Morphological and molecular study of Didymodiclinus marginati n. sp. (Trematoda: Didymozoidae) gill parasite of Epinephelus marginatus from the central and western Mediterranean Sea.

The current study provides a morphological and molecular characterization of a new species of Didymodiclinus (Trematoda: Didymozoidae) infecting the dusky grouper, Epinephelus marginatus (Teleostei: Serranidae) from the Mediterranean Sea. A total of 279 dusky grouper specimens were examined for didymozoid gill parasites from the Mediterranean Sea between 1998 and 2020. New species differs from the most similar congeneric species by the rudiments of female reproductive organs in functional male specimens, and the seminal receptacle, Mehlis gland and accessory gland cells in functional female specimens, not observed in Didymodiclinus branchialis (Yamaguti, 1970), Didymodiclinus epinepheli (Abdul-Salam, Sreelatha and Farah, 1990) and Didymodiclinus pacificus (Yamaguti, 1938), respectively. These species are also characterized by their different hosts and location within the host tissues, being from other geographical localities. Moreover, this is the first species reported in E. marginatus from the central and western Mediterranean Sea. Genetic analyses were performed on partial 28S and partial internal transcribed spacer-2 ribosomal RNA regions and the mitochondrial cytochrome oxidase 1 (cox1) gene by polymerase chain reaction. Comparison of genetic sequences of Didymodiclinus marginati n. sp. with the available deposited sequences of 28S revealed that the new isolates cluster with several unidentified didymozoids and groups as a sister clade of the Nematobothrinae subfamily. Moreover, 28S and cox1 phylogenetic trees evidenced that Didymodiclinae is well separated from Didymozoinae and other gonochoric didymozoids. Following both morphological and genetic results, a key of identification for the genus Didymodiclinus is proposed.

Survey on parasitic infections in wildcat (Felis silvestris silvestris Schreber, 1777) by scat collection.

Wildcats are endangered felid species living in Europe, Asia, and Africa. Regrettably, scientific information on parasites of wildcats is particularly meager and they often rely on data gained by necropsies of a small number of animals. In the present study, scat collection was used to assess the parasite spectrum of European wildcats living in the Etna Park (Sicily, Italy). Scat collection was performed from May to September 2010 by weekly walking four transects for a total of 391 km. Samples were then analyzed by flotation and sedimentation techniques to investigate wildcat parasitic fauna. A total of 121 scats of wildcats were collected, and parasitic forms (i.e., oocysts, eggs, and larvae) were retrieved in 110 (90.9 %) of the samples. Parasites found were Physaloptera sp. (52.1 %), tapeworms (45.5 %), Toxocara cati (43.8 %), Eucoleus aerophilus (27.3 %), Ancylostoma sp. (22.3 %), Troglostrongylus brevior (15.7 %), trematodes (9.9 %), Isospora felis (4.1 %), Cylicospirura sp. (1.7 %), and Acanthocephala (0.8 %). The prevalence of endoparasitic infections herein recorded is similar to that described in other studies conducted using necropsy technique. The species richness of parasites found in the present survey, with a total of nine helminths and one protozoon, is the highest ever reported for wildcat in Europe. Scat collection and examination are reliable and rapid non-invasive tools which can be used in a systematic survey design to study the parasite spectrum of wildcat as well as that of other endangered wild species.

Development of Acanthocheilonema reconditum (Spirurida, Onchocercidae) in the cat flea Ctenocephalides felis (Siphonaptera, Pulicidae).

To investigate larval development of Acanthocheilonema reconditum in the cat flea Ctenocephalides felis, fleas were fed through an artificial feeding system with dog blood containing different concentrations of microfilariae (i.e. low, group L = 250; medium, group M = 500; high, group H = 1500 microfilariae per mL) or no microfilariae (group C). Fleas were sampled at 12 different time-points throughout the study period (D1-D28) and A. reconditum was detected by dissection, PCR and histology. Of 2105 fleas fed with infected dog blood, 891 (38·7%) died during the study before being sampled whilst the remaining (n = 1214) were examined for A. reconditum. Upon dissection, first-stage larvae (L1) were identified after 2 days post infection (D2), second-stage (L2) at D13 and infective third-stage larvae (L3) at D15. Eighteen (30%) of 60 pools of fleas molecularly examined tested positive. Histologically, L2 were detected at D13 in the sub-cuticle region embedded in the back muscle of one female flea. This study provides original data on larval development of A. reconditum in C. felis and reports on the usefulness of the artificial feeding system.

