Dermatophytosis in animals: epidemiological, clinical and zoonotic aspects.

AIM: Dermatophytosis are the most frequent fungal infections of pets and livestock and play an important role in animal and human health due to their zoonotic potential. Another important aspect of these infections is linked to the economic consequences in farm animal and fur production systems. An overview of dermatophytosis in animals is described in this paper. Epidemiological, clinical and zoonotic aspects are addressed, considering individual species, both pets and farmed animals. METHODS: In particular, most recent investigations in the field of animal mycology, carried out in Central Italy, are reported, with particular reference to rabbit, ruminants, horse, dog, cat and some wild species. RESULTS: The information in this article show how dermatophytes infect a wide range of animals which may be in contact with human beings either directly or indirectly. Consequently they are frequently a source of infection for human beings who, vice versa, may sometimes become contagious for animals. CONCLUSION: Fungal pathogens derive their power to spread from contamination of the animal's habitat - whether the animal is a conventional pet or not, a farm animal or living in the wild. Thus if treatment of the animal or human patient is to achieve optimal efficacy, it needs to be associated with adequate environmental measures.

Diagnostic and clinical features of animal malasseziosis.

Malassezia yeasts infection represents a common clinical concern with a special regard to canine dermatology. The Authors review the main clinical features of malasseziosis in canine and feline medicine, summarizing predisposing factors and aetiopathogenesis of the yeasts' infection. A special reference was given to clinical and microscopical diagnosis.

A simple duplex-PCR protocol for routine diagnosis and follow up of canine leishmaniasis.

The introduction of PCR has efficiently improved diagnosis of canine leishmaniasis. In order to provide a robust, efficient and reliable diagnostic method, a duplex PCR assay targeting the Leishmania infantum kDNA minicircle and the canine GAPDH gene as inner control was designed. Sensitivity of the assay reached 0.15 parasites/ml blood. Development, testing and application of this system on a group of 10 dogs during therapy administration (60 days) are also described. Six dogs (out of eight that have been showing a positive PCR result on peripheral blood during the study) were tested negative at day 62, indicating a reduction of parasitaemia at the end of the treatment period. All the animals had a positive PCR on lymph node aspirate both at the beginning and at the end of treatment. These findings seem to suggest that, in order to test therapy efficacy, PCR on whole blood could be a useful assay in dogs that have a positive PCR at the beginning of the treatment, while PCR positivity on lymph nodes lasts longer than the observation period during therapy administration. The presence of the GAPDH inner control band efficiently contributed to prevent false negatives, by highlighting samples affected by haemoglobin inhibition or inappropriate DNA isolation.

Neospora caninum in Wild Waterfowl: Occurrence of Parasite DNA and Low Antibody Titers.

Thirty-five adult waterfowl (14 males and 21 females) representing various orders and species were sampled during the hunting season from 2015 to 2016. Antibodies to Neospora caninum were detected by IFAT on blood samples, while heart and brain were subjected to molecular analysis for the detection of Neospora caninum DNA. Twelve birds (34.3%) (6 Anas crecca , 3 Anas platyrhynchos , 2 Anas penelope , 1 Anas acuta ) showed antibodies versus N. caninum , while 10 animals out of 35 (4 A. crecca , 2 A. platyrhynchos , 2 A. penelope , 1 A. acuta , and 1 Vanellus vanellus ) scored positive for at least 1 DNA sample, with an overall prevalence of 28.6%. The present report shows for the first time the occurrence of antibodies and N. caninum DNA in waterfowl. The avian species investigated in the present report, which feed directly from the soil and/or water, would be able to ingest oocysts excreted by final canid hosts and could contribute to parasite transmission in the sylvatic cycle. To achieve a definitive result about the role of these avian species in the epidemiology of this protozoan, the presence of viable parasites should be demonstrated by bioassay and/or culture, as well as histological evidence of N. caninum cysts in avian tissues.

Sarcoptic mange and other ectoparasitic infections in a red fox (Vulpes vulpes) population from central Italy.

