M. Arboix

Prevalence of Leishmania infantum Infection in Dogs Living in an Area of Canine Leishmaniasis Endemicity Using PCR on Several Tissues and Serology

ABSTRACT We studied and compared the prevalence of Leishmaniainfection and the seroprevalence and the prevalence of canine leishmaniasis in an area where canine leishmaniasis is endemic. One hundred dogs living on the island of Mallorca (Spain) were studied. In this study, we clinically examined each dog for the presence of symptoms compatible with leishmaniasis, determined the titer of anti-Leishmania antibodies, and investigated the presence of Leishmania DNA by PCR in skin, conjunctiva, and bone marrow samples of each dog. The prevalence of the disease and the seroprevalence were 13 and 26%, respectively. In 63% of the dogs,Leishmania DNA could be detected by PCR in at least one of the tissues studied. The results of positive PCR in the bone marrow, the conjunctiva, and the skin were 17.8, 32, and 51%, respectively. The prevalence of the infection, 67%, was calculated using all animals that were seropositive and/or positive by PCR with any tissue. The results showed that the majority of dogs living in an area where canine leishmaniasis is endemic are infected by Leishmania and that the prevalence of infection is much greater than the prevalence of overt Leishmania-related disease.

The Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection

Veterinarians working in the Balearic Islands (Mallorca), an endemic region of canine leishmaniosis, have reported very few cases of leishmaniosis in Ibizian hounds while concurrently observing that dogs of other breeds had a high incidence of clinical canine leishmaniosis. To further investigate this observation, two populations of dogs from the Balearic Islands were examined for the presence of Leishmania-specific cellular immunity using a delayed type hypersensitivity test (DTH) to leishmanin and for the presence of Leishmania-specific humoral immunity using an ELISA. Fifty-six asymptomatic dogs, 31 Ibizian hounds and 25 dogs belonging to other breeds were examined. Seventy-seven percent of the dogs demonstrated a specific immune response against Leishmania, either humoral or cellular. This finding suggests that the infection rate (77%) was higher than previously considered. For Ibizian hounds 81% were DTH positive while only 48% of the other dogs were DTH positive. A statistical association between Ibizian hounds and positive DTH response was found. A specific humoral response was found in 48% of Ibizian hounds and in 56% of the other dogs. No statistical association relative to the Leishmania-specific IgG1 and IgG2 levels were found between the two groups. The Ibizian hound has been reported to be more resistant to Leishmania infection and we found that the Ibizian hound mounts a significant cellular response to infection. Thus, the Ibizian hound may be an interesting canine model for the investigation of protective anti-Leishmania immune response.

Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate.

Six healthy beagle dogs were infected with Leishmania infantum (MCAN/ES/92/BCN-83/MON-1) by intravenous inoculation of 5 x 10(7) promastigotes and two others were used as controls. When animals showed clinical signs of disease at 29, 37, 41 and 45 weeks post-infection (p.i.), they were treated with meglumine antimoniate (20.4 mg Sb/kg/12 h) subcutaneously for two periods of 10 days each. Sera were tested periodically for Leishmania antibodies by Dot-ELISA, ELISA and Western blot (WB). Aspirates of popliteal lymph node (PLN), peripheral blood sample (PB) and healthy skin were cultured in NNN and Schneiders medium. PLNs were positive between 8 and 20 weeks p.i. and in one animal PB was positive 6 weeks p.i. Samples of healthy skin, obtained before treatment, were also positive. Dot-ELISA and ELISA detected specific antibodies at an early stage between 4 and 12 weeks p.i and surpassed the cut-off between 16-24 weeks p.i., while the WB was positive between 10-19 weeks p.i. The pattern of bands revealed during the first stages of infection was variable and only in two cases did the positivity start with bands of low molecular weight (12-14 kD); the number of bands increased until 15-24 weeks p.i., after which sera revealed a complete pattern of bands, from 12 to 85 kD, in the antigen of Leishmania. After treatment the clinical improvement of the animals was accompanied by a decrease in antibody titers (Dot-ELISA and ELISA) although the parasites remained in the PLN. This was reflected in the WB by a decrease in the intensity of bands, especially those in the region of 12-30 kD. A new increase in the antibody levels between 3 and 5 months after terminating the therapy was detected in the WB by a restoration of the initial complete pattern of bands.

Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas: Evolution in the course of infection and after treatment

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.

Long term improvement in the treatment of canine leishmaniosis using an antimony liposomal formulation.

Pharmacokinetic and clinical effectiveness of liposome-encapsulated N-methylglucamine antimoniate (LMA) was performed in dogs suffering from experimental leishmaniosis. LMA was compared with N-methylglucamine antimoniate (MGA), the same drug in its free form. Sb plasma concentrations for LMA were always higher than those for MGA. Mean residence time (MRT), half-life time (t(1/2)) and clearance (Cl) showed that Sb was eliminated slower after liposome administration. The high volume of distribution (Vd) obtained with LMA suggests that Sb could achieve therapeutic concentrations in parasite-infected tissues. Average plasma concentration at steady state (Css(ave)) shows that Sb body concentrations after LMA treatment (9.8 mg/kg Sb, each 24h) would be effective in Leishmania infantum canine infection. Comparing LMA with MGA in a 1-year follow-up we observed no relapses for LMA and total protein and gammaglobulin concentrations were within normal range, while for MGA both began to rise 3 months after treatment. Use of antimonial liposomal formulations may restore effectiveness to an existing drug and reduce toxicity.

