The incidence of clinical and clinicopathological signs associated with the progression of infection was evaluated prospectively in 329 naïve young dogs exposed to Leishmania infantum transmission and examined periodically during 22 months (M). The dogs were part of Leishmania vaccine investigations performed under natural conditions. Vaccinated groups were considered in the evaluation when the vaccine resulted non-protective and the appearance and progression of signs did not differ statistically from controls at each time point, otherwise only control groups were included. 115 beagles were part of 3 studies (A to C) performed in the same kennel; 214 owned dogs (29 breeds, 2.3% beagles) were included in a study (D) performed in 45 endemic sites. At M22 the prevalence of any Leishmania infection stage classified as subpatent, active asymptomatic, or symptomatic was 59.8% in studies A–C and 29.2% in study D. Despite different breed composition and infection incidence, the relative proportion of active infections and the progression and type of clinical and clinicopathological signs have been similar in both study sets. All asymptomatic active infections recorded have invariably progressed to full-blown disease, resulting in 56 sick dogs at M22. In these dogs, lymph nodes enlargement and weight loss — recorded from M12 — were the most common signs. Cutaneous signs were seen late (M18) and less frequently. Ocular signs appeared even later, being sporadically recorded at M22. Most clinicopathological alterations became evident from M12, although a few cases of thrombocytopenia or mild non-regenerative anemia were already observed at M6. Albumin/globulin inversions were recorded from M12 and urea/creatinine increase appeared mostly from M18. Altogether our findings indicate that any susceptible young dogs naturally infected by L. infantum present a common pattern of progression of signs during 2 years post infection, providing clues for medical and epidemiological applied aspects.

Canine leishmaniasis (CanL) is a disease characterized by a large variety of clinical alterations, the majority of which being due to immune mediated mechanisms. Sick dogs usually produce high levels of Leishmania-specific immunoglobulins which may give rise to circulating immune complexes (CICs) whose defective clearance by scavenging macrophages induces vasculitis and their deposition in specific organs. The aim of this study was to assess the serum level of CICs in dogs exposed to natural and experimental infection. Fifty-two sera were examined, belonging to untreated groups of naïve beagles previously studied to assess the performance of anti-leishmanial vaccines under natural (no. 22 dogs) or experimental (no. 30 dogs) transmission. Sera were classified in five groups according to the dog's health condition, IFAT titer, and the bone marrow (BM) nested (n)-PCR result. A: no.10 healthy dogs before the experimental infection; B: no.10 clinically healthy dogs infected experimentally, IFAT negative (= reciprocal titer <160) and n-PCR positive; C: no.10 clinically healthy dogs naturally infected, IFAT positive at titers 160-320 and n-PCR negative; D: no.10 sick dogs experimentally infected, IFAT positive at titer >320 and n-PCR positive; E: no.12 sick dogs naturally infected, IFAT positive at titer >320 and n-PCR positive. CICs levels were assessed by ELISA method (canine CIC assay-Cloude-Clone Corporation, USA). The two groups characterized by negative IFAT (A and B) had the lowest median level of CICs (16.09 and 12.78 µg/ml, respectively). CICs value increased progressively in the group C and reached the highest levels in the groups D and E, both characterized by high antibodies titer and severe disease, independently from the mode of infection. Significant differences in CICs concentration (p < 0.0001) were demonstrated between A, B, and C groups when compared with D or E groups of dogs. No differences were found inside the first three groups, while differences were recorded between the last two groups of sick dogs. CICs serum concentration increased with the progress of leishmaniasis, being significantly correlated with the increase of specific antibodies over time. High CICs levels detectable by commercial ELISA proved specific to an established Leishmania infection in dogs in the absence of other concomitant infections, as demonstrated by the similar trend assessed in experimentally and naturally infected dogs.

