Linda Mugnaini

Seroprevalence, Detection of DNA in Blood and Milk, and Genotyping of Toxoplasma gondii in a Goat Population in Italy

Toxoplasma gondii is the causative agent of a major zoonosis with cosmopolitan distribution and is known to be transmitted mainly by the ingestion of undercooked or raw animal products. Drinking unpasteurized goats milk is a risk factor associated with human toxoplasmosis. However, very little is known about the excretion of DNA in goat milk. Aim of the present study was to determine the seroprevalence of T. gondii infection using a modified agglutination test (MAT), to detect T. gondii DNA by nested-PCR (n-PCR) in samples of blood and milk from seropositive goats, and to genotype DNA isolates using 11 molecular markers in 127 adult lactating goats from 6 farms in Italy. Positive MAT results were found in 60.6% of goats while 13% of blood and milk samples from seropositive goats were positive to n-PCR. A kappa coefficient of 1 indicated a perfect agreement between blood and milk n-PCR. Genetic characterization of isolates revealed the occurrence of genotype III (n = 7), genotype I (n = 1), and atypical genotypes with hints for genotype I (n = 2). Our results suggest that the risk of excretion of Toxoplasma tachyzoites might frequently occur in milk of seropositive goats testing positive to n-PCR on blood.

Prevalence of Toxoplasma gondii infection in Myocastor coypus in a protected Italian wetland.

BackgroundToxoplasma gondii is the causative agent for a major zoonosis with cosmopolitan distribution. Water has been implicated in outbreaks of toxoplasmosis in recent years. Coypus (Myocastor coypus), commonly nutria, are large semi-aquatic invasive rodents, naturalized throughout European countries, including most wetlands of Central Italy. The habitat of these animals is both terrestrial and aquatic, making them a species highly exposed to the parasite.FindingsThe occurrence of the infection was evaluated using a modified agglutination test (MAT) in 74 adult coypus from a naturalized population living in a wetland of Central Italy. Nested PCR (n-PCR) assay was carried out on some of them. Positive T. gondii MAT results were found in 44 animals (59·4%), 30 males (68·2%) and 14 females (31·8%). Antibody titers were ranging from 20 to 40960, while 12 out of 23 (52·2%), examined animals, 8 males (66·7%) and 4 females (33·3%), resulted positive to n-PCR. All n-PCR positive animals were seropositive, showing antibody titers ranging from 640 to 40960.ConclusionsOur results indicate that examined animals are heavily parasitized with Toxoplasma. This suggests that coypus could be a reservoir of this parasite, because they can be eaten both by scavenger animals and by humans, and that these animals would play a role in maintaining the cycle of T. gondii.

Detection and genotyping of Toxoplasma gondii DNA in the blood and milk of naturally infected donkeys (Equus asinus).

BackgroundToxoplasma gondii is a worldwide zoonotic protozoan. Consumption of raw milk from infected animals is considered a risk factor for acquiring toxoplasmosis in humans. Recently, donkey milk has been indicated for therapeutic and nutritional purposes and T. gondii infection is common in donkeys. The purpose of the present paper was to detect the presence of parasite DNA in milk of T. gondii positive donkeys.FindingsAntibodies to T. gondii were found in 11 out of 44 healthy lactating donkeys by IFAT. T. gondii DNA was detected by PCR in blood of 6 and milk of 3 seropositive jennies. Results of limited RFLP-PCR genotyping indicated the presence of T. gondii genotype II or III, commonly found in Europe.ConclusionsThe occurrence of T. gondii DNA in milk suggests that the consumption of raw milk from seropositive donkeys could be a potential source of human infection.

SEROLOGIC, MOLECULAR, AND PATHOLOGIC SURVEY OF TOXOPLASMA GONDII INFECTION IN FREE-RANGING RED FOXES (VULPES VULPES) IN CENTRAL ITALY

We tested 191 sera of red foxes (Vulpes vulpes; 78 females and 113 males) for antibodies to Toxoplasma gondii using an indirect immunofluorescent test. Tissue samples of myocardium, lymph nodes, and brains from antibody-positive animals were tested for T. gondii DNA using specific PCR and processed for histopathology and immunohistochemistry (IHC) to detect T. gondii antigen. Of 192 (53.4%) antibody-positive animals, eight were positive by PCR on myocardium and one on brain. All DNA extracts were genotyped. Histopathology showed lesions characteristic of protozoan encephalitis; IHC did not show T. gondii antigen in examined tissues. The high antibody prevalence found in our study, which is the first in Italy, and the occurrence of polymorphic strains (combination of different type I and III alleles) divergent from typical T. gondii strains, suggests red foxes may be a sentinel of T. gondii in the environment.

