The in vitro life cycle of zoonotic helminths is an essential tool for -omic translational studies focused on disease control and treatment. Anisakiosis is an emerging zoonosis contracted by the ingestion of raw or undercooked fish infected with the third stage larvae (L3) of two sibling species Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii, the latter being the predominant species in the Mediterranean basin. Recently, in vitro culture of A. pegreffii has been developed to enable fast and large-scale production of fertile adults. However, the conditions for larval development from hatching to infective L3 were not fulfilled to complete the cycle. Herein, we used a Drosophila medium supplemented with chicken serum and adjusted different osmolarities to maintain the culture of L3 hatched from eggs for up to 17 weeks. The highest survival rate was observed in the medium with the highest osmolarities, which also allowed the highest larval exsheathment rate. Key morphological features of embryogenesis and postembryogenesis studied by transmission electron microscopy revealed that the excretory gland cell is differentiated already up to 48h post-hatching. Extracellular vesicles and cell-free mitochondria are discharged between the two cuticle sheets of the secondstage larvae (L2). Contemporarly cultivated, two populations of adult A. simplex s.s. and A. pegreffii reached an average production of 29,914.05 (±27,629.36) and 24,370.96 (±12,564.86) eggs/day/female, respectively. The chromosome spreads of A. pegreffii obtained from mature gonads suggests a diploid karyotype formula of 2n=18. The development of a reliable protocol for the in vitro culture of a polyxenous nematode such as Anisakis spp. will serve to screen for much needed novel drug targets, but also to study the intricated and unknown ecological and physiological traits of these trophically transmitted marine nematodes.

Animal infection models are crucial for studying various aspects of Ehrlichia canis infections. To understand the pathogenesis of the first Chinese isolate of E. canis and simulate the natural progression of canine ehrlichiosis, we developed a model with 18 Beagle dogs that consisted of E. canis initial infection (days 0-17), treatment with doxycycline or rifampicin (days 18-32), recovery (days 33-66), E. canis reinfection (days 67-91), and Babesia vogeli superinfection (days 92-116). We measured body weight and rectal temperature every other day, drew blood every 4 days for routine hematology and biochemistry tests, and for quantification of E. canis and B. vogeli by quantitative PCRs. In this study, the first isolate of E. canis from China was used to experimentally infect dogs, and the infected dogs exhibited clinical signs of acute severe ehrlichiosis, including high fever, loss of appetite, dehydration, and body weight loss, confirming the similar pathogenicity of E. canis in China as compared to isolates from other regions. Infection with E. canis and B. vogeli led to reduced body weight and fever in dogs. Doxycycline treatment led to absence of E. canis DNA in infected dogs, while rifampicin treatment lowered the blood E. canis copy number up to 1.5 folds. E. canis-free infected dogs after doxycycline treatment were successfully re-infected with E. canis, indicating dogs with antibodies are still at risk of re-infection. Super-infection with B. vogeli resulted in higher fever, more severe anemia, and a reduced number of platelets. Splenectomized dogs showed significantly higher E. canis numbers during recovery and re-infection than intact dogs. The histological changes were observed in brain, lung, kidney, liver and spleen of the infected dogs. The findings in this study provide insights into clinical and hematologic responses, as well as effective treatment options, for dogs infected with the first Chinese isolate of E. canis, and may contribute to our understanding of the diagnosis and prevention of tick-borne diseases in dogs, including canine monocytic ehrlichiosis.

