Molecular detection and identification of piroplasms (Babesia spp. and Theileria spp.) and Anaplasma phagocytophilum in questing ticks from northwest Spain.

Anaplasma phagocytophilum and some piroplasm species are pathogens mainly transmitted by Ixodes ricinus. Considering that this tick species is predominant in north-western Spain, individual specimens (652 nymphs, 202 females and 202 males) and 23 larval pools were processed to determine the prevalence of these pathogens in questing I. ricinus from that region. Additionally, Dermacentor marginatus, Dermacentor reticulatus, Ixodes frontalis and Ixodes acuminatus were individually analysed. The groESL operon as well as the 16S rRNA and msp2 genes of Anaplasma were analysed. Similarly, piroplasms were identified at the 18S rRNA gene and the ITS1 of Babesia spp. and Theileria spp. Babesia venatorum (1.5%), A. phagocytophilum (0.7%), Babesia microti (0.3%) and Theileria sp. OT3 (0.2%) were detected in I. ricinus. A single I. frontalis (8.3%) tested positive to A. phagocytophilum. Although a low percentage of I. ricinus were infected with A. phagocytophilum and piroplasms, a potentially human pathogenic variant of A. phagocytophilum was detected, and both Babesia species found were zoonotic. Since the vector of Theileria sp. OT3 remains unknown, further investigations are needed to unravel the role of I. ricinus in the transmission of this piroplasm.

Effect of different temperatures on survival and development of Aelurostrongylus abstrusus (Railliet, 1898) larvae.

Aim of the study was to get in-depth information on Aelurostrongylus abstrusus first-stage larvae (L1s) survival at different temperatures and to assess the capability of these larvae to develop into the third infective stage (L3s). Faeces of a naturally infected cat were split into two aliquots: the first was divided in subsamples assigned to four groups (F1-F4); from the second aliquot, L1s were extracted by Baermann technique, suspended in water and divided into four groups (W1-W4). Groups were stored at different temperatures (F1/W1 -20 ± 1°C; F2/W2 +4 ± 1°C; F3/W3 +14 ± 1°C; and F4/W4 +28 ± 1°C) and L1s vitality assessed every seven days. The capability of L1s stored in water to develop into L3s in snails was evaluated at the beginning and every 21 days. The L1s of W2 and F2 groups remained viable for a longer period (231 and 56 days, respectively) compared to those of other groups. The capability of L1s to moult into L3s in snails showed a decreasing trend; the group W2's L1s maintained the capability to moult into L3s for the longest time (day 189) compared to the other groups. The time of survival of A. abstrusus L1s is influenced by temperature. However, the species seems to be more resistant to temperature variations than other feline lungworms, and this may explain its wider distribution across Europe.

Endoparasites detected in faecal samples from dogs and cats referred for routine clinical visit in Sardinia, Italy.

This study aimed to update data on the prevalence of intestinal and lung parasitic infections in owned dogs and cats in Sardinia, Italy. Examinations on faecal samples from 619 dogs and 343 cats routinely referred to the Veterinary Teaching Hospital of the University of Sassari were performed between the years of 2011 and 2015. Individual faecal samples were analysed using the Wisconsin technique for copro-microscopic examination and the Baermann technique for the presence of lungworm larvae. Endoparasites were found in 34.9% and 43.4% of examined dogs and cats, respectively. Helminthic infections (21.2% in dogs and 32.6% in cats) occurred more frequently than protozoan infections (17.9% in dogs and 17.8% in cats). In both dogs and cats, the most common parasites were ascarids (12.1% and 15.7%), Cystoisospora spp. (10.2% and 10.8%), Giardia duodenalis (9.4% and 8.5%), and hookworms (7.9% and 5.5%). Evidence of bronchopulmonary nematode infections were found in 0.8% of examined dogs and in 15.8% of examined cats. Age was identified as a risk factor, with animals younger than 6months more frequently infected than older animals, while no significant association was observed for gender. This study demonstrated that endoparasites in owned dogs and cats of Sardinia have considerably high prevalence. Veterinary practitioners and pet owners should be more aware of these infections and should adopt more effective and standardized control practices.

Cross-sectional survey on Tritrichomonas foetus infection in Italian cats.

