Potential role of ATP-binding cassette transporters against acaricides in the brown dog tick Rhipicephalus sanguineus sensu lato.

ATP-binding cassette (ABC) transporters have been shown to be involved in pesticide detoxification in arthropod vectors and are thought to contribute to the development of drug resistance. Little is currently known about the role they play in ticks, which are among the more important vectors of human and animal pathogens. Here, the role of ABC transporters in the transport of fipronil and ivermectin acaricides in the tick Rhipicephalus sanguineus (Ixodida: Ixodidae) was investigated. Larvae were treated with acaricide alone and acaricide in combination with a sub-lethal dose of the ABC transporter inhibitor cyclosporine A. The LC50 doses and 95% confidence intervals (CIs) estimated by mortality data using probit analysis were 67.930 p.p.m. (95% CI 53.780-90.861) for fipronil and 3741 p.p.m. (95% CI 2857-4647) for ivermectin. The pre-exposure of larvae to a sub-lethal dose of cyclosporine A reduced the LC50 dose of fipronil to 4.808 p.p.m. (95% CI 0.715-9.527) and that of ivermectin to 167 p.p.m. (95% CI 15-449), which increased toxicity by about 14- and 22-fold, respectively. The comparison of mortality data for each separate acaricide concentration showed the synergic effect of cyclosporine A to be reduced at higher concentrations of acaricide. These results show for the first time a strong association between ABC transporters and acaricide detoxification in R.sanguineus s.l.

Efficient identification of Malassezia yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

BACKGROUND: Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in neonates, children and adults and can be life-saving for those patients. Ma-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique. OBJECTIVES: To create an extensive database of main mass spectra (MSPs) that will allow quick identification of Malassezia species by MALDI-TOF MS. METHODS: An in-house library of 113 MSPs was created from 48 reference strains from the CBS-KNAW yeast collection. The in-house library was challenged with two test sets of Malassezia strains, namely 165 reference strains from the CBS collection and 338 isolates collected in Greece, Italy, Sweden and Thailand. RESULTS: MALDI-TOF MS allowed correct identification of all 14 Malassezia spp. MALDI-TOF MS results were concordant with those of sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2 domains of the large subunit of the ribosomal DNA. CONCLUSIONS: Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks.

Physiological and molecular characterization of atypical lipid-dependent Malassezia yeasts from a dog with skin lesions: adaptation to a new host?

Three lipid-dependent Malassezia isolates (here named 114A, 114B and 114C) recovered from a dog with skin lesions were phenotypically and genotypically characterized. All presented ovoid cells and buds formed on a narrow base. Most of the results from physiological tests were consistent with those of Malassezia furfur. The phylogenetic analysis of ITS-1 and LSU nucleotide sequences was concordant in placing all three clinical Malassezia isolates close to M. furfur. However, the phylogenetic data on the chs-2 sequence revealed that clinical isolate 114A is distinct from M. furfur and was closely affiliated to the sequence of M. pachydermatis with high nodal support. In particular, lipid-dependent isolates 114A displayed chs-2 sequences similar (100%) to that of the non-lipid dependent species Malassezia pachydermatis. The presence of the genetic and physiological polymorphisms detected in these three isolates of M. furfur could have resulted from a process of adaptation of this anthropophilic species to a new host.

Epidemiology and risk factors for dermatophytoses in rabbit farms.

While dermatophytoses of several animal species have been extensively investigated, information on their occurrence and epidemiology in rabbits is limited. We carried out a study from October 2006 to February 2007 of 23 rabbit farms in Apulia and Basilicata regions (southern Italy) in order to investigate the occurrence and risk factors associated with dermatophytoses in breeding rabbits. Dermatophytes were isolated from 86.9% (20/23) of the farms and from 51.8% (420/810) of the animals sampled. Trichophyton mentagrophytes (47.9%) and Microsporum canis (3.2%) were isolated from diseased (71.7%) and healthy (48.4%) animals as well from the surrounding environment (7.5%). The occurrence of lesions, the age of rabbits, and farm management (e.g., temperature, humidity and methods and frequency of disinfection practices) were identified as the most significant risk factors (P < 0.05) for the occurrence of dermatophytes. Animals in fattening and finishing stages were the most frequently infected (i.e., 58.2 and 61.6% respectively). Dermatophyte prevalence was significantly (P < 0.05) higher in areas with higher temperature (>20°C) and relative humidity ranging from 62-65%. The results of the present investigation suggest that zoonotic dermathophytes are present in rabbit farms and highlight the importance of correct management procedures for the control of the infections.

