RReevviieeww ooff LLiitteerraattuurree

II. REVIEW OF LITERATURE

2.1 The Mycoplasmas

Mycoplasma is a trivial name for a group of microorganisms that belongs to the

class Mollicutes. The name Mollicutes, from Latin mollis meaning “soft” and cutis

meaning “skin” indicating their characteristic lack of a cell wall (Razin et al., 1998). The

trivial terms “mycoplasmas” or “mollicutes” have been interchangeably used to denote

any species included in Mollicutes. Excluding Mycoplasma the class mollicutes consists

of eight other genera: Ureaplasma, Entomoplasma, Mesoplasma, Spiroplasma,

Acholeplasma, Anaeroplasma, Asteroleplasma and Phytoplasma.

Mycoplasmas are the smallest and simplest self-replicating prokaryotes, which

lack a rigid cell wall and are bound by a single membrane, the plasma membrane.

Compared to the genome of Escherichia coli (4.64 Mb), the genome sizes of

mycoplasmas are extremely small (0.58–1.35 Mb).

Owing to their small genomic size, these microorganisms appear to have limited

metabolic options for replication and survival. Often portrayed as minimal bacteria,

mycoplasmas have evolved from low G+C content Gram-positive ancestors by massive

losses of genetic material and extensive genome downsizing. The significant genome

compaction that occurred in mollicutes was made possible by adoption of a parasitic

mode of life. To keep to the parasitic mode of life, mycoplasmas have developed rather

sophisticated mechanisms to colonize their hosts and resist the host immune system

(Razin et al., 1998).

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Most mycoplasmas are parasites of many animal species, including humans,

exhibiting strict host and tissue specificities. They enter an appropriate host, in which

they multiply and survive for extended periods of time. They have a predilection for

moist mucosal surfaces and often colonize the respiratory and urogenital tracts of their

hosts. Other anatomical sites, such as joints, are frequently involved. Although

mycoplasmas often enter into a commensal relationship with their hosts, many

mycoplasmal species are potentially pathogenic and may, in fact, behave as frank

pathogens. Mycoplasmas cause a variety of diseases, including respiratory, arthritic, and

urogenital infections. Clinically, these disease states may be either acute and self-limiting

or chronic, with a varied pattern of remission and reactivation. Further, complications and

sequelae that appear to be autoimmune in nature may follow the initial acute infection.

The clinical picture of mycoplasma infections in humans and animals is more suggestive

of damage due to host immune and inflammatory responses rather than to direct toxic

effects by mycoplasmal cell components (Rottem and Naot, 1998).

Pathogenic mechanisms of mycoplasmas are partly attributed to competing with

their host cells for metabolic substrates such as lipid precursors, purines and pyrimidines

and to their ability to attach (adhesion), fuse and invade the host cells causing variation of

their phenotype (phase variation/antigenic variation/size variation), thereby evading the

host immune response by means of mimicry of host antigens and generation of

phenotypic plasticity and cytopathic effects (Baranowski et al., 2010). Biofilm formation

by some mycoplasmas has been documented recently, and is suggested to play a role in

their pathogenesis and persistence in the host and/or environment (Noormohammadi,

2007). Apart from providing a specific anti-mycoplasmal defense, the host immune

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system is also involved in the development of pathogenic lesions and exacerbation of

mycoplasma-induced diseases (Rottem, 2003).

2.2 Mycoplasmas affecting small ruminants

Mycoplasmas are small, fastidious, wall-less bacteria which can cause disease in

all major species of animals including humans. Many mycoplasmas have been isolated

from sheep and goats but only a few have been linked directly to disease (Table 1).

Table 1 : Mycoplasmas causing disease in small ruminants

MYCOPLASMA HOST DISEASE

M. agalactiae Sheep/Goats CA

M. capricolum subsp. capripneumoniae Goats (Sheep) CCPP

M. capricolum subsp. capricolum Goats (Sheep) Pneumonia, CA

M. conjunctivae Sheep/goats Keratoconjunctivitis

M. mycoides subsp. mycoides (large colony) Goats (Sheep) Pneumonia, CA

M. mycoides subsp. capri Goats Pneumonia

M. ovipneumoniae Sheep/goats Pneumonia

M. putrefaciens Goats (Sheep) Mastitis, arthritis, CA

Acholeplasma oculi Sheep/goats Keratoconjunctivitis

CA - Contagious agalactia; CCPP- Contagious caprine pleuropneumonia

In small ruminants, they cause respiratory disease, mastitis, arthritis, genital

disease and eye lesions. The most important of these diseases are contagious caprine

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pleuropneumonia (CCPP) and contagious agalactia (CA) which are designated by the

Office of International Epizooties as List B diseases because of their economic impact on

livestock. Some of the mycoplasmas causing CCPP and CA are members of the

“Mycoplasma mycoides” cluster which consists of six mycoplasmas (Mycoplasma

mycoides subsp. mycoides Large Colony (MmmLC), Mycoplasma mycoides subsp.

mycoides Small Colony (MmmSC), M. mycoides subsp. capri (Mmc), M. capricolum

subsp. capricolum (Mcc), M. capricolum subsp. capripneumoniae (Mccp), and

Mycoplasma species bovine serogroup seven (Cottew, 1974), all of which are important

pathogens of small and large ruminants.

2.2.1 Contagious agalactia (CA)

Contagious agalactia of sheep and goats has been known for about two centuries.

Contagious agalactia is a disease of sheep and goats caused by mycoplasmas, which is

clinically manifested as mastitis, arthritis, keratoconjunctivitis and pneumonia (Nicholas,

1996). Mycoplasma agalactiae, the main causal organism of CA of sheep and goats was

initially isolated by Bridre’ and Donatien in 1923 and was the first caprine and ovine

Mycoplasma species established (Bridre’ and Donatien, 1923).

CA may be caused by four different mycoplasma species: Mycoplasma agalactiae

(Ma), Mycoplasma mycoides subsp. mycoides LC (Mmm LC), Mycoplasma capricolum

subsp. capricolum (Mcc) and Mycoplasma putrefaciens (Mp). All these species contribute

significantly to economic losses (Nicholas, 1995).

Contagious agalactia (CA) is a disease predominantly of milking sheep and goats

herd in the spring season soon after lactation. The young ruminants become infected

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directly at sucking while the adults are contaminated via the milker's hands, milking

machines or by bedding which often provides a rich source of mycoplasmas. The CA

infection frequently occurs as an enzootic disease. In lactating female animals, it is

usually manifested by mastitis, whereas in males, young animals and non-lactating

females the disease is manifested as arthritis, keratoconjunctivitis and respiratory

problems (Madanat et al., 2001).

2.2.2 Contagious caprine pleuropneumonia

CCPP caused by M. capricolum subsp. capripneumoniae (formerly Mycoplasma

F38) has been known for many years causing major losses in goat herds. In addition two

other “M. mycoides cluster” members, M. mycoides subsp. mycoides LC and M. mycoides

subsp. capri, were also implicated in the aetiology of the disease because of their ability

to cause pleuropneumonia in small ruminants that resembles CCPP. This confusion was

compounded by the difficulty of isolating and growing the highly fastidious F38 in vitro.

Classical CCPP is characterised by its readily contagious nature and histopathologically,

by an interstitial intralobular edema of the lung rather than thickening of the interlobular

septa which is seen with M. m. mycoides LC and M. m. capri. Naive herds can suffer

losses of up to 80 per cent mortality and 100 per cent morbidity when exposed for the

first time (Nicholas et al., 2008).

2.2.3 M. ovipneumoniae

M. ovipneumoniae causes an atypical pneumonia with high levels of morbidity

and up to 20 per cent mortality in small ruminants and is often associated with

Pasteurella haemolytica infection. Clinical signs include coughing, dyspnoea, pyrexia,

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inappetance and profound depression. Post mortem lesions are confined to the thoracic

cavity and includes fibrinopurulent pleurisy and pericarditis with copious amounts of

yellow oily fluid in the pericardial sac and free in the thoracic cavity. On histopathology,

severe suppurative pleurisy, widespread suppurative alveolitis with necrosis, and severe

necrotizing bronchiolitis are appreciable (Nicholas et al., 2008)

2.3 Isolation of mycoplasma

The first report of isolation of mycoplasma species was made by Greig (1955) in

Canada from respiratory tract of sheep suffering from respiratory illness. The disease

primarily affects the mammary glands, joints, eyes and, to a lesser degree, respiratory

tract. It is clinically manifested as mastitis, arthritis, conjunctivitis and pneumonia

(Nicholas, 1996). Several reports of isolation of mycoplasma have been reported in sheep

and goat. In sheep and goat mycoplasma has been isolated from respiratory tract, eyes of

healthy animals, conjunctival sacs, pneumonic lungs and from animals showing

respiratory illness.

