Retrospective Long-Term Evaluation of Miltefosine-Allopurinol Treatment in Canine Leishmaniosis

: Miltefosine-Allopurinol (MIL-AL) combination is reported to be one of the most effective treatments for canine leishmaniosis, thanks to its oral administration and MIL-documented low impact on renal function. However, MIL-AL is considered a second-choice treatment when compared to meglumine-antimoniate-allopurinol combination, mainly due to the risk of earlier relapses. The aim of this study was to evaluate the efficacy of the MIL-AL protocol during a long-term follow-up with an average duration of nine years. Dogs were living in Southern Italy (Puglia, Italy) in an area considered endemic for Canine leishmaniosis (CanL). Inclusion criteria were clinical and/or clinicopathological signs consistent with CanL; positive result to Leishmania quantitative ELISA; and negativity to the most frequent canine vector-borne infections. All dogs received 2 mg/kg MIL for 28 days, and 10 mg/kg AL, BID, for a period varying between 2 and 12 months. Ancillary treatments were allowed according to the clinical condition of the dog. A total clinical score and a total clinicopathological score were calculated at each time point by attributing one point to each sign or alteration present and then by adding all points. Improvement after each treatment was defined by the reduction of at least 50% of the total score. A survival analysis (Kaplan-Meier curve) was performed for quantifying the probability of the events occurring during the study follow-up. The following events were considered: decreased and negative ELISA results; improvement/recovery of the clinical and clinicopathological alterations; and relapse of leishmaniasis. One hundred seventy-three dogs (75f and 98m) were retrospectively included in the study by examining their clinical records since the first diagnosis of CanL. One hundred forty-three (83%) dogs were under five years of age. The mean duration of the follow-up period was 5.4 (±1.1) years with a minimum of 3.2 years and a maximum of 9 years. All dogs received a first treatment of MIL-AL at inclusion; then, during the follow-up course, 30 dogs required a second treatment, 2 dogs required a third treatment and 1 dog required a fourth and a fifth treatment. The mean time interval between the first and the second treatment was 27.2 (±18.3) months. After the first treatment, all dogs had decreased ELISA levels, in an average interval of 2.6 (±1.6) months. One hundred seventy dogs (98%) experienced a clinical improvement (mean time 3.0 ± 4.9 months); 152 (88%) dogs were considered clinically recovered after a mean time of 16.7 ± 13.5 months. A similar trend was observed for clinicopathological alterations; interestingly, proteinuria decreased in most dogs (p < 0.0001-Chi-square for trends). Thirty dogs experienced relapses, the earliest after 4.8 months. The mean time without relapse was 90.4 (±2.5) months. In relapsed dogs, the mean time for clinical improvement after the second treatment was 8.6 (±12.6) months, whereas it was 11.0 (±15.4) months for clinicopathological alterations. Five dogs had limited gastrointestinal side effects associated with MIL treatment. The present study confirms that the MIL-AL protocol can be considered one of the most effective treatments for CanL therapy, mainly for its capacity to provide a long-time clinical improvement in a large majority of treated dogs. As reported in the literature, the clinical stabilization of dogs does not occur immediately after treatment, probably due to the particular pharmacokinetic properties of MIL. The efficacy of MIL-AL decreases in dogs that need more than one treatment, suggesting the necessity to alternate anti-Leishmania drugs for the treatment of relapses. Side effects were transient and slight, even in dogs that required several treatments

An investigation of polymorphisms in innate and adaptive immune response genes in canine leishmaniosis

