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In the field of veterinary medicine, the detection of parasite eggs in the fecal samples of livestock animals represents one of the most challenging tasks, since their spread and diffusion may lead to severe clinical disease. Nowadays, the scanning procedure is typically performed by physicians with professional microscopes and requires a significant amount of time, domain knowledge, and resources. The Kubic FLOTAC Microscope (KFM) is a compact, low-cost, portable digital microscope that can autonomously analyze fecal specimens for parasites and hosts in both field and laboratory settings. It has been shown to acquire images that are comparable to those obtained with traditional optical microscopes, and it can complete the scanning and imaging process in just a few minutes, freeing up the operator's time for other tasks. To promote research in this area, the first AI-KFM challenge was organized, which focused on the detection of gastrointestinal nematodes (GINs) in cattle using RGB images. The challenge aimed to provide a standardized experimental protocol with a large number of samples collected in a well-known environment and a set of scores for the approaches submitted by the competitors. This paper describes the process of generating and structuring the challenge dataset and the approaches submitted by the competitors, as well as the lessons learned throughout this journey.

Parasites and parasitologists have always lived together in good and bad luck in a sort of forced marriage. In recent times bad luck certainly prevailed, because of increasing man-made emergencies such as wars, chemical disasters, but also because of natural disasters, amplified by climate change, that condition more and more parasite–host equilibrium. The symposium at the National Congress of the Italian Society for Parasitology, was a first occasion for Italian parasitologists to reason about ‘disaster parasitology’ and researchers’ responsibilities. Extreme weather events and their impacts on parasites’ epidemiology are illustrated, comparing disasters that recently occurred in Italy with literature data. In particular, the Sardinian Island was hit subsequently by fires and floods exacerbating the effects on ecosystems and parasite–host-relationships. Examples of Cryptosporidium outbreaks in man and Fasciola hepatica infections in various hosts after heavy rains are reviewed and effects of droughts on pasture borne parasites, such as gastro-intestinal nematodes of ruminants are discussed. Heavy rains may also cause dissemination of toxic substances released accidentally from chemical plants as happened e.g. in Milan province (IT) after the dioxin hazard. The overlapping effects of strictly man-made disasters with climate change dependent extreme weather events is further challenging the understanding of what are the consequences of disasters on ecosystems and parasite epidemiology.

Anthelmintic resistance in small ruminants is a serious worldwide problem. To reduce their spread, it is essential to know the prevalence of helminths on farms and the control practices adopted. As these studies in the Calabria region of southern Italy are fragmentary and outdated, a study on the prevalence of helminths in small ruminant holdings in this area has been conducted. The measures implemented to control helminths were also evaluated through questionnaires administered to farmers. In particular, on 90 farms (45 sheep and 45 goats), 1800 faecal samples from 900 sheep and 900 goats were collected in the spring. Using the FLOTAC dual technique, parasitological examinations demonstrated the presence of gastrointestinal nematodes in 100% of sheep and goat farms, followed by Nematodirus spp. (84.44% sheep and 48.89% goats), Moniezia spp. (73.33% sheep and 35.56% goats), Trichuris ovis (48.89% sheep and 42.22% goats), lungworms (28.89% sheep and 42.22% goats), Strongyloides papillosus (40% sheep and 26.67% goats), Dicrocoelium dendriticum (13.33% sheep and 26.67% goats), Calicophoron daubneyi (6.67% sheep and 31.11% goats), Fasciola hepatica (6.67% sheep and 4.44% goats), and Skrjabinema ovis (4.44% sheep and goats). The questionnaires showed that 82% and 85% of the farmers had applied pasture rotation, and that 93.3% and 86.6% had used anthelmintics in the previous year for sheep and goats, respectively. Only 24.4% of sheep farmers and 11.3% of goat farmers had carried out parasitological tests prior to treatments. The most used classes of anthelmintics were macrocyclic lactones and benzimidazoles, and only in 21.6% and 15.6%, for sheep and goats, respectively, was drug rotation carried out. These results denote that helminths represent a health problem for small ruminants and highlight a lack of knowledge of parasite control strategies among farmers. In these conditions, anthelmintic resistance phenomena could develop over time. Therefore, it is necessary to implement all possible strategies for the control of helminths, and to prevent the spread of anthelmintic resistance phenomena on farms in southern Italy.