Canine leishmaniosis (CanL) is caused by protozoans of the genus Leishmania and characterized by a broad spectrum of clinical signs in dogs. Early diagnosis is of great importance in order to perform an appropriate therapy and to prevent progression towards severe disease. The aim of this study was to compare a point-of-care molecular technique, i.e., the loop-mediated isothermal amplification (LAMP), with a real-time polymerase chain reaction (Rt-PCR), and three serological techniques, i.e., immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and a rapid SNAP Leishmania test, to develop an integrated approach for the diagnosis of CanL. Sixty dogs were chosen after physical examination and collection of blood and sera samples, fine-needle aspiration of lymph nodes, and conjunctival swabs were performed. Lymphadenopathy (82.3%), as well as clinicopathological alterations of total proteins (70.6%), were the most frequent signs. Forty-one (68.3%) samples resulted positive at least to one technique. IFAT resulted in the best serological diagnostic method (specificity = 100%, sensitivity = 97.2%), detecting a higher number of positive samples than those revealed by other techniques. Among the samples used for molecular analysis, fine-needle aspiration of lymph nodes was revealed as the best sample source. LAMP showed a substantial agreement (κ = 0.80; p <0.0001) with Rt-PCR; therefore, it could be promising for the rapid diagnosis of CanL. Nevertheless, further studies should be performed to confirm these findings.

Bone-marrow aspirate material is commonly considered as one of the most sensitive tissues for a reliable diagnosis of leishmaniasis. The procedure herein described may permit less experienced veterinarians to be familiar with a quick and safe assessment method for leishmaniasis diagnosis in their patients. Animals are positioned in right lateral recumbency, and the area corresponding to the second, third, or fourth sternebra is identified and aseptically prepared. A 18-gauge needle connected to a 10 mL syringe is driven through the skin, up to the bone wall, and firmly pushed forward while rotating. Entry into the sternebra's cavity is clearly perceived by the fall of resistance offered by the cortex. Some 2,500 sternal bone-marrow samplings were safely and efficiently performed on 887 dogs of different breeds and aging from 6 months to 14 years, during eight years of clinical activity for routine diagnosis of canine leishmaniasis in pets or for the efficacy evaluation of anti-Leishmania immunobiologicals in dogs naturally exposed to parasite transmission. Most of the samples (1716) were from 387 dogs enrolled for anti-Leishmania vaccine studies. The safety of the method was particularly assessed on these dogs that as per study protocol were submitted to repeated bone-marrow aspirations (2–4 per year) in follow-up examinations.

Canine vector-borne diseases (CVBDs) are a spectrum of diseases caused by different pathogens transmitted by blood-feeding arthropoda. The aim of this study was to investigate leishmaniosis, babesiosis, and filarial infections in dogs with three different lifestyles (hunting, stray, and sheep dogs) in Molise, the smallest region of southern Italy, where data available about these parasitic infections are very scant. A cross-sectional survey was conducted on 318 hunting, 180 stray, and 218 sheep dogs. Immunofluorescence antibody test, blood smear, molecular techniques and Knott's test were performed to detect Leishmania infantum, Babesia spp. and filarial nematodes. Association between positivity to CVBDs, age, sex, and living conditions was evaluated. An overall prevalence of 12.3% of CVBDs caused by L. infantum (10.2%), B. canis canis (0.3%) and filarial nematodes (2.1%) was detected. Three dogs showed co-infections of L. infantum and B. c. canis (0.1%) or Acanthocheilonema reconditum (0.3%). A significantly association was found only for filarial infection in hunting dogs. These parasites were reported also in dogs without clinical signs. It is very important to plan effective control programs for CVBDs to guarantee not only the health and welfare of pets, but also the public safety, because some of mentioned parasites are of zoonotic importance.

