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Abstract The enteric protozoan parasites Giardia duodenalis and Cryptosporidium spp. are common cause of diarrhea in pet dogs and cats, affecting primarily young animals. This comparative study evaluates the diagnostic performance of conventional and molecular methods for the detection of G. duodenalis and Cryptosporidium spp. infection in dogs and cats. The compared diagnostic assays included merthiolate-iodine-formalin (MIF) method, lateral flow immunochromatography rapid test (ICT) and real-time PCR; using direct immunofluorescence assay (DFA) as golden standard. The study included the analysis of 328 fecal samples from different dog (n = 225) and cat (n = 103) populations. According to DFA, the overall prevalence of G. duodenalis was 24.4% (80/328, 95% CI: 19.8–29.4), varying from 11.6% (12/103, 95% CI: 6.2–19.5) in cats to 30.2% (68/225, 95% CI: 24.3–36.7) in dogs. The overall prevalence of Cryptosporidium spp. was 4.0% (13/328, 95% CI: 2.1–6.7), varying from 2.9% (3/103, 95% CI: 0.6–8.3) in cats to 4.4% (10/225, 95% CI: 2.1–8.0) in dogs. MIF was only used for the detection of G. duodenalis, which was identified by this method in 22.7% of dogs and 7.8% of cats, respectively. DFA was the most sensitive technique for detecting G. duodenalis in samples from dogs and cats (p-value: < 0.001), followed by real-time PCR. Identification of Cryptosporidium infections was most effectively accomplished by the combination of DFA and PCR technique (p-value: < 0.001). In addition, epidemiological (sex, age, origin) and clinical (fecal consistency) variables were collected to assess their potential associations with an increased likelihood of infection by G. duodenalis and/or Cryptosporidium spp. Breeder dogs were more likely to harbor G. duodenalis infection (p-value: 0.004), whereas female cats were significantly more infected with Cryptosporidium (p-value: 0.003). In conclusion, DFA (alone or in combination with PCR) has been identified as the most accurate and cost-effective method for detecting G. duodenalis and Cryptosporidium spp. in fecal samples from pet dogs and cats. This highlights their importance in both veterinary and clinical settings for enabling prompt treatment and preventing potential transmission to humans.

Abstract Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens. Graphical Abstract

Abstract Background Cats are now recognized as competent hosts for Leishmania infantum and a blood source for sand fly vectors. Although canine leishmaniosis (CanL) is endemic in Mediterranean Basin countries, large-scale epidemiological studies are lacking for feline leishmaniosis (FeL). This study aimed to assess the prevalence of L. infantum infections, associated risk factors, clinical signs, and clinicopathological abnormalities in domestic cat populations from six Mediterranean Basin countries. Methods From 2019 to 2022, blood and serum samples of cats (n = 2067) living in Italy (n = 300), Greece (n = 297), Portugal (n = 295), France (n = 231), Israel (n = 313), and Spain (n = 631) were collected along with animal data (i.e., age, sex, breed, housing conditions, and geographical origin), clinical signs, and laboratory blood test parameters. Cats were grouped according to their age as kittens (up to 1 year), young (older than 1 and younger than 7 years), mature (between 7 and 10 years), and senior (older than 10 years). Serum samples were tested for L. infantum by immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), and blood samples of seropositive cats were tested for L. infantum kinetoplast deoxyribonucleic acid (kDNA). Viral infection by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) was molecularly addressed in all cats enrolled. Statistical analysis was performed to evaluate the association between the risk of L. infantum infection and independent variables, and among co-infection of L. infantum with FIV and/or FeLV, clinical signs, and clinicopathological abnormalities. Results Overall, 17.3% (358/2067) of cats scored positive for L. infantum by serological tests. Specifically, 24.7% were from Portugal, 23.2% from Greece, 16.6% from Israel, 15% from Spain, 13.3% from France, and 12.6% from Italy. Leishmania infantum DNA was detected in 15 seropositive animals. Housing condition and FIV infection proved to be risk factors for FeL. Leishmania seropositivity was significantly associated with weight loss, lymphadenomegaly, gingivostomatitis, and oral ulcers, as well as with reduced albumin and albumin/globulin ratio, increased total globulins and total proteins, leukocytosis, and thrombocytosis. Conclusions This study provides, for the first time, a large-scale epidemiological survey on FeL and its clinical presentation, revealing that L. infantum circulates among domestic cats, especially shelter/free-roaming and FIV-infected animals, living in CanL endemic countries of the Mediterranean Basin. Graphical Abstract

Abstract Background Canine leishmaniosis (CanL), caused by Leishmania infantum, is an important vector-borne parasitic disease in dogs with implications for human health. Despite advancements, managing CanL remains challenging due to its complexity, especially in chronic, relapsing cases. Mathematical modeling has emerged as a powerful tool in various medical fields, but its application in understanding CanL relapses remains unexplored. Methods This retrospective study aimed to investigate risk factors associated with disease relapse in a cohort of dogs naturally infected with L. infantum. Data from 291 repeated measures of 54 dogs meeting the inclusion criteria were included. Two logistic mixed-effects models were created to identify clinicopathological variables associated with an increased risk of clinical relapses requiring a leishmanicidal treatment in CanL. A backward elimination approach was employed, starting with a full model comprising all potential predictors. Variables were iteratively eliminated on the basis of their impact on the model, considering both statistical significance and model complexity. All analyses were conducted using R software, primarily employing the lme4 package, and applying a significance level of 5% (P

Abstract ParSCo (Parasitology Summer Course) is an intense, 1-week-long summer course organized by the Parasitology Unit of the Department of Veterinary Medicine, University of Bari, Italy, with the support of the World Association for the Advancement of Veterinary Parasitology (WAAVP), the European Veterinary Parasitology College (EVPC) and Parasites and Vectors. The course, which is conducted in southern Italy, is planned for parasitologists and post-graduate students working in the field of parasitology. The course consists of theoretical and practical lessons, which include the collection, identification and diagnosis of parasites of pets, livestock and wildlife. The participants in ParSCo are afforded the opportunity to be involved in clinical examination and sample collection for the diagnosis of parasitic diseases (e.g. leishmaniosis, thelaziosis and many tick-borne diseases) present in the Mediterranean Basin. The course is conducted at Casa di Caccia, a hunting lodge situated in the Gallipoli Cognato Forest near the Basento River in the Basilicata region in southern Italy. In addition to the training purpose, ParSCo is a great opportunity for sharing knowledge and expertise while becoming part of the parasitology community in a pleasant environment. In this editorial, we share some information and celebrate 10 years of ParSCo, looking forward to forthcoming sessions of this unique parasitology summer course. Graphical Abstract

Gelatin is a promising biopolymer for edible coatings thanks to its low cost and gelling properties. However, its weak mechanical properties limit its use. This study aimed to develop a gelatin coating with tomato extract, analyzing its antioxidant activity and rheological properties for food applications. Gelatin concentrations (2, 5, and 7%) were evaluated, and it was determined that 7% with 7.5% glycerol was the optimal mixture. Three concentrations of tomato extract (0.5, 1, and 1.5%) were added, and antioxidant activity was evaluated using the ABTS technique, as well as the interaction of components through FT-IR and physicochemical analysis. The results showed that there were no significant differences in terms of their physicochemical characterization, maintaining a pH of 5 and a yellowish hue. The FT-IR spectra indicated there were hydrogen bond interactions between gelatin and the extract. The antioxidant capacity was higher with the 1.5% extract, achieving an inhibition of 58.9%. It was found that the combination of the different materials used improved the rheological (specifically the viscosity and stability of the material) and antioxidant properties of the gelatin. These findings suggest that modified gelatin coatings may be effective in extending the shelf life of foods.