Our laboratory's vaccine development strategy against the livestock parasite Fasciola hepatica centres around disrupting key biological processes by combining groups of antigens with similar/complementary functional actions into a single vaccine cocktail. In this study the focus was on antioxidant protein vaccines and a protease inhibitor vaccine aimed at disrupting the parasite's ability to defend against oxidative stress and protease-inhibitor balance, respectively. Two combinations of recombinantly expressed antioxidants were assessed, namely peroxiredoxin (rFhPrx), thioredoxin (rFhTrx) and thioredoxin-glutathione reductase (rFhTGR) (Group 1) and rFhPrx, rFhTrx, and two superoxide dismutases (rFhSOD1 and rFhSOD3) (Group 2). The protease inhibitor vaccine cocktail included representatives of each of the key secreted protease inhibitor families, namely a Kunitz-type inhibitor (rFhKT1), a serpin (rFhSrp1) and a stefin, (rFhStf1) (Group 3). The vaccine combinations were formulated in adjuvant Montanide 61VG administered at five timepoints; two before experimental challenge with 60F. hepatica metacercariae and three after infection. The vaccine combinations did not reduce the liver fluke burden, and only Group 2 displayed a marginal reduction in egg viability (8.2%). Despite previous results showing an effect of liver fluke vaccines on overall weight gain in infected animals, no significant (P value >0.05) impact on weight gain was observed in this study. Antibodies were elicited against all the vaccine antigens within the cocktails and were maintained at high levels to the end of the trial, due to our strategy of continuing vaccine administration after infection. However, these responses were not boosted by the challenge F. hepatica infection. A comparative analysis with previous vaccine data using a protease inhibitor vaccine found no repeat of the promising outcomes associated with this vaccine, indicating that the addition of rFhSrp1 to the vaccine cocktail did not improve vaccine efficacy. Assessment of liver pathology across the two trials using a modified liver enzyme score (glutamate dehydrogenase to platelet ratio) at eight weeks post infection suggests an association with liver fluke burden above 45 flukes, which could be used to predict liver pathology in future trials. The results reported in this study highlight the ambiguousness in liver fluke vaccine development and the difficulty in obtaining consistent and repeatable protection. This work stresses the need for repetition of trials and the use of sufficiently sized groups to assess vaccine efficacy with adequate statistical power.

Equine piroplasmosis (EP) is caused by Theileria equi and Babesia caballi, transmitted by tick vectors. Horses can suffer an acute, subacute, and chronic forms of the disease, with clinical signs such as poor performance, fever, pale mucosal membranes, and jaundice. The diagnosis of EP subclinical cases is complex due to the sensitivity of real-time PCR and the limited parasite load in some carriers, making it challenging to differentiate them from seropositive, PCR negative (S+PCR-) individuals. This study aimed to describe haematological and biochemical changes in asymptomatic EP carriers, EP S+PCR- horses and control horses (EP seronegative and PCR negative). It also investigated potential haemato-biochemical markers to aid in distinguishing true EP carriers alongside molecular and serological tests. A comprehensive haematology and biochemistry profile was conducted on 410 sera and EDTA blood samples, comprising 130 EP positives by real-time PCR and competitive ELISA (cELISA) (carriers), 130 EP negatives by real-time PCR but positive to cELISA (S+PCR-) and 150 EP negative horses to real-time PCR and c-ELISA (controls). Our study confirmed that a haematological and biochemistry profile could help to differentiate between EP carriers/S+PCR- from healthy horses. Carriers and S+PCR- horses showed significant increases in the white blood cell count (WBC), high total proteins (TP) and total globulins (GLOB) concentration, and liver function markers compared to controls. Additionally, the evaluation of uric acid (UA) suggested oxidative stress in carrier horses. However, no useful haemato-biochemical diagnostic markers were identified to aid the challenging differentiation of EP carriers and S+PCR- horses, highlighting the need for improvement in molecular/serological diagnosis for these horses.

