lower genital tract infections when inoculated iatrogenically into chimpanzees (Tully et al., 1986). Stipkovits et al. (1989) reported that the respiratory tracts of 13 monkeys which died of severe pneumonia during transportation were
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colonised with M. pneumoniae. Furthermore the human urogenital species M. hominis or U. urealyticum colonised the urethra of monkeys following intra-urethral inoculations that led to a mild urethritis (Stipkovits et al., 1990). An unusual case where it was not possible to state whether the transfer occurred from human to animal or vice versa occurred in a Budapest school, where 30 students became ill with fever and respiratory tract catarrhal symptoms; two students developed pneumonia that was serologically shown to be due to M. pneumoniae. This organism was also isolated from the lungs of a number of hamsters that were being cared for in the classroom of the students (Balogh et al., 1997). A previous study has shown that hamsters are susceptible to experimental infection with M. pneumoniae (Collier and Clyde, 1974). Reports of infections in man with animal species of mycoplasma are rare, but probably occur more frequently than is generally recognised, the majority of cases remaining undiagnosed because so few laboratories are equipped to detect and identify mycoplasmas, or infection may present mild or no symptoms. In some cases there is no certain evidence that the infecting organism was acquired from contact with another animal species. This is the case with M. fermentans, a frequent contaminant of laboratory cell lines. The species has been isolated from man on rare occasions but is very commonly detected by PCR in the synovial fluid of patients with rheumatoid arthritis (Schaeverbeke et al., 1996) and has been associated with infections in patients with AIDS (Dawson et al., 1993) and Gulf War Syndrome (Nicolson and Nicolson, 1996). Despite the apparent frequency with which M. fermentans is detected by PCR, no evidence of the species natural habitat has been reported and it is far from certain that humans are its only host. Recently, Nicholas et al. (1998) reported the isolation of M. fermentans from genital infections in sheep; since then further isolations have been made from both rams and ewes with genital lesions in three other sheep flocks in England (Nicholas et al., 2000a). No route of transmission from sheep to man or vice versa has yet been identified though it is possible to speculate. Occasionally, a foreign species may colonise human body sites as secondary invaders without causing disease. Madoff et al. (1979) reported a case, where M. bovis a cause of mastitis, and infections of the joints and respiratory tract in cattle, was isolated from the sputum of a woman with bronchopneumonia and central nervous system abnormalities. The only contact the patient had with cattle was exposure to cow manure during gardening activities three weeks before developing the symptoms. There was serological evidence of M. pneumoniae but this species was not isolated. Attempts to identify M. bovis antibodies in the patientÕs blood were
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unsuccessful. The disease subsided following tetracycline therapy. The patientÕs symptoms were consistent with M. pneumoniae as the primary aetiological agent and the presence of M. bovis could not be explained. In another case of M. bovis infection in a human that responded to tetracycline treatment, there was no evidence of M. pneumoniae (HJ Ball, personal communication). Introduction of almost any foreign mycoplasma species through skin trauma may be a potential hazard in apparently healthy individuals even when the organism is not known to be pathogenic in its natural host. A curious case of laboratory-acquired infection occurred in 1971 when a microbiologist accidentally injected a thumb with a viable suspension of M. caviae with FreundÕ s adjuvant while working with guinea pigs. This species is found in guinea pigs but apparently nonpathogenic for rodents. Within four days, the hand became swollen and painful and the subject acquired serum antibodies to the organism but fortunately the condition was cured after oral tetracycline therapy (Hill, 1971). Probably the most documented observations of nonhost mycoplasma infections in humans are those received from seal bites (Hartley and Pitcher, 2002). M. phocicerebrale is a species frequently isolated from the wounds of injured harbour seals in the UK (Ayling et al., 2001). This species has been isolated from the hands of patients with ‘‘seal finger’’, a painful condition common in sealers which results from bites or from skinning or handling sea mammals (Stadtlander and Madoff, 1994). Supporting evidence for the implication of this species was produced when the mycoplasma was isolated both from the swollen finger of a young female trainer and the front teeth and pharynx of a seal which had bitten her (Baker et al., 1998). The condition is treatable with tetracycline but not with penicillin or erythromycin backing the argument that mycoplasmas may be a cause. Mycoplasma infections are particularly common in immuno-compromised patients and cases of human infection with mycoplasmas normally inhabiting domestic animals are probably due to persistent and intimate contact between them in these susceptible patients. Armstrong et al. (1971) demonstrated colonisation of the throat by M. canis in several family members who were in very close contact with their dog. One family member, who was receiving anti-neoplastic chemotherapy, developed a respiratory infection that was treatable by tetracycline. A case of septic arthritis due to M. felis has been reported in a woman with hypo-gammaglobulinaemia. The woman had worked in an animal shelter and had kept cats for 20 years. She had received a cat bite shortly before her illness and the wound had been treated with antibiotics and healed (Bonilla et al., 1997).
