Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing.

AIMS: To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau-PCR and amplified fragment length polymorphism (AFLP). METHODS AND RESULTS: Eighty-one isolates from bovine and caprine dairy products (n = 53) and from diseased rainbow trouts and other fishes (n = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84.4% to 97.5% and discriminatory powers from 0.798 to 0.986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau-PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes. CONCLUSION: L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks. SIGNIFICANCE AND IMPACT OF THE STUDY: L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau-PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.

Identification of enteric Helicobacter in avian species.

The presence of enteric Helicobacter species was investigated in poultry (n=130) and in pet and ornamental birds (n=50) using a PCR sequencing method which permits the differentiation of many Helicobhacter species derived from animal tissues. All samples were of Italian origin, except for 21 Guinea fowl from a French flock. About 80% of poultry (chickens, laying hens, Guinea fowl) were positive to Helicobacter DNA. H. pullorum was most frequently (62.1%) identified whereas H. pylori and 3 H. sp. hamster B strains were seen in only 3 cases each. Pet and ornamental birds were all negative. H. canadensis was found in all Guinea fowl from a French farm. This is the first report on the occurrence of this bacterium in poultry.

Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections.

Among animal lentiviruses, Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV) and Small ruminant lentiviruses (SRLV) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia. The detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life. Capsid antigen (CA) is the major viral core protein and specific antibodies against this antigen are usually first recognised in infected sheep, goat and horse, remaining detectable for long period. Transmembrane (TM) domain of envelope glycoprotein contains a well conserved motif known to form an immunodominant epitope in several lentiviruses. In this study a simple strategy was developed to express the entire CA and the TM epitope in a single fusion protein from equine, feline and small ruminant lentiviruses in prokaryotic system and evaluated the diagnostic utility of a purified preparation in an indirect ELISA for each of the three infections. Results demonstrate that, for FIV and SRLV infections, the combination of CA and TM fractions increases the sensitivity of diagnostic tests based only on CA. The corresponding CA/TM antigen from EIAV showed excellent agreement with Coggins test.

Prokaryotic expression and antigenic characterization of three recombinant Leishmania antigens for serological diagnosis of canine leishmaniasis.

Three recombinant antigens of Leishmania chagasi (= L. infantum) were expressed in prokaryotic systems and evaluated (using a panel of dog sera characterized by parasitological and serological immunofluorescent antibody test [IFAT] techniques) as diagnostic markers of infection. The whole open reading frame encoding K9, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment encoding a single 39-amino-acid subunit of the kinesin-related protein K39 (K39sub) were amplified from L. infantum DNA and cloned into a pGEX-2T expression vector in frame with glutathione S-transferase (GST). The sensitivity and specificity of enzyme-linked immunosorbent assays (ELISAs) using K26 as an antigen (evaluated with sera from 20 parasitologically positive and 20 parasitologically negative dogs) were both 100% (95% confidence interval [CI] = 83.2 to 100). When K9 and K39sub were used, sensitivity was 95% (95% CI = 75.1 to 99.9) and specificity was 100% (95% CI = 83.2 to 100). Using 182 field sera, a good agreement was found between the recombinant K26 ELISA and IFAT (K = 0.92; 95% CI = 0.86 to 0.98) results and between the K9 and K39sub ELISA (used in parallel) and IFAT (K = 0.87; 95% CI = 0.80 to 0.95) results. The results demonstrate that each antigen carries immunodominant epitopes and that their combination may further increase the sensitivity of currently available serological tests.