Detection and Identification of Mycoplasma conjunctivae in Infectious Keratoconjunctivitis by PCR. Based on the16S. rRNA. Gene

Abstract

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).

Isolation, identification and molecular characterization of Mycoplasma bovis in mastitic dairy cattle by PCR and culture methods

“…The bacteria infection can also cause abortion, infertility, arthritis, kerato-conjunctivitis, otitis media, pneumonia, and subcutaneous abscesses which lead to huge economic losses worldwide (Giacometti et al, 1999;Nicholas and Ayling, 2003). There are at least 11 other Mycoplasma species associated with mastitis outbreaks in cattle, but the infection induced by M. bovis is characterized by the increased number of severe clinical mastitis that are mostly non-responsive to treatment.…”

Detection of specific Mycoplasma conjunctivae antibodies in the sera of sheep with infectious keratoconjunctivitis

“…The percentages of animals with clinical symptoms in these herds are given in Table I. In these herds, M. conjunctivae was detected by PCR in the eyes of sheep [7].…”

“…To detect M. conjunctivae infections we used a nested PCR test based on the 16S rRNA gene, performed on eye swabs from behind the third eyelid [7]. Furthermore, a Western blot technique was used to detect M. conjunctivae antibodies from serum [5].…”

“…The term IKC is used to describe a clinical condition which can apparently not always be ascribed to the same infectious agent [11]. Mycoplasma conjunctivae has often been involved as the causative agent of IKC in both domestic [16,18] and wild ruminants [7,9,13], but its prevalence is not well known in most countries. This may be due to the lack of techniques for the detection of M. conjunctivae infections.…”

“…However, this method is cumbersome and requires specialised technical experience. Recently, a PCR method based on the 16 S rRNA gene, for the detection and identification of the agent from clinical material was described as well as a Western blot technique for the detection of M. conjunctivae antibodies in infected animals [5,7]. In contrast, no method is available for the rapid detection of specific M. conjunctivae antibodies in sera.…”