Cystoisospora spp. infection at a dog breeding facility in the Madrid region: Infection rate and clinical management based on toltrazuril metaphylaxis.

Canine coccidiosis caused by Cystoisospora canis and Cystoisospora ohioensis-complex is common in kennels. While often underestimated, coccidiosis may cause severe clinical signs in puppies and sometimes even lead to death, so preventative measures are important. This study examines Cystoisospora spp. infection at a Labrador retriever breeding facility in Madrid, Spain. To identify environmental factors associated with infection, dams were examined throughout a reproductive cycle (from oestrus to 60 days postpartum) and their puppies during their first 60 days of life. Also assessed was the efficacy of combined treatment with emodepside (0.9 mg/ml) and toltrazuril (18 mg/ml) at a dose of 0.5 ml/kg of weight, equivalent to 0.45 mg/kg and 9 mg/kg, respectively, in puppies on day 35 of life. Oocyst shedding was detected in 4.6-18.6% of 45 dams examined and in 2.2-9.1% of their litters (315 puppies). In both cases, peak opg elimination was recorded on day 30 postpartum/of life. The species of Cystoisospora detected were C. canis (91.3%) and C. ohioensis-complex (8.7%). While in both dams and puppies opg counts were higher in autumn when rainfall was at its highest, correlation between opg and rainfall emerged as significant only in puppies (p = 0.031). The treatment of 35 day-old puppies with toltrazuril was 100% effective in controlling this infection in the kennel. Our findings therefore suggest the need for a strict hygiene regime and the use of toltrazuril as blanket treatment to reduce Cystoisospora transmission in dog breeding facilities.

Intensive training programme for ultrasound-guided minimally invasive synovial tissue biopsy on knees and wrists in different phases of inflammation.

OBJECTIVES: To develop an intensive training programme for ultrasound (US)-guided synovial tissue (ST) biopsy on knees and wrists in inflammatory arthritis and to assess the learning curve, patient tolerability, sample quality and trainees' expectations. METHODS: Active or remission rheumatoid arthritis patients were enrolled. Nine trainees joined the 4-month programme in a centre experienced in performing US-guided ST biopsies consisting of four sequential phases: (1) observation, (2) performance of guided step-by-step phases, (3) execution of the whole procedure on paired joints (knees or wrists) of the same patient in parallel with the trainer and (4) performance of the procedure autonomously. Sample representativity was assessed by histology, and procedure-related adverse events were recorded. Before and after the programme, trainees' expectations and perceptions were collected. RESULTS: 328 ST biopsy procedures were included. The rate of trainees' informative samples was: (1) comparable to the trainers in active and remission knees, but lower in active wrists (70% for trainees vs 100% for trainers, p=0.06) in phase 3; (2) excellent on active knees and wrists (91.9% and 90.9% respectively) but lower (77.6%, p=0.0089) on remission knees in phase 4. Procedures performed by trainees did not affect patient tolerability. Trainees' expectations about procedure-related invasiveness and pain infliction decreased while the difficulty of procedure execution on active wrists and remission knees remained perceived as moderately difficult. CONCLUSIONS: This intensive training programme develops advanced skills in the performance of US-guided ST biopsy on knees and wrists, yielding high-quality specimens available for basic and translational studies on inflammatory joint diseases.

The assessment of the drug retention rate of secukinumab in patients with psoriatic arthritis in a real-life multicentre cohort.

