A cross-sectional study on performance evaluation in Italian standardbred horses' real-time PCR-positive for Theileria equi.

BACKGROUND: Inflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared between PCR-positive and -negative horses. RESULTS: This cross-sectional study included 120 well-trained IS racehorses and was performed over a two-years period. The prevalence of T. equi was 36.3%, whereas all samples were negative for B. caballi. Red blood cells count, haemoglobin concentration, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase activities were significantly higher in PCR-positive horses, whereas blood urea nitrogen, globulin concentration and globulin-to-albumin ratio were significantly lower in PCR-positive horses compared to PCR-negative ones. Nonetheless, all values fell within the physiological range. The best racing time, which was selected as the most representative of the performance metrics at the principal component analysis, was not affected by PCR positivity, the muscle mass score or the training yard. The best racing time was significantly better in horses with a mild or no signs of muscular atrophy, within the PCR-positive group. The muscle mass score was associated with the training yard in PCR-negative horses. CONCLUSIONS: Prevalence of T. equi was high in IS racehorses in southern Italy. The absence of obvious changes in haematological and biochemical parameters, as well as performance metrics in positive horses, highlights the need for specific diagnostic tests to identify chronically infected horses.

Serological and entomological survey of canine leishmaniasis in Lampedusa island, Italy.

BACKGROUND: During 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. RESULTS: Seroprevalence 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). CONCLUSION: The 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.

First detection of Leishmania major in dogs living in an endemic area of zoonotic cutaneous leishmaniasis in Tunisia.

BACKGROUND: Dogs are considered the main domestic animals that may be a reservoir for Leishmania infantum, the agent of zoonotic visceral leishmaniasis (ZVL) in several countries of the world. The dog may host other Leishmania species, but its epidemiological role in the maintenance and spreading of these parasites is not completely elucidated. Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major, affects thousands of people every year and is particularly diffused in many countries of North Africa and Middle East Asia. In ZCL endemic countries, few reports of L. major-positive dogs have been reported, probably because most human cases occur in poor rural areas where the social role of the dog and its medical management is not well considered. The aim of the present study is to better understand the possible involvement of domestic dogs in the epidemiology of ZCL. METHODS: Our research focused on a well-established endemic focus of ZCL, in the area of Echrarda, Kairouan Governorate, central Tunisia. A total of 51 dogs with no or mild clinical signs of vector borne diseases were selected in small villages where human cases of ZCL are yearly present. All dogs were sampled for the Leishmania spp. diagnosis, by using the following procedures: blood sample for serology and buffy coat quantitative polymerase chain reaction (qPCR), popliteal fine needle aspiration, and cutaneous biopsy punch for lymph node and skin qPCR. RESULTS: The results demonstrated a high percentage (21.6%) of dogs positive at least at one or more test; the most sensitive technique was the lymph node qPCR that detected 8/11 positive dogs. Nine, out of the eleven positive dogs, resulted as infected by Leishmania infantum; ITS1-PCR-sequencing allowed Leishmania major identification in the remaining two cases, both from the popliteal lymph node samples, which can suggest a possible visceral spread of a cutaneous Leishmania species in the dog. Interestingly, one of the two L. major-positive dogs was living in the same house where 6-year-old children showed cutaneous lesions referred to as ZCL. CONCLUSIONS: To our knowledge, this is the first report of L. major-positive dogs in Tunisia, the  epidemiological role of which remains under investigation.

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.

Effect of diet on postprandial glycemic and insulin responses in healthy dogs.