Adult of Dirofilaria repens in a dog with recurrent multiple subcutaneous nodular lesions.

Subcutaneous dirofilariosis is a zoonosis parasitizing the dog, transmitted by Culicidae mosquitoes. An increasing number of new cases have been reported both in dogs and in humans all over the world. The aim of this work was to describe an uncommon case of recurrent multiple subcutaneous nodules by Dirofilaria repens in a 2-year-old dog. Surgical excision of two nodules on both hind limbs revealed a single thread-like live nematode and multiple parasite fragments, respectively. The parasite was morphologically identified as an adult female of D. repens. Haematoxylin-eosin tissue sections of nodules revealed a diffuse pyogranulomatous angiocentric dermatitis with an intense eosinophilic infiltrate throughout the dermis. One month post-surgery, a new nodular lesion was detected on the right thigh and resolved spontaneously in a few weeks. During the following 3 months, two other nodules were briefly noted both on the right hip and on the left side of the chest. The dog was periodically treated with milbemycin oxime and praziquantel, starting a week before the appearance of the first nodule. Microfilariae were not detected neither in the bloodstream nor in fine-needle aspirates at any time, and the diagnosis was made only after surgery. Practitioners should consider dirofilariosis in the differential diagnosis of subcutaneous swellings, especially if they have a recurrent behaviour, previously described in humans but not in dogs.

Feline lungworm Oslerus rostratus (Strongylida: Filaridae) in Italy: first case report and histopathological findings.

Oslerus rostratus syn. Anafilaroides rostratus (Strongylida: Filaroididae) is a metastrongyloid transmitted by snails, which localizes in peri-bronchial tissues and in the lung parenchyma of wild as well as domestic cats. In Europe, this nematode has been reported only on two occasions, being diagnosed in cats from Majorca Island and in northern Spain. Here, we describe a case of O. rostratus infection in a necropsied 4-year-old cat in Sicily (southern Italy). At the inspection of lungs, slender and greyish nematodes (four females and two males) were found embedded in the peri-bronchial tissues and in the bronchial walls. Parasites were morphological and molecularly identified as O. rostratus, with their 18S sequences being identical among them and showing a high homology (99%) with those available in public databases. At the histology, nematodes were encapsulated in a pseudo-cystic formation surrounded by an interstitial inflammatory process and fibrous tissue. Lung lesions were mainly represented by peri-luminal fibrosis, hyperplasia and hypertrophy of the bronchial mucosa and glands, respectively. This first record of O. rostratus infection from Italy indicates that this parasite should be included in the differential diagnosis of feline of lungworm infection.

A case of abnormal swimming patterns in juvenile Oblada melanura naturally infected with Philometra obladae (Nematoda: Philometridae) in the Tyrrhenian Sea off Sicily, Italy.

The saddled seabream, Oblada melanura (L.), is a common seawater species present in the Mediterranean. Between July and August 2023, during diving activities along the Sicilian coast of Italy, we recorded with a digital camera several specimens of O. melanura showing an abnormal pattern of swimming in the water column. The unusual swimming behaviour was characterized by fast, uncoordinated directional changes and isolation from the remaining fishes in the shoal. Four dead fish were found and collected for necropsy and parasitological evaluation/examination. Upon gross examination, all fish showed an evident coelomic distension; the coelomic cavity of two fish was filled with nematodes that dislocated the coelomic organs, and the other two had degraded nematodes and a conspicuous quantity of fluid in the coelomic cavity. All collected parasites were identified as Philometra obladae (Nematoda: Philometridae) according to morphological criteria. Here, we describe the unusual swimming behaviour of O. melanura naturally infected with Ph. obladae and the results of the examination of dead fish infected with this parasite. Future studies are needed to better evaluate and describe the dynamics and the epidemiology of Ph. obladae infection in wild O. melanura.

Evidence for direct transmission of the cat lungworm Troglostrongylus brevior (Strongylida: Crenosomatidae).