2.3.1 Isolation of mycoplasma from respiratory illness

Cottew and Lloyd (1965) and Okoh and Ochol (1986) isolated mycoplasma from

goats and sheep suffering from respiratory illness characterized by coughing and nasal

discharges respectively. They used lungs and thoracic fluid from the affected flock for

isolation of mycoplasma.

Ungureanu and Cirstet (1969), Cottew (1971), Erdag (1972), Livingston (1973)

and Mahi and Nayil (1975) have reported isolation of mycoplasma strains from

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pneumonic lungs of sheep. Leach (1970) isolated M. arginini from the nostrils of sheep in

a flock showing outbreak of coughing.

Talavera et al. (1985) reported that five of 12 normal adult slaughtered sheep

from different flocks yielded mycoplasma. Three cultures were from vagina, two from

nasal cavities and one from ear canal. By immunofluorescence, two isolates were

identified as M. mycoides subsp. mycoides (large colony type), two as M. arginini, one as

M. agalactiae and one as mixed M. agalactiae and M. mycoides.

Houshaymi et al. (2002) isolated Mycoplasmas from freeze-dried lung samples of

goats from the Western lowlands of Eritrea suspected of being affected by contagious

caprine pleuropneumonia (CCPP). The goats belonged to two herds in which mortality

and morbidity rates were high. Mycoplasma capricolum subsp. capripneumoniae was

identified in most samples by the polymerase chain reaction. Following cloning, M.

capricolum subsp. capripneumoniae isolates were analysed biochemically and shown to

be metabolically similar.

Ikheloa et al. (2004) screened forty-two (2.65 per cent) pneumonic lungs in 1580

goats slaughtered at three different locations in Northern Nigeria to identify the possible

Mycoplasma species in the animals. Twenty-seven isolates of Mycoplasma isolated from

the pneumonic lungs of goats, were characterized biochemically and identified

serologically and included Mycoplasma arginini (10); Mycoplasma capricolum;

Mycoplasma mycoides subsp. mycoides LC (2); Mycoplasma capricolum subsp.

capripneumoniae (1); Mycoplasma bovis (1) and and Mycoplasma V (10).

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Adehana et al. (2006) recovered fifty Mycoplasma strains from pneumonic lungs

of sheep and goats from Cotonou abattoirs. Mycoplasma strains from sheep were

serologically identified as follows: group A, M. ovipneumoniae; group B, M. mycoides

subsp.mycoides LC; group C, M. capricolum subsp. capripneumoniae and group D, M.

arginini, respectively. In goats, they were serologically identified as follows: group A, M.

mycoides subsp. mycoides LC; group B, M. capricolum subsp. capricolum; group C, M.

capricolum subsp. capripneumoniae and group D, M. arginini, respectively.

Srivastava et al. (2010) reported the first recognised outbreak of CCPP in

Mauritius, in which around 300 goats from 20 herds had died. Acutely affected goats

showed overt clinical signs of respiratory disease. M. capricolum subsp.

capripneumoniae was found to be the causative agent.

2.3.2 Isolation of mycoplasma from mastitis and arthritis

Damass (1983) isolated Mycoplasma agalactiae in udder secretion from a goat

with mastitis, indicating active infection unlike the avirulent isolates obtained previously

and concluded that the presence of high concentration of isolate indicates about its

pathogenicity.

Aarabi and Sotodehina (1984) observed that tests on milk samples from sheep and

goats yielded 23 strains of M. agalactiae and three strains of M. mycoides sub. sp.

mycoides. The later was isolated from sheep and goats in Iran for the first time.

Hazell et al. (1985) reported outbreak of mastitis, pneumonia and arthritis

following introduction of two goats to a herd. Four of the 23 does and eight kids died

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Lesions of severe fibrinous polyarthritis, synovitis and interstitial pneumonia were found.

No bacterial pathogens were cultured but M. mycoides mycoides was isolated from lung,

spleen and mammary gland of a doe and from joints of two kids.

Gaillard et al. (1986) observed agalactia and arthritis in a herd of 65 goats in Midi

Pyrenees and in 136 milking goats in Poitevine region. M. putrefaciens was isolated from

milk samples from both herds and M. mycoides subsp mycoides was isolated from

synovial fluid from a goat with arthritis in the first herd and from ear swabs from animals

in the second herd.

Montagna (1988) reported that out of 121 mycoplasma strains isolated from the

milk of sheep and goats suspected of contagious agalactia, 84 were M. agalactiae, 34

were M. capricolum, 2 were M. mycoides sub. sp. mycoides and one was M. mycoides

subsp capri.

Egwu et al. (2001) investigated the status of intramammary mycoplasmosis in

caprine udders in Nigeria. A total of 57 and 24 milk samples were collected from udders

of goats affected by mastitis and from apparently normal goats’ udders, respectively.

Acute and chronic mastitis were more commonly observed in goats between one and

three years old. Mycoplasma agalactiae and Mycoplasma capricolum occurred at a

significantly higher rate (P<0.05) in udders affected by mastitis than in normal healthy

udders. Other mycoplasmas occurring in low prevalence include Mycoplasma bovis and

Mycoplasma mycoides subsp. mycoides LC.

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Al-Momani et al. (2006) surveyed 104 flocks of local sheep and goats (17 sheep,

27 goat and 60 mixed flocks) during the period of 2002–2003 for the occurrence of

mycoplasma infections in Northern Jordan. The clinical signs seen in the studied flocks

were varying degree of mastitis in sheep and goats, arthritis, mainly in kids, and

pneumonia in both sheep and goats of most age groups. Mycoplasmas were isolated from

17 (26 per cent) of the 62 milk samples and 12 (3.9 per cent) of the 310 nasal swab

samples collected from goats and from eight (13 per cent) of the 62 milk samples and

seven (2.3 per cent) of the 310 nasal swabs collected from sheep. Forty four isolates were

identified which included Mycoplasma mycoides subsp. mycoides large colony type

(Mmm LC) (n = 9), Mycoplasma capricolum subsp. capricolum (Mcc) (n = 13),

Mycoplasma putrefaciens (n = 21) and Mycoplasma agalactiae (n = 1). Several isolates

could not be identified.

De la Fe et al. (2007) reported the first isolation of Mycoplasma capricolum

subsp. capricolum, on the island of Lanzarote (Spain) from goats with clinical and

subclinical mastitis and some cases of arthritis and pneumonia. Mycoplasma capricolum

subsp. capricolum was isolated as the main causal agent of the outbreaks, associated with

M. mycoides subsp. mycoides ‘‘large colony type’’ (Mmm LC) in two flocks.

Contreras et al. (2008) performed a cross-sectional study on dairy goat herds to

establish the relationship between the presence of Mycoplasma species in bulk-tank milk

samples from different farms and the bulk-tank milk somatic cell count (BTMSCC) in an

area where contagious agalactia (CA) is endemic. Of the 1068 milk samples tested, 7.9

per cent (n = 84) showed the presence of Mycoplasma spp. (Mycoplasma agalactiae 82

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per cent and Mycoplasma mycoides subsp. mycoides large colony 17 per cent). Somatic

cell counts for bulk-tank samples containing mycoplasmas were higher than those

recorded for negative samples (1,176,000 cells/ml vs. 875,000 cells/ml; P < 0.001).

2.3.3 Isolation of mycoplasma in India

In India, isolation of mycoplasma in sheep and goat has been reported by several

workers. Kumar and Pathak (1978) and Banerjee et al. (1979) reported that M. arginini

was isolated from nasal swabs of sheep with respiratory affections.

Haribabu et al. (1982a) isolated Mycoplasma from pneumonic lungs of sheep

from different farms in Andhra Pradesh and identified them as M. ovipneumoniae and M.

arginini based on growth inhibition test and other biochemical tests.

