Hepatic fasciolosis represents a major health and production concern in sheep farming. Although ultrasonography has been used to evaluate fasciolosis in ruminants, the feasibility of a rapid ultrasonographic protocol performed in standing sheep under field conditions has not been specifically described. This study aimed to provide a preliminary, descriptive evaluation of the feasibility of a fast on-farm ultrasonographic protocol for the assessment of hepatic alterations in sheep with suspected chronic fasciolosis. Sixteen Sarda sheep with Fasciola hepatica fecal eggs count > 100 eggs per gram underwent clinical examination, hematological tests, and liver ultrasonography. Three sheep were slaughtered, and their livers were examined by gross pathology and histopathology. The median duration of the ultrasonographic examination was 2.5 min per animal. Recurrent ultrasonographic features included heterogeneous parenchymal echotexture and echogenicity (87.5 %), irregular diaphragmatic liver surface (68.8 %), moderate to severe bile duct dilatation (50 %), and thickened gallbladder walls (56 %). In a subset of animals (37.5 %), intraluminal structures compatible with adult flukes were visualized within the biliary tract. Pathological and histological findings in the necropsied animals were consistent with the ultrasonographic features observed in vivo. This study provides preliminary descriptive evidence that a rapid hepatic ultrasonography protocol is feasible under field conditions in standing sheep with suspected chronic fasciolosis. Further prospective studies are required to clarify the diagnostic performance and clinical utility of ultrasonography in ovine fasciolosis.
The availability of laboratory tests to screen and diagnose migrants and travellers for neglected and tropical parasitic diseases significantly impacts individual and public health. Italian scientific societies for parasitology, tropical diseases, and global health developed a survey to assess number and geographical localisation of laboratories able to carry out adequate diagnostics. An open-ended and multiple-choice questionnaire was constructed and sent to 752 members working in Italian microbiology laboratories via scientific societies' mailing lists. Data concerning malaria, cystic echinococcosis, leishmaniasis, schistosomiasis, strongyloidiasis, and Chagas disease were included. Members from 96 laboratories replied. At least one laboratory responded from 18 out of 20 Italian regions. Serological tests for Schistosoma spp., Strongyloides stercoralis, Trypanosoma cruzi, Echinococcus spp., and Leishmania spp. are performed in <50% of responding laboratories. Only 56.6% of labs provide all three recommended tests for malaria diagnosis in the emergency room. Direct identification methods availability varies for Schistosoma eggs (75-95.8%), S. stercoralis larvae (53.1%), trypomastigotes (59.4%), and Leishmania amastigotes (53.1%). Geographical differences (mainly northern versus southern regions) were evident. The survey underlines the need to improve diagnosis for neglected and tropical diseases, to define a network of reference laboratories for testing less prevalent diseases, and to share information, education, and training for both clinicians and microbiologists/parasitologists.
Barcoding studies have provided significant insights into phylogenetic relationships among species belonging to the genus Ligia (Crustacea, Isopoda). Herein the diversity of the Italian sea slater Ligia italica from Tunisia is studied for the first time. Samples were collected from 18 localities in Tunisia, and the analysis included previously published sequences from Italy and Greece available in GenBank. Bayesian and Maximum Likelihood phylogenetic analyses were carried out using a fragment of the mitochondrial COI gene. Putative cryptic species were explored using the 'barcode gap' approach in the software ASAP. A genetic landscape shape analysis was carried out using the program Alleles in Space. The analyses revealed highly divergent and well-supported clades of L. italica dispersed across Tunisia (Clades A1 and A2), Greece (Clade B) and Italy (Clades C1 and C2). High genetic dissimilarity among clades suggested that L. italica constitute a cryptic species complex. Divergence among different L. italica lineages (Clades A, B and C) occurred around 7-4.5 Ma. The detected high genetic distances among clades did not result from atypical mitochondrial DNAs or intracellular infection by Wolbachia bacteria. The complex history of the Mediterranean Sea appears to have played a significant role in shaping the phylogeographic pattern of Ligia italica. Additional morphological and molecular studies are needed to confirm the existence of cryptic species in Ligia italica in Mediterranean.