Background: The aim of this study was to determine exposure to vector-borne pathogens (VBPs) in populations of dogs living on Greek islands in the Ionian and Aegean seas. Methods: In total, 1154 dogs with different lifestyles and of varying ages and breeds were randomly sampled and examined for the presence of clinical signs compatible with canine vector-borne diseases (CVBDs). Blood was collected from each individual animal. For the detection of antibodies against Leishmania spp., the WITNESS ® Leishmania test was performed, and positive samples were further examined with indirect enzymatic immunoassay (ELISA). Antibodies to Borrelia burgdorferi, Ehrlichia canis or E. ewingii, as well as Anaplasma phagocytophilum or A. platys were investigated using the Snap ® 4Dx ® Plus test. Positive Ehrlichia spp. and Anaplasma spp. samples were further examined using an indirect ELISA for further identification of the species. Results: In total, 25.6% of dogs were exposed to at least one of the pathogens investigated, with seroprevalences varying regionally. Of these seropositive dogs, 27.4% displayed clinical signs suggestive of CVBDs, such as cutaneous lesions, enlarged lymph nodes, pale mucous membranes, onychogryphosis and weight loss. The overall seroprevalence detected using the rapid tests was 15.3% for Leishmania spp., whereas 2.3% of the examined dogs were found to be positive for Anaplasma spp. and 7.5% for Ehrlichia spp. while B. burgdorferi was not detected. Twenty-four samples positive to A. phagocytophilum by ELISA were analysed by PCR for the presence of Anaplasma DNA. PCR and sequencing results showed the presence of A. platys DNA in 4 samples and E. canis DNA in 4 samples. The remaining samples (66.7%) were negative. Conclusions: In the present study, exposure of dogs to VBPs was shown in the geographical areas investigated. Results confirm that on Greek islands VBPs represent a constant health risk for both native and visiting dogs, suggesting the presence of distinct "hot-spots" of VBP infections on different islands. In order to reduce the risk of transmission and the spread to non-endemic regions, the protection of dogs through use of repellents and vaccines, together with owner education, seem to be of paramount importance.

BackgroundDuring last decade Lampedusa island (Italy) has been interested by a deep social change caused by the massive arrival of migrants from north Africa. The goal of this study was to evaluate current CanL burden and risk factors for Visceral Leishmaniosis (VL) on Lampedusa, actually based on very few data obtained in a previous study performed fifteen years ago. Two hundred and forty-two dogs were enrolled for the detection of Leishmania infantum infection by serology. In addition, an entomological investigation was performed to confirm the presence of Leishmania-vectors.ResultsSeroprevalence was of 54.13%. 223 sand flies specimens were collected. Among them, 4 species were identified: Phlebotomus perniciosus, P. papatasi, P. neglectus, Sergentomia minuta, with P. perniciosus the most abundant (67.7%; p < 0.01).ConclusionThe high proportion of seropositive dogs together with the presence of the most competent vector for L. infantum, P. perniciosus, demonstrate that L. infantum abundantly circulates in the island and may constitute a risk for people, particularly for hosted migrants.

Infected dogs are considered the main domestic animal reservoirs for Leishmania infantum parasite. Infectiousness to competent phlebotomine vectors has been associated with many factors, the main being the severity of the disease exhibited by infected dogs. This study examines the relationship between different clinical parameters and the infectiousness to colonized Phlebotomus perniciosus sand flies having a blood meal on dogs. Data obtained in the present study come from an untreated group of Leishmania sick dogs submitted to xenodiagnosis for the evaluation of a spot on insecticide solution. Seventeen dogs were diagnosed as affected by leishmaniasis through clinical examination, immunofluorescence antibody test (IFAT) serology, and loop-mediated isothermal amplification (LAMP). The disease severity (clinical score) was staged by using a numeric value derived from eight clinical and parasitological parameters. Xenodiagnosis was performed on caged dogs exposed for 1.5 h to sand-fly bites. The following parameters related to sand flies were examined: blood feeding (% of blood engorged females), promastigote detection (% of promastigote-positive sand flies), promastigote burden, and the promastigote stage maturation (potential transmissibility rate). Statistical relationship between the clinical score and entomological parameters was investigated, as well as the possible correlation between each clinical and laboratory parameters and sand fly infection/infectivity. The severity of clinical score may influence the blood feeding by, and the probability of promastigote detection in, sand flies; skin lesions seem to be the main factor that influences the rate of blood feeding. Promastigote burden is related to IFAT titer, skin lesions, and clinical score. All entomological parameters are strongly related among them. This study confirms that both P. perniciosus infection and infectivity are influenced by a dog's clinical condition.