Diagnosis of mycoplasmosis in goats

1. Diagnosis of Mycoplasmosis in goats
Presented by :Mohammad iliyas
Presented to : Dr.Mohammad Iqbal yatoo

2. Diagnosis
• The diagnosis of mycoplasmoses under routine Indian
conditions involves history, clinical signs, physical examination
followed by postmortem examination and histopathology.
• Clinical diagnosis of the respiratory mycoplasmoses is
confounded by immense variations which may be observed in
symptom expression,morbidity and mortality, depending on the
genetic constitution, age and managementof the animals at risk,
the novelty of the organism to them, the strain of the organism
involved, stress factors (e.g. dehorning, castration) and the
occurrence of intercurrent diseases such as P P R , heartwater,
coccidiosis and type B Clostridium perfringens enterotoxaemia.

4. Commonly used diagnostic
techniques
• complement fixation test (CFT),
• Enzymelinked immunosorbent assay (ELISA),
• polymerase chain reaction(PCR),
• sodium dodecyl sulfate polyacrylamide gel
electrophoresis(SDS-PAGE),
• Restriction Fragment Length Polymorphism(RFLP)
• DNA sequencing
• For diagnosis of mycoplasmosis the conventional
methods include isolation in specific media, followed by
biochemical characterization and staining (Chakraborty
et al., 2014)

5. Collection of samples
• Samples from live animals comprise nasal and ear canal
swabs;
• As with pasteurellae, nasal carriage of mycoplasmas can, and
often does,occur in the absence of pneumonia.
• Immediately after collection, all swabs should be placed in
transport or mycoplasma medium.
• The best necropsy samples are lung lesions, particularly from the
interface between consolidated and unconsolidated areas, and
pleuritic fluid.
• If microbiological examination cannot be performed
immediately, samples or indeed whole lungs can be stored
deep-frozen at - 20°C for considerable periods (months) with
little apparent loss in viability of the pathogens.

6. Microscopy
• Seen in lesion exudates or suspensions by dark ground
microscopy .
• Alternatively they can be rendered visible in impression
smears from cut lesions by staining method of May-
Grunwald-Giemsa (P.C.LEFEVRE,G.E.JONES)
• M. capricolum and M. ovipneumoniae both produce
coccobacillary or very short filamentous forms in broth
cultures.
• The two M. mycoides subspecies and F38-like
organisms all show branching filamentous
morphology in vivo.

7. Media for isolation
• Modified Hank's Balanced Salt Solution Liquid Media (MBHS-L) for ovine
and caprine mycoplasma (Nicholas, 2002; Kumar et al., 2012; Kumar et al., 2014).
• MacOwan and Minette’s media to culture Mccp organisms (MacOwan &
Minette, 1976)
• Other media are WJ (Jones & Wood, 1988), modified Hayflick’s, and
modified Newing’s tryptose broth (Kibor & Waiyaki, 1986)
• Mohan et al.(1990) and Thiaucourt et al. (1992) noted that media
enriched with 0.2 % (or up to 0.8 %) sodium pyruvate are better
for
primary isolation and antigen production of Mccp.

8. Cultivation
• Cultures are incubated at 37°C. Plates are best incubated in a
humidified atmosphere of 5 % C 0 2 , 9 5 % air or N 2 , or in a
candle jar with moisture source .
• Plate cultures are generally examined every two or three days
(mycoplasmas remain viable longer on agar than in broth)
using a stereobinocular microscope .
• The morphology of colonies varies with the medium used, the
mycoplasma species, its passage level and age of culture.
• The other classical mycoplasma species found in goats and
sheep
demonstrate the conventional 'fried egg' colony morphology,
though all can produce bizarre-looking colonies in early
passage levels.

9. Biochemical tests
• The tests most commonly used are
• glucose breakdown,
• arginine hydrolysis,
• reduction of tetrazolium chloride (aerobically and
anaerobically),
• 'film and spots‘ formation,
• phosphatase activity,
• serum digestion and
• digitonin sensitivity.

11. Serological methods of identification
• The traditional, and still current, basis of
mycoplasma identification is by serology.
• Latterly, however, the isolation and recognition of
increasing numbers of species and strains has, in its
wake, revealed the existence of considerable
serological cross-reactivity between some
mycoplasmas.

