Cyclosporine induced generalized hyperkeratosis in a dog.

INTRODUCTION: Cyclosporine is a potent immunosuppressive agent used in veterinary medicine to treat a variety of inflammatory or immune mediated conditions. Many adverse effects are associated with this medication, however most of them rarely occur. A 5-year-old, female intact French bulldog was presented with multiple, multifocally distributed, severe hyperkeratotic and papillomatous/verrucous plaques. The dog was on long-term immunosuppressive treatment with cyclosporine for meningoencephalitis of unknown origin (MUO). It had an history of atopic dermatitis and calcinosis cutis. A papillomavirus infection was excluded by polymerase chain reaction (PCR), and histopathologic analysis revealed a chronic lymphoplasmacytic non-specific dermatitis, perifolliculitis and periadnexitis and focal folliculitis with papillomatous epidermal hyperplasia and orthokeratotic hyperkeratosis. The diagnosis of "cyclosporine-induced epidermal hyperplasia with secondary pyoderma" was made. Cyclosporine was discontinued and as an alternative mycophenolate mofetil was started to control the MUO. An antimicrobial treatment was prescribed for three weeks. After four months, the skin lesions had healed completely. To date after 2 years, the dog is still in remission. The occurrence of hyperplastic lesions associated with cyclosporine therapy have already been described in previous reports. Most of them resemble those of psoriasiform lichenoid dermatitis, although papilloma virus may be detected in some instances. The dog of the present case showed some peculiarities in the histopathological findings, and a papillomavirus involvement was ruled out with PCR. Like observed in a previous report, there was no correlation between cyclosporine blood level and the severity of dermatological changes. A discontinuation of cyclosporine resulted in complete healing in 4 months. This case highlights the importance of regular monitoring and follow-ups in patients on immunosuppressive therapy. Even rare side effects should always be considered in these cases.

INTRODUCTION: La cyclosporine est un puissant agent immunosuppresseur utilisé en médecine vétérinaire pour traiter une variété de conditions inflammatoires ou à médiation immunitaire. De nombreux effets indésirables sont associés à ce médicament, mais la plupart d'entre eux se produisent rarement. Un bouledogue français intact, âgé de 5 ans, a été présenté avec de multiples plaques hyperkératosiques et papillomateuses/verruqueuses sévères, réparties de manière multifocale. Le chien suivait un traitement immunosuppresseur à long terme à base de cyclosporine pour une méningo-encéphalite d'origine inconnue (MUO). Il avait des antécédents de dermatite atopique et de calcinosis cutis. Une infection à papillomavirus a été exclue par réaction en chaîne par polymérase (PCR) et l'analyse histopathologique a révélé une dermatite chronique lymphoplasmocytaire non spécifique, une périfolliculite et une périannexite ainsi qu'une folliculite focale avec hyperplasie épidermique papillomateuse et hyperkératose orthokératosique. Le diagnostic d'¼hyperplasie épidermique induite par la cyclosporine avec pyodermie secondaire¼ a été posé. La cyclosporine a été stoppée et on a commencé à administrer du mycophénolate mofétil comme alternative pour contrôler l'OMU. Un traitement antimicrobien a été prescrit pendant trois semaines. Après quatre mois, les lésions cutanées étaient complètement guéries. À ce jour, après deux ans, le chien est toujours en rémission. L'apparition de lésions hyperplasiques associées au traitement par la cyclosporine a déjà été décrite dans des rapports précédents. La plupart d'entre elles ressemblent à celles de la dermatite lichénoïde psoriasiforme, bien que le virus du papillome puisse être détecté dans certains cas. Le chien du cas présent présentait quelques particularités dans les résultats histopathologiques et une implication du papillomavirus a été exclue par PCR. Comme observé dans un rapport précédent, il n'y avait pas de corrélation entre le taux sanguin de cyclosporine et la sévérité des altérations dermatologiques. L'arrêt de la cyclosporine a permis une guérison complète en 4 mois. Ce cas souligne l'importance d'une surveillance et d'un suivi réguliers des patients sous traitement immunosuppresseur. Les effets secondaires, même rares, doivent toujours être pris en compte dans ces cas.

