Biotic and abiotic factors shape the microbiota of wild-caught populations of the arbovirus vector Culicoides imicola.

Biting midges of the genus Culicoides are known vectors of arboviruses affecting human and animal health. However, little is known about Culicoides imicola microbiota and its influence on this insect's biology. In this study, the impact of biotic and abiotic factors on C. imicola microbiota was characterized using shotgun-metagenomic sequencing of whole-body DNA samples. Wild-caught C. imicola adult nulliparous females were sampled in two locations from Sicily, Italy. The climatic variables of temperature and soil moisture from both localities were recorded together with potential host bloodmeal sources. Shared core microbiome among C. imicola populations included Pseudomonas, Escherichia, Halomonas, Candidatus Zinderia, Propionibacterium, and Schizosaccharomyces. Specific and unique taxa were also found in C. imicola from each location, highlighting similarities and differences in microbiome composition between the two populations. DNA and protein identification showed differences in host preferences between the two populations, with Homo sapiens and Canis lupus familiaris L. being the preferred bloodmeal source in both locations. A principal component analysis showed that the combined effect of host preferences (H. sapiens) and local soil moisture factors shape the microbiome composition of wild-caught populations of C. imicola. These results contribute to characterizing the role of the microbiome in insect adaptation and its utility in predicting geographic expansion of Culicoides species with potential implications for the control of vector-borne diseases.

Allele frequencies of microsatellite loci for genetic characterization of a Sicilian bovine population.

Short tandem repeats are used as an effective method to trace DNA markers in genotyping. Using a standardized kit, we tested 11 microsatellite markers recommended by the International Society for Animal Genetics (ISAG) in a sample of 495 Sicilian cattle. The aim of this study was to investigate the allele frequencies in the Sicilian cattle population to provide a reference database and at the same time to assess the use of the ISAG microsatellite panel for pedigree analysis. DNA samples were collected from blood and amplified in an 11-plex polymerase chain reaction (PCR); PCR products were injected in a 3130 Genetic Analyzer. All loci showed high mean polymorphism information content (0.768), and the observed mean heterozygosity was less than the expected value (0.732 vs 0.794, respectively). The exact test for Hardy-Weinberg proportions, allele number, and inbreeding coefficient were calculated. Our results indicated that equilibrium was not always maintained. The observed mean homozygote value exceeded the expected value (132.81 vs 102.14), but no evidence for allele dropout was found. These results could be explained by a non-random mating; further studies using a larger number of animals could confirm or invalidate this hypothesis. The probability of identity and exclusion of a locus were also estimated and proved to be useful in paternity testing. The ISAG microsatellite panel is useful to screen the Sicilian bovine kinship. Currently, an allele frequency database is being constructed.

Putative hybrids between two Anisakis cryptic species: molecular genotyping using High Resolution Melting.

The genus Anisakis includes nine recognized species and the complex of cryptic species Anisakis simplex s. l. is often associated with the human disease known as anisakiasis. During the last decades the use of nuclear ribosomal ITS allowed the identification and description of numerous anisakid nematodes and the discovery of recombinant genotypes or putative hybrids even in other parasitic helminths, such as those between A. simplex sensu stricto and A. pegreffii. The existence of pure hybrids of the two sibling species has been long debated due to the large recovery of larval forms from sympatric areas and the rare observation of adult hybrids. The aims of the present report were to identify anisakid nematodes collected from Stenella coeruleoalba using PCR-RFLP of ITS and to focus the interest on hybrid forms using a High Resolution Melting (HRM) and direct sequencing analyses, since the new record of putative hybrid at adult stage. The PCR-RFLP analysis enabled to identify A. simplex s.s., A. pegreffii, the heterozygous genotype of the two species and A. physeteris. The use of the genotyping approach based on HRM confirmed the profiles of the two species A. simplex s.s. and A. pegreffii, and of the hybrid individuals. The new record of adult hybrids in definitive hosts rekindles the long debate about their existence and their evolutionary meaning. Since the reproductive isolation between A. simplex s.s. and A. pegreffii is the assumption for their existence as separated species, the use of alternative molecular markers and population genetic studies on adult anisakids are recommended.