OBJECTIVES: We aimed to evaluate the drug retention rate (DRR) of secukinumab, an anti-IL-17A monoclonal antibody, in patients with psoriatic arthritis (PsA) in a real-life cohort, and to assess the impact of comorbidities and patient clinical characteristics on the DRR of secukinumab. METHODS: A retrospective study of prospective followed-up patients was performed to evaluate the DRR of secukinumab on patients with PsA attending the recruiting centres between January 2016 and June 2022. RESULTS: In 207 patients with PsA, a 60-month DRR of secukinumab of 57.0% was estimated (mean time of administration of 21.5±17.1 months). Male gender, age ≥65 years, disease duration ≥5 years and ≥10 years did not influence the DRR of secukinumab. The presence of comorbidities, considering any concomitant disorder, did not affect the DRR of secukinumab. In patients with cardiometabolic multimorbidity, a trend toward a better DRR of secukinumab was recorded. In fact, patients with high blood pressure, dyslipidaemia, and type 2 diabetes showed a trend toward an improved DRR of secukinumab. Furthermore, the presence of obesity did not influence the DRR of secukinumab. Different dosages, previous bDMARDs, and concomitant therapy with csDMARDs did not influence the DRR of secukinumab. CONCLUSIONS: A cumulative 60-month DRR of secukinumab of 57.0% in patients with PsA was retrieved. The presence of cardiometabolic multimorbidity could be associated with an improved DRR of secukinumab, whereas obesity did not affect this feature in our cohort. Previous bDMARDs, concomitant csDMARDs, and different drug dosages could not influence the DRR of secukinumab over time.

Determining the PASS cut-off points for the FIQR, FASmod and PSD in patients with fibromyalgia: a registry-based study.

OBJECTIVES: To determine the cut-off values of Patient Acceptable Symptom State (PASS) for the revised Fibromyalgia Impact Questionnaire (FIQR), the modified Fibromyalgia Assessment Scale (FASmod), and the Polysymptomatic Distress scale (PSD) and to determine the predictors of PASS in patients with fibromyalgia (FM). METHODS: FM patients belonging to the Italian Fibromyalgia Registry (IFR) completed the FIQR, the FASmod and the PSD. The PASS was assessed using a dichotomous answer. The cut-off values were obtained through the receiver operating characteristic curve (ROC) analyses. A multivariate logistic regression analysis was performed to determine predictors of achieving the PASS. RESULTS: 5545 women (93.7%) and 369 males (6.3%) were included in the study. The 27.8% of patients reported an acceptable symptom state. Patients in PASS differed in all patient-reported outcome measures (p <0.001). The FIQR PASS threshold was ≤58 (area under the ROC curve [AUC] = 0.819). The FASmod PASS threshold was ≤23 (AUC = 0.805) and the PSD PASS threshold was ≤16 (AUC = 0.773). In the pairwise AUC comparison, the discriminatory power of the FIQR PASS outperforms both FASmod PASS (p = 0.0124) and PSD PASS (p <0.0001). Multivariate logistic analysis showed that FIQR items related to memory and pain were the only predictors of PASS. CONCLUSIONS: The FIQR, FASmod, and PSD PASS cut-off points for FM patients have never been determined before. This study provides additional information to facilitate interpretation of the severity assessment scales in daily practice and clinical research related to FM patients.

Mothers and fathers of pre-school children: a study on parenting stress and child's emotional-behavioral difficulties.

The present study aimed to analyze the differences between maternal and paternal parenting stress and children's behavioral functioning, as determined by teacher and parent reports. In addition, it sought to evaluate the presence of clusters based on parenting stress and to determine whether perceptions of children's behavioral functioning varied across these clusters. The sample was composed of the parents and teachers of N = 201 children. Parents completed a self-report questionnaire on parenting stress and parents and teachers filled out report-form questionnaires assessing children's emotional and behavioral functioning. The results showed that mothers had higher levels of parenting stress than fathers, and both parents reported more prosocial behavior in children than did teachers. Furthermore, middle stressed parents had children who expressed more prosocial behavior than did parents in other clusters. The findings also showed that parenting stress influenced partners' perceptions of children's behavioral functioning. The multiplicity of child observers facilitated a better understanding of how parenting stress, linked to parent-child interactions, may play an important role in shaping parents' perceptions of their children.

Multicenter Real-World Study on Effectiveness and Early Discontinuation Predictors in Patients With Non-small Cell Lung Cancer Receiving Nivolumab.

