Fatigue in Multiple Sclerosis: A Resting-State EEG Microstate Study.

Fatigue affects approximately 80% of people with Multiple Sclerosis (PwMS) and can impact several domains of daily life. However, the neural underpinnings of fatigue in MS are still not completely clear. The aim of our study was to investigate the spontaneous large-scale networks functioning associated with fatigue in PwMS using the EEG microstate approach with a spectral decomposition. Forty-three relapsing-remitting MS patients and twenty-four healthy controls (HCs) were recruited. All participants underwent an administration of Modified Fatigue Impact scale (MFIS) and a 15-min resting-state high-density EEG recording. We compared the microstates of healthy subjects, fatigued (F-MS) and non-fatigued (nF-MS) patients with MS; correlations with clinical and behavioral fatigue scores were also analyzed. Microstates analysis showed six templates across groups and frequencies. We found that in the F-MS emerged a significant decrease of microstate F, associated to the salience network, in the broadband and in the beta band. Moreover, the microstate B, associated to the visual network, showed a significant increase in fatigued patients than healthy subjects in broadband and beta bands. The multiple linear regression showed that the high cognitive fatigue was predicted by both an increase and decrease, respectively, in delta band microstate B and beta band microstate F. On the other hand, higher physical fatigue was predicted with lower occurrence microstate F in beta band. The current findings suggest that in MS the higher level of fatigue might be related to a maladaptive functioning of the salience and visual network.

Baiting not-owned dogs against Echinococcus granulosus: innovative tools for integrated control.

Cystic echinococcosis (CE), caused by the larval stage of the cestode Echinococcus granulosus, is one of the most widespread zoonoses in Mediterranean countries. Baiting not-owned dogs with praziquantel (PZQ), due to their key role in the maintaining the transmission of CE, currently appears to be the most effective way to limit the transmission of CE, as well as an important aspect to introduce for the control of this parasitic disease. Therefore, this study aims to test 3 types of PZQ-based baits by evaluating different parameters (integrity over time, attractiveness and palatability for dogs, and mechanical resistance after release to different altitudes) and the bait acceptance in field by target animals, i.e. not-owned dogs, by using camera traps. The double PZQ-laced baits (with a double layer of highly palatable chews) showed the greatest resistance in the environment while also preserving the attractiveness and palatability up to 10 days, also withstood heights of 25 m, thus resulting as the most suitable also for drone delivery. The results on the field showed that most of the baits were consumed by not-owned dogs (82.2%), while the remaining were consumed by wild boars (8.9%), foxes (6.7%), badgers (1.1%) and hedgehogs (1.1%), confirming the specific and high attractiveness of the double PZQ-laced baits for the target population and highlights how an anthelmintic baiting programme may be a viable tool for the management of E. granulosus among free-ranging dog populations in endemic rural areas.

Control of gastrointestinal helminths in small ruminants to prevent anthelmintic resistance: the Italian experience.

Helminth infections are ubiquitous in grazing ruminants and cause significant costs due to production losses. Moreover, anthelmintic resistance (AR) in parasites is now widespread throughout Europe and poses a major threat to the sustainability of modern ruminant livestock farming. Epidemiological data on the prevalence and distribution of gastrointestinal nematodes, cestodes and liver- and rumen-flukes in Italian small ruminants are outdated and fragmentary. However, anthelmintics are commonly used to control these infections and often without prior diagnosis. Compared to other European countries, few reports of AR in small ruminants against levamisole, ivermectin and benzimidazoles have been published in Italy, but recent studies suggest that this phenomenon is spreading. Increased and integrated research efforts in developing innovative approaches to control helminth infections and AR are needed and must be tailored to the peculiarities of each context in order to be effectively implemented. This manuscript provides an overview on helminth prevalence and distribution, sustainable treatment strategies and integrated control approaches in small ruminants in Italy. The implementation of targeted treatments and targeted selective treatments is discussed based on different parameters, such as fecal egg count, degree of anaemia (FAMACHA© method), milk production and body condition score. In addition, several Italian studies have also investigated the implementation of alternative strategies such as the use of different natural bioactive compounds or genetic selection for resistance and resilience to helminth infections. These concrete solutions for helminth management in small ruminant farms in the country are reported and discussed, representing a valid example for other Mediterranean countries.