Metastrongyloids of cats are emerging pathogens that may cause fatal broncho-pulmonary disease. Infestation of definitive hosts occurs after ingestion of intermediate or paratenic hosts. Among metastrongyloids of cats, Troglostrongylus brevior and Troglostrongylus subcrenatus (Strongylida: Crenosomatidae) have recently been described as agents of severe broncho-pulmonary disease. Here, we provide, for the first time, observational evidence suggesting the direct transmission of T. brevior from queen cat to suckling kittens. This new knowledge will have a significant impact on current scientific information of this parasite and shed new light into the biology and epidemiology of metastrongyloid nematodes.

Helminths of the Bluntnose Sixgill Shark, Hexanchus griseus (Bonnaterre, 1788), from the Strait of Messina (Sicily, Southern Italy).

Bluntnose sixgill shark, Hexanchus griseus (Bonnaterre, 1788), is a little-known elasmobranch in the Mediterranean Sea. Given the lack of information about H. griseus, the aim of our study was to describe the helminth fauna of this species. In March 2023, one H. griseus juvenile female specimen was found off the coast of Messina (Italy) and referred by the Italian Coast Guard to our laboratory for necropsy and parasitological evaluation. After necropsy, the specimen's gills, stomach and spiral valve were investigated for parasite presence. All collected parasites were stored in 70% ethanol for routine parasitological analysis. No lesions due to parasites were found in the gills or skin. Three species of helminths were found in one studied female specimen of Hexanchus griseus, namely, two cestode species (Phyllobothrium sinuosiceps and Nybelinia sp., larvae) and one trematode (Otodistomum veliporum). Among them, five Trypanorhyncha plerocercoid larvae were found attached to the stomach mucosa, and six adult cestodes and one digenean trematode were collected from the spiral valve. No other parasite taxa were found in the celomic organs. This study reports new information regarding the parasitic fauna of H. griseus from the central Mediterranean Sea.

First Report of Proteocephalus longicollis (Zeder, 1800) in Alosa fallax lacustris (Fatio, 1890) from Lake Garda (Italy): Morphological and Molecular Study.

Twaite shad (Alosa fallax) includes two subspecies, the anadromous Alosa fallax nilotica, and the landlocked species Alosa fallax lacustris, representing the only Clupeidae inhabiting Lake Garda. Study of the parasitic fauna of this species in this area is still limited. For this reason, the aim of the present study was to evaluate the presence of tapeworms from A. fallax lacustris sampled in Lake Garda. Sixty-six A. fallax lacustris specimens were collected at the Milan fish market (Lombardy, Italy); specifically, an evaluation of the gastrointestinal contents was carried out for the presence of helminths. All parasites found were fixed in 70% ethanol and transferred to the laboratories of the University of Messina for morphological and molecular analysis. Parts of the parasites were stained with Semichon's carmine red technique. Molecular analysis was carried out using LSU rRNA and ITS2 region genes. Eight specimens out of sixty-six (12.1%) were positive for adult cestodes within the pyloric caeca. Morphological and molecular analysis could to identify the parasites found to be Proteocephalus longicollis. This parasite species is not considered a zoonotic agent, representing a low risk of parasitic fish-borne zoonosis for consumers of this appreciated fish from Lake Garda.

Reaction of Cornu aspersum Immune System against Different Aelurostrongylus abstrusus Developmental Stages.

Cornu aspersum, the land snail, is recognized as a suitable intermediate host of Aelurostrongylus abstrusus; however, there is little information both on larval development as well as on the intermediate host's immune system reaction to the parasite. The aim of the study was to assess the histological reaction of C. aspersum's immune system against A. abstrusus. Sixty-five snails were provided by a snail farm. Five of them were digested to assess the absence of natural parasitic infections. The remaining sixty were divided into five groups. Three groups of snails were infected with A. abstrusus using by-contact or injection methods; one group was injected only with saline solution and one group was left untreated as the control. The snails of group A were sacrificed and digested on study days 2, 10, and 18; snails of the other groups were collected and examined for histopathological analysis on study days 2, 10, and 18. On study day 2, in the infected snails, several free L1s were observed along with the absence of immune system reactions. On day 10, the L2s elicited an intense reaction in the internal layer of the muscular foot. On day 18, all L3s partially encapsulated by the snail's immune system were observed in the outermost part of the muscular foot, which is near and among the goblet cells. This last finding suggests that L3s could be shed with the snail's mucus and spread in the environment, representing an alternative route of transmission for this feline lungworm.