Fifty red foxes (Vulpes vulpes) from the district of Pisa (central Italy) were examined for ectoparasites. Sarcoptic mange was diagnosed on the presence of clearly visible skin lesions with confirmatory demonstration of Sarcoptes scabiei at parasitological and histopathological analysis. Ticks and fleas were collected directly from the carcases during post mortem examination, fixed and identified by morphological examination. For the detection of ear Malassezia and mite infections, cytological and parasitological examinations of ear wax samples were performed. All data were statistically analysed using a χ2 test with the Yates correction. An overall prevalence of 84% for ectoparasitic infections was found in examined subjects. In regard to isolated ectoparasites, 38%, 8%, 82%, 6% and 8% of foxes resulted positive for S. scabiei, Otodectes cynotis, Malassezia spp., fleas (Archaeopsylla erinacei, Pulex irritans, Ctenocephalides canis) and ticks (Ixodes ricinus and Rhipicephalus sanguineus), respectively. Malassezia ear infection was significantly more prevalent in animals older than 1 year (P < 0.01). Prevalence (38%), severity of lesions and poor body conditions observed in most Sarcoptes-infected animals indicate that sarcoptic mange should be considered the most important ectoparasitic infection of red foxes in the examined area.

Phoretic association of mites and mallophaga with the pigeon fly Pseudolynchia canariensis.

Myialges anchora Trouessart, 1906 and M. lophortyx (Furman & Tarshis, 1953) gravid females, surrounded by clusters of eggs, were found strongly inserted into the cuticle of head, thorax, abdomen, femurs and wings of Pseudolynchia canariensis (Macquart, 1840), a hippoboscid fly parasite of the pigeon. This lousefly results obligatory host for ovigerous females of Myialges and for the development of their eggs, and phoretic host because the dispersal of hatching larvae to new hosts may then occur with dispersal of fly carriers. Together with the Myiolges species, not ovigerous females of Ornithocheyletia hallae Smiley, 1970 and Columbicola columbae (Linnaeus, 1758) were found on the pigeon fly.

Studies on canine leishmaniasis control. 2. Effectiveness of control measures against canine leishmaniasis in the Isle of Elba, Italy.

A serological and parasitological survey carried out in 1985 in the Isle of Elba, Italy, revealed a high prevalence of canine leishmaniasis. This focus was considered ideal for the evaluation of effectiveness of treating infected dogs with meglumine antimoniate, applied as a control measure during 1985 and 1986. New data on prevalence were obtained for the years 1986 and 1987. Incidence of new canine leishmaniasis cases after the transmission seasons 1984, 1985 and 1986 were determined by examining 2 cohorts: cohort I, dogs born within 2 transmission seasons, and cohort II, adult dogs examined and found to be negative before each transmission season. Over 2000 tests were carried out on 1500 dogs. Prevalence analysis showed a reduction of infective dogs (symptomatic and oligosymptomatic) from 14.4% in 1985 to 5.2% in 1987. Incidence analysis showed a decrease of new cases from 12.4% after transmission season 1984 to 4.6% after transmission season 1987. The results indicate a two-thirds reduction of the disease frequency in dogs of the Isle of Elba after a 2-year period of control measures.

Studies on canine leishmaniasis control. 1. Evolution of infection of different clinical forms of canine leishmaniasis following antimonial treatment.

81 dogs naturally infected with Leishmania infantum in the Isle of Elba, Italy, were treated with meglumine antimoniate (Glucantime). 36 of them (45.5%) were asymptomatic cases. 4-24 months after treatment the dogs were clinically and serologically examined; the recovery rates were 47.2% for asymptomatic cases, 33.3% for oligosymptomatic cases, and 11.1% for symptomatic cases. Furthermore, treatment had prevented the development of patent disease in 90% of non-recovered asymptomatic cases, whereas it had produced only slight improvement of clinical condition in patent dogs which were still infected after drug administration. Treatment with antimonial drugs is therefore recommended in canine leishmaniasis control if non-patent or sub-clinical forms of the disease are detected by seroepidemiological surveys.

Evaluation of dot enzyme-linked immunosorbent assay (dot-ELISA) for the serodiagnosis of canine leishmaniosis as compared with indirect immunofluorescence assay.