The outcome of infection with Leishmania infantum in dogs is variable, which is thought to be due to the nature of the immune response mounted by the host. As a consequence, the clinical signs and severity of canine leishmaniosis vary between individual dogs. Host immunogenetic factors might play an important role in determining the outcome of infection. The aim of this study was to examine polymorphisms in innate and adaptive immune response genes, to determine whether any of these were associated with susceptibility or resistance to L. infantum infection. Genomic DNA was obtained from two groups: pet dogs in endemic regions of Europe and a group of Beagles exposed to sand fly infection as part of a vaccine study. Genotyping was performed using a SNP (single nucleotide polymorphism) array for selected immune response genes. The first part of the study compared 62 clinical cases with 101 clinically unaffected dogs that were seronegative for Leishmania antibodies. One SNP in the CIITA gene demonstrated a significantly higher minor allele frequency in the case group, compared with the control group at the individual SNP level after permutation, but was not significant after correction for multiple testing. The second part of the study examined 48 Beagle dogs exposed to L. infantum over two transmission seasons. Twenty-seven dogs with a resistant phenotype (no evidence of clinical disease, seronegative at the end of the study period, negative on lymph node culture and only transiently PCR positive in bone marrow) were compared with 21 dogs demonstrating a susceptible phenotype (clinical disease, seropositive, positive lymph node culture and consistently PCR positive in bone marrow). Three SNPs in TLR3, two SNPs in PTPN22 and one SNP in TLR4 and IL1A were associated with the susceptible phenotype in the Beagle group at the individual SNP level after permutation analysis, but were not significant after correction for multiple testing. Further validation of these SNPs is required in a larger cohort of dogs, ideally with extreme phenotypes to confirm an association with the outcome of L. infantum infection

Comparison of Two Dosing Regimens of Miltefosine, Both in Combination With Allopurinol, on Clinical and Parasitological Findings of Dogs With Leishmaniosis: A Pilot Study

Miltefosine (MIL)–allopurinol combination therapy administered at standard dosage is effective to treat canine leishmaniosis, nevertheless for some dogs the digestive tolerance of MIL is not acceptable. This study evaluates an alternative therapeutic protocol by using a modified dosage of MIL to increase its effectiveness and improve the digestive tolerance. Thirty-four Leishmania infantum owned naturally infected dogs were included and monitored for 180 days. The dogs were allocated in two randomized groups: Group X−18 dogs treated with MIL registered dose of 2 mg/kg, oral administration, once daily, for 28 days; Group Y−16 dogs treated with 1.2 mg/kg for 5 days followed by 2.5 mg/kg for 25 days. Both groups were also treated with allopurinol. Digestive tolerance was monitored by adverse events observation. Treatments effectiveness was evaluated by monitoring the reduction of clinical score, the improvement of clinicopathological abnormalities, the reduction of parasitological load by PCR and the number of relapses. 16.6% dogs of group X and 12.5% dogs of group Y showed treatment associated adverse events. The reduction of clinical score was 61.7% for group X and 71.6% for group Y. All dogs showed an improvement of laboratory parameters after treatment. Quantitative PCR showed better results in group Y compared to group X; relapses were only registered in four dogs of group X. The modified protocol demonstrates a better trend of results in term of tolerance, clinical effectiveness, parasitological load reduction and relapses control, suggesting it could be considered for new large-scale studie

Determination of the effect of collars containing 10% w/w imidacloprid and 4.5% w/w flumethrin (Seresto®) on the incidence of Leishmania and other canine vector-borne pathogen infections in Greece

Background: The objective of this field study was to assess the effect of treating a considerable portion of a dog population naturally exposed to canine vector-borne pathogens (CVBPs) in endemic areas with a 10% w/w imidacloprid/4.5% w/w flumethrin collar (Seresto®) on the transmission of CVBPs and the resulting incidence of infection. Methods: A total of 479 dogs from two sites were enrolled in the study. Collars were placed on all dogs continuously for 21 months, with replacement of the collar every 7 months. All dogs were examined, including body weight and blood/conjunctival swab collections, every 7 months. Serum samples were analysed for the presence of antibodies against Leishmania infantum, Ehrlichia canis and Anaplasma phagocytophilum. PCR assays were also performed on blood samples and conjunctival swab collected from the dogs for the presence of L. infantum, and on blood samples only for the presence of Ehrlichia spp. and Anaplasma spp. Sand flies were collected, identified to species level and molecularly tested for L. infantum throughout two vector activity seasons. Results: The results showed that the Seresto collar was safe with continuous use. At study inclusion, 419, 370 and 453 dogs tested negative for L. infantum, Ehrlichia spp. and Anaplasma spp., respectively (353 dogs tested negative for any pathogen). Overall, 90.2% of the dogs were protected from L. infantum infection on both sites combined. The entomological survey confirmed the presence of competent vectors of L. infantum at all monitored locations, namely the sand flies Phlebotomus neglectus and Phlebotomus tobbi, both of which are regarded as the most important competent vectors in the Mediterranean basin. All captured sand flies tested negative for L. infantum. Protection against ticks and fleas was high, with only two dogs showing a low number of ticks and seven dogs having low numbers of fleas at single evaluation time points. Across the entire study population, a number of dogs became infected with tick-transmitted pathogens, but prevention of transmission was 93% for E. canis and 87.2% for Anaplasma spp. when all cases from both sites were combined. Conclusions: The Seresto® (10% w/w imidacloprid/4.5% w/w flumethrin) collar significantly reduced the risk of CVBP transmission when compared to previously observed incidences of CVBP infections in two highly endemic areas under field conditions