Autism spectrum disorder (ASD) involves social communication difficulties and repetitive behaviors, and it has a growing prevalence worldwide. Symptoms include cognitive impairments, gastrointestinal (GI) issues, feeding difficulties, and psychological problems. A significant concern in ASD is food selectivity, leading to nutrient deficiencies. Common GI issues in ASD, such as constipation and irritable bowel syndrome, stem from abnormal gut flora and immune system dysregulation. Sensory sensitivities and behavioral challenges exacerbate these problems, correlating with neurological symptom severity. Children with ASD also exhibit higher oxidative stress due to low antioxidant levels like glutathione. Therapeutic diets, including ketogenic, high-antioxidant, gluten-free and casein-free, and probiotic-rich diets, show potential in managing ASD symptoms like behavior, communication, GI issues, and oxidative stress, though the evidence is limited. Various studies have focused on different populations, but there is increasing concern about the impact among children. This review aims to highlight the food preferences of the ASD population, analyze the effect of the physicochemical and nutritional properties of foods on the selectivity in its consumption, GI problems, and antioxidant deficiencies in individuals with ASD, and evaluate the effectiveness of therapeutic diets, including diets rich in antioxidants, gluten-free and casein-free, ketogenic and essential fatty acids, and probiotic-rich diets in managing these challenges.

IntroductionCanine leishmaniosis (CanL) is a systemic disease that affects dogs. When multiplication of the parasite cannot be controlled, dogs consistently show high levels of antigen and IgG antibodies, which lead to the formation of circulating immune complexes (CIC). Timely intervention to reduce the parasite load and CIC levels is crucial for preventing irreversible organ damage. However, a diagnostic test to quantify CIC levels is currently lacking.MethodsIn this real-world study, we aimed to examine the performance of a new ELISA to measure CIC levels in dogs naturally infected with Leishmania infantum. Thirty-four dogs were treated according to their clinical condition and followed for 360 days. Before (day 0) and after treatment (days 30, 90, 180, 270, and 360), all dogs underwent a physical examination, and blood samples were obtained for CBC, biochemical profile, serum protein electrophoresis and IFAT. Serum PEG-precipitated CIC were determined by ELISA.ResultsOur results indicate higher CIC levels in dogs in advanced disease stages showing higher antibody titres (p

Though scantly investigated, Leishmania infantum infection and clinical cases of leishmaniasis in cats have been recently reported in several countries of the Mediterranean basin, with large variability in prevalence data. A major limitation in the comparability of the data available is attributed to the differences in diagnostic techniques employed and cat populations sampled. The aim of this study was to assess the prevalence of L. infantum infection in owned cats across Italy by serological and molecular tests and the identification of potential risk factors. Blood samples from 2,659 cats from northern (n = 1,543), central (n = 471) and southern (n = 645) Italy were tested for antibodies against L. infantum, by an immunofluorescence antibody test and for the parasites' DNA, by real-time PCR. Samples were additionally screened for feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) proviral DNAs. An overall cumulative L. infantum prevalence of 3.9% was recorded by serology (3.3%) and/or qPCR (0.8%), with a higher rate (10.5%) in southern Italy. The risk of L. infantum infection in cats was significantly associated to the geographical areas (South vs North and Centre; p

Abstract Background Strongyloides stercoralis is a soil-transmitted intestinal nematode with a complex life cycle that primarily affects humans, non-human primates, dogs, and occasionally cats. This study presents, to the best of our knowledge, the first case of S. stercoralis infection and its genotyping in a domestic dog from Argentina. Methods The patient was a female wired-haired Teckel dog exhibiting recurrent coughing. Coproparasitological analysis using the Baermann technique revealed the presence of rhabditiform larvae morphologically compatible with S. stercoralis. To confirm this finding, molecular diagnosis (18S ribosomal RNA) and analysis of the cox1 gene were performed. Results We identified a haplotype (HP20) that has previously only been related to S. stercoralis infection in dogs, but was found in the present study to be highly related to the haplotype (HP16) of a zoonotic variant and divergent from those previously described from human patients in Argentina. Furthermore, unlike in human cases following treatment with ivermectin, the dog was negative after moxidectin treatment according to polymerase chain reaction of the sampled faeces. Conclusions This case report shows the importance of further investigation into potential transmission events and prevalences of S. stercoralis in dogs and humans in South America. The results reported here should also encourage future work that examines different scenarios of infection with S. stercoralis in dogs and humans with the aim of integrating clinical management, diagnosis, treatment and follow-up strategies in the quest for new approaches for the treatment of this disease in animals and humans. The findings support the adoption of a One Health approach, which recognizes the interconnectedness between animal and human health, in addressing parasitic infections such as strongyloidiasis. Graphical Abstract

Abstract Background Domperidone (Leisguard®) is an immunomodulatory drug used as a preventive measure in healthy dogs. However, no studies have been published in healthy Leishmania infantum-seropositive dogs. The aim of this study was to evaluate the clinical efficacy and safety of domperidone as immunotherapy in Leishmania-seropositive healthy dogs. Methods Sixty-seven dogs were treated with domperidone at 0.5 mg/kg and 44 dogs received placebo, once daily for 4 consecutive weeks. Monthly treatments were repeated every 4 months until the end of the 1-year follow-up period. Veterinary examinations were performed on days 0, 30, 120, 150, 240, 270 and 360. Samples of blood and urine were collected on days 0, 120, 240 and 360 for routine laboratory tests and quantitative in-house ELISA for the detection of L. infantum-specific antibodies. Furthermore, Leishmania real-time PCR and IFN-γ ELISA were performed at day 0 and the end of the study. Dogs that developed disease were withdrawn from the study and classified as sick dogs. Adverse drug reactions were reported. Results Thirty dogs developed disease during the follow-up period: 13/67 (19.4%) in the group treated with domperidone and 17/44 (38.6%) in the placebo-treated group (P = 0.03). Low-seropositive dogs treated with domperidone (4/40, 9.1%) were significantly less likely to develop disease compared to low-seropositive dogs treated with placebo (7/24, 29.2%; P = 0.04), while no differences were found between domperidone (9/23, 39.1%) and placebo (10/20, 50%) in medium- to high-seropositive dogs. At the end of the study, a higher proportion of Leishmania PCR-positive dogs was observed in the placebo-treated group (16/33, 48.5%) compared to the domperidone group (13/51, 25.5%; P = 0.04). Furthermore, low-seropositive dogs treated with domperidone with an increase of IFN-γ concentration presented a higher increase than those treated with placebo at the end of the study. Four dogs treated with domperidone presented self-limiting diarrhea. Conclusions Healthy dogs with low L. infantum antibody levels treated with domperidone were less likely to develop disease compared to placebo-treated dogs. Furthermore, domperidone presented a good safety profile. Graphical abstract

Abstract Background Canine leishmaniosis caused by Leishmania infantum is an endemic disease in Spain. The dog is considered the main reservoir, and the detection of specific serum antibodies against L. infantum antigen is the most used technique for diagnosing this infection. The LEISCAN LEISHMANIA ELISA test is a commercialized enzyme‐linked immunosorbent assay for the detection and measurement of canine anti‐Leishmania serum antibodies. Objectives The aim of this study was to assess seroprevalence results of apparently healthy dogs in different areas of Spain using LEISCAN. Methods Collection of sera from 5451 apparently healthy dogs was performed between 2020 and 2021 in different areas of Spain. Dogs were of adult age (≥12 months), were not previously diagnosed with clinical leishmaniosis or vaccinated against Leishmania and did not present clinical signs compatible with L. infantum infection. LEISCAN was performed following the manufacturer's protocol. Results The overall seroprevalence was 5.5%. The highest seroprevalences were found in the Southeast of Spain: Comunidad Valenciana (14%) and Región de Murcia (14%), whereas the lowest seroprevalences were found in Northern Spain: Galicia (1%), Navarra (2%) and Castilla y León (2%) (p‐value

Tick-borne diseases (TBDs) are caused by pathogens of human and veterinary concern representing a major public health issue worldwide. Although feline medicine has progressed much in the recent decades, data on feline TBDs (FeTBDs) remain scant. Therefore, this study aimed to assess the prevalence of apicomplexan parasite infections, associated risk factors and clinical-hematological abnormalities in domestic feline populations from countries of the Mediterranean basin. Blood and serum samples from cats (n = 600) living in France, Greece, Israel, Italy, Portugal and Spain were collected along with animal data (i.e., age, sex, breed, housing conditions and geographical origin), clinical signs and laboratory blood test parameters. Cats were grouped according to their age as kitten (up to one year), young (between one and six years), mature (between seven and ten years) and senior (older than ten years). Blood samples were tested for Hepatozoon spp. and piroplasmids by conventional PCR targeting 18S rRNA gene. The overall prevalence of Hepatozoon spp. infection was 14.5%, being significantly higher in cats from Greece (30%) and Portugal (23%), followed by Spain (15%), Israel (15%) and France (4%). Cats from Italy scored negative. Hepatozoon felis was identified in 86 animals, with three different sequence types and H. silvestris was detected in one shelter cat from Portugal. No piroplasmid DNA was amplified. The risk of Hepatozoon spp. infection was related to feline geographical provenience, housing condition and age. No statistical correlation was reported with any clinical signs, while increased alanine aminotransferase (ALT) activity was the only laboratory abnormality significantly associated (p = 0.03) with the infection. Data suggest a high circulation of H. felis, and only occasionally of H. silvestris, within domestic feline populations in the Mediterranean basin, mainly in shelter or free roaming and young cats with asymptomatic or subclinical infection.