A dot enzyme-linked immunosorbent assay (dot-ELISA) was developed and compared with a standard indirect immunofluorescence assay (IFA) for the rapid serodiagnosis of canine leishmaniosis. The two tests were used to examine sera from Leishmania infantum-infected and control dogs. Using the dot-ELISA, 137 of 149 sera (91.9%) from infected animals gave a clearly positive reaction, whereas in the standard IFA technique 147 (98.7%) were positive at a reciprocal titer of 40 or over (titer range 40-10,240). Control sera from 75 healthy dogs, not living in endemic areas, and 11 dogs with other diseases (babesiosis, cryptococcosis, ehrlichiosis, dermatitis, and chronic hepatitis) but Leishmania-negative were used to determine the specificity of the assays. All the sera were negative with IFA (100%), whereas using the dot-ELISA only 74 sera (86%) from controls gave a negative result. In the standard IFA no cross-reactivity was noted, in the dot-ELISA a weak cross-reaction was observed with a serum sample from a dog with babesiosis. The interpretation of dot-ELISA could be easily performed by visual inspection of the nitrocellulose disks. The most remarkable feature of dot-ELISA was the high sensitivity (91.9%) and positive predictive value (92.6%), even if the test showed a specificity lower than IFA. Because of its easy performance and high sensitivity, the dot-ELISA may be a useful test to be executed in the field for the diagnosis of canine leishmaniosis.

Renal involvement in mice experimentally infected with Toxocara canis embryonated eggs.

Histological examination of kidneys from mice experimentally infected with Toxocara canis embryonated eggs demonstrated the presence of a segmental or diffuse mesangioproliferative glomerulonephritis. Immunohistochemical studies established that renal alterations were associated with glomerular deposits of IgG, IgM and third component of complement (C3). These findings suggest that an immunomediated mechanism might possibly be involved in the genesis of kidney damage observed in mice infected with T. canis embryonated eggs.

Comparison between an enzyme-linked immunosorbent assay using a detergent-soluble Leishmania infantum antigen and indirect immunofluorescence for the diagnosis of canine leishmaniosis.

Serum samples collected from 290 dogs--186 Leishmania-infected and 104 control animals--were screened to detect the presence of specific antibodies to Leishmania infantum antigens in Tuscany, Italy. Two different techniques were compared: an indirect immunofluorescence assay (IFA) and an indirect enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using a detergent-soluble antigen of L. infantum promastigotes. Triton X-100 and protease inhibitors were used as detergent and to increase reproducibility of the assay, respectively. A strong correlation between the anti-Leishmania antibody levels obtained by ELISA and those obtained using IFA was observed. The ELISA appeared to be somewhat more sensitive than IFA (99.5% vs. 98.4%), while its specificity was lower (97.1% vs. 100%), even if not significantly different. Immunoblotting analysis, using the detergent-soluble L. infantum antigen, demonstrated that proteins of M(r) 30 and 73 kDa were recognized by all positive sera, regardless of the serum titre. Furthermore, antigens of M(r) 16, 18, 26, 33, 50 and 117 kDa were also frequently reactive with a large proportion of sera from infected dogs. This ELISA demonstrated a high sensitivity and specificity as well as the IFA, and it seems to be a suitable assay for large scale epidemiological studies.

Comparison of aminosidine (paromomycin) and sodium stibogluconate for treatment of canine leishmaniasis.

Twelve dogs naturally infected with Leishmania infantum were treated subcutaneously with aminosidine at a dose of 10 mg kg-1 per day for four weeks. Antimonial compounds were used as reference drugs in twelve Leishmania-infected dogs. Eleven of the twelve dogs submitted to aminosidine therapy responded within 30 days. The treatment with the aminoglycoside antibiotic presented a marked decrease of anti-Leishmania antibody titres than the controls. Aminosidine also reduced urinary protein, serum IgG, and circulating immune complex concentrations. Side effects were observed only in a dog with pre-existent renal lesions. This study proved that aminosidine is an effective, tolerable and safe drug for the treatment of canine leishmaniasis and that it could be used as a suitable substitute for antimonial therapy.

Neospora caninum infection in a Bernese cattle dog from Italy.