Abstract Background Fasciolosis (Fasciola hepatica) and paramphistomosis (Calicophoron daubneyi) are two important infections of livestock. Calicophoron daubneyi is the predominant Paramphistomidae species in Europe, and its prevalence has increased in the last 10–15 years. In Italy, evidence suggests that the prevalence of F. hepatica in ruminants is low in the southern part, but C. daubneyi has been recently reported at high prevalence in the same area. Given the importance of reliable tools for liver and rumen fluke diagnosis in ruminants, this study evaluated the diagnostic performance of the Mini-FLOTAC (MF), Flukefinder® (FF) and sedimentation (SED) techniques to detect and quantify F. hepatica and C. daubneyi eggs using spiked and naturally infected cattle faecal samples. Methods Briefly, negative bovine faecal samples were artificially spiked with either F. hepatica or C. daubneyi eggs to achieve different egg count levels: 10, 50 and 100 eggs per gram (EPG) of faeces. Moreover, ten naturally infected cattle farms from southern Italy with either F. hepatica and/or C. daubneyi were selected. For each farm, the samples were analysed individually only with MF technique and as pools using MF, FF and SED techniques. Bayesian latent class analysis (LCA) was used to estimate sensitivity and accuracy of the predicted intensity of infection as well as the infection rate in the naturally infected farms. Results The outcome of this study showed that the highest number of eggs (F. hepatica and C. daubneyi) recovered was obtained with MF, followed by FF and SED in spiked infected samples at 50 and 100 EPG, while at lower infection levels of 10 EPG, FF gave the best results. Moreover, the sensitivity for all the techniques included in the study was estimated at > 90% at infection levels > 20 EPG for both F. hepatica and C. daubneyi eggs. However, MF was the most accurate of the three techniques evaluated to estimate fluke infection intensity. Nevertheless, all three techniques can potentially estimate infection rate at farm level accurately. Conclusions Optimization and standardization of techniques are needed to improve the FEC of fluke eggs. Graphical Abstract

Anthelmintic drug resistance has proliferated across Europe in sheep gastrointestinal nematodes (GINs). Sheep welfare and health are adversely impacted by these phenomena, which also have an impact on productivity. Finding alternatives for controlling GINs in sheep is thus of utmost importance. In this study, the anthelmintic effectiveness (AE) of a Calabrian ethnoveterinary aqueous macerate based on Punica granatum (whole fruits) was assessed in Comisana pregnant sheep. Furthermore, an examination, both qualitative and quantitative, was conducted on milk. Forty-five sheep were selected for the investigation. The sheep were divided by age, weight, physiological state (pluripara at 20 days before parturition), and eggs per gram of feces (EPG) into three homogeneous groups of 15 animals each: PG received a single oral dosage of P. granatum macerate at a rate of 50 mL per sheep; AG, treated with albendazole, was administered orally at 3.75 mg/kg/bw; and CG received no treatment. Timelines were as follows: D0, treatments, group assignment, fecal sampling, and AE assessment; D7, D14, D21, fecal sampling, and AE evaluation. The FLOTAC technique was used to evaluate the individual GIN fecal egg count (FEC) using a sodium chloride flotation solution (specific gravity = 1.20) and 100 × (1-[T2/C2]) as the formula for evaluating FEC reduction. Following the lambs’ weaning, milk was collected on the following days (DL) in order to quantify production: DL35, DL42, DL49, DL56, DL63, DL70, DL77, and DL84. The amount of milk produced by every animal was measured and reported in milliliters (ml) for quantitative evaluations. Using MilkoScan TM fT + foss electric, Denmark, the quality of the milk (casein, lactose, protein concentration, and fat, expressed as a percentage) was assessed. The macerate demonstrated a considerable AE (51.8%). Moreover, its use has resulted in higher milk production rates quantitatively (15.5%) and qualitatively (5.12% protein, 4.12% casein, 4.21% lactose, and 8.18% fat). The study showed that green veterinary pharmacology could be the easiest future approach to counteracting anthelmintic resistance in sheep husbandry.

