BackgroundThere is limited information available on cytokine profiles in dogs with different degrees of disease severity due to natural infection of Leishmania infantum. The aim of this study was to investigate L. infantum-specific IFN-γ and IL-10 production in blood from dogs with leishmaniosis at diagnosis and correlate these findings with disease severity, humoral immune response and blood parasitemia.MethodsSixty dogs were diagnosed based on physical examination, routine laboratory tests, L. infantum-specific antibody levels measured by quantitative ELISA and blood parasitemia by real-time PCR. Heparin whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ and IL-10 concentrations were measured in supernatants with sandwich ELISAs.ResultsThe majority of dogs (n = 36) were classified as LeishVet stage II (moderate disease). The rest of the dogs were classified as stage I (n = 10), III (n = 10) and IV (n = 4). Dogs classified with stage I and IIa presented significantly higher (P = 0.02) LSA IFN-γ concentrations, lower (P <0.0001) antibody levels and a tendency for lower blood parasitemia (P = 0.1) than dogs classified with stages IIb, III or IV while no differences in ConA IFN-γ or IL-10 concentrations were observed among groups. Thirty-five dogs produced significantly higher LSA IFN-γ (mean ± SD: 2320 ± 3960 pg/ml) and ConA IFN-γ (mean ± SD: 7887 ± 7273 pg/ml) when compared with 25 dogs that did not produce detectable LSA IFN-γ but produced ConA IFN-γ (mean ± SD: 4917 ± 5233 pg/ml). IFN-γ producer dogs presented lower (mean ± SD: 5750 ± 14,082 ELISA units (EU), P = 0.001) antibody levels and blood parasitemia (mean ± SD:   5 ± 10 parasites/ml, P = 0.001) when compared with IFN-γ non-producers (mean ± SD: 19,638 ± 28,596 EU and 1100 ± 5112 parasites/ml), respectively. LSA IL-10 was not detectable in 34 dogs while 49 dogs secreted ConA IL-10 (mean ± SD of 90 ± 103 pg/ml). LSA IFN-γ concentration was negatively correlated with blood parasitemia and antibody levels and positively correlated with ConA IFN-γ and LSA IL-10 concentrations.ConclusionsThe results of this study demonstrate that sick dogs lacking L. infantum specific IFN-γ production in stimulated whole blood produce a strong humoral response, have a high blood parasitemia and severe clinical disease. IL-10 does not appear to be a marker of disease severity.

BackgroundThe severity of canine leishmaniosis (CanL) due to Leishmania infantum might be affected by other vector-borne organisms that mimic its clinical signs and clinicopathological abnormalities. The aim of this study was to determine co-infections with other vector-borne pathogens based on serological and molecular techniques in dogs with clinical leishmaniosis living in Spain and to associate them with clinical signs and clinicopathological abnormalities as well as disease severity.MethodsSixty-one dogs with clinical leishmaniosis and 16 apparently healthy dogs were tested for Rickettsia conorii, Ehrlichia canis, Anaplasma phagocytophilum and Bartonella henselae antigens by the immunofluorescence antibody test (IFAT) and for E. canis, Anaplasma spp., Hepatozoon spp., Babesia spp. and filarioid DNA by polymerase chain reaction (PCR).ResultsAmong the dogs examined by IFAT, the seroprevalences were: 69% for R. conorii, 57% for E. canis, 44% for A. phagocytophilum and 37% for B. henselae; while the prevalences found by PCR were: 8% for Ehrlichia/Anaplasma, 3% for Anaplasma platys and 1% for H. canis. No other pathogen DNA was detected. Statistical association was found between dogs with clinical leishmaniosis and seroreactivity to R. conorii antigen (Fisher’s exact test: P = 0.025, OR = 4.1, 95% CI = 1–17) and A. phagocytophilum antigen (Fisher’s exact test: P = 0.002, OR = 14.3, 95% CI = 2–626) and being positive to more than one serological or molecular tests (co-infections) (Mann-Whitney test: U = 243, Z = -2.6, n1 = 14, n2 = 61, P = 0.01) when compared with healthy dogs. Interestingly, a statistical association was found between the presence of R. conorii, E. canis, A. phagocytophilum and B. henselae antibodies in sick dogs and some clinicopathological abnormalities such as albumin and albumin/globulin ratio decrease and increase in serum globulins. Furthermore, seroreactivity with A. phagocytophilum antigens was statistically associated with CanL clinical stages III and IV.ConclusionsThis study demonstrates that dogs with clinical leishmaniosis from Catalonia (Spain) have a higher rate of co-infections with other vector-borne pathogens when compared with healthy controls. Furthermore, positivity to some vector-borne pathogens was associated with more marked clinicopathological abnormalities as well as disease severity with CanL.

