CLINICAL PATHOLOGY

Culture and Nucleic Acid Recognition

Culture of the organism is the reference method for detection of the pathogen. However,
mycoplasmas are labile, making it necessary to use a special transport medium protecting this
microorganism and preventing proliferation of other bacteria. Longdistance transport of
samples, particularly when unrefrigerated drastically affects the viability of the bacteria
rendering them unfit for culture.16 Frequently, attempts to isolate MmmSC fail because the
organism is labile, is present in too little quantities, and is so demanding in its growth
requirements. Negative results should therefore always be regarded as inconclusive.1
In case of successful culture final identification of mycoplasmas is usually made by
means of a biochemical test such growth inhibition, the fluorescent antibody test (FAT) or the
immunofluorescence tests (IMF). Specific nucleic acid recognition using the polymerase chain
reaction (PCR) has become common practice over the last two decades. Although most PCR
protocols rely on previous culture, preenrichment, or extraction of mycoplasma, PCR is also used
without prior culture, directly using samples taken from nasal swabs, bronchioalveolar lavage or
transtracheal wash fluid, pleural fluid, blood, urine, or pulmonary tissue. The PCR can identify
the organism in bacterial isolates or clinical material within 2 days of extraction and is sensitive
and highly specific.1 An inconvenience of the PCR results from its high sensitivity, which makes
it susceptible to false-positive results caused by contamination. More recently isothermal
loopmediated amplification (LAMP) of DNA sequences specific for MmmSC has been
developed. The LAMP assay detects MmmSC DNA directly form crude samples of pulmonary or
pleural fluid and serum or plasma within 1 hour using a simple dilution protocol.16
Immunologic Tests
A number of immunologic test to identify the causative agent or its antigen in tissue,
biological fluids, or cultures are available. Such tests include the indirect fluorescent antibody
test (IFA) and the fluorescent antibody test (FAT), which both use hyperimmune rabbit serum
against MmmSC and labeled antibovine IgG. The growth inhibition test (GIT) is based on
direct inhibition of growth of MmmSC by a specific hyperimmune serum. Although this is a
simple test to perform cross-reactions within the mycoides cluster are common.1 The antigen
immunodiffusion test (AGID) has also been used to detect specific antigens present on the
surface of MmmSC. The AGID is considered to lack sensitivity, and little is known about its
specificity.1 Because all these tests depend on the presence of a minimum number of organisms,
only positive results should be considered conclusive.
Serologic Tests
Serologic tests that identify an immune reaction of an individual animal to infection with
MmmSC include the complement fixation test (CFT) and the competitive enzymelinked
immunosorbent assay (C-ELISA). Both are prescribed tests for international trade according the
OIE. This group of diagnostic tests has important limitations because of the nature of the
pathogenesis of the disease with its long incubation period and the relatively rapid decline of the
antibody titer.The complement fixation test (CFT) is rapid to perform and easy to interpret.
With a sensitivity in the range of 70% to 80% and specificity of 98% it is best suited to diagnose
clinically affected animals with acute lesions but less suitable to identify either animals in early
stage of the disease, chronically infected or carrier animals with low antibody titers.1,17 The
therapeutic use of antimicrobials further increases the risk of a false-negative test results.
Vaccinated animals give a positive reaction for about 6 weeks, although this period may be much
longer if severe vaccination reactions occur. Because of the limited sensitivity the CFT is
considered unreliable on an individual animal level, but it is deemed to be highly effective in
detecting infected herds when testing the entire population. The test is widely used in to
determine freedom of disease on a herd level.1 Because false positive results caused by serologic
cross-reactions with other species of the mycoides cluster can occur, it is advisable to confirm a
positive test result by postmortem and bacteriologic examination. The C-ELISA has a similar or
even greater specificity than the CFT.1,17 The sensitivity of the C-ELISA was found to be
superior to the CFT particularly to detect animals in the chronic stage of the disease, whereas the
CFT appears to outperform the C-ELISA in the detection of animals in the acute phase of the
disease.17,18 An indirect ELISA based on a recombinant protein, LppQ-NX (LppQ ELISA),
has been developed and provides good sensitivity and specificity for the diagnosis of CBPP and
is robust under harsh climatic conditions. The CFT, competitive ELISA, and LppQ ELISA, all
used for detection of antibodies to MmmSC, were compared with postmortem inspection for the
diagnosis of CBPP in naturally infected cattle in an endemic area in Zambia between 2007 and
2008.17 The percentage of positive sample was 67.5% for post postmortem examination, 59.0%
for the C-ELISA, 52.6% for the CFT, and 44.4% for the LppQ ELISA. Of the three serologic
tests the CFT identified the largest number of animals in the acute phase of the disease, whereas
the C-ELISA was the most sensitive test to detect animals in advanced stages of the disease. The
LppQ ELISA had a very poor sensitivity (10.8%) to identify animals in the early stage of the
disease, whereas in the chronic stage it had a sensitivity ranging above the CFT but below the C-
ELISA.
