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.

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.

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.

LEISHMANIA INFANTUM INFECTION IN BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS RUFOGRISEUS) IN A SPANISH WILDLIFE PARK.

Although dogs are the main reservoir for human Leishmania infantum infection, the disease has also been reported in other domestic and wild mammals. In 2011, a fatal case of naturally acquired leishmaniosis was described for the first time in a Bennett's wallaby (Macropus rufogriseus rufogriseus) kept in a wildlife park in Madrid (Spain). This study was designed to assess the infection status of twelve Bennett's wallabies in the same park one year after this incident. Phlebotomus perniciosus, the main vector of L. infantum in Spain, was screened for using sticky and Centers for Disease Control miniature light traps. L. infantum infection was confirmed by molecular diagnosis in four animals, but only one wallaby returned a positive serology result. The presence of the sand fly vector was also confirmed in this habitat. These results suggest that the first case of L. infantum in a wallaby in this park was not an isolated incident and stress the need for further work to determine the role of this parasite in the morbidity and mortality of these macropods. Madrid was recently the scene of an outbreak of human cutaneous and visceral leishmaniosis. Epidemiological studies have so far revealed the widespread presence of L. infantum infection in animals other than the dog. Our ongoing work suggests a risk of L. infantum infection not only among captive animals in Madrid, but also among threatened species or even species that are already extinct in the wild.

First detection of Onchocerca lupi infection in dogs in southern Spain.

BACKGROUND: Onchocerca lupi causes ocular pathology of varying severity in dogs from south-western United States, western Europe and northern Asia. This filarioid has also been recognized as a zoonotic agent in Tunisia, Turkey, Iran and the USA, though the information about the biology and epidemiology of this infection is largely unknown. In Europe, O. lupi has been reported in dogs from Germany, Greece, Hungary, Portugal and Romania and in a cat from Portugal. The present study was designed to establish the occurrence of O. lupi in dogs in southwestern Spain. In the present study a total of 104 dogs of different breed, sex, and age living in a shelter in Huelva (SW Spain) were examined. Skin snip samples were collected using a disposable scalpel in the forehead and inter-scapular regions and stored as aliquots in saline solution (0.5 ml) before light microscopy observation of individual sediments (20 µl) and molecular examination. RESULTS: Of the 104 dogs examined, 5 (4.8 %) were skin snip-positive for O. lupi: two by microscopy and three by PCR. One of the O. lupi infected dogs showed neurological signs but ocular ultrasonography and/or MRI detected no abnormalities. CONCLUSIONS: This first report of O. lupi infection in dogs in southern Spain expands the range of geographical distribution of this parasite and sounds an alarm bell for practitioners and physicians working in that area.

Course of experimental infection of canine leishmaniosis: follow-up and utility of noninvasive diagnostic techniques.

This study compares the utility of a molecular diagnosis of experimental CanL on non-invasive samples (urine, conjunctival (CS), oral (OS) and vulvar (VS) swabs) with that of traditional invasive techniques during the course of infection. Eight dogs were experimen-tally infected with Leishmania infantum and followed monthly for 12 months to assess clinical, clinicopathological, immunological and parasitological variables. Active infection was produced in 100% of the dogs. The animals showed positive bone marrow (BM) cytologies and cultures, clinical signs, clinicopathological abnormalities and a high specific humoral immune response. The infection was detected at 90 days post-infection (p.i.) by real-time quantitative PCR (rtQ-PCR) on BM in all dogs and in blood in 2 dogs, while anti-L. infantum antibody seroconversion occurred between Days 120 and 180 days p.i. The tissue with the highest L. infantum kDNA load, as detected by rtQ-PCR, was BM (range 381.5­70,000 parasites/ml at the study end), this sample type showing greater sensitivity than peripheral blood (PB). The vulvar swabs used here for the first time to quantify para-site loads in dogs revealed a greater load than oral and conjunctival swabs at one year p.i. Urine samples showed the lowest concentrations of L. infantum DNA (maximum: 8.57 par-asites/ml). Our results suggest that for the early detection of infection, adding to serology a test such as rtQ-PCR on OS or VS improves sensitivity and specificity.

Current status of L. infantum infection in stray cats in the Madrid region (Spain): implications for the recent outbreak of human leishmaniosis?

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.

First study on efficacy and tolerability of a new alkylphosphocholine molecule (oleylphosphocholine-OlPC) in the treatment of canine leishmaniosis due to Leishmania infantum.

The alkylphosphocholine oleylphosphocholine (OlPC) represents a potential new therapy for the treatment of canine leishmaniosis caused by Leishmania infantum. The aim of the present study was to evaluate the efficacy and safety of OlPC in a small cohort of dogs naturally infected with L. infantum and defined as clinically sick (LeishVet stages II and III). A total of eight dogs were included in the study and were treated orally with 4 mg/kg OlPC for 14 days. Dogs were assessed at the clinical and parasitological level at four time points during a total follow-up period of 90 days (before treatment and at 15, 30, and 90 days post-treatment onset). Ln-PCR, real-time quantitative PCR, antibody testing (IFAT), and culture of bone marrow aspirates were evaluated at the four time points. OlPC treatment induced a rapid and satisfactory clinical recovery in terms of clinical score reduction and weight gain, and treatment efficacy was found to be associated with a decrease in bone marrow parasitic load. Serological titers measured by IFAT were stable in any of the treated dogs at any time point after treatment. OlPC was well tolerated and no severe adverse events were noted in any of the treated dogs; even some dogs showed slight intestinal disorders. This proof-of-principle study is the first to show that short oral treatment with OlPC improves clinical signs of canine L. infantum leishmaniosis, highlighting the need to perform additional studies to optimize the dosing regimen and to assess long-term treatment efficacy of this drug.