Expression of the micro-opioid receptor on Malassezia pachydermatis and its effect in modulating phospholipase production.

Malassezia spp. may act as opportunistic skin pathogens in humans and animals. Malassezia pachydermatis proliferation and phospholipase production may play a pathogenic role in the occurrence of skin lesions in dogs. This study investigates the presence of mu-opioid receptor (MOR) in M. pachydermatis strains isolated from healthy dogs and dogs with skin lesions and its effects on phospholipase activity (p.a.). P.a. of 64 M. pachydermatis isolates was evaluated using different concentrations of naloxone (Nx), a MOR antagonist. Isolates were divided into Group A (i.e., 40 isolates from 26 dogs with dermatitis) and Group B (i.e., 24 isolates from 12 healthy dogs). The MOR expression was analyzed by Western blot and immunofluorescence. A statistically higher p.a. than that of the controls was found with isolates in Group A at a Nx concentration of 10(-6) M (P<0.05). No isolate in Group B displayed p.a. in either control samples or in the presence of any Nx concentration. Immunoblotting revealed two positive MOR immunoreactive bands of approximately 65 and 98 kDa. MOR expression and localization was also demonstrated by immunofluorescence in isolates from Groups A and B. This study provides the first evidence of MOR expression on M. pachydermatis cell membranes pointing to its possible role in modulating p.a. production in isolates from dogs with skin lesions.

Competence of the housefly, Musca domestica, as a vector of Microsporum canis under experimental conditions.

The role of Musca domestica Linnaeus as a vector of the dermatophyte Microsporum canis was investigated under experimental laboratory conditions. About 400 4-day-old M. domestica flies were divided into two groups. Group A consisted of about 200 infected flies and group B comprised about 200 uninfected flies that were used as controls. Each trial was run three times. Flies from group A were fed for 24 h with a solution of ultra-high temperature-treated (UHT) milk containing about 10(6) colony-forming units (CFU) per mL of M. canis (infected milk inoculum [IMI]). The control group (group B) was fed with only UHT milk spiked with a teaspoon of honey. Microsporum canis was detected from faeces, vomitus, external surfaces and internal organs of 20 adult flies, eggs, first-, second- and third-stage (L1, L2, L3) larvae and pupae of each group, as well from 20 adult newly emerged flies (NEFs; from infected generations only). Samples were collected at 2, 4, 6 and 24 h post-infection (p.i.) (i.e. the times at which IMI was available) and on 2, 5, 7 and 8 days p.i. from adult flies, faeces and vomitus. Eggs, L1, L2, L3 and pupae were processed as soon as they appeared. Equivalent samples were taken from group B. All the samples were individually cultured. Microsporum canis was not isolated from the control group, from eggs, larvae, pupae or NEFs, or from faeces and vomitus, although it was detected on the body surface (26.2%) and internal organs (26.9%) of adult flies. The highest positivity for M. canis was detected on flies within the first 6 h p.i. (i.e. 57.2% on the body surface and 71.6% in the internal organs). No M. canis was isolated at 24 h p.i., but it was isolated from the body surface only at 2 and 5 days p.i. The results presented provide evidence that M. domestica transmits M. canis mechanically with its outer body surface for up to 5 days p.i., but does not do so through its vomitus and faeces or transovarially. The role played by M. domestica in the epidemiology of human and animal dermatophytoses is discussed.

The pathogenesis of Malassezia yeasts.

The genus Malassezia includes twelve species of yeast, many of which have been mainly associated with human and canine diseases. Malassezia pachydermatis colonizes the skin and mucosal sites of healthy dogs and cats. Despite being part of the normal cutaneous microflora, Malassezia spp. yeast may become pathogenic under certain circumstances. This article reviews the factors related to both host and yeast which affect the pathogenical or commensal phenotypes of Malassezia yeasts.