BACKGROUND: Real-world (RW) evidence on nivolumab in pretreated patients with non-small cell lung cancer (NSCLC) by matching data from administrative health flows (AHFs) and clinical records (CRs) may close the gap between pivotal trials and clinical practice. METHODS: This multicenter RW study aims at investigating median time to treatment discontinuation (mTTD), overall survival (mOS) of nivolumab in pretreated patients with NSCLC both from AHF and CR; clinical-pathological features predictive of early treatment discontinuation (etd), budget impact (BI), and cost-effectiveness analysis were investigated; mOS in patients receiving nivolumab and docetaxel was assessed. RESULTS: Overall, 237 patients with NSCLC treated with nivolumab were identified from AHFs; mTTD and mOS were 4.2 and 9.8 months, respectively; 141 (59%) received at least 6 treatment cycles, 96 (41%) received < 6 (etd). Median overall survival in patients with and without etd were 3.3 and 19.6 months, respectively (P < .0001). Higher number, longer duration, and higher cost of hospitalizations were observed in etd cases. Clinical records were available for 162 patients treated with nivolumab (cohort 1) and 83 with docetaxel (cohort 2). Median time to treatment discontinuation was 4.8 and 2.6 months, respectively (P < .0001); risk of death was significantly higher in cohort 2 or cohort 1 with etd compared with cohort 1 without etd (P < .0001). Predictors of etd were body mass index <25, Eastern Cooperative Oncology Group performance status >1, neutrophile-to-lymphocyte ratio >2.91, and concomitant treatment with antibiotics and glucocorticoids. The incremental cost-effectiveness ratio of nivolumab was 3323.64 euros ($3757.37) in all patients and 2805.75 euros ($3171.47) for patients without etd. Finally, the BI gap (real-theoretical) was 857 188 euros ($969 050.18). CONCLUSION: We defined predictors and prognostic-economic impact of nivolumab in etd patients.

Internet Addiction: The Role of Parental Care and Mental Health in Adolescence.

Internet addiction in adolescence is a social issue which is being increasingly discussed worldwide. Hence, deepening the knowledge of its development is necessary to prevent short- and long-term negative outcomes. This study involving 266 adolescents (mean age = 16.1 years, SD = 1.4) aimed at analyzing the relationship between quality of parental care, Internet addiction and adolescents' mental health, by means of the following self-report tools: the Parental Bonding Instrument, the Internet Addiction Test, and the Strengths and Difficulties Questionnaire. The findings, collected through structural equation model analyses, showed that perceived care from both father and mother had significant indirect effects on Internet addiction problems through adolescents' mental health problems. Furthermore, Internet addiction problems were demonstrated to be negatively associated with maternal care but not with paternal care. The study provides empirical support to the need of family-based prevention and intervention programs to take care of Internet addiction.

Feline thelaziosis (Thelazia callipaeda) in Spain: state-of-the-art and first prophylactic trial in cats.

OBJECTIVES: Thelaziosis caused by the nematode Thelazia callipaeda (Spirurida, Thelaziidae) affects the eyes of domestic and wild carnivores, lagomorphs and even humans. The role of cats in spreading thelaziosis remains unclear. The present study assesses the current status of feline T callipaeda infection in Spain and presents the results of the first prophylactic trial conducted in this species. METHODS: We retrospectively analysed the occurrence of feline thelaziosis (study 1), examined its prevalence based on cross-sectional sampling of endemic areas (study 2), and assessed the therapeutic/prophylactic benefits of a spot-on (moxidectin) and therapeutic efficacy of an eye drop (ivermectin) formulation (study 3). RESULTS: In study 1, 69 Thelazia-infected cats were examined. Autochthonous cases were detected in 18/26 municipalities surveyed in Spain, which corresponds to 88.4% (n = 61/69) of the total number of cases. In study 2, 74 cats (20 from La Vera region and 54 from Orense province) were examined from 2011 to 2013. Ten of these cats (13.5%) were infected with T callipaeda. The infection prevalence was 40% (n = 8/20) in La Vera and 3.7% (n = 2/54) in Orense. The therapeutic efficacy of ivermectin was assessed over a summer (2011) in 12 cats, while that of moxidectin was examined over a 17-month period in seven cats. Four of these seven cats were subsequently included in an all-year-round prophylaxis trial. Treatment efficacy ranged from 91.7% (ivermectin) on day 28 after treatment to 100% (moxidectin) on day 14. Moxidectin prevented reinfections in cats. CONCLUSIONS AND RELEVANCE: Our data confirm the endemic status of T callipaeda infection in Spain, revealing its high prevalence in cats living in known endemic areas. Moxidectin was effective in treating and preventing infection. We strongly recommend the inclusion of thelaziosis in the differential diagnosis of pets and humans presenting with ocular manifestations, along with adequate preventive measures.

