An update and ecological perspective on certain sentinel helminth endoparasites within the Mediterranean Sea.

The Mediterranean Sea is recognized as a marine biodiversity hotspot. This enclosed basin is facing several anthropogenic-driven threats, such as seawater warming, pollution, overfishing, bycatch, intense maritime transport and invasion by alien species. The present review focuses on the diversity and ecology of specific marine trophically transmitted helminth endoparasites (TTHs) of the Mediterranean ecosystems, aiming to elucidate their potential effectiveness as 'sentinels' of anthropogenic disturbances in the marine environment. The chosen TTHs comprise cestodes and nematodes sharing complex life cycles, involving organisms from coastal and marine mid/upper-trophic levels as definitive hosts. Anthropogenic disturbances directly impacting the free-living stages of the parasites and their host population demographies can significantly alter the distribution, infection levels and intraspecific genetic variability of these TTHs. Estimating these parameters in TTHs can provide valuable information to assess the stability of marine trophic food webs. Changes in the distribution of particular TTHs species can also serve as indicators of sea temperature variations in the Mediterranean Sea, as well as the bioaccumulation of pollutants. The contribution of the chosen TTHs to monitor anthropogenic-driven changes in the Mediterranean Sea, using their measurable attributes at both spatial and temporal scales, is proposed.

Pearl formation associated with gymnophallid metacercariae in Mytilus galloprovincialis from the Northwestern Adriatic coast: Preliminary observations.

Gymnophallids of the genus Parvatrema are small trematodes infecting waterbirds in their adult stage. Several species of clams and mussels have been found to act as first and second intermediate hosts, in which the trematode larval stages induce the formation of pearls. In this study, a wild population of Mytilus galloprovincialis was sampled along the Northwestern coast of the Adriatic Sea to evaluate the origin and extent of visible pearls. Parasitological investigations, including morphological and molecular analyses, and histopathology were carried out on a representative sample of mussels (n = 158) from June to September 2021. The overall prevalence of infection reached 75.3 %, and the intensity of infection ranged from a few trematodes to thousands per mussel, mostly occurring in the mantle and surrounded by variable numbers of conchiolin layers. Morphological studies allowed classification of the metacercariae as belonging to the genus Parvatrema, and the pairwise comparison of the obtained sequences, encompassing the internal transcribed spacer (ITS) region, revealed a genetic similarity (96.8 %) to Parvatrema duboisi. However, the phylogenetic analysis demonstrated the independent clustering of the obtained ITS sequences compared to other available Parvatrema species. For the relevant commercial impact that pearl formation may have on farmed mussels, ecological and epidemiological aspects of this infection would deserve further investigation in the area.

Molecular and morphological characterization of Bolbosoma balaenae (Acanthocephala: Polymorphidae), a neglected intestinal parasite of the fin whale Balaenoptera physalus.

Post-mortem examination of a fin whale Balaenoptera physalus stranded in the Mediterranean Sea led to the finding of Bolbosoma balaenae for the first time in this basin. In this work, we describe new structural characteristics of this parasite using light microscopy and scanning electron microscopy approaches. Moreover, the molecular and phylogenetic data as inferred from both ribosomal RNA 18S-28S and the mitochondrial DNA cytochrome oxidase c subunit 1 (cox1) for adult specimens of B. balaenae are also reported for the first time. Details of the surface topography such as proboscis's hooks, trunked trunk spines of the prebulbar foretrunk, ultrastructure of proboscis's hooks and micropores of the tegument are shown. The 18S + 28S rRNA Bayesian tree (BI) as inferred from the phylogenetic analysis showed poorly resolved relationships among the species of Bolbosoma. In contrast, the combined 18S + 28S + mtDNA cox1 BI tree topology showed that the present sequences clustered with the species of Bolbosoma in a well-supported clade. The comparison of cox1 and 18S sequences revealed that the present specimens are conspecific with the cystacanths of B. balaenae previously collected in the euphausiid Nyctiphanes couchii from the North Eastern Atlantic Ocean. This study provided taxonomic, molecular and phylogenetic data that allow for a better characterization of this poor known parasite.

A pilot study on molecular diagnosis of Hapalotrema mistroides (Digenea: Spirorchiidae) infection in blood samples of live loggerhead turtles Caretta caretta.