Epidemiological and molecular updates on hookworm species in dogs from southern Italy.

BACKGROUND: The zoonotic hookworms Ancylostoma caninum and Uncinaria stenocephala are widespread soil-transmitted helminths in dogs in Europe. Given the veterinary and public health importance of hookworms in dogs and the recent changes in the molecular epidemiology of some species, there is a need to continuously monitor the epidemiological and molecular prevalence of these parasites also at the "local" level. The present study aimed to update the epidemiological scenario of hookworm infections in both owned and stray dogs in southern Italy and to discriminate between different hookworm species (A. caninum and U. stenocephala) through molecular analyses. For this purpose, a retrospective analysis was performed over 10 years (2011-2021), including a total of 7008 owned dogs and 5642 stray dogs referred to our laboratory for copromicroscopic examinations. Moreover, 72 faecal samples, from dogs naturally infected by hookworms, were used to discriminate between A. caninum and U. stenocephala using two PCR protocols. Prior to molecular analyses, a subsample of 40/72 positive faecal samples was used for morphometric investigations on hookworm eggs. RESULTS: The results of the ten-year retrospective analysis (2011-2021) showed an overall prevalence of hookworm infection of 9.16%, specifically 5.1% in owned dogs and 14.2% in stray dogs. Logistic regression showed a significant association between positivity to hookworms and the variable "puppies" both in stray (13.84%; OR = 2.4) and owned (7.07%; OR = 2.2) dogs. The results of molecular analyses showed that positivity was confirmed only in 21/72 samples, specifically, 6 samples using protocol A and 19 with protocol B. Sequencing revealed 15 samples positive to U. stenocephala and 6 to A. caninum. CONCLUSIONS: The findings of this study showed a high prevalence of hookworm infections in dogs in southern Italy, updating the epidemiological scenario of the last decade. Moreover, the results of the study revealed the first identification of hookworm species in dogs in Italy by molecular studies, highlighting that U. stenocephala is more prevalent than A. caninum.

Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples.

Toxoplasmosis is a zoonotic disease caused by the protozoan parasite Toxoplasma gondii. Infection in humans has usually been related to the consumption of raw, undercooked or cured meat. The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR)-based assay for the detection and quantification of T. gondii in meat samples. To optimize the ddPCR, T.gondii reference DNA aliquots at five known concentrations: 8000 cg/µl, 800 cg/µl, 80 cg/µl, 8 cg/µl were used. Moreover, results obtained by ddPCR and quantitative PCR (qPCR) were compared using 80 known samples (40 positive and 40 negative), as well as 171 unknown diaphragm tissue samples collected at slaughterhouses. The ddPCR showed a sensitivity of 97.5% and a specificity of 100%, with a detection limit of 8 genomic copy/µl of T. gondii. A nearly perfect agreement (κ = 0.85) was found between results obtained by ddPCR and qPCR for both positive and negative known samples analysed. On the 171 diaphragm tissue samples from field, 7.6% resulted positive by ddPCR and only 1.2% by qPCR. Therefore, this innovative method could be very useful for the detection of T. gondii in meat samples, aiming to prevent human infections.

The Kubic FLOTAC microscope (KFM): a new compact digital microscope for helminth egg counts.

The Kubic FLOTAC microscope (KFM) is a compact, low-cost, versatile and portable digital microscope designed to analyse fecal specimens prepared with Mini-FLOTAC or FLOTAC, in both field and laboratory settings. In this paper, we present the characteristics of the KFM along with its first validation for fecal egg count (FEC) of gastrointestinal nematodes (GINs) in cattle. For this latter purpose, a study was performed on 30 fecal samples from cattle experimentally infected by GINs to compare the performance of Mini-FLOTAC either using a traditional optical microscope (OM) or the KFM. The results of the comparison showed a substantial agreement (concordance correlation coefficient = 0.999), with a very low discrepancy (−0.425 ± 7.370) between the two microscopes. Moreover, the KFM captured images comparable with the view provided by the traditional OM. Therefore, the combination of sensitive, accurate, precise and standardized FEC techniques, as the Mini-FLOTAC, with a reliable automated system, will permit the real-time observation and quantification of parasitic structures, thanks also to artificial intelligence software, that is under development. For these reasons, the KFM is a promising tool for an accurate and efficient FEC to improve parasite diagnosis and to assist new generations of operators in veterinary and public health.

