Validation of an internal method for the diagnosis of infections with Chlamydophila abortus and Coxiella burnetii by real-time multiplex PCR.

Chlamydophila abortus and Coxiella burnetii are one of the major pathogens implicated in abortion in cattle. Their characteristic of obligate intracellular bacteria, and of zoonotic agents, makes their culture difficult in diagnostic laboratories, and the traditional tools of diagnosis (detection of sera antibodies by ELISA, Stamp's coloration) encounter specificity, sensitivity and interpretability limits. Individual PCR have recently been developed. Nevertheless, their income/cost is a limiting factor for breeders. As the symptoms are not specific, the request for analysis often concerns the two valences. Consequently, the development and the validation of an internal multiplex PCR appears to be a suitable solution.

Fibrinogen Bastia (gamma 318 Asp-->Tyr) a novel abnormal fibrinogen characterized by defective fibrin polymerization.

A new congenital dysfibrinogen, Fibrinogen Bastia, was discovered in a 20-year-old woman with no clinical symptoms. The plasma thrombin-clotting time was severely prolonged. The functional plasma fibrinogen concentration was low (0.2 mg/ml), whereas the immunological concentration was normal (2.9 mg/ml). Purified fibrinogen Bastia displayed a markedly prolonged thrombin-clotting time related to a delayed thrombin-induced fibrin polymerization. Both the thrombin-clotting time and the fibrin polymerization were partially corrected by the addition of calcium ions. The anomaly of fibrinogen Bastia was found to be located in the gamma-chain since by SDS-PAGE performed according to the method of Laemmli two gamma-chains were detected, one normal and one with an apparently lower molecular weight. Furthermore, analysis of plasmin degradation products demonstrated that calcium ions only partially protect fibrinogen Bastia gamma-chain against plasmin digestion, suggesting that the anomaly is located in the C-terminal part of the gamma-chain. Sequence analysis of PCR-amplified genomic DNA fragments of the propositus demonstrated a single base substitution (G-->T) in the exon VIII of the gamma chain gene, resulting in the amino acid substitution 318 Asp (GAC)-->Tyr (TAC). The PCR clones were recloned and 50% of them contained the mutation, indicating that the patient was heterozygous. These data indicate that residue Asp 318 is important for normal fibrin polymerization and the protective effect of calcium ions against plasmin degradation of the C-terminal part of the gamma-chain.