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BackgroundNormal-looking skin of dogs with leishmaniosis frequently shows microscopic lesions along with the presence of Leishmania amastigotes. However, histological lesions with or without detection of amastigotes might not occur in less severe clinical cases. In addition, comparative studies between paired clinically-lesioned and normal-looking skin samples from dogs with different disease severity are lacking. The objective of this study was to compare histological and parasitological findings by Leishmania immunohistochemistry (IHC) and quantitative PCR (qPCR) on paired clinically-lesioned and normal-looking skin biopsies from 25 dogs with different clinical stages of leishmaniosis, 11 with stage I-mild disease (papular dermatitis) and 14 with stage II-III (ulcerative or exfoliative dermatitis).ResultsThe study demonstrated microscopic lesions in 14 out of 25 (56%) samples from normal-looking skin biopsies. In those samples, perivascular to interstitial dermatitis composed by macrophages with lymphocytes and plasma cells was observed mainly in the superficial and mid-dermis. The intensity of the dermatitis was mild to moderate and always less prominent than in the clinically-lesioned skin. In normal-looking skin samples, the presence of parasites was detected by histology, IHC and qPCR in 5/25 (20%), 8/25 (32%) and 18/25 (72%), respectively. Leishmania was encountered in 11/25 (44%), 23/25 (92%) and 25/25 (100%) of clinically-lesioned skin samples by histology, IHC and qPCR, respectively. Normal-looking skin from dogs with stage I-mild disease was less frequently inflamed (P = 0.0172). Furthermore, Leishmania was more easily demonstrated by histology (P = 0.0464), IHC (P = 0.0421) or qPCR (P = 0.0068) in normal-looking skin of dogs with stage II-III-moderate to severe disease. In addition, in the latter group, there was a significantly higher parasite load studied by means of qPCR than in dogs with less severe disease (P = 0.043). Clinically-lesioned skin from dogs with stage I disease was more frequently characterised by the nodular to diffuse pattern and granuloma formation (P = 0.0166) and by a lower parasite load studied by means of qPCR (P = 0.043) compared with more diseased dogs.ConclusionsNormal-looking skin from dogs with stage I is less likely to present histological lesions as well as harbour the parasite when compared with dogs with moderate to severe leishmaniosis.

BackgroundVenezuela is an endemic area for human and canine leishmaniosis due to Leishmania infantum and parasites of the Leishmania braziliensis and L. mexicana complexes. Limited data are available on feline leishmaniosis (FeL) in this region. The aim of this study was to describe clinical and diagnostic aspects of FeL in Venezuela.ResultsThirty-one domestic cats from urban areas of Lara State in Venezuela were enrolled. Twenty-five were healthy. Six other cats had solitary or multiple nodular lesions, which were located on the nose, ears, tail and lower limbs. Cutaneous lesions were characterized by diffuse pyogranulomatous infiltrate in all sick cats with numerous intracellular and extracellular amastigotes, and immunohistochemistry was positive for Leishmania in five sick cats. All healthy cats were seronegative for L. infantum and L. braziliensis antigens by ELISA. Two out of five sick cats yielded a positive ELISA result to both Leishmania antigens with higher antibody levels to L. braziliensis compared to L. infantum. Significantly higher antibody levels by ELISA as well as a higher number of bands by Western blot (WB) were found for L. braziliensis when compared to L. infantum antigens in all sera from Venezuelan sick and healthy cats. All healthy cats were blood Leishmania spp. qPCR negative while three out of six sick cats were blood qPCR positive. All paraffin-embedded skin biopsies (n = 4) as well as cutaneous cytology (n = 3) were positive by Leishmania spp. qPCR in sick cats. Leishmania speciation was obtained only from the cutaneous lesion samples from cytological preparations of two out of three sick cats which were identified as infected with L. mexicana or a closely related specie.ConclusionsFeline leishmaniosis should be included in the differential diagnosis list of nodular-ulcerative lesions. The most reliable diagnostic technique in sick cats is cytological or histopathological examination along with immunohistochemistry, since blood PCR and serology by ELISA might be negative. WB appears to be more sensitive in detecting infection. Cats with leishmaniosis from Venezuela are most likely infected with species of L. mexicana or a closely related species or the L. braziliensis species complex and not with L. infantum.