Chlamydial infections in animals
Chlamydophila abortus : Laboratory
diagnosis
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 and Cordy, 1962; Wilson and
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 and
Storz, 1985b). 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. More recently, ELISA tests based on recombinant antigens of the polymorphic membrane protein
(POMP) family have 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; Souriau 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].
Nucleic acid based methods
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). Recently, 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 shoed the chlamydiae involved to be closely related to the pigeon serovar B group.
[Comment: A variety of
"home made" nucleic acid amplification based assays for the diagnosis of C.
abortus have been described. The need now is for comparison of published
methodologies in order to identify a validated method for general use].
[PG] Updated [MEW] July 2003
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