A rare case of deradelphus cephalo-thoracoomphalopagus in lamb.

Male cross-breed lamb conjoined twins were sent for examination. The twins came from a farm located in the outskirts of Palermo. The twins were spontaneously delivered and found dead by the farmer. X-ray examination and necropsy were performed. The deformity was identified as deradelphus cephalo-thoracoomphalopagus. This condition has never been reported in veterinary literature. Our case reports of male conjoined twins characterized by two separated hearts with their own pericardial sacs and two separated livers. These findings represent a rare condition in conjoined twinning.

Detection of natural killer T cells in mice infected with Rickettsia conorii.

Little information is available regarding the role of natural killer T (NKT) cells during the early stage of Rickettsia conorii infection. Herein, C3H/HeN mice were infected with the Malish 7 strain of R. conorii. Splenocytes from these mice were analysed in the early stage of the infection by flow cytometry and compared with uninfected controls. Our results showed an increase in NKT cells in infected mice. Additionally, NKT interleukin (IL)-17(+) cells increased three days after infection, together with a concurrent decrease in the relative amount of NKT interferon (IFN)-γ(+) cells. We also confirmed a higher amount of NK IFN-γ(+) cells in infected mice. Taken together, our data showed that NKT cells producing Il-17 increased during the early stage of rickettsial infection. These results suggest a connection between IL-17(+) NKT cells and vasculitis, which is the main clinical symptom of rickettsiosis.

A Molecular survey of Anaplasma spp., Rickettsia spp., Ehrlichia canis and Babesia microti in foxes and fleas from Sicily.

Fleas (Insecta: Siphonaptera) are obligate bloodsucking insects, which parasitize birds and mammals, and are distributed throughout the world. Several species have been implicated in pathogen transmission. This study aimed to monitor red foxes and the fleas isolated from them in the Palermo and Ragusa provinces of Sicily, Italy, as these organisms are potential reservoirs and vectors of pathogens. Thirteen foxes (Vulpes vulpes) and 110 fleas were analysed by polymerase chain reaction (PCR) to detect DNA of the pathogens Ehrlichia canis, Babesia microti, Rickettsia spp., Anaplasma phagocytophilum, Anaplasma platys, Anaplasma marginale and Anaplasma ovis. In the foxes, A. ovis was detected in only one animal, whereas the prevalence of the E. canis pathogen was 31%. B. microti and Rickettsia spp. were not detected. Of all of the collected fleas, 75 belonged to the species Xenopsylla cheopis, 32 belonged to Ctenocephalides canis, two belonged to Ctenocephalides felis and one belonged to Cediopsylla inaequalis. In the fleas, the following pathogens were found: A. ovis (prevalence 25%), A. marginale (1%), A. phagocytophilum (1%), Rickettsia felis (2%) and E. canis (3%). X. cheopis was the flea species most frequently infected with Anaplasma, in particular A. ovis (33%), A. marginale (1%) and A. phagocytophilum (1%). Both C. felis exemplars were positive for R. felis. E. canis was found in the lone C. inaequalis and also in 3% of the X. cheopis specimens. No fleas were positive for B. microti or A. platys. As foxes often live in proximity to domestic areas, they may constitute potential reservoirs for human and animal parasites. Further studies should be performed on fleas to determine their vectorial capacity.

Prevalence of tick-borne pathogens in ticks in Sicily.