Evaluation of Plasma C-Reactive Protein as a Biomarker in Dogs with Atopic -Dermatitis Receiving Allergen-Specific Immunotherapy: A Pilot Study.

INTRODUCTION: In this pilot study, we wished to determine if C-reactive protein (CRP) levels could be a useful severity or treatment biomarker for canine atopic dermatitis (AD). Nine atopic dogs received allergen immunotherapy for 1 year. Blood was collected before and at four re-evaluation visits. At each time point, the skin lesions were graded with the Canine Atopic Dermatitis Extent and Severity Index (CADESI) 4, and the plasma CRP levels were measured by Enzyme-linked Immunosorbent Assay (ELISA). We found a significant yet minimal correlation between the CRP levels and the CADESI4 scores. The CRP levels were not significantly different between dogs with AD of increasing severity. Finally, there was no correlation between the percentage change in CADESI4 and CRP values during immunotherapy. In conclusion, the lack of significant difference in CRP levels between dogs of increasing AD severity and lack of correlation between percentage changes in skin lesion and CRP values suggest that this protein would not be a clinically-useful biomarker in atopic dogs.

INTRODUCTION: Dans cette étude pilote, nous avons souhaité déterminer si les niveaux de protéine C-réactive (CRP) pourraient être un biomarqueur de gravité ou de traitement utile pour la dermatite atopique canine (DA). Neuf chiens atopiques ont reçu une immunothérapie allergénique spécifique pendant un an. Du sang a été prélevé avant et lors de quatre visites de réévaluation. À chacune de ces visites, les lésions cutanées ont été classées au moyen de l'indice d'étendue et de gravité de la dermatite atopique canine (CADESI) 4 et les taux plasmatiques de CRP ont été mesurés par dosage immuno-enzymatique (ELISA). Nous avons trouvé une corrélation significative mais minime entre les niveaux de CRP et les scores CADESI4. Les niveaux de CRP n'étaient pas significativement différents entre les chiens atteints de DA de gravité croissante. Enfin, il n'y avait pas de corrélation entre le pourcentage de changement des valeurs de ­CADESI4 et de CRP pendant l'immunothérapie. En conclusion, l'absence de différence significative des taux de CRP entre les chiens de gravité croissante de DA et le manque de corrélation entre les changements de ­pourcentage de lésion cutanée et les valeurs de CRP suggèrent que cette protéine ne serait pas un biomarqueur cliniquement utile chez les chiens atopiques.

Impact of the early-life skin microbiota on the development of canine atopic dermatitis in a high-risk breed birth cohort.

Canine atopic dermatitis (CAD) is a prevalent inflammatory skin disease of dogs worldwide. Certain breeds such as the West Highland White Terriers (WHWT) are predisposed to suffer from CAD. Microbial dysbiosis is known to play a significant role in the pathogenesis of the disease, which is similar to its human counterpart, atopic dermatitis (AD). To date, no large cohort-study has been conducted in a predisposed dog breed to study the impact of the early-life microbiota on the development of CAD, as well as the possible implication of factors such as hygiene and access to the outdoors. In this study skin samples of 143 WHWT, including 109 puppies up to three weeks old and 34 parent dogs, from 17 breeders, were subjected to 16S rRNA gene and ITS2 amplicon sequencing to disclose the bacterial and fungal oral and skin microbiota, respectively. The oral samples served as a control group to confirm differences between haired and mucosal surfaces. The cutaneous microbiota differed between sample sites and age of the dogs. The season of sampling, geographical origin as well as hygiene status of the household and the access to the outdoors shaped the skin microbiota of the puppies significantly. However, we found that the individual early-life microbiota did not predispose for the later development of CAD.