Gupta et al. (1984) carried out an investigation into an outbreak of contagious

caprine pleuropneumonia (CCPP) with typical symptoms in kids in an organized goat

farm in Bihar. Kids below six months of age were highly susceptible. Out of 91 isolates

from nasal swabs, nasopharynx, trachea, and lungs, 50 were identified by biochemical

and growth inhibition test using specific antiserum. Among them, 17 were M. agalactiae,

22 were M. mycoides subsp. mycoides, two were M. arginini and 11 were M.

ovipneumoniae.

Prasad et al. (1984) recovered mycoplasmas from the mammary glands of

slaughtered goats with mastitis and from milk samples from animals suffering from

mastitis and the isolates were identified as M. arginini.

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Kumar and Chandiramani (1987) isolated three M. agalactiae strains from the

liver and lungs of 30 premature or still born kids. This was the first report of M.

agalactiae from these organs of premature kids.

Sreeramulu and Krishnaswamy (1987) screened 41 nasal swabs and 33 lung

samples collected from ill and dead lambs two to 4 months old suffering from pneumonia

or other respiratory signs in three organized sheep farms and free flocks in Andhra

Pradesh. A total of 30 isolates (18 nasal swabs and 12 lungs) were identified as

mycoplasma of which 21 were M. arginini, five were M. mycoides sub. sp. capri and four

were M. alkalescense.

Konsam et al. (1989) collected nasal swabs from a flock of 183 kids aged 6

months suffering from pneumonia and 37 tracheal swabs and samples of lung tissues

from animals that died. Of the 45 mycoplasma species isolated, 11 (24.4 per cent) were

M. mycoides subsp. mycoides, 9 (20 per cent) were M. agalactiae and three (6.8 per cent)

were M. capricolum.

Mukherji et al. (1990) isolated Mycoplasma mycoides subsp. mycoides (large

colony type) in 16 (9.94 per cent) out of 161 (15.3 per cent) cases of pneumonia in the

goats. The isolates were identified by cultural, biochemical and growth inhibition tests.

Nayak and Bhowmik (1990) made an attempt to screen mycoplasma from 289

cases of septicaemic polyarthirtis in young goats in four geoclimatic zones of West

Bengal. All the secretions and excretions, heart blood and tissues infected (4.42 per cent)

were positive for Mycoplasma isolates which were identified as Mycoplasma mycoides

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subsp. mycoides Large colony type on the basis of cultural, morphological, biochemical

and growth inhibition test.

Rahaman and Singh (1990) examined bronchial exudates for mycoplasma from

100 pneumonic lungs from goats slaughtered in Ludhiana and New Delhi. Mycoplasma

capri was isolated from two samples, Acholeplasma laidlawii from two, and an

unidentified mycoplasma from one. Serogroup 11 mycoplasma were demonstrated in 4

cases by the FAT.

Chaturvedi et al. (1991) examined 175 samples including 32 nasal swabs, 41

tracheal swabs, 102 specimens of lung tissues taken from sheep with respiratory diseases

or slaughtered sheep with evidence of pneumonia. Forty one yielded Mycoplasma and

Acholeplasma spp. The rate of isolation from the three sites was 28.1, 26.8 and 21.5 per

cent respectively.

Panda et al. (1997) collected the cervico-vaginal swabs from sheep and goats with

a history of repeat breeding or cervicitis, vaginitis, pyometra and abortion. Among 97

samples examined bacteriologically, 6 (6.1 per cent) contained mycoplasma and 10 (10.1

per cent) Acholeplasmas. Mycoplasma mycoides subsp. mycoides, M. arginini, M.

capricolum and Acholeplasma ladlawii were identified.

Singh et al. (2004) isolated and characterized an Indian strain of Mycoplasma

mycoides subsp. mycoides LC from a case of caprine arthritis. The isolate was identified

based on biochemical, digitonin sensitivity, growth inhibition tests and PCR.

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Suryanarayana et al. (2009) isolated Mycoplasma from pneumonic sheep in

Karnataka in PPLO broth and characterized it as M. mycoides ssp. mycoides (LC) by

biochemical tests and PCR.

2.4 Serological methods to identify mycoplasma species

Carmichael et al. (1972) conducted serological tests like complement fixation test,

growth inhibition test (GI) and metabolic inhibition (MI) test and biochemical tests like

glucose fermentation, arginine utilization and tetrazolium hydrochloride reduction to

identify and characterize ovine mycoplasma.

In Sudan, Mahi and Nayil (1978) used growth inhibition and metabolic inhibition

tests for identification of mycoplasma from pneumonic sheep lungs and found that all

strains except one failed to react with antisera to M. mycoides var capri, M. mycoides var

mycoides, while isolates in the group reacted strongly with antisera to M. arginini.

Sikdar (1979) applied disc growth inhibition, IHA, CF, counter

immunoelectrophoresis (CIE) and Epi-immunofluorescence test for the detection of

different mycoplasma isolates and confirmed that the disc growth inhibition was simplest

and sensitive. Quick results were observed with CIE while the epi-immunofluorescence

was found to be highly specific for quick identification of unknown isolates.

Srinivas (1987) observed that the double immunodiffusion, CIE, IEP and IHA

tests may be used for identification of various fractioned antigens of M. capri. He further

reported about the ability of antigens for the diagnosis of antibodies in disease.

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Thaker (1987) used IHA, CF, CIE, tube agglutination and imunodiffusion tests

with hyperimmune serum prepared against various fractions of M. capri in rabbits for the

detection of antibodies. He found better results while using antiserum against whole cell

in GI, CIE, immunodiffusion and CF tests, while the antisera against whole cell and

membrane antigens were found to be equally good in IHA and tube agglutination.

2.5 Polyacrylamide gel electrophoresis

Razin (1968) studied the taxonomy of mycoplasma by electrophoresis of cell

proteins. He recorded that the polyacrylamide gel electrophoretic patterns of the cell

proteins of five M. hominis strains showed difference corresponding with their known

serological and nucleic acid heterogenicity. The patterns of three M. mycoides var

mycoides strains isolated in different countries were identical. The electrophoretic

patterns of several caprine strains resembled those of M. mycoides var capri.

Zola et al. (1970) studied the polyacrylamide gel electrophoresis of lysates of

mycoplasmas and reported that different strains of M. gallisepticum, M. pneumonis, M.

ladlawii, Acholeplasma granulosum, A. neurolyticum and bovine group, could be

distinguished by means of a technique of electrophoresis on flat gel slab though the

patterns of M. granulosum and M. ladlawii were similar.

Sodium dodecyl sulphate (SDS) dissociated proteins into their constituent

polypeptide chains. Polyacrylamide gel electrophoresis in the presence of SDS separated

polypeptide chains according to their molecular weights. Thus the size of the polypeptide

chains of a given particle could be determined by comparing their electrophoretic

mobilities on SDS gel to the motilities of marker proteins with well characterized

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polypeptide chain molecular weights. Weber et al. (1972) measured molecular weight by

electrophoresis with SDS-acrylamide gel and found that this technique was both reliable

and reproducible and the results were easy to interpret.

Paroz et al. (1977) studied mycoplasmas and Acholeplasma from cattle, sheep,

goats and pigs by electrophoresis in SDS-polyacrylamide gel with the help of the slab

technique. This method was used for identification of 47 isolates of M. ovipneumoniae

and three isolates of M. arginini.

Haribabu et al. (1982b) reported that SDS-PAGE of M. ovipneumoniae yielded

three to six bands. Five isolates showed similar patterns of bands when compared with

the known strain of M. ovipneumoniae.

Costas et al. (1987) characterized twenty six isolates belonging to the

Mycoplasma mycoides cluster by one dimensional SDS-PAGE of their cellular proteins.

It is concluded that high resolution SDS-PAGE combined with computerized analysis of

protein patterns is effective for the investigation of taxonomic relationship within this

group of mycoplasma.

Leach et al. (1989) classified and compared twenty five stains of M. mycoides

subsp. mycoides LC or subsp. capri by one dimensional SDS PAGE of their proteins. The

results confirmed earlier studies indicating the protein pattern inseparability of subsp.

capri and subsp. mycoides LC strains and their distinctiveness from the classical M.

mycoides subsp. A.B.C strains and other members of M. mycoides cluster.

