Mycoplasma bovis, A Multi Disease Producing Pathogen: An Overview
Received: June 22, 2010;
Accepted: August 05, 2010;
Published: November 24, 2010
Mycoplasma bovis, first isolated in the USA from the milk of a mastitic cow
in 1961 (Hale et al., 1962) belongs to the class
Mollicutes, order Mycoplasmatales, family Mycoplasmataceae and
the genus Mycoplasma (Razin et al., 1998).
Initially it was named as Mycoplasma bovimastitidis then it was classified
in group 5, M. agalactiae var bovis, on the basis of its antigenicity
and biochemical tests (Leach, 1973) and due to similarity
with the clinical signs of contagious agalactia in sheep caused by M. agalactiae.
Both the species are identical in cell-and colony-form as well as in their metabolic
behavior with the sharing of high number of antigens. It is difficult to differentiate
them by usual morphological, metabolical and serological methods (Gonzalez
et al., 1995; Gummelt et al., 1996;
Kumar, 2000). Later with the advancement of techniques
it was ranked as species and named Mycoplasma bovis based on 16S ribosomal
RNA sequences (Askaa and Erno, 1976).
Among pathogenic mycoplasma species which mainly colonize bovine respiratory
mucous membrane (Ter Laak et al., 1992) Mycoplasma
bovis is the most important and most pathogenic bovine mycoplasma causing
respiratory, venereal and other diseases of bovines all over the world mainly
in Europe and North America. Mycoplasma bovis is very versatile pathogen
and has been reported from the cases of genital disorders and abortions (Langford,
1975; Ruhnke, 1994; Byrne et
al., 1999) bovine pneumonia (Srivastava, 1982),
reduction of semen fertility (Kissi et al., 1985),
arthritis and meningeal abcesses (Stipkovits et al.,
1993), decubital abscesses (Kinde et al., 1993),
keratoconjonctivitis (Jack et al., 1977; Kirby
and Nicholas, 1996), otitis (Walz et al., 1997),
poly arthritis (Henderson and Ball, 1999) and mastitis
(Byrne et al., 2000; Hirose
et al., 2001).
Economic losses: There is as such no survey available for the economic
losses due to M. bovis infections, although it is estimated to cause
at least for the quarter or third of total losses approximately a sum of 576
million Euros per year in Europe due to respiratory diseases in cattle (Nicholas
and Ayling, 2003). However, more comprehensive data is available for the
losses caused by this organism due to mastitis ($ 108 million) and loss of the
weight gain and the diminished carcass value ($ 32 million) in the USA (Rosengarten
and Citti, 1999).
According to Campbell (2009) average of a minimum of
$100.00 expended per case of M. bovis on treatment costs/animal health
in Canada with the conservative estimate of approximately $20,000 in expenses
due to Mycoplasma bovis cases in a 10,000 head feedlot per year.
Source of infection: Mycoplasma bovis is supposed to be a natural
habitat of respiratory tract in healthy bovines without showing any clinical
symptoms and is shed through their nasal discharges for month or years. The
genital tract of both male and female animals can also harbor M. bovis
and can be a source of the infection through coitus and natural service (Kreusel
et al., 1989) or through artificial insemination with deep frozen
bull semen (Jurmanova and Sterbova, 1977) as mycoplasmas
can survive in frozen semen for several years. Milk may also be a source of
infection which acts as major source of infection for suckling calves (Pfutzner,
1990; Hirose et al., 2001). It has also been
reported from sheep (Bocklisch et al., 1987),
goat (Egwu et al., 2001), rabbits (Boucher
et al., 2001), Poultry (Hasan et al.,
2008) and can be transmitted from these carrier animals and birds. Isolation
of M. bovis from human respiratory disease (Madoff
et al., 1979) is suggestive of human carriers. Mycoplasma bovis
are quite resistant to environmental conditions (Nagatomo
et al., 2001) therefore, the transmission through fomites and mechanically
can also not ignored.