The feline genotype of Tritrichomonas foetus is a widespread cause of large-bowel diarrhoea in cats. The aim of this study was to determine the prevalence of the T. foetus infection in cat populations across Italy. Fresh, individual faecal samples were collected from 267 cats, kept in different environments (i.e., private households, breeding structures, municipal catteries and colonies) in three different sites across Italy. The faecal samples were tested by PCR to detect T. foetus. Moreover, the same samples were subjected to a concentration-flotation technique and a commercial direct fluorescent-antibody (DFA) test to detect additional enteric parasites, including Giardia duodenalis. The overall prevalence of T. foetus infection was 5.2%. All the infected cats showed diarrhoea at the time of sampling: 9 out of 14 positive cats were co-infected with G. duodenalis, 1 with Toxocara cati and 3 with Dipylidium caninum. The risk factor analysis showed that not only the breed, but also co-infections with G. duodenalis and Dipylidium caninum were significantly associated with the presence of T. foetus. This study confirms the presence of T. foetus in cats living in Italy, suggesting that this protozoan parasite should always be included in the differential diagnosis of patients referred with large-bowel disease symptoms, especially if they were purebred animals, or affected by other enteric protozoa, such as G. duodenalis.

Simultaneous infection by four feline lungworm species and implications for the diagnosis.

Besides Aelurostrongylus abstrusus, other parasites belonging to the superfamily Metastrongyloidea, namely Oslerus rostratus, Troglostrongylus brevior and to the family Trichuridae, i.e. Eucoleus aerophilus (syn. Capillaria aerophila), have also been reported as agents of respiratory infection in domestic cats. A case of simultaneous infection by four feline lungworm species in Sardinia is herein described. An adult female cat (Felis silvestris catus), road-killed in the southeast part of Sardinia (municipality of Villacidro, province of Cagliari), Italy, was referred to the Laboratory of Parasitology of the Veterinary Teaching Hospital in Sassari. At necropsy, the lungs were examined and dissected under a stereomicroscope for the presence of parasites, and first-stage larvae (L1) of broncho-pulmonary nematodes were searched for in a faecal sample using the Baermann method. Parasites collected in the lungs were morphologically identified as A. abstrusus, E. aerophilus, and O. rostratus. In addition to the above species, L1s of Troglostrongylus spp. were detected at coproscopy but no adult specimen was found in the lungs. The morphological identification was confirmed by the molecular amplification and sequencing of cox1 mitochondrial gene, 18S and ITS2 ribosomal DNA. This finding stands as the first simultaneous infection by four feline lungworm species in the same animal, and as the first report of O. rostratus and E. aerophilus in Sardinia.

The European wildcats (Felis silvestris silvestris) as reservoir hosts of Troglostrongylus brevior (Strongylida: Crenosomatidae) lungworms.

The increasing reports of Troglostrongylus brevior lungworm in domestic cats from Italy and Spain raised questions on its factual distribution and on the role wildcats play as reservoirs of these parasites. Carcasses of 21 wildcats were collected in natural parks of southern Italy (i.e., Catania, Sicily n=5 and Matera, Basilicata n=16) and biometrically and genetically identified as Felis silvestris silvestris, but two as hybrids. Troglostrongylus brevior and Eucoleus aerophilus lungworms were found in 15 (71.4%) and 7 (33.3%) individuals, respectively, being five (23.8%) co-infected by the two species. Both lungworms showed an aggregated distribution in the host population, assessed by k-index (i.e., 0.69 for T. brevior and 0.42 for E. aerophilus). Although no statistical significant difference was recorded among age, gender and geographical location of wildcats, a larger rate of infection by T. brevior was assessed in yearlings (85.7%) than adults (64.3%). This is the first epidemiological study reporting T. brevior infection in the European wildcat and discusses the potential threat this may represent for the conservation of this endangered species of felids. In addition, given the large frequency of lungworm infection herein recorded, the role of wildcats as reservoir hosts of these parasites to domestic cats is discussed.

A retrospective and geographical epidemiological survey of traumatic myiasis in southern Italy.

