[PMC free article] [PubMed] [Google Scholar] 18. detection of antibodies in sera from convalescent pigs showed no correlation with clinical and pathological findings. is the causative agent of enzootic pig pneumonia (15, 21), a disease found on pig farms worldwide which is characterized by high morbidity and low mortality rates (19, 25). Coughing LY500307 is the principal clinical sign, and retarded growth and LY500307 poor food conversion may result in considerable economic losses. Furthermore, this agent predisposes the pigs to secondary pulmonary infections, hence, increasing the mortality among the herds and the financial problems associated with such losses (19, 25). encodes several characterized immunodominant proteins, among which are the p36 cytosolic protein (28, 29), the p46, p65, and p74 membranous proteins (3, 17, 18, 22), and the p97 adhesin (31). The functions of these proteins have not been yet elucidated, but specific reactants may eventually be useful tools for the diagnosis of (10). Furthermore, p36 is apparently highly conserved among different strains from various parts of the world (28, 29). Hyperimmune sera that have been produced against the recombinant p36 protein showed no reactivity against other porcine mycoplasmas, including and (29). Furthermore, no cross-reactivity was demonstrated against or species isolated from humans, other farm animals, cats, and dogs (29). The diagnosis of is usually done by cultivation of the organism or by immunofluorescence tests performed on frozen thin lung sections using polyclonal antibodies (1, 7, 19, 20, 25). However, because of the fastidious nature of this microorganism, its culture and identification may take up to 1 1 month. The cultures are also often contaminated with in primary isolation attempts (9). Moreover, the overall efficacy of serological detection methods, such as enzyme-linked immunosorbent assays (ELISAs), is often hampered because of the potentially cross-reacting antigenic relationships that exist between (2, 9). The indirect immunofluorescence (IIF) assay is still widely used for the diagnosis of because it is a rapid and convenient technique and allows histopathological observations. However, the use of polyclonal antisera may result in the nonspecific detection of other pathogens, namely, and (2, 9, 24, 32). The use of monoclonal antibodies (MAbs) increases the specificity of serological and immunohistochemical tests (10, 26). This paper describes the cloning and was used as the reference strain in this study. Other strains of (ATCC 25095 and J), as well as (ATCC 27399), (ATCC 23838), (ATCC 17981), and (ATCC 23206), were obtained from the American Type Culture Collection, Manassas, Va., and used for comparative antigenic studies. was cultivated from a field case of polyarthritis and was kindly provided to us by Claude Montpetit, Ministre de l’Agriculture, des Pcheries et de l’Alimentation du Qubec. The mycoplasmas were grown in modified Friis medium (11), containing 20% porcine serum (Gibco-BRL), 5% fresh yeast extract (Gibco-BRL), methicillin (0.15 mg/ml; Sigma-Aldrich Canada, Oakville, Ontario, Canada), bacitracin (0.15 mg/ml; Sigma-Aldrich), and thallium acetate (0.08 mg/ml; Sigma-Aldrich). The cells were harvested by centrifugation at 12,000 for 30 min at 4C, washed three times, and suspended in 0.1 M phosphate-buffered saline (PBS), pH 7.4. Field isolates of were cultivated from lung homogenates of pigs suffering from acute or chronic respiratory problems that had been forwarded to our laboratory for confirmation by PCR of outbreaks of enzootic pneumonia in pig herds in Southern Quebec (4). DNA extraction and PCR conditions. Genomic DNA from was extracted and purified, as previously described (4). The oligonucleotide primers used for enzymatic amplification of the entire open reading frame (ORF) of the p36 gene (948 bp) of were selected from the previously published DNA sequence (16) of the strain ATCC 25934 (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”X67286″,”term_id”:”49109″,”term_text”:”X67286″X67286). The sequence of the forward primer, FSp36, was Rabbit Polyclonal to CEBPD/E 5 GGG CCG ATG AAA CCT ATT AAA ATA GCT 3, and that of the reverse primer, RSp36, was 5 GCC GCG AAA TTA AAT ATT TTT AAT TGC ATC CTG 3. LY500307 The sequence analysis for the primer selection was performed using the McVector 3.5 (International Biothechnologies) and Gene Works 2.2 (IntelliGeneticsInc., Mountain View, Calif.) programs. The oligonucleotide primers were synthesized in an LY500307 automated Gene Assembler DNA synthesizer (Pharmacia LKB). The PCR protocol used for amplification of the p36 gene.