Unveiling the relationship between autonomic involvement, fatigue, and cognitive dysfunction in early relapsing-remitting multiple sclerosis.

BACKGROUND: Fatigue is a common, yet disabling, symptom in patients with multiple sclerosis (PwMS). Fatigue has shown to be associated with self-reported autonomic nervous system (ANS) symptoms, particularly for cognitive fatigue; however, the question whether ANS involvement is related to cognitive impairment has never been addressed. We performed a study to unveil the complex relationship between fatigue, ANS symptoms, and cognitive impairment. METHODS: We prospectively recruited early PwMS that were tested with Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS), State-Trait Anxiety Inventory (STAI), Beck Depression Inventory (BDI), Modified Fatigue Impact Scale (MFIS) and Composite Autonomic Symptoms Scale-31 (COMPASS-31) scale. We performed a comparison between fatigued and non-fatigued patients and between cognitive unimpaired and impaired patients. We evaluated the association of COMPASS-31, MFIS, BDI, STAI, and BICAMS scores, and the analysis was repeated for each scale's sub-scores. A multivariable analysis was performed to elucidate predictors of fatigue. RESULTS: Forty-four patients were recruited. Fatigued patients had higher COMPASS-31 total, orthostatic intolerance, secretomotor, and pupillomotor scores. No differences in fatigue and ANS symptoms were found between cognitive impaired and unimpaired patients. MFIS total score correlated with STAI state (p = 0.002) and trait (p < 0.001), BDI (p < 0.001), COMPASS-31 total (p < 0.001), orthostatic intolerance (p < 0.001), pupillomotor scores (p = 0.006). Multivariable analysis showed that BDI (p < 0.001) and COMPASS-31 (p = 0.021) predicted MFIS score. Sub-scores analysis showed that orthostatic intolerance has a relevant role in fatigue. CONCLUSION: ANS symptoms are closely related with fatigue. Orthostatic intolerance may have a predominant role. Cognitive impairment seems not to be associated with ANS symptoms.

Nabiximols and botulinum toxin injections for patients with multiple sclerosis: efficacy on spasticity and spasms in a single-centre experience.

BACKGROUND: Spasticity is a common and disabling symptom in patients with multiple sclerosis (PwMS): as highlighted by many epidemiological studies, it is often a severe and not well treated. Despite the availability of evidence-based spasticity management guidelines, there is still great variability in everyday therapeutic approach, especially for the most complex cases. METHODS: In our single-centre study, we retrospectively evaluated PwMS-treated nabiximols and botulinum toxin injections (BTI) from July 2015 to April 2019. Clinical and demographic data were collected. The severity of spasticity and spasms was recorded by modified Ashworth Scale (mAS) and Penn Spasm Frequency Scale (PSFS) at baseline and after 1 month of treatment. RESULTS: We evaluated 64 treatments for MS-related spasticity: 28 patients were treated with BTI and 36 patients with nabiximols. We found that both BTI and nabiximols are effective in reducing mAS (nabiximols, BTI: p < 0.001), PSFS frequency (nabiximols: p = 0.001, BTI: p = 0.008) and intensity (nabiximols: p = 0.001, BTI p = 0.016). No differences were found when directly comparing the efficacy of the two treatments, except for a statistical trend favouring BTI on spasms intensity (p = 0.091). Eleven patients were treated with both BTI and nabiximols, and only four patients continued both treatments. All dropouts were due to inefficacy of at least one of the two therapies. CONCLUSIONS: Our single-centre experience highlights that both BTI and nabiximols are effective in treating multiple sclerosis-related spasticity; however, BTI treatment may be more effective on spasms intensity. Combined nabiximols and BTI treatment could represent a therapeutic option for severe spasticity.

Early putamen hypertrophy and ongoing hippocampus atrophy predict cognitive performance in the first ten years of relapsing-remitting multiple sclerosis.