Laboratory evidence that dinotefuran, pyriproxyfen and permethrin combination abrogates Leishmania infantum transmissibility by sick dogs

Dogs are reservoir hosts of leishmaniasis caused by Leishmania infantum and transmitted by phlebotomine vectors. The effect of dinotefuran, pyriproxyfen and permethrin spot-on solution (Vectra®3D, Ceva Santé Animale, Libourne, France) on Leishmania transmissibility by naturally infected dogs via reared Phlebotomus perniciosus, was assessed. Dogs affected by leishmaniasis were submitted to xenodiagnosis and 6 infecting >10% of insects were treated topically on day 0. Antifeeding, insecticidal and anti-transmissibility effects were evaluated through xenodiagnoses performed on days 1, 7 and 28, using individual pre-treatment parameters as control. Feeding and mortality rates were assessed at 24 h, whereas promastigote infection, maturation and burden were assessed up to 96 h post blood meal (potentially infectious rate). On day 1, the anti-feeding efficacy was >95% in 4 dogs, insecticidal efficacy 100% in 4 dogs, and anti-transmissibility effect 100% in 6 dogs. Efficacy rates recorded on day 7 were very similar to day 1. On day 28, anti-feeding and insecticidal efficacy values were much broader, ranging 32.6–100% and 7.7–94.4%, respectively. Potentially infectious insects were recorded from two dogs, with sharp decrease in transmissibility rate as compared with pre-treatment condition. Altogether, Vectra®3D abrogated by >98% the potential Leishmania transmissibility by the examined pool of infected dogs over 1 month

Prevalence of Proteinuria in Owned Dogs from Italy : a Multicentric Study

Even though proteinuria is related to different causes, when it is persistent and associated with inactive urinary sediment, it is primarily due to kidney disease. Early detection of proteinuria allows us to identify several pathological conditions. The aim of the study was screening a canine population not known as being proteinuric, by the urinary dipstick. The study was carried out in seven Italian veterinary clinics during a period of six weeks. Dogs were enrolled with no restriction of sex or age. Females in estrus, dogs with signs of genitourinary diseases, or those previously diagnosed with proteinuric nephropathy were excluded. Dogs were considered \u201cnonproteinuric\u201d (NP) in case of negative dipstick test or \u201csuspected proteinuric\u201d (SP), if positive at the dipstick. When possible, proteinuria was confirmed by UPC ratio. A total of 1156 dogs were evaluated: 414 were from northern Italy and 742 from southern Italy. Based on dipstick test, 655 (56.6%) dogs were NP, while 501 (43.3%) were SP. Among the NP dogs 225 out of 414 (54.3%) were in northern Italy and 430 of 742 (57.9%) in southern Italy. One hundred eighty-nine of 414 (45.7%) SP dogs were identified in northern Italy and 312 of 742 (42.1%) in southern Italy. No statistical difference was found between the North and the South of Italy. UPC was available in 412 out of 501 SP samples: proteinuria was confirmed in 263 (63.86%) samples. Results from our study showed a high percentage of suspected proteinuric dogs, apparently not affected by renal diseases, together with the absence of statistically significant differences based on geographical area

Influence of Clinical Status and Parasite Load on Erythropoiesis and Leucopoiesis in Dogs Naturally Infected with Leishmania (Leishmania) chagasi

