The principal methods applied to the laboratory diagnosis of enzootic abortion in sheep traditionally include demonstrating the organism in impression smears of placental cotyledons and foetal membranes by modified Ziehl-Nielson straining, centrifuge-assisted isolation in cell culture, and the detection of antibodies. A presumptive diagnosis may also be made based on flock and farm history, and gross placental pathology.
Chlamydiae have been reported in large numbers in the foetal membranes of aborting ewes (Stamp et al., 1950), and similarly in the faeces of aborting ewes and in apparently healthy ewes and lambs (Dungworth & Cordy, 1962; Wilson & Dungworth, 1963). Although culture has long been considered the 'gold standard' and most sensitive method of diagnosis, it has significant disadvantages. A cold chain is required to protect the viability of the organism during specimen transport. Moreover modern molecular methods of diagnosis based on nucleic acid amplification are potentially much more sensitive than either culture or ELISA-based assays for chlamydial antigen.
In sheep, serological confirmation of chlamydial infections in abortion-affected flocks is generally the method used because isolation is a specialised and time consuming technique. High and rising antibody levels to chlamydial antigen can be demonstrated in post-abortion ewes by the complement fixation test (CFT). Despite its shortcomings, the test has been widely used and has been useful for diagnosis on a flock basis. The disadvantage of the CFT is that it is genus-specific response and cannot distinguish between antibodies to C. abortus or C. pecorum infection.
Antibodies to C. pecorum cross-react in the micro-immunofluorescence test with antigens of other mammalian chlamydiae including C. abortus (Perez-Martinez & Storz, 1985bb). These cross-reactive, genus-specific antibodies interfere in the interpretation of test results and pose particular problems in sera from animals infected with strains of both species, or colonized in the intestine with clinically inapparent C. pecorum strains. Some distinction can be achieved in indirect immunofluorescence assays (IFA) by comparing the end-point titers of heterologous sera with chlamydial whole inclusion antigens (Griffiths et al., 1992; 1996; Markey et al., 1993). However, these tests are too complex for large-scale routine serology. Immunoblotting of sera can also be used to demonstrate serological distinctions between C. abortus and C. pecorum infection.
Several ELISA-based serological tests have been reported for the diagnosis of enzootic abortion of ewes. One study examined 5 different tests for the detection of antibodies to C. abortus abortion disease in sheep. The tests included ELISAs using LPS or solubilised protein antigens, and immunoblotted chlamydial antigens. Sera were examined from naturally or experimentally infected C. abortus- or C. pecorum-infected sheep. None of these tests proved satisfactory for diagnostic purposes. An ELISA test based on monoclonal antibodies which recognised motifs in the VS1 and VS2 regions of C. abortus MOMP discriminated antibodies to C. abortus but not C. pecorum (Salti-Montesanto et al., 1997). A detergent solubilised protein ELISA test was reported by Donn et al., 1997, but it performed less well than the CFT in detecting abortion positive sera. The main disadvantage of these types of ELISA test is that they are not based on defined or unequivocal antigens. Tests based on defined antigens might permit the establishment of standardised, comparatively evaluated tests.
An ELISA based on MOMP peptides, which differentiated between abortion sera and sera from SPF lambs, has been described by Kaltenboeck et al., 1997. ELISA tests based on recombinant antigens of the polymorphic membrane protein (POMP) family have also been reported (Buendia et al., 2001; Longbottom et al., 2001). This group of outer membrane proteins has been shown to react strongly with sera from C. abortus infected sheep but not with sera from C. pecorum infected flocks (Griffiths et al., 1992; Soriau et al., 1994). Longbottom et al., 2002 prepared a series of overlapping recombinant antigens representing the polymorphic outer membrane protein (POMP90) of C. abortus. These were assessed by enzyme-linked immunosorbent assay (ELISA) against a panel of 143 serum samples from sheep experimentally infected with C. abortus, from sheep clinically free of ovine abortion, and from specific-pathogen-free lambs experimentally infected with different subtypes of C. pecorum. The results were compared to those obtained by complement fixation test (CFT) and another recently described test, an indirect ELISA (iELISA) using recombinant OMP91B (rOMP91B) fragment (rOMP91B iELISA) (D. Longbottom, E. Psarrou, M. Livingstone, and E. Vretou, FEMS Microbiol. Lett. 195:157-161, 2001). ELISAs based on rOMP90-3 and rOMP90-4 were identified as being more sensitive and specific than conventional CFT. Assays with both fragments were evaluated further with a panel of 294 serum samples from flocks with documented histories of abortion, from flocks with no clinical histories of abortion but which had a high proportion of samples seropositive by CFT, and from animals with no histories of abortion but from which various C. pecorum subtypes had been isolated. ELISAs with both POMP90 fragments outperformed the CFT. Importantly, serum samples from C. pecorum_-infected animals produced no false-positive results. The ELISA based on the rOMP90-4 fragment was more sensitive and was also able to identify apparently healthy animals that were infected with an enteric strain of _C. abortus in flocks that were probably infected with both enteric C. abortus and C. pecorum strains. The identification of animals infected with enteric C. abortus is important for controlling the spread of ovine abortion. Overall, the new rOMP90-4 ELISA was found to be a more sensitive and specific test than CFT for differentiating animals infected with C. abortus from those infected with C. pecorum [Longbottom et al., 2002].
The polymerase chain reaction (PCR) test permits the sensitive detection of chlamydial DNA in clinical samples and the identification of isolates from a range of chlamydial infections in animals. These include sheep (Thiele et al., 1992; Hewinson et al., 1991b; 1991c), cattle (Wittenbrink et al., 1993; Domeika et al., 1994), pigs (Kaltenboeck et al., 1992; Schiller et al., 1997), horses, koalas (Girjes et al., 1993), and various bird species (Hewinson et al., 1991). Kaltenboeck et al., 1991 and Rasmussen et al., 1992 reported the detection of as few as 10 chlamydial genomes, by PCR. In a PCR test used routinely on avian clinical samples to detect C. psittaci, between 6-60 genomes were detectable, confirmed by DNA titration (Hewinson et al., 1991). A PCR test based on detection of C. abortus and C. psittaci pmp genes has been described by Lauroucau et al., 2001 as being more sensitive than PCR tests based on ompA genes. A touchdown enzyme time release modification of a PCR for the diagnosis of C. abortus infections has also been described (Amin 2003). The assay was specific for C. abortus versus C. pecorum and had a sensitivity of 0.25 of an inclusion forming unit.
In one investigation, of an epizootic of chlamydial abortions in cattle in a herd in Cumbria, U.K., a chlamydial agent was recovered and shown by sequencing of the ompA gene to be identical to the type strain of ovine enzootic abortion (Griffiths et al., 1995). Apparently, close contact between sheep and cattle on the affected farm resulted in transmission of the C. abortus infection. In two other cases of suspected sporadic bovine encephalomyelitis, chlamydial DNA was detected in brain samples in a C. psittaci PCR test (Hewinson et al., 1997). In these two clinical cases, one of which was reported by Piercy et al., 1999, sequencing of the 16S-23S intergenic spacer region of ribosomal DNA showed the chlamydiae involved to be closely related to the pigeon serovar B group.
[PG] Updated [MEW] July 2003
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