An unknown mycoplasma, later shown to be part of the mycoplasma flora of the feline respiratory tract, was the only bacterial species isolated from the infected hand of a veterinarian with soft tissue cellulitis following a cat scratch (McCabe et al., 1987). Treatment with vibramycin was eventually successful after erythromycin had failed to control the initial infection. Mycoplasmas should be considered as potential pathogens in disease following any animal bite and scratch events where other common causes such as bartonellosis can be ruled out. A fatal infection as a result of pneumonia and septicaemia in an abattoir worker with advanced non-HodgkinÕs lymphoma due to Mycoplasma arginini has been reported (Yechouron et al., 1992). The natural habitat of M. arginini is unknown and the species has been isolated from a wide range of domestic animals but most commonly from sheep and goats and is probably one of the least host specific mycoplasmas known. It is also one of the commonest species to infect laboratory cell lines.
3. Mycoplasma transfer between animal species The canine species, M. canis, appears to be accepted as a part of the bovine flora where there is contact between dogs and cattle (ter Laak et al., 1993). Interestingly while it mainly causes reproductive disease in dogs, in cattle, respiratory disease is most frequent. Its widespread occurrence in pneumonic calves in the UK since its first isolation in 1995 indicates its successful colonisation of this host (Nicholas et al., 2000b) M. felis, normally found in cats has several times been reported in outbreaks of respiratory disease in horses (Wood et al., 1997) though firm evidence for its role as a pathogen is lacking. M. gallisepticum is an important respiratory pathogen of poultry. It is found in many birds and may be transmitted by songbirds (Hartup et al., 2000). It is a close relative in evolutionary terms to M. pneumoniae on the basis of 16S rRNA sequence homology. M. pneumoniae is genetically very homogeneous in contrast to M. gallisepticum that is a heterogeneous species as shown by genetic typing (Santha et al., 1988). Comparison of these two mycoplasmas suggests that genetic diversity within a species may be an indication of a wider host range and is independent of its evolutionary relationships with other mycoplasmas. The presence of the goat pathogen, M. mycoides subsp. mycoides LC, in cattle has been described previously but was thought to be avirulent for this species (Nicholas and Bashiruddin, 1995). However, the spread of LC from goats to calves through infected unpasteurised goat milk in New Zealand recently led to severe out-
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breaks of polyarthritis, septicaemia and deaths (Jackson and King, 2002). LC and M. capricolum subsp. capricolum have also been detected serologically in South American camelids (Hung et al., 1991; Nicholas, 1998) but isolates of these mycoplasmas from these animals remain elusive. M. bovis, a major cause of calf pneumonia and mastitis, was originally classified as a subspecies of the small ruminant pathogen M. agalactiae because of its close immunological and biochemical relationship. However, distinct genetic differences particularly in the uvr C gene have been exploited to develop a PCR capable of distinguishing the two species (Subramaniam et al., 1998). This PCR was used to identify M. bovis in goats with mastitis in the UK (Ayling et al., in press); without this test there would have been real concerns that the UK was experiencing its first case of contagious agalactia. Since its first isolation in 1972 in Australia (Carmichael et al., 1972) the unclassified mycoplasma, M. ovine/caprine serogroup 11, has been found sporadically in sheep and goats with a variety of clinical conditions including infertility and vulvovaginitis (Nicholas et al., 2002). The biochemically similar M. bovigenitalium causes similar conditions in cattle. In a comparison of strains from these two species, Nicholas et al. (2002) demonstrated very close genetic, biochemical and immunological relationships between the two species and recommended that the M. serogroup 11 should be reclassified as M. bovigenitalium. One of the recommended sampling sites for pathogenic mycoplasmas in sheep and goats is the ear canal and is believed to be due the presence of ear mites (Psoroptes cuniculi or Railleria caprae) from which mycoplasmas have been isolated (Da Massa and Brooks, 1991). Goat fleas (order Siphonaptera) too have been implicated in disease transmission; Nayak and Bhowmik (1990) showed that the blood taken from polyarthritic goat kids contained up to 105 viable cells per mL of M. mycoides LC organisms and when the fleas were placed on unaffected kids led to polyarthritis with septicaemia. What is not clear from either study is whether this represents just the passive carriage of mycoplasmas from the blood of acutely affected animals or whether mites and fleas act as reservoirs and in which mycoplasmas replicate.