Abstract Large populations of unowned cats constitute an animal welfare, ecological, societal and public health issue worldwide. Their relocation and homing are currently carried out in many parts of the world with the intention of relieving suffering and social problems, while contributing to ethical and humane population control in these cat populations. An understanding of an individual cat’s lifestyle and disease status by veterinary team professionals and those working with cat charities can help to prevent severe cat stress and the spread of feline pathogens, especially vector-borne pathogens, which can be overlooked in cats. In this article, we discuss the issue of relocation and homing of unowned cats from a global perspective. We also review zoonotic and non-zoonotic infectious agents of cats and give a list of practical recommendations for veterinary team professionals dealing with homing cats. Finally, we present a consensus statement consolidated at the 15th Symposium of the Companion Vector-Borne Diseases (CVBD) World Forum in 2020, ultimately to help veterinary team professionals understand the problem and the role they have in helping to prevent and manage vector-borne and other pathogens in relocated cats. Graphical Abstract

Abstract Background Canine vector-borne disease (CVBD) has been an area of increasing interest in Europe over the last few decades, and there have been changes in the prevalence and distribution of many of these diseases. Monitoring CVBD infections in Europe is often done by individual countries, but aggregated data for the European countries are helpful to understand the distribution of CVBDs. Methods We used an extensive retrospective database of results from point-of-care rapid enzyme-linked immunosorbent assay (ELISA) tests on dogs across Europe to identify distribution and seropositivity in animals tested for selected CVBDs (Anaplasma spp., Ehrlichia spp., Borrelia burgdorferi, Leishmania spp., and Dirofilaria immitis) from 2016 through 2020. Geographic distribution of positive tests and relative percent positive values were mapped by the Nomenclature of Territorial Units for Statistics classification for regions with sufficient test results for reporting. Results A total of 404,617 samples corresponding to 1,134,648 canine results were available from dogs tested in 35 countries over the 5-year study period. Over this period the number of test results per year increased whereas test positivity decreased. Leishmania spp. had the largest increase in total test results from 25,000 results in 2016 to over 60,000 results in 2020. Test positivity for Leishmania spp. fell from 13.9% in 2016 to 9.4% in 2020. Test positivity fell for Anaplasma spp. (7.3 to 5.3%), Ehrlichia spp. (4.3 to 3.4%), and Borrelia burgdorferi (3.3 to 2.4%). Dirofilaria immitis test positivity trended down with a high of 2.7% in 2016 and low of 1.8% in 2018. Leishmania spp. test positivity was highest in endemic areas and in several non-endemic countries with low numbers of test results. Co-positivity rates were significantly higher than expected for all pathogen test positive pairs except for Ehrlichia spp. with Borrelia burgdorferi and D. immitis with Borrelia burgdorferi. Conclusions This study represents the largest data set on CVBD seropositivity in Europe to date. The increase in the number of test results and decreasing test positivity over the study period may reflect changes in testing behavior and increased screening of healthy animals. The Europe-wide mapping of CVBD provides expected test positivity that can help inform veterinarians’ decisions on screening and improve prevention and identification of these important, sometimes zoonotic, diseases. Graphical Abstract

IntroductionCoronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available.MethodsHerein, the first comparative study of six “in house” and two commercial serological tests developed to monitor SARS-CoV-2 infection in pets was performed with a well-coded panel of sera (61 cat sera and 74 dog sera) with a conservative criterion (viral seroneutralisation and/or RT–qPCR results) as a reference. Four “in house” tests based on either the RBD fragment of the spike protein (RBD-S) or the N-terminal fragment of the nucleoprotein (N) were developed for the first time. The analytical specificity (ASp) of those tests that showed the best diagnostic performance was assessed. The validation included the analysis of a panel of sera obtained pre-pandemic from cats and dogs infected with other coronaviruses to determine the analytical Sp (17 cat sera and 41 dog sera).Results and discussionELISAS based on the S protein are recommended in serosurveillance studies for cats (RBD-S SALUVET ELISA, ELISA COVID UNIZAR and INgezim® COVID 19 S VET) and dogs (INgezim® COVID 19 S VET and RBD-S SALUVET ELISA). These tests showed higher diagnostic sensitivity (Se) and DSp in cats (>90%) than in dogs. When sera obtained prior to the pandemic and from animals infected with other coronaviruses were analyzed by RBD-S and N SALUVET ELISAs and INgezim® COVID 19 S VET, a few cross reactors or no cross reactions were detected when dog and cat sera were analyzed by tests based on the S protein, respectively. In contrast, the number of cross reactions increased when the test was based on the N protein. Thus, the use of tests based on the N protein was discarded for serodiagnosis purposes. The results obtained revealed the most accurate serological tests for each species. Further studies should attempt to improve the diagnostic performance of serological tests developed for dogs.

Abstract The Companion Vector-Borne Diseases (CVBD) World Forum is a working group of leading international experts who meet annually to evaluate current scientific findings and future trends concerning the distribution, pathogenesis, clinical presentation, diagnosis and prevention of vector-borne infections of dogs and cats. At the 14th Symposium of the CVBD World Forum in Trieste, Italy (March 25–28, 2019), we identified the need to (i) bring attention to the potential spread of parasites and vectors with relocated dogs, and (ii) provide advice to the veterinary profession regarding the importance of surveillance and treatment for parasites and vector-borne infections when rehoming dogs. This letter shares a consensus statement from the CVBD World Forum as well as a summary of the problem faced, including the role of veterinary professionals in parasite surveillance, causal issues, and the importance of interdisciplinary cooperation in addressing the problem. To limit opportunities for dissemination of parasites and vectors, whenever possible, underlying problems creating the need for dog rehoming should be addressed. However, when it is necessary to rehome dogs, this should ideally take place in the country and national region of origin. When geographically distant relocation occurs, veterinary professionals have a vital role to play in public education, vigilance for detection of exotic vectors and infections, and alerting the medical community to the risk(s) for pathogen spread. With appropriate veterinary intervention, dog welfare needs can be met without inadvertently allowing global spread of parasites and their vectors.

Abstract Background Canine leishmaniosis (CanL) is a parasitic zoonotic disease, endemic in the Mediterranean basin including Spain. While knowledge about CanL, its management, treatment, prevention and control mounts, it remains unclear whether all clinical veterinarians follow the same international recommendations, such as those of the LeishVet group. This study was thus designed to assess recent trends in the clinical management of CanL in veterinary clinics across Spain through a questionnaire-based survey. Results were compared with those of a prior national multicenter questionnaire administered by our research team in 2005. Methods A questionnaire consisting of 28 questions about CanL was developed using Google Forms and distributed by email to 1428 veterinary clinics in Spain. Questions were designed to obtain data on common clinical signs, techniques and complementary exams used to diagnose the disease, and on its monitoring, treatment and control measures. Data were collected in a database for statistical analysis. Results Completed questionnaires were returned by 295 clinics. Compared to the situation in 2005, responses indicate that clinical signs of CanL have not changed significantly, cutaneous lesions being still the most prevalent sign observed by practitioners. Quantitative serological techniques are considered an adequate approach to diagnosis, provided their results are supported by the findings of a thorough physical exam, as well as complementary tests (complete blood count, biochemical profile, plasma protein electrophoretogram and complete urinalysis). Treatment protocols and check-ups follow international recommendations. Finally, a multimodal approach is being endorsed to adequately control CanL including preventive measures such as annual serological check-ups and the combination of repellents and vaccines. Additionally, owners are being better informed about CanL by veterinarians, which translates to the improved control of this zoonosis. Conclusions The clinical management of CanL has recently undergone significant changes owing to improvements in clinical knowledge of the disease, more unified international criteria, improved diagnostic techniques and their adequate interpretation, as well as a greater awareness of the disease transmitted to owners.

