Lluís Ferrer

Canine leishmaniosis - new concepts and insights on an expanding zoonosis: part one

Recent research has provided new insights on the epidemiology, pathology and immunology of canine leishmaniosis (CanL) and its genetic basis. The prevalence of infection in endemic areas is considerably higher than that of apparent clinical illness. In addition, infection spreads rapidly among dogs in the presence of optimal conditions for transmission. Infection involves a variety of granulomatous and harmful immune-mediated responses, and susceptibility to the disease is influenced by a complex genetic basis. These concepts will be instrumental for devising control programs. This review, the first in a series of two articles on CanL, presents an updated view on progress in elucidating the epidemiology and pathogenesis of this challenging disease, and the second part focuses on advances in diagnosis, treatment and prevention.

Prevalence of Leishmania infantum Infection in Dogs Living in an Area of Canine Leishmaniasis Endemicity Using PCR on Several Tissues and Serology

ABSTRACT We studied and compared the prevalence of Leishmaniainfection and the seroprevalence and the prevalence of canine leishmaniasis in an area where canine leishmaniasis is endemic. One hundred dogs living on the island of Mallorca (Spain) were studied. In this study, we clinically examined each dog for the presence of symptoms compatible with leishmaniasis, determined the titer of anti-Leishmania antibodies, and investigated the presence of Leishmania DNA by PCR in skin, conjunctiva, and bone marrow samples of each dog. The prevalence of the disease and the seroprevalence were 13 and 26%, respectively. In 63% of the dogs,Leishmania DNA could be detected by PCR in at least one of the tissues studied. The results of positive PCR in the bone marrow, the conjunctiva, and the skin were 17.8, 32, and 51%, respectively. The prevalence of the infection, 67%, was calculated using all animals that were seropositive and/or positive by PCR with any tissue. The results showed that the majority of dogs living in an area where canine leishmaniasis is endemic are infected by Leishmania and that the prevalence of infection is much greater than the prevalence of overt Leishmania-related disease.

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.

A novel unstable duplication upstream of HAS2 predisposes to a breed-defining skin phenotype and a periodic fever syndrome in Chinese Shar-Pei dogs.

Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (praw = 2.3×10−6, pgenome = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p<0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation.

Canine mast cell tumors express stem cell factor receptor

c-kit protooncogene encodes a type III transmembrane receptor kinase, the stem cell factor receptor, or KIT. The ligand of the KIT. stem cell factor, is a cytokine that stimulates mast cell growth and differentiation. We have studied immunohistochemically KIT expression in 23 canine mast cell tumors (MCTs), 10 histiocytomas, 5 malignant melanomas, and in 2 cell lines derived from mast cells (HMC-1, human and C2, canine). As expected, KIT was detected both in the human mast cell leukemia cell line (HMC- ) and in the canine mastocytoma cell line C2. In normal canine skin, KIT expression was confined to mast cells. All canine MCTs expressed KIT, although the intensity of the staining reaction varied considerably among the 23 neoplasms. Grade III tumors showed the highest expression of KIT, whereas grade I tumors showed the lowest expression of KIT. Two patterns of KIT expression were detected in mast cells. In normal canine mast cells and in some neoplastic mast cells, KIT appeared mainly on the cell membrane. However, in many canine MCTs, KIT is accumulated in the cytoplasm, usually near the cell nucleus. The meaning of these two patterns is not clear. Expression of KIT could not be detected immunohistochemically in any of the other neoplasias investigated. According to our results, it can be concluded that most, if not all, canine MCT express KIT. Furthermore, there is an inverse correlation between the degree of differentiation and the expression of KIT. Moreover, according to our results, KIT can be used as a reliable immunohistochemical marker for canine mast cells and undifferentiated mast cell tumors.

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.

Canine leishmaniasis associated with systemic vasculitis in two dogs

Two cases of canine leishmaniasis associated with systemic necrotizing vasculitis are described. The main macroscopic lesions were haemorrhagic in type; histopathological changes confirmed a vascular lesion affecting small arteries of several organs (skin, intestinal tract, kidney, urinary bladder, mesenteric lymph nodes, adrenal gland, myocardium, lung, eye and choroid plexus). The presence of the parasite was confirmed with a specific immunocytochemical stain. The possibility of an immunological aetiology of the vasculitis and its classification is discussed.

Treatment of demodicosis in dogs: 2011 clinical practice guidelines

BACKGROUND AND OBJECTIVES These guidelines were written by an international group of specialists with the aim to provide veterinarians with current recommendations for the diagnosis and treatment of canine demodicosis. METHODS Published studies of the various treatment options were reviewed and summarized. Where evidence in form of published studies was not available, expert consensus formed the base of the recommendations. RESULTS Demodicosis can usually be diagnosed by deep skin scrapings or trichograms; in rare cases a skin biopsy may be needed for diagnosis. Immune suppression due to endoparasitism or malnutrition in young dogs and endocrine diseases, neoplasia and chemotherapy in older dogs are considered predisposing factors and should be diagnosed and treated to optimize the therapeutic outcome. Dogs with disease severity requiring parasiticidal therapy should not be bred. Secondary bacterial skin infections frequently complicate the disease and require topical and/or systemic antimicrobial therapy. There is good evidence for the efficacy of weekly amitraz rinses and daily oral macrocyclic lactones such as milbemycin oxime, ivermectin and moxidectin for the treatment of canine demodicosis. Weekly application of topical moxidectin can be useful in dogs with milder forms of the disease. There is some evidence for the efficacy of weekly or twice weekly subcutaneous or oral doramectin. Systemic macrocyclic lactones may cause neurological adverse effects in sensitive dogs, thus a gradual increase to the final therapeutic dose may be prudent (particularly in herding breeds). Treatment should be monitored with monthly skin scrapings and extended beyond clinical and microscopic cure to minimize recurrences.

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.

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.