BACKGROUND: Parasites of the family Spirorchiidae cause disease and mortality in marine and freshwater turtles; two species, Hapalotrema mistroides and Neospirorchis sp., are reported in the resident population of loggerhead turtles of the Mediterranean Sea, with the first being the most widespread. In vivo diagnosis of spirorchidiasis can represent a challenge in guaranteeing prompt control and treatment of the disease and is currently limited to copromicroscopy. The aim of this study was the development of a real time PCR assay with TaqMan probe for the detection of H. mistroides infection in the blood of live loggerhead turtles, Caretta caretta, hospitalized in rehabilitation centres. Its potential use for in vivo diagnosis is explored. RESULTS: The developed real time PCR successfully detected H. mistroides DNA from both positive controls and experimental blood samples of live loggerhead sea turtles, showing good specificity, sensitivity and good reaction efficiency. Two out of three turtles which had demonstrated positivity at copromicroscopy also tested positive to this blood assay; DNA of H. mistroides was detected within the blood of one sea turtle, which tested negative for copromicroscopy. CONCLUSIONS: This study describes a specific and rapid molecular assay to detect H. mistroides infection from live sea turtles and highlights for the first time the presence of DNA of this species in turtle blood samples. Since this assay is able to detect low amounts of the parasitic free DNA in blood samples, its application could be helpful for in vivo diagnosis of H. mistroides infection as well as for epidemiological purposes.

Epidemiology of Sulcascaris sulcata (Nematoda: Anisakidae) ulcerous gastritis in the Mediterranean loggerhead sea turtle (Caretta caretta).

Sulcascaris sulcata Rudolphi 1819 is a gastric nematode parasite of sea turtles. Here, we report the occurrence and describe for the first time the pathological changes caused by S. sulcata in the Mediterranean loggerhead sea turtle (Caretta caretta) stranded along the Tyrrhenian coast and northern Adriatic coast of Italy. Prevalence of infection was significantly higher in loggerhead sea turtles from the Adriatic Sea. Both prevalence and abundance of infection showed an increasing trend along with host age classes from both geographical localities. Nevertheless, while many small loggerhead sea turtles were found infected from the Adriatic Sea, only bigger individuals were infected from the Tyrrhenian Sea. The most common gross pathological change was a mucous gastritis with focal to multifocal raised ulcerous lesions roundish to irregular in shape ranging from 1 to over 20 cm in length, and cream-yellowish to greenish in color. The severity grade of gastritis increased with higher number of S. sulcata individuals. Microscopic pathological changes ranged from atrophic gastritis with heterophilic infiltration in the lamina propria to the destruction of the mucosal and sub-mucosal surfaces and necrosis. Results here obtained demonstrate that S. sulcata may cause ulcerous gastritis in both samples of loggerhead sea turtles studied from the Mediterranean Sea. Observed differences in S. sulcata infection among the different host age classes and between the two studied basins are likely linked to the differences of regional habitat and intermediate prey host availability.

Correction to: Epidemiology of Sulcascaris sulcata (Nematoda: Anisakidae) ulcerous gastritis in the Mediterranean loggerhead sea turtle (Caretta caretta).

The original version of this article contained a mistake in the value of Bar in figure 3 caption. It should be Bar = 200 µm instead of Bar = 500 µm.

Gastrointestinal Parasites in Non-Human Primates in Zoological Gardens in Northern Italy.

Non-human primates (NHPs) host a variety of helminth and protist parasites that are able to cause infection in humans. Gastrointestinal parasites in NHPs living in two zoological gardens of Northern Italy were studied. An total of 96 faecal pools were collected from 26 groups of NHPs. The mini-Flotac method was applied to fecal samples to detect gastrointestinal helminthiases, while the detection of the protists Cryptosporidium spp., Blastocystis sp. and Giardia duodenalis was performed by targeting SSU rRNA through nested PCR and real-time PCR; they were further studied by sequencing the same gene for Blastocystis and ßgiardine and triosephosphate isomerase (TPI) genes for Giardia. Twenty-two out of the 96 examined fecal pools (22.9%) were positive for one or more helminth species, including Hymenolepis diminuta, Trichurid, Capillariid and Strongylid eggs. All samples were negative for Cryptosporidium spp., while 16/26 (61.5%) animals were positive for G. duodenalis in the real-time PCR; the sequences obtained assigned them all to sub-assemblage BIV. Blastocystis sp. was detected in 22/26 of the NHPs (84.6%); molecular analyses attributed the isolates to ST 4, allele 92. Analyses of the feces of sympatric rats revealed the presence of the same allele, as well as of Hymenolepis diminuta eggs, raising concern about their role as parasite reservoirs in the facilities.