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De la Fe et al. (2006) characterized protein and antigen variability among

Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by

SDS-PAGE and immunoblotting. A total of 25 field strains from Spain and the two type

strains were analysed by SDS PAGE and immunoblotting. Two polyclonal antisera

(PAbs) raised against a pool of strains of each mycoplasma species were used. The

results revealed a high degree of protein variability among the field strains. The type

strain of Mmm LC appeared to be representative of the field strains of this species,

whereas this was not the case with the M. agalactiae type strain.

Amit et al. (2010 b) carried out polyacrylamide gel electrophoresis of two Indian

isolates of M. agalactiae (RPNS 200 and RPNS 216) and one isolate of M. bovis

(NC317) using two types of antigens viz. whole cell antigens (WCA) and sonicated

supernatant antigen (SSA) under non-reducing and non-denaturating conditions. The

results revealed 5 to 9 polypeptides in the region of 160.32 to 19.95 kDa. Protein profiles

of WCA of M. agalactiae and M. bovis were almost similar and revealed 5 polypeptides

namely, 160.32, 107.15, 97.70, 44.66 and 28.84 kDa. The SSA of both the species has the

difference in polypeptides, as the bands of 70.70, 33.88 and 20.89 kDa were present only

in the isolates of M. agalactiae, while the polypeptides of 20.50 and 19.95 were present

in M. bovis. They opined that polypeptides could be used for species identification and

differentiation.

Kathiresan et al. (2010) characterized the surface lipoprotein P48 in Indian

isolates of Mycoplasma agalactiae, which has been reported to play an important role in

immune response of infected animals and has also been designated as one of the most

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potent candidates for the diagnosis of contagious agalactia. SDS-PAGE analysis of the

four Indian isolates along with the standard strain of M. agalactiae revealed a major band

of 48 kDa. Subsequently, immunoblot assay using polyclonal serum against standard

strain of M. agalactiae showed that 48 kDa protein was the major immunodominant

protein. Further, immunoblot analysis with monospecific serum against recombinant P48

confirmed the presence of P48 in all the Indian isolates.

2.6 Molecular methods for identification of mycoplasmas

Tola et al. (1994) devised a species-specific DNA probe for the detection of

Mycoplasma agalactiae. A 4.5 kb M. agalactiae DNA fragment present in strains from

different areas of Sardinia was cloned and used to detect M. agalactiae DNA in sheep

milk samples by dot blot hybridization. The probe recognized only M. agalactiae strains

and did not cross-hybridize with other mycoplasmas (M. capricolum, M. mycoides subsp,

capri, M. mycoides subsp, mycoides, M. putrefaciens and M. arginini) or bacteria ( E.

coli, P. hemolytica, S. aureus, S. epidermis, L casei, S. durans, S. lactis and S.

thermophilus) which might be found in sheep milk.

Laurence et al. (1995) reported application of PCR for detection of mycoplasmas

causing contagious agalactia, M. agalactiae, M. capricolum subsp. capricolum and M.

mycoides spp. mycoides LC. It was based on two polymerase chain reaction assays: the

Ma-PCR for the detection of M. agalactiae and the MYC-PCR for ‘mycoides cluster’,

including M. capricolum subsp. capricolum and M. mycoides spp. mycoides LC. An M.

agalactiae strain was identified by a 933-bp Ma-PCR product and no amplification with

the MYC-PCR. In contrast, a 460-bp MYC_PCR product and a negative or a 350-bp Ma-

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PCR product characterized a ‘mycoides cluster’ strain. M. capricolum subsp. capricolum

and M. mycoides spp. mycoides LC were identified by their species-specifc Asel pattern

of the 456- bp MYC-PCR product.

Hotzel et al. (1996) devised a PCR scheme for differentiation of organisms

belonging to the Mycoplasma mycoides cluster. A set of primer combinations derived

from the CAP-21 genomic regions of member organisms of the Mycoplasma mycoides

cluster was selected by means of which complete differentiation within the cluster could

be accomplished. Nested PCR involving cluster-specific amplification at the first stage

and group-specific amplification using internal primers at the second stage was shown to

be applicable for identification of all six groups forming the cluster. For example,

external primers Pl/P2 and internal primers P6/P7 were used to distinguish M. mycoides

subsp. mycoides SC from LC strains.

Tola et al. (1996) developed a polymerase chain reaction (PCR)-based test for the

detection of Mycoplasma agalactiae in sheep milk samples. Two oligonucleotide primers

were designed to amplify a 375 bp fragment of M. agalactiae chromosomal DNA.

Amplified products were analyzed by agarose gel electrophoresis and Southern blot

hybridization using a fluorescein labeled 528 bp probe. The primers allowed the

amplification of a fragment of M. agalactiae DNA and did not amplify any specific

fragment of other mycoplasmal DNAs.

Tola et al. (1997) developed a simple and rapid method for DNA extraction from

sheep milk to be used for polymerase chain reaction (PCR) diagnosis of Mycoplasma

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agalactiae. PCR results were compared with those of conventional culture and found that

PCR was more specific and sensitive compared to cultural techniques.

Greco et al. (2001) optimized a multiplex polymerase chain reaction (m-PCR)

assay for the simultaneous detection of several species of small ruminant mycoplasmas.

Two sets of oligonucleotide primers specific for Mycoplasma agalactiae (Ma) and

Mycoplasma “mycoides” cluster (M.m. cluster) were used in the test. The m-PCR was

able to amplify a 375-bp fragment of Ma chromosomal DNA and a 257-260-bp fragment

of M.m. cluster chromosomal DNA.

Benedicte et al. (2001) cloned the gene for a 30-kDa immunodominant

transmembrane protein antigen, P30, of Mycoplasma agalactiae from type strain PG2

and expressed it in Escherichia coli. Immunoblot analysis using the monospecific

polyclonal anti-P30-HIS serum indicated that P30 is specific to M. agalactiae.

Furthermore, PCR amplification with specific primers for P30 and Southern blot analysis

revealed the presence of the gene in all M. agalactiae strains tested and its absence in the

other mycoplasma species.

Bashiruddin et al. (2005) evaluated PCR systems for the identification and

differentiation of Mycoplasma agalactiae and Mycoplasma bovis. Specificity of four

different PCR systems for M. agalactiae and three systems for M. bovis on a total of 41

strains of the two Mycoplasma species was studied. As the vast majority of PCR

examinations (97.1 per cent of all tests) correctly identified the strains the specificity of

all seven detection systems appears to be high.

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McAuliffe et al. (2005) developed a new diagnostic test based on PCR of the 16S

rRNA gene with Mycoplasma-specific primers and separation of the PCR product

according to primary sequence using denaturing gradient gel electrophoresis (DGGE).

DGGE enabled the differentiation of 67 Mycoplasma species of human and veterinary

origin and represented a significant improvement on current tests as diagnosis of

Mycoplasma infection could be made directly from clinical samples in less than 24 h.

Mahdavi et al. (2009) carried out a comparative study of homology of

cytoplasmic membrane protein 40 KDa of Mycoplasma agalactiae in isolated strains in

Iran. Cytoplasmic membrane Protein 40 KDa (P40) in this bacterium is a strong

immunogenic protein, major virulence factor and is specific for M. agalactiae. The P40

gene of isolated M. agalactiae strains was amplified with PCR technique and for further

studies this fragment was cloned and sequenced. In comparative study of P40 gene

sequence of isolated strains in Iran with other strains in gene bank with the use of BLAST

program showed the homology (Identities 100 per cent) of Lorestan strain with French

strains 4258, 4021 and Spanish strain PG2 and as for Taleghan and Shiraz strains the

most homology (Identities 99 per cent) was achieved with French Strains 4258, 4021 and

Spanish strains PG2, 6968, 5225 and Swiss strain 7375 and Italian strain 9.

Oravcova et al. (2009) evaluated the capacity of the Mycoplasma agalactiae p40

gene as a diagnostic marker for contagious agalactia in sheep by quantitative real-time

PCR. They evaluated the capacity of the assay to detect Mycoplasma agalactiae in 797

milk samples (373 raw sheep milk samples from refrigerated tanks of different farms and

424 milk samples from individual sheep of a flock positive for M. agalactiae). While the

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assay was able to detect 57 (15.28 per cent) positive samples of the 373 milk samples

from different farms, identification by microbiological isolation coupled with microscopy

detected only 36 (9.65 per cent) samples, and the conventional PCR detected 31 (8.31 per

cent) samples.