The prevalence of Anaplasma, Ehrlichia, Rickettsia and Babesia/Theileria species was analysed in questing and feeding adult ticks in Sicily. A total of 678 ticks were collected and analysed in this study. Of these, 29 were questing ticks and 649 were collected from infested cattle, sheep, goats or dogs. Tick species analysed included Rhipicephalus bursa, R. turanicus, R. sanguineus, Hyalomma lusitanicum, H. marginatum, Dermacentor marginatus, Ixodes ricinus, R. (Boophilus) annulatus and Haemaphysalis punctata. With the exception of R. annulatus and H. punctata for which only eight and 15 ticks were analysed, respectively, all tick species were found to be infected. Most ticks were found to be infected with a single pathogen genus. Data obtained from questing ticks was analysed to test for differences between tick species in the prevalence of infection for different pathogens. These preliminary results suggested that the most important vectors of pathogens that may affect human and/or animal health in Sicily are R. turanicus for Anaplasma spp. and D. marginatus for Rickettsia spp. For Ehrlichia spp. and Babesia/Theileria spp., R. turanicus/D. marginatus and H. lusitanicum may be the most important vectors but additional studies are needed to confirm these results.

Characterization of the apical membrane antigen-1 in Italian strains of Babesia bigemina.

Babesia bigemina is a parasite endemic in different parts of the world, including Europe and the Americas. One of the few genes characterized in this species codifies for the Apical Membrane Antigen 1 (AMA-1), a trans-membrane antigen recently identified. In this research, we characterized the ama-1 gene from three Italian B. bigemina strains, two B. bigemina strains obtained from Ragusa, Sicily (ITA1 and ITA3) and a third one obtained from Benevento, Campania (ITA2). Italian sequences were compared with those of the Australian strain obtained from the Sanger Institute web site and to strains from different parts of the world. The results obtained confirmed that this newly described ama-1 gene is highly conserved among Italian and foreign strains which has implications for vaccine development.

Characterization of anaplasma infections in Sicily, Italy.

This study aimed to characterize infection with Anaplasma marginale, A. phagocytophilum, A. ovis, and A. platys in humans, animals, and ticks in Sicily, Italy, during 2003-2006. Serologic (competitive ELISA [cELISA]) and indirect immunofluorescence antibody [IFA]; N= 1990) and DNA (polymerase chain reaction [PCR]; N= 2788) tests were conducted on horse, donkey, cattle, sheep, goat, pig, dog, cat, roe deer, wild boar, human, and tick samples. The results reported herein suggested that in Sicily cattle are a major reservoir for A. marginale, dogs for A. platys, and sheep and goats for A. ovis. Domestic animals, such as cattle, horses, donkeys, sheep, dogs, and cats, may serve as reservoir for A. phagocytophilum, but different strains may infect ruminants and humans. All Anaplasma spp. characterized in Sicily had some distinctive genotypes for this region. Low genetic diversity was observed in A. ovis and A. platys, whereas A. marginale and A. phagocytophilum strains showed high genetic diversity. These results expanded our knowledge about the prevalence of Anaplasma spp. in Sicily and provided information to understand the epidemiology of these infections and implement measures to diagnose, treat, and control transmission to humans and animals in this region.

Anaplasmosis in cattle in Italy.

Bovine anaplasmosis caused by Anaplasma marginale is a disease transmitted by ticks belonging to the Ixodidae family. Southern Italy is considered an endemic zone but environmental and social factors are changing the epidemiology of the disease to expand to previously anaplasmosis-free regions. The available data of published reports of anaplasmosis in Italy together with the data obtained by the National Centre of Reference for Anaplasma, Babesia, Rickettsia and Theileria (C.R.A.Ba.R.T.), allowed to report A. marginale infection in different Italian regions (Lazio, Marche, Campania, Puglia, Basilicata, Calabria, Lombardy, Tuscany, Umbria and Sicily). Cattle are also subject to infection with the related Ixodes ricinus-transmitted pathogen, Anaplasma phagocytophilum that results in reduced milk production in cattle. A. phagocytophilum infect also small ruminants, domestic and wild animals and causes the human granulocytic anaplasmosis. Different studies have been conducted on the presence of A. phagocytophilum in Italy both in the tick vectors and in the wild and domestic reservoirs. Contrary to A. marginale, the prevalence of A. phagocytophilum embraces the whole Italian territory from the Alps to the southern and insular regions.

