Seroprevalence and risk factors associated with Leishmania infantum in dogs in Sardinia (Italy), an endemic island for leishmaniasis.

Leishmaniasis is a widespread, vector-borne parasitosis causing clinical manifestations in animals and in humans. In dogs, Canine Leishmaniasis has been reported in as much as 50 countries and the Mediterranean basin is known to be one of the most affected zones. Within these areas, the Island of Sardinia (Italy) has long been considered endemic for leishmaniasis and the presence of two arthropod vectors has recently been reported there. Nevertheless, to date, no epidemiological surveys regarding CanL have been carried out on the island. Hence, for the first time, the seroprevalence and the risk factors were investigated. Blood samples, as well as clinical and general information from 1.147 dogs, were collected and analyzed. Dogs consisted of two distinct populations, namely "owned dogs" and "kennel dogs." Anti-Leishmania IgG antibodies were detected using IFAT and samples were scored as positive at a cut-off dilution of 1:80. Data was analyzed using a Chi-squared test and bivariate and multivariate analyses were performed. Overall, 15.4% of dogs were found to be infected with CanL while only 44.1% of these animals exhibited clinical signs. Owned dogs (27.2%) were found to be infected more often than kennel dogs (10.6%); male dogs were found to be more frequently infected than female dogs and the number of infected animals increases with age. The present survey confirmed the endemic nature of leishmaniasis in Sardinia with a similar seroprevalence as mainland Italy. The results obtained serve as validation for the hypothesis that, in endemic areas, clinical CanL representations constitute only a fraction of the leishmaniasis cases.

Species delimitation based on mtDNA genes suggests the occurrence of new species of Mesocestoides in the Mediterranean region.

BACKGROUND: This study is the first contribution to the molecular taxonomy of Mesocestoides spp. from domestic and wild carnivores in the Mediterranean area. A total of 13 adult worms and 13 larval stages of Mesocestoides spp. were collected from domestic and wild carnivore hosts in Italy and Tunisia. Samples collected in the Slovak Republic were used as comparative samples from outside the Mediterranean. The genes cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) of the mitochondrial genome were used as molecular markers to investigate the presence of cryptic Mesocestoides species in the area analysed. RESULTS: Results were consistent in showing three well-supported clusters of Mesocestoides spp. in southern Italy and Tunisia, which were strongly divergent from Mesocestoides litteratus, M. corti and M. lineatus. High levels of genetic variation and no evidence of geographical structuring was found between the clusters. CONCLUSIONS: Considering the low dispersal capability of the intermediate hosts of Mesocestoides spp., the lack of geographical structuring among the Mediterranean regions could be due to a high potential for dispersion of the definitive hosts. This study provides a foundation for future formal descriptions of new species of the genus Mesocestoides in the Mediterranean area.

Retrospective study on Cystic Echinococcosis in cattle of Italy.

INTRODUCTION: Cystic Echinococcosis (CE) is one of the most widespread zoonosis of veterinary and medical importance still constituting a sanitary, economic and socio-cultural problem in Italy. METHODOLOGY: The aim of this study was to update epidemiological data on cattle CE in Italy. Data on CE positivity of 5,336 cattle were acquired from abattoir registers between January 2009 and July 2010. Morphobiological characterization of hydatids was performed by direct examination of liver and lungs of 1,664 animals butchered in the same slaughterhouses in 2010. Strain typing of parasites was carried out through the amplification and sequencing of nd1 and cox1 mitochondrial genes. RESULTS: Overall CE prevalence was of 8.1% (430/5,336). Parasitological examination of hydatids showed an overall prevalence of 8.6% with a fertility rate of 0.7% (12/1,664). Regarding localization, hydatids were found in 8% of the livers and in 7.6% of the lungs, respectively. Among positive animals, higher prevalence was observed in the liver (93%) compared to lungs (88.1%) (p > 0.05). CONCLUSION: The economic loss due to organs condemnation related to CE in cattle amounted to almost € 24,000 per year in the examined abattoir during 2010. Sequence analysis showed the presence of G1 (sheep strain) or Echinococcus granulosus sensu strictu in all examined samples. The G1 confirmed, once more, its possible development into several intermediate hosts such as cattle, especially in areas like southern Italy and Sardinia where the lifecycle of the parasite is still to date carried on by sheep and dogs.

Cystic echinococcosis in cattle dairy farms: spatial distribution and epidemiological dynamics.