Transmission: The main route of transmission is aerosol route by the
small, inhaled droplets from infected animals and contaminated dust particles
(Pfutzner et al., 1983; Stipkovits
et al., 2000). The hematogenous transmission is essential for joint
affections (Romvary et al., 1977). Calves are
affected in both the ways via horizontal transmission through aerosol infection
of respiratory tract and vertical transmission as milk is as one of major source
of infection particularly from the mastitic udder. Moreover, newborn calves
can become infected vertically from the uterus (Pfutzner
and Schimmel, 1985).
As small number of living organisms is enough to cause the infection of the
teat canal (Bennett and Jasper, 1980); the udder may
be infected through teat canal and the milking process (Thomas
et al., 1981) due to contaminated milking pens, milking machines,
wiping cloths, milkers and fomites.
Both male and female genital tracts are affected through ascending infections
from the contaminated environment or by direct contact of other animals shedding
M. bovis. In females artificial insemination of cows with infected semen
are major source of infection (Eaglesome and Garcia, 1990).
Uterine discharges and aborted fetuses may be source of infection (Bocklisch
et al., 1987; Pfutzner and Schimmel, 1985).
Diagnosis of the M. bovis infection: The M. bovis organism remains infective in the respiratory tract and can be transmitted to the next generation. Moreover, frequent way of vertical infection for suckling calves is the milk of asymptomatic shedder or mastitic cows. Thus, the diagnosis of infection is an essential part to reduce the economic losses caused by M. bovis infection.
The nasal mucus of other affected calves or cows is an important factor in
horizontal spreading of M. bovis. The type and site of sample should
be selected depending upon the clinical signs. Nasal, genital, conjunctival,
tracheal swabs and samples of milk, synovia, semen and other liquid samples
are collected into Medium-B broth containing phenol red and glucose (Erno
and Stipkovits, 1973a) and kept at 4°C and rushed to the diagnostic
laboratory at the earliest. However, from respiratory and uterine affections
M. bovis can be better recovered from broncho-alveolar lavages and deep
intra uterine sampling. Tissues are collected aseptically in small pieces and
after homogenization with Phosphate Buffer Saline (PBS) of pH 7.2 to 7.6 are
submitted as previously mentioned method of culturing.
CULTURE AND IDENTIFICATION
Mycoplasma bovis grow well in suitable culture media and produces typical
fried-egg shape colonies after 72 to 96 h of incubation. Medium B and Hayflicks
medium are two commonly used media although certain variants are also available
with certain modification and are used to identify the M. bovis on the
basis of cultural characteristics eg. detection of M. bovis colonies
based on the lipase reaction (Shimizu, 1983) and red
color reaction of M. bovis colonies (Windsor and Bashiruddin,
1999). Then biochemical properties are assessed as per the method of Erno
and Stipkovits (1973a, b). Lauerman
(1994) recommended growth inhibition test, metabolic inhibition test and
indirect immno fluorescence test with anti M. bovis hyper immune sera.
Specific Mab-based sandwich or capture ELISA (Ball et
al., 1994) or dot immunobinding test using polyclonal sera (Poumarat
et al.,1991; Kumar, 2000) are also used to
confirm M. bovis from culture.
Antibodies to M. bovis can be detected within two week of infection
and persist for several months therefore, can be detected with various immunological
methods (Kumar et al., 2004) viz., counter current
electrophoresis, ELISA, immunoblotting, Immunobinding and immunohistochemistry
methods. The use of these tests is particularly useful, when the isolation of
the agent is difficult due to chronic infection or regular treatment with antibiotics
at a high dosage (Nicholas and Ayling, 2003). The presence
of M. bovis in nasal cavity may not be immunogenic so these tests are
effective whenever there is invasion of M. bovis.
