Mycoplasma Affecting Domestic Animals with Special Reference to Their Economic Importance
ISOLATION OF MYCOPLASMAL AGENTS:
Mycoplasmas can be isolated from infected tissues using medium containing penicillin and thallium acetate. The isolate can then be identified by serological methods using standard antiserum for agglutination and complement fixation test (CFT), or the supernatant fluid from the broth culture can be used for carrying out a precipitation test. Growth and metabolic inhibition tests are additional methods used for identification of newly isolated strains. The procedure for isolation and identification mentioned so far are applied for Mycoplasma mycoides. M. bovigenitalium can be identified microscopically in stained preparations of milk from mastitis cases and the organism can also be isolated from these samples.
IDENTIFICATION OF MYCOPLASMAL AGENTS:
Mycoplasmas are fastidious organisms and unlike bacteria they require cholesterol or a related sterol incorporated in the medium for growth.
There is absence of cell wall in mycoplasmal agents, so they are not rigid and show a great deal of plasticity and variety of bizarre shapes like granules or, cocci of tear drop cells etc. may develop during their life cycle. They may assume filamentous or, ring forms. Variations in morphology are related to composition of media in which the mycoplasmal agents grow.
Mycoplasmas are Gram-negative organisms, but stain poorly with aniline dyes.They can be stained by Geimsa’s method after fixation with methyl alcohol or after staining negatively with nigrosin. Geimsa is also satisfactory for staining sections, particularly if they are treated with potassium permanganate (1%) for 2 minutes which increase the intensity of subsequent staining. Dienes’ staining method is suitable for examination of colonies on agar. The stain consists of azure II (0.25g), methylene blue (0.50g), maltose (2.0g), Na2CO3 (0.05g), benzoic acid (0.04g) and distilled water (20 ml). A small piece of agar containing the colony is placed on a slide and covered by coverslip containing the stain in contact to the colony. The colony stains moderately, while the surrounding agar remains unstained.
Mycoplasmas are well spread in nature in both pathogenic and non-pathogenic forms. They can cause diseases in cattle, sheep, goats etc. 10% carbon dioxide supplement is required in culture media for their growth. They get entry in the host through respiratory, gastrointestinal routes etc. and cause chronic pneumonia, respiratory ailments, and arthritis in animals. Mycoplasmas pose to be a serious problem in West Bengal and Brahmaputra valley of Assam. Mycoplasma are designated as pleuropneumonia like organisms (PPLO).
Growth in solid medium
A general suitable medium for the isolation of mycoplasmas consist of 20% unheated horse serum, 10% fresh yeast extract, which has not been heated above 75oC, 20 μg/ml of deoxyribonucleic acid together with 50 units/ml of penicillin and 0.25 mg/ml of thallous acetate (1 : 4000) to control the growth of bacterial contaminants. The optimum temperature for growth is 36-38oC for pathogenic strains of mycoplasma and after 3-4 days of incubation some colonies measure about 0.5-1 mm in diameter with a yellowish, opaque centre giving rise to a poached egg like appearance. Smaller colonies can only be seen under low-power microscope.
Growth in fluid medium
This consists of serum broth similar to the solid medium referred to as above with the omission of agar. Growth may be difficult to observe unless by lateral illumination against dark background.
Cultivation in embryonated eggs
Cultivation of mycolasma in embryonated hen’s egg is of particular importance. Inoculation into the yolk sac of 7 days old embryonated eggs, particularly for pathogenic avian strains which cause death of the embryos in 2-4 days after inoculation is characterized by extensive cutaneous hemorrhages and generalized edema.
Cultivation in cell culture
A variety of cell culture systems have been employed including monolayer of HeLa cells, chicken heart fibroblasts and human conjunctival cells. M. bovigenitalium can grow on bovine cell culture media. Mycoplasma may grow intracellularly or extracellularly showing extensive cytopathic effects.
Carbohydrate fermentation tests using glucose enable mycoplasma to be divided into two groups- fermenting and non-fermenting species. Mycoplasma associated with animal infections ferment glucose, fructose, mannose and maltose with the production of acid.
Resistance to physical and chemical agents
Mycoplasmas are labile to temperature exposure of 55oC for 15 minutes and 60oC for 5 minutes. The organism remains viable for several months in frozen lung and freeze dried cultures. Disinfectants like 1% phenol, 0.5% formalin or 0.01% mercuric acid are highly lethal to the organism.
ANTIGENS AND TOXINS:
- mycoides possess at least three antigenic components constituting only one antigenic type and the serological activity is known to be associated with galactan which can be detected in the blood of acutely infected cattle. A neurotoxin is produced by strain of M. neurolyticum. M. gallisepticum produces a soluble exotoxin. Some experiments have suggested that M. mycoides may produce a diffusible toxin.
Serological tests which can be used for diagnosis of mycoplasmal infections are as follows:
(b) Agar gel test
(c) Rapid slide serum agglutination test
(d) Rapid slide whole blood agglutination test
(e) Indirect haemagglutination test
(f) Growth and metabolic inhibition test
(g) Fluorescent antibody test
(h) Allergic (skin) test
ECONOMIC SIGNIFICANCE OF MYCOPLASMAL AGENTS:
- i) The incubation period of mycoplasmas are very long (nearly 1-4 months) during which the cattle becomes unproductive. Cattle which recover are also weak and emaciated and are never able to regain their previous productivity.
- ii) Pregnant cattle which become infected abort leading to prenatal deaths causing losses to farmers.
iii) M. bovigenitalium causes mastitis in dairy cattle with swollen fibrosed udder and sudden decrease in milk yield.
- iv) In sheep and goats the mortality may be as high as 90%. During the acute phase of disease, death occurs in 10-14 days.
- v) agalactiae causes mastitis in lactating ewe and doe.
- vi) hyorhinis causes pneumonia and arthritis in pigs.
vii) M. canis causes throat and urogenital tract problems in dogs. As a result, the bitches which become affected in adolescence are not able to produce offspring and dogs become sterile in most cases.
viii) M. gallisepticum causes chronic respiratory disease in poultry along with sinusitis and arthritis of limb joints. In causes serious drop in egg production which affects national economy in adverse manner.
- ix) Public health importance:
- pneumoniae causes pneumonia in human beings. Many mycoplasmal agents are commensals in throat and mouth of human beings.
Buxton, A., Fraser, G., 1977. Animal Microbiology. 1st edn. Blackwell Scientific Publication Ltd., Victoria, Australia.