A study monitoring cystic echinococcosis (CE) in adult dairy cattle from intensive livestock farms located in the municipality of Arborea (Sardinia, Italy) was carried out between 2012 and 2015. A retrospective study of veterinary reporting forms of post-mortem inspections in 10 different Italian slaughterhouses was also performed. In addition, data on viability and molecular characterisation of hydatid cysts removed from parasitised organs in cattle was carried out. A geographical information system (GIS) with data layers of the study area and the geo-referenced points of 160 cattle farms was constructed. CE was found in 21.9% (35/160) of the surveyed farms. The retrospective study revealed that 0.05% (13/23,656) of adult slaughtered animals (over one year of age) from Arborea had tested positive to CE. The results stratified per year showed the following CE prevalences: 0.09% (5/5673) in 2012; 0.02% (1/5682) in 2013; 0.08% (5/6261) in 2014; and 0.03% (2/6040) in 2015 (χ2 with 3 degrees of freedom=3.81; P=0.282). The E. granulosus sensu stricto (formerly called G1 or sheep strain) was detected in all cysts subjected to molecular analysis. The GIS analysis showed that CE is fairly resilient in the Arborea territory where most of cattle farms are located, while a small cluster of cases was found located in the southeastern part of Arborea, close to districts where sheep farms are situated. The present survey reports the presence of CE in Sardinian dairy cattle intensive farms and suggests that the parasitic pressure of CE in the island continues to be very strong.

Morphology and genetic variability within Taenia multiceps in ruminants from Italy.

The aim of this study was to investigate the genetic variability and population structure of Taenia multiceps, and to correlate morphological features of individual coenuri with haplotypes. A total of 92 animals (86 sheep; 4 goats; 1 cattle; 1 mouflon, Ovis musimon) aged between 6-36 months showing clinical symptoms of cerebral coenurosis were included in this study. T. multiceps coenuri (n=118) sampled from live animals during routine surgery procedures or at post-mortem inspections were examined morphologically and molecularly identified. Morphological features of the 52 coenuri selected for this study (number and size of large and small hooks) were within the range reported in the literature. Fifty-two of the molecularly confirmed T. multiceps coenuri harboured by 47 animals (sheep=41; cattle=1; goats=4; mouflon=1) were used to determine gene genealogies and population genetic indices and were compared to the 3 T. multiceps genetic variants, Tm1-Tm3 previously described from Sardinia, Italy. For the 379 bp cox1 dataset we identified 11 polymorphic sites of which 8 were parsimony informative. A high haplotype diversity (0.664±0.067) was recorded for the cox1 sequences defining 10 haplotypes (TM01-TM10). The comparison of haplotypes generated in this study with published T. multiceps Tm1 variant pointed to the possible existence of a common lineage for T. multiceps. No correlation was detected between the size of the small and large hooks and the cox 1 haplotypes. Polycystic infestation (2-9 coenuri) was recorded in 27.7% of animals (13/47). No statistical correlation between polycystic T. multiceps infection and haplotypes was detected.

A life-threatening brainstem compression by cerebral Echinococcus granulosus.

Cystic echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus. It is of worldwide importance, and is widespread in the Mediterranean region and Middle East. This tapeworm shows great intraspecific variation in relation to host specificity, epidemiology and morphology. This variability led in previous years to the identification of ten (G1-G10) different genotypes of the parasite. Cerebral localization of E. granulosus is not common: it especially affects children and is more frequently located in the supratentorial region. It can be life-threatening due to its localization in eloquent areas especially in the posterior fossa. Despite the benign nature of hydatid cyst, invasion of critical areas may cause significant mortality and morbidity in some patients. Urgent surgical decompression and adjuvant medical treatment must be employed as soon as possible in these patients. We present a clinical case of life-threatening brainstem compression in a child due to a rare form of CE which was confirmed with biomolecular techniques. She presented with respiratory distress and progressive quadriparesis. All cysts were removed by microsurgical technique and albendazole was given postoperatively for one year with regular follow-ups.

Clinical evolution of cerebral coenurosis from invasive to chronic infection in sheep and a goat.

This survey describes the evolution in vivo of Coenurus cerebralis in small ruminants. At presentation, neurological signs and cerebrospinal fluid (CSF) features were suggestive of multifocal or diffuse inflammatory reaction. Magnetic resonance imaging (MRI) captured the transition between the invasive and quiescent phase of the infection, revealing the concurrent presence of meningitis and small cysts. During the quiescent phase, in all animals, neurological symptoms disappeared, and cerebrospinal fluid was unremarkable while cysts grew progressively. Subsequently, the onset of neurological symptoms coincided with MRI signs of diffuse or localized increase of intracranial pressure, as confirmed by direct intracranial pressure measuring. All the animals had an excellent post-surgical recovery. This is the first report describing the evolution of coenurosis in vivo. Sequential imaging allowed describing interesting such as the death of some coenuri and different parasite growth rate in the same host.

