The 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. Sixty-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). Among 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, n 1 = 14, n 2 = 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. This 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.

Background: Canine leishmaniosis (CanL) due to Leishmania infantum remains common, and veterinarians do not always follow scientifically sound approaches for diagnosis, treatment and prevention. Objectives: To provide consensus guidelines for diagnosis and evidence‐based guidelines for treatment and prevention of CanL. Methods and Material: Clinical consensus guidelines for the diagnosis were structured based on literature and authors' experience. Three electronic databases were searched for randomised controlled trials, systematic reviews and meta‐analyses on treatment and prevention. Results, Conclusions and Clinical Importance: Diagnosis should be based on compatible clinical signs and/or clinicopathologic abnormalities, exclusion of differentials, demonstration of infection and increased concentration of anti‐ Leishmania IgG (quantitative serology). Euthanasia for public health purposes is not recommended and drugs with anti‐ Leishmania activity should be avoided in subclinically infected dogs. Recommended treatments include meglumine antimoniate‐allopurinol (first‐line treatment), miltefosine‐allopurinol (first‐line treatment) and aminosidine‐allopurinol (second‐line treatment); marbofloxacin may be considered in dogs with advanced chronic kidney disease. In endemic areas, recommended measures for prevention include deltamethrin 4% collar, flumethrin 4.5%‐imidacloprid 10% collar or permethrin 50%‐imidacloprid 10% spot‐on, not using infected blood products for transfusion, not breeding seropositive bitches or dogs with CanL, administration of domperidone (seronegative dogs) and dietary nucleotides‐active hexose correlated compound (subclinically infected, seropositive dogs). Vaccination with Li ESP with MDP may be considered, whereas protein Q vaccine is recommended in areas with very high rates of seroconversion. In non‐endemic areas, recommended measures include not using infected blood products for transfusion and removal of infected female dogs from reproduction.

Normal-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). The 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. Normal-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.

A wide spectrum of clinical manifestations and immune responses exist in canine L. infantum infection. Ibizan hounds are more “resistant” to disease than other dog breeds. Recognition of pathogen-associated molecule patterns by toll like receptors (TLRs) rapidly triggers a variety of anti-microbial immune responses through the induction of pro-inflammatory cytokines such as TNF-α and IL-6 which may play an important role in controlling Leishmania infection. The main objective of this study was to investigate and compare the effect of a TLR2 agonist (TLR2a) alone or in combination with L. infantum antigen (LSA) on ex vivo whole blood cytokine production from healthy seronegative IFN-γ non-producer dogs from an area of low in canine leishmaniosis endemicity (n = 11); sick seropositive dogs with low production of IFN-γ (n = 17) and healthy seronegative or low positive Ibizan hounds with a predominant IFN-γ production (n = 21) from a highly endemic area. Whole blood was stimulated with medium alone (Ø), LSA, concanavalin A, TLR2 (Pam3CSK4) receptor agonist (Ø + TLR2a) and TLR2a and LSA (LSA + TLR2a) for 48 h. Supernatants were harvested for measurement of canine TNF-α and IL-6 cytokines by ELISA. A significant increase of TNF-α was found in the supernatants of stimulated blood from all groups (Ø + TLR2a and LSA + TLR2a) when compared with medium alone. A similar pattern was observed for IL-6. Interestingly, a significant increase of TNF-α production was only observed when stimulation with LSA + TLR2a was compared with TLR2a alone in Ibizan hounds. A significant increase of TNF-α production was observed with stimulation of LSA + TLR2a when compared with LSA in all groups. Significantly higher concentrations of TNF-α and IL-6 were detected in Ibizan hounds, especially for the Ø + TLR2a and LSA + TLR2a treatments compared with other groups. This study demonstrated that TLR2a alone enhances the production of the inflammatory cytokines TNF-α and IL-6 in sick, “resistant” and healthy non-infected dogs. In addition, a combination of LSA+TLR2a promoted a synergistic pro-inflammatory effect with TNF-α in Ibizan hounds but not in seropositive sick dogs and seronegative healthy dogs. These findings might suggest the importance of Pam3CSK4 as a possible immunomodulator for CanL.