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.

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.

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.

Elimination of Dirofilaria immitis Infection in Dogs, Linosa Island, Italy, 2020-2022.

On Linosa Island, Italy, Dirofilaria immitis infection has been hyperendemic in dogs and seroprevalent among islanders. In 2020, we implemented a heartworm disease elimination program on Linosa Island. Of 54 dogs tested for D. immitis antigen and microfilariae, 28 had positive results and received treatment with oral doxycycline twice daily for 4 weeks plus topical imidacloprid/moxidectin monthly for 12 months. The 26 dogs with negative results received monthly topical imidacloprid/moxidectin as preventive. During month 1, the number of microfilaremic dogs was reduced by 76.5%. From month 2 on, all animals were microfilariae negative, and during months 3 to 9, the number of antigen-positive dogs decreased progressively. Treatment of positive dogs coupled with chemoprophylaxis for noninfected dogs was effective, protecting them from new infections. The elimination program reduced the risk for human infection, representing a One Health paradigm. Monitoring and chemoprophylaxis are advocated to maintain the status of heartworm disease-free area.

New distribution patterns of Dirofilaria immitis in Italy.

In recent decades, the number of autochthonous cases and foci of Dirofilaria immitis in dogs from southern regions has increased considerably, suggesting that the distribution of the species is not limited to northern Italian regions. This epidemiological picture emerges from case reports or studies in specific locations where outbreaks of heartworm disease have occasionally been reported together with the presence of mosquito vectors. To obtain a more comprehensive picture of the current distribution of D. immitis in southern Italy, a multicenter cross-sectional survey of canine filariasis was conducted. Owned and sheltered dogs (n = 1,987) were included in the survey regardless their breed, attitude and/or sex. All included dogs were older than 1 year and had no history of chemoprophylactic treatment against filarioses. A blood sample was collected from enrolled dogs and screened by modified Knott's test and, when positive, tested using D. immitis specific ELISA rapid test (SNAP 4DX, IDEXX). The overall microfilaremia prevalence was 17% (n = 338) being single-species infection (92.6%) more common that mixed (7.4%). Remarkably, D. immitis was the most frequent species detected with an overall prevalence of 11.4% (n = 227), followed by Dirofilaria repens (n = 74; 3.7%), and Acanthocheilonema reconditum (n = 12; 0.6%). Sheltered dogs were significantly more infected by D. immitis, as well as mongrel dogs and animals housed in rural areas. Data here reported indicate that D. immitis is largely present in southern Italy, raising awareness about the necessity of proper screening and chemoprophylactic treatments in exposed animals.

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.

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.

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.

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.

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.

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.

Bovine besnoitiosis in a cattle herd in Sicily: an isolated outbreak or the acknowledgment of an endemicity?

Bovine besnoitiosis is a debilitating infectious disease caused by Besnoitia besnoiti (Apicomplexa; Sarcocystidae). The disease is mainly characterized by cutaneous and systemic signs, infertility in bulls, and abortion in cows. The current study describes an autochthonous outbreak of bovine besnoitiosis in Sicily, Southern Italy, being the first report of B. besnoiti infection in the island so far. In a cattle farm located in Syracuse province, a 4-year-old Belgian blue bull born in Sicily displayed typical clinical signs of the sub-acute/chronic disease phase with thickening of the skin of the scrotum and testicular degeneration. Histological examination of scrotal biopsies revealed the presence of several tissue cysts of B. besnoiti. The serological analysis of the herd using a commercial ELISA revealed a high seroprevalence (45 out of 55; 82%) of antibodies against B. besnoiti. Few seropositive animals (5 out of 45; 11%) showed clinical signs, cysts in vestibulum vaginae (1 out of 31; 3.2%), and testicular degeneration (4 out of 14; 28.6%) assessed by ultrasonographic investigations. The paucity of clinical signs associated with the high seroprevalence in the farm led to hypothesize that bovine besnoitiosis is endemic in the area though further studies are needed. Local practitioners should be more aware of the disease to facilitate the early detection of cases, prevent the spread of infection, and avoid economic losses and animal health problem.