A survey on the prevalence and geographical distribution of traumatic myiasis in sheep, and the risk factors for the disease, was carried out in a region of southern Italy. A total of 138 sheep flocks were selected and visited to acquire data on the presence or absence of traumatic myiasis using both a questionnaire for retrospective analysis and animal inspection. Prevalences registered at the farm and animal levels, respectively, were 8.7% and 6.3% in 2010, and 5.8% and 5.0% in 2011. Records of the occurrence of the parasitic disease in this region are recent: a statistically significant (P < 0.01) progressive increase in the number of farms affected (from 0.7% to 8.7%) has been registered since 2007. Wohlfahrtiosis was found in 11 of 95 (11.6%) geographical units sampled and three significant (P < 0.05) clusters of spatial farm aggregation were identified in the southern part of the study area. A total of 158 presently uninfested farms were considered to be at high risk for transmission as a result of their proximity to infested farms. The spreading of Wohlfahrtia magnifica (Diptera: Sarcophagidae) in southern regions of Italy represents a warning that the risk for infestation may become more significant in other Italian regions, as well as other European countries.

New insights on metastrongyloid lungworms infecting cats of Sardinia, Italy.

In addition to the well-known Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been diagnosed as a causative agent of bronco-pulmonary infections of cats in Spain and Italy. However, information concerning the impact of this species of lungworm on feline population is limited to a few case reports. From July 2011 to May 2013 an epidemiological survey was carried out on Sardinia island (Italy), where 107 individual faecal samples were examined by Baermann technique, and first-stage larvae were identified based on their morphology and characterization of molecular markers. The 29.9% (32/107) of cats examined were infested by broncho-pulmonary nematodes and, although A. abstrusus was the most frequently detected (n=27; 25.2%), larvae of T. brevior were also found (n=7; 6.5%). In addition, two cats (1.9%) were co-infested by both species. Overall metastrongyloid infection was higher in female cats (n=22; 38.6%) than in males (n=10; 20%) (χ(2)=4.39; p=0.036). The mean age of positive animals was 21.1 (±29.8) months, being infected animals from 2 months to 10 years of age. Of the 32 animals that scored positive for lungworms only 6 (18.8%) displayed a respiratory condition associated with lungworm infestations. Biomolecular characterization confirmed the morphological diagnosis of A. abstrusus. Positive samples that were identified at genus level as Troglostrongylus spp. were molecularly characterized as T. brevior. This study represents the first epidemiological survey on metastrongyloid lungworms of domestic cats in Sardinia and the first report of T. brevior on this island.

New insights onto cardiopulmonary nematodes of dogs in Sardinia, Italy.

Dog heartworms Angiostrongylus vasorum and Dirofilaria immitis cause severe parasitological diseases; the importance of these parasitosis is growing due to their health impact on animals, the possible zoonotic implications and the recent spreading across several European countries and previously non-endemic areas. The aim of this study is to update the epidemiological scenario of cardiopulmonary nematodes A. vasorum and D. immitis in dogs of Sardinia island and to perform a morphological identification of larvae by the use of the Baermann and Knott techniques respectively and the molecular characterization of the mitochondrial cytochrome c oxidase subunit I (cox1) and the second ribosomal transcribed spacer region (ITS-2) of larvae L1 of A. vasorum. In the present study, 3.4% (5/146) of dogs resulted positive at Baermann technique for A. vasorum while 8.9% (61/684) to D. immitis. If on one side A. vasorum can be considered an emerging parasite in Sardinia, the parasitic pressure and the risk of infection for D. immitis in the island seems to be increased compared with the recent past.

Lungworms of the genus Troglostrongylus (Strongylida: Crenosomatidae): neglected parasites for domestic cats.

Feline lungworms belonging to the Troglostrongylus genus have been neglected for a long time. However, recent reports of Troglostrongylus brevior and Troglostrongylus subcrenatus in domestic cats have stimulated the interest of the scientific community on these lungworms. Troglostrongylus spp. have an indirect life cycle, which overlaps that of the better known Aelurostrongylus abstrusus. Nonetheless, adult worms of both genera have a distinct morphology and localization within the respiratory system of definitive hosts and, potentially, a different epidemiology and pathogenicity. As copromicroscopy has a low specificity for metastrongyloids due to the similarities of first-stage larvae, specific morphometrical keys and/or PCR diagnostic tools are advocated. Accordingly, more clinical studies and necropsy data are needed to elucidate the impact of Troglostrongylus spp. on the health of domestic cats. This article reviews current information on Troglostrongylus spp. as well as data on their occurrence in Europe and reports specific key morphological characters for the identification of adults and larvae, which is important to refine their diagnosis and for a better understanding of the feline lungworm infections.