A cutaneous nodule associated with Neospora caninum infection was diagnosed in a 5-year-old male Bernese cattle dog from Italy. The ulcerative lesion was 2-3 cm wide located in the skin of the tarsal region. Haematological values were normal and the dog did not show any neurological abnormalities. The dermal lesion consisted of a diffuse necrotic dermatitis with a dense infiltrate of mostly neutrophils and macrophages, surrounded by a fibrous wall. Histological sections revealed numerous tachyzoites of N. caninum scattered throughout the tissue. Diagnosis was confirmed both by immunohistochemical staining and electron microscopic examination. The dog had a 1:640 IFAT titre to N. caninum. Four weeks after surgical excision new subcutaneous nodules reappeared. The cutaneous lesions resolved following 21 days of therapy with clindamycin hydrochloride. These observations demonstrate the presence of N. caninum in Italy and confirm that neosporosis should be considered in the differential diagnosis of pyogranulomatous dermatitis in dogs. Clindamycin may be an effective treatment for cutaneous neosporosis.

[Feline leishmaniasis: what's the epidemiological role of the cat?]. / Leishmaniosi felina: quale ruolo epidemiologico?

Feline leishmaniasis (FL) is a quite uncommon feature. Clinical disease has been described in cats since nineties begin. More than 40 reports in world literature have been referred, but the clinical cases have been only recently well defined. Most of the reports focus on infected cats living in endemic areas, even if, more recently FL due to Leishmania infantum was found in Sao Paulo State, in Brazil where autochthonous human or canine leishmaniasis cases have never reported. In Europe clinical cases of FL have been described from Portugal, France, Spain and Italy from 1996 to 2002. When a typing of the etiological agent was performed L. infantum was identified in all reported cases. In some endemic areas serological surveys have also been carried out in cats, using IHAT in Egypt, Western blot in France or IFAT in Italy. Sixty Egyptian cats had low serological antibody titers, from 1/32 to 1/128, in the endemic focus of canine leishmaniasis of Alpes Maritimes 12 out of 97 (12.5%) cats showed antibodies versus antigens 14 and/or 18 kDa of L. infantum. A previous survey by means of IFAT in Liguria and Toscana on 110 and 158 feline sera respectively reports a seroprevalence of 0.9% with low titer, while sera from Sicily seem to be positive at higher dilutions. Animals living in an endemic area can develop specific antibodies against leishmania and, in our experience, they can be evidentiated by means of IFAT. The antibody titers appear to be lower in affected cats than in dogs, even if the number of clinical cases is very scanty. PCR tests on feline blood samples are in progress, but preliminary results confirm the presence of leishmania DNA in such specimens. Cutaneous leishmaniasis is the more frequent form in cats and it was reported from several countries. Typical signs include nodular to ulcer or crusty lesions on the nose, lips, ears, eyelids, alopecia: clinical signs of cutaneous FL are unspecific and in endemic area this infection must be taken into account. Visceral leishmaniasis is not common in cats: this form shows visceral involvement: liver and spleen are interested, with lymph nodes and kidney. The cat probably has to considerate to play an active role in the disease, in contrast to goats, calves and horses who could act as accidental reservoirs of leishmania, while sheep appears to be not susceptible to experimental infection. In endemic foci for kala-azar in Sudan cows, goats and donkeys had a high prevalence of specific antibodies. Recently in Europe sporadic cases of equine leishmaniasis have been reported: L. infantum was the causative agent. Equine leishmaniasis appears as a self-healing skin-dwelling disease, with a massive accumulation of parasites. The animals do not often show detectable specific antibodies and recover without any chemotherapy. Untreated affected cats can frequently die and we also observed lymph nodes and blood involvement indicating a spread of leishmania in feline hosts. The epidemiological role of the cat has never been clarified due also to lack of xenodiagnosis trials. This species is believed to have a high degree of natural resistance, as observed following experimental infection. Some of the affected cats were FIV and/or FeLV positive and these viroses such as stress may induce an impaired cellular immune response, even if leishmania infected cat was not submitted to CD4+, CD8+ lymphocyte counts nor other immunological test. However the resistance of the cat to leishmania infection probably depends on genetic factors, not strictly related to cell mediated immunity, taking into account the high seroprevalence of FIV infections (30%) in our country versus the number of clinical cases.

Analysis of renal immune-deposits in canine leishmaniasis. Preliminary results.