BACKGROUND: The first years of relapsing-remitting multiple sclerosis (RRMS) constitute the most vulnerable phase for the progression of cognitive impairment (CImp), due to a gradual decrease of compensatory mechanisms. In the first 10 years of RRMS, the temporal volumetric changes of deep gray matter structures must be clarified, since they could constitute reliable cognitive biomarkers for diagnostic, prognostic, and therapeutic purposes. METHODS: Forty-five cognitively asymptomatic patients with RRMS lasting ≤ 10 years, and with a brain MRI performed in a year from the neuropsychological evaluation (Te-MRI), were included. They performed the Brief International Cognitive Assessment battery for MS. Thirty-one brain MRIs performed in the year of diagnosis (Td-MRI) and 13 brain MRIs of age- and sex-matched healthy controls (HCs) were also included in the study. The relationships between clinical features, cognitive performances, and Te- and Td-MRI volumes were statistically analyzed. RESULTS: Cognitively preserved (CP) patients had significantly increased Td-L-putamen (P = 0.035) and Td-R-putamen volume (P = 0.027) with respect to cognitively impaired (CI) ones. CI patients had significantly reduced Te-L-hippocampus (P = 0.019) and Te-R-hippocampus volume (P = 0.042) compared, respectively, with Td-L-hippocampus and Td-R-hippocampus volume. Td-L-putamen volume (P = 0.011) and Te-L-hippocampus volume (P = 0.023) were independent predictors of the Symbol Digit Modalities Test score in all patients (r2 = 0.31, F = 6.175, P = 0.001). CONCLUSION: In the first years of RRMS, putamen hypertrophy and hippocampus atrophy could represent promising indices of cognitive performance and reserve, and become potentially useful tools for diagnostic, prognostic, and therapeutic purposes.

Effects of a cooling vest with sham condition on walking capacity in heat-sensitive people with Multiple Sclerosis.

PURPOSE: Heat sensitivity is a common contraindication in people with Multiple Sclerosis (pwMS), and physical fatigue is one of the most frequently reported symptoms that can affect quality of life. Increases in body temperature may exacerbate fatigue and heat-related symptoms. Decreasing body temperature via cooling devices may mitigate disease symptoms and improve physical abilities and quality of life. This study evaluates the effects of a cooling vest with sham condition on walking capacity using a commercially-available cooling vest specifically designed for pwMS. METHODS: A counter-balanced, cross-over design was used to assess the effects of a cooling vest (CryoVest Comfort, CryoInnov, France) (COLD) from a menthol-induced sham condition (CON) on ground walking time to exhaustion (Tex, s) and distance at exhaustion (Dex, m) in ambulatory pwMS. Secondary outcomes were heart rate (HR, bpm), thermal sensation (Tsens), skin chest (Tchest) and back (Tback) temperature. RESULTS: Ten females with Multiple Sclerosis (59 ± 9 years, EDSS 3.0-5.5) participated to the study. During COLD, pwMS walked significantly longer (1896 ± 602 vs. 1399 ± 404 s, p < 0.001) and farther (1879 ± 539 vs. 1302 ± 318 m, p < 0.001) than CON. Importantly, Tsens and HR at exhaustion were not significantly different between conditions, although Tchest (- 2.7 ± 1.8 °C, p < 0.01) and Tback (- 3.9 ± 1.8 °C, p < 0.001) were lower at volitional fatigue during COLD. CONCLUSION: The lightweight cooling vest improved total walking time and distance in heat-sensitive pwMS. These physiological improvements were likely due to feeling perceptually cooler in the COLD trial, compared to the corresponding point of fatigue in the CON condition.

Efficacy of fluralaner spot-on solution for the treatment of Ctenocephalides felis and Otodectes cynotis mixed infestation in naturally infested cats.

BACKGROUND: Cats can be infested with several ectoparasite species, especially Ctenocephalides felis and Otodectes cynotis. The aim of this study was to evaluate the efficacy of a single topical application of fluralaner against C. felis and O. cynotis natural infestation in stray (study 1) and owned (study 2) cats in central and southern Italy. RESULTS: The number of live fleas found on each cat on Day 0 ranged from 1 to more than 30 (arithmetic mean live flea count = 11.9 in study 1; 14.6 in study 2) while no live fleas were found on days 7 and 84 post topical application of fluralaner. The number of live mites found on each cat on Day 0 ranged from 1 to 42 (arithmetic mean live mite count = 6.4 in study 1; 8.9 in study 2) while no live mites were found on days 7 and 84 post topical application of fluralaner. CONCLUSIONS: Topical fluralaner completely eliminated fleas and ear mites from infested cats and was 100% effective against both parasites up to 84 days after treatment.

The recovery of added nematode eggs from horse and sheep faeces by three methods.