Cercopithifilaria spp. in dogs in Sardinia Island (Italy).

A survey on Cercopithifilaria spp. was carried out on owned and kennelled dogs in Sardinia, Italy. A total of 180 dogs were sampled and tested by microscopic detection or PCR of dermal microfilariae in skin snip sediments. The overall prevalence for Cercopithifilaria spp. at both microscopy and molecular tests was 9.4 % (17/180), while 8.3 % (15/180) of dogs scored positive at microscopic detection of sediments only. Of the 225 microfilariae measured, 212 were identified as Cercopithifilaria bainae and the remaining as Cercopithifilaria sp. II. All samples were molecularly processed for specific amplification of cytochrome oxidase subunit 1 (cox1) and ribosomal 12S gene fragments. The Basic Local Alignment Search Tool analysis of the cox1 and 12S sequences here obtained showed a high nucleotide similarity (99 and 100 %, respectively) with those of C. bainae available in GenBank. In particular, cox1 haplotype I (HI; n=14), haplotype HXVIII (n=2), and a new haplotype, named HXIX (n=1), differing for a single polymorphism from HI, were detected. This study reports data on the occurrence, distribution, and genetic makeup of C. bainae and Cercopithifilaria sp. II infesting dogs in Sardinia, suggesting that these filarioids are spread in areas where Rhipicephalus sanguineus sensu lato ticks occur.

Rhipicephalus sanguineus (Ixodida, Ixodidae) as intermediate host of a canine neglected filarial species with dermal microfilariae.

The life cycles of filarioids of dogs presenting dermal microfilariae have been little studied. Following the recent retrieval of dermal microfilariae identified as Cercopithifilaria sp. in a dog from Sicily (Italy), this study was designed to assess the role of the brown dog tick Rhipicephalus sanguineus as an intermediate host of this filarial species. An experimental tick infestation was performed on an infected dog using 300 nymphs of R. sanguineus. Engorged nymphs were collected and examined by both microscopic dissection and molecular analysis at five time points (i.e., the same day of tick detachment and 10, 20, 30 and 50 days post-detachment) to detect the presence and developmental stage of filariae in the ticks. A total of 270 engorged nymphs were collected from the dog and developing filarioid larvae detected in 10 (5%) out of 200 ticks dissected. Infective third-stage larvae were observed in 4 (2%) of the all dissected ticks, 30 days post-detachment. Twelve (6.6%) out of 181 samples molecularly tested were positive for Cercopithifilaria sp. This study demonstrates that nymphs of R. sanguineus feeding on a dog naturally infected by Cercopithifilaria sp. can ingest microfilariae, which develop up to the third infective stage thus suggesting that this tick species might act as an intermediate host of this little known canine filarioid.

Risk for the introduction of exotic ticks and pathogens into Italy through the illegal importation of tortoises, Testudo graeca.

In April 2008, 585 tortoises illegally imported into Italy from North Africa were examined for the presence of ticks. Of these, 221 tortoises (37.8%) were infested with a mean intensity of 3.9 +/- 3.1 ticks (range 1-17 ticks). A total of 798 ticks (672 males, 125 females and one nymph) were collected and identified as Hyalomma aegyptium (L.) (Acari: Ixodidae). The overall male : female ratio observed was 5 : 1. The prevalence and mean intensity [+/-standard deviation (SD)] of ticks were higher among male (67.4%, 4.0 +/- 3.2) than female (55.6%, 3.8 +/- 3.1) tortoises, although this difference was not significant. By contrast, the prevalence and mean intensity of ticks were significantly higher on tortoises weighing >100 g (61.5%, 4.0 +/- 3.2) compared with tortoises weighing <100 g (12.1%, 2.1 +/- 1.1). Of the infested tortoises, 89.8% had ticks on their hind limbs, 21.0% on forelimbs, 18.6% on the tail and pre-anal area, and 4.8% on the head; thus the hind limbs were evidently the preferred attachment site. The present report highlights the need to develop surveillance systems to prevent the introduction and spreading of exotic ticks and tick-borne pathogens in Italy and other European countries.

