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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.

Leishmaniases comprise a broad spectrum of human and animal diseases caused by infection of the mononuclear phagocyte system with protozoan parasites of the genus Leishmania. These agents are transmitted by the bite of phlebotomine sandflies in tropical, subtropical, and temperate zones of the world. Although humans are the sole reservoir hosts for some Leishmania species, in most cases other animals play a major role in the maintenance of infections. In countries of the Mediterranean basin, Middle East, and Latin America, wild canids and domestic dogs are the main reservoirs of zoonotic visceral leishmaniasis (ZVL), a severe disease caused by Leishmania infantum (synonym: Leishmania chagasi) (9). Over the past decade, the frequency of ZVL increased due to climate changes and human factors (5, 10).The diagnosis of L. infantum infection in dogs (canine leishmaniasis) is important in veterinary practice and in surveillance of ZVL. The immunofluorescent antibody test (IFAT) is routinely used for the detection of specific antibodies. IFAT is difficult to standardize and to interpret, however, and it is too laborious for screening large numbers of sera.Immunoenzymatic assays such as the enzyme-linked immunosorbent assay (ELISA) are easier to standardize and more practical as routine laboratory tools. The performance of ELISA tests is greatly affected by the quality of the antigens used, however, and test specificity limitations are the main drawback when crude antigen preparations are used.Recombinant technology, together with the characterization of specific immunodominant antigens at the genetic level, allowed the development of a second generation of diagnostic immunoassays, and the recent validation of a recombinant K39 ELISA as a diagnostic marker for canine leishmaniasis represents a good example (11). The rK39 (recombinant K39) antigen is a repetitive immunodominant B-cell epitope of the 230-kDa kinesin-related protein of L. chagasi (3, 13). In Leishmania spp., K39 antigen is mainly expressed in the amastigote stage and elicits a strong immunoresponse in both asymptomatic and clinically infected dogs.In addition to K39, two other antigens of L. chagasi (K9 and K26) have been genetically characterized (1): K26 shows a central 14-amino-acid repeat region which is specific to L. chagasi and L. donovani and a flanking region homologous to K9. Since these two antigens had not been evaluated as diagnostic markers for canine leishmaniasis, in this work we characterized the recombinant K9 and the recombinant repeat region of K26 expressed in Escherichia coli. In addition, since it was not clear whether the single repetition unit of K39 antigen carried an immunodominant epitope, the 39-aminoacid subunit of this kinesin-related protein was expressed in a similar way. The three recombinant antigens were then employed in a multiple-well ELISA to screen a panel of wellcharacterized dog sera.DNA was extracted from promastigotes of the L. infantum reference strain MHOM/TN/80/IPT1 (zymodeme MON-1) by conventional procedures. ...