Morphological and Molecular Identification of Mullet Helminth Parasite Fauna from Ganzirri Lagoon (Sicily, Southern Italy).

Mullets (Osteichthyes: Mugilidae) are a euryhaline species widely distributed all over the world, thus representing an excellent study model for host-parasite interactions. From March to June 2022, 150 mullets, belonging to Chelon labrosus (n = 99), Chelon auratus (n = 37), and Oedalechilus labeo (n = 14) species, were caught to identify the helminth parasite fauna of the different mullet species present in the Ganzirri Lagoon (Messina, Sicily, Italy). A parasitological evaluation of the gastrointestinal tract (GIT) was carried out with a total worm count technique (TWC) to detect helminth presence. All collected parasites were stored in 70% ethanol until morphological evaluation, and frozen at -80 °C for subsequent molecular analysis, using 28S, ITS-2, 18S primers. The morphological evaluation allowed for the identification Acanthocephalan parasites (Neoechinorhynchus agilis) from two C. labrosus specimens. Sixty-six samples were positive for adult digenean trematodes (C. labrosus, 49.5 %; C. auratus, 27%, and O. labeo, 50%), molecularly identified as Haploporus benedeni. This study represents the first survey of helminthic parasite fauna of mullets from the south of Italy. The presence of Hydrobia sp. in the stomach contents of mullets allowed us to infer the H. benedeni life cycle in the Ganzirri lagoon.

New insights into the ecology and biology of Acanthocheilonema reconditum (Grassi, 1889) causing canine subcutaneous filariosis.

In spite of its wide distribution among dogs and the evidence of its implication as a zoonotic agent, scant information is available on the biology of Acanthocheilonema reconditum (Spirurida, Onchocercidae). In this study, blood samples from 152 Sicilian dogs were examined for A. reconditum microfilariae at the beginning of the study and 1 year later. The periodicity of microfilaraemia was investigated by bleeding 2 highly microfilaraemic dogs twice a day for 10 days and, later on, every 2 weeks for 1 year and a third animal every 3 h for 96 h. Fleas and ticks infesting dogs were collected and dissected for the detection of A. reconditum larvae. The prevalence of infestation was 11·2% (17/152) and 13·3% (16/120) at the beginning and at the end of the study, with a 1 year cumulative incidence of 5·9%. Although dogs bled twice a day showed a higher number of microfilariae in most of the morning samples, the absence of any circadian rhythm was suggested by data of the third experiment conducted by bleeding a dog every 3 h for 4 days. A. reconditum developing forms were detected in 5·1% (4/78) of dissected fleas, but not in any of the 272 ticks. The study provides new insights into the biology and ecology of this dog filarioid in its definitive and intermediate hosts.

Unusual Localization of Hysterothylacium incurvum in Xiphias gladius (Linnaeus 1758) Caught in the Atlantic Ocean.

This study represents the first report of Hysterothylacium incurvum within swordfish (Xiphias gladius) heart chambers. Swordfish is a large pelagic teleost, considered one of the most appreciated fish worldwide. Among swordfish parasites, Anisakis sp. and Hysterothylacium sp. have been used to evaluate biological and ecological aspects of this teleost. Between 2021 and 2022, 364 X. gladius hearts, caught from the Atlantic Ocean (FAO 27.IXa and FAO 34 areas), were collected at the Milan fish market (Lombardy, Italy). Three specimens from FAO 27.IXa was positive for seven adult nematodes (p = 1.55%) within the heart chambers. Of these, three specimens were found within the bulbus arteriosus and 4 in the ventricle. All parasites were stored in 70% ethanol and processed for parasitological and molecular analysis using Cox2, ITS regions/ITS-I-5.8S-ITS-II, and rrnS genes. The analysis allowed us to identify the retrieved parasite as H. incurvum. According to our evaluation, the final localization is due to the movement of L3 larvae from the coelomic cavity to the bloodstream, with consequent development to the adult stage within the heart. Finally, the parasite localization, considered non-marketable fish parts, does not pose a significant risk to consumers, also considering the low zoonotic potential of H. incurvum.

Clinical Case of Feline Leishmaniosis: Therapeutic Approach and Long-Term Follow-Up.