Introduction: Dog owners have gradually changed their approach, paying more attention to the nutrition and health of their animals. Various pet foods with different ingredients and nutritional characteristics are available on the market. The present study aimed to evaluate the administration of three diets, namely, two grain-free (GF1 and GF2) and one grain-based (CB), with different sources of carbohydrates that can influence the glycemic and insulin postprandial responses in healthy dogs. Materials: Fifteen healthy dogs were dived in three groups and alternatively fed each diet for 50 days. Blood samples were collected at beginning of each feeding period. Glycemia and insulin were measured before and after 120, 240 and 360 minutes diet administration to evaluate postprandial responses. Results: GF2 diet showed the highest level of albumin and mean insulin concentration (p < 0.001). Furthermore, the GF1 diet caused the smallest (p < 0.001) glucose and insulin area under the curve (AUC) and the lowest (p < 0.05) glucose nadir. Otherwise, GF1 showed the highest (p < 0.01) insulin time to peak. The GF2 diet showed the highest level of albumin while reporting the lowest amount of fructosamine (p < 0.05). The diet GF2 registered the highest (p < 0.001) level of insulin zenith. The cereal-based (CB) diet reported the highest amount of fructosamine (p < 0.05). The CB diet had the highest levels of glucose and the highest (p < 0.001) glucose and insulin mean concentrations. Diet CB reported the lowest (p < 0.001) insulin nadir. Discussion: Diets with different carbohydrate sources and chemical compositions could modulate the glycemic response in healthy dogs. Bearing in mind that glycemic/insulin postprandial responses influence energy availability and that different dogs have specific lifestyles, it may be preferable to also consider these aspects when choosing a maintenance diet for animals.

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.

Examining the Veterinary Electronic Antimicrobial Prescriptions for Dogs and Cats in the Campania Region, Italy: Corrective Strategies Are Imperative.

Companion animals are increasingly being recognised as important contributors to the spread of antimicrobial-resistant bacteria. The present work aimed to measure the antimicrobial drug prescribing in dogs and cats in the Campania Region, Italy by analysing the Veterinary Electronic Prescriptions (VEPs) between 2019 and 2020. The medical records associated with antimicrobial drug prescriptions were collected according to the drug administration (systemic or topical) and the rationale for the treatment chosen. In the period under investigation, 166,879 drugs were prescribed of which 129,116 (73.4%) were antimicrobial. A total of 83,965 (65%) antibiotics were prescribed to dogs, 40,477 (31.4%) to cats, and 4674 (3.6%) to other companion animals. In dogs, 90.5% of VEPs prescribed for systemic treatment included an antimicrobial Critically Important or Highly Important or Important for human medicine (WHO, 2018). The most widely prescribed class was fluoroquinolones. The antimicrobials prescribed were mainly metronidazole-spiramycin (29.7%), amoxicillin-clavulanic (19.6%), enrofloxacin and cephalexin in dogs (16.5%) and enrofloxacin (22.6%) and amoxicillin-clavulanic acid (21.4%) in cats. Based on the results, the widespread use of broad-spectrum antimicrobials and the use of molecules for which limitations should be observed according to the EMA guidelines has emerged.

Monitoring and detection of new endemic foci of canine leishmaniosis in northern continental Italy: An update from a study involving five regions (2018-2019).

Canine leishmaniosis (CanL) is an emerging zoonosis caused by Leishmania infantum and transmitted in southern Europe by phlebotomine sand flies of the subgenus Phlebotomus (Larroussius). Endemic foci of CanL have been recorded in northern continental Italy since early 1990s and attributed to the northward expansion of vector populations due to climatic changes in association with travelling/relocated infected dogs from the southern Mediterranean littoral. In this study, further spread of endemic Leishmania foci was monitored during 2018-2019 in five regions (Aosta Valley, Piedmont, Lombardy, Veneto and Friuli-Venezia Giulia), with focus to territories where investigations were not performed, or they have been inconclusive. Clinical cases of CanL identified by local veterinary practitioners and confirmed by reference diagnosis centers were regarded as autochthonous if their origin from, or travel to, areas endemic for CanL were excluded in the previous ≥2 years. Around these index cases, i) serosurveys for L infantum were carried out where indicated, ii) sampling from potential autochthonous cases in healthy or clinically-suspected resident dogs was intensified by collaborating veterinary practitioners, and iii) suitable sites were investigated for the presence of competent phlebotomine vectors. Fifty-seven municipalities whose enzootic status of CanL was unreported before 2018, were identified as endemic. The stability of 27 foci recorded over the past decade, was also confirmed. Competent phlebotomine vectors, mainly Phlebotomus perniciosus, were collected for the first time in 23 municipalities. The newly recorded endemic municipalities appear to be distributed over a west-to-east decreasing gradient: 30 in Piedmont, 21 in Lombardy, 4 in Veneto and 2 in Friuli-Venezia Giulia. As regards Veneto, it should be noted that a relatively restricted territory was investigated as several municipalities of the region had already been surveyed and detected as endemic for CanL in the past. Cold climate conditions of the easternmost region of Friuli-Venezia Giulia bordering non-endemic territories of Slovenia, are probably less favorable to L infantum transmission.