Shahram et al. (2009) suggested the reassignment of Mycoplasma mycoides

subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri based

on analysis using the polymerase chain reaction(PCR), restriction enzyme endonuclease

analysis(REA), protein profile patterns, random amplification of polymorphic

DNA(RAPD) fingerprinting, 16SrRNA gene sequencing and antisera growth inhibition

tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony

type(MmmLC) and eight strains of M. mycoides subsp. capri (Mmc).

2.7 Studies on seroprevalence studies of mycoplasma

Levisohn et al. (1991) used an ELISA specific for M. agalactiae and MmmLC for

differential diagnosis of these infections in naturally infected goat herds. The specificity

of the antigens was demonstrated by immunoblotting and by ELISA using monospecific

hyperimmune rabbit sera. Results indicated the ability to detect subclinical mycoplasma

infection and individual carrier goats on the basis of ELISA, a finding which will assist

control procedures.

Rosati et al. (2000) analyzed recombinant P48, a major surface lipoprotein of M.

agalactiae which plays an important role in the immune response of infected animals.

P48 expressed in E. coli was used in western blot and indirect ELISA using well-

characterized sheep sera. They demonstrated that specific antibodies against P48 were

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detected 3 weeks after onset of clinical disease and the recombinant P48 was a

diagnostically relevant marker of M. agalactiae infection.

Zendulkova et al. (2004) used monoclonal antibody- based sandwich ELISA to

detect Mycoplasma agalactiae antigen in sheep and goats. A total of 99 animals were

examined. Biological materials for examination included 353 swabs (133 from sheep and

220 from goats) collected from conjunctival, nasal and vaginal mucosae and the external

ear canal. The results were positive in 11 animals, ambiguous in 10 animals and negative

in the rest of them (78).

Fusco et al. (2007) developed a new recombinant ELISA for sero-diagnosis of

contagious agalactia (CA) based on two M. agalactiae surface proteins, namely, P80 and

P55. Identification of these immunodominant and common antigens was accomplished

by examining the antibody response elicited in sheep during experimental infection and

comparing it to the protein expression profiles of 75 M. agalactiae field strains.

Al-Momani et al. (2008) carried out serological detection of M. agalactiae in

104 small ruminants flocks consisting of 18 sheep, 27 goats and 59 flocks containing

both sheep and goats in northern Jordan between 2002 and 2003 using an indirect ELISA.

To increase the chances of detecting this mycoplasma, sick or older animals were

sampled. A high seropositivity to M. agalactiae was found in small ruminants suggesting

a major role for M. agalactiae in contagious agalactia in northern Jordan. There was no

significant difference in the seroprevalence of M. agalactiae in sheep and goats at flock

level.

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Ingle et al. (2008) screened sheep for Contagious Caprine Pleuro Pneumonia

antibodies by slide agglutination test using colored CCPP antigen. Out of the 294 serum

samples screened, 99 were positive indicating an overall seroprevalence of 33.67 per

cent. Slide agglutination test for CCPP detection using colored antigen was found to be

quick, simple, and of low cost with ease of application in the field without the need for

any specialized training and equipments.

Jagdev et al. (2008) recorded the seroprevalence of Mycoplasma in sheep and

goats of Himachal Pradesh based on agglutination test using Mycoplasma mycoides

subsp. capri colored antigen. Out of the 314 serum samples screened, 15 (4.77 per cent)

were found positive for mycoplasmosis. The prevalence was found to be slightly higher

in goats (5.02 per cent) compared to sheep (4.44 per cent). The seroprevalence of

mycoplasmosis was the highest in Kangra district (11.59 per cent) followed by Mandi

district (9.67 per cent) and Hamirpur district (2.17 per cent). The high seroprevalence of

mycoplasmosis in Kangra district may be attributed to the migratory flocks as the district

falls along the route of migration followed by the nomadic tribes.

Dahiya et al. (2009) compared capture-ELISA with cultural isolation for detection

of Mycoplasma mycoides subsp. mycoides (LC type, M-30) in experimentally infected

lambs. The organism could be recovered from 65.55 per cent of the specimens examined

for cultural reisolation. Capture-ELISA detected MmmLC antigens in 73.33 per cent of

the examined tissues. Comparison of results of both the techniques revealed a 90 per cent

agreement and 10 per cent disagreement. It was concluded from the present studies that

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capture-ELISA was a more sensitive technique than reisolation of organism for detection

of MmmLC in the tissues of the animals.

2.8 Pathology of field cases of mycoplasmosis

St. George et al. (1971) described the proliferation of septal cells, leucocytic

infiltration generally restricted to a few lymphocytes in the alveolar septa and isolated

foci of neutrophils in consolidated areas in pneumonic lungs of sheep suspected of

mycoplasmosis. Lymphoid proliferation was not a prominent feature. The microscopic

lesions in lungs of experimental lambs were indistinguishable from the cases of field

outbreaks.

Lesions such as hyperplasia of alveolar and bronchial epithelium, hyperplasia of

peribronchial follicles, desquamation of alveolar epithelial cells and proliferative

interstitial pneumonia were frequently reported in mycoplasmosis in sheep (Stipkovits et

al., 1975; Nicolet et al., 1979; Bolske et al., 1982).

Moustaff et al. (1977) studied the histopathology of 52 lung pieces suspected for

mycoplasmal pneumonia, 22 of which yielded M. arginini and 4 glucose fermenting

mycoplasma. The main pathological changes observed microscopically were proliferative

interstitial pneumonia with excessive lymphoid hyperplasia. Thickening of the alveolar

wall, lymphoid infiltration in the peribronchiolar and perivascular tissue, proliferation

and desquamation of bronchial and bronchiolar epithelium were noted. It was concluded

that mycoplasma infection in lungs of sheep cause proliferative interstitial pneumonia

with lymphoid hyperplasia.

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Jones et al. (1979) reported that on histopathological examination of lungs from

34 lambs, 15 showed proliferate exudative pneumonia and 11 showed lymphoid

hyperplasia sometime associated with interstitial pneumonia. In 8 samples no significant

pathological changes could be observed.

Haribabu et al. (1982b) reported the pathological changes in lungs of sheep from

which mycoplasma were isolated. The gross lesions in lungs were reddish grey to purple

consolidations involving whole of apical and cardiac lobes of both the lungs and whole of

mediastinal lobe on right side in all animals. The antero-ventral portions of diaphragmatic

lobes were also involved in three sheep. The alveoli were filled with macrophages,

lymphocytes and a few neutrophils. There was infiltration of round cells in the interstitial

tissue thereby increasing its thickness. Peribronchial, peribronchiolar and perivascular

lymphoid aggregations were observed in all the cases. The bronchial and bronchiolar

exudates consisted of considerable number of degenerative neutrophils, desquamated

epithelial cells and cellular debris. In two cases, there was focal cornification, while in a

few there was focal subpleural fibrosis. Areas of collapse were also noted.

Damass and Porter (1987) observed serosal petechiation, excess of pericardial,

peritoneal, pleural and fibrinous synovial fluid, moderate pulmonary hyperplasia and

enlarged liver and spleen in four lambs of three weeks age which had developed clinical

symptoms like pneumonia, pyrexia, and lameness with enlarged, painful joints.

Mycoplasma capricolum or a related mycoplasma was isolated from most joints.

Kinde et al. (1994) investigated a commercial dairy goat herd of 600 animals

which experienced sudden onset of arthritis/polyarthritis, clinical mastitis, and sudden

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death in does. The offending infectious agents were found to be Mycoplasma agalactiae

and M. mycoides subsp. mycoides (caprine biotype). Gross necropsy of the does affected

with M. mycoides subsp. mycoides showed purulent discharges from the udders, enlarged

supramammary lymph nodes, enlarged and firm spleens and swollen livers. Microscopic

findings were characterized by a loss of vascular integrity and diffuse fluid leakage in

multiple organs.

Rodriguez et al. (1996) isolated M. agalactiae from the conjunctival sac of 20

adult sheep and three lambs in a herd of 200 Spanish sheep where 20 per cent of the

animals developed unilateral or bilateral keratoconjunctivitis affecting young and adult

animals. The cornea showed an erosive to ulcerative keratitis diagnosed either clinically

or histologically. Histologically, an abundant infiltration of neutrophils, determined areas

of liquefactive necrosis. Neovascularization was also noticed in superficial and deep

areas of the cornea. These vessels were associated with the migration of neutrophils and

mononuclear cells, inducing an acute to subacute keratitis. At the ultrastructural level,

using SEM and TEM, mycoplasma were detected either in neutrophils or free on the

surface of the cornea.

