Manolis N. Saridomichelakis

A cross-sectional study of Leishmania spp. infection in clinically healthy dogs with polymerase chain reaction and serology in Greece.

A total of 73 clinically healthy hunting dogs, experiencing an outdoor lifestyle and originating from an area where canine leishmaniasis is endemic, were included in the study. Polymerase chain reaction (PCR) and indirect immunofluorescent antibody test (IFAT) for Leishmania spp. were done on bone marrow and serum samples, respectively, obtained from all 73 dogs, just before the beginning of the sandfly season. PCR was found positive in 46/73 (63%) whereas, IFAT only in 9/73 (12.3%) of the dogs. The prevalence and the incidence of Leishmania infection by PCR were 61.9 and 47.1%, respectively. No association was found between the breed, age, sex, length of hair coat of the dog, urban or rural life and the presence of ample vegetation and water collections in the proximity of their living quarters, and the result of PCR. These findings clearly demonstrate that most of the dogs residing areas where leishmaniasis is endemic become infected but usually remain seronegative. Serological screening of the general canine population in these areas may result in an underestimation of the true prevalence of the infection rate.

A randomised, blinded, placebo-controlled clinical trial with allopurinol in canine leishmaniosis.

A total of 45 non-uremic dogs, with clinical signs indicating leishmaniosis, entered the study. Diagnosis was confirmed by indirect immunofluorescence assay (IFA) on serum and polymerase chain reaction (PCR) on bone marrow samples. The dogs were randomly allocated into Group A (n=37) that received allopurinol (10mg/kg B.W., per os, twice daily) for 4 consecutive months, and Group B (n=8) that were placebo-treated. Clinical signs were scored just before and at monthly intervals throughout the study period, in a blinded and independent fashion. Complete blood count, serum biochemistry profile, urinalysis, lymph node and bone marrow parasitology, IFA and enzyme-linked immunosorbent assay (ELISA) serology and bone marrow PCR were carried out at the beginning and at the end of the trial. A total of three Group A and one Group B dogs died of end stage kidney disease that developed during the trial. In Group A animals that endured the trial there was a significant improvement in the general body condition, conjunctivitis, peripheral lymphadenopathy, splenomegaly, masticatory muscle atrophy, ulcerative stomatitis, epistaxis, exfoliative dermatitis, cutaneous ulcerations, blepharitis and nasodigital hyperkeratosis. The same observation was made for anemia, lymphopenia, hyperproteinemia, hyperglobulinemia, hyperphosphatemia, increased alkaline phosphatase activity and the low albumin/globulin ratio. By contrast, no improvement of any kind was seen in Group B dogs. Lymph node and bone marrow parasite numbers were significantly decreased in Group A animals. In Group B, that occurred only in the lymph nodes. Apart from remission of clinical signs and restoration to normal of clinicopathological abnormalities, allopurinol did not eliminate Leishmania organisms, as the PCR result on bone marrow was still positive in all the dogs that finished the trial.

Advances in the pathogenesis of canine leishmaniosis: epidemiologic and diagnostic implications

Canine leishmaniosis caused by Leishmania infantum (Syn. L. chagasi) is an important zoonosis with a complex pathogenesis. Parasite transmission occurs via female sandflies that inject promastigotes into the skin of the host. The interaction between the parasite and skin immune system is influenced by the repeated infectious bites and the simultaneous intradermal injection of sandfly saliva. Amastigotes are transported via infected macrophages to the regional lymph nodes and finally dissemination may occur. The outcome of the infection depends on host factors (genetic background, cell-mediated and humoral immune response, cytokine milieu, concurrent diseases) and parasite virulence. Resistance may be breed-associated; it is characterized by low to undetectable antibody production and effective cell-mediated immunity, and is orchestrated by cytokines such as interleukin-2, interferon-gamma and tumour necrosis factor-alpha. Susceptibility may be genetically determined or acquired (advanced age, concurrent diseases); in these dogs, parasite multiplication goes unrestricted and overproduction of specific and nonspecific antibodies occurs, leading to multiple organ pathology. Resistance or susceptibility is not an all-or-nothing phenomenon and many intermediate phenotypes may be found. From a diagnostic point of view, although clinical cases are readily identified using microscopy and serology, investigation should not stop at this point and an extensive search for underlying diseases is advised, especially in aged dogs. Conversely, microscopy and conventional serology are frequently negative in asymptomatic infected dogs; to identify such dogs, polymerase chain reaction, evaluation of cutaneous delayed-type hypersensitivity, in vitro lymphocyte proliferation test to Leishmania antigen, and Western blotting may be employed.