Update on the treatment and prevention of ocular thelaziosis (Thelazia callipaeda) in naturally infected dogs from Spain.

This study examines the therapeutic and year-round prophylactic efficacy of different formulations used in dogs in three Spanish areas where canine thelaziosis is endemic. The study was conducted as a Good Clinical Practice, multicentre, randomised field study in privately owned outdoor dogs naturally infected with Thelazia callipaeda. The active pharmaceutical ingredients tested were: an oral formulation of milbemycin oxime 12.5 mg combined with praziquantel 125 mg (A), a subcutaneous sustained-release formulation of moxidectin 10 g (B), a moxidectin 2.5% weight/volume (w/v) spot-on formulation combined with imidacloprid 10% w/v (C), and an eye drop formulation (6 µg) of ivermectin 10 mg/ml diluted 10% in propylene glycol (D). Infected dogs were randomly allocated to treatment Groups A, B, C and D. Dogs testing negative for T. callipaeda inspection in two visits (Day 7/Day 14 and D30) were enrolled in the prophylaxis trial and reallocated to the corresponding study group (A, B, C or D). Treatment efficacy ranged from 70.4% recorded in Group A 1 week after treatment, to 100% recorded in Group C on Day 30 and in Group B on Day 60. Treatment was more efficacious in Group D (85.7% 1 week after treatment) than A, but was never 100% efficacious as in Groups B and C. Year-round prophylactic efficacy was 83.3% in Group A, 100% in Group B, 93.5% in Group C and 87.5% in Group D. In conclusion, products containing moxidectin were highly efficacious both in treating and preventing canine thelaziosis. Milbemycin also emerged as a good option. However, the off-label use of topical or subcutaneous ivermectin should be avoided due to possible adverse reactions such as pruritus, irritation or redness. In endemic areas, monthly prophylaxis to limit the spread of T. callipaeda to new areas across Europe and reduce zoonotic risks is essential.

Psychological effects of the COVID-2019 pandemic: Perceived stress and coping strategies among healthcare professionals.

Starting from China, the coronavirus disease (COVID-19) contagion spread unexpectedly and quickly all over the world, particularly affecting Italy. In the early stages of the epidemic, healthcare professionals have been in the front-line to manage the infection. The current study aimed to analyse the impact of COVID-19 outbreak on healthcare professionals and to detect some risk and protective factors of their distress levels, with regard to socio-demographic variables, direct exposure to COVID-19 and the coping strategies used to deal with stress. The data were collected during the peak of the infection. A total of 595 healthcare professionals enrolled in the study and completed the measures of socio-demographical and professional data, perceived stress (PSS) and coping strategies (COPE- NVI-25). Overall, we found that a positive attitude towards the stressful situation was the main protective factor, while female gender, seeking social support, avoidance strategies and working with COVID-19 patients were risk factors. Economic status, problem solving ability and turning to religion were not associated with stress levels. This study, one of the first on this topic, highlighted the main coping strategies used by healthcare professionals in facing the highly stressful situation caused by the pandemic.

