Xavier Roura

Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas: Evolution in the course of infection and after treatment

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.

Diagnosis of canine leishmaniasis by a polymerase chain reaction technique

A polymerase chain reaction (PCR) technique for the diagnosis of canine leishmaniasis on bone marrow samples was developed which amplified a 120 bp DNA fragment of the Leishmania kinetoplast DNA, common to all Leishmania species. Forty-five of 46 dogs in which leishmaniasis had been diagnosed were positive with the PCR technique, whereas none of 41 healthy dogs gave a positive result. Fifteen dogs with leishmaniasis that had been treated for six months with N-methylglucamine antimoniate and allopurinol were also investigated. Seven were positive, implying that they remained infected despite the resolution of their clinical signs.

Prevalence of Hemotropic Mycoplasmas in Healthy and Unhealthy Cats and Dogs in Spain

The aims of the present study were to determine the prevalence of hemoplasmas in cats and dogs from the Barcelona area of Spain with the use of species-specific quantitative polymerase chain reaction (qPCR) assays and to evaluate any associations between hemoplasma infection, clinical presentation, and vector-borne infections. Blood samples from cats (191) and dogs (182) were included and were classified as healthy (149) or unhealthy (224). Ethylenediamine tetra-acetic acid blood samples underwent DNA extraction and qPCR analysis. Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, and ‘Candidatus Mycoplasma turicensis’ were detected in cats, whereas Mycoplasma haemocanis and ‘Candidatus Mycoplasma haematoparvum’ were detected in dogs, with prevalences of 3.7%, 9.9%, 0.5%, 14.3%, and 0.6%, respectively. In cats, no association between hemoplasma infection and health status, age, breed, presence of anemia, Feline leukemia virus status, and other vector-borne infections was found, but outdoor access (P = 0.009), male sex (P = 0.01), and Feline immunodeficiency virus status (P < 0.001) were significantly associated with hemoplasma infection. In dogs, sex, age, health status, presence of anemia, and breed were not significantly associated with hemoplasma infection, but a significant association was found between hemoplasma infection and vector-borne infections (P < 0.001). The present report documents the occurrence of feline ‘Candidatus M. turicensis’ and canine ‘Candidatus M. haematoparvum’ infections in Spain.

Guideline for veterinary practitioners on canine ehrlichiosis and anaplasmosis in Europe

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.

Canine leishmaniasis: the key points for qPCR result interpretation

BackgroundDiagnosis and follow up of CanL is difficult since the range of clinical signs is varied and seroprevalence is high in endemic areas. The aims of this study were: i) demonstrate the advantages of Leishmania qPCR to diagnose and control CanL and highlight its prognostic value and ii) propose guidelines for tissue selection and infection monitoring.FindingsThis study included 710 dogs living in an endemic area of leishmaniasis. Forty percent (285/710) exhibited clinical signs consistent with CanL. Infection was detected in 36.3% (258/710) of the dogs of which 4.5% (32/710) were detected by qPCR, 16.2% (115/710) detected by ELISA and 15.6% (111/710) tested positive for both tests. Only 17.9% (127/710) of the dogs were classified sick (affected) with CanL.All symptomatic dogs with medium or high ELISA titers were qPCR-positive in blood samples. All dogs with inconclusive or low ELISA results with high or medium qPCR parasitemia values developed the disease. Seventy one percent of asymptomatic ELISA-positive dogs confirmed by qPCR (medium to high parasitemia) developed the disease.Bone marrow or lymph node aspirate should be selected to ensure the absence of the parasite in asymptomatic dogs: 100-1,000 parasites/ml in bone marrow are detectable in blood, whereas lower parasite loads are usually negative. Almost 10% of negative samples in blood were positive in conjunctival swabs.ConclusionsBecause qPCR allows parasite quantification, it is an effective tool to confirm a diagnosis of CanL in (i) cases of inconclusive ELISA results, (ii) when the dog has not yet seroconverted, or (iii) for treatment monitoring.

Emesis in dogs: a review

Emesis is a common presenting sign in small animal practice. It requires a rational approach to management that is based upon a sound understanding of pathophysiology combined with logical decision making. This review, which assesses the weight of available evidence, outlines the physiology of the vomiting reflex, causes of emesis, the consequences of emesis and the approach to clinical management of the vomiting dog. The applicability of diagnostic testing modalities and the merit of traditional approaches to management, such as dietary changes, are discussed. The role and usefulness of both traditional and novel anti‐emetic drugs is examined, including in specific circumstances such as following cytotoxic drug treatment. The review also examines areas in which common clinical practice is not necessarily supported by objective evidence and, as such, highlights questions worthy of further clinical research.

PCR survey of vectorborne pathogens in dogs living in and around Barcelona, an area endemic for leishmaniosis

Blood samples from 153 dogs living in and around Barcelona were assayed for Leishmania infantum and Ehrlichia, Anaplasma, Rickettsia, Bartonella, Hepatozoon, Babesia and Theileria species by PCR amplification of DNA, and the amplicons obtained were sequenced. The prevalence of the infectious agents was L infantum (29·4 per cent), Ehrlichia and Anaplasma species (4·0 per cent), Hepatozoon canis (3·3 per cent), Babesia canis vogeli (2·0 per cent), Babesia gibsoni (2·0 per cent), Babesia canis canis (1·3 per cent) and Theileria annae (0·7 per cent). Coinfections were present in seven of the dogs and they were significantly associated with L infantum infection (P=0·024). There was a significant correlation between clinical signs of illness and the load of L infantum.