Abstract Background This report describes L. infantum infection seroprevalence in dogs in Spain through data obtained from peer-reviewed literature and a cross-sectional serological survey assessing epidemiological and habitat variables as risk factors for infection. The study also provides preliminary sand fly species distribution data and indicates factors affecting their distribution and density. Methods Three different studies were conducted in Spain: (i) a peer-reviewed literature seroprevalence survey (1985–2019); (ii) a cross-sectional serological survey (2011–2016); and (iii) a preliminary entomological survey (2013–2014). In the cross-sectional serological survey, 1739 dogs from 74 different locations including 25 Spanish provinces were tested for L. infantum by indirect immunofluorescence antibody test (IFAT) (antibody titre ≥ 1:100). Seroprevalence of L. infantum infection was analysed by province and bioclimatic zone. Statistics were used to analyse relationships between several dog- and environment-related variables and L. infantum seroprevalence. In parallel, during 2013–2014, sand flies were collected across the Iberian Peninsula and the Balearic Islands using CDC light traps to examine relationships between habitat-related factors and sand fly species densities (number of sand flies per trap per hour). Results The literature review revealed that the provinces showing the highest seroprevalence were Balearic Islands (57.1%), Ourense (35.6%), Málaga (34.6%) and Cáceres (34.2%), and those showing the lowest seroprevalence were Vizcaya (0%), Cantabria (2.0%) and Álava (3.3%). In our survey, anti-Leishmania IgG antibodies were detected in 176 of the 1739 dogs rendering a seroprevalence of 10.12%. Percentage seroprevalence distributions significantly varied among bioclimatic belts. Seropositivity for L. infantum was related to size (large breed dogs versus small) and were significantly higher in younger dogs (≤ 1 years-old). In the entomological survey, 676 sand flies of five species were captured: 562 (83.13%) Phlebotomus perniciosus; 64 (9.47%) Sergentomyia minuta; 38 (5.62%) P. ariasi: 6 (0.89%) P. sergenti; and 6 (0.89%) P. papatasi. Phlebotomus perniciosus showed a greater density in the thermo-Mediterranean than in the meso-Mediterranean zone. Densities of S. minuta and P. ariasi were significantly higher in rural habitats. Conclusions This updated seroprevalence map of L. infantum infection in dogs in Spain defines non-endemic, hypoendemic, endemic and hyperendemic areas, and confirms P. perniciosus as the most abundant sand fly vector in Spain.

Abstract Background The drosophilid Phortica variegata is known as vector of Thelazia callipaeda, the oriental eyeworm native to Asia that has become an emergent zoonotic agent in several European regions. Unlike almost all other arthropod vectors of pathogens, only P. variegata males feed of lachrymal secretions of animals, ingesting first-stage larvae (L1) of the worm living in the orbital cavities of the host, and allowing with the same behaviour the introduction of infective L3. Despite the increased detection of T. callipaeda in many European countries, information about the length of the lachryphagous activity period of P. variegata and a deep knowledge of the environmental and climatic variables involved are still limited. Methods We herein present the results of a multicentre study involving five sites from four different countries (Italy, Spain, UK and USA) where canine thelaziosis is endemic and/or where it has already been ascertained the presence of P. variegata. Field data have been obtained on a fortnightly basis from mid-April to the end of November 2018 from a contemporary standardized sampling (same sampling effort and time of collection in all sites) of lachryphagous flies collected around the eyes of a human bait using an entomological net. These data have been associated to data collection of local climatic variables (day length, temperature, wind speed, barometric pressure and relative humidity). Results Overall, a total of 4862 P. variegata flies (4637 males and 224 females) were collected, with high differences in densities among the different sampling sites. Significant positive correlations were found between P. variegata male density and temperature and wind speed, while negative correlations were observed for barometric pressure and relative humidity. However, the above significant differences are confirmed in each sampling site separately only for the temperature. Conclusions This multicentre study highlights that temperature is the major common environmental driver in describing the lachryphagous activity of P. variegata in Europe and USA and, therefore, the transmission risk of thelaziosis.

Abstract Background Pets may be carriers of infectious agents including parasites. As part of a larger-scale study covering the whole of Europe, this study examines deworming measures reported by Spanish pet owners and identifies risk factors. Methods An online questionnaire was administered to cat and dog owners in Spain. The replies provided were used to obtain information about the petsʼ living conditions and to accordingly classify each pet into one of the four ESCCAP infection risk categories (A, B, C or D) for which different deworming frequencies are recommended. Questions were also asked about pet care and ownersʼ attitude toward their pets. The Kruskal-Wallis test was used to correlate risk groups with deworming frequencies. Results Completed questionnaires were returned by 500 cat owners and 501 dog owners. According to responses, 96.21% of dogs were assigned to risk category D (maximum risk), and only 1.2%, 2.2% and 0.4% to A, B and C, respectively. Almost all cats were assigned to the minimum risk category A (indoor cats, 62%) or maximum risk category D (outdoor cats, 32.8%); only 3.4% and 1.8% of cats were classified as risk B and C respectively. More dogs were allocated to the higher risk group compared to cats, which were more frequently kept indoors. Cats were reportedly dewormed less frequently than dogs (2.56 and 3.13 times per year respectively), consistent with their different infestation risk. Thus, pets in the lower risk group A were either adequately dewormed or treated more often than necessary. Only a small proportion of cats were not dewormed at all (n = 14). Alarmingly, almost all pets in risk groups B, C or D (representing 95% of dogs and 39% of cats) were dewormed less often than recommended. Conclusions More effective health education is required for the management of zoonotic endoparasite diseases under the umbrella of One Health targeted at owners, veterinarians, general practitioners, and health authorities. To align deworming frequency with infection risk, pet owners should be provided with clear, compelling instructions.

Leishmania infantum, the etiological agent of canine leishmaniosis (CanL) in Europe, was responsible of the largest outbreak of human leishmaniosis in Spain. The parasite infects and survives within myeloid lineage cells, causing a potentially fatal disease if left untreated. The only treatment option relies on chemotherapy, although immunotherapy strategies are being considered as novel approaches to prevent progression of the disease. To this aim, a deeper characterization of the molecular mechanisms behind the immunopathogenesis of leishmaniosis is necessary. Thus, we evaluated, for the first time, the host immune response during L. infantum infection through transcriptome sequencing of the popliteal lymph nodes aspirates of dogs with CanL. Differential expression and weighted gene co-expression network analyses were performed, resulting in the identification of 5,461 differentially expressed genes (DEGs) and four key modules in sick dogs, compared to controls. As expected, defense response was the highest enriched biological process in the DEGs, with six genes related to immune response against pathogens (CHI3L1, SLPI, ACOD1, CCL5, MPO, BPI) included among the ten most expressed genes; and two of the key co-expression modules were associated with regulation of immune response, which also positively correlated with clinical stage and blood monocyte concentration. In particular, sick dogs displayed significant changes in the expression of Th1, Th2, Th17 and Tr1 cytokines (e. g. TNF-α, IFN-γ, IL-21, IL-17, IL-15), markers of T cell and NK cell exhaustion (e. g. LAG3, CD244, Blimp-1, JUN), and B cell, monocyte and macrophage disrupted functionality (e. g. CD40LG, MAPK4, IL-1R, NLRP3, BCMA). In addition, we found an overexpression of XBP1 and some other genes involved in endoplasmic reticulum stress and the IRE1 branch of the unfolded protein response, as well as one co-expression module associated with these processes, which could be induced by L. infantum to prevent host cell apoptosis and modulate inflammation-induced lymphangiogenesis at lymph nodes. Moreover, 21 lncRNAs were differentially expressed in sick dogs, and one key co-expression module was associated with chromatin organization, suggesting that epigenetic mechanisms could also contribute to dampening host immune response during natural L. infantum infection in the lymph nodes of dogs suffering from clinical leishmaniosis.