Babesiosis in Italy: an overview.

Babesiosis is a tick-transmitted disease caused by hematotropic parasites of the genus Babesia. Tick-borne diseases (TBD) have increasingly been recognized in the world as public health problems. The piroplasms are transmitted by ticks and are able to infect a wide variety of vertebrate hosts which are competent in maintaining the transmission cycle. Babesiosis occurrence is usually linked to seasonal variations that affect the vector, but climatic changes have not been common in the subtropical regions of Italy, especially during the last few years. This paper is aimed at compiling information about babesiosis in Italy both from the available literature and from the records of our Centre. Recent biotechnological approaches have aid to the detection of parasites and the monitoring of tick vectors. Moreover, our research has lately been focused on the investigation of the presence of Babesia parasites in wild animals, yielding very interesting results.

Incomplete caudal duplication in a puppy: gross and radiological observations.

Caudal duplication is a rare type of conjoined twinning. The aim of this case report is to describe a case of caudal duplication in a male crossbreed puppy. The puppy was macroscopically and radiographically examined after death. External features included a supernumerary limb projecting from the perineum, complete lack of tail, anal atresia, double penis and absence of scrotal sacs. Internal examination showed anomalies involving the intestine and the urinary system. At x-ray examination, the supernumerary limb showed features similar to that of the normal right hindlimb but showed absence of fibula and tibia and presence of three metatarsi and phalanges. The authors discuss the pathogenic mechanisms of this condition, infrequently reported in veterinary practice, pointing out the importance of embryonic duplications commonly associated with dystocia.

Observed prevalence of tick-borne pathogens in domestic animals in Sicily, Italy during 2003-2005.

The objective of this study was to characterize the observed prevalence of tick-borne pathogens (TBP) in domestic animals in Sicily, Italy during 2003-2005. Serological (competitive ELISA and indirect immunofluorescence antibody, n = 3299) and DNA tests (polymerase chain reaction and reverse line blot, n = 2565) were conducted on horse, donkey, cattle, sheep, goat, pig and dog samples. Pathogens analysed included Anaplasma, Ehrlichia, Rickettsia, Babesia and Theileria species, and Coxiella burnetii. The most prevalent TBP were Anaplasma and Babesia species. The results reported herein suggested that cattle could serve as the major reservoir for Babesia and Theileria spp. while for Anaplasma spp. cattle, dogs, sheep and goats may be the most important reservoir species. These results expanded our knowledge about the prevalence of TBP in Sicily and provided information to understand the epidemiology of tick-borne diseases and may help to implement measures to diagnose, treat and control transmission to humans and animals in this region.

Toxoplasma gondii infections in sheep in Sicily, southern Italy.

The aim of the study was to determine the burden of Toxoplasma gondii-infections in sheep in Sicily, southern Italy and the risk factors for infection. Sera from 1961 sheep were collected just before slaughtering from 62 farms located in 8 out of 9 Sicilian administrative districts. The sera were analysed for Toxoplasma-specific IgG antibodies using commercially available enzyme-linked immunosorbent assay. Sheep less than 4 weeks old were further analysed by ELISA for Toxoplasma-specific IgM-antibodies. Data on farm size and location were obtained from slaughterhouse sanitary reports and through structured telephone interviews of the veterinary officers from public health districts. The overall seroprevalence of Toxoplasma-specific IgG-antibodies were 49.9% (937/1876) by ELISA. Eighty-seven (54/62) percent of the farms had at least one Toxoplasma-positive animal. All the farms fed the animals outdoor on pasture and only one was claiming organic farming. Having cats on the farm, age of the animals, farm size and the use of surface water sources for drinking were all significantly associated with T. gondii-infected animals on the farm. T. gondii infection in mutton used for human consumption is very prevalent, and eating unprocessed sheep and lamb meat has a high risk of transmitting infections to humans. The presence of cats on the farm, farm size and using surface water as drinking water for the animals were risk factors for infection in sheep, with age as a significant confounder.