4. Uncultured agents Recently, it has been established through 16S rDNA sequencing that the haemotrophic, unculturable bacteria previously known as Haemobartonella and Eperythrozoon spp., are in fact species of Mycoplasma and several species have been renamed as such (Neimark et al., 2002a). These organisms, commonly referred to
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by the trivial name ‘‘haemoplasmas’’, have been detected within the erythrocytes of cats, dogs, pigs, rodents and cattle where they may cause anaemia. There have been sporadic reports of similar infections in man, but these have been poorly characterised (Kallick et al., 1972; Archer et al., 1979; Dooley, 1980; Puntaric et al., 1986; Duarte et al., 1992; Yang et al., 2000) and to date, they have not been subjected to similar analysis. It has been shown that haemoplasmas can infect primates since asymptomatic carriage of a haemotrophic mycoplasma has been confirmed in squirrel monkeys (Neimark et al., 2002b). There is some evidence to suggest that Eperythrozoon-like species can be experimentally transmitted by arthropods (ticks) (Berkenkamp and Wescott, 1988) but further studies are needed to confirm this. Haemoplasmas appear to show the same host specificity as other mycoplasmas, as judged by their identification through 16S rDNA sequencing (Tasker et al., 2003) but few strains have been studied in detail. In view of the apparent wide distribution of these organisms among host mammals and the current lack of knowledge of their biology or how they are acquired, it is possible that they may transfer between mammals and should be considered potential hazards in the interspecies transfer of biological material.
5. Conclusions Well over 100 Mycoplasma species have been described to date, yet very few laboratories are equipped to test for these organisms and for those that can, the gradual increase in new species means that identification becomes more laborious, as traditionally it is necessary to test each species against new antisera. The task becomes even more difficult if laboratories have to test for mycoplasma species outside their area of general interest because of species crossing between hosts. The advent of PCR has to some extent improved the possibility of detecting them, but where many PCR studies have been carried out there have not been any concurrent attempts to culture mycoplasmas that should be the ‘‘gold standard’’ for the detection of unusual species. Furthermore it should be noted that the PCR can only detect mycoplasmas whose partial or complete sequences are known and the appropriate primers have been designed. For unknown mycoplasmas, 16S rDNA sequencing remains the only technique capable of identification but is presently time consuming and expensive and can sometimes fail to differentiate related species as occurs with M. gallisepticum from poultry and Mycoplasma imitans from ducks which though clearly different on other criteria, their 16S rRNA sequences differ by only two nucleotides (Harasawa et al., 2004).
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A promising new technique, which requires only a single PCR using mycoplasma-specific primers, is denaturing gradient gel electrophoresis (DGGE). This method proved useful in a recent study in which 32 animal mycoplasmas were tested and shown to produce 27 distinct profiles; only members of the M. mycoides cluster, with the exception of M. capricolum subsp. capricolum, and M. mycoides subsp. mycoides SC, showed identical profiles though this may be overcome in future by designing primers from other genes (McAuliffe et al., 2003). Since this time, the DGGE has become a routine technique at the Veterinary Laboratory Agency. It has the distinct advantage over other identification methods in being able to detect and identify mycoplasmas including mixed species directly from clinical material within 2–3 days. In view of their small genome, limited biosynthetic capability and parasitic life style, it is perhaps not surprising that mycoplasmas are often restricted to a single host species. In contrast, another Mollicute genus, Acholeplasma, have relatively large genomes (>1500 kbp), exhibit more metabolic activity than mycoplasmas, are less fastidious and have a wider range of hosts. A. laidlawii, for example, has been isolated from cattle, sheep, horses, cats, pigs, and several avian species (Ernø, 1994). M. genitalium, on the other hand, the smallest mycoplasma known (genome size 580 kbp), has only ever been detected in man. However, the numerous reports of mycoplasmas crossing the species barriers may reflect a greater than previously thought adaptability to different hosts possibly brought out about by their high mutation rates. Furthermore, humans or animals that have deficient immune systems or, perhaps, have undergone physiological change such as stress may be more susceptible to acquiring foreign mycoplasmas with potentially pathogenic consequences. Improved detection methods, like PCR and gene sequencing as well as better mycoplasma medium enabling the growth of more species, are providing a greater insight into host colonisation.
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