Canine atopic dermatitis in Greece: clinical observations and the prevalence of positive intradermal test reactions in 91 spontaneous cases

Atopic dermatitis was diagnosed in a total of 91 dogs by combining the compatible historical evidence and clinical signs with the presence of one or more positive intradermal test reactions well correlated with the exposure to the aeroallergens and the seasonality of the clinical signs. Compared to the general hospital population Yorkshire terriers, Chinese Shar-Peis and cocker spaniels showed a strong predilection. No such predilection was found regarding the sex of the animals. The age of the dogs at the onset of the clinical signs ranged from 2 months to 8 years (median: 2.5 years). Moderate to severe pruritus, noticed in all the 91 dogs, was either localized (29/91) or generalized (64/91) and non-seasonal (43/91), seasonal (19/91) or of unknown seasonality (29/91). The most common cutaneous lesions included erythema, hyperpigmentation, hypotrichosis and crusts; their body distribution was generalized (64%) or localized (36%) with the feet as the most common site of involvement. Five dogs that had unlesional skin were significantly younger and had been pruritic for a shorter period of time compared to the majority of our study population. Otitis externa (43/91) and bacterial pyoderma (30/91) were the most common conditions associated with atopic dermatitis, while the prevalence of Malassezia dermatitis was very low (2/91). Of the other allergic skin diseases flea allergic dermatitis was the most common (29/91) followed by food hypersensitivity (2 out of the 15 dogs tested). The majority of the dogs demonstrated multiple sensitivities to the 50 aeroallergens tested, while domestic mites (77/91), and particularly Dermatophagoides farinae (64/91), were the most commonly implicated. The total number of the positive intradermal test reactions was increasing parallel to the age of the dogs but it was negatively associated with the presence of skin lesions on the carpal and tarsal joints.

Masticatory and skeletal muscle myositis in canine leishmaniasis (Leishmania infantum)

Twenty-four dogs with a parasitologically and serologically established diagnosis of leishmaniasis were studied to investigate the atrophy of the masticatory muscles which commonly occurs in this disease, and to compare the lesions in the masticatory muscles with those in the cranial tibial muscles. The 24 animals were divided into three groups of eight, group A dogs with no muscular atrophy, group B dogs with different degrees of atrophy in the masticatory and skeletal muscles, and group C dogs with similar degrees of atrophy in the masticatory and skeletal muscles. Increased activities of creatine phosphokinase and lactate dehydrogenase were recorded in only some of the dogs in groups B and C, but there were no significant differences between the mean activities in the three groups. Electromyographic changes indicating myopathy and involving both the temporalis and cranial tibial muscles, were observed in two of the dogs in group A, seven of those in group B, and in all the dogs in group C. Muscle histopathology revealed a variable degree of muscle fibre necrosis and atrophy, mononuclear infiltrates and neutrophilic vasculitis in all the dogs except two in group A. Leishmanial amastigotes were found within macrophages and myofibres in 16 of the dogs, some in each group. IgG immune complexes were detected in muscle samples, and circulating antibodies against myofibres were detected in serum samples from all the 24 dogs.

Effects of allopurinol treatment on the progression of chronic nephritis in canine leishmaniosis (Leishmania infantum)

Forty dogs with canine leishmaniosis (CL) participated in this study, which was designed to investigate the effect of allopurinol on the progression of the renal lesions associated with this disease. The animals were allocated into 5 groups. Group A dogs (n = 12) had neither proteinuria nor renal insufficiency, group B dogs (n= 10) had asymptomatic proteinuria, and group C dogs (n = 8) were proteinuric and azotemic. Two more groups, CA and CB, comprising 5 dogs each, served as controls for groups A and B, respectively. Group A, B, and C dogs received allopurinol PO (10 mg/kg q12h) for 6 months, whereas group CA and CB dogs were placebo-treated. Serum biochemistry profile, urinalysis, urine protein/creatinine ratio, and glomerular filtration rate (GFR) measurements were carried out at the beginning of the study, the 3rd month, and the 6th month, whereas renal biopsies were carried out only at the beginning and the end of the trial. Membranoproliferative glomerulonephritis was the most common cause of chronic renal failure. Mesangioproliferative and tubulointerstitial nephritis were detected even in group A and CA dogs. Allopurinol not only lowered proteinuria in group B dogs but also prevented the deterioration of GFR and improved the tubulointerstitial, but not the glomerular, lesions in both group A and group B dogs. Further, it resolved the azotemia in 5 of the 8 dogs admitted with 2nd stage chronic renal failure (group C). Consequently, treatment with allopurinol is advisable in CL cases with asymptomatic proteinuria or 1st-2nd stage chronic renal failure.

Factors associated with the occurrence of epistaxis in natural canine leishmaniasis (Leishmania infantum)

BACKGROUND Canine leishmaniasis (CanL) is a common cause of epistaxis in dogs residing in endemic areas. The pathogenesis of CanL-associated epistaxis has not been fully explored because of the limited number of cases reported so far. HYPOTHESIS Epistaxis in CanL could be attributed to more than 1 pathomechanism such as hemostatic dysfunction, biochemical abnormalities, chronic rhinitis, and coinfections occurring in various combinations. ANIMALS Fifty-one dogs with natural CanL. METHODS The allocation of 51 dogs in this cross-sectional study was based on the presence (n = 24) or absence (n = 27) of epistaxis. The potential associations among epistaxis and concurrent infections (Ehrlichia canis, Bartonella spp., and Aspergillus spp.), biochemical and hemostatic abnormalities, and nasal histopathology were investigated. RESULTS Hypergammaglobulinemia (P= .044), increased serum viscosity (P= .038), decreased platelet aggregation response to collagen (P= .042), and nasal mucosa ulceration (P= .039) were more common in the dogs with epistaxis than in those without epistaxis. The other significant differences between the 2 groups involved total serum protein (P= .029) and gamma-globulin (P= .013) concentrations, which were higher, and the percentage platelet aggregation to collagen, which was lower (P= .012) in the epistaxis dogs. CLINICAL IMPORTANCE CanL-associated epistaxis appears to be the result of multiple and variable pathogenetic factors such as thrombocytopathy, hyperglobulinemia-induced serum hyperviscosity, and nasal mucosa ulceration.