The immunoblotting test (IB) is based on an immunoenzymatic reaction with higher
sensitivity and specificity than the CFT. The IB is recommended as a confirmatory test on
positive samples previously analyzed with another test because IB is not suitable for mass
screening and may be difficult to standardize.1 No single serologic test is capable of detecting all
CBPP affected animals in the field. These tests are most useful for diagnosis at the herd level. In
the absence of a gold standard test for the serologic diagnosis of CBPP, some uncertainties
remain unresolved. Suspicious CBPP cases identified by positive serology must be confirmed by
further investigations that demonstrate the presence of antigen in the respiratory tissues of
animals.
TREATMENT
Official conventional wisdom in the past held that treatment of clinical cases of CBPP
with antimicrobials is counterproductive to contain the disease because it gives rise to persistent
infection and may produce symptomless carrier animals.5 Accordingly, the use of antimicrobials
is legally banned in many endemically affected countries. Nevertheless, the use of antimicrobials
in affected regions is widespread, mainly because, with limited availability of vaccines, it is
considered the only available and effective treatment and control measure.5,19 In recent years
the popularity of antibiotic treatment and the perception of positive results led to some research
activity suggesting that antimicrobial use may be of value primarily to control disease
transmission.5 Despite the perception of veterinarians and farmers that antibiotics can alleviate
the clinical course of the disease, enabling some improvement in condition, field studies suggest
that antimicrobial therapy had little to no effect on severity of signs, course of the disease, and
mortality rate in clinically affected animals.20,21 Treatment failures in clinically affected
animals may be attributable to inadequate dosage or duration of treatment and to the chronic
nature of the condition. Treatment success of antimicrobial therapy to treat mycoplasma infection
greatly depends on a timely initiation of the treatment, but clinically affected animals in endemic
areas frequently are in an advanced or even chronic stage of disease and thus are unlikely to
show strong treatment response.21 Because of the generally poor treatment response and because
these animals present a source of infection for herdmates, clinically affected animals should
rather be culled than treated. In contrast the treatment of in-contact animals appeared to
considerably reduce disease transmission, which resulted in a marked decrease in the disease
occurrence, morbidity, and mortality rates in affected herds.21 An increasing body of evidence
suggests that use of antimicrobials, primarily as a part of a disease control program, should be
reconsidered.5,15,19 The major classes of antimicrobials that are effective against mycoplamsas
are tetracyclines, macrolides, florfenicol, and fluoroquinolones. A number of in vitro and in vivo
studies have been published in recent years with results supporting the use of fluoroquinolones,
several different macrolides, and tetracyclines to reduce the shedding of MmmSC.21-24 Beta-
lactam antibiotics and sulfonamides are inherently ineffective against the Mycoplasmas that do
not have a cell wall and do not synthesize folic acid.
CONTROL
There are four essential tools in CBPP control and eradication: livestock movement control,
stamping out, vaccination, and treatment.3 The possible strategies used for control in affected
countries or regions are as follows:
Slaughter of all sick and in-contact cattle. This requires full cooperation of cattle owners and
an adequate and timely compensation system. This strategy is impractical in developing
countries with a pastoral economy.
Slaughter of all sick cattle and vaccination of in-contact cattle. This strategy is used
frequently and usually perpetuates the disease.
Vaccination of healthy cattle with slaughter of sick cattle in an epidemic and revaccination
of cattle at risk. This method depends on the ability of the authorities to detect epidemics
rapidly, most effectively, by abattoir surveillance and to maintain vaccination for at least 3 years.
Vaccination in endemic areas must be done annually, whereas newly infected areas require repeat
vaccinations aimed at eradication of the disease.