Leishmania infantum-Derived Glycoinositolphospholipids in the Immunodiagnosis of Subclinically Infected Dogs.

Lipophosphoglycan (LPG), when used as an ELISA target, confers high specificity and sensitivity to the detection of Leishmania infantum antibodies in dogs. Glycoconjugates are economically viable because the yield is very high after extraction/purification. In addition, they are very stable, which allows their use in point-of-care testing without special storage conditions. During the glycoconjugate extraction, a glycoinositolphospholipid (GIPL)-enriched fraction is obtained in similar quantities as LPG. Since GIPLs can be extracted from the same parasite pellet as LPGs, this work aimed to evaluate the immune recognition of GIPLs by Leishmania infantum-infected dogs and its use for canine leishmaniasis (CanL) immunodiagnosis. Like LPG, GIPLs were recognized by sera from L. infantum-infected dogs, but with less sensitivity (83.8%). However, 80% (16/20) of subclinically infected dogs were detected as positive in the assay. Different from LPG, the GIPL-based assay achieved a lower specificity (73.7%) and cross-reactions occurred with T. cruzi and L. braziliensis-infected dogs. Although GIPLs exhibited a similar performance to LPG for subclinically L. infantum-infected dogs, the occurrence of cross-reactivities with other protozoa and a lower sensitivity hinders its use for an immunodiagnostic test. In places where those diseases do not co-exist such as in the Mediterranean region, its use for subclinically dogs could be an alternative.

Leishmania infantum-derived lipophosphoglycan as an antigen in the accurate serodiagnosis of canine leishmaniasis.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania protozoan and has an important biological role in host-parasite interactions both in the midgut epithelium of the sand fly vector and in the vertebrate macrophages. Canine leishmaniasis (CanL) is a chronic infectious disease predominantly caused by Leishmania infantum. An early and accurate immunodiagnosis of the disease is crucial for veterinary clinical practice and for disease control. In this work, we evaluated L. infantum LPG as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for CanL immunodiagnosis (LPG-ELISA) by testing serum samples from 97 naturally infected dogs with diverse clinical presentations ranging from subclinical infection to severe disease, as evaluated by veterinarian infectologists. Serum samples from healthy dogs from non-endemic areas (n = 68) and from dogs with other infectious diseases (n = 64) were used as controls for assay validation. The performance of the LPG-ELISA was compared with that of an ELISA using the soluble fraction of L. infantum total lysate antigen (TLA). LPG-ELISA presented a superior performance in comparison to TLA-ELISA, with 91.5% sensitivity, 98.5% specificity and 99.7% accuracy. A distinguishing feature of the LPG-ELISA compared to the TLA-ELISA was its higher ability to identify subclinical infection in clinically healthy dogs, in addition to the absence of cross-reactivity with other canine infectious diseases. Finally, LPG-ELISA was compared to TR DPP visceral canine leishmaniasis test, the immunochromatographic test recommended by the Brazilian Ministry of Agriculture. LPG-ELISA exhibited higher values of specificity (98.5% versus 93.1%) and sensitivity (91.5% versus 90.6%) compared to TR DPP. In conclusion, L. infantum-derived LPG was recognized by antibodies elicited during CanL in different infection stages and was shown to be a suitable antigen for specific clinical settings of veterinary diagnosis and for public health usage.

Isolation of naturally infecting Leishmania infantum from canine samples in Novy-MacNeal-Nicolle medium prepared with defibrinated blood from different animal species.

The most commonly used culture medium for the in vitro isolation of Leishmania spp. from canine biological samples is biphasic Novy-MacNeal-Nicolle (NNN) medium, whose solid phase is prepared using rabbit blood. Leishmania infantum parasites from natural infections are highly sensitive and demanding for growth in axenic conditions when firstly obtained from the dog's body. The objective of this study was to evaluate whether NNN medium (NNN-test) prepared with chicken blood (NNN-C), ox blood (NNN-O), horse blood (NNN-H) or sheep blood (NNN-S) was viable for the isolation of parasites from naturally infected dogs, in an endemic area for visceral leishmaniasis caused by L. infantum. Spleen aspirates from six dogs previously diagnosed as infected by parasitological methods were simultaneously inoculated in each NNN-test medium, including the conventional medium prepared with rabbit blood (NNN-R), and the cultures were examined for three weeks under optic microscopy. Spleen samples were also analyzed for parasite loads by quantitative PCR (qPCR). Cultures from three of the six dogs (50%) were positive in at least one of the NNN-test media: one sample presented the highest spleen parasite load by qPCR (1.19 × 104 parasites/mL) and was positive in all test media; the second sample presented parasitic isolation in the first week of culture in all inoculated media, of which the NNN-C medium had the highest mean parasite count (NNN-C = 23.5 × 104/mL vs. NNN-R = 3.25 × 104/mL); the third sample was positive only in the NNN-S medium besides the conventional control NNN-R. Cultures from the three remaining dogs were negative in all NNN media, including the control and test media; of those three dogs, two presented the lowest spleen parasitic loads according to qPCR. Blood from chicken, ox, horse and sheep shown to be viable for the preparation of NNN culture medium for the primary isolation of L. infantum from samples of naturally infected dogs and can be considered as an alternative to rabbit blood when necessary.