Dogs are the main reservoir of Leishmania infantum and in some countries have been regularly culled as part of government policy to control visceral leishmaniasis. At the 13th Symposium of the Companion Vector-Borne Diseases World Forum in Windsor, UK, March 19–22, 2018, we consolidated a consensus statement regarding the usefulness of dog culling as a means of controlling visceral leishmaniasis. The statement highlighted the futility of culling infected dogs, whether healthy or sick, as a measure to control the domestic reservoir of L. infantum and reduce the risk for visceral leishmaniasis.

In this study, we describe SARS-CoV-2 infection dynamics in one cat and three dogs from households with confirmed human cases of COVID-19 living in the Madrid Community (Spain) at the time of expansion (December 2020 through June 2021) of the alpha variant (lineage B.1.1.7). A thorough physical exam and nasopharyngeal, oropharyngeal, and rectal swabs were collected for real-time reverse-transcription PCR (RT-qPCR) SARS-CoV-2 testing on day 0 and in successive samplings on days 7, 14, 21, and 47 during monitoring. Blood was also drawn to determine complete blood counts, biochemical profiles, and serology of the IgG response against SARS-CoV-2. On day 0, the cat case 1 presented with dyspnea and fever associated with a mild bronchoalveolar pattern. The dog cases 2, 3, and 4 were healthy, but case 2 presented with coughing, dyspnea, and weakness, and case 4 exhibited coughing and bilateral nasal discharge 3 and 6 days before the clinical exam. Case 3 (from the same household as case 2) remained asymptomatic. SARS-CoV-2 detection by RT-qPCR showed that the cat case 1 and the dog case 2 exhibited the lowest cycle threshold (Ct) (Ct < 30) when they presented clinical signs. Viral detection failed in successive samplings. Serological analyses revealed a positive IgG response in cat case 1 and dog cases 3 and 4 shortly after or simultaneously to virus shedding. Dog case 2 was seronegative, but seroconverted 21 days after SARS-CoV-2 detection. SARS-CoV-2 genome sequencing was attempted, and genomes were classified as belonging to the B.1.1.7 lineage.

Lynx pardinus is one of the world’s most endangered felines inhabiting the Iberian Peninsula. The present study was performed to identify the presence of microsporidia due to the mortality increase in lynxes. Samples of urine (n = 124), feces (n = 52), and tissues [spleen (n = 13), brain (n = 9), liver (n = 11), and kidney (n = 10)] from 140 lynxes were studied. The determination of microsporidia was evaluated using Weber’s chromotrope stain and Real Time-PCR. Of the lynxes analyzed, stains showed 10.48% and 50% positivity in urine and feces samples, respectively. PCR confirmed that 7.69% and 65.38% belonged to microsporidia species. The imprints of the tissues showed positive results in the spleen (38.46%), brain (22.22%), and liver (27.27%), but negative results in the kidneys. PCR confirmed positive microsporidia results in 61.53%, 55.55%, 45.45%, and 50%, respectively. Seroprevalence against Encephalitozoon cuniculi was also studied in 138 serum samples with a positivity of 55.8%. For the first time, the results presented different species of microsporidia in the urine, feces, and tissue samples of Lynx pardinus. The high titers of anti-E. cuniculi antibodies in lynx sera confirmed the presence of microsporidia in the lynx environment. New studies are needed to establish the impact of microsporidia infection on the survival of the Iberian lynx.

Cardiopulmonary nematodes are highly pathogenic parasites affecting domestic and wild canids. As the result of conservation programs, the Iberian wolf (Canis lupus signatus) population has recently expanded, and its distribution range covers lands from where it had long disappeared. However, the exact epidemiological role of the wolf in the life cycle of zoonotic parasites causing diseases transmissible to pets and/or humans is largely unknown. This study sought to determine the diversity of cardiopulmonary nematode parasite species that affect wolves inhabiting northwestern areas of the Iberian Peninsula, and to estimate their prevalence and the relationship between these parasites and several epidemiological variables. For this purpose, we examined the cardiopulmonary systems of 57 wolves from Galicia (from the provinces A Coruña n = 15, Lugo n = 21, Ourense n =15 and Pontevedra n = 6) using techniques of dissection and cup sedimentation. Collected worms were then identified under a light microscope according to their morphological features. Three species of nematodes were detected: Angiostrongylus vasorum (the “French-heartworm”), Crenosoma vulpis and Eucoleus aerophilus, the latter being of zoonotic interest. The prevalence was 24.5% (14/57; 95% CI 13.3–35.6%) overall, 19.3% for A. vasorum (11/57; 95% CI 8.8–29.2%), 7% for C. vulpis (4/57; 95% CI 0.4–13.6%) and 3.5% for E. aerophilus (2/57; CI −1.1–9.1%). A significant relationship (p = 0.002) was found between age and the presence of C. vulpis, which was only found in juvenile animals. Furthermore, a higher prevalence of A. vasorum and/or C. vulpis was observed in wolves with a lower body condition score (40% and 20%, respectively), though the difference was not significant (p = 0.221 and p = 0.444, respectively). Our findings indicate a high “French-heartworm” and lungworm burden in the wolf population of northern Spain, and they identify a need for studies designed to elucidate the epidemiological role played by the Iberian wolf and to identify possible risks for veterinary and public health.

Abstract Background While in Europe Babesia canis has been traditionally held responsible for canine piroplasmosis, Babesia microti-like piroplasm (Bml) infection is being ever more observed in dogs, with the first clinical cases reported in northwestern Spain. This study examines the epidemiological role of healthy dogs living in endemic areas of Bml infection in Spain. The data obtained were used to describe the clinical status and map the geographical distribution of Bml infection in healthy dogs in northwestern Spain. Results Blood samples and ticks were taken from 756 healthy dogs representatively across the whole Galicia region (northwestern Spain): stray (n = 211), hunting dogs (n = 333) and pets (n = 212). Blood samples were tested by microscopy parasite observation, nested PCR-RFLP and sequencing. Piroplasm infection prevalences in healthy dogs from northwestern Spain were 17.1% (129/756) by PCR and 3.4% (26/756) by microscopy observation. The species found by PCR were: 2.2% (17/756) for B. canis and 15.1% (114/756) for Bml. Co-infection with B. canis and Bml was noted in 2 dogs. The higher prevalences detected were Bml in hunting dogs (25.5%; 85/333) and B. canis in stray dogs (6.6%; 14/211). In fox-hunting dogs from any area and dogs from the A Coruña Province, significantly higher prevalences of Bml infection were detected (P

Abstract Background The fruit fly Phortica variegata (Drosophilidae: Steganinae) feeds on the ocular secretions of animals and humans, and has been described as an intermediate host of the eye worm Thelazia callipaeda (Spirurida: Thelaziidae) in Italy. Despite the increased detection of T. callipaeda in many European countries, information about its vector role in natural conditions is still limited. In the Iberian Peninsula, thelaziosis caused by T. callipaeda has been reported in dogs, cats, red foxes, wild rabbits and humans. Methods In the last seven years, we have detected increased numbers of cases of canine thelaziosis at three locations in mainland Spain: Site 1, La Vera region (Cáceres Province, central-western Spain; 51 cases); Site 2, El Escorial municipality (Madrid Community, central Spain; 23 cases); and Site 3, Miraflores de la Sierra municipality (Madrid Community, central Spain; 41 cases). Site 1 is considered endemic for T. callipaeda while the other two sites have been recently recognised as risk zones for T. callipaeda infection. Results From June 2016 to September 2017, 2162 flies were collected and morphologically identified as Phortica spp. (Site 1, n = 395; Site 2, n = 1544; and Site 3, n = 223). Upon dissection, third-stage T. callipaeda larvae were found in two out of 155 flies examined from Site 1, and both these larvae tested molecularly positive for the eye worm. Of the 395 flies collected from Site 1, 371 were molecularly processed for arthropod species identification and T. callipaeda detection. All 371 flies were identified as P. variegata and 28 (7.5%; 95% CI: 4.8–10%) tested positive for T. callipaeda DNA haplotype 1. Conclusions Our findings indicate that T. callipaeda circulates among dogs and P. variegata in Spain, where zoonotic cases have been also reported. The co-existence of canine thelaziosis and Phortica spp. in geographical areas previously considered free of the eye worm indicates a risk of infection for both animals and humans living in this region.