The red fox (Vulpes vulpes) as a potential natural reservoir of human cryptosporidiosis by Cryptosporidium hominis in Northwest Spain.

Giardia duodenalis and Cryptosporidium spp. are ubiquitous intestinal protozoa that parasitize domestic and wild animals, as well as human beings. Due to their zoonotic potential, the objective of the present study was to determine the presence of these pathogens in the fox population (Vulpes vulpes) located in Northwest Spain. A total of 197 faecal samples from legally hunted foxes were collected in the autonomous region of Galicia. The presence of G. duodenalis and Cryptosporidium spp. was investigated by PCR-based methods amplifying the small subunit ribosomal RNA (ssu rRNA) gene of the parasites. Attempts to genotype obtained positive samples were subsequently conducted at the glutamate dehydrogenase (gdh) and ß-giardin (bg) genes of G. duodenalis, and the 60 kDa glycoprotein (gp60) gene of Cryptosporidium. Giardia duodenalis and Cryptosporidium spp. were identified in 19 (9.6%) and 12 (6.1%) of the investigated samples, respectively. However, five Cryptosporidium species were detected at the ssu rRNA locus: C. hominis (33.4%, 4/12), C. canis (25.0%, 3/12), C. parvum (16.7%, 2/12), C. ubiquitum (8.3%, 1/12) and C. suis (8.3%, 1/12). An additional Cryptosporidium-positive sample was identified at the genus level only. Typing and subtyping of Giardia- and Cryptosporidium-positive samples were unsuccessful. The detection of C. hominis in wild foxes indicates the probable overlapping of sylvatic and domestic cycles of this parasite in rural settings. Besides, this finding raises the question of whether red foxes may act as natural reservoirs of C. hominis. The detection of C. parvum and C. suis is suggestive of active transmission events between farm and wild animals, opening up the possibility of transmission to human beings.

Latest trends in L. infantum infection in dogs in Spain, Part II: current clinical management and control according to a national survey of veterinary practitioners.

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.

Latest trends in Leishmania infantum infection in dogs in Spain, Part I: mapped seroprevalence and sand fly distributions.

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.

Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations.

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.

Spain as a dispersion model for Thelazia callipaeda eyeworm in dogs in Europe.

Thelazia callipaeda (Spirurida, Thelaziidae) causes ocular infection in carnivorous animals and humans. While growing numbers of companion dogs and sometimes cats are being diagnosed with thelaziosis, little is known about its real spread. As it has been also diagnosed in wild animals and human beings, it is considered a potential emerging zoonotic disease. This study provides information about the spread of the parasite in dogs in Spain since its initial description in 2010 until 2018. The first detection of T. callipaeda in the Principality of Andorra in 2017 is also reported. Two different studies were conducted: a) a survey in which clinical cases from veterinary practices were collected and b) a prevalence study in two endemic areas in western and central Spain (Site 1 La Vera region, Cáceres, and Site 2 El Escorial municipality, Madrid). In total, 1114 cases of thelaziosis were detected in 121 municipalities of Spain and 6 municipalities of Andorra. In 92 out these 127 municipalities, reports were of autochthonous cases. Six hundred twenty-three out of 1114 presented data collection sheet and were included in the statistical analysis: 510 cases identified by veterinarians in Spain and Andorra in Study 1, and 113 cases detected among the 234 dogs (48.3 %) examined in the prevalence study (Study 2). Prevalences were 61.3 % (84/137) for Cáceres and 29.9 % (29/97) for Madrid, being Site 1 significantly more risky (P <  0.0001, odds ratio: 3.72, CI: 2.14-4.47 %) compared to Site 2. Our study updates data for canine thelaziosis reported in the last decade in Spain and Andorra. Results highlight the urgent need for prevention strategies to control the spread of this potential zoonotic disease.

The role of healthy dog carriers of Babesia microti-like piroplasms.