Abstract Background Several studies in both experimentally and naturally infected dogs have reported the adulticide effect of a combination of macrocyclic lactones and doxycycline against Dirofilaria immitis, showing that these protocols can be used as an alternative to melarsomine. The present study evaluated the efficacy of oral, topical and extended-release injectable formulations of moxidectin when combined with doxycycline in dogs naturally infected with D. immitis from a shelter located in southern Italy. Methods Thirty dogs with naturally acquired D. immitis infection were divided in three groups (G) and treated with oral moxidectin (G1) once a month for 9 consecutive months, topical moxidectin (G2) once a month for 9 consecutive months or extended release moxidectin injectable (G3) at enrolment and again at 6 months (Day 180). All treatment groups received doxycycline for the first 30 days. Microfilarial concentrations in 1 ml (mff/ml) blood were determined monthly for 9 months with the modified Knott’s test. A clinical scoring system was employed for each dog enrolled in the study based on thoracic radiography and cardiac ultrasound (CU) examinations performed at Day − 15 (before treatment) and at Day 180. Results Results from the present study suggest that the majority of dogs from all treatment groups became antigen negative, as evaluated at Day 270: 9/10 dogs (90.0%) from G1, 6/10 dogs (60.0%) from G2 and 8/10 dogs (80.0%) from G3. Improvement of radiographic alterations was observed in all treatment groups, and almost all dogs were cleared of pulmonary abnormalities by 6 months from the beginning of treatment (P = 0.000). Cardiac ultrasound examination showed a progressive improvement of cardiac function in a limited number of animals (4/30). Conclusions The combination of doxycycline and three different formulations of moxidectin leads to antigen-negative status in naturally infected dogs.

Nowadays, the exclusive use of commercial anthelmintics for the treatment of gastrointestinal nematode infections in ruminants is less sustainable due to anthelmintic resistance, as well as the problem of drug residues in animal products and the environment. Therefore, an integrated therapeutic approach is needed, including the search for alternatives to synthetic anthelmintic drugs. The aim of this study was to evaluate the possibility of using the essential oil of peppermint (Mentha x piperita L.) in the control of gastrointestinal nematodes in sheep. For this purpose, the in vitro and in vivo anthelmintic efficacy of this oil and the toxic effects on the hosts were examined. In the in vitro egg hatch test, ovicidal activity varied from 21.0–90.3% depending on the concentration of essential oil used (0.0125, 0.025, 0.049, 0.195, 0.781, 3.125, 12.5, and 50 mg/mL). To some extent, anthelmintic efficacy was confirmed in the in vivo fecal egg count reduction test at a mean dose of 150 mg/kg, with an average reduction of nematode eggs of 26.9 and 46.0% at Days 7 and 14 after treatment, respectively. Furthermore, no toxic effects of applied oil were observed on sheep behavior, kidney, or liver function. The main compounds identified by gas chromatography–mass spectrometry analyzes were menthol (32.6%), menthone (22.0%), menthyl-acetate (10.0%), and isomenthone (9.39%). Due to their complex chemical compositions, numerous bioactive ingredients, and natural origin, herbal formulations represent a potentially valuable alternative for the control of gastrointestinal nematodes in sheep. In this context, the results of the present study showed that peppermint essential oil is one of the promising candidates. Further studies should be performed to collect more data on the safety profile of M. piperita EO in treated animals to find the most appropriate formulation for use in field conditions and to test it against resistant gastrointestinal nematode populations.