The response to allopurinol treatment and survival time of a case of feline leishmaniosis in a FIV co-infected cat is herein reported. In May 2019, a 13-year-old neutered European Shorthair male was referred due to weight loss and exfoliative dermatitis. Lymphadenomegaly and splenomegaly were detected upon clinical inspection, while the presence of several Leishmania infantum amastigotes were detected on splenic and lymphonodal fine needle aspiration (FNA). Allopurinol (10 mg/kg PO q 12 h) was administered. After two months, the cat's clinical symptoms disappeared, and the owners decided to interrupt the therapy. In February, two reddish nodular fleshy neoformations appeared in both eyes, and amastigotes of Leishmania were detected by cytology on conjunctival swabs. Allopurinol treatment was re-started at the same rate; the ocular lesions regressed in two weeks, and the owner again decided to interrupt the therapy. In July, the patient had a new relapse, but the owner, tired of continuous relapses, refused further treatment of the disease. The cat's health condition continued to worsen: in October 2021, the ocular lesions appeared again, and in November the patient died. This case underlines the effectiveness of allopurinol and highlights how interruption of treatment frequently leads to relapse, impairing the animal's health condition and prognosis.

Dirofilaria immitis infection in the Pelagie archipelago: The southernmost hyperendemic focus in Europe.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioids of zoonotic concern, which primarily cause canine heartworm disease (HWD) and subcutaneous dirofilariosis, respectively. The geographical distribution of these nematodes is constantly changing, due to many factors such as the colonization by new invasive mosquito species, the increased movement of animals and the absence of chemoprophylactic strategies in non-endemic regions. Although HWD has always been considered endemic in northern Italy, an increase in the prevalence of this disease has been recorded in the last decades in central and southern regions. We describe the southernmost hyperendemic European focus of heartworm disease in the Pelagie archipelagos. From June to November 2020, 157 dogs and 46 cats were sampled in Linosa and Lampedusa islands for the detection of Dirofilaria immitis and Dirofilaria repens by modified Knott's, rapid antigen and molecular tests. Mosquitoes were collected using CDC light and BG sentinel-2 traps and aspirators, and tested for host blood meal and Dirofilaria spp. DNA. Out of 56 dogs in Linosa and 101 in Lampedusa, 33 (i.e. 58.9%) and 8 (i.e. 7.9%) were positive to D. immitis and D. repens, respectively. Three cats scored positive to D. immitis (i.e. 17.6%) in Linosa. Six mosquito species were identified, and the abdomen of a non-engorged Aedes albopictus was positive for D. immitis and human DNA. The results suggest that D. immitis infection could spread to new previously non-endemic territories in southern Europe, representing a real threat to animal and human health.

External ophthalmomyiasis by Oestrus ovis in tourists visiting Italy. Report of three cases and a literature review.

BACKGROUND: Oestrus ovis is a dipteran parasite responsible of myiasis in small ruminants and reported as the causative agent of myiasis in humans particularly in sheep farming areas. In recent years, several human cases had been reported without any previous history of exposure to livestock or visit to rural areas. CASE PRESENTATION: We describe three clustered cases of ophthalmomyiasis externa in tourists in Sardinia and Sicily and discuss them in the light of the published literature. The patients presented ocular irritation, foreign body sensation, pain, redness, and lacrimation of the eyelids. After extraction, all the larvae were morphologically and molecularly confirmed as L1 instar of O. ovis larvae. CONCLUSIONS: The present study points out the need to draw attention and awareness among the physicians, and to consider ophthalmomyiasis when consulted for conjunctivitis in the summer and spring seasons.

Genital myiasis by Wohlfahrtia magnifica (Diptera, Sarcophagidae) in Sicily (Italy).

Wohlfahrtia magnifica (Schiner, 1862; Diptera: Sarcophagidae), known also as flesh fly or screwworm, is a fly distributed especially in south Europe, Middle East, North Africa and China. Maggots of W. magnifica are responsible for traumatic myiasis in warm-blooded vertebrate animals and humans. In Italy data on wohlfahrtiosis in animals and humans are scant. This paper reports three cases of genital myiasis by W. magnifica in Sicily that occurred in a goat, a ram and a dog, respectively. Maggots were found in the vulva of a goat, in a wound localised in the scrotum of a ram and in the prepuce of a dog. Although the disease was not recorded before in the region, data collected from a questionnaire specifically designed for the study suggest that the disease is endemic in the area for more than 20 years. Larger epidemiological studies in the region are needed to get more data on disease prevalence and pattern of infestation as well as more information and education is needed for both farmers and veterinarians on wohlfahrtiosis and its strategies of control and treatment.