Differential diagnosis of Eimeria species in farmed Japanese quails (Coturnix japonica).

Similarly to poultry industry, coccidiosis may cause significant economic losses also in the commercial quail industry, an emerging sector undergoing uneven development around the world. Although scant and mostly dated, the available literature reports detailed morphological and morphometric features of both oocysts and sporocysts of the Eimeria species hitherto recognized in Japanese quails, i.e. E. tsunodai, E. uzura, E. bateri, and E. fluminensis. Mixed infections are very common in the field and require an accurate differential diagnosis of diverse species of coccidia, identifying the highly pathogenic ones, in particular E. tsunodai (localized in the caeca), and E. uzura (localized in both caeca and small intestine). This goal is hampered by time-consuming laboratory procedures involving highly qualified staff and facilities, and poorly compatible with routine management practices in farmed quails. A supplemental difficulty is represented by the lack of nucleotide sequences available in GenBank. To overcome these issues, copromicroscopic and molecular analyses (amplifying the 18S rRNA region, and the internal transcribed spacers regions ITS1-5.8rRNA-ITS2) were performed on oocysts populations separately isolated from pools of 12 caecal and 12 cloacal contents collected from 240 naturally infected laying Japanese quails. Data on morphological and morphometric features of 1,000 sporulated oocysts were statistically compared, demonstrating the presence of different Eimeria species colonizing the 2 intestinal tracts. This result was also confirmed by PCR and phylogenetic analysis of the 18S rRNA gene. Overall results allowed to hypothesize the presence of E. uzura in our Japanese quails. Although a certain identification at species level was not obtained, the present study demonstrates that reasonable turnaround times of monitoring procedures performed on Japanese quail farms, shedding light on the in vivo and post-mortem differential diagnosis of coccidiosis can be achieved, and provide obvious benefits in disease understanding and control.

Faecal egg count reduction test in goats: Zooming in on the genus level.

The faecal egg count reduction test (FECRT) is the most widely used method to assess treatment efficacy against gastrointestinal nematodes (GIN). Information on genera composition of the GIN community is not available with this test and it is commonly obtained by identifying cultured third-stage larvae (L3) or through molecular assays in the post-treatment survey, but results provided are usually only qualitative or semi-quantitative. The updated WAAVP guidelines now recommend assessing anthelmintic efficacy for each GIN genus/species separately (genus-specific FECRT), but this approach is poorly employed in Europe and in goats especially. For this reason, four FECRT trials were conducted using oxfendazole and eprinomectin in two Italian goat farms. Samples were processed individually using the McMaster technique and then pooled to create two samples from faeces of 5 animals each. Pooled samples were analysed using the McMaster and cultured for seven days at 26°C to obtain L3s. The genus-specific FECRT was based on larval identification, integrating coproculture and FEC results. Larvae were identified as Haemonchus, Trichostrongylus, Teladorsagia, Oesophagostomum / Chabertia and Bunostomum. Molecular assays (a multiplex real-time PCR and two end-point PCRs) were also implemented on pooled samples to support the morphological identification. The Spearmann Rho test confirmed a high correlation between the two approaches (Rho = 0.941 and Rho = 0.914 respectively for Haemonchus and Trichostrongylus, the two most common genera). Both oxfendazole and eprinomectin were effective in one farm, while none in the other farm (FECR = 75.9% and 73.3% respectively). In the second farm, the genus-specific FECRT highlighted a different response to treatment among genera: oxfendazole lacked efficacy against both Haemonchus and Trichostrongylus spp., eprinomectin only against Haemonchus, while all other genera were susceptible to both drugs. This study brings new attention on the importance of adopting a genus-specific approach to identify and quantify differences in susceptibility to anthelmintics among genera in goats, providing support for FECRT interpretation, anthelmintic resistance evaluation and evidence-based GIN control.

Comparison of diagnostic methods for laboratory diagnosis of the zoonotic tapeworm Dipylidium caninum in cats.