BACKGROUND: Nematode infections in horses are widespread across the world. Increasing levels of anthelmintic resistance, reported worldwide in equine parasites, have led to the creation of programs for the control of nematodes based on faecal egg counts (FEC). To improve nematode egg counting in equine faecal samples and establish whether the matrix of equine faeces or the eggs affect the counts, the analytical sensitivity, accuracy and precision of Mini-FLOTAC (combined with Fill-FLOTAC), McMaster and Cornell-Wisconsin techniques were compared. Known numbers of eggs extracted from equine or ovine faeces were added to egg free ovine and equine faeces to give counts of 10, 50, 200 and 500 eggs per gram (EPG) of faeces. RESULTS: The Cornell-Wisconsin significantly underestimated egg counts and McMaster showed a low analytical sensitivity, revealing 100% of sensitivity only for concentrations greater than 200 EPG. EPG values detected by Mini-FLOTAC did not differ significantly from expected counts at any level of egg density. CONCLUSIONS: Mini-FLOTAC combined to Fill-FLOTAC which provides an accurate method of weighing without need for a balance and filtering out debris, could be used for FEC on the farm as well as in the laboratory.

Innovative tools for the diagnosis of Echinococcus granulosus in definitive hosts.

The aim of this study was to develop and validate an innovative protocol for the diagnosis of Echinococcus granulosus and other Taeniidae in dogs. For this purpose, three experiments were performed, using faecal samples from naturally infected dogs. Firstly, the FLOTAC technique was calibrated with five flotation solutions: saturated sodium chloride (specific gravity, s.g. = 1.20), zinc sulphate (s.g. = 1.35), zinc chloride (s.g. = 1.45), Breza (s.g. = 1.30) and modified Breza (s.g. = 1.40). Then, FLOTAC was compared with four techniques of flotation in centrifuge using: zinc sulphate (s.g. = 1.20), Breza (s.g. = 1.30), modified Breza (s.g. = 1.40), and zinc chloride (s.g. = 1.45). Finally, four different protocols of DNA extraction were compared for Taeniidae identification: QIAamp Tissue Kit and QIAamp Stool from eggs; QIAamp Stool and Wizard Magnetic Purification System for Food from faeces. FLOTAC with zinc sulphate was the most efficient method to detect Taeniidae eggs, showing highest mean of eggs per gram (EPG) of faeces. The QIAamp Stool, using eggs concentrated by FLOTAC, was the best method for DNA extraction. The combination of these protocols provided the highest number of positive samples with PCR, i.e., 47/50 (94.0%). The three negative samples showed a low faecal egg count (2 EPG) below the detection limit (4 EPG) of the protocol. From sequencing of the 47 positive samples: 6 samples were identified as E. granulosus sensu stricto (s.s.), 28 as Taenia hydatigena and 6 as T. pisiformis. A co-infection between different genera of Taeniidae was found in 7 samples.

Eprinomectin pour-on (EPRINEX® Pour-on, Merial): efficacy against gastrointestinal and pulmonary nematodes and pharmacokinetics in sheep.

BACKGROUND: The anthelmintic efficacy of the 0.5% w/v topical formulation of eprinomectin (EPN), EPRINEX® Pour-on (Merial) when administered at 1 mg/kg body weight was evaluated in sheep in two dose confirmation laboratory studies and one multicenter field study. In addition, the pharmacokinetics of EPN when administered at that dosage to adult sheep was determined. RESULTS: In the two dose confirmation studies, which included 10 sheep each, sheep treated with topical EPN had significantly (p < 0.05) fewer of the following nematodes than the untreated sheep with overall reduction of nematode counts by >99%: adult Dictyocaulus filaria, Haemonchus contortus, Teladorsagia circumcincta(pinnata/trifurcata), Trichostrongylus axei, T. colubriformis, T. vitrinus, Cooperia curticei, Nematodirus battus, Strongyloides papillosus, Chabertia ovina and Oesophagostomum venulosum, and inhibited fourth-stage Teladorsagia larvae. A total of 196 sheep harboring naturally acquired gastrointestinal nematode infections were included in the field efficacy study at two sites each in Germany (48 Merino x Ile de France lambs, 52 adult Merino females) and in Italy (adult male and female Bagnolese, Lacaune, Lacaune x Bagnolese, Bagnolese x Sarda sheep; 48 animals per site). Animals were blocked on pre-treatment body weight and within each block, one animal was randomly assigned to the control (untreated) group and three animals were randomly assigned to be treated with topical EPN. Examination of feces 14 days after treatment demonstrated that, relative to the controls, topical EPN-treated sheep had significantly (p < 0.0001) lower strongylid egg counts. Reduction was ≥97% at each site and 98.6% across all sites. Pharmacokinetics of EPN following single treatment with topical EPN were determined in eight ~4.5 year old female Merino cross sheep based on the analysis of plasma samples which were collected from two hours to 21 days following treatment. The main pharmacokinetic parameters were: Cmax 6.20 ± 1.71 ng/mL, AUClast 48.8 ± 19.2 day*ng/mL, Tmax 3.13 ± 2.99 days and T1/2 6.40 ± 2.95 days. No treatment-related health problems or adverse drug events were observed in any study. CONCLUSION: These studies demonstrated 0.5% w/v EPN administered topically at 1 mg/kg body weight to be highly efficacious against a broad range of ovine gastrointestinal nematodes and D. filaria lungworms and well tolerated by sheep of different ages, breeds, gender and physiological status.