The present contribution discusses the possible defensive camouflage strategy adopted by Leiopleura venustula (Gory, 1841), a South American leafmining buprestid beetle (Buprestidae, Agrilinae, Tracheini). This species presents a coloration pattern resembling older dry bird droppings and displays the capability to secrete a large amount of highly convoluted wax filaments on its dorsum. These filaments, specifically secreted by a series of pores present in the rough areas of pronotum and elytra, apparently improve the quality of the camouflage mimicking the (white) uric acid commonly found in bird faeces. SEM images of distribution of pores and wax filaments are presented.
To develop a multiplex RT-quantitative PCR (RT-qPCR) assay to quantify sex-specific Plasmodium falciparum gametocyte transcripts (pfCCp4, pfMGET), for evaluating the impact of drug treatments on gametocyte viability for malaria transmission-blocking drug development. We optimized an RT-qPCR assay incorporating a normalization transcript to use the ΔΔCt method (differences in Cycle threshold) to quantify gametocyte transcript levels. The assay was used on ex vivo gametocytes from P. falciparum natural infections exposed for 24 h to six drugs impairing mosquito transmission, as measured by the direct membrane feeding assay. Follow-up in vitro experiments showed that an additional 48 h incubation, following drug wash-out, was required to monitor decline in transcript levels and potential sex-specific effects. The optimized assay revealed efficacy of drug treatment as a reduction in transcript levels for two of the six drugs tested: 30% for pfMGET and 80% for pfCCp4 in methylene blue (5 µM)-treated samples, and 75% for both sex-specific transcripts in samples treated with P218 (0.25 µM). In the remaining drugs, a 48 h incubation period post drug wash-out was required to measure a decline in transcript levels. Furthermore, a differential reduction in the levels of male versus female gametocyte transcripts suggested sex-specific effects for two of the drugs. The multiplex RT-qPCR assay provides a reliable method to assess the inhibitory effects of drug treatments on P. falciparum gametocytes, with the potential to evaluate both overall and sex-specific impacts on gametocyte viability. This assay represents a valuable tool in the development and evaluation of transmission-blocking drugs, particularly in distinguishing effects on male and female gametocytes.
Cardio-pulmonary parasites, such as Angiostrongylus vasorum, Crenosoma vulpis, and Eucoleus aerophilus, pose a significant concern on account of pulmonary and cardiac problems they induce in dogs. While the red fox is known to be a key reservoir host for A. vasorum and may also play a role in transmitting C. vulpis and E. aerophilus, there has been no recent research on these parasites in foxes from Sardinia, with the most current studies dating back to 1986. A survey was conducted on red foxes in Sardinia, where a total of 51 foxes were collected, necropsied, and examined for adult worms in their hearts and lungs. The worms were identified using morphometric analysis and molecular methods. The results showed a 54.9% overall prevalence at dissection: 45.1% of the foxes were positive for E. aerophilus, 17.6% for C. vulpis, and 13.7% for A. vasorum. The molecular analyses validated the morphological characterization. In comparison to previous research, which found 13 out of 85 foxes to be positive for A. vasorum with a prevalence rate of 15.3% and 1 for E. aerophilus with a prevalence of 1.2%, this study showed an increased prevalence of E. aerophilus and C. vulpis, and a decrease in the prevalence of A. vasorum. These results indicate that the red foxes in Sardinia represent a reservoir host for cardio-pulmonary nematodes and it should be considered in the differential diagnosis of respiratory distress syndrome in dogs.