Seropositivity rates for agents of canine vector-borne diseases in Spain : a multicentre study

BackgroundControlling canine vector-borne diseases (CVBD) is a major concern, since some of these diseases are serious zoonoses. This study was designed to determine seropositivity rates in Spain for agents causing the following five CVBD: leishmaniosis (Leishmania infantum: Li), heartworm (Dirofilaria immitis: Di), ehrlichiosis (Ehrlichia canis: Ec), anaplasmosis (Anaplasma phagocytophilum/Anaplasma platys: An) and Lyme disease (Borrelia burgdorferi: Bb).MethodsAnti-An, -Bb, and -Ec antibodies and the Di antigen were determined using the 4DX SNAP® Test (IDEXX Laboratories) and anti-L. infantum (Li) antibodies using the Leishmania SNAP® Test (IDEXX Laboratories) in blood and/or serum samples.ResultsAmong 1100 dogs examined, overall seropositivity rates were: Li (15.7%), Ec (5%), An (3.1%), Di (1.25%) and Bb (0.4%). While seropositivity towards Bb and Di was similar in all geographic regions, rates were significantly higher in the east of Spain (8.3%) for An, significantly higher in the north (20%) for Ec, and significantly higher in the Southeast (46.6%) and South (27.4%), and significantly lower in the north (0%) for Li.No statistical associations were observed between sex and the CVBD analyzed (p ≥ 0.05) while the following associations with other variables were detected: a higher seropositivity to Ec (40%) and Bb (6.7%) in dogs under one year of age compared with adults (p < 0.05); and a higher seropositivity to An and Li in dogs that lived outdoors versus indoors (p = 0.01; p < 0.001, respectively). Seropositivity rates of 2.1%, 0%, 1.7%, 0.5% and 4.2% were recorded respectively for An, Bb, Ec, Di and Li in dogs with no clinical signs (n = 556) versus 3.8%, 0.6%, 7.5%, 1.8% and 25.9% for those with signs (n = 507) suggestive of a CVBD.ConclusionThe data obtained indicate a risk for dogs in Spain of acquiring any of the five CVBD examined. Veterinarians in the different regions should include these diseases in their differential diagnoses and recommend the use of repellents and other prophylactic measures to prevent disease transmission by arthropod vectors. Public health authorities also need to become more involved in the problem, since some of the CVBD examined here also affect humans.

Long term follow-up of dogs diagnosed with leishmaniosis (clinical stage II) and treated with meglumine antimoniate and allopurinol

Twenty-three dogs with a diagnosis of leishmaniosis (clinical stage II) were treated with meglumine antimoniate and allopurinol and were followed up for 2-9 years. The treatment showed efficacy and the clinical condition of the dogs improved markedly in the first 3 months of treatment. Anti-Leishmania antibody titres declined slowly although most dogs remained seropositive 1 year after beginning treatment. Inter-individual variability in the evolution of the titres was very high. The dogs presented with three types of complications during the follow-up period. (1) Three dogs experienced relapses characterized by clinical signs, high anti-Leishmania titres and high parasitaemia. (2) Eight dogs presented immune-mediated lesions, such as uveitis, arthritis and cutaneous vasculitis; in all of these cases, the dogs had high titres of anti-Leishmania antibodies at diagnosis and during follow-up. (3) Three dogs presented xanthine urolithiasis most likely due to the allopurinol treatment. In one case the xanthine uroliths led to hydronephrosis and nephrectomy. The study demonstrated a long survival for dogs with leishmaniosis treated with the combination of meglumine antimoniate and allopurinol. Clinicians should pay special attention to the appearance of immune-mediated lesions, especially in dogs with sustained high antibody titres, and to urolithiasis.

Therapy of ocular and visceral leishmaniasis in a cat

Abstract An 8‐year‐old, spayed female Domestic Short‐haired cat was referred for further evaluation of chronic lymphocytic–plasmacytic stomatitis and bilateral ocular disease. The cat had been treated with systemic glucocorticoids for several months. Initial ophthalmic examination revealed bilateral deep stromal corneal ulcers, exudative panuveitis and secondary glaucoma. Mature mild neutrophilia and monocytosis were detected on complete blood cell count. Abnormalities in the serum profile were hyperglycemia, mild azotemia, hyperglobulinemia and moderate polyclonal gammapathy. Urinalysis revealed glucosuria without ketonuria. Diabetes mellitus was diagnosed and treatment with long‐acting insulin was started. An enzyme‐linked immunosorbent assay was highly positive for leishmaniasis, and treatment with allopurinol was started. Although specific topical treatment was applied, melting ulcers progressed to corneal perforation and both eyes were enucleated. Ocular histology showed large numbers of intracellular organisms compatible with amastigotes of the genus Leishmania located in the uveal tract, cornea, sclera and retina. Results of inmunohistochemistry staining on ocular samples were positive for Leishmania. Bone marrow cytology demonstrated numerous macrophages with intracytoplasmatic Leishmania. Polymerase chain reaction results on bone marrow for Leishmania were positive. Three weeks later, hypoglycemic episodes permitted withdrawal of the insulin therapy. To the authors’ knowledge this is the first case of ocular and visceral leishmaniasis diagnosed in vivo and under systemic treatment in a cat.