1.5.2.4.1. Serological identification:
Serological tests are either used for identification of isolates (growth inhibition test and immunofluorescence) or detection of antibodies in goat sera. The role of the later group in diagnosis of CCPP is limited due to the fact that animals die in the acute stage of the disease before they develop antibodies. Accordingly, serology should be applied on a herd level, not on individual basis, and that whenever possible, paired serum samples collected 3-8 weeks apart, should be examined.
1.5.2.4.1.1. Immunofluorescence (IF) and growth inhibition (GIT):
Identification of mycoplasmas to species level has relied heavily on serological methods with antisera against whole mycoplasma cell. Mainly outer membrane antigens are detected with IF and GIT which explains their high specificity (Erno, 1983). These tests are therefore recommended for species identification of mycoplasma (Freundt et al., 1979; Subcommittee on the taxonomy of Mollicutes, 1979).
1.5.2.4.1.2. Complement fixation test (CFT):
Complement fixation test was used for the detection of CCPP infection (MacOwan, 1976; MacOwan and Minette, 1977). It was found to be more specific, though less sensitive, when compared to indirect haemagglutination test (Muthomi and Rurangirwa, 1983). Its main disadvantage is the high level of technical expertise required to perform the test.
1.5.2.4.1.3. Polysaccharide-specific antibody detection latex agglutination test (LAT):
In this test, latex beads are sensitized with the polysaccharide produced by Mccp in culture supernatant. The reagent is then used in a slide agglutination test to detect antibodies (Rurangirwa et al., 1987). At present the test is routinely used in Kenya. It is a very useful test in outbreaks because it can be performed at the pen-side using a drop of whole blood.
Both CF test and IHA test findings emphasize the difficulties inherent in the serological diagnosis of CCPP when using whole cell or membrane antigen. The use of more defined antigen such as the polysaccharide provides greater specificity without cross-reactivity with sera against the other three principal caprine mycoplasmas.
1.5.2.4.1.4. Indirect haemagglutination test (IHA):
The test was used for diagnosis of CCPP (Muthomi and Rurangirwa, 1983). The specificity of this test for the M. mycoides cluster had been evaluated and results showed clear cross reactivity between these organisms (Jones and Wood, 1988). Abbas et al. (1987) used membrane antigens of Mmm LC type, Mmc, Mccp and Mcc strains in IHA test and applied it in CCPP in infected and healthy goats in the Sudan. Among animals with signs of CCPP, 72% had antibodies to the two subspecies of M.Mycoides, but only 7% had antibodies to M. capripneumoniae.
1.5.2.4.1.5. Enzyme linked immunosorbent assay (ELISA):
Indirect ELISA was developed to screen goat sera at a single dilution of antibody to Mccp (WamWayi et al., 1989). The test was more sensitive than CFT in detecting antibodies in goat serum. Its specificity was not investigated, but the author reported that cross-reactivity in this test had been observed between Mccp and Mmm (SC) antigens. In attempts to overcome
such inspecificity problems, a competitive ELISA (cELISA) was tried. The test was done using monoclonal antibodies (mAb) for the diagnosis of both CBPP (Abusurga et al., 1997; Goff and Thioucourt, 1998) and CCPP (Rurangirwa et al., 1997; Thioucourt et al., 1994). It has been described as a promising tool for the detection of CBPP and investigations are in progress to evaluate the test under the field conditions. Blocking ELISA was found also highly specific for CCPP and it detected antibodies in sera of naturally infected or artificially immunized animals while remained negative with hyperimmune sera to related strains such as PG 50 of Group 7 of Leach (Thioucourt et al., 1994).
1.5.2.4.1.6. Polyacrylamide gel electrophoresis (PAGE) and Immunoblotting:
Whole-cell protein analysis by sodium dodecyl sulphate–
polyacrylamide gel electrophoresis [SDS-PAGE] and/or Immunoblotting is widely used to characterize bacteria, including mycoplasma isolates (Costas et al., 1987; khan et al., 1987). The procedure is characterized by high specificity which allows the differentiation of distinct groups within a species.
For CCPP, Monoclonal Antibody to Mccp, designated E8-18, was tested against different strains in the Mycoplasma mycoides cluster with western blotting (Rurangirwa et al., 1997). E8-18 reacted with a 24-kDa antigen of Mccp but did not with any antigens of the other strains in the cluster. The identified antigen was found to be an integral protein on the surface of live Mccp. According to this finding, western blotting with monoclonal antibodies can be used in differentiating Mccp from closely related and difficult-to-distinguish Mycoplasma species.
1.5.2.5. Molecular Biology:
In recent years, there has been a virtual revolution in methods for the detection of bacteria, viruses and other microorganisms, as a consequence of the use of new molecular biology techniques.
The PCR is an in vitro cyclic repetitive reaction which allows the amplification of a specific deoxyribonucleic acid (DNA) region that located between two regions of known DNA sequence (Newton and Graham, 1997).
It was originally invented by a scientist called kary Mullis and described by Saiki et al. (1985) and Mullis and Fallona (1987). It was considered that the work of Saiki et al (1988) has greatly improved the technique.
A diagnostic method for CCPP based on PCR of the 16S rRNA genes from Mccp and restriction enzyme analysis of the PCR product has been developed (Ros Boscϋnana et al., 1994). The members of the M. mycoides cluster have two rRNA operons, designated rrnA and rrnB (Chrestiniansin et al., 1990); and the above diagnostic method for CCPP was based on a polymorphism (nucleotide difference between the two 16S rRNA genes in the same strain) which was found to be unique for Mccp. This polymorphism can therefore be easily used as a molecular marker for this subspecies, because it is localized in a restriction site for PstI (Ros Boscϋnana et al., 1994). This unique polymorphism has been shown to be present in 16 strains of Mccp from different parts of the world, but it was not present in 39 strains representing other species or subspecies of the M. mycoides cluster.
This system can be applied directly on various clinical samples to detect DNA from Mccp (Bölske, 1995). It is proved to be useful for the diagnosis of CCPP and it has now been applied in identification of several Mccp isolates recovered from goats in many countries.