Bergonier et al. (1997) reported that sheep affected by contagious agalactia

caused by M. agalactiae showed elevated temperature, inappetance and alterations in the

consistency of the milk in lactating ewes with decline and subsequent failure of milk

production, often within two to three days as a result of interstitial mastitis. Lameness and

keratoconjunctivitis were seen in about five to ten percent of infected animals. Fever was

common in acute cases and was accompanied by nervous signs, but both signs were rare

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in the more frequently observed sub-acute and chronic infections. Pregnant animals may

abort. M. agalactiae may occasionally be found in lung lesions, but pneumonia is not a

consistent finding.

Sanchis et al. (2000) reported that sheep affected by contagious agalactia caused

by M. agalactiae suffered from mastitis and or agalactia, keratoconjunctivitis and

arthritis.

Madanat et al. (2001) concluded that contagious agalactia was usually manifested

as mastitis in lactating females. Males, young animals and non-lactating females suffered

from arthritis, keratoconjunctivitis and respiratory problems.

Janovsky et al. (2002) observed that infectious keratoconjunctivitis caused by

Mycoplasma conjunctivae was a highly contagious ocular infection and was characterized

by inflammation of the conjunctiva and cornea, and in the most advanced stages, the

cornea was opaque or even perforated.

Gil et al. (2003) reported genital lesions in adult male and female goats from a

commercial flock in the Extremadura region of southwestern Spain, following an

outbreak of contagious agalactia syndrome caused by Mycoplasma agalactiae and M.

putrefaciens. Although both species were isolated from several organs, M. putrefaciens

was the only agent isolated from the genital lesions reported here, characterized by

desquamative salpingitis and cystic catarrhal metritis in females and by testicular

degeneration in males. Mycoplasma putrefaciens was isolated from the testes of only one

of the males examined.

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Mondal et al. (2004) studied the clinico-haematology and pathology of caprine

mycoplasmal pneumonia in rain fed tropics of West Bengal. Three hundred and ten

isolates were obtained from 4500 nasal swabs, and were identified as Mycoplasma

agalactiae (Ma, 102), Mycoplasma mycoides subsp. capri (Mmc, 124) and M. mycoides

subsp. mycoides LC (Mmm, 84) by biochemical tests and GIT. The clinical

manifestations were rise of temperature (40–43 ◦C), marked depression, tremor,

reluctance to move, painful accelerated respiration, cough, partial to complete closure of

nostril, nasal discharge, anorexia, low tone bleating, recumbency and few cases of

arthritis and corneal opacity. Haematologically, the animals were found anaemic, with

leucocytosis followed by leucopenia. Pathomophological study of dead animals showed

congested trachea with the presence of frothy exudates and some cases showed chronic

tracheitis. Pneumonia with unilateral or bilateral involvement of the lungs was

characteristic. Histopathologically trachea was erosive, edematous and haemorrhagic.

The lung blood vessels were haemorrhagic, necrosis of lining cells, infiltration with

neutrophils, lymphocytes and macrophages were also observed along with thrombosis

and emphysema.

Adehan et al. (2006) recovered fifty Mycoplasma strains from pneumonic lungs

of sheep and goats from Cotonou abattoirs. In this study, histopathological findings

suggestive of CCPP, characterized by oedema mixed with fibrin fluid and inflammatory

cells, mainly polymorphonuclear neutrophils and lymphocytes in the alveolar spaces and

interstitial septae, were encountered in the pneumonic lungs of sheep and goats.

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Azevedo et al. (2006) reported mastitis, agalactia and polyarthritis in does and

polyarthritis and conjunctivitis in kids and lambs during two outbreaks of contagious

agalactia by Mycoplasma agalactiae in Paraiba state, northeastern region of Brazil.

Morbidity ranged from 26.1 per cent to 100 per cent in does, 36.5 per cent to 100 per

cent in kids and 49 per cent in lambs.

Corrales et al. (2007) stated that contagious agalactia is an infectious syndrome

caused by several species of mycoplasma and classically characterized by the triad of

mammary, joint and eye symptoms, although further symptoms may also appear.

Zendulkova et al. (2007) stated that contagious agalactia is a highly infectious

disease of sheep, caused by Mycoplasma agalactiae. It is characterised by arthritis,

keratoconjunctivitis, pneumonia and in females by mastitis and occasional abortion.

De la Fe et al. (2009) studied the effects of the presence of Mycoplasma spp. on

goat milk quality in herds without symptoms of contagious agalactia in 26 herds on the

island of Lanzarote (Spain), where CA was endemic. Five hundred and seventy

individual milk samples and 266 bulk tank milk (BTM) samples were microbiologically

analysed for the presence of Mycoplasma spp. and infection was confirmed in 13 herds.

A total of 31, 10 and 11 strains of Mycoplasma mycoides subsp. mycoides LC (MmmLC),

M. agalactiae and M. capricolum subsp. capricolum were isolated.

Rodriguez et al. (2010) investigated a goat pleuropneumonia outbreak in a herd in

the province of Ciudad Real, Spain. Severe respiratory signs and high mortality were the

most significant clinical observations. The adult goats presented mainly respiratory

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symptoms and/or mastitis, whereas the young animals died showing arthritis and/or

keratoconjunctivitis. A focal extensive fibrino-necrotic pleuropneumonia was

macroscopically seen, and the histopathological analysis confirmed a fibrinopurulent and

necrotic pleuropneumonia with areas of acute pyogenic bronchopneumonia and fibrinous

pericarditis associated with a multifocal purulent mastitis and/or a fibrinopurulent

arthritis in some goats. Microbiologically, the mycoplasmas isolated grew rapidly (18–24

h), and, after 48 h, there were colonies of 1–1.5 mm diameter. These isolations were

biochemically characterized as Mycoplasma mycoides spp. and showed serological

characteristics corresponding to Mycoplasma mycoides subsp. mycoides Large Colony.

As per OIE Terrestrial Manual, 2008, contagious agalactia is a serious disease

syndrome of sheep and goats that is characterized by mastitis, arthritis,

keratoconjunctivitis and, occasionally, abortion. Mycoplasma agalactiae is the main

cause of the disease in sheep and goats, but M. capricolum subsp. capricolum, M.

mycoides subsp. capri and M. putrefaciens produce clinically similar disease, more often

in goats, which may be accompanied by pneumonia.

2.9 Experimental induction of mycoplasmosis using small ruminant

mycoplasmas

2.9.1 Clinical symptoms

Ojo (1976) experimentally infected goats with M. mycoides subsp. mycoides and

M. mycoides subsp. capri by the endobronchial route. The infected animals were dull,

unable to eat and had a high temperature. The mean mortality rate for all the infected

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animals was 74 per cent, the highest mortality being 100 per cent for animals infected

with the Smith strain of M. capri.

Macowan et al. (1984) compared the infection with an ovine field isolate of

Mycoplasma agalactiae in castrated lambs following inoculation by different routes. The

prolonged infections produced were symptomless apart from limited arthritis in one

animal inoculated with the isolate from sheep and increased lacrymation in another

associated with the goat isolate.

Guha and Verma (1987 a) infected kids intratracheally with Mycoplasma

mycoides subsp. mycoides isolated from kids with CCPP and observed clinical signs

characterized by respiratory symptoms, nasal discharge, coughing, dullness and

depression.

Guha and Verma (1987 b) observed fever, anorexia, occasional coughing,

sneezing and dyspnoea in kids inoculated with 24 hours old culture of M. agalactiae

intramuscularly.

DaMassa et al. (1992) inoculated a mycoplasma designated strain GM790A,

isolated from milk and internal organs of two lactating goats that showed mastitis and

arthritis, into the teat canal of two lactating goats. It resulted in an abrupt diminution of

lactation leading to mastitis and agalactia in about three days. Milk production partially

resumed at a low level three weeks pi, but the milk still contained 1 x 102 CFU of the

agent.