BackgroundSoil-transmitted helminth (STH) control programs currently lack evidence-based recommendations for cost-efficient survey designs for monitoring and evaluation. Here, we present a framework to provide evidence-based recommendations, using a case study of therapeutic drug efficacy monitoring based on the examination of helminth eggs in stool.MethodsWe performed an in-depth analysis of the operational costs to process one stool sample for three diagnostic methods (Kato-Katz, Mini-FLOTAC and FECPAKG2). Next, we performed simulations to determine the probability of detecting a truly reduced therapeutic efficacy for different scenarios of STH species (Ascaris lumbricoides, Trichuris trichiura and hookworms), pre-treatment infection levels, survey design (screen and select (SS); screen, select and retest (SSR) and no selection (NS)) and number of subjects enrolled (100-5,000). Finally, we integrated the outcome of the cost assessment into the simulation study to estimate the total survey costs and determined the most cost-efficient survey design.Principal findingsKato-Katz allowed for both the highest sample throughput and the lowest cost per test, while FECPAKG2 required both the most laboratory time and was the most expensive. Counting of eggs accounted for 23% (FECPAKG2) or ≥80% (Kato-Katz and Mini-FLOTAC) of the total time-to-result. NS survey designs in combination with Kato-Katz were the most cost-efficient to assess therapeutic drug efficacy in all scenarios of STH species and endemicity.Conclusions/significanceWe confirm that Kato-Katz is the fecal egg counting method of choice for monitoring therapeutic drug efficacy, but that the survey design currently recommended by WHO (SS) should be updated. Our generic framework, which captures laboratory time and material costs, can be used to further support cost-efficient choices for other important surveys informing STH control programs. In addition, it can be used to explore the value of alternative diagnostic techniques, like automated egg counting, which may further reduce operational costs.Trial registrationClinicalTrials.gov NCT03465488.

The aim of this study was to assess the single and synergistic effects of fenbendazole (Fenb) and metronidazole (Metro) for the treatment of Giardia duodenalis infection in different species of non-human primates (NHPs) housed in a zoological garden of southern Italy. Moreover, the study also aimed to better define the circulation of G. duodenalis zoonotic assemblages in NHP and the potential occurrence of zoonotic transmission between the staff from the zoo and NHP. Briefly, six species that belonged to four families (Lemuridae, Cercopithecidae, Atelidae, and Hylobatidae) of NHP and housed in six cages (CG) were identified as Giardia positive and divided into two groups. Group F (N = 16 animals) was treated with Fenb (50 mg/kg, every 24 h for 5 consecutive days) and Group M (N = 7 animals) was treated with Metro (25 mg/kg, two times a day for 5 consecutive days). After the first round of therapy, all the animals were retreated for 5 days by inverting the drugs in each group. On each sampling day [study days (SDs) 3–24], the samples were tested for the presence of Giardia cysts using the FLOTAC technique. Multiple fecal tests for the antigen detection of Giardia, such as rapid ELISA and direct immunofluorescence (IFA), were performed at each sampling point only on samples that resulted in positive for Giardia cysts with FLOTAC. The efficacy of Fenb ranged from 30 to 67% and for Metro ranged from 82 to 96%. The results showed the synergistic effects of Metro and Fenb (98–100%) over the combination of Fenb and Metro (52–90%) against the infection by Giardia in NHPs. The overall k agreement between FLOTAC and IFA was reached 0.858 (p = 0.0001). In contrast, all the samples had a negative antigen result when using ELISA. At molecular analysis, six samples were confirmed positive for Giardia by nested PCR. Only two positive samples were successfully sequenced that showed 100% of identity with assemblage B. All the samples from the humans included in the study resulted in negative for Giardia cysts. Overall, the study emphasizes the need for regular monitoring of Giardia infections in NHP housed in zoos by traditional diagnostic tools combined with molecular characterization of the parasite.

Soil-transmitted helminth (STH) infections are among the most common neglected tropical diseases worldwide causing high morbidity and mortality rates in endemic areas. Preventive chemotherapy (PC) programmes and health education are recommended by the World Health Organization (WHO) to reduce the impact of STH in endemic countries. Following our role as WHO collaborating centre (WHO CC ITA-116), we have developed a WebGIS and a dataset to support PC programmes to monitor the impact of STH control. This vHealth presentation shows the potentiality of these tools in improving communication among WHO’s regional and country offices, Ministries of Health, pharmaceutical industries and other partners.