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 < 0.001). Upon physical examination, tick infestation was observed: 130 ticks in 18 hunting and three pet dogs. These were subsequently identified as Rhipicephalus sanguineus (s.l.) (49.2%), Ixodes hexagonus (38.5%), Ixodes ricinus (6.9%) and Dermacentor reticulatus (5.4%). Among the more prevalent ticks infesting healthy carrier dogs were I. hexagonus, followed by D. reticulatus and I. ricinus. CONCLUSIONS: Babesia canis and Bml were the only piroplasm species found infecting healthy dogs in Galicia, the prevalence of Bml being higher than of B. canis. Factors correlated with a higher Bml infection risk were being a hunting dog and living in the A Coruña Province. Healthy dogs travelling to other countries could act as carriers and probably contribute to the spread of Bml infection in dogs and wild carnivores throughout Europe.

Detection of Thelazia callipaeda in Phortica variegata and spread of canine thelaziosis to new areas in Spain.

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.

Babesia microti-like piroplasm (syn. Babesia vulpes) infection in red foxes (Vulpes vulpes) in NW Spain (Galicia) and its relationship with Ixodes hexagonus.

Piroplasmosis is caused by several species of protozoa such as the Babesia microti-like piroplasm (Bml), an emerging blood protozoan also known as Theileria annae or Babesia vulpes. Infection by Bml was first reported in dogs in Spain where it is endemic today. Recently, a high prevalence of Bml has been increasingly detected in red foxes (Vulpes vulpes) in European countries. The objective of this study was to determine infection levels of this parasite in foxes from Galicia, NW Spain, and ticks species infestation in these carnivores, where they are so far unknown. Samples of blood, spleen and ticks (if present) were taken from 237 hunted red foxes in the Galicia region. Blood smears were prepared for direct parasite observation, and spleen and tick samples were examined by nested PCR. Prevalences of Bml infection in Galician red foxes were estimated at 72% (171/237) by PCR and 38.23% (26/68) by direct observation. Among 837 ticks collected, the main tick identified was Ixodes hexagonus (present in 82.4% of the foxes) followed by Ixodes ricinus (12.3%), Dermacentor reticulatus (12.3%) and Rhipicephalus sanguineus sensu lato (3.5%). From 34 foxes testing positive for Bml, 616 ticks were collected: positive Bml PCR results were obtained in 55.6% (227/408) of ticks collected from 9 foxes, while the 208 ticks from the remaining 25 infected foxes returned negative PCR results. Given that canine piroplasmosis is endemic in this area, our observations point to the red fox as the main reservoir for Bml infection and the high proportion of I. hexagonus among ticks collected from red foxes suggests its likely role as vectors of B. microti-like piroplasm in this region. Further studies are needed for a better understanding of the link between the wild and domestic life cycles of this piroplasm.

First report of Leishmania infantum infection in the endangered orangutan (Pongo pygmaeus pygmaeus) in Madrid, Spain.

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.

Detection and molecular characterization of Acanthamoeba spp. in stray cats from Madrid, Spain.

Acanthamoeba spp. is a widespread protozoan that has been isolated from air, dust, soil, water and biological samples. An opportunistic pathogen of humans and animals, it may cause ocular keratitis, encephalitis, and even multisystem disease. The frequency of Acanthamoeba in animals is unknown. The aim of present study was determine the presence of Acanthamoeba spp. in immunocompromised stray cats - animals possibly more likely to harbour the infection given their immunocompromised status and frequenting of contaminated environments. Of 307 cats examined, 55 were positive for feline immunodeficiency virus and/or feline leukaemia virus and therefore included in the study. Corneal scrapings were obtained to isolate Acanthamoeba spp. by culture and molecular detection by conventional and real time PCR. None of the samples examined directly by molecular methods were positive for Acanthamoeba spp. However, two (3.6%) cases of the cultured samples provided positive results, which were confirmed by subsequent molecular analysis. Sequencing assigned one isolate to genotype T4 and the other to T2. Since Acanthamoeba spp. may also infect animals and humans, the present findings may raise some public health and veterinary concerns.