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Rana et al. (1992) experimentally induced caprine mastitis with mycoplasma

ovine/caprine serogroup 11 through the teat canal. The infected glands were hot, tender,

painful and swollen from the first day pi. The mastitic milk showed characteristic

physical changes and marked increase in somatic cell count. Subsequently, there was

reduction in gland size and agalactia.

Hasso et al. (1993) studied the severity of contagious agalactia in goats as related

to the route of infection and pregnancy following inoculation of a local isolate of

Mycoplasma agalactiae, by four different routes of inoculation (intravenous,

intramammary, subcutaneous and oral) each. Major clinical signs throughout the

experiment were mastitis, arthritis, and conjunctivitis. Clinical signs were most obvious

in the subcutaneously inoculated group which also yielded the highest number of M.

agalactiae recoveries before and after sacrifice, followed by the intramammary,

intravenous and lastly the orally inoculated group. Pregnant animals were affected more

severely than non-pregnant animals.

Munish et al. (2002) reported the clinicohematological parameters in

experimental Mycoplasma mycoides subsp. mycoides (LC) pneumonia in lambs by

respiratory route. Clinical signs constituted rise in temperature, nasal and ocular

discharge, dullness, depression, coughing, sneezing, laboured breathing and arching of

back.

Munish et al. (2003) observed the clinicohematological parameters in

experimental pneumonia due to concurrent infection with Mycoplasma mycoides ssp

mycoides (LC) and Pasteurella hemolytica infection in lambs. High rise in temperature,

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nasal and ocular discharge, dullness, depression, coughing, sneezing, laboured breathing,

arching of back, exhaustion, loss of muscular and eye reflexes, submandibular edema and

blood tinged nasal discharge in terminal stages of disease were the manifestation.

Singh et al. (2004) studied the virulence of an Indian strain of Mycoplasma

mycoides subsp. mycoides LC isolated from a case of caprine arthritis by pathogenicity

test in mice and goat. The inoculated kid became off-fed by third-day pi with serous

nasal discharge, which later on turned muco-purulent. There was elevated body

temperature with cough and respiratory distress and the animal succumbed by the seventh

day.

Di Provvido et al. (2009) experimentally infected goats with a mycoplasma

(isolated previously from a goat with contagious agalactia in northern Jordan, by either

intratracheal or by aerosol route and by placing “in-contact” with other goats. After two

weeks, those infected intratracheally became febrile, showing a nasal discharge and slight

conjunctivitis, followed a week later by respiratory distress and polyarthritis. Lesions

seen at necropsy included coagulative necrotic pneumonia, fibrinous pleurisy with pleural

exudate, and inflammatory exudates, necrosis and fibrosis in the joints. Animals infected

by aerosol showed much milder clinical signs, including nasal discharge and occasional

swollen joints. In the “in-contact” goats, seroconversion was first seen after seven weeks,

accompanied by coughing and laboured respiration. Lesions in this group consisted of

fibrinous pneumonia with focal areas of necrosis and abundant pleural exudate.

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2.9.2 Haematology

Guha and Verma (1987a) reported an elevated neutrophil count in kids infected

intratracheally with Mycoplasma mycoides subsp. mycoides. There was little change in

hemoglobin level, TLC and body weight.

Nayak and Bhowmik (1990) conducted experimental transmission of Mycoplasma

mycoides subsp. mycoides (large colony type) to weaned Black Bengal goat kids by

placing infected fleas on each kid's body surfaces. The kids developed characteristic

clinical signs and showed leucopenia with neutropenia, an increased amount of

fibrinogen and mortality with lesions of suppurative polyarthritis associated with

septicaemia.

Rana et al. (1992) experimentally induced caprine mastitis with mycoplasma

ovine/caprine serogroup 11 through the teat canal. Haematological analysis revealed

marked leucocytosis with neutrophilia in initial stages and lymphocytosis in later stages.

Gutierrez et al. (1999) conducted a clinico-pathological and haematological study

in kids experimentally infected simultaneously with Mycoplasma mycoides subsp. capri

and Mycoplasma mycoides subsp. mycoides (large colony-type). The experimental

infection showed an acute septicaemic clinico-pathological picture with lethal outcome.

The haematological data were as follows: lymphocytic and neutropenic leukopenia, an

increase in prothrombin time and activated partial thromboplastin time, a decrease in

Antithrombin III, fibrinogen and thrombocytopenia.

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Munish et al. (2002) studied the clinicohematological parameters in experimental

Mycoplasma mycoides subsp. mycoides (LC) pneumonia in lambs by respiratory route.

Hb, PCV and TEC values declined. A significant neutrophilia along with

lymphocytopenia was also noticed.

Munish et al. (2003) studied the clinicohematological parameters in experimental

pneumonia due to concurrent infection with Mycoplasma mycoides ssp mycoides (LC)

and Pasteurella hemolytica infection in lambs. A significant neutrophilia along with

lymphocytopenia upto 10 dpi was also observed. Hb, PCV and TEC values were in

normal range.

2.9.3 Gross and histopathology

Watson et al. (1968) inoculated sheep and goats with 6 type N, 5 type A and 2

type C mycoplasma strains. The results indicated that type N strains of mycoplasma were

non-pathogenic when inoculated subcutaneously into goats. Type A strains produced the

syndrome of contagious agalactia in sheep and goats as observed in Turkey, but without

the keratitis. The characteristic lesion produced by these strains was a subcutaneous

oedema and necrosis at the site of injection.

Lloyd (1970) injected Mycoplasma mycoides subcutaneously into rabbits, and

produced an oedematous lesion heavily infiltrated with leucocytes and perivascular

haemorrhage.

Piercy (1970) studied the reaction in joints after intra articular inoculation of a

killed suspension of Mycoplasma mycoides var. mycoides in specifically sensitized and

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non-sensitized calves. When the histopathological features of joint lesions in each group

of calves were compared, it showed that an immediate-type allergic reaction had occurred

in the joint capsule of sensitized calves was obtained. Persistence of fibrin deposits and

progression to a chronic synovitis in the joint capsule of sensitized calves suggested that

the allergic reaction delayed resolution of the inflammatory reaction induced by the killed

M. mycoides.

Ojo (1976) experimentally infected goats with M. mycoides subsp. mycoides and

M. mycoides subsp. capri by the endobronchial route. Gross lesions were confined to the

lungs, pleura and pericardium. Histologically the important lesions were congested

alveolar septae, acute pyogenic bronchopneumonia and acute purulent pleurisy.

Sreeramulu et al. (1987) made an experimental study with Mycoplasma arginini,

M. alkalescenes and M. mycoides subsp. capri in five month old lambs. Histological

examination of tissues from natural and experimental pneumonic cases revealed

aggregates of the leucocytes around the glands and blood vessels of the lungs and

trachea. In experimental infection the changes in the lungs were mainly of interstitial

pneumonia, whereas in natural or field pneumonic cases, fibrinous and suppurative

changes were predominant.

Rana et al. (1992) experimentally induced caprine mastitis with mycoplasma

ovine/caprine serogroup 11 through the teat canal. Histopathologic examination indicated

that the acute phase of the induced mastitis was characterised by vacuolation and

degeneration of secretory epithelium with a marked neutrophil and macrophage response.

Subsequently, the chronic interstitial mastitis was characterized by lymphocytic

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infiltration, chronic galactophoritis and extensive fibrosis leading to reduction in

glandular parenchyma which caused agalactia in the infected glands.

Wesonga et al. (1993) studied the relationship between clinical signs and early

lesions of experimental CCPP caused by Mycoplasma strain F38 using intratracheal and

endobronchial route. Lung lesions were characterized by hyperaemia and edema, and

mild clinical signs were characterized by an occasional cough. Goats sacrificed after

fever showed lung consolidation characterized by firmness. The area of consolidation

increased in diameter from day 1 up to day 5 of fever.

Hasso et al. (1994) produced contagious agalactia experimentally in four groups

of goats using a local isolate of Mycoplasma agalactiae and employing four different

routes of inoculation (intravenous, intramammary, subcutaneous and oral).

Pathologically, there was acute to chronic mastitis in the intramammary and

subcutaneously inoculated groups; acute synovitis in the intravenously inoculated group

and to a lesser extent in the intramammary inoculated group; and subacute enteritis in the

orally inoculated group. No changes were detected in the eyes. It was concluded that the

used M. agalactiae isolate could cause synovitis and severe mastitis, the impacts of

which on the general health and productivity of the animals were evident.