Phospholipase activity of yeasts from wild birds and possible implications for human disease.

Over the last decades, reports on yeast infections in humans have increased especially with respect to immunocompromised individuals. Phospholipases are enzymes which may be associated with pathogenic processes caused by opportunistic yeasts. Phospholipase activity (ph.a.) was investigated in 163 isolates of 13 species of yeasts. A total of 133 isolates were obtained through the screening of a total of 768 cloacae of wild birds (Group I: 182 birds of prey; Group II: 165 passeriformes and Group III: 421 other wild migratory birds), while 30 isolates were recovered from the droppings of birds housed in 32 distinct aviaries (Group IV). Phospholipase production was evaluated and quantified at 2 and 5 day pre-incubation (Pr.t) and incubation times (I.t). Isolates from cloacae (48.1%) and excreta (73.3%) produced ph.a. with the highest values registered after 5 days of I.t. Candida albicans, C. tropicalis, C. glabrata, C. lusitaniae, C. pelliculosa, Cryptococcus albidus, C. laurentii, Trichosporon beigelii, and Saccharomyces cerevisiae displayed the highest ph.a. after 2 days of Pr.t while Candida famata, C. guilliermondii and Cryptococcus neoformans after 5 days of Pr.t. Ph.a. was never found in Rhodotorula rubra isolates recovered from the cloacae of wild birds. Isolates (73.3%) from bird droppings showed a higher ph.a. than those from cloacae thus indicating that wild birds not only act as carriers but may also spread phospholipase-producing yeasts in the environment.

Dermatophytoses in cats and humans in central Italy: epidemiological aspects.

Two hundred hair/skin samples were collected from 2002 to 2004 from two groups of cats (privately owned and stray cats from a shelter) and 165 samples were obtained during the same period from persons in whom dermatophyte infection was highly suspected. The epidemiological data were statistically evaluated. Thirteen of the 100 privately owned cats (13%) and 100% of the stray cats were positive; of the 165 human samples examined 109 (66%) were positive for dermatophytes. Microsporum canis was the most common dermatophyte isolated in both cat groups while Trichophyton mentagrophytes was the most common in humans. Interestingly, a geophylic dermatophyte species (Microsporum gypseum) was found to be present and associated with clinical signs. Living in the countryside proved to be a risk factor for dermatophytoses in privately owned cats while in humans the main risk factor for M. canis was contact with animals followed by young age. None of the variables considered was associated with positivity for T. mentagrophytes while positivity for other fungi was correlated with life in the countryside.

Role of beta-endorphin on phospholipase production in Malassezia pachydermatis in dogs: new insights into the pathogenesis of this yeast.

Malassezia spp. are lipophilic yeasts that are part of the normal cutaneous microflora and sometimes act as pathogens causing dermatitis. This study investigated the interactions occurring between beta-endorphin and phospholipase activity in isolates of M. pachydermatis in dogs presenting cutaneous lesions. Phospholipase production was evaluated and quantified on 144 isolates suspended in Dixon broth to which different beta-endorphin concentrations (from 600 to 0.6 pM) were added. The isolates were divided into three groups: group A comprised isolates from lesional skin of dogs with dermatitis confined to one site, group B consisted of isolates from the healthy skin of the same dogs with localized lesions, and group C was made up of isolates from assorted skin sites of healthy dogs. A statistically higher phospholipase activity than that of the controls was recorded in group B at all tested beta-endorphin concentrations. In groups A (Pz=0.62) and C (Pz=0.62) phospholipase activity was statistically higher than the controls only at a concentration of 600 pM. This study suggests that beta-endorphin plays an important role in the production of phospholipase in M. pachydermatis isolates and provides evidence that beta-endorphin concentrations affect the number but not the Pz value of phospholipase-producing isolates. B-endorphin concentrations may play a relevant role in inducing M. pachydermatis cell differentiation towards the production or non-production of phospholipase.