Abstract Background Some wild animals have been recognized as potential reservoirs of Leishmania infantum infection (e.g. carnivores, lagomorphs, rodents, etc.). Leishmania infantum was also identified infecting humans and lagomorphs (i.e. hares and rabbits) over the period of 2009–2016, with the latter acting as the main reservoirs involved in the human leishmaniosis outbreak in Madrid. Results Two cases of clinical leishmaniosis are reported in orangutans (Pongo pygmaeus pygmaeus) housed at two different centres in Madrid. The first is the case of a 36-year-old male orangutan with severe weight loss and apathy. A complete blood count and biochemical profile revealed anaemia, neutropenia, hypoalbuminaemia and elevated transaminases. Hepato-splenomegaly was also observed. Four months later, due to worsening of clinical signs (mainly bilateral epistaxis), blood and bone marrow samples were collected. Amastigotes of L. infantum were detected in macrophages from a bone marrow aspirate and by specific polymerase chain reaction. The second case was a 34-year-old female orangutan with severe weight loss and apathy and no other apparent clinical signs. A complete blood count and biochemical profile revealed anaemia, pancytopenia and hypoalbuminaemia. Splenomegaly and pericardial effusion were also observed. As leishmaniosis was included in the differential diagnosis, both blood and bone marrow samples were collected. Leishmania infantum infection was confirmed by microscopy, molecular diagnosis and serology (immunofluorescence antibody test). Both animals were treated daily with oral miltefosine for 28 days; allopurinol was also given uninterruptedly in Case 2 for at least 6 months. During follow-up, though good clinical recovery was clear, a lack of parasitological cure was confirmed molecularly in both blood and bone marrow samples from the two orangutans. In both habitats, the presence of the sand fly vector identified as Phlebotomus perniciosus was confirmed. Conclusions To our knowledge, this is the first report of L. infantum infection in great apes and in the endangered species P. p. pygmaeus. We are presently looking for L. infantum in other non-human primates living in the same peri-urban areas. If detected, we will examine the impacts of this serious disease on these critically endangered species.

Abstract Background The prevalence of Leishmania infantum infection in clinically healthy dogs can be several times higher than that of clinical disease in endemic areas. Although treatment is not recommended in dogs with subclinical infection, these animals should be managed to prevent disease progression and parasite transmission to human beings or to other dogs. Dietary nucleotides and active hexose correlated compound (AHCC) have been shown to modulate the immune response. A recent study in dogs with clinical leishmaniosis receiving an initial 28-day course of methylglucamine antimoniate showed that six-month administration of a dietary supplement containing nucleotides plus AHCC achieves similar efficacy to allopurinol. Since the type of immune response plays a key role in the evolution of patients with leishmaniosis, the present study was aimed at evaluating the preventive effect of this supplement in avoiding or delaying disease progression in clinically healthy Leishmania-infected dogs. Methods Forty-six dogs were included in this multicenter, randomized, double-blind, placebo-controlled trial. Dogs received once-daily oral administration of a placebo or a dietary supplement containing nucleotides plus AHCC. Disease progression was monitored throughout the study in both groups. At 0, 60, 180 and 365 days of treatment, clinical signs were evaluated using a validated clinical scoring system, and several analytes were measured from blood, urine, and bone marrow samples. Results During the study, a significantly lower (P = 0.047) proportion of dogs changed their clinical status and became sick in the supplement group (3/20; 15%), compared to the placebo group (10/22; 45.5%). ELISA-determined antibody titers were significantly reduced compared to baseline at all time points with the supplement (P

Abstract Background The cat flea, Ctenocephalides felis, is the most prevalent flea species detected on dogs and cats in Europe and other world regions. The status of flea infestation today is an evident public health concern because of their cosmopolitan distribution and the flea-borne diseases transmission. This study determines the spatial distribution of the cat flea C. felis infesting dogs in Spain. Using geospatial tools, models were constructed based on entomological data collected from dogs during the period 2013–2015. Bioclimatic zones, covering broad climate and vegetation ranges, were surveyed in relation to their size. Results The models builded were obtained by negative binomial regression of several environmental variables to show impacts on C. felis infestation prevalence: land cover, bioclimatic zone, mean summer and autumn temperature, mean summer rainfall, distance to urban settlement and normalized difference vegetation index. In the face of climate change, we also simulated the future distributions of C. felis for the global climate model (GCM) “GFDL-CM3” and for the representative concentration pathway RCP45, which predicts their spread in the country. Conclusions Predictive models for current climate conditions indicated the widespread distribution of C. felis throughout Spain, mainly across the central northernmost zone of the mainland. Under predicted conditions of climate change, the risk of spread was slightly greater, especially in the north and central peninsula, than for the current situation. The data provided will be useful for local veterinarians to design effective strategies against flea infestation and the pathogens transmitted by these arthropods.

Abstract Background Canine leishmaniosis (CanL) has been in the spotlight since the 2009 outbreak of human leishmaniosis in Madrid. In the framework of the Leishmaniosis Surveillance Programme set up in Madrid, this study examines Leishmania-specific seroprevalences in stray dogs for the outbreak area and rest of the Madrid region over the period spanning from the outbreak to the present (2009–2016). These data are of interest because stray dogs could be sentinels for disease surveillance in endemic areas. Since 2011, we have also been monitoring owned dogs in the outbreak area. Methods Over the study period, Leishmania infantum seroprevalence was determined in 2,123 stray dogs from the outbreak and non-outbreak areas. A serological study was also performed for owned dogs in the outbreak area: high-risk dogs such as hunting or farm dogs (n = 1,722) and pets (n = 1372). All dogs were examined and blood was collected. The variables recorded for each animal were: breed, age, sex, and clinical history indicating if the animal was healthy or clinically suspected of having any disease, and if they showed a clinical picture compatible with CanL. Results Seroprevalences of L. infantum in stray dogs were similar in the two areas examined: 4.7% (20 out of 346) in the outbreak area and 5.4% (96 out of 1,777) in the remaining Madrid region (χ 2 = 0.080, P = 0.777). A significant association was found between seroprevalence and age (z = -6.319; P

The main hypothesis for the aetiology of meningoencephalitis of unknown origin (MUO) in dogs is an autoimmune or genetic cause that is associated with a triggering event (environmental factors/infectious agents). The aim of this ambispective cohort study was to test for Leishmania infantum infection in the blood and cerebrospinal fluid (CSF) of dogs with MUO in an endemic area of canine leishmaniosis. Dogs with MUO were selected amongst all dogs undergoing blood anti-L. infantum antibody testing (control group). The blood plasma or serum samples from all dogs were analysed for anti- L. infantum antibodies by a quantitative indirect fluorescent assay (IFAT). In dogs with MUO, CSF samples were obtained for analysed by PCR detection of L. infantum DNA. Forty-four percent and 22% of the dogs in the MUO group featured magnetic resonance imaging (MRI) findings and CSF cytology respectively, consistent with L. infantum infection. IFAT, PCR, and histological findings were negative for L. infantum. A significant difference in L. infantum infection prevalence was found between the control and MUO group (p = 0.0022). While it seems unlikely that L. infantum plays a role in the aetiology of MUO, in endemic areas, this pathogen should be included in the differential diagnosis of this neurological disorder.

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV), two of the most important pathogens of cats, produce chronic systemic diseases with progressive death of cells involved in the immune response, ultimately leading to death. Immunostimulants is one of the few alternatives to the symptomatic treatment. In this study, 27 naturally FeLV-infected (FeLV+) and 31 naturally FIV-infected (FIV+) cats were administered orally by their owners 60 IU/day of recombinant human interferon alpha (rHuIFN-α) for four months in alternate weeks. Clinical status was evaluated and blood samples collected at four different visits or months (M): pretreatment (M0), mid-treatment (M2), end of treatment (M4), and 4–8 months after end of treatment (M10). Most cats ostensibly improved their clinical status, and many became asymptomatic. rHuIFN-α treatment improved the anemic processes observed at M0 (at least in cats with mild or moderate anemia) and leukocyte counts, including a more favorable CD4+/CD8+ ratio. An increase in the serum gammaglobulin concentration was seen in 80% of the cats. Despite observing an obvious favorable progress in the clinical, biopathological, and CD4+/CD8+ values during treatment, almost invariably all the parameters analyzed worsened after treatment discontinuation (M10), which suggests that the interferon-α protocol should be either extended or include additional cycles for a long-lasting benefit in FeLV+ and FIV+ cats.