Martrenchar et al. (1995) reported death in two goats with pneumonic, arthritic

and mastitic symptoms following experimental inoculation with MmmLC. Necropsy

observations of infected animals included areas of pulmonary hepatization with

macroscopic thickening of the interlobular septa in one goat and invasion of the lung by

numerous small abscesses with no macroscopic thickening of the interlobular septa in the

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other animal. In both cases, white fibrinous plaques were present in the thoracic cavity

but no pleural fluid was observed.

Rodriguez et al. (1996) conducted a pathological and immunohistochemical study

of caprine pleuropneumonia induced by subspecies of Mycoplasma mycoides.

Mycoplasma mycoides subsp. mycoides (Large Colony) (MmmLC), Mycoplasma

mycoides subsp. capri (Mmc), and Pasteurella multocida. Gross and microscopical

lesions were typical of caprine pleuropneumonia (CPP), with bronchopneumonia,

fibrinopurulent or fibrinonecrotic pleuropneumonia and dilatation of the interlobular

septa and pleura.

Darzi (1998) induced clinical mastitis in lactating goats by intracisternal

inoculation with Mycoplasma capricolum subsp. capripneumoniae (Mcc).

Histopathology revealed that the mastitis was acute and purulent initially, followed by

infiltration of lymphonuclear cells and fibroplasia in the interacinar tissue, and later by

massive fibrosis.

Rodriguez et al. (2000) induced lung lesions using Mycoplasma agalactiae and

Mycoplasma bovis in goats by a combined (intratracheal + intranasal) route. Both

Mycoplasma spp. induced moderate bronchointerstitial pneumonia, characterized by

lymphoid hyperplasia of the BALT and infiltration of mononuclear cells into the alveolar

walls. The predominant phagocytic cell in the pulmonary parenchyma and the airways

was the macrophage. The main cellular type in the BALT was the CD3+T lymphocyte,

and the ratio of CD4+: CD8+cells was >1.

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2.9.4 Immunohistochemical demonstration of mycoplasma antigens

Rodriguez et al. (1996) conducted a pathological and immunohistochemical study

of caprine pleuropneumonia. Immunohistochemical examination with antisera against

MmmLC and Mmc showed mycoplasma antigens in the lumina of the airways and

alveoli, mainly inside the cytoplasm of neutrophils and macrophages, but extracellular

antigen was demonstrated in areas of necrosis.

Darzi (1998) demonstrated the presence of mycoplasma-like bodies localized

mainly on the surface of acinar/duct epithelial cells by IHC, following intrammary

inoculation of Mycoplasma capricolum subsp. capripneumoniae (Mcc) in goats

Wesonga et al. (2004) demonstrated M. capripneumoniae antigen in the lungs of

goats infected with Mycoplasma capricolum subsp. capripneumoniae by

immunohistochemistry.

Castro-Alonso et al. (2009) demonstrated mycoplasma antigen in the degenerated

acinar epithelium of goats within 5 dpi with Mycoplasma agalactiae by intramammary

route.

Castro-Alonso et al. (2010) explored the ability of Mycoplasma agalactiae to

modulate the immune system in host tissues by immunohistochemically and

chronologically characterizing the main cell subsets present during the mammary

immunoinflammatory response. Results indicated an innate immune response in animals

sacrificed at 5dpi, characterized by an abundance of myeloid-histiocyte antigen Mac387+

and lysozyme+ cells that was unable to block or control Ma infection. The chronic stage

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of the inflammatory process observed in the goats killed at 45dpi was mainly

characterized by expansion of the CD8 compartment at the expense of the CD4 subset

leading to a reduced CD4/CD8 ratio.

2.9.5 Reisolation studies

Guha and Verma (1987a) infected kids intratracheally with Mycoplasma mycoides

subsp. mycoides isolated from kids with CCPP. They reported that the nasal discharge of

infected kids became positive for M. mycoides subsp. mycoides by the 4th dpi.

Guha and Verma (1987b) inoculated kids with 24 hours old culture of M.

agalactiae intramuscularly. M. agalactiae was isolated from lungs and heart of a kid that

died after 48 hours.

Nayak and Bhowmik (1990) established experimental transmission of

Mycoplasma mycoides subsp. mycoides (large colony type) in goat kids by placing

infected fleas on each kid's body surfaces. The organisms were recovered in high

numbers from heart blood, body fluids, and infected organs and joints.

Kaur et al. (1998) tested Mycoplasma capricolum subsp. capripneumoniae of

cow-udder origin for its mastitogenic capability in rabbit mammary-glands.

Establishment of mycoplasma organisms and presence of histopathological lesions in

mammary glands were the parameters for describing mastitogenic potential. The

reisolations of injected Mccp organisms in the pure form from the infected glands along

with the occurrence of histopathological changes were suggestive of mastitis during the

entire 8-days period of observation. Based on their observations they recommended that

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rabbit mammary-gland was a potential in vivo experimental laboratory model to screen

the mastitogenic potential of mycoplasmas of animal-udder origin.

Rodriguez et al. (2000) induced lung lesions experimentally using Mycoplasma

agalactiae and Mycoplasma bovis in goats by a combined (intratracheal + intranasal)

route. M. agalactiae or M. bovis was recovered from the respiratory tract and lung of all

but two infected animals.

Garg et al. (2004) experimentally tested Mycoplasma canadense, a clinical isolate

from milk of a mastitic buffalo, for its pathogenic potential in rat and rabbit mammary

gland models. In the rat and rabbit mammary glands, M. canadense organisms persisted

upto 6-day and 7-day postinfection, respectively and caused histopathological changes

suggestive of subacute to chronic mastitis during the experimental period.

Singh et al. (2004) studied the virulence of an Indian strain of Mycoplasma

mycoides subsp. mycoides LC by pathogenicity test in mice and goat. MmmLC was re-

isolated from lungs, liver, spleen, and lymph nodes of the infected animals.

Castro-Alonso et al. (2009) inoculated Mycoplasma agalactiae in goats by the

intramammary route and reported shedding of Mycoplasma agalactiae organisms in milk

upto 37 dpi.

De la Fe et al. (2010) examined the ability of M. agalactiae to colonize the ears of

goats infected experimentally by the intramammary route. M. agalactiae was detected in

19/20 (95 per cent) ear swabs from goats sampled at 15 and 45 dpi, whereas all ear swabs

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collected before inoculation, ear swabs collected from the group sampled at 5 dpi and ear

swabs from control goats at the time of sacrifice were negative for M. agalactiae.

2.9.6 Studies on humoral immune response in mycoplasma infected animals

Lloyd (1970) injected Mycoplasma mycoides subcutaneously into rabbits.

Agglutinating antibody occurred in all the rabbits and some evidence for an actively

acquired immunity to further infection was apparent after one injection of Mycoplasma

mycoides.

Gilmour et al. (1982) injected lambs intra-tracheally with a lung suspension

containing Mycoplasma ovipneumoniae and Pasteurella haemolytica serotype A2. Sera

collected pre-inoculation and at necropsy and when examined by a micro enzyme-linked

immunosorbent assay, demonstrated an increase in antibody to both organisms in all

animals.

Hasso and Al-Omran (1994) used the enzyme linked immuno-sorbent assay

(EL1SA) to evaluate humoral immune response to Mycoplasma agalactiae. Four groups

of goats were inoculated via different routes with the microorganism. Serum samples

were collected at monthly intervals for five successive months. Peak antibody levels were

detected at 3-4 months in the subcutaneously and orally inoculated groups, while a higher

peak since the first month was detected in the intramammary inoculated group. The

intravenously inoculated group revealed very slight increase in antibody levels. The best

route of inoculation for high and protective antibody levels was the intramammary route.

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Dahiya et al. (2004) demonstrated Mycoplasma mycoides subsp. mycoides (LC,

M-30) antigen in experimentally infected lamb tissues by IFAT (indirect

immunofluorescent technique). Antigens were detected, starting from 3 dpi to the end of

the experiment i.e. 28 dpi in lungs, bronchial and mediastinal lymph nodes, upto 12 dpi in

spleen and liver and at 28 dpi in synovial membrane. IFAT could be a valuable tool for

studying the immunopathogenesis of mycoplasmal infection in lambs.