Dirofilaria immitis and Angiostrongylus vasorum: the contemporaneous detection in kennels.

BACKGROUND: The cardiopulmonary nematodes Dirofilaria immitis and Angiostrongylus vasorum are increasingly reported in dogs and are responsible for two diseases with overlapping endemic areas, especially in Europe: dirofilariosis and angiostrongylosis. The reasons for their apparent emergence are unknown, but several factors (e.g. increased disease awareness, better diagnostic tools, climatic changes, seasonal population dynamics and movements of animals) may play a role in the recent rise in reports of infection in the various countries of Europe. The aim of this study was to investigate the seroprevalence of D. immitis (by DiroCHECK® ELISA) and the fecal presence of first stage larvae (L1) of A. vasorum (by FLOTAC) in dogs from 68 kennels of the Campania region (southern Italy). The fecal samples were collected from pooled samples using the box as epidemiological unit. To the authors's knowledge, this is the first cross-sectional survey conducted at regional-scale in Italy and in Europe on the contemporaneous detection of D. immitis antigens and A. vasorum L1 in kennels. RESULTS: Antigens of D. immitis were detected in 24/537 (4.4%; 95% Confidence Interval = 3.0-6.7) dogs in 6 out of the 68 kennels (8.8%; 95% CI = 3.6-18.9). The 24 positive samples for D. immitis antigen were tested also with AngioDetect® and only 1 sample was seropositive for A. vasorum with a prevalence of 4.2%. A. vasorum L1 were detected in dogs from 9 out of the 68 kennels (13.2%; 95% CI = 21.8-44.9). Pooled fecal samples from 25 boxes out of the 1360 analyzed resulted positive to A. vasorum L1 (1.8%; 95% CI = 1.2-2.7). CONCLUSIONS: The present study indicates that cardiopulmonary nematodes are present in Campania region in symptomatic dogs as well as in asymptomatic ones. Therefore, regular parasitological surveillance, appropriate treatment strategies and high quality standard of hygiene are required to guarantee the health and welfare of kennel dogs.

Comparison of different molecular protocols for the detection of Uncinaria stenocephala infection in dogs.

The present study aims to assess the performance of different molecular targets using various matrices of samples for the detection of Uncinaria stenocephala (US) in hookworm infected dogs. To this end, the DNA extraction was performed on the following matrices of samples: (i) larvae of US obtained from experimentally infected dogs with US with different larvae counts per microliter (µl); (ii) pure US eggs suspension in distilled water with different egg counts per µl; (iii) spiked dog fecal samples with different US eggs per gram (EPG) of feces; (iv) feces from dogs naturally infected with hookworm eggs; (v) fecal suspension with hookworm eggs recovered from the FLOTAC apparatus. All the samples were tested with four different PCR protocols targeting specific regions for the detection of both hookworms US and AC as follows: Protocol A (ITS1, 5.8 S, ITS2) and Protocol B (18 S) for the detection of both species, Protocol C (ITS1) for the detection of AC and Protocol D (ITS1) for the detection of US. The best results were obtained with DNA extracted from US larvae matrix obtained from experimentally infected dogs, showing a detection limit of 3.5 larvae/ml for the protocols A, B and D. A moderate correlation was found between the FLOTAC technique and PCR protocols B and D with respect to fecal samples from dogs naturally infected with hookworms. Indeed, PCR protocols B (18 S) and D (ITS1) gave the best results for feces and fecal suspension from naturally infected dogs. However, all the PCR protocols used showed lower sensitivity than FLOTAC technique. Perhaps, isolating US eggs in advance could help to obtain better quality and quantity of DNA, avoiding some notable factors such as inhibitors present in faecal samples. However, a further study is needed to evaluate and standardise a protocol for the recovery of parasitic elements, that could be applied prior to DNA extraction. Therefore, this could lead to a better amplification of US eggs DNA. In conclusion, our results showed that the type of sample (sample-matrix) used for the DNA extraction samples is crucial, as this affects the diagnostic sensitivity of the technique.