Role of birds of prey as carriers and spreaders of Cryptococcus neoformans and other zoonotic yeasts.

In the last 20 years, cases of human cryptococcosis, have increased in immunocompromised patients. In several instances, the cases have been associated with the exposure of the patients to bird droppings. In order to investigate birds of prey as potential carriers and spreaders of Cryptococcus neoformans and other yeasts of importance in human infections, 182 swab samples were collected from the cloacae of several species of birds of prey (Group I) and 32 faecal samples from aviaries in which the birds were housed (Group II). Samples were also taken from digestive tract of 60 dead birds (Group III). A total of 454 samples were cultured from which 215 colonies of yeastlike fungi were recovered and identified. Cryptococcusneoformans var. grubii was isolated from three cloacae samples (4.8%) collected from Falco tinnunculus and from one sample (3.1%) obtained from Buteo buteo, as well as from samples collected at the aviaries in which these birds were kept. Overall, 18 samples (9.9%) from Group I, 13 (40.6%) from Group II, 12 crops (20%), three proventriculi (5%) and 12 cloacae (20%) from Group III yielded positive cultures for yeasts. The results indicate that birds of prey and in particular, F. tinnunculus and B. buteo, may act as carriers and spreaders of C. neoformans and other zoonotic yeasts.

Incidences of canine leishmaniasis in an endemic area of southern Italy.

Canine leishmaniasis (CanL), caused by Leishmania infantum, is widely distributed in many Mediterranean countries and is considered endemic in southern and central Italy with prevalence reaching up to 48.4%. Determination of the incidence would be useful as a measure of the risk of infection, then to evaluate the usefulness of control measures and to estimate whether a new focus is autochthonous or imported. This study was performed on two sites in the Apulia region of southern Italy, namely sites A and B. A total of 262 dogs were included in the evaluation of incidence, 94 farm dogs from site A and 168 dogs (92 farm and 76 kennel dogs) from site B. The incidence of infection was determined by using two different approaches: in site A by means of incidence density rate (IDR); in site B by the yearly seroconversion rate. In site A, the IDR was calculated at 4.25% dog-years; in site B the yearly incidence rate was of 9.52% (6.5% and 13.1% in farm and kennel dogs, respectively). The strength and weakness of the two different approaches (i.e. annual monitoring or monthly interval monitoring) for calculating the incidence of CanL in an endemic area have been discussed.

Occurrence of yeasts in cloacae of migratory birds.

Several species of yeast have been reported as pathogens in humans based on increases in immunodeficiency syndromes and as a result of immunosuppressant chemotherapy in cancer treatment. Domestic and wild birds are known to act as carriers of human pathogenic fungi. To gain additional information on the yeasts present in the cloacae of some species of migratory birds, 421 wild birds (24.39% out of 1726 birds caught in Romania, Hungary and Bulgaria) were sampled with the permission of the local judicial authority. The state of conservation of the birds (i.e. post-mortem alterations, colour of the mucosae etc.), along with their age and sex were determined. Samples were collected directly from the cloacae and cultured, and colonies were identified in each positive sample. Yeasts were isolated from 15.7% of the animals sampled, with the highest percentage found in coots (Fulica atra -58.8%) and the lowest in quails (Coturnix coturnix -1.7%). A total of 131 isolates belonging to 15 species of yeast were identified. Rhodotorula rubra was the yeast with the highest number of isolates (28.2%), followed by Cryptococcus albidus (18.4%), Candida albicans (9.2%), Trichosporon cutaneum (8.4%), Candida guilliermondii (6.1%), Candida tropicalis (6.1%) and other species. The present study represents the first survey on the occurrence of yeasts in the cloacae of migratory birds. The prevalence and species of yeasts isolated is discussed on the basis of the ecology, diet, and habitat of the birds.

The epidemiology of canine and feline dermatophytoses in southern Italy.