Assessment of cross‐reactivity among five species of house dust and storage mites

In vitro cross-reactivity among two house dust (Dermatophagoides farinae, D. pteronyssinus) and three storage (Acarus siro, Tyrophagus putrescentiae, Lepidoglyphus destructor) mites was examined in 20 mite-sensitive dogs with natural occurring atopic dermatitis (group A), 13 high-IgE beagles experimentally sensitized to D. farinae (group B), and five healthy beagles (group C). Intradermal testing (IDT) and serology for allergen-specific IgE demonstrated that co-sensitization for all possible pairs of the five mites was generally 45% or higher among group A dogs. In the same dogs, enzyme-linked immunosorbent assay cross-inhibition results indicated that each one of D. farinae, A. siro and T. putrescentiae was a strong inhibitor of all the remaining mites, whereas D. pteronyssinus was a strong inhibitor of L. destructor. A high number of positive IDT and serology test results for D. pteronyssinus, A. siro, T. putrescentiae and L. destructor were recorded among group B dogs. No conclusive evidence of exposure to these mites was found upon analysis of dust samples from their environment and their food for the presence of mites and guanine. Also, the number of positive test results was generally higher among group B than among group C dogs. Enzyme-linked immunosorbent assay cross-inhibition revealed that D. farinae was a strong inhibitor of D. pteronyssinus, A. siro and T. putrescentiae. Collectively, these results demonstrated extensive in vitro cross-reactivity among house dust and/or storage mites that can explain false-positive results upon testing of dust mite-sensitive dogs with atopic dermatitis.

Validation of the Canine Atopic Dermatitis Extent and Severity Index (CADESI)-4, a simplified severity scale for assessing skin lesions of atopic dermatitis in dogs

BACKGROUND Severity scales are used to grade skin lesions in clinical trials for treatment of dogs with atopic dermatitis (AD). At this time, only two scales have been validated, namely the Canine Atopic Dermatitis Extent and Severity Index (CADESI)-3 and the Canine Atopic Dermatitis Lesion Index (CADLI). However, the high number of assessed sites makes the CADESI-3 impractical. HYPOTHESIS/OBJECTIVES The aim of this study was to develop and validate a fourth version of the CADESI that is simpler and quicker to administer. METHODS Body sites, lesions and severity grades were revised by members of the International Committee on Allergic Diseases of Animals (ICADA). The newly designed CADESI-4 was tested for its validity (i.e. content, construct and criterion), reliability (i.e. inter- and intra-observer reliability and internal consistency), responsiveness (i.e. sensitivity to change) and time to administer. Disease severity benchmarks were chosen using receiver operating characteristic methodology. RESULTS The CADESI-4 was simplified in comparison to its previous version to comprise 20 body sites typically affected in atopic dogs. Three lesions (erythema, lichenification and alopecia/excoriation) were scored from 0 to 3 at each site. The CADESI-4 had satisfactory validity, reliability and sensitivity to change. On average, the time to administer a CADESI-4 was one-third that of a CADESI-3. Proposed benchmarks for mild, moderate and severe AD skin lesions are 10, 35 and 60, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE The CADESI-4 is simpler to use and quicker to administer than its previous version. The ICADA recommends the CADESI-4 instead of the CADESI-3 to score skin lesions of AD in dogs enrolled in clinical trials.

A cross-sectional sero-epidemiological study of canine leishmaniasis in Greek mainland.

Canine leishmaniasis is endemic in Greece as in other countries of the Mediterranean basin. In this study, the regional prevalence of canine seropositivity to Leishmania spp. in Greek mainland was simultaneously assessed in 7 different regions. A total of 2620 serum samples were collected from clinically healthy dogs and were tested for anti-L. infantum antibodies by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). A high degree of agreement (κ=0.96) was observed between these two tests. The seroprevalence rate in the whole study population was nearly 20%, being highest among dogs living in Attiki (30.12%) and lowest for those living in Florina (2.05%). There was no difference in terms of the gender of the dogs, their length of hair coat or their utility; on the contrary, seroprevalence rates were significantly higher among dogs in the age groups of 1-3 years (23.39%) and 3-9 years (23.35%) than in younger (2.26%) or older (6.03%) dogs. Epidemiological data on the seroprevalence of canine leishmaniasis provide indirect information on the prevalence of the infection and the disease and are necessary to implement and then to evaluate the effectiveness of control measures.