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Numerous reports of resistance to terbinafine in Trichophyton spp. from all over the world are arousing justified attention and concern. Point mutations in the gene that encodes the squalene epoxidase (SQLE) enzyme are responsible for these therapeutic resistances. Primary objective of the study was to describe first isolates of Trichophyton spp. resistant to terbinafine among the patients treated between September 2019 and June 2022 at the Dermatology Units of Ospedale Maggiore Policlinico and San Bortolo Hospital. Secondary objective was to study the resistance mechanism. Patients with confirmed Trichophyton spp. infection has been treated with systemic and topical terbinafine. Patients were then re-evaluated 12 weeks after the therapy. Patients with incomplete or absent response to terbinafine underwent a new skin scraping for direct mycological examination, new identification of dermatophyte species from culture and MALDI-TOF, molecular species identification, antifungal susceptibility testing and molecular analysis of SQLE gene. We identified five patients without clinical response to treatment with terbinafine. The DNA sequencing of the ITS region identified one Trichophyton rubrum and four Trichophyton indotineae. The T. rubrum strain showed minimum inhibitory concentration (MIC) (90% growth inhibition) of 4 mg/L for terbinafine. The four T. indotineae strains showed a MICs range of 0.25-4 mg/L for terbinafine. The analysis of the SQLE gene in the T. rubrum strain showed a nucleotide substitution generating a missense mutation (L393F). The SQLE gene sequencing in the T. indotineae strains showed a nucleotide substitution generating a missense mutation (F397L) in two strains, a nucleotide substitution L393S in one strain and a nucleotide substitution F415C in another strain. We report the first cases of terbinafine-resistant Trichophyton isolates in the Italian population. Solid antifungal management programs will be needed to promote more responsible use of antimycotics and preserve their therapeutic efficacy to control antifungal resistance.
Numerous biting and nuisance insects are a noted cause of discomfort and stress to horses. Pyrethrins and pyrethroids have been used for many years in numerous formulations for the control of insect pests in animals, humans and environment. There are, however, few studies reporting their field efficacy in horses. The aim of the present study was to evaluate the repellent activity of a spray formulation based on prallethrin and permethrin synergized with piperonyl butoxide (BRONCO® Equine Fly Spray, Farnam Companies, Inc., USA) against annoying and harmful insects for horses in field conditions. Nine horses of mixed breed were divided into 2 groups (treatment and control). Pre-treatment insect counts were compared to daily counts for 4 days post-treatment (pt). One minute after the administration of the product (day 0), all the horses were negative for the presence of insects. All counts up to the 6-h pt check remained negative for Hippobosca equina, tabanid flies and Simulium spp., showing 100% efficacy. This remained above 90% throughout the study. For the H. equina, the repellent efficacy remained > 99.7% for all 4 days pt, for tabanid flies > 93.3% and for Simulium spp. > 97.4%. The efficacy against Musca spp. decreased from 82.2% at day 0 to 62.2% at day 3. Treatment was well-tolerated. In conclusion, despite the low number of tested horses, Bronco® has demonstrated high insecticide and repellent efficacy and a good persistence, maintained for up to 4 days post-treatment, against the most common species of insects harmful for horses.
The gill monogenean Sparicotyle chrysophrii (Van Beneden & Hesse, 1863) Mamaev, 1984 is a specific and common parasite of wild and cultured gilthead sea bream Sparus aurata Linnaeus, 1758, able to cause disease and mortality in aquaculture systems. Few molecular studies have been carried out on this monogenean, and its population structure and genetic diversity are barely known. This study provides the first contribution to the population genetic variation of S. chrysophrii, based on two molecular markers - the structural ribosomal RNA (rRNA) for the large subunit (28S) and the cytochrome c oxidase subunit I (COI) gene. Samples were collected from the gills of farmed and wild S. aurata from Italy and the Spanish Mediterranean. The analysis included previously published sequences. The 28S rDNA analysis was consistent with previous studies of specimens isolated from S. aurata and confirmed the presence of only one species on the gills of this host in the Mediterranean Sea. The COI sequences analysis suggested that the samples isolated in a previous study from a different host species, wild Boops boops (Linnaeus, 1758) in the Adriatic Sea, may represent a new undescribed sister species of S. chrysophrii. The low nucleotide diversity of S. chrysophrii isolated only from S. aurata versus the high haplotype diversity revealed small differences between haplotypes. The haplotypes shared between wild and farmed hosts from Spain provided the first molecular evidence of the possible transfer of S. chrysophrii between wild and farmed populations of S. aurata. The mtDNA COI analysis did not show a clear genetic structure, probably the result of several factors including coevolution, wild and farmed host interactions, and host population structure in space and time.