Background: The bone marrow is considered to be an important storage of parasites in Leishmania-infected dogs, although little is known about cellular genesis in this organ during canine visceral leishmaniasis (CVL). Methodology/Principal Findings: The aim of the present study was to evaluate changes in erythropoiesis and leucopoiesis in bone marrow aspirates from dogs naturally infected with Leishmania chagasi and presenting different clinical statuses and bone marrow parasite densities. The evolution of CVL from asymptomatic to symptomatic status was accompanied by increasing parasite density in the bone marrow. The impact of bone marrow parasite density on cellularity was similar in dogs at different clinical stages, with animals in the high parasite density group. Erythroid and eosinophilic hypoplasia, proliferation of neutrophilic precursor cells and significant increases in lymphocytes and plasma cell numbers were the major alterations observed. Differential bone marrow cell counts revealed increases in the myeloid:erythroid ratio associated to increased numbers of granulopoietic cells in the different clinical groups compared with non-infected dogs. Conclusions: Analysis of the data obtained indicated that the assessment of bone marrow constitutes an additional and useful tool by which to elaborate a prognosis for CVL

LeishVet guidelines for the practical management of canine leishmaniosis

The LeishVet group has formed recommendations designed primarily to help the veterinary clinician in the management of canine leishmaniosis. The complexity of this zoonotic infection and the wide range of its clinical manifestations, from inapparent infection to severe disease, make the management of canine leishmaniosis challenging. The recommendations were constructed by combining a comprehensive review of evidence-based studies, extensive clinical experience and critical consensus opinion discussions. The guidelines presented here in a short version with graphical topic displays suggest standardized and rational approaches to the diagnosis, treatment, follow-up, control and prevention of canine leishmaniosis. A staging system that divides the disease into four stages is aimed at assisting the clinician in determining the appropriate therapy, forecasting prognosis, and implementing follow-up steps required for the management of the leishmaniosis patient

Seropositivity rates for agents of canine vector-borne diseases in Spain : a multicentre study

Background: Controlling canine vector-borne diseases (CVBD) is a major concern, since some of these diseases are serious zoonoses. This study was designed to determine seropositivity rates in Spain for agents causing the following five CVBD: leishmaniosis (Leishmania infantum: Li), heartworm (Dirofilaria immitis: Di), ehrlichiosis (Ehrlichia canis: Ec), anaplasmosis (Anaplasma phagocytophilum/Anaplasma platys: An) and Lyme disease (Borrelia burgdorferi: Bb). Methods: Anti-An, -Bb, and -Ec antibodies and the Di antigen were determined using the 4DX SNAP® Test (IDEXX Laboratories) and anti-L. infantum (Li) antibodies using the Leishmania SNAP® Test (IDEXX Laboratories) in blood and/or serum samples. Results: Among 1100 dogs examined, overall seropositivity rates were: Li (15.7%), Ec (5%), An (3.1%), Di (1.25%) and Bb (0.4%). While seropositivity towards Bb and Di was similar in all geographic regions, rates were significantly higher in the east of Spain (8.3%) for An, significantly higher in the north (20%) for Ec, and significantly higher in the Southeast (46.6%) and South (27.4%), and significantly lower in the north (0%) for Li. No statistical associations were observed between sex and the CVBD analyzed (p ≥ 0.05) while the following associations with other variables were detected: a higher seropositivity to Ec (40%) and Bb (6.7%) in dogs under one year of age compared with adults (p < 0.05); and a higher seropositivity to An and Li in dogs that lived outdoors versus indoors (p = 0.01; p < 0.001, respectively). Seropositivity rates of 2.1%, 0%, 1.7%, 0.5% and 4.2% were recorded respectively for An, Bb, Ec, Di and Li in dogs with no clinical signs (n = 556) versus 3.8%, 0.6%, 7.5%, 1.8% and 25.9% for those with signs (n = 507) suggestive of a CVBD. Conclusion: The data obtained indicate a risk for dogs in Spain of acquiring any of the five CVBD examined. Veterinarians in the different regions should include these diseases in their differential diagnoses and recommend the use of repellents and other prophylactic measures to prevent disease transmission by arthropod vectors. Public health authorities also need to become more involved in the problem, since some of the CVBD examined here also affect humans