Iván Ravera

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.

Small Demodex populations colonize most parts of the skin of healthy dogs

BACKGROUND It is unproven that all dogs harbour Demodex mites in their skin. In fact, several microscopic studies have failed to demonstrate mites in healthy dogs. HYPOTHESIS/OBJECTIVES Demodex canis is a normal inhabitant of the skin of most, if not all, dogs. This hypothesis was tested using a sensitive real-time PCR to detect Demodex DNA in the skin of dogs. ANIMALS One hundred dogs living in a humane society shelter, 20 privately owned and healthy dogs and eight dogs receiving immunosuppressive or antineoplastic therapy. METHODS Hair samples (250-300 hairs with their hair bulbs) were taken from five or 20 skin locations. A real-time PCR that amplifies a 166 bp sequence of the D. canis chitin synthase gene was used. RESULTS The percentage of positive dogs increased with the number of sampling points. When a large canine population was sampled at five cutaneous locations, 18% of dogs were positive for Demodex DNA. When 20 skin locations were sampled, all dogs tested positive for mite DNA. Our study indicates that Demodex colonization of the skin is present in all dogs, independent of age, sex, breed or coat. Nevertheless, the population of mites in a healthy dog appears to be small. Demodex DNA was amplified from all 20 cutaneous points investigated, without statistically significant differences. CONCLUSIONS AND CLINICAL IMPORTANCE Using a real-time PCR technique, Demodex mites, albeit in very low numbers, were found to be normal inhabitants of haired areas of the skin of healthy dogs.

Immunology and pathogenesis of canine demodicosis

Demodex mites colonized the hair follicles and sebaceous glands of mammals millions of years ago and have remained relatively unchanged in this protected ecologic niche since then. The host immune system detects and tolerates their presence. Toll-like receptor-2 of keratinocytes has been demonstrated to recognize mite chitin and to elicit an innate immune response. The subsequent acquired immune response is poorly understood at present, but there is experimental and clinical evidence that this is the main mechanism in the control of mite proliferation. A transgenic mouse model (STAT(-/-) /CD28(-/-) ) has demonstrated that the immune response is complex, probably involving both cellular and humoral mechanisms and requiring the role of co-stimulatory molecules (CD28). It is known that a genetic predisposition for developing canine juvenile generalized demodicosis exists; however, the primary defect leading to the disease remains unknown. Once the mite proliferation is advanced, dogs show a phenotype that is similar to the T-cell exhaustion characterized by low interleukin-2 production and high interleukin-10 and transforming growth factor-β production by lymphocytes, as described in other viral and parasitic diseases. Acaricidal treatment (macrocyclic lactones) decreases the antigenic load and reverses T-cell exhaustion, leading to a clinical cure. Although in recent years there have been significant advances in the management and understanding of this important and complex canine disease, more research in areas such as the aetiology of the genetic predisposition and the immune control of the mite populations is clearly needed.

Phylogenetic relationships in three species of canine Demodex mite based on partial sequences of mitochondrial 16S rDNA

BACKGROUND The historical classification of Demodex mites has been based on their hosts and morphological features. Genome sequencing has proved to be a very effective taxonomic tool in phylogenetic studies and has been applied in the classification of Demodex. Mitochondrial 16S rDNA has been demonstrated to be an especially useful marker to establish phylogenetic relationships. HYPOTHESIS/OBJECTIVES To amplify and sequence a segment of the mitochondrial 16S rDNA from Demodex canis and Demodex injai, as well as from the short-bodied mite called, unofficially, D. cornei and to determine their genetic proximity. METHODS Demodex mites were examined microscopically and classified as Demodex folliculorum (one sample), D. canis (four samples), D. injai (two samples) or the short-bodied species D. cornei (three samples). DNA was extracted, and a 338 bp fragment of the 16S rDNA was amplified and sequenced. RESULTS The sequences of the four D. canis mites were identical and shared 99.6 and 97.3% identity with two D. canis sequences available at GenBank. The sequences of the D. cornei isolates were identical and showed 97.8, 98.2 and 99.6% identity with the D. canis isolates. The sequences of the two D. injai isolates were also identical and showed 76.6% identity with the D. canis sequence. CONCLUSION Demodex canis and D. injai are two different species, with a genetic distance of 23.3%. It would seem that the short-bodied Demodex mite D. cornei is a morphological variant of D. canis.