A Toxocara canis infection influences the immune response to house dust mite allergens in dogs.

BACKGROUND: The "hygiene hypothesis" suggests that a western way of life, including the extended use of anti-infective drugs, a high standard of hygiene and the resulting reduced exposure to microorganisms, could be one of the possible explanations for the increasing prevalence of allergic diseases in humans and animals. OBJECTIVES: we wished to evaluate if a nematode infection influenced IgE sensitization and allergic reactions to house dust mites in an experimental atopic dog model. METHODS: Twelve 10-week-old beagles were included: six of them were inoculated orally withToxocara canis (Tc) while six served as non-infected. Tc-specific IgE and IgG against Tc L3 E/S antigen (TcE/S antigen) were measured before and after Tc infection. All twelve dogs were sensitized epicutaneously to Dermatophagoides farinae (Df) house dust mites and then challenged twice epicutaneously with the mite. Total IgE and Df-specific IgE were measured before/after sensitization and after challenge. Local skin lesion scores were assessed before/after sensitization and after challenge while the duration of pruritus manifestations was measured by video after the second challenge. RESULTS: Toxocara canis -infected dogs exhibited higher levels of IgG and IgE levels against Tc, Df-specific IgE, total IgE but lower skin lesion scores and pruritus durations after challenge, compared to dogs not infested with this nematode. CONCLUSION & CLINICAL RELEVANCE: These observations suggest that a Tc infection increases the sensitization to Df in dogs. The possible protective effect against Df-induced clinical signs after allergen challenge should be confirmed in larger studies.

[Intralymphatic immunotherapy: An effective and safe alternative route for canine atopic dermatitis]. / Intralymphatische Immuntherapie: Eine sichere und effektive Methode zur Desensibilisierung der caninen atopischen Dermatitis.

INTRODUCTION: Allergen-specific immunotherapy (ASIT) is the only etiologic treatment of atopic dermatitis in dogs. In humans it has been shown that intralymphatic immunotherapy (ILIT) enhanced efficacy and patient compliance and reduced treatment time from 3 years to 8 weeks. As only safety data have been published yet, the aim of this study was to evaluate the clinical efficacy of ILIT in dogs. 20 atopic dogs underwent ILIT with alum-precipitated allergens administered every 4 weeks for 3 to 7 times in the popliteal lymph node. Pruritus (Hill score), CADESI (canine atopic dermatitis severety index), concurrent medications and adverse reactions were recorded initially and every 4 weeks for a total period of 24 weeks. The observed clinical response was good in 12/20 (60%) patients and improvement could be seen in some dogs already after 4 weeks. The median number of injections was 5.6. All dogs tolerated the procedure well and no adverse effects were recognized during or after ILIT. Therefore ILIT should be regarded as a safe alternative to subucaneous ASIT, enabling a faster clinical improvement with the same response rate.

INTRODUCTION: L'immunothérapie spécifique de l'allergène est le seul traitement étiologique de la dermite atopique du chien. On a pu montrer que, chez l'homme, l'immunothérapie intralymphatique (ITIL) augmente la fiabilité du traitement et permet de réduire sa durée de 3 ans à 8 semaines. Comme jusqu'à ce jour seules des données relatives à la tolérance avaient été publiées, la présente étude a pour but d'examiner l'efficacité clinique de l'ITIL chez les chiens. Vingt chiens atopiques ont été désensibilisés au moyen d'allergènes précipités à l'aluminium par ITIL dans les ganglions poplités toutes les 4 semaines. Le prurit (Hill score), le CADESI (canine atopic dermatitis severity index), les médicaments appliqués et les effets secondaires observés ont été enregistrés au début du traitement puis toutes les 4 semaines durant au total 24 semaines. 12/20 (60%) des patients ont bien répondu au traitement. L'amélioration clinique a pu être partiellement constatée après 4 semaines déjà. En moyenne, 5.6 injections ont été nécessaires. Tous les chiens ont bien toléré l'ITIL et il n'a pas été observé d'effet secondaire pendant ou après le traitement. L'immunothérapie intralymphatique semble donc une alternative sure à l'immunothérapie spécifique de l'antigène sous-cutanée et permet d'obtenir un effet plus rapide avec le même taux de réponse.