Cross-sectional investigation on sheep sarcosporidiosis in Sardinia, Italy.

An epidemiological survey on sarcosporidiosis was carried out with a cross sectional investigation on macroscopic and microscopic Sarcocystis spp. in Sarda breed sheep slaughtered in different abbatoirs of Sardinia, Italy. For the macroscopic survey, muscular samples (diaphragm, abdominal and intercostals muscles, cutaneous muscles and muscles of the thigh) from 769 slaughtered Sarda sheep, oesophagus (n=365) and laryngeal and pterygoid muscles (n=521) were macroscopically investigated and Polymerase Chain Reaction (PCR) on selected macroscopic cysts was performed for a molecular identification of macroscopic Sarcocystis species. For the microscopic investigation 112 heart samples from slaughtered Sarda sheep were collected and investigated with two different protocols: unstained (compression) examination and a molecular technique. The overall prevalence of infection for macroscopic forms of sarcocysts was of 23.3% (179/769) with prevalences higher in the oesophagus (31.6%; 125/395) compared with the other investigated tissue type; two different morphotypes, classified as large oval (LO) macroscopic cysts, identified as Sarcocystis gigantea, and slender fusiform (SF) sarcocysts, were identified. The examination of heart samples revealed an overall prevalence of 77.7% (87/112) for Sarcocystis spp.; the nested-PCR analysis of heart samples allowed to identify the microscopic species, Sarcocystis tenella and Sarcocystis arieticanis, with prevalences of 95.5% (107/112) and 17.8% (5/112) respectively. Reported results highlight the high prevalence of Sarcocystis infection in the island and suggests the need of an improvement of control and prevention strategies for this parasitosis.

Is the red fox (Vulpes vulpes) a competent definitive host for Taenia multiceps?

BACKGROUND: Shepherd and stray dogs are thought to represent the primary definitive hosts of Coenurosis by Taenia multiceps, due to their feeding habits which translate into high chances of coming into contact with infected intermediate hosts. Nonetheless, little attention has been paid to the role of the red fox (Vulpes vulpes) in the epidemiology of coenurosis. In fact a knowledge gap exists on the role played by red foxes in the epidemiology of Taenia multiceps and the capability of this parasite to produce fertile and viable eggs in this wild canid, i.e. on the occurrence of a sylvatic cycle. This study investigates the role of the red fox (Vulpes vulpes) in the epidemiology of T. multiceps and related metacestodoses. METHODS: The small intestine of 63 red foxes was macroscopically examined for the presence of cestodes. Adult parasites were identified morphologically as being T. multiceps. Tapeworm eggs were counted and stored at 4 °C in physiological saline solution prior to experimental infection of four sheep and one goat. Sheep were inoculated orally on Day 0 with 3000 (sheep 1), 5000 (sheep 2 and 3) or 7000 eggs (sheep 4), while the goat was infected with 5000 eggs of T. multiceps. The animals were followed-up regularly by MRI and underwent surgical treatment between days 180 to day 240 post infection. Collected coenuri were identified using morphological and molecular methods. RESULTS: A total of 6.3 % of red foxes were found infected with T. multiceps and the eggs obtained from the worms were determined to have a viability of 45.4 %. Two of the challenged sheep and the goat developed disease compatible with T. multiceps. Morphometrical features of the cysts were consistent with those of T. multiceps; nucleotide amplification and sequencing of mitochondrial genes (i.e., cox1 and Nd1) from the metacestode material confirmed the identification. CONCLUSIONS: The present study is the first to provide evidence of the role of the red fox as a competent definitive host for T. multiceps, thus changing the epidemiological scenarios of infections by this cestode.

A preliminary investigation into the genetic variation and population structure of Taenia hydatigena from Sardinia, Italy.

Cysticercosis caused by the metacestode stage of Taenia hydatigena is endemic in Sardinia. Information on the genetic variation of this parasite is important for epidemiological studies and implementation of control programs. Using two mitochondrial genes, the cytochrome c oxidase subunit 1 (cox1) and the NADH dehydrogenase subunit 1 (ND1) we investigated the genetic variation and population structure of Cysticercus tenuicollis from Sardinian intermediate hosts and compared it to that from other hosts from various geographical regions. The parsimony cox1 network analysis indicated the existence of a common lineage for T. hydatigena and the overall diversity and neutrality indices indicated demographic expansion. Using the cox1 sequences, low pairwise fixation index (Fst) values were recorded for Sardinian, Iranian and Palestinian sheep C. tenuicollis which suggested the absence of genetic differentiation. Using the ND1 sequences, C. tenuicollis from Sardinian sheep appeared to be differentiated from those of goat and pig origin. In addition, goat C. tenuicollis were genetically different from adult T. hydatigena as indicated by the statistically significant Fst value. Our results are consistent with biochemical and morphological studies that suggest the existence of variants of T. hydatigena.