The tapeworm Dipylidium caninum is the most widely distributed cestode infecting dogs, cats, and sometimes humans, worldwide. The diagnosis of the infection caused by D. caninum is achieved via the visualization of proglottids in feces or with traditional microscopic tests, but both lack sensitivity. The present study has evaluated and compared the diagnostic performance of a PCR protocol on different feline biological samples to detect D. caninum. A sample of feces, a Scotch tape test from the perianal area, and a rectal swab were collected from a total of 100 privately owned cats from Italy and Greece. All fecal samples were subjected to macroscopic examination and to floatation. Based on the results of the above tests the cats were divided in three groups, i.e. (i) cats positive for D. caninum (regardless of positivity for other endoparasites (Group A; n = 50 cats), (ii) cats negative for D. caninum but infected by other helminths (Group B; n = 25 cats), and (iii) cats negative for intestinal endoparasites (Group C; n = 25 cats). For each sample, the DNA was extracted from feces, floatation supernatant, Scotch tape test and rectal swabs and subjected to PCR. For 33 cats from Group A, at least one sample type scored positive at PCR. Of these, all were PCR-positive in the floatation aliquot, while nine and one cats were positive by PCR on feces and Scotch tape test, respectively. Swabs were negative by PCR for all the cats. None of the samples from cats of Groups B and C was positive by any PCR. Sequences obtained from amplicons generated from samples of cats enrolled in Italy had 99-100 % identity with the recently described D. caninum feline genotype. The data presented here suggest that PCR could be a useful tool for diagnosing D. caninum infections, under certain circumstances, e.g. when proglottids are unidentified, unseen or overlooked, even though it has limitations, e.g. false negative results due to fecal PCR inhibitors, uneven distribution of parasitic elements, or to intermittent proglottid and/or egg shedding. Thus, it may not be, currently, the best diagnostic choice for dipylidiosis.

Neuropathic pain following sagittal split ramus osteotomy of the mandible: prevalence, risk factors, and clinical course.

PURPOSE: To estimate the prevalence of, risk factors for, and clinical course of neuropathic pain (NPP) after sagittal split ramus osteotomy (SSRO) of the mandible in a large cohort of patients. MATERIALS AND METHODS: A retrospective cohort of all patients who underwent SSRO at 2 medical centers within Kaiser Permanente Northern California from January 2007 through September 2012 was assembled. Demographic, clinical, and surgical factors were abstracted from medical records and relevant comorbidities were identified. The prevalence of NPP in the cohort was calculated and the clinical signs, symptoms, temporal characteristics, and treatment response in affected patients were noted. RESULTS: The authors identified 1,778 patients who underwent SSRO and excluded 107 patients according to predefined criteria. The remaining 1,671 patients had a median age of 24 years (interquartile range, 19 to 35 yr) and 62.4% were women. Seven patients developed NPP after SSRO, which was an overall prevalence of 0.42%. All patients with NPP in this cohort were women and had a median age of 49 years. The risk factors for developing NPP after this surgery were older age (P = .0098), depression (P = .0100), and female gender. NPP developed an average of 30 days postoperatively (range, 18 to 56 days) and persisted for a median duration of 52 days (range, 30 to 69.5 days). All patients responded favorably to anticonvulsant (n = 6) or tricyclic (n = 1) medications, and no patients developed chronic postsurgical pain. CONCLUSIONS: NPP was an infrequent complication after SSRO, occurring in 1 of 238 patients in this cohort. The short duration and positive response to medication are reassuring findings. The results of this investigation highlight the need for prospective studies to further understand the spectrum of postoperative NPP.

Comparing pooled and individual samples for estimation of gastrointestinal strongyles burden and treatment efficacy in small ruminants.

Monitoring endoparasite burden (FEC) and treatment efficacy (FECR) is a key element of sustainable parasite control. However, the costs of the analysis often discourage their implementation by farmers and veterinary practitioners. Pooling samples is considered to be a good alternative to reduce time and monetary costs, but limited data are available on the use of pooled samples in small ruminants, especially for goats. In this study, data collected over the years in sheep and goat farms were analyzed, and results obtained from individual and pooled analysis were compared for the purposes of FEC and FECR assessment. A total of 801 individual and 134 pooled samples (composed of 3-12 individual samples) were included. For FECR testing, 2 pools of 5 samples each were created per trial and the same animals were sampled at day 0 (D0 - treatment day) and 14 days after (D14). Samples were analyzed by McMaster technique (limit of detection 20 EPG). Results from pooled and individual FEC were not significantly different (Wilcoxon signed-rank test) and correlation (Spearman's rank test) was high for all sub-categories, although agreement (Lin's concordance correlation) was often classified as poor. Results were not influenced by the pool size (<6 or ≥6). Interpretation of treatment efficacy between the two methods was comparable for all sheep trials, while it differed for goats in 4 out of 10 trials. Wilcoxon signed-rank test indicated a non significant difference between pooled and individual FECR. However, correlation and agreement between FECR were considerably better for sheep compared to goats, for which they were very limited, despite the correlation between FEC at D0 and D14 was always high. According to our results, pooled FECR can be a good option but the absence of 95 %CI represents a major drawbacks in the interpretation of results. Further studies on the topic for goats are needed.