[Clinical symptomps, diagnosis and therapy of feline allergic dermatitis]. / Klinische Merkmale, Diagnose und Therapie des felinen Atopie Syndroms.

Allergies are often suspected in cats and they are mainly hypersensitivity reactions against insect bites, food- or environmental allergens. Cats, with non flea induced atopic dermatitis, normally present with one oft he following reaction patterns: miliary dermatitis, eosinophilic dermatitis, selfinduced alopecia or head and neck excoriations. None of these reaction patterns is nevertheless pathognomonic for allergic dermatitis, therefore the diagnosis is based on the one hand on the exclusion of similar diseases on the other hand on the successful response on a certain therapy. Recently a study on the clinical presentation of cats with non flea induced atopic dermatitis was published. In this study certain criteria for diagnosing atopy in cats were proposed. For therapy of allergic cats cyclosporin, glucocorticoids, antihistamines, hypoallergenic diets and allergen specific immunotherapy are used. This article should provide a recent overview on the clinical symptoms, diagnosis and therapy of feline allergic dermatitis.

Quantitative real time polymerase chain reaction assays for the sensitive detection of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely understood. Sensitive and quantitative DNA detection methods are needed to determine whether serologically positive animals are infectious and to examine the role of vectors (e.g. haematophagous insects) in the transmission of the parasite. To this end, we established two different 5'-nuclease quantitative assays to detect B. besnoiti infection in cattle and to estimate the parasite load in samples (BbRT1 and BbRT2). These PCRs are based on the sequence of the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene. Tests with serial dilutions of B. besnoiti genomic DNA in a buffer containing 100 ng/µl bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification was possible in samples containing ≥1 pg B. besnoiti genomic DNA with a coefficient of variation of ≤ 2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used, 90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings, were significantly correlated. The specificity of the PCRs was confirmed using genomic DNA from related parasites, including genomic DNA of Besnoitia spp., Neospora caninum, Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Isospora spp., Sarcocystis spp., Eimeria bovis, Cryptosporidium parvum, and Trypanosoma brucei brucei. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis.

Evaluation of a commercial ELISA for the specific detection of antibodies against Besnoitia besnoiti.

Bovine besnoitiosis is an economically important disease in cattle caused by the protozoan parasite Besnoitia besnoiti, which occurs endemically in many countries of Africa and Asia and is spreading in Europe. Serological identification of subclinically infected cattle is important to avoid the introduction of infected animals into naive herds. Here we determine the sensitivity and specificity of the PrioCHECK(®) Besnoitia Ab, a serological test recently introduced into the European market. Analytical specificity was examined using sera from animals experimentally infected with parasites related to B. besnoiti (n=27). Three animals experimentally infected with Neospora caninum or Toxoplasma gondii showed inconclusive reactions in the ELISA (percent positivity relative to the positive control [PP] 10% ≤ 20%) while all other sera reacted negative (PP<10%). An estimate of the diagnostic specificity was obtained by analysing field sera from bovine herds without besnoitiosis but with abortion problems associated to N. caninum (n=403). The analysis revealed a specificity of 94.3% or 96.8% depending on the applied cut-off (PP 10% or 20%, respectively). Sensitivity was assessed with sera from 110 animals of a herd in Germany where clinical bovine besnoitiosis was first diagnosed in September 2008. A positive serological reference standard was defined regarding sera from animals as reference positive, if these animals had tested positive in at least two of a panel of three other serological tests (two different B. besnoiti immunoblots and one immunofluorescence antibody test) on both of two sampling dates, November 2008 and April 2009. A diagnostic sensitivity of 91.8% or 75.5% was determined for sera collected in November 2008 and a sensitivity of 82.7% or 50% for sera collected in April 2009 (cut-off PP 10% or PP 20%, respectively). The marked drop in sensitivity from November 2008 to April 2009 was predominantly observed in reference-positive cattle without clinical signs. We conclude that PrioCHECK(®) Besnoitia Ab is a valuable diagnostic tool to detect clinically infected animals. Thus it may be used to support control measures, e.g., for the separation of infected animals from the remaining herd to avoid a further transmission of the infection within the herd.