ELISA: The ELISA tests are useful diagnostic methods but only for screening
purposes (Pfutzner and Sachse, 1996). There are commercially
available tests with mixed antigens in order to minimize the false negative
reactions due to the antigenic variability of M. bovis (Le
Grand et al., 2001). There are also non-commercial ELISA tests developed
to detect M. bovis infection (Boothby et al.,
1981; Uhaa et al., 1990; Byrne
et al., 2000). The ELISA tests performed from milk can also be applied
in the examination of mastitis outbreaks (Byrne et al.,
SDS-PAGE and western blot: This technique can be used to compare the
antigenic structure of the strains or for the examination of the humoral immune
response patterns of the host animal. However, the use of monoclonal antibodies
specific to M. bovis can be used for the diagnostic purpose. Rosengarten
et al. (1994) observed 12 major strain variable antigens whereas
Sachse et al. (1992) identified 34 isolates of
M. bovis with a high degree of similarity in most of the isolates. Boothby
et al. (1983) used SDS-PAGE and GED ELISA for the characterization
of antigens from mycoplasmas of animal origin and observed certain areas of
homology without any common protein band in different species along with high
reactivity of proteins with homologous and heterologous antisera. Poly acrylamide
gel electrophoresis (PAGE) used for separation of native proteins of M. bovis
revealed protein profiles almost similar to M. agalactiae (Kumar
et al., 2001b). However, Kumar et al.
(2000) characterized Sonicated Supernatant Antigen (SSA) and Whole Cell
Antigens (WCA) of an Indian isolate of M. bovis (NC317) by SDS-PAGE and
immunoblotting. SDS-PAGE profile revealed 21 and 22 polypeptides in the region
of 181.97 to 20.89 kDa in WCA and SSA, respectively. On immunoblotting, with
polyclonal hyper immune serum raised against M. bovis NC317, only 12
and 14 polypeptides were found immunogenic in WCA and SSA, respectively. Alberti
et al. (2006) reported that Western blotting might represent a useful
tool for discriminating M. bovis from M. agalactiae after the
PCR RFLP test. Rifatbegovic et al. (2009) studied
the isolates of Bosnia and Herzegovina by SDS PAGE and Immuoblotting to find
out immunogenic and protein similarity.
Immunobinding assay (IBA): Immunobinding tests were also used for the
diagnosis of M. bovis. Kumar et al. (2002)
used immuno binding assay of M. bovis but it showed cross reactivity
with M. agalactiae. Recently an immuno binding test was developed on
nitrocellulose paper with monoclonal antibody to diagnose Mycoplasma bovis
in cultural isolates from the genital tract of artificially-infected heifers
polymerase chain reaction was used as the gold standard (Flores-Gutiérrez
et al., 2009).
Immunohistochemistry: The use of specific antibodies either by Immuno
Fluroscence (Knudtson et al., 1986) or Immuno
Histochemical (Adegboye et al., 1995) can be
used for the in situ detection of M. bovis. However, IH enables
the visualization of the antigen together with the specific lesions (Rodriguez
et al., 1996).
Monoclonal antibodies: Monoclonal antibodies can differentiate between
strains belonging to the same species (Poumarat et al.,
1994) but the monoclonal antibodies produced against the M. bovis antigen
react with antigens prepared from other Mycoplasma species (Berthold
et al., 1992; Rasberry and Rosenbusch, 1995)
due to genetic similarity between the so-called PvpA membrane protein of M.
gallisepticum and the variable surface lipoproteins (Vsps) of M. bovis
(Yogev et al., 1994). Mab against the 27 kDa
antigen determinant of M. bovis is responsible for adherence to cells
(Sachse et al., 1993). This surface glycoprotein
enables M. bovis bacteria to adhere to alveolar phagocytes (macrophages)
(Howard et al., 1976) and to neutrophilic granulocytes
(Thomas et al., 1991).
Variable surface lipoproteins (Vsps)-is closely related to the polymorphism
of M. bovis. The family of the Vsp antigens comprises three members:
VspA, VspB and VspC (Behrens et al., 1994; Rosengarten
et al., 1994; Beier et al., 1998).
The produced Vsps-specific antibodies allow us to study the host cell-M.
bovis relationship, the differences between the isolated strains and their
association with virulence.