A pivotal strategy to decrease the risk of visceral leishmaniasis in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum (L. chagasi). Immunotherapy is a viable approach to treat sick dogs because cell-mediated immunity is the principal defense mechanism against L. infantum. Domperidone is an immune-stimulatory drug increasingly used in veterinary medicine as a prophylactic or immunotherapeutic agent. Domperidone treatment has shown to prevent overt disease or improve the clinical condition of infected dogs. However, veterinarians should be aware of the potential cardiotoxicity of domperidone when given together with drugs that inhibit CYP450s liver enzymes or those that prolong the QT interval. On the other hand, learning whether domperidone treatment significantly decreases dog infectivity to sand fly vectors is of capital importance since this result should have a palpable impact on the infection risk of humans living in regions endemic for visceral leishmaniasis.

This article reviews essential topics of canine visceral leishmaniasis (CVL) due to Leishmania infantum infection. It focuses on the current serological and molecular diagnostic methods used in epidemiological research and veterinary clinics to diagnose CVL and includes new point-of-care (POC) tests under development. The efficacy of different treatment regimens on the clinical improvement and infectiousness of dogs is also addressed. In the last section, the review provides a critical appraisal of the effectiveness of different control measures that have been implemented to curb disease transmission.

Abstract Limited data is available on feline leishmaniosis (FeL) caused by Leishmania infantum worldwide. The LeishVet group presents in this report a review of the current knowledge on FeL, the epidemiological role of the cat in L. infantum infection, clinical manifestations, and recommendations on diagnosis, treatment and monitoring, prognosis and prevention of infection, in order to standardize the management of this disease in cats. The consensus of opinions and recommendations was formulated by combining a comprehensive review of evidence-based studies and case reports, clinical experience and critical consensus discussions. While subclinical feline infections are common in areas endemic for canine leishmaniosis, clinical illness due to L. infantum in cats is rare. The prevalence rates of feline infection with L. infantum in serological or molecular-based surveys range from 0 % to more than 60 %. Cats are able to infect sand flies and, therefore, they may act as a secondary reservoir, with dogs being the primary natural reservoir. The most common clinical signs and clinicopathological abnormalities compatible with FeL include lymph node enlargement and skin lesions such as ulcerative, exfoliative, crusting or nodular dermatitis (mainly on the head or distal limbs), ocular lesions (mainly uveitis), feline chronic gingivostomatitis syndrome, mucocutaneous ulcerative or nodular lesions, hypergammaglobulinaemia and mild normocytic normochromic anaemia. Clinical illness is frequently associated with impaired immunocompetence, as in case of retroviral coinfections or immunosuppressive therapy. Diagnosis is based on serology, polymerase chain reaction (PCR), cytology, histology, immunohistochemistry (IHC) or culture. If serological testing is negative or low positive in a cat with clinical signs compatible with FeL, the diagnosis of leishmaniosis should not be excluded and additional diagnostic methods (cytology, histology with IHC, PCR, culture) should be employed. The most common treatment used is allopurinol. Meglumine antimoniate has been administered in very few reported cases. Both drugs are administered alone and most cats recover clinically after therapy. Follow-up of treated cats with routine laboratory tests, serology and PCR is essential for prevention of clinical relapses. Specific preventative measures for this infection in cats are currently not available.

Abstract Background In north-western Spain, piroplamosis caused by Theileria annae is now recognized as a serious problem because veterinarians, despite being aware of the clinical signs of piroplasmosis, lack the necessary information on its epidemiology or specific diagnostic tools for its management. This, along with the fact that T. annae infection is also refractory to current piroplamosis treatments, prompted this study designed to assess the clinical presentation and diagnosis of this largely unknown parasitic disease in dogs. Methods One hundred and twenty dogs in NW Spain suspected clinically of having piroplasmosis were examined and piroplasm species detected by light microscopy (LM) observation of Giemsa-stained blood smears, immunofluorescent antibody test (IFAT), and PCR plus sequencing. Results Seventy five of the sick dogs were confirmed to be infected with T. annae by PCR (designated “true infection cases”). Intraerythrocytic ring-shaped bodies morphologically compatible with small piroplasms were observed by LM in 59 (57 true infections) of the 120 blood samples. Anti-Babesia antibodies were detected by IFAT in 59 of the 120 sera (55 of which were “true infections”). Using PCR as the reference method, moderate agreement was observed between positive LM vs PCR and IFAT vs PCR results (kappa values: 0.6680 and 0.6017, respectively). Microscopy examination and IFAT were moderately sensitive in detecting the pathogen (76% and 73.3%, respectively). In the 75 cases of “true infection”, the most common clinical signs observed were pale mucous membranes, anorexia and apathy. Blood cell counts consistently revealed severe regenerative anaemia and thrombocytopenia in dogs with piroplasmosis due to T. annae. Young dogs (≤3 year) (p = 0.0001) were more susceptible to the disease. Conclusion Microscopy showed moderate diagnostic sensitivity for acute T. annae infection while IFAT-determined antibody titres were low (1/64 to 1/128). The infecting species should be therefore confirmed by molecular tests. Our results suggest that the disease affects dogs in regions of Spain bordering the endemic Galicia area where this piroplasm has not been previously reported (Asturias, northern Spain). Further epidemiological surveys based on serological and molecular methods are required to establish the current geographical range of T. annae infection.

Abstract Canine ehrlichiosis and anaplasmosis are important tick-borne diseases with a worldwide distribution. Information has been continuously collected on these infections in Europe, and publications have increased in recent years. Prevalence rates are high for Ehrlichia and Anaplasma spp. infections in dogs from different European countries. The goal of this article was to provide a practical guideline for veterinary practitioners on the diagnosis, treatment, and prevention of ehrlichiosis and anaplasmosis in dogs from Europe. This guideline is intended to answer the most common questions on these diseases from a practical point of view.

Abstract Background Domestic cats can be infested by a large range of parasite species. Parasitic infestations may cause very different clinical signs. Endoparasites and ectoparasites are rarely explored in the same study and therefore multiparasitism is poorly documented. The present survey aimed to improve knowledge of the prevalence and risk factors associated with ecto- and endoparasite infestations in owned cats in Europe. Methods From March 2012 to May 2013, 1519 owned cats were included in a multicenter study conducted in 9 veterinary faculties throughout Europe (Austria, Belgium, France, Hungary, Italy, Romania and Spain). For each cat, ectoparasites were checked by combing of the coat surface associated with otoscopic evaluation and microscopy on cerumen samples. Endoparasites were identified by standard coproscopical examinations performed on fresh faecal samples. Risk factors and their influence on parasitism were evaluated by univariate analysis followed by a multivariate statistical analysis (including center of examination, age, outdoor access, multipet status, and frequency of treatments as main criteria) with logistic regression models. Results Overall, 50.7% of cats resulted positive for at least one internal or one external parasite species. Ectoparasites were found in 29.6% of cats (CI95 27.3-32.0%). Otodectes cynotis was the most frequently identified species (17.4%), followed by fleas (15.5%). Endoparasites were identified in 35.1% of the cats (CI95 32.7-35.7%), including gastro-intestinal helminths in 25.7% (CI95 23.5-28.0), respiratory nematodes in 5.5% (CI95 4.2-7.0%) and protozoans in 13.5% (CI95 11.8-15.3%). Toxocara cati was the most commonly diagnosed endoparasite (19.7%, CI95 17.8-21.8%). Co-infestation with endoparasites and ectoparasites was found in 14.0% of the cats, and 11.9% harbored both ectoparasites and gastro-intestinal helminths. Age, outdoor access, living with other pets, and anthelmintic or insecticide treatments were significantly associated with the prevalence of various parasites. Conclusions This survey demonstrates that parasitism is not a rare event in European owned cat populations. The prevalence of multi-parasitism is significantly greater than expected by chance and hence there is tendency for some individual cats to be more prone to infestation by both endo- and ectoparasites due to common risk factors.