Comparative evaluation of immunofluorescent antibody and new immunoblot tests for the specific detection of antibodies against Besnoitia besnoiti tachyzoites and bradyzoites in bovine sera.

Besnoitia besnoiti, an apicomplexan parasite causes economically important disease in cattle in many countries of Africa and Asia is re-emerging in Europe. Serological identification of infected cattle is important because introduction of these animals into naive herds seems to play a major role in the transmission of the parasite. We report new, simplified immunoblot-based serological tests for the detection of B. besnoiti-specific antibodies. Antigens were used under non-reducing conditions in the immunoblots, because reduction of the antigen with beta-mercaptoethanol diminished the antigenicity in both, tachyzoites and bradyzoites. Ten B. besnoiti tachyzoite and ten bradyzoite antigens of 15-45 kDa molecular weight were recognized by B. besnoiti infected cattle, but not or only weakly detected by cattle infected with related protozoan parasites, Neospora caninum, Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, or Sarcocystis hirsuta. The sensitivity and specificity of B. besnoiti immunoblots were determined with sera from 62 German cattle with clinically confirmed besnoitiosis and 404 sera from unexposed German cattle including 214 sera from animals with a N. caninum-specific antibody response. Using a new scoring system, the highest specificity (100%) and sensitivity (90%) of the immunoblots were observed when reactivity to at least four of the ten selected tachyzoite or bradyzoite antigens was considered as positive. When a cut-off based on this scoring system was applied to both the tachyzoite- and the bradyzoite-based immunoblots, there was an almost perfect agreement with the indirect fluorescent antibody test with a titre of 200 as the positive cut-off. We identified and partially characterized 10 tachyzoite and 10 bradyzoite B. besnoiti antigens which may help to develop new specific and sensitive serological tests based on individual antigens and in the identification of possible vaccine candidates.

First in vitro isolation of Besnoitia besnoiti from chronically infected cattle in Germany.

Besnoitia besnoiti was in vitro isolated during the first recorded outbreak of bovine besnoitiosis in Germany. Molecular characterization of the new isolate, named Bb-GER1, revealed almost 100% identity with other B. besnoiti isolates obtained in Portugal, Spain, Israel or South Africa, when partial sequences of the 18S ribosomal RNA gene, of the internal transcribed spacer 1 and of the 5.8S RNA gene were compared. Cystozoites obtained from skin tissue of one bull were infectious for gamma-interferon knockout (GKO) mice by intraperitoneal (ip) inoculation. Tachyzoites were detected in the peritoneal cavity, spleen, liver and lung of the mice 5 days post-infection. The parasite could be maintained in GKO mice by ip inoculation for at least 5 passages. Peritoneal washings containing tachyzoites were obtained from infected mice and used to infect five cell lines (Vero, MARC-145, NA42/13, BHK(21), KH-R). The best growth of tachyzoites was observed in BHK(21) cells, but replication occurred to a smaller extent also in MARC-145, NA42/13 and KH-R cells. Subsequent comparative analyses revealed that after direct infection of these cell lines with cystozoites derived from bovine skin, the growth was best in NA42/13 cells. Considerable replication was also observed in the BHK(21) and KH-R cell lines. Our observations on the growth characteristics of Bb-GER1 partially contrast those for other isolates. The preferential growth in particular cell lines may be characteristic for particular B. besnoiti isolates. A potential association between growth properties and differences in virulence remains to be established. This is the first in vitro isolation of B. besnoiti from cattle in Germany.