Natural Products against Sand Fly Vectors of Leishmaniosis: A Systematic Review.

Leishmaniosis is a vector-borne disease transmitted to animals and humans by the bite of blood-sucking phlebotomine sand flies. These small insects play a crucial role in the diffusion of the disease. To date, the sole strategy recognized for the prevention of leishmaniosis is the use of topical repellent compounds against sand fly bites. Several synthetic insecticides and repellents have been developed; however, the wide and unprejudiced use of these formulations have led to the loss of their effectiveness and the development of resistance phenomena. Moreover, some of these synthetic repellents have severe detrimental effects on the environment and could represent a serious threat to both animal and human health. Recently, an increased interest in the research on alternative approaches to sand fly control has been expressed. In this study, we systematically reviewed the efforts of the scientific community to individuate a phytochemical alternative for the control of sand fly species recognized as vectors of Leishmania spp. Based on literature research using different electronic databases, a total of 527 potentially relevant studies were screened and narrowed down to a final 14 eligible scientific reports. Our analysis suggests that although there is a rapidly growing body of literature dedicated to botanical insecticides and repellents against sand fly vectors of Leishmania spp., much of this literature is limited to in vitro studies conducted in laboratory conditions, and only a few of them investigated the repellency of plant-based products. These studies highlighted that natural compounds display a really short period of action and this significantly limits the use of these products as an alternative to chemical-based repellents.

Parasite Fauna of the Dusky Grouper (Epinephelus marginatus, Lowe 1834) from the Central Mediterranean Sea.

This study aimed to investigate parasite fauna of E. marginatus from the central Mediterranean Sea between Messina and Syracuse. In the present survey; parasite fauna of dusky grouper was investigated for two main reasons: the economic value of this species and the current lack of studies regarding the capture area. Seventy dusky groupers were caught from May 2018 to February 2020. Forty-seven out of the 70 specimens (67.2%) were infected with one or more parasite species. The most abundant species was Prosorhynchus caudovatus (42.9%), followed by Podocotyle temensis (28.6%), Didymodiclinus sp. (18.6%), Philometra jordanoi (5.7%), Anisakis Type II larvae (5.7%). Higher prevalence of infection of P. jordanoi and Contracaecum sp. was found in warm months (March to September), while P. caudovatus and P. temensis were mostly found during cold months. Weight and total length of E. marginatus were positively correlated with the parasitic load of P. jordanoi and Didymodiclinus sp. The different prevalence of parasite infection found between warm and cold months is probably related to the diet of the dusky grouper; which is characterized by mollusks that are intermediate hosts for parasite species found. None of the parasites found in the present survey is responsible for zoonosis.

Anisakis pegreffii Larvae in Sphyraena viridensis and Description of Granulomatous Lesions.

The aim of the present study was to describe gastric granuloma caused by Anisakis pegreffii in Sphyraena viridensis caught in the central Mediterranean Sea. Sixty-eight S. viridensis specimens were collected from different fish markets on the east coast of Sicily. Coelomic organs were observed both macroscopically and with the aid of stereomicroscope. Parasite specimens and lesioned tissues were collected for identification, histological and molecular analyses. Twelve specimens (p = 17.6%) were positive for the presence of nematode larvae, morphologically identified as larvae of Anisakis sp., with values of mean abundance and mean intensity of 0.9 and 4.8, respectively. One large female specimen showed massive parasite infection associated with nodular lesions of the gastric wall. By histology, several nematode larvae encysted through the gastric wall were found. The parasite bodies were surrounded by a granulomatous reaction made up of macrophages, epithelioid cells, some lymphocytes and an external connective sheet. Molecular analysis of 18S rRNA and cox2 genes from Anisakis sp. collected larvae, identified them as A. pegreffii. The lesions here described, though macroscopically superimposable on human eosinophilic granuloma, microscopically showed significant differences in the inflammatory cells involved and in the type of immune reaction.