Previous studies carried out on 34 dogs spontaneously infected by Leishmania infantum showed the presence of kidney lesions characterized by immunologically mediated glomerular and tubular damage. Glomerular immune-deposits were studied in 13 of these dogs. Immunoglobulins were isolated from kidney tissues by acid elution; IgG fractions from eluates, obtained by ammonium sulfate precipitation, were subjected to clonotypic analysis by autoradiography after isoelectrofocusing (IEF) using 125I radiolabelled goat IgG fraction-anti Fab2 of dog IgG. Idiotypic characterization of IgG eluted from kidney tissues was performed by IEF and autoradiography using both 125I radiolabelled membrane antigens of L. infantum extracted by Triton x 100 and 125I radiolabelled dog IgG for rheumatoid or anti-idiotypic activity. The IgG deposited in the kidney tissues of examined dogs were polyclonal and a specific activity against Leishmania membrane antigens was revealed. Meanwhile an anti-IgG activity of deposited immunoglobulins was not observed.

Efficacy of oral terbinafine in feline dermatophytosis due to Microsporum canis.

Microsporum canis is the dermatophyte most commonly responsible for ringworm in cats. The purpose of this paper was to evaluate the in vivo efficacy of oral terbinafine (Lamisil; Sandoz) in the treatment of feline ringworm caused by M canis, and to consider this drug as an alternative to griseofulvin or imidazoles. Fifteen cats infected with M canis were treated orally once daily with 30 mg/kg of terbinafine over a 2-week period. All treated animals were checked for dermatophytes on the last day of treatment, a month later and 3 months after the last administration of the drug. Only 12 cats could be used in the whole trial and 11 of these (92%) showed a complete cure. Terbinafine could be an effective alternative to griseofulvin when fungal resistance or idiosyncrasic intolerance are shown and, compared with griseofulvin, could give a faster rate of cure and less relapses.

Feline cutaneous phaeohyphomycosis due to Cladophyalophora bantiana.

A case of feline cutaneous phaeohyphomycosis due to Cladophyalophora bantiana is described. The cat was presented with breathing difficulty and a swollen, ulcerated nodule on the dorsal nose and left nostril. Histological examination of the nodule revealed a cystic granulomatous dermatitis characterised by neutrophils, macrophages and giant cells. Pigmented, yeast-like fungus cells and hyphal elements were easily identified in haematoxylin-eosin stained tissue sections. Cladophyalophora bantiana was isolated from a tissue specimen. This organism, primarily known to cause cerebral infection in humans and cats, only rarely causes cutaneous infection. Despite anti-fungal chemotherapy two relapses occurred. The cat was feline immunodeficiency virus- and feline leukemia virus-negative and even if the owner was unaware of trauma, the hypothesis of wound contamination is the most likely.

Environmental detection of Microsporum canis arthrospores in the households of infected cats and dogs.

Microsporum canis is the dermatophyte most frequently recovered from canine and feline ringworm cases. The household environment can be contaminated both by symptomatic animals and through asymptomatic M canis carriage, resulting in a potential human health risk. The load of M canis arthrospores was determined in households harbouring infected pets, in order to evaluate the infectivity of the animals versus the environment. The environments inhabited by 30 symptomatic animals (21 cats and 9 dogs) infected by M canis were examined by sampling both surfaces and indoor air. The surfaces were examined by means of contact plates; the air sampling was performed with a Sas super-100 AIR SAMPLER (PBI, Italy). Environmental contamination was detected in all households with cats, while only four out of nine houses harbouring dogs were found positive. The frequence of isolation in each sampling, and the results in terms of colony forming units per plate in the different houses appeared to be quite homogeneous. Heavily infected environments harboured kittens only. Infected owners were observed in eight households, in all of which at least one infected cat was present. No history of human dermatophytosis in households harbouring dogs was found. On the basis of our results, infected cats appear to cause substantial environmental contamination, and provoke a substantial presence of viable airborne fungal elements. Dogs seem to be of lower importance in the spread of M CANIS: they contaminated surfaces, but they never contaminated the air. The results of this study confirm the potential leading role of the feline species in the environmental spread of M canis.

[Quantitative PCR in the diagnosis of Leishmania]. / PCR quantitativa nella diagnosi di Leishmania.

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.