In addition to the Vsp lipoproteins occurring in the membrane of M. bovis,
the membrane protein of 67 kDa molecular weight (pMB67) also has an important
role: it induces antibody response during natural infection or disease and may
be suitable for the development of vaccines or diagnostic preparations (Behrens
et al., 1996).
Isolation of M. bovis is difficult, time consuming and cumbersome to
perform. Moreover, the cross reactivity of M. bovis with many other mycoplasma
species dragged the attention on the DNA-based techniques (Kumar
et al., 2001a). DNA-based techniques, especially PCR can yield rapid
and specific diagnosis of infections caused by M. bovis (Hirose
et al., 2001).
Plasmid probes containing random genomic fragments used in dot blot hybridization
tests produced cross reaction with M. agalactiae or M. arginini
(McCully and Brock, 1992). Synthetic oligonucleotide
probes from the 16 S RNA gene were applied by Mattsson et
al. (1994) are not specific and sensitive enough for the routine diagnosis.
The use of PCR in the diagnosis of M. bovis reduced the time of diagnosis
from organs or the broth cultures even contaminated with bacteria.
The use of 16 S ribosomal RNA gene in PCR (Chavez Gonzalez
et al., 1995) also produced cross reactivity with M. agalactiae.
Thereafter, Ghadersohi et al. (1997) designed
PCR primers from sequences obtained from a M. bovis specific dot blot
hybridization probe which was further modified by Hayman
(2003) into a seminested setup. The PCR system designed for UvrC gene sequences
provide high specificity and clear distinction of M. bovis thus UvrC
gene could distinguish between M. bovis and M. agalactiae (Subramaniam
et al., 1998).
The Vsp gene based system described by Ghadersohi et
al. (1997) modified by Hayman (2003) by adding
a new forward primer (MbF) to the system-provided rapid detection of this organism.
The semi-nested system has been developed by Hayman and
Hirst (2003) whereas, Pinnow et al. (2001)
developed a specific nested PCR test, with which the preservative-treated milk
samples can also be examined. Alberti et al. (2006)
used 16S rDNA sequence to establish the relatedness of strains. A Restriction
Fragment Length Polymorphism (RFLP) strategy directed to the identification
of phylogenetic clusters was designed to restrict the diagnostic investigation
to a few bovine mycoplasma species (Alberti et al.,
2006). Reverse-transcription PCR and primer extension analysis indicated
that both p68 and p48 are transcribed in M. bovis under in vitro
growth conditions. Mycoplasma bovis is the first mycoplasma species in
which two malp-related genes have been identified (Lysnyansky
et al., 2008). Rossetti et al. (2010)
described a new specific real-time PCR assay targeting the UvrC gene that was
developed to directly detect M. bovis from milk and tissue samples without
laborious DNA purification.
Mycoplasma bovis infection can be diagnosed in several ways. Out of them isolation and identification not only requires specially equipped laboratories but also difficult due to secondary bacterial infections or inhibitory effects of antibiotics. Normal presence of M. bovis in nasal mucosa cannot be detected due to lack of antibodies. Moreover, serological tests particularly ELISA are useful tools for the herd diagnosis but cross-reactivity of M. bovis with other mycoplasmas and individual variation of immune response may sometime produce doubtful results.
Molecular tests particularly Polymerase Chain Reaction (PCR) with specific primers have better chance for the detection of the organism both early and chronic infections. The PCR systems targeting the 16S RNA gene M. bovis specific dot blot hybridization probe improve the specificity and sensitivity. The assay targeting the UvrC gene is also very sensitive and specific. Moreover, a conservative prokaryotic lipoprotein signal sequence of N-terminal part of variable cell surface lipoproteins (Vsp) can be an ideal region to be targeted with PCR assays to detect M. bovis. Polymerase chain reaction (PCR) and sequencing-based methods are very specific but require a massive amount of work if a wide range of species has to be investigated with dedicated assays.
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