Corrigendum: Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance.

[This corrects the article DOI: 10.3389/fvets.2022.1085996.].

Balaenophilus manatorum in Debilitated and Bycatch-Derived Loggerhead Sea Turtles Caretta caretta from Northwestern Adriatic Sea.

Balenophilus manatorum (Copepoda: Harpaticoida) is one of the few components of the epibiontic fauna of Caretta caretta that show a "true" parasitic association with their host. From rrosive to ulcerative cutaneous lesions may seldom appear as a consequence of the copepod feeding on keratin on turtles' skin. Debilitating Turtle Syndrome (DTS) is the final outcome of a chronic insufficient assumption of nutrients, generally occurring with the impairment of immune functions and high epibiota burdens. In this survey, the presence of B. manatorum in C. caretta from the Northwestern Adriatic Sea was investigated and the relation between infection indices and the co-occurrence of DTS was studied. Clinical examination was performed at the time of rescue, including routine hematological assessment; external parasites were isolated mechanically from turtles' skin and morphologically identified through observation with an optic microscope and SEM. Ten turtles were classified as affected by DTS, all of them being small juveniles with typical clinical and clinicopathological presentation. A higher prevalence, abundance, and density of infection were found in turtles affected by the syndrome. The presence of massive skin coverage by the burrowing barnacle Pletylepas hexastylos prevented a proper evaluation of the pathology associated with B. manatorum in turtles affected by DTS. In any event, eventual skin damages caused by the parasite may represent a port of entry for secondary infections in such immunocompromised animals. Therefore, infection by B. manatorum should not go overlooked in debilitated turtles and should be opportunely treated.

Molecular survey of Cytauxzoon spp. and Hepatozoon spp. in felids using a novel real-time PCR approach.

Tick-transmitted apicomplexans of the genera Cytauxzoon and Hepatozoon affect a wide range of felids worldwide, but little is known about them. Recently, several studies addressed the species circulating in Europe, their distribution, and their hosts. Molecular assays are the method of choice for their detection. Unfortunately, conventional PCRs already described are time- and cost-consuming and specific for either Hepatozoon or Cytauxzoon detection. This study was developed to evaluate (i) the occurrence of Cytauxzoon and Hepatozoon in felids using a fast and cost-saving real-time PCR capable of detecting both protozoa simultaneously, (ii) the distribution of Cytauxzoon and Hepatozoon species in north-eastern Italy, and (iii) the involvement of other susceptible felid hosts in the same area. An SYBR® Green-based real-time PCR with primers targeting the 18S-rRNA was validated and applied to 237 felid samples, i.e., whole blood from 206 domestic cats and 12 captive exotic felids, and tissues from 19 wildcats. Positive results were obtained by melting temperature curve analysis due to the specific melting peak (i.e., 81°C Cytauxzoon spp.; 78-78.5°C Hepatozoon spp.). Positive samples were subjected to conventional PCR, followed by sequencing for species identification. Phylogenetic analyses were performed to assess relatedness among European isolates. Data on domestic cats (age class, sex, origin, management, and lifestyle) were recorded, and statistical analyses were performed to identify potential risk factors. A total of 31 (15%) domestic cats were positive for Hepatozoon spp. (i.e., 12 for H. felis, 19 for H. silvestris), while six (2.9%) for C. europaeus. The prevalence of Hepatozoon felis was significantly (p < 0.05) higher in domestic cats, while H. silvestris was higher in strays and animals from the Eastern region (i.e., Friuli-Venezia Giulia). Cytauxzoon europaeus was detected only in stray cats from Friuli-Venezia Giulia (province of Trieste). Among captive felids, one tiger was infected with H. felis and another with H. silvestris; eight out of 19 (42%) wildcats were positive for Hepatozoon spp. (i.e., six with H. felis, two with H. silvestris) and four out of 19 (21%) for Cytauxzoon europaeus. Outdoor lifestyle and origin (i.e., Friuli-Venezia Giulia region) were the most relevant risk factors for H. silvestris and C. europeus infections. Conversely, H. felis was most frequently isolated from domestic cats, suggesting different modes of transmission.