Dog tick-borne diseases in Sicily.

In Sicily many tick borne diseases are endemic, in particular way those that see like main carrier ticks that prefer, for their vital cycle, climatic conditions characterized by high temperatures and a warmth-humid atmosphere. The more important pathologies transmitted by ticks causing diseases in dogs are babesiosis and ehrlichiosis. Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia conorii, Coxiella burnetii and tick transmitted encephalitis virus assume particular relevance because they are agents of zoonosis. Our centre, C.R.A.Ba.R.T, have conducted many researches and carried out many tests for diagnostic aim in order to estimate the spread of the main tick borne diseases in Sicilians' dogs. A study lead on 342 dogs has evidenced seroprevalence for Babesia canis, Ehrlichia canis and Rickettsia respective of 5.17%, 21.70% and 53.43%. A study on zoonotic agent seroprevalences in dogs gave the following percentages: C. burnetii 31.50%, R. conorii 73.60% and A. phagocytophilum 32.80%. The data carried out from IZS Sicily diagnostic service on 5,634 tests done in 2004-2005, confirm the experimental results on the presence of B. canis, E. canis, R. conorii, A. phagocytophilum, C. burnetii, Rickettsia spp., Anaplasma spp. and Ehrlichia spp. in all the Sicilian areas.

Infection with Anaplasma phagocytophilum in a seronegative patient in Sicily, Italy: case report.

BACKGROUND: Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe. Although about 65 cases of HGA have been reported in Europe, some of them do not fulfill the criteria for confirmed HGA. Confirmation of HGA requires A. phagocytophilum isolation from blood, and/or identification of morulae in granulocytes and/or positive PCR results with subsequent sequencing of the amplicons to demonstrate specific rickettsial DNA. Seroconversion or at least fourfold increase in antibody titers to A. phagocytophilum has been used as criteria for confirmed HGA also. CASE PRESENTATION: Infection with A. phagocytophilum was confirmed by PCR in a patient in Sicily, Italy, who had negative serology for A. phagocytophilum. A fragment of A. phagocytophilum 16S rDNA was amplified by two independent laboratories and sequenced from two separate patient's blood samples. The 16S rDNA sequence was identical in both samples and identical to the sequence of the A. phagocytophilum strain USG3 originally obtained from a dog. CONCLUSION: Infection with A. phagocytophilum was confirmed in a patient without a detectable antibody response against the pathogen. The results reported herein documented the first case of confirmed HGA in Sicily, Italy. These results suggested the possibility of human infections with A. phagocytophilum strains that result in clinical symptoms and laboratory findings confirmatory of HGA but without detectable antibodies against the pathogen.

Genetic diversity of Anaplasma marginale strains from cattle farms in the province of Palermo, Sicily.

Bovine anaplasmosis, caused by the tick-borne rickettsia Anaplasma marginale, is endemic in Sicily and results in economic loss to the cattle industry. This study was designed to characterize strains of A. marginale at the molecular level from cattle in the Province of Palermo, Sicily. Seropositivity of cattle >or=1 year old for A. marginale in the study area ranged from 62% to 100%. The observed prevalence of A. marginale infections in cattle herds ranged from 25% to 100%. Two predominant A. marginale msp4 genotypes were found. A positive correlation was found between the prevalence of infection and the presence of Rhipicephalus (Boophilus) annulatus. Phylogenetic analysis of msp4 sequences of European strains of A. marginale did not provide phylogeographical information. These results suggest that development of farm husbandry systems and vaccines for genetically heterogeneous populations of A. marginale are needed for control of anaplasmosis in this region of Sicily.