Ascaris suum is one of the most important parasites of pigs. Apart from liver condemnation due to lesions caused by migrating larvae ("milk spots"), A. suum infections can compromise weight gain, feed conversion efficacy, as well as meat quality. The true prevalence of infection depends on the diagnostic test used and is often underestimated. We compared liver inspection at slaughter with serology, based on the recognition of a purified A. suum haemoglobin or complete homogenate of the 3rd stage larvae isolated from lungs, in nine pig farms in northern Italy. Liver lesions were found on all farms with prevalence ranging from 3.8% to 98.3%. All farms were also positive for circulating antibodies against As-Hb and As-Lung-L3, with prevalence among pigs on each farm ranging from 36.4-100% and 54.5-100%, respectively. Seroprevalence was consistently higher when compared to the prevalence of milk spots at slaughter. The higher sensitivity of the ELISA tests combined with their ease of use makes them an interesting tool to evaluate A. suum infection levels.
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Throughout history, wildlife has been regarded as a major source of infectious diseases. Rodentia, the most speciose order of mammals, whose members are recognised hosts of more than 60 zoonotic diseases, represent a potential threat to human health. Recently, epidemiological data from Saudi Arabia indicated an actual growth in the number of emerging and/or re-emerging cases of several zoonoses. However, there is a lack of studies focusing on the molecular taxonomy of rodents and the pathogens they may harbour in this region. In this study, the first molecular characterisation of six rodent taxa in this region is provided, based on partial Cyt B and 16S genes. The data confirm the spread of rodent-associated C. burnetii strains in Jazan, southwestern Saudi Arabia. The PCR targeting IS111, the multi-copy transposase gene, revealed 17.5% (36/205) positive samples, whereas the second nested PCR, targeting the single-copy Com1 gene, revealed 16.6% (34/205) positive samples. Phylogenetic and network analyses indicated the presence of four haplotypes of C. burnetii within the studied localities. One major haplotype (H-2) was observed in all rodent species and from 18 localities. The infection rates of C. burnetii among studied species, localities and habitats were not significantly different (>0.05). Our results facilitate the assessment of the health risk associated with rodents and the development of strategies to control the increasing impacts of Q fever.
In the fight against anthelmintic resistance, targeted selective treatments (TSTs), where only a small percentage of a flock receives treatment, have become increasingly popular. Overall, implementation of such treatments can be based on various parameters including Body condition score (BCS). As infection with non-bloodsucking nematodes, frequently encountered on sheep farms in the central Mediterranean basin, commonly causes bodyweight reduction, the aim of this research is to evaluate the effectiveness of BCS as a parameter for the implementation of TSTs in lactating dairy sheep with subclinical gastrointestinal nematode (GIN) infections from the island of Sardinia, Italy. Faecal samples from 1012 ewes divided into 2 groups (third and fifth month of lactation) were collected and their BCS recorded. Faecal egg counts and coprocultures were performed for the assessment of the GIN burden and identification of present species. An overall GIN prevalence of 85.4% with a mean eggs per gram (EPG) of faeces of 210.1 ± 347.3 was found. Teladorsagia spp. and Trichostrongylus spp. were the GIN genera most identified. Overall, animals with the lowest BCS had the highest EPG values and a negative correlation (r = - 0.163) between the EPG values and BCS of the studied animals was found, which was most significant for older sheep. This research confirmed BCSs and EPG values for GIN in sheep to be negatively correlated, particularly in older ewes. Application of TSTs for lactating sheep with a BCS < 2.25, especially to older ewes, could be beneficial in case of subclinical GIN infections, although further studies are needed to work out precise recommendation.