Clinical Significance of ROMs, OXY, SHp and HMGB-1 in Canine Leishmaniosis.

This study aimed to investigate the role of oxidative stress parameters (ROMs, OXY, SHp), the Oxidative Stress index (OSi), and High Mobility Group Box-1 protein (HMGB-1) in canine leishmaniosis (CanL). For this study, thirty dogs, naturally infected with Leishmania spp. (Leishmania Group, LEISH) and ten healthy adult dogs (control group, CTR) were included. The diagnosis of CanL was performed by a cytological examination of lymph nodes, real time polymerase chain reaction on biological tissues (lymph nodes and whole blood), and an immunofluorescence antibody test (IFAT) for the detection of anti-Leishmania antibodies associated with clinical signs such as dermatitis, lymphadenopathy, onychogryphosis, weight loss, cachexia, lameness, conjunctivitis, epistaxis, and hepatosplenomegaly. The HMGB-1 and oxidative stress parameters of the LEISH Group were compared with the values recorded in the CTR group (Mann Whitney Test, p < 0.05). Spearman rank correlation was applied to evaluate the correlation between the HMGB-1, oxidative stress biomarkers, hematological and biochemical parameters in the LEISH Group. Results showed statistically significant higher values of SHp in the LEISH Group. Specific correlation between the ROMs and the number of red blood cells, and between HGMB-1 and SHp were recorded. These preliminary data may suggest the potential role of oxidative stress in the pathogenesis of CanL. Further studies are undoubtedly required to evaluate the direct correlation between inflammation parameters with the different stages of CanL. Similarly, further research should investigate the role of ROMs in the onset of anemia.

Clinical Findings in Healthy Dogs Fed With Diets Characterized by Different Carbohydrates Sources.

In recent years, pet owners have become more interested in the ingredients, and quality of pet-food, and several studies have demonstrated that feed management could affect healthy status. Recently, some authors indicated that commercial diets formulated without cereals, or using unconventional protein, and starch sources, can cause a reduction in taurine levels in both whole blood, and plasma. Nevertheless, the specific mechanism by means of which nutritional factors determine this reduction is not completely clear. Thirty neutered half-breed dogs were recruited at a kennel in the province of Naples (Italy) to investigate the influence of carbohydrates sources, and dietary density of nutrients on healthy status of dogs in terms of blood count, and biochemical parameters. The dogs were housed in the kennel and divided into three distinct groups. Three iso-energy, and iso-nitrogen commercial kibble diets (named GF1, GF2, and CB) with different protein, and carbohydrates contents, and carbohydrates sources were chosen for the trial. The chemical composition and amino acid profile of each of the three tested diets were analyzed. Moreover, blood samples of each dog were collected to evaluate the hematological and biochemical profiles. The taurine level was determined both on plasma and whole blood. The effect of the diets was analyzed statistically, and all tested diets were compared to the control one. There were significant differences between the three tested diets as regards their chemical composition. The concentrations of all amino acids seem to reflect protein content diets. The hematological profile resulted within the ranges considered physiological for the canine species for all subjects. Compared to the control diet, the three tested diets showed significant differences in blood count for MCHC and platelets. The biochemical profile showed significant differences between the diets, particularly their AST, fructosamine, lipase, and triglycerides values. The diets did not affect the blood and plasma taurine levels. They resulted in higher than optimal reserve levels. Preliminary results showed that the sources of carbohydrates and use of balanced diets affected only some biochemical parameters and did not alter the levels of taurine in healthy adult dogs.