The study presents the results of a cross-sectional survey to describe the epidemiology of ascarid and strongylid nematodes in horses, the impact of diverse climatic conditions on parasite diversity and the levels of faecal egg shedding in different age groups of managed Thoroughbred horses. Individual faecal samples (n=1377) collected from 62 Thoroughbred farms across four climatic zones in Australia were analysed using the modified McMaster technique for faecal egg counts (FECs) and strongylid nematodes were identified utilising PCR-directed next-generation sequencing (NGS) of the second internal transcribed spacer of the nuclear ribosomal DNA (ITS-2). Across all age groups, the prevalence of ascarid and strongylid nematodes was 12% (95% confidence interval 10-14%) and 72% (70-74%), respectively. Based on strongylid FECs, yearlings had the highest prevalence (89%) followed by weanlings (83%), foals (79%), wet mares (61%), dry mares (59%) and stallions (54%). However, for Parascaris spp., foals had the highest prevalence (46%) followed by weanlings (32%) and yearlings (13%). The highest mean FECs for Parascaris spp. were observed in foals (418 eggs per gram [EPG] of faeces) while those for strongylids were in yearlings (1002 EPG). Of the adult horses (mares and stallions), 67% (489 of 729) and 11% (77 of 729) were low (i.e., ≤250 EPG) and moderate (i.e., 251-500 EPG) strongylid egg-shedders, respectively. Strongylid egg shedding varied across climatic zones, with the highest mean FECs in the summer rainfall (723 EPG) followed by non-seasonal rainfall (629 EPG), winter rainfall (613 EPG), and Mediterranean (606 EPG) rainfall zones. Twenty-three nematode species were detected using NGS, with Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%) and Coronocyclus coronatus (23%), being the most abundant species. Three species of Strongylus (i.e., S. vulgaris, S. equinus and S. edentatus) were also detected. The nemabiome composition, species richness and relative abundance varied within horse age and between climatic zones. These empirical findings provide a comprehensive understanding of the prevalence of parasites within horse populations and the multifaceted factors that influence their occurrence, thereby allowing for the formulation of tailored strategies aimed at parasite control in domestic horses.

We aimed to validate a targeted selective treatment (TST) methodology for treating parasitic gastrointestinal infections in ewes in different physiological states using parasitological and hematological parameters. Forty ewes were monitored from December 2021 to June 2022 and evaluated during various physiological stages in their life cycle. Before starting the experiment, a fecal egg count (FEC) reduction test was performed to evaluate the efficacy of the anthelmintic (AH) treatment. Weekly assessments were performed based on the Famacha© (F) system and body condition score (BCS), and ewes were subjected to AH treatment when necessary, with their physiological states recorded. Ewes were treated when they presented F≥3, BCS≤2.0 (when F=2), or submandibular edema. Parasitological, i.e., FEC, and hematological, i.e., hematocrit (Ht), parameters were evaluated monthly to determine the efficiency of the TST methodology. Comparisons between the mean Ht and FEC values in ewes subjected to AH treatment and untreated ewes were performed using analysis of variance, followed by Tukey's test. Spearman's correlation was performed to determine the correlation between the variables, i.e., F scores, BCS, Ht, and FEC. All tests were performed at a significance level of 5 %. During the experimental period, 1138 evaluations were performed. The main reason for AH treatment was F≥3. Ewes in early pregnancy, lactation and late pregnancy received comparatively more AH treatments than the other physiological states. Ewes in late pregnancy and lactation exhibited lower mean Ht values (23.5 % and 22.9 %) and higher mean FEC values (3269 and 1426) compared with those in early pregnancy (30.2 % and 727 EPG). In addition, a statistically significant difference was observed in the Ht and FEC values of ewes that presented submandibular edema (P<0.001) compared with those that did not exhibit submandibular edema. The genus Haemonchus sp. showed a 96.4 % prevalence in coprocultures. A positive correlation existed between F scores and FEC (r=0.3819) and a negative correlation between F scores and Ht (r=-0.4728). Ewes that needed AH treatment had lower mean Ht values than ewes that did not need the treatment (19.2 % × 29.3 %; P<0.001) and higher mean FEC values (8747× 1163; P<0.001), confirming that these ewes needed AH treatment. The TST methodology based on F scores, BCS, and submandibular edema could effectively identify individuals in the herd needing AH treatment, identifying 13 % additional cases requiring treatment than using only the F score criterion.