Angiostrongylus chabaudi Biocca, 1957: a new parasite for domestic cats?

BACKGROUND: Natural infection with a species of Angiostrongylus has been reported only once in wildcats from central Italy by Biocca in 1957. The causative species of this infection was identified as Angiostrongylus chabaudi. Following this report, this parasite had never been found in either wild or domestic cats. FINDINGS: The lungs and the pulmonary arteries of an adult female cat (Felis silvestris catus), road-killed in Sardinia, Italy, were macroscopically examined and dissected under a light microscope for the presence of parasites. A slender nematode was detected and its morphometrical features were consistent with those of A. chabaudi. Morphological data were supplemented by sequencing of the partial cytochrome oxidase c subunit 1 (cox1) gene, as well as the internal transcribed spacer 2 (ITS2) of the rDNA. Nucleotide sequences displayed 99% homology with the ITS2 sequence [GenBank KM216825.1] of a specimen of Angiostrongylus sp. recovered recently from the pulmonary artery of a wildcat in Germany and 91% with cox1 sequence [GenBank GU138118.1] of Angiostrongylus vasorum. CONCLUSION: The results of the present study indicate, for the first time, that A. chabaudi may also infect domestic cats, and thus should be considered in the diagnosis of metastrongyloid species infecting their cardio-pulmonary system.

Canine giardiosis in Sardinia Island, Italy: prevalence, molecular characterization, and risk factors.

INTRODUCTION: The flagellate protozoan Giardia duodenalis causes infection in humans and in various animals. Eight distinct assemblages (A-H) have been identified within G. duodenalis; assemblages A and B are those specific to humans and animals, and assemblages C to H are restricted to animal hosts. METHODOLOGY: The present study estimated the prevalence of G. duodenalis assemblages in dogs living in the Sardinia region and evaluated the related risk factors. Individual fecal samples were collected from 655 dogs between January 2007 and December 2010, and a form was filled out for each animal to analyze historic data that were available at the time of sampling. Fecal samples were subjected to microscopic and genetic investigations. RESULTS: Cysts of G. duodenalis were found in 172 (26.3%) samples, with significant values in puppies between three and nine months of age, and in kennelled and hunting dogs. The molecular characterization showed the presence of assemblages D (49%), C (36.1%), and subtype A2 (4.2%). CONCLUSION: The present survey contributes to the knowledge of the occurrence of canine giardiosis in Italy in a region with a high number of dogs and numerous animal movements, which is especially relevant for touristic reasons.

Toltrazuril and sulphonamide treatment against naturally Isospora suis infected suckling piglets: is there an actual profit?

A study was carried out to assess the efficacy and the economic profit of prophylactic treatment against Isopsora suis with toltrazuril or with a sulfamethazine/trimethoprim combination in piglets from an intensive pig farm. Thirty-one litters were included in study. Eight litters were treated once with toltrazuril (20 mg/kg b.w.) at 3 days of age (Toltra group); 8 litters were treated with 2 ml/animal of a [corrected] sulphonamide combination (sodium sulfamethazine 250 [DOSAGE ERROR CORRECTED] mg and trimethoprim 50 [DOSAGE ERROR CORRECTED] mg/kg b.w.) for 3 consecutive days starting at 3 days of age (Sulfa group), and 15 litters were untreated (control group). Counts of oocyst per gram on pooled feces sampled from each litter were carried out on Days 7, 14, 21 and 28 and diarrhea was registered daily from pooled samples. Piglets were weighed on Days 1, 7 and 28 and mean weight gain (WG) and daily weight gain (DWG) were evaluated. The economic profit of treatment was evaluated comparing the WG of piglets of each treatment group from the day of birth to Day 28. On Days 14, 21 and 28, toltrazuril showed a better efficacy in controlling fecal oocyst output, diarrhea and weight gain compared with sulphamidic treatment (P<0.001). The budgeting analysis showed a return of economic benefit of euro 0.915 per toltrazuril-treated piglets and an additional cost of euro 1.155 per sulphonamide-treated piglets.