A total of 424 animals (268 dogs and 156 cats) with skin lesions (alopecia and peripheral scaling) were examined from January 1999 to December 2002. Of the 424 samples examined, 99 (23.3%) yielded a positive culture and, in particular, 20.5% of the dog samples and 28.2% of the cat samples. Microsporum canis was the most common dermatophyte isolated from dogs and cats (77.7%), followed by geophilic dermatophyte species (M. gypseum, Trichophyton terrestre). Young dogs and cats, especially those younger than 1 year, showed a statistically significant higher prevalence of M. canis infection than older animals. No statistically significant association was found between infection and sex in cats, while male dogs were more affected by dermatophytes. Among breeds, Yorkshire terriers showed the highest positivity (50%) caused mainly by M. canis (46.6%), while no differences were noticed for cats. A significantly higher prevalence of positive samples was registered in summer and in autumn for cats. The presence of dermatophytes was not associated with itching. The diagnostic value of Wood's lamp fluorescence and microscopic examination proved to be scarce compared with fungal cultures as only 45.5% of the 77 samples that tested positive for M. canis at the cultural examination was positive under Wood's lamp florescence and 53.2% at microscopic examination.

Association between phospholipase production by Malassezia pachydermatis and skin lesions.

An evaluation was made of the phospholipase activities of Malassezia pachydermatis strains isolated from healthy dogs versus those from dogs with dermatitis and otitis. A high percentage of strains of M. pachydermatis obtained from lesion sites (93.9%) produced phospholipase, compared to the strains obtained from healthy skin of the same dog with localized lesions (41.4%) and healthy dogs (10.6%).

Antifungal activity of essential oils from leaves and flowers of Inula viscosa (Asteraceae) by Apulian region.

Some essential oils from several plants (Artemisia verlotorum, Lavandula augustifolia, Ocimum gratissimum) have proved to have acaricidal, antifungal and antibacterial activity. Inula viscosa Ait. (Asteraceae), a plant growing spontaneously in the Mediterranean area, is currently used by popular medicine for its therapeutic effects. Flavonoids, azulenes, sesquiterpenes, and essential oils have been isolated and identified from its leaves. This paper reports the results of the composition and antifungal activity in vitro against dermatophytes and Candida spp. of the four essential oils obtained by steam distillation of the leaves, flowers, whole plant and whole plants without flower extracts of I. viscosa. All the extracts proved to have a significant antifungal activity against dermatophytes even at low concentrations (0.01 mg/ml). The leaf extracts exhibited the greatest antifungal efficacy. The high concentration of the sesquiterpene (carboxyeudesmadiene), occurring in the leaf extracts, may explain its greater antifungal activity.

[Fungistatic activity of a sesquiterpene lactone (tomentosin) isolated from fresh Inula viscosa (Asteraceae) flowers from the Puglia region]. / Attività fungistatica di un sesquiterpene lattone (tomentosina) isolato dai fiori freschi di Inula viscosa (Asteraceae) della regione Puglia.

A sesquiterpene lactone, tomentosin, has been isolated and identified form Inula viscosá Ait (Asteraceae) flowers. The molecule, at 1 mg/ml concentration, showed to be active in vitro against Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes.

[Research on antifungal activity of flowers and leaves of Inula viscosa (Asteraceae)]. / Ricerche sull'attività fungistatica di fiori e foglie di Inula viscosa (Asteraceae).

The authors carried out a preliminary screening about the in vitro antifungal activity of some extracts of flowers and leaves of Inula viscosa obtained with different solvents. All extracts showed antifungal activity against dermatophytes and Candida species. The best results were obtained with Inula viscosa flowers extracts. These results may be ascribed to the different flavonoids and different flavonoid concentrations in our samples.

Antifungal activity of Apulia region propolis.

A study was carried out to assess the in vitro antifungal activity of some natural Apulian propolis extracts of different origin. Their antifungal activity was compared to the antifungal activity of conifers and commercial propolis extracts. All extracts revealed antifungal activity against dermatophytes and Candida species. The antifungal activity differences found depended on the origin of the propolis and the solvent used for extraction. The best antifungal activity was given by the 'Orimini' propolis. The antifungal activity may have been influenced by the presence of different cinnamic and flavonoid components and their different concentration in the extracts. Further investigations are needed to validate this hypothesis.