A retrospective molecular study of select intestinal protozoa in healthy pet cats from Italy

The feline gut can harbour a number of protozoan parasites. Recent genetic studies have highlighted new epidemiological findings about species of Cryptosporidium, assemblages of Giardia duodenalis and Toxoplasma gondii. Furthermore, epidemiological studies suggest the occurrence of Tritrichomonas foetus in cats is on the increase worldwide. The prevalence of selected intestinal protozoa was determined by PCR using DNA previously extracted from the faeces of 146 privately owned healthy cats from Italy. Molecular genotyping on T gondii, G duodenalis and Cryptosporidium DNA was achieved. PCR assays were positive in 32 (22.9%) samples. Three animals (2.0%) were positive for T foetus and Cryptosporidium DNA, 15 specimens (10.3%) were positive for T gondii and 11 (7.5%) for G duodenalis. Co-infections were never observed. Results of the typing analysis allowed the identification of Cryptosporidium felis in all cases. The specimens positive for T gondii hinted at clonal genotype I (n = 7), genotype II (n = 1) and genotype III (n = 7). The G duodenalis isolates were referable to assemblages F (n = 9) and C (n = 2). In conclusion, the results obtained in this study add to the literature regarding the epidemiology of these parasites by confirming their presence in the faeces of healthy pet cats.

Canine and feline dermatophytosis due to Microsporum gypseum: A retrospective study of clinical data and therapy outcome with griseofulvin

OBJECTIVE Microsporum gypseum is a common inhabitant of the soil, occasionally responsible for human and animal ringworm. Few reports describe the treatment of dermatologic diseases due to M. gypseum. The objective of this study was to evaluate retrospectively cases of M. gypseum infection in dogs and cats. MATERIAL AND METHODS The occurrence of infection by this dermatophyte was retrospectively evaluated in dermatological specimens from 15,684 dogs and cats dermatologically diseased from Italy. Clinical outcome after treatment with griseofulvin combined with topical enilconazole was evaluated in 41 dogs and, out of label, 10 cats. Furthermore, in vitro susceptibility to griseofulvin and enilconazole was evaluated on 31 clinical isolates of M. gypseum. RESULTS One hundred and eighty-five specimens out of 15,684 (1.1%) scored positive for M. gypseum. The treatment failed to achieve both mycological and clinical cure in 16 dogs (39%) and four cats (40%), as well as fungal isolates demonstrated a very poor in vitro sensitivity when tested versus griseofulvin: the MIC value was 150 μg/mL. The ED50 value was calculated at 66 μg/mL. CONCLUSION Blind treatments with griseofulvin in ringworm due to M. gypseum should be avoided.

Toxoplasma gondii in Waterfowl: The First Detection of this Parasite in Anas crecca and Anas clypeata from Italy

Abstract: Understanding the spread of Toxoplasma gondii in waterfowl is of interest for elucidating the potential involvement of these birds in maintaining the parasitic life cycle because birds are exposed to these parasites. Sera from 103 adult, free-range game birds, representing 13 different species living in the Italian wetlands, were examined using modified agglutination tests for antibodies specific to T. gondii. In seropositive birds, the brain and heart were homogenized and DNA was extracted to perform nested polymerase chain reaction (nPCR) and genotyping. Out of the 103 serum samples, 9 (8.7%) were scored as positive with titers ranging from 1/20 to 1/160. The seropositive animals corresponded to 4 of the species: Anas platyrhynchos (mallard; 2/17), Anas clypeata (northern shoveller; 2/11), Anas crecca (common teal; 3/41), and Gallinago gallinago (common snipe; 2/8). Parasite DNA was detected in 3 out of 9 brain samples, while the PCR results from the heart specimens were negative in all of the birds. The occurrence of non-clonal types of T. gondii was suggested in all cases. In conclusion, we describe the first documented detection of DNA of T. gondii in tissues from the northern shoveller and common teal, thereby extending the range of intermediate hosts for this parasite.