Development of a PCR technique specific for Demodex injai in biological specimens

The identification of Demodex injai as a second Demodex species of dog opened new questions and challenges in the understanding on the Demodex–host relationships. In this paper, we describe the development of a conventional PCR technique based on published genome sequences of D. injai from GenBank that specifically detects DNA from D. injai. This technique amplifies a 238-bp fragment corresponding to a region of the mitochondrial 16S rDNA of D. injai. The PCR was positive in DNA samples obtained from mites identified morphologically as D. injai, which served as positive controls, as well as in samples from three cases of demodicosis associated with proliferation of mites identified as D. injai. Furthermore, the PCR was positive in 2 out of 19 healthy dogs. Samples of Demodex canis and Demodex folliculorum were consistently negative. Skin samples from seven dogs with generalized demodicosis caused by D. canis were all negative in the D. injai-specific PCR, demonstrating that in generalized canine demodicosis, mite proliferation is species-specific. This technique can be a useful tool in the diagnosis and in epidemiologic and pathogenic studies.

Identification of a third feline Demodex species through partial sequencing of the 16S rDNA and frequency of Demodex species in 74 cats using a PCR assay

BACKGROUND Demodex cati and Demodex gatoi are considered the two Demodex species of cats. However, several reports have identified Demodex mites morphologically different from these two species. The differentiation of Demodex mites is usually based on morphology, but within the same species different morphologies can occur. DNA amplification/sequencing has been used effectively to identify and differentiate Demodex mites in humans, dogs and cats. HYPOTHESIS/OBJECTIVES The aim was to develop a PCR technique to identify feline Demodex mites and use this technique to investigate the frequency of Demodex in cats. METHODS Demodex cati, D. gatoi and Demodex mites classified morphologically as the third unnamed feline species were obtained. Hair samples were taken from 74 cats. DNA was extracted; a 330 bp fragment of the 16S rDNA was amplified and sequenced. RESULTS The sequences of D. cati and D. gatoi shared >98% identity with those published on GenBank. The sequence of the third unnamed species showed 98% identity with a recently published feline Demodex sequence and only 75.2 and 70.9% identity with D. gatoi and D. cati sequences, respectively. Demodex DNA was detected in 19 of 74 cats tested; 11 DNA sequences corresponded to Demodex canis, five to Demodex folliculorum, three to D. cati and two to Demodex brevis. CONCLUSIONS AND CLINICAL IMPORTANCE Three Demodex species can be found in cats, because the third unnamed Demodex species is likely to be a distinct species. Apart from D. cati and D. gatoi, DNA from D. canis, D. folliculorum and D. brevis was found on feline skin.

Efficacy of a new topical cyclosporine A formulation in the treatment of atopic dermatitis in dogs.

Topical treatment with cyclosporine A (CsA) has recently become possible with the development of novel nanotechnology pharmaceutical formulations of CsA able to penetrate through the epidermis providing good absorption and dermal action. The aim of this multicentre, blinded, parallel, randomized, placebo controlled trial was to evaluate the efficacy of a new topical CsA formulation in dogs with atopic dermatitis (AD). Dogs (n=32) with severe and moderate clinical signs of non-seasonal AD, but few localized lesions, were randomly allocated to receive topical CsA (17 dogs) or placebo (15 dogs) and were treated twice a day for 6 weeks. Before and 21 and 45 days after starting the treatment, the severity of a previously selected skin lesion was evaluated according to a dermatological scoring system. Owners using a visual analogue scale also assessed pruritus weekly and effectiveness of the treatment was defined as a reduction of at least 50% in these variables after 45 days. After 21 and 45 days the lesion severity score in animals treated with CsA was significantly lower than at baseline (P<0.01, both times). In contrast, the animals on placebo showed no significant improvement at days 21 or 45. The percentage of dogs with an effective reduction in pruritus at the end of the trial was 87.5% and 28.6% in the CsA and placebo groups, respectively. These results suggest that topical administration of CsA is effective in reducing the severity of skin lesions and pruritus in dogs with moderate to severe AD as soon as 3 weeks after starting treatment.

Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis.

Pyogranulomatous mural folliculitis in a cat treated with methimazole

An 11-year-old spayed female domestic shorthair cat was presented for polydipsia, hyperactivity and bilateral thyroid gland enlargement. Total T4 (TT4) was in the upper interval range; therefore, an early hyperthyroidism was suspected. A treatment trial with methimazole was started, as the owner refused further tests. Six months later the owner stopped the treatment. One year later, clinical signs persisted and TT4 was still in the upper interval range. Methimazole was re-introduced but 48 h later the cat presented non-pruritic alopecia with erythema, scales and perilesional yellowish crusts. Pyogranulomatous mural folliculitis was diagnosed by histopatological examination of the skin biopsies. Methimazole was withdrawn and macroscopic lesions healed and disappeared histologically in 15 days. An idiosyncratic drug reaction to methimazole was suspected. To the best of our knowledge, this is the first report of feline pyogranulomatous mural folliculitis likely secondary to an adverse drug reaction to methimazole administration.