Mar Bardagí

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.

Cutaneous mucinosis in shar-pei dogs is due to hyaluronic acid deposition and is associated with high levels of hyaluronic acid in serum

Cutaneous mucinosis affects primarily shar-pei dogs. Hyaluronic acid (HA) is considered to be the main component of mucin and CD44 is the major cell surface receptor of HA, necessary for its uptake and catabolism. The aims of this study were to identify the composition of the mucin in cutaneous mucinosis of shar-pei dogs, investigate the correlation between the deposition of HA and the expression of CD44, and determine whether shar-pei dogs with cutaneous mucinosis presented with elevated levels of serum HA. In skin biopsies, the mucinous material was stained intensely with Alcian blue and bound strongly by the hyaluronan-binding protein. No correlation was found between the degree of HA deposition in the dermis and the expression of CD44 in the skin of shar-pei dogs affected or unaffected by cutaneous mucinosis. A clear positive correlation was found between the existence of clinical mucinosis and the serum HA concentration. In control dogs, serum HA ranged from 155.53 to 301.62 microg L(-1) in shar-pei dogs; without mucinosis it ranged from 106.72 to 1251.76 microg L(-1) and in shar-pei dogs with severe mucinosis it ranged between 843.51 to 2330.03 microg L(-1). Altogether, the results reported here suggest that mucinosis of shar-pei dogs is probably the consequence of a genetic defect in the metabolism of HA.

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.

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.

Demodex injai infestation and dorsal greasy skin and hair in eight wirehaired fox terrier dogs

Demodex injai mites were detected on trichoscopic examinations and/or deep skin scrapings in eight wirehaired fox terrier dogs with dorsal greasy skin and hair. Histological examination performed in five dogs revealed marked sebaceous gland hyperplasia with lympho-plasmacytic periadnexal dermatitis in all of them. One mite section was observed in one patient. Seven dogs were parasitologically cured after 2 to 7 months of oral ivermectin treatment. Greasy skin and hair resolved in four dogs, was partially reduced in two dogs and persisted in the remaining dog. Skin biopsies were repeated after parasitological cure in two dogs and revealed the persistence of sebaceous gland hyperplasia with mild lympho-plasmacytic periadnexal dermatitis and no parasites. Based on the findings in this case series, the terrier dog breed might be at increased risk for the development of D. injai mite infestation associated with dorsal greasy skin and hair, and microscopically with sebaceous gland hyperplasia. Persistence of sebaceous gland hyperplasia after parasitological cure in some patients suggested that this histological finding may not always be resulting from Demodex infestation. Moreover, low numbers of adult mites and variable clinical responses to acaricidal therapy suggested a contributory rather than a major role of D. injai in this skin condition. Dermatopathological diagnosis of sebaceous gland hyperplasia, particularly in case of dorsal trunk specimens from terrier dog breeds, warrants the search for D. injai mites on trichoscopic examinations and/or deep skin scrapings.

Hereditary cutaneous mucinosis in shar pei dogs is associated with increased hyaluronan synthase-2 mRNA transcription by cultured dermal fibroblasts.

Shar pei dogs are known for the distinctive feature of thick, wrinkled skin as a consequence of high dermal mucin content. Excessive dermal deposition of mucinous substance leading to severe skin folding, and/or to the more severe vesicular form characterized by dermal vesicles or bullae, is highly prevalent in this breed and is known as idiopathic mucinosis. Hyaluronic acid (HA) is the main component that accumulates in the dermis, and high levels of HA have also been detected in the serum of shar pei dogs. In this study, the cellular and molecular mechanisms underlying cutaneous mucinosis of shar pei dogs were investigated. Thirteen shar pei dogs and four control dogs of other breeds were included. In primary dermal fibroblast cultures, transcription of the family of hyaluronan synthases (HAS) involved in HA synthesis, and of hyaluronidases (HYAL) involved in HA degradation, were studied by reverse transcriptase polymerase chain reaction. The location of HA in cell cultures was studied by immunofluorescence and confocal laser microscopy. Dermal fibroblasts transcribed HAS2, HAS3, HYAL1 and HYAL2, but no amplification for HAS1 was found. A higher transcription of HAS2 was demonstrated in shar pei dogs compared with control dogs. By confocal microscopy, HA was detected as a more diffuse and intense network-like pattern of green fluorescence in the fibroblast cells of shar pei dogs in comparison with control dogs. Together, these results provide additional evidence that hereditary cutaneous mucinosis in shar pei dogs may be a consequence of over-transcription or increased activity of HAS2.

Immunohistochemical detection of COX-2 in feline and canine actinic keratoses and cutaneous squamous cell carcinoma.

Cyclooxygenase-2 (COX-2) overexpression and its causal role in epidermal carcinogenesis have been demonstrated in human actinic keratoses (AK) and cutaneous squamous cell carcinoma (SCC). The aim of this study was to determine immunohistochemically the level of expression of COX-2 in feline and canine AK (n=18), SCC (n=36) and inflammatory dermatoses (n=24). COX-2 immunoreactivity was detected in all feline and canine SCC. In all specimens, labelled basal and suprabasal neoplastic keratinocytes were localized within and below areas of superficial erosion or ulceration and only scattered deeper tumour cells were positively labelled. In most cases, positive immunoreactivity of keratinocytes was associated with the presence of granulocytes. COX-2 expression was detected in 3/9 canine and 4/9 feline cases of AK and in only one case was associated with inflammation. Inflammatory dermatoses were characterized by positively labelled epidermal and follicular basal and suprabasal keratinocytes that were always associated with granulocyte exocytosis. These results indicate that further study of the effect of using COX-2 inhibitors in the management and prevention of feline and canine cutaneous SCC is warranted. The association between inflammatory cells and COX-2 expressing epidermal cells opens a new line of research regarding the role of COX-2 in SCC oncogenesis. Moreover, further studies should investigate the role of COX-2 in the pathogenesis and management of AK in animals.

Dermatophagoides farinae-specific immunotherapy in atopic dogs with hypersensitivity to multiple allergens: A randomised, double blind, placebo-controlled study

A randomised, placebo-controlled, double blind study was conducted on 25 dogs that had atopic dermatitis, together with skin test reactivity and elevated serum IgE to Dermatophagoides farinae (Df) and at least one additional allergen. Dogs were treated with either a Df-restricted immunotherapy solution (n=14) or a placebo (n=11) and evaluated 6 weeks and 3, 5, 7 and 9 months after the initiation of treatment using a clinical scoring system (SASSAD) and pruritus analogue scale scores. The Df-restricted solution and the placebo had an equal effect on both pruritus and the skin manifestations (P>0.05). The results of this study indicate that in dogs with atopic dermatitis based on hypersensitivity to environmental allergens in addition to D. farinae, Df-restricted immunotherapy is insufficient to control the disease. Consequently, a solution for allergen-specific immunotherapy should remain customised.

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.