Parasitic diseases are considered to be a cause of oxidative stress which leads to oxidative damage of various molecules including DNA. This can result in mutations, replication errors, and genome instability. Therefore, aim of this study was to measure DNA damage induced by Dirofilaria immitis in the single cells such as dogs' leukocytes using the comet assay. Also, we monitored the effects of antiparasitic treatment on mitigation of sensitivity to DNA damage in leukocytes treated with H2O2 using the in vivo and ex vivo comet assay. The whole blood samples from 34 dogs from Serbia were used, both males and females, from one to 13 years old, both pure and mixed-breeds. A rapid immunochromatographic test (Antigen Rapid Heartworm Ag 2.0 Test Kit, Bionote, Minnesota, USA) was used for the detection of D. immitis antigens. The modified Knott's test and PCR were used in the aim of detecting D. immitis microfilariae in dogs' blood, and evaluating the number of circulating microfilariae during the treatment. The genotoxicity evaluation showed that D. immitis infection resulted in DNA damage in naturally infected dogs, with the highest DNA damage occurring in the group of dogs with severe clinical signs. Treatment with ivermectin and doxycycline decreased DNA damage in leukocytes of dogs in all groups, as the intensity of infection decreased due to applied therapy. Ex vivo comet assay results showed that leukocytes exhibited decreased sensitivity to H2O2-induced DNA damage during treatment. The results of the modified Knott's test and PCR in our study showed that treatment with ivermectin and doxycycline was successful in decreasing the average number of microfilariae during the time and at the end eliminating them from the dogs' blood.

Fasciolosis is a widely distributed zoonosis reported over 81 countries around the world. Good and early diagnostic method is critical in controlling this disease and prevention of injury to the liver and bile ducts. In this study, we identified a novel member (cathepsin L7) of cathepsin family from Fasciola spp.. Firstly, the biological character of CL7 was analyzed according to the information of cathepsin L family, and then rCL7 was expressed and purified, a new iELISA based on CL7 was developed. The results exhibited CL7 iELISA had 100% sensitivity 100% specificity in sheep (cut-off 1.329) and 100% sensitivity 93.75% specificity in cattle (cut-off 0.756). Moreover, anti-Fasciola CL7 antibodies could be detected in early Fasciola gigantica infected buffaloes, as early as 3 week-post-infection (WPI). In conclusion, it is suggested that CL7 with low cost, early detection, good specificity and sensitivity could be used as a candidate antigen for detection of ruminant fasciolosis.

Cyathostominae are ubiquitous to grazing horses and regarded the most prevalent internal parasite in the horse. Unfortunately, decades of indiscriminate use of anthelmintic drugs have resulted in the development of resistance in cyathostomins to all currently available drug groups, the most recent being a documented lack of efficacy to the macrocyclic lactones (ML). In vivo determination of anthelmintic resistance in horses most often utilises the faecal egg count reduction test (FECRT). Further, a shortened egg reappearance period (ERP) can indicate a change in response to the applied treatment and suggest an upcoming reduction of efficacy. Although both true resistance as demonstrated by the FECRT and shorter ERPs after ML treatment have now been shown in cyathostomins worldwide, the efficacy of ML as regards to cyathostomins in Sweden is currently unknown. The aim of the present study was therefore to determine FECRTs and ERPs after ivermectin (IVM) treatment in Swedish horses. Sixteen equestrian establishments with a minimum of six horses excreting at least 150 eggs per gram faeces (EPG) at screening were selected. For each establishment, FECRTs and ERPs were determined by collecting faecal samples prior to and 14 days after IVM treatment (200µg/kg), and thereafter at weekly intervals for a total of eight weeks. All participants responded to a questionnaire detailing pasture management methods and anthelmintic routines.Questionnaire results showed that the majority of establishments (69%) only treated horses with anthelmintic drugs if indicated by faecal diagnostics and all of the establishments had a mean FECRT exceeding 99.0% and ERPs ranging from six to over eight weeks. The ERP was shown to increase with age as young individuals were shown to excrete cyathostomin eggs earlier after treatment compared with older horses (R = 0.21, p=0.015). Riding schools, stud farms and those declaring not to use separate summer and winter paddocks had significantly shorter ERPs (p <0.01).In conclusion, retained ERPs and no confirmed resistance to IVM were found in Swedish equine establishments practising selective anthelmintic treatment, and supports the use of selective deworming regimens as a means of reducing the risk of anthelmintic resistance development.