Pharmacokinetics of meglumine antimoniate after administration of a multiple dose in dogs experimentally infected with Leishmania infantum

Pharmacokinetics of meglumine antimoniate in dogs with experimentally induced leishmaniosis has been investigated. After infection, dogs received a dose of 75 mg kg-1 of meglumine antimoniate twice daily by subcutaneous injection for 10 days. Blood samples were collected throughout the treatment. No statistical differences were found in the kinetic behaviour of the drug administered as a single dose to healthy dogs and that administered as a multiple dose to infected animals. However, peak plasma concentrations (Cmax) of 30.8 +/- 12.8 micrograms ml-1 found after this dosage regimen were higher than those observed after the single dose administration of 100 mg kg-1 24 h-1. Furthermore, sustained antimony concentrations of 1.14 +/- 0.52 micrograms Sb ml-1 were detected throughout the treatment. No signs of toxicity were found in the animals treated indicating that this regimen would be very appropriate to treat canine leishmaniosis.

Disposition of antimony after the administration of N-methylglucamine antimoniate to dogs

A study was carried out in dogs to define the pharmacokinetic profile of antimony and to define a better therapeutic protocol for the treatment of canine leishmaniasis. Six healthy beagle dogs received 100 mg/kg of N-methylglucamine antimoniate containing 27.2 per cent of antimony intravenously, intramuscularly and subcutaneously. After intravenous administration the plasma concentration of antimony decreased rapidly and after 240 minutes it was lower than the ED50 values suggested for Leishmania donovani. The pharmacokinetic parameters and bioavailability of antimony were calculated after each route of administration in each dog. The curves of plasma concentrations vs time were best described by a triexponential open model with a mean (sd) half life t½α of 9.4 (4.4) min, a t½pβ of 45.3 (4.5) min and a t½γ of 618.0 (93.5) min. The mean volume of distribution at steady state was 0.25 (0.03) litres/kg and the total body clearance was 0.25 (0.04) litres/h/kg. The peak plasma concentration (Cmax) after intramuscular administration was 27.2 (3.1)μg/ml, and after subcutaneous administration it was 25.5 (4.5) μg/ml; they were reached after 73.6 (11.9) min and 85.6 (11.3) min, respectively. The bioavailabilities after intramuscular and subcutaneous administration were 91.7 (7.1) and 92.2 (7.1) per cent, respectively. More than 80 per cent of the antimony was excreted in the urine in the first nine hours.

Skin mast cell releasability in dogs with atopic dermatitis.

Isolated dermal mast cells from atopic dogs are a valuable tool for the analysis of their functional properties in atopic dermatitis. We have characterized the histamine secretory pattern of mast cells enzymatically dispersed from the skin of dogs naturally suffering from this condition. The total histamine content found per isolated skin mast cell was higher in the allergic dogs than in nonatopic (control) animals (8.7 pg/mast cell versus 5.2 pg/mast cell). This phenomenon together with the well known higher concentration of skin mast cell number in atopic dermatitis lesions might account for the observed increase in local histamine concentration (15.0 μg/g versus 9.0 μg/g). Atopic dog-derived mast cells were highly reactive to both non-immunological (ionophore A23187) and an immunological-like (concanavalin A) stimulus. Furthermore, histamine net release induced by concanavalin A (1 mg/ml) stimulation was clearly enhanced in the atopic dogs (33.3% net release versus 15.4% in controls). These results have not been described in dermal mast cells dispersed from the skin of individuals with atopic dermatitis and clearly support the hypothesis that mast cells play a major role in causing and possibly modulating atopic dermatitis, through enhanced sensitivity or releasability. however, whether these two phenomena are primary abnormalities of atopic dermatitis, or only secondary changes, remains undetermined.

Rapid headspace gas chromatographic method for the determination of liquid/gas partition coefficients

A rapid, efficient and low-cost headspace technique useful for the determination of liquid/gas partition coefficients of gases and volatile substances of low and intermediate solubility is described. The equilibration step is carried out at constant pressure using glass syringes, with a ratio of liquid/gas phase volumes of ca. 1:3; after 30 min at the desired temperature, the headspace is recovered by transfer into another syringe and analyzed by gas chromatography. A study of the partition coefficients in water at 37 degrees C of 27 volatile compounds demonstrated that the method is fully applicable for all gases, with exception of those with a partition coefficient higher than 300.

Anthelmintic induced congenital malformations in sheep embryos using netobimin

Benzimidazole compounds have teratogenic effects in domestic and experimental animals. In this study, 14 Manchega ewes were treated orally, under controlled conditions, with 20 mg netobimin (a prodrug of a benzimidazole compound) per/kg bodyweight on the 17th day of pregnancy. Congenital malformations and abortions affected 60 per cent of the lambs. The main malformations were skeletal and renal, but vascular malformations were observed for the first time. The abnormalities were investigated using radiological, dissection and vascular injection techniques, and associations among them were recorded. The anomalies are discussed in terms of embryological considerations.