Mature albacore tuna (Thunnus alalunga) are expected to have high energy requirements at the time of breeding. However, there are no descriptions of the diet of albacore in the Mediterranean Sea that can help us to understand if such requirements can be obtained from feeding during reproduction. In this study, we analysed the stomach contents of reproductively active albacore captured from 2010 to 2015 in the oligotrophic waters of the western Mediterranean Sea, one of their main spawning grounds. Estimates of stomach fullness revealed intense feeding activity, and prey composition indicated important consumption of mesopelagic fish, including barracudinas, myctophids and small pelagic crustaceans. Plastic debris occurred in 25%-53% of the stomachs sampled across all years. Prey composition was not different between males and females. However, females fed at higher rates and had higher hepatosomatic index values than males, suggesting that increased feeding could contribute to meet their higher energy demand associated with offspring production. We observed a diet shift from small crustaceans to fish prey along fish size. During the spawning period, albacore showed a specialist feeding behaviour by preying on aggregations of vertically migrating myctophids and small crustaceans, probably when they were near the surface. This study provides information and biological data to support ecosystem modelling and to increase the understanding of albacore ecology.
The microcotylid Sciaenacotyle pancerii is a pathogenic monogenean infecting Argyrosomus regius, a candidate for species diversification in the Mediterranean aquaculture. Life-history stages of S. pancerii commonly co-occur in field infections, but to date, morphological data have only been provided for oncomiracidia and adults although identifying life-history stages can be useful in infection management. A total of 114 specimens of S. pancerii were analysed to characterize the developmental events and to assess morphological and morphometric variations before and after maturity. The post-larval development of S. pancerii is characterized by: expansion and bifurcation of the gut, loss of the larval haptor, protandrous development of the genitalia and vitellaria formation. The size variability of larval hooks, hamuli and germanium of S. pancerii is firstly reported and dimensional ranges of parasite body, haptor, testes, posteriormost clamps and eggs are widened. The size of most of the diagnostic features of S. pancerii significantly increases after parasite maturity and therefore, only those specimens with more than 116 clamps should be considered for minimising development-related variability in size. The high number of clamps, their fast development and the asymmetry in their size and arrangement suggest that S. pancerii may use a mixed attachment strategy between the closely related microcotylids and heteraxinids. This combination of features may be host related and linked to the gill structure of the sciaenid fish and the phylogenetic position of the genus Sciaenacotyle; distant from other microcotylids while close to heteraxinid species.
Several viruses can be transmitted by mosquitoes. We searched some of these viruses in 20,778 mosquitoes, collected in 95 traps on the plains of Emilia-Romagna (North of Italy) in 2021. We detected West Nile virus (WNV) and Usutu virus (USUV) in pools of Culex (Cx.) pipiens. In addition, we detected two insect-specific flaviviruses in three pools of Aedes (Ae.) caspius and in two of Ae. vexans. Tahyna virus (TAHV) was detected in six pools, three of Ae. caspius and three of Cx. pipiens, and one isolated strain was obtained from one of the Ae. caspius pools. Moreover, we detected TAHV in pools of several mosquito species (Ae. caspius, Ae. vexans, Ae. albopictus, Anopheles maculipennis s.l.) collected in the previous year of surveillance. Our data indicate Ae. caspius as the species most infected with TAHV in the surveyed area. Together with the likely plasticity of the cycle, we reported strong genome stability of the TAHV, probably linked to a successful adaptation of the virus to its ecological niche. Interestingly, in six pools of Cx. pipiens we detected two associated viruses among USUV, WNV, TAHV and all the three viruses in two pools. This result allows us to assume the presence of particular conditions that prompt the circulation of arboviruses, creating the conditions for viral hot spots. While no human diseases related to Tahyna virus were reported in Italy, its detection over the years suggests that it is worth investigating this virus as a potential cause of disease in humans in order to assess its health burden. IMPORTANCE We reported in this work the detection of three Arboviruses (Arthropod-borne viruses) in mosquitoes collected in Emilia-Romagna in 2021. In addition to West Nile and Usutu viruses, which were reported from more than 10 years in the study area, we detected and isolated Tahyna virus (TAHV). We also reported detections of TAHV obtained in previous years of surveillance in different species of mosquitoes. TAHV is the potential causative agent of summer influenza-like diseases and also of meningitis. Even if human cases of disease referable to this virus are not reported in Italy, its relevant presence in mosquitoes suggests investigating the possibility they could.