Abstract Background Since 2009, the incidence of human leishmaniosis in the SW of the Madrid region has been unusually high. Although dogs are the main reservoir for this disease, a role played by dogs in this outbreak has been ruled out and investigators are now considering other hosts (eg. cats, rabbits, hares) as possible alternative reservoirs. This study was designed to examine the Leishmania infantum status of stray cats in Madrid to assess its possible implications in the human leishmaniosis outbreak. Methods 346 captured stray cats were tested for antibodies against L. infantum by the indirect fluorescent antibody technique (IFAT) and nested-PCR methods were used to detect Leishmania DNA in blood samples of cats testing seropositive for L. infantum and/or retroviruses infection. Cats were also tested for Toxoplasma gondii using the direct agglutination test (DAT) and feline leukemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies (PetChek* FIV/FeLV). The presence of intestinal parasites was determined using a routine coprological method. Results The seroprevalence of L. infantum infection (cut off ≥ 1/100) was 3.2% (11/346). However, it was not possible to amplify Leishmania DNA in any of the blood samples. Seropositivity was not associated with sex, age, capture site, clinical status, retrovirus infection or T. gondii seropositivity. Of the 11 cats seropositive for L. infantum, 3 also tested positive for FIV, none for FeLV and 6 for T. gondii. It should be mentioned that the prevalence of FeLV p27 antigen was 4% and of FIV antibody was 9.2%. Although the seroprevalence of T. gondii was quite high at 53.5%, no T. gondii oocysts were found in any of the faeces samples analysed (n = 287). In contrast, intestinal parasites were detected in 76 (26.5%) samples, Toxocara cati being the most prevalent. Conclusions Our results suggest a stable L. infantum infection situation among the stray cats of the Madrid area; the disease is uncommon and no clinical cases have been reported to date. The detection of other zoonotic parasites such as T. gondii and T. cati in stray cats indicates a need to adopt strict control measures in this population.

Abstract Background Canine leishmaniosis (CanL) caused by Leishmania infantum is a widespread endemic disease in SW Europe. This study was designed to determine how veterinarians clinically manage CanL in this region by analysing information collected in a questionnaire completed by local veterinarians working in clinics in France, Portugal, Greece, Spain, Italy and Slovenia. Methods Over the period 2004–2011, a questionnaire on CanL was sent to 12,546 small animal clinics located in the six countries surveyed. The questionnaire with 10 items comprising open and closed questions sought to obtain comparable data regarding the main clinical manifestations of CanL, the diagnostic methods used, the treatment regimens selected, recommended preventive measures and awareness of the important public health implications of CanL. Results The data collected reflect similarities in the clinical manifestations reported although there was some variation in the concurrent diseases described, and wide variation in the clinical management of CanL among the countries examined in terms of dosing regimens, therapeutic agents and the criteria used to diagnose CanL. Most veterinarians properly informed dog owners about the preventive measures available and about the zoonotic implications of CanL. Conclusions This survey describes the current situation in SW endemic countries in Europe regarding the clinical management of CanL. The data collected reveal a need to unify criteria from evidence-based medicine to determine and similarly apply the best diagnostic and treatment methods available for this disease in the different countries.

Specific treatments for the long-life infections by feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are either toxic, expensive or not too effective. Interferon α (IFN-α) is an immunomodulatory molecule which has been shown in vitro to decrease the release of infective particles. The aim of this study was to follow the progress of the clinical score and viral parameters of FeLV- and FIV-naturally infected privately owned cats treated with recombinant human IFN-α (rHuIFN-α, Roferon-A). Twenty-seven FeLV-infected cats (FeLV+) and 31 FIV-infected cats (FIV+) were enrolled in the study. Owners were instructed to orally administer 1 mL/day of 60 IU rHuIFN-α/mL in alternating weeks for four months. Blood samples were taken at the beginning of the study (M0), mid-treatment (M2), end of treatment (M4), and 6−10 months later (M10). Clinical status at these time points improved notably with rHuIFN-α treatment, regardless of the initial severity of the disease, an effect which lasted throughout the study in most animals (15 of the 16 FeLV+ symptomatic cats; 20 of the 22 FIV+ symptomatic cats) improved markedly their clinical situation. In FeLV+ cats plasma antigenemia (p27CA), reverse transcriptase (RT) activity, and proviral load decreased at M2 and M4 but increased again at M10 (“rebound effect”). The level of antigenemia or RT activity was below the detection limits in FIV+ cats, and the effect on proviral load was less marked than in FeLV+ cats. Taken together, these results indicate that rHuIFN-α is a good candidate for treating FeLV+ cats, but the “rebound effect” seen when treatment was discontinued suggests that additional studies should be conducted to clarify its effect on progression of the infection in cats.

Type-I interferons (IFN-I) play an important role in the innate immune response to several retroviruses. They seem to be effective in controlling the in vivo infection, though many of the clinical signs of retroviral infection may be due to their continual presence which over-stimulates the immune system and activates apoptosis. IFN-I not only affect the immune system, but also operate directly on virus replication. Most data suggest that the in vitro treatment with IFN-I of retrovirus infected cells inhibits the final stages of virogenesis, avoiding the correct assembly of viral particles and their budding, even though the mechanism is not well understood. However, in some retroviruses IFN-I may also act at a previous stage as some retroviral LTRs posses sequences homologous to the IFNstimulated response element (ISRE). When stimulated, ISREs control viral transcription. HIV-1 displays several mechanisms for evading IFN-I, such as through Tat and Nef. Besides IFN-α and IFN-β, some other type I IFN, such as IFN-τ and IFN-ω, have potent antiviral activity and are promising treatment drugs.

Microsporidia are ubiquitous parasites infecting all animal phyla and we present evidence that supports their zoonotic potential. Fecal samples taken from domestic (cats and dogs), farm (pigs, rabbits and ostriches) and wild animals (foxes) from different provinces of Spain were evaluated for microsporidia infection by light microscopy and PCR. After Microsporidia species identification, E. bieneusi genotypes were additionally studied by sequence analysis of the ITS region. Eighty-five samples out of 159 exhibited structures that were compatible with microsporidia spores by Webeŕs stain with 37 of them being confirmed by PCR. Microsporidia species identified included E. bieneusi, E. intestinalis and A. algerae. We report the first diagnosis of E. intestinalis and E. bieneusi in ostriches and A. algerae in pigs. We also provide new information on the molecular characterization of E. bieneusi isolates both in rabbits and ostriches. All of the E. bieneusi genotypes identified belonged to the zoonotic group of genotypes (Group I) including genotypes A (dogs), I (pigs), D (rabbits and foxes) and type IV (ostriches). Our results demonstrate that microsporidia are present in domestic, farm and wild animals in Spain, corroborating their potential role as a source of human infection and environmental contamination.

In marine organisms primarily intended for human consumption, the quality of the muscle and the extracted oils may be affected by lipid oxidation during storage, even at low temperatures. This has led to a search for alternatives to maintain quality. In this sense, antioxidant compounds have been used to prevent such lipid deterioration. Among the most used compounds are tocopherols, which, due to their natural origin, have become an excellent alternative to prevent or retard lipid oxidation and maintain the quality of marine products. Tocopherols as antioxidants have been studied both exogenously and endogenously. Exogenous tocopherols are often used by incorporating them into plastic packaging films or adding them directly to fish oil. It has been observed that exogenous tocopherols incorporated in low concentrations maintain the quality of both muscle and the extracted oils during food storage. However, it has been reported that tocopherols applied at higher concentrations act as a prooxidant molecule, probably because their reactions with singlet oxygen may generate free radicals and cause the oxidation of polyunsaturated fatty acids in fish oils. However, when tocopherols are included in a fish diet (endogenous tocopherols), the antioxidant effect on the muscle lipids is more effective due to their incorporation into the membrane lipids, which can help extend the shelf life of seafood by reducing the lipid deterioration that occurs due to antioxidant synergy with other phenolic compounds used supplements in fish muscle. This review focuses on the most important studies in this field and highlights the potential of using tocopherols as antioxidants in marine oils.