Comparison of Direct and Indirect Detection of Toxoplasma gondii in Ovine Using Real-Time PCR, Serological and Histological Techniques.

Toxoplasma gondii is a zoonotic pathogen and the ingestion of tissue cysts by consumption of lamb or mutton has been identified as a possible cause of infection in humans. Many serological surveys in sheep have been performed, showing relevant serological rates; however, while the detection of antibodies indicates an exposure to T. gondii, this does not necessarily imply the presence of tissue cysts in edible tissue. The current study aims to provide further understanding on the occurrence of T. gondii in sheep muscles and the strength of correlation between serological positivity and presence of the parasite in sheep. From 349 sheep, samples (i.e., blood, heart and diaphragm) were collected and subjected to ELISA tests, real-time PCR and histological tests. Despite the high seroprevalence, T. gondii DNA was detected in the heart and/or the diaphragm from 13 out of the 349 tested sheep (3.7%); all were adults (13/191). Furthermore, the histological tests did not reveal the presence of T. gondii tissue cysts in any of the examined portions of interventricular septum. It should be considered that the likelihood of detecting genetic material of the parasite is probably influenced by the uneven distribution of the tissue cysts in the carcass as well as the methodology applied. The findings of this study support the importance of describing the uncertainty associated with the data used for risk assessment to reduce inaccurate estimation or risk overestimation.

Prevalence of Helminths in Small Ruminant Farms and Evaluation of Control Practices Used to Counter Anthelmintic Resistance in Southern Italy.

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.

Effect of pomegranate (Punica granatum) anthelmintic treatment on milk production in dairy sheep naturally infected with gastrointestinal nematodes.

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.

An innovative strategy for deworming dogs in Mediterranean areas highly endemic for cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus sensu lato, is a zoonotic parasitic disease of economic and public health importance worldwide, especially in the Mediterranean area. Canids are the main definitive hosts of the adult cestode contaminating the environment with parasite eggs released with feces. In rural and peri-urban areas, the risk of transmission to livestock as well as humans is high because of the free-roaming behavior of owned/not owned dogs. Collecting data on animal movements and behavior using GPS dataloggers could be a milestone to contain the spread of this parasitosis. Thus, this study aims to develop a comprehensive control strategy, focused on deworming a dog population in a pilot area of southern Italy (Campania region) highly endemic for CE. METHODS: Accordingly, five sheep farms, tested to be positive for CE, were selected. In each sheep farm, all shepherd dogs present were treated every 2 months with praziquantel. Furthermore, 15 GPS dataloggers were applied to sheep and dogs, and their movements were tracked for 1 month; the distances that they traveled and their respective home ranges were determined using minimum convex polygon (MCP) analysis with a convex hull geometry as output. RESULTS: The results showed that the mean daily walking distances traveled by sheep and dogs did not significantly differ. Over 90% of the point locations collected by GPS fell within 1500 mt of the farm, and the longest distances were traveled between 10:00 and 17:00. In all the sheep farms monitored, the area traversed by the animals during their daily activities showed an extension of < 250 hectares. Based on the home range of the animals, the area with the highest risk of access from canids (minimum safe convex polygon) was estimated around the centroid of each farm, and a potential scheme for the delivery of praziquantel-laced baits for the treatment of not owned dogs gravitating around the grazing area was designed. CONCLUSIONS: This study documents the usefulness of geospatial technology in supporting parasite control strategies to reduce disease transmission.