The development of anthelmintic resistance in sheep gastrointestinal nematodes (GINs) requires novel strategies for the sustainable control of these parasites. This study aimed to evaluate the anthelmintic efficacy of the Origanum vulgare (L.) essential oil (EO) against sheep GINs and to evaluate the possibility of its use in control practice. The in vitro egg hatch test was conducted at eight different concentrations (50, 12.5, 3.125, 0.781, 0.195, 0.049, 0.025, and 0.0125 mg/mL) of the tested EO. For the in vivo fecal egg count reduction test, the EO of O. vulgare was administrated orally at a mean single dose of 150 mg/kg to sheep from two farms in Southern Italy, whereby potential toxic effects to the hosts were also evaluated. In the egg hatch test, the inhibition of egg hatchability varied from 71.3% to 93.7%, depending on the concentration used. The high anthelmintic potential was confirmed in the fecal egg count reduction test with an average reduction of nematode eggs in feces of 43.2% and 60.1% on days 7 and 14 after treatment, respectively. In addition, no toxic effects were noticed during the clinical examination of sheep or by observing blood count and liver or kidney function test results. The obtained results suggest the strong activity of the O. vulgare EO against sheep GINs, probably due to a high percentage of carvacrol (76.21%), whereby it can be considered safe for sheep at the dose tested in vivo. Therefore, it is suitable for use in veterinary practice as a part of an integrated strategy for the control of sheep GINs.

Parasites, in particular, gastrointestinal nematodes (GINs) represent one of the main burdens affecting small ruminant farming and pose a serious threat to their health, welfare, productivity, and reproduction. The correct management of animals and the correct use of anthelmintic drugs are the pillars of the GIN control programs for small ruminants. However, globally due to the indiscriminate use of synthetic anthelmintics, there is a significant increase in anthelmintic resistance phenomena to one or more classes of drugs. Even if such a problem never represented a serious threat in southern Italy because of the favourable environmental conditions and because of the good farm management, the phenomenon is actually showing a steep increasing trend and requires alternative treatment measures and constant monitoring. The use of phytotherapies is considered a valuable alternative approach for GIN control in small ruminants and could help with reducing the amount of synthetic drugs used and the forthcoming anthelmintic resistance. From this perspective, the Calabria territory offers a wide number of plants with anthelmintic efficacy that could be helpful for this purpose. The aim of this study was to evaluate the anthelmintic efficacy of aqueous pomegranate (Punica granatum L.) macerate compared to the treatment with Ivermectin and Albendazole in sheep naturally infected with GINs. The pomegranate macerate derives from the ethnoveterinary knowledge of the Calabria region, Southern Italy. The anthelmintic efficacy was evaluated according to the faecal egg count reduction test (FECRt) using the FLOTAC techniques in two sheep farms in Southern Italy. The FECR was calculated from individual samples using the formula FECR = 100 × (1 − [T2/C2]). The treatment with Albendazole in the first farm showed an efficacy of 99.8% after 14 days and 94.8% after 21 days, while the treatment with Ivermectin in the second farm showed an efficacy of 99.9% after 14 days and 96.5% after 21 days of treatment. The pomegranate macerate, in both farms, showed a value of efficacy of around 50% from day 7 to day 21 after the treatment. Previous studies highlighted the presence of gallic acid as the main component in the pomegranate macerate, and its efficacy in nematode control has been as well previously demonstrated in other plant extracts. This in vivo study demonstrated the unequivocal efficacy of plant macerate in easily reducing 50% of the number of GIN eggs in sheep faeces. These results, obtained without the use of synthetic anthelmintics, indicate the use of green veterinary pharmacology as a sustainable alternative to the use of synthetic drugs to reduce the increase in drug resistance phenomena and the environmental impact.

Herbal products such as essential oils may play a promising role in the treatment of infections caused by gastrointestinal nematodes (GINs). The aim of this study was to evaluate the in vitro potential of 11 essential oils (EOs) and one binary combination of isolated EO compounds, as well as the in vivo anthelmintic efficacy of two EO formulations. Four GIN genera were identified in the coproculture examination: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. The in vitro egg hatch test (EHT) was performed at six different concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL) for each EO, whereas in the in vivo faecal egg count reduction test (FECRT), each EO sample was diluted in sunflower oil and orally administrated at a dose of 100 mg/kg to the different group of animals. In the EHT, the EOs of Origanum vulgare, Foeniculum vulgare, Satureja montana, Satureja hortensis and two types of Thymus vulgaris were the most effective. The dominant compounds of these EOs were carvacrol, thymol, anethol, p-cymene and γ-terpinene, indicating their importance for the anthelmintic activity. In the FECRT, both T. vulgaris EO type 1 and linalool:estragole combination show an anthelmintic potential with a mean effect on FECR of approximately 25%. The results suggest the possible role of tested EOs as anthelmintic agents in sheep farms, although further in vivo tests are needed.