Leishmania spp.-Infected Dogs Have Circulating Anti-Skeletal Muscle Autoantibodies Recognizing SERCA1.

Leishmania spp. infection is associated with an inflammatory myopathy (IM) in dogs. The pathomechanism underlying this disorder is still elusive, however, the pattern of cellular infiltration and MHC I and II upregulation indicate an immune-mediated myositis. This study aimed to investigate the presence of autoantibodies targeting the skeletal muscle in sera of leishmania-infected dogs and individuate the major autoantigen. We tested sera from 35 leishmania-infected dogs and sera from 10 negative controls for the presence of circulating autoantibodies with indirect immunofluorescence. Immunoblot and mass spectrometry were used to identify the main target autoantigen. Immunocolocalization and immunoblot on immunoprecipitated muscle proteins were performed to confirm the individuated major autoantigen. We identified circulating autoantibodies that recognize skeletal muscle antigen(s) in sera of leishmania-infected dogs. The major antigen was identified as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1). We also found that canine SERCA1 presents several identical traits to the calcium-translocating P-type ATPase of Leishmania infantum. In the present study, we defined circulating anti-SERCA1 autoantibodies as part of the pathogenesis of the leishmania-associated IM in dogs. Based on our data, we hypothesize that antigen mimicry is the mechanism underlying the production of these autoantibodies in leishmania-infected dogs.

Examining the Relationship of Clinical and Laboratory Parameters With Infectiousness to Phlebotomus perniciosus and Its Potential Infectivity in Dogs With Overt Clinical Leishmaniasis.

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.

Assessment of Circulating Immune Complexes During Natural and Experimental Canine Leishmaniasis.

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.

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 studies.

Mapping the canine vector-borne disease risk in a Mediterranean area.

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.

Clinical, Molecular and Serological Diagnosis of Canine Leishmaniosis: An Integrated Approach.

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.

Simultaneous Detection of Parasitic Vector Borne Diseases: A Robust Cross-Sectional Survey in Hunting, Stray and Sheep Dogs in a Mediterranean Area.

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.

Nostril mass caused by Leishmania spp. in a dog.

Canine leishmaniasis (CanL) is a systemic zoonotic disease caused by the protozoan Leishmania, an intracellular macrophage parasite, transmitted by the bite of phlebotomine sandflies. In dogs, the clinical disease is mostly characterised by symptoms associated with viscerocutaneous lesions such as lymphadenopathy, splenomegaly, skin lesions, and renal and ocular disease caused by the deposition of immune complexes. The parasite may provoke mucosal lesions which cause atypical clinical signs. The aim of this study is to describe an atypical nostril mass in a dog infected by Leishmania. Clinical examination did not show any systemic clinical signs, while haematological, biochemical, and urinary parameters demonstrated a mild disease stage. Diagnosis was confirmed through the isolation of cultured live parasites by biopsy. The dog was treated with a combination of miltefosine and allopurinol, showing full remission of clinical symptoms after 2 months. The authors outline the importance of considering CanL in the differential diagnosis of mucous and tumour-like lesions.

Sternal Aspiration of Bone Marrow in Dogs: A Practical Approach for Canine Leishmaniasis Diagnosis and Monitoring.

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.

Prospective study on the incidence and progression of clinical signs in naïve dogs naturally infected by Leishmania infantum.

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.