There 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. Sixty 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. The 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. The 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.

Leishmania infantum-specific antibodies are used extensively for the diagnosis and monitoring of treatment in canine leishmaniosis. Different views have been described for the measurement of L. infantum antibody levels for the monitoring of anti-leishmanial treatment. In addition, molecular techniques using blood are frequently employed in the clinical setting. However, there are not enough studies to prove the usefulness of PCR in diagnosis, treatment monitoring and in assessing the prognosis of the disease. The objectives of this study were to evaluate L. infantum-specific antibodies and blood parasitemia at the time of diagnosis and during treatment and to correlate these with the dog’s clinical status. Thirty-seven dogs were diagnosed and followed-up during treatment (days 30, 180 and 365). The treatment protocol consisted of a combination of meglumine antimoniate for one month and allopurinol for at least one year. Leishmania infantum-specific antibodies and blood parasitemia were assessed by an end point sera dilution ELISA and by real-time PCR, respectively. The majority of dogs were classified as LeishVet stage II (moderate disease) at the time of diagnosis (86 %) and the rest as stage III. Results showed variable levels of specific antibodies at the time of diagnosis [median ± interquartile range (IQR): 1372 ± 8803 ELISA units (EU)]. Twenty-three seropositive dogs (64 %) were detected as PCR-positive at the time of diagnosis. Interestingly, a rapid significant antibody level reduction was observed by day 30 of treatment (median ± IQR: 604 ± 2168 EU). A continuing significant decrease of specific antibodies was also found at days 180 (median ± IQR: 201 ± 676 EU) and 365 (median ± IQR: 133 ± 329 EU) in association with clinical improvement. A significant blood parasitemia reduction was also observed at all time points studied. Mean parasites/ml ± SD were 19.4 ± 79.1 on day 0, 2.2 ± 11.7 on day 30, 0.9 ± 2.9 on day 180, and 0.3 ± 0.7 on day 365. This study reports a significant reduction of L. infantum antibodies measured by an end point sera dilution ELISA method after 30 days of treatment associated with clinical improvement. A low proportion of sick dogs with moderate disease were negative by blood real-time PCR at the time of diagnosis.

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.

Repurposing clinically available drugs to treat the new coronavirus disease 2019 (COVID-19) is an urgent need in the course of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) pandemic, as very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases, such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated in the micromolar range for SARS-CoV-2 in vitro. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious viruses. The mechanism resides in the inhibitory activity toward α-glucosidases that are involved in the early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. Indeed, the antiviral potential of protein glycosylation inhibitors against SARS-CoV-2 is further highlighted by the low-micromolar activity of the investigational drug Celgosivir. These data point to a relevant role of this approach for the treatment of COVID-19.