A Comparative Study of Mini-FLOTAC With Traditional Coprological Techniques in the Analysis of Cetacean Fecal Samples.

Free-ranging cetaceans are considered sentinels for the marine ecosystem's health. New and non-invasive methods have been set up for the collection of fecal samples from free-ranging big whales at sea, permitting to gain an excellent epidemiological picture of parasitic infections in wild populations. To select the best protocol to be used for copromicroscopic examination in cetaceans stool samples, we evaluated the sensitivity of two commonly used techniques, i.e., a sedimentation-floatation method and the Mini-FLOTAC through validation by helminth isolation from the digestive tract. For this aim, gastrointestinal content and fecal samples were collected during necropsy from 44 cetaceans, including bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalba), sperm whales (Physeter macrocephalus), Risso's dolphins (Grampus griseus), Cuvier's beaked whales (Ziphius cavirostris), pilot whale (Globicephala melas), and fin whales (Balaenoptera physalus). Helminths were recovered through washing and filtering of the gastrointestinal contents and morphologically identified. Copromicroscopic examinations were performed on formalin-preserved fecal samples, using a sodium nitrate, sodium thiosulphate, and sucrose solution (s.g. = 1.450) for both methods. Helminths belonging to 9 taxa (i.e., the trematodes Synthesium tursionis, Synthesium delamurei, Campula palliata, Braunina cordiformis, Pholeter gastrophilus, the nematode Anisakis sp., cestodes of the family Tetrabothriidae and the acanthocephalan Bolbosoma sp.) were isolated. Eggs referable to the same taxa, with the exception of cestodes, were found in copromicroscopic analyses. Sensitivity of the Mini-FLOTAC method appeared higher or equal for all taxa, proving superior to the sedimentation-flotation method for the detection of all except Anisakis sp. The concordance of the two tests indeed revealed a moderate to perfect agreement (kappa values 0.42-1). Not excluding the limitations inherent to the techniques themselves, explanations for false-negative results at copromicroscopy could be linked to parasite-related factors, including prepatent infections, low parasitic burdens, or intermittent egg shedding. Notwithstanding these limitations, this study evidenced that the Mini-FLOTAC protocol approximates more accurately the composition of the gastrointestinal helminthic community of cetaceans from copromicroscopic examination, providing at the same time a quantitative estimation.

Management of giant osteoma in the mandible associated with minor trauma: a case report.

BACKGROUND: Osteoma is a benign tumor of the bones, which can be classified as central or peripheral. The occurrence in the jawbones is uncommon, but when it occurs, there is a greater prevalence of the mandible. The etiology is still unknown, and the hypothesis of its development is debated. CASE PRESENTATION: A 35-year-old Caucasian man presenting a tumor lesion in the right jawbone that had been growing for 8 years sought medical service complaining of speaking impairment. According to the patient, the tumor appeared shortly after a minor trauma caused by tooth extraction. The diagnosis of the lesion was made through clinical, radiographic, and histological methods, and the surgical treatment was successful and satisfactory for the patient as well as the surgical team, despite a short follow-up. CONCLUSION: Etiopathogenesis of osteoma is not determined in the majority of cases. In the present report, it was possible to hypothesize the association between a minor trauma and the development of the tumor, reinforcing the reactive theory of tumor development. The uncommon location of the osteoma, as well the possibility of identifying the possible cause of the lesion, makes this case particularly interesting.

Evidence of Dirofilaria immitis in Felids in North-Eastern Italy.