Trematode blood flukes of the genus Cardicola are potentially lethal in bluefin tuna cultures. The present study proposed a new method to detect aporocotylid eggs in tuna gills. Aporocotylid eggs were detected by analysing a pair of gill filaments of five transversal areas of the eight holobranches of one hundred Atlantic bluefin tuna and observed with glycerol and a stereomicroscope with an oblique brightfield. Data were gathered according to holobranches, transversal areas and their combination. Eggs were uniformly distributed among the holobranches, but they had the highest prevalence in the second and fifth transversal areas, which is controversial with respect to previous studies of egg distribution. An abbreviated method called the T-two test, which had the highest sensitivity (96.8%), is proposed for the detection of Cardicola spp. infections instead of the analysis all the holobranches. The T-two test limits the time and cost of the egg parasite screening analysis. The analyses of ten samples could be sufficient to detect the presence of parasites in farmed bluefin tuna; fish from the wild are expected to be less infected and more samples (45) would therefore be necessary.
The larval stages of tapeworms in the species complex Echinococcus granulosus sensu lato cause a zoonotic disease known as cystic echinococcosis (CE). Within this species complex, genotypes G6 and G7 are among the most common genotypes associated with human CE cases worldwide. However, our understanding of ecology, biology and epidemiology of G6 and G7 is still limited. An essential first step towards this goal is correct genotype identification, but distinguishing genotypes G6 and G7 has been challenging. A recent analysis based on complete mitogenome data revealed that the conventional sequencing of the cox1 (366 bp) gene fragment mistakenly classified a subset of G7 samples as G6. On the other hand, sequencing complete mitogenomes is not practical if only genotype or haplogroup identification is needed. Therefore, a simpler and less costly method is required to distinguish genotypes G6 and G7. We compared 93 complete mitogenomes of G6 and G7 from a wide geographical range and demonstrate that a combination of nad2 (714 bp) and nad5 (680 bp) gene fragments would be the best option to distinguish G6 and G7. Moreover, this method allows assignment of G7 samples into haplogroups G7a and G7b. However, due to very high genetic variability of G6 and G7, we suggest to construct a phylogenetic network based on the nad2 and nad5 sequences in order to be absolutely sure in genotype assignment. For this we provide a reference dataset of 93 concatenated nad2 and nad5 sequences (1394 bp in total) containing representatives of G6 and G7 (and haplogroups G7a and G7b), which can be used for the reconstruction of phylogenetic networks.
Lutzomyia migonei is incriminated as a vector of Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. Recently, this phlebotomine sand fly species has been suggested as a vector for Leishmania infantum, which causes zoonotic visceral leishmaniasis. Considering the widespread distribution of Lu. migonei in South America, the existence of isolated populations has been hypothesized. Three Lu. migonei populations, two from north-eastern Brazil (Machados, Pernambuco State, and Baturité, Ceará State) and other from the south-eastern region (Niterói, Rio de Janeiro State) were analysed both morphologically and genetically. Though no significant morphological differences were found amongst the sand fly specimens analysed, discriminant analysis based on specific morphometric characters (i.e., length of wing, antennal segment 3 and coxite for males, and length of wing and antennal segment 3 for females), showed that specimens from Machados were closer to Baturité than to Niterói. The molecular analysis of cytochrome c oxidase subunit I gene sequences also supported this observation by the distinct separation of two monophyletic clades, grouping specimens from Machados and Baturité separately from those of Niterói. Our results suggest the existence of different populations within the distribution range of Lu. migonei. Whether these populations are reproductively isolated and/or present differences in terms of vector competence/capacity for L. braziliensis and L. infantum needs to be further investigated.