Antimycotic Activity of Some Aromatic Plants Essential Oils Against Canine Isolates of Malassezia pachydermatis: An in vitro Assay

The antifungal activity and the chemical composition of essential oils (EOs) from some Mediterranean autochthonous plants were investigated against Malassezia pachydermatis, a fastidious opportunistic yeast usually involved in canine external otitis. Minimum inhibitory concentrations (MICs) of Anthemis nobilis, Citrus limon, Citrus paradisi, Illicium verum, Lavandula hybrida, Mentha piperita, Ocimum basilicum, Origanum vulgare, Origanum majorana, Rosmarinus officinalis, Salvia sclarea, Thymus serpillum were assessed by microdilution test; minimum fungicidal activity (MFC) was also determined. O. vulgare, T. serpillum and O. basilicum showed the lowest MIC and MFC values (0.8%) followed by C. limon and M. piperita (1%). EOs from the tested plants showed variable degrees of anti-malassezia activity, putatively related to their chemical composition. The effectiveness, manageability and pleasant organoleptic properties of O. vulgare, T. serpillum, O. basilicum, C. limon and M. piperita EOs make them advisable as promising new natural antifungal drugs in the management of M. pachydermatis otitis in dog.

Clinical and mycological evaluation of an herbal antifungal formulation in canine Malassezia dermatitis

OBJECTIVE Malassezia pachydermatis is a common cause of more widespread dermatitis in dogs (CMD). Recurrences are common, and this disorder can be very troubling for both dogs and for the pet owner. MATERIAL AND METHODS The treatment of 20 dogs affected by dermatitis due to M. pachydermatis, with Malacalm(®), a commercially available mixture consisting of essential oils (Citrus aurantium 1%, Lavandula officinalis 1%, Origanum vulgare 0.5%, Origanum majorana 0.5%, Mentha piperita 0.5% and Helichrysum italicum var. italicum 0.5%, in sweet almond oil and coconut oil) is reported. The effectiveness of the whole mixture, of component essential oils and of their more represented compounds against clinical isolates was evaluated by a microdilution test. Twenty animals were topically administered the mixture twice daily for 1 month. Ten animals were treated with a conventional therapy based on ketoconazole 10mg/kg/day and chlorhexidine 2% twice a week for 3 weeks. At the end of both treatments animals significantly improved their clinical status. Adverse effects were never noticed. Follow-up visit performed on day 180th allowed to observe a recurrence of clinical signs in all the subjects treated conventionally, while not significant clinical changes were referred in dogs treated with Malacalm(®). The overall MIC value of Malacalm(®) was 0.3%. O. vulgare showed the lowest minimum inhibitory concentrations (MIC), being active at 0.8%, followed by M. piperita (1%), O. majorana (1.3%), C. aurantium (2%) and L. officinalis (4%) while H. italicum did not yield any antimycotic effect up to 10%. Active major compounds were thymol, carvacrol, p-cymene, 1,8-cineol, limonene and menthol. CONCLUSION The phytotherapic treatment achieved a good clinical outcome, and no recurrence of skin disorders on day 180th was recorded. This herbal remedium appeared to be a safe tool for limiting recurrences of CMD.

A herbal antifungal formulation of Thymus serpillum, Origanum vulgare and Rosmarinus officinalis for treating ovine dermatophytosis due to Trichophyton mentagrophytes

A number of herbal products with anti‐inflammatory, antiseptic and antimycotic properties are available for dermatological usage. The successful treatment of 13 sheep affected by ringworm due to Trichophyton mentagrophytes with a mixture consisting of essential oils (EOs) of Thymus serpillum 2%, Origanum vulgare 5% and Rosmarinus officinalis 5% in sweet almond (Prunus dulcis) oil. The effectiveness of EOs and of the major components of the mixture (thymol, carvacrol, 1,8 cineole, α‐pinene, p‐cymene, γ‐terpinene) against the fungal clinical isolate was evaluated by a microdilution test. Thirteen animals were topically administered with the mixture twice daily for 15 days. The other sheep were administered with a conventional treatment (seven animals) or left untreated (two animals). Minimum inhibitory concentration (MIC) values were 0.1% for T. serpillum, 0.5% for O. vulgare, 2.5% for I. verum and 5% for both R. officinalis and C. limon. Thymol and carvacrol showed MICs of 0.125% and 0.0625%. A clinical and aetiological cure was obtained at the end of each treatment regimen in only the treated animals. Specific antimycotic drugs licenced for food‐producing sheep are not available within the European Community. The mixture tested here appeared to be a versatile tool for limiting fungal growth.