Dirofilaria immitis is a mosquito-borne nematode, causing heartworm (HW) disease in wild and domestic canids. HW can also affect felids with different clinical patterns from asymptomatic pictures to sudden death, making the monitoring and diagnosis complicated. Canine HW is endemic in North-eastern Italy; however, very little information has been recorded for felids. This study aims to provide new information on HW in felids in North-eastern Italy. Two hundred and six domestic cats from Veneto, Friuli-Venezia Giulia, Trentino Alto-Adige regions (North-eastern Italy), nine captive felids from zoological parks from Veneto, and nineteen European wildcats from Friuli Venezia Giulia were recruited. Sera/plasma was analysed for the detection of anti-HW antibodies (Ab) and HW antigens (Ag); positive blood samples were molecularly analysed, targeting the HW DNA (5S-rRNA gene). Twelve out of two hundred and six (5.8%) cats presented with Ab, and three out of two hundred and six (1.5%) presented with Ag, mainly those from the Veneto region, already known as a canine HW-endemic area. Among Ab-positive cats, two were from Belluno, a mountain province previously considered free, suggesting the expansion of HW into the northern areas. No cats were positive for both Ab and Ag. Three out of nineteen (15.8%) wildcats were Ag-positive, constituting the first HW report in Italy. No captive felids were positive. Dirofilaria immitis DNA was not amplified in positive samples, suggesting the low sensitivity of PCR on blood. This study provides new data on the occurrence of HW in domestic cats and wildcats in North-eastern Italy.

Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance.

The red fox acts as reservoir for several helminthic infections which are of interest for both public and animal health. Huge efforts have been made for the assessment of the sensitivity of coprological tests for the detection of Echinococcus multilocularis, while less attention has been paid to other helminthic species. This study aimed at assessing the performance of two copromicroscopic techniques in the detection and prevalence estimation of gastrointestinal helminths in the red fox. Helminths were isolated from the small intestines of 150 red foxes from Bolzano province, Italy, with a scraping, filtration and counting technique (SFCT) and morphologically identified. Rectal contents were collected and submitted to simple flotation (FT) and, only for Taenids, a method based on the concentration of eggs and identification with multiplex PCR (CMPCR). Using SFCT as a reference standard, we estimated the sensitivity of the copromicroscopic tests. Three species of nematodes (namely, Toxocara canis, Uncinaria stenocephala and Pterygodermatites sp.) and five species of cestodes (E. multilocularis, Taenia crassiceps, T. polycantha, Hydatigera taeniaeformis, Mesocestoides sp.) were identified with SFCT, whereas eggs referable to the same taxa were detected with fecal diagnostics, except for Pterygodermatites sp. and Mesocestoides sp. The sensitivity of FT was low for all taxa, ranging from 9.8 to 36.3%, with lower values for Taeniidae. CMPCR was confirmed to perform better for the detection of Taeniidae eggs (23.5%) and the multiplex PCR on retrieved eggs was effective in the identification of the species. A meta-analysis of literature also suggested that our results are consistent with existing data, indicating that copromicroscopy tends to underestimate the prevalence of helminthic infections. The extent of such underestimation varies with taxon, being higher at high prevalence levels, in particular for cestodes. Irregular dynamics of egg shedding, and routine deep freezing of red fox feces may explain the frequency of false negatives with copromicroscopy. Low sensitivity of copromicroscopic tests should be accounted for when estimating prevalence and when defining the correct sample size for the detection of the parasites.

Cytauxzoon sp. and Hepatozoon spp. in Domestic Cats: A Preliminary Study in North-Eastern Italy.

Knowledge on the presence of Cytauxzoon sp. and Hepatozoon spp. in Italy is scant and mostly limited to a few areas of Northern and Southern regions, respectively. The present study updated the current epidemiological scenario by investigating the occurrence of these protozoa in domestic cats from three broad regions of North-Eastern Italy. Blood samples from cats at risk of vector-borne diseases were processed by PCR to detect Cytauxzoon and Hepatozoon DNA. Blood smears were observed for haemoparasite inclusions. The influence of cat individual data (e.g., provenance, management, indoor/outdoor lifestyle) on the prevalence of haemoprotozoan infections was statistically evaluated. Among 158 cats, Cytauxzoon and Hepatozoon DNA were detected in 6 (3.8%) and 26 (16.5%) animals, respectively. No Hepatozoon gamonts were detected in blood smears, whereas all Cytauxzoon PCR-positive samples were microscopically positive, though with low levels of parasitaemia. Two species of Hepatozoon were identified, Hepatozoon felis (n = 10) and Hepatozoon silvestris (n = 16). Hepatozoon silvestris prevalence values were significantly (p < 0.05) higher in the region Friuli Venezia Giulia and in stray cats. Cytauxzoon sp. was detected in 6/39 (15.4%) stray cats from Friuli Venezia Giulia (Trieste province). These data add new information on the occurrence of these neglected protozoa in domestic cats' populations.