12. Serological tests
• Growth inhibition test (GIT) It depends on the direct inhibition of growth
on solid media by specific hyperimmune serum of high potency.
• Growth precipitation test (GPT) The G P T detects soluble cytoplasmic
and extramembraneous antigens released by viable cultures and allowed to
diffuse through solid mycoplasma growth medium towards various
mycoplasma antisera .
• The interaction of antigens and specific antiserum produces one or more
precipitin lines in the agar medium.
• Indirect fluorescent antibody test (IFAT) The direct fluorescent antibody
test (DFAT) and the IFAT are probably the most
effective of the various serological methods available for mycoplasma
identification
• Metabolism inhibition (MIT)and tetrazolium reduction inhibition (TRIT)
Metabolising glycolytic mycoplasmas change the p H of media to acid
through breakdown of glucose to (mainly) lactic acid. Arginine-hydrolyzing
mycoplasmas create an alkaline change by producing, finally, ammonia

13. • The three most frequently used serological tests in veterinary
mycoplasmology have been the complement fixation (CFT), indirect
haemagglutination (IHAT) and slide agglutination (SAT) tests.
• More recently described tests are the enzyme-linked immunosorbent assay
(ELISA), the single reverse radial immunodiffusion test (SRRIT)
and the similar single radial haemolysis test (SRHT) .
• The CFT (prescribed test in OIE Terrestrial manual)
• The CFT is more specific, though less sensitive, than the I H A T in the assay of
antibodies both to subsp. mycoides (LC) and to strain F38 .
• The I H A T
• The I H A T is most commonly performed with erythrocytes which are either fresh
and tanned, or treated with glutaraldehyde.
• Chima and Onoviran , who used the test for M. mycoides
subsp. mycoides (SC), also considered that glutaraldehyde treatment
reduced crossreactions by making the erythrocytes bind the more specific
protein antigens.

14. The SAT
• This test has been mainly used in veterinary
mycoplasmology in the diagnosis of CBPP in
cattle, and of chronic respiratory disease in
poultry caused by M. gallisepticum.
• It depends on the clumping of whole
mycoplasma cell antigen by specific 780
antibody, and can be performed in tubes, on
slides or on a white tile using stained antigen.
• Its disadvantages are lack of sensitivity and
specificity.

15. • The ELISA
• This test was first applied in veterinary mycoplasmology to
the diagnosis of enzootic pneumonia of swine caused by M.
hyopneumoniae .
• The test depends on the adherence of proteins to plastic
surfaces. Several forms of the test have been described;
most now use microtitration plates .
• PCR is the technique of choice for diagnosis of CCPP.however
isolation remains the confirmatory test.
• Latex agglutination test and IHA are being increasingly used.

16. Some specific tests with
references
• For highly specific diagnostic test especially based on monoclonal antibody highly specific and
potent antigenic protein is isolated in SDS-PAGE followed by western blotting using homo- and
heterologous sera against different Mycoplasma species and subspecies (Srivastava et al., 2000;
Chakraborty et al., 2014; Kumar et al., 2014; OIE, 2017a).
• PCR as well as PCR-RFLP are being used for molecular diagnosis of several clusters as well as
groups using species specific primers (Chakraborty et al., 2014).
• Restriction Fragment Length Polymerase (RFLP) assay (Kumar et al., 2011b)
• Lateral Flow assay (Arun et al., 2014),
• Loop mediated Isothermal Amplification (LAMP) test (Rekha et al., 2014; Rekha et al., 2015)
• These tests help in differentiating M. capricolum and M. putrefaciens(Chakraborty et al., 2014)
• Recently multiplex PCRs are being used for diagnosis of mycoplasma infections (Chakraborty et
al.,2014; Settypalli et al., 2016).
• Specific genes like arcD, polC and fusA are being explored for diagnostic purpose (Woubit et al.,
2004; Chakraborty et al., 2014; Ambroset et al., 2017)

17. • Kashoo et al. (2013) conducted a study for amplification as well as cloning of the M.
agalactiae p80 gene for which PCR assays (specific for M. agalactiae) have been
employed.
• In accordance with Rurangirwa & McGuire (1996), the general diagnosis of members
of Mollicutes is based primarily on the following criteria: absence of a cell wall,
typical "fried egg“ appearance of the minute colonies, filtrability through a
membrane filter of 450 nm pore diameter, and absence of reversion of fresh isolates to
bacteria of divisions I, II or IV under appropriate conditions.
• Though in India the usual test used in most of the studies on mycoplasmosis is rapid
plate agglutination test also called as slide agglutination test (Ravishankar et al. 2011;
Awati & Chavhan, 2013; Gupta et al., 2016) but has been misinterpreted for
diagnostic test of CCPP in most of these studies and is not confirmatory for CCPP.
• PCR was used for confirmation of CCPP in goats in Kerala (Abraham et al., 2015).
• A genome fragment of Mycoplasma conjunctivae, causative agent of
pink eye in sheep as well as goat, which is 750 base pairs in length has
been amplified by PCR by using organism specific primers (Kumar et
al., 2011a; Kumar et al., 2011b; Shahzadet al., 2013)

18. • A suitable LAMP(Loop-Mediated Isothermal Amplification) test was
developed for the diagnosis of Mycoplasma agalactiae the causative agent of
contagious agalactia in goats with the detection level of 20 fg DNA. The test
could be performed with 70 min. at 58°C constant temperature (Rekha et al.,
2015).