Use of the FLOTAC technique for the diagnosis of Aelurostrongylus abstrusus in the cat.

Aelurostrongylus abstrusus is a lungworm of the domestic cats distributed worldwide. Its presence, however, is often underestimated due to limitation of common coprological diagnostic techniques. The present study reports, for the first time, the use of the FLOTAC technique for A. abstrusus diagnosis in feline faecal samples with results compared to the following three standard copromicroscopic techniques: the Baermann technique, the McMaster technique and the Wisconsin technique. The comparison study showed that the FLOTAC technique produced mean larvae per gram of faeces significantly (P<0.05) greater than that produced by the other more widely used diagnostic tools. Thus, the FLOTAC technique can be utilised for quantifying lungworm larva burdens in feline faecal samples because of its high sensitivity, as already shown for various parasites of veterinary and human importance.

Cystic echinococcosis in equids in Italy.

Between March 2003 and February 2007, the livers and the lungs of 2,231 horses from various Italian regions were examined for cystic echinococcosis presence at the time of slaughter. Hydatid cysts were found in six horses, namely four from Sardinia, one from Sicily, and one from Tuscany. The location, number, morphology, and fertility of the cysts found were determined. DNA was extracted from the germinal layers and protoscoleces of the fertile cysts and polymerase chain reactions (PCR) were performed in order to strain type DNA isolates for reduced nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1), cytochrome c oxidase subunit 1 (CO1) and 12S partial genes. The PCR products were then purified and sequenced in forward and reverse. Hydatid materials obtained from positive animals were identified as Echinococcus granulosus s.s. (old G1, sheep strain) and Echinococcus equinus (old G4, horse strain) for ND1, CO1, and 12S partial genes. This allowed us to record the presence of the E. equinus in Italy for the first time with molecular tools and also to report new data on the epidemiological situation of this parasite in Italy.

Efficacy of oxfendazole and fenbendazole against tortoise (Testudo hermanni) oxyurids.

Thirty-six tortoises (Testudo hermanni) with naturally acquired oxyurids infections were used to assess the anthelmintic efficacy of oxfendazole (Dolthene; Merial) and fenbendazole (Panacur; Hoechst Roussel Vet). Animals were randomly assigned to three groups (A, B, and C) based on sex and weight. Animals in group A (seven males and six females) were orally treated with oxfendazole at dose rate of 66 mg/kg, group B animals (nine males and eight females) were orally treated with fenbendazole at dose rate of 100 mg/kg, and group C animals (three males and three females) were not treated and served as controls. All animals were individually stabled in plexiglas boxes under controlled conditions of temperature, humidity, and light beginning 7 days pretreatment and continuing for the duration of the trial. Individual tortoises feces were examined daily by the McMaster technique and drugs efficacy was assessed by the fecal eggs count reduction (FECR) test. Both drugs showed 100% of FECR. However, oxfendazole reached this level 12 days after treatment, whereas 31 days after treatment were necessary to obtain the same stable result with fenbendazole. The two drugs were well tolerated by all the animals and no adverse reactions were observed after treatment.

Agrometeorology and models for the parasite cycle forecast.

Insects are strongly influenced by meteorological variables in their natural environment. In agriculture, mathematical models have been developed to understand and forecast the cycle of pests based on climate data. By this manner, with the goal of reduce and rationalize plant chemical treatments, agrometeorological models have been realized to estimate the length and starting times of parasites phenological phases. In Sicily a new network of 95 GSM meteorological stations and a specific mathematical model for Aonidiella aurantii are used by Sicilian Agrometeorological Information System (SIAS) for the integrated pest management program of citrus orchards in the Island. As the plants parasites, vector borne diseases are influenced by climate in their appearance and abundance. In lights of the benefits that could derive from a model for the control of Leishmania vectors, SIAS experiences in modelling were used to develop a deductive model for Phlebotomus perniciosus which represents the major vector of human and canine leishmaniasis in Sicily.