Getting around the roundworms: Identifying knowledge gaps and research priorities for the ascarids.

The ascarids are a large group of parasitic nematodes that infect a wide range of animal species. In humans, they cause neglected diseases of poverty; many animal parasites also cause zoonotic infections in people. Control measures include hygiene and anthelmintic treatments, but they are not always appropriate or effective and this creates a continuing need to search for better ways to reduce the human, welfare and economic costs of these infections. To this end, Le Studium Institute of Advanced Studies organized a two-day conference to identify major gaps in our understanding of ascarid parasites with a view to setting research priorities that would allow for improved control. The participants identified several key areas for future focus, comprising of advances in genomic analysis and the use of model organisms, especially Caenorhabditis elegans, a more thorough appreciation of the complexity of host-parasite (and parasite-parasite) communications, a search for novel anthelmintic drugs and the development of effective vaccines. The participants agreed to try and maintain informal links in the future that could form the basis for collaborative projects, and to co-operate to organize future meetings and workshops to promote ascarid research.

Repeated imidocarb treatment failure suggesting emerging resistance of Babesia canis in a new endemic area in north-eastern Germany.

Canine babesiosis has been increasingly diagnosed in various regions of Germany such as north-eastern Germany in recent years. A dog with several relapses of Babesia canis infection after treatment with imidocarb is described. A 9-year-old male Magyar Viszla with B. canis infection was referred after two treatments with imidocarb (dosage 2.1 mg/kg SC) because of lethargy, fever and pancytopenia (additional treatments with prednisolone and doxycycline). Merozoites were detected in the blood smear and imidocarb treatment was repeated. Clinical signs, pancytopenia and a positive B. canis PCR occurred after the 3rd (6 mg/kg SC), 4th (7.7 mg/kg SC) and 5th (7.5 mg/kg SC and doxycycline for 4 weeks in addition) imidocarb injection and thorough tick prevention with isoxazoline and permethrin products. 12 days after the 5th injection, the PCR was negative for the first time. The dog was again presented with fever 35 days after the 5th injection. The B. canis PCR was positive and laboratory examination revealed pancytopenia. Treatment with atovaquone/azithromycin for 18 days was performed and no further relapse occurred for 32 weeks. In the case of suspected imidocarb resistance in B. canis infection, treatment with atovaquone/azithromycin can be an alternative.

Identification of tungiasis infection hotspots with a low-cost, high-throughput method for extracting Tunga penetrans (Siphonaptera) off-host stages from soil samples-An observational study.

BACKGROUND: The sand flea, Tunga penetrans, is the cause of a severely neglected parasitic skin disease (tungiasis) in the tropics and has received little attention from entomologists to understand its transmission ecology. Like all fleas, T. penetrans has environmental off-host stages presenting a constant source of reinfection. We adapted the Berlese-Tullgren funnel method using heat from light bulbs to extract off-host stages from soil samples to identify the major development sites within rural households in Kenya and Uganda. METHODS AND FINDINGS: Simple, low-cost units of multiple funnels were designed to allow the extraction of >60 soil samples in parallel. We calibrated the method by investigating the impact of different bulb wattage and extraction time on resulting abundance and quality of off-host stages. A cross-sectional field survey was conducted in 49 tungiasis affected households. A total of 238 soil samples from indoor and outdoor living spaces were collected and extracted. Associations between environmental factors, household member infection status and the presence and abundance of off-host stages in the soil samples were explored using generalized models. The impact of heat (bulb wattage) and time (hours) on the efficiency of extraction was demonstrated and, through a stepwise approach, standard operating conditions defined that consistently resulted in the recovery of 75% (95% CI 63-85%) of all present off-host stages from any given soil sample. To extract off-host stages alive, potentially for consecutive laboratory bioassays, a low wattage (15-25 W) and short extraction time (4 h) will be required. The odds of finding off-host stages in indoor samples were 3.7-fold higher than in outdoor samples (95% CI 1.8-7.7). For every one larva outdoors, four (95% CI 1.3-12.7) larvae were found indoors. We collected 67% of all off-host specimen from indoor sleeping locations and the presence of off-host stages in these locations was strongly associated with an infected person sleeping in the room (OR 10.5 95% CI 3.6-28.4). CONCLUSION: The indoor sleeping areas are the transmission hotspots for tungiasis in rural homes in Kenya and Uganda and can be targeted for disease control and prevention measures. The soil extraction methods can be used as a simple tool for monitoring direct impact of such interventions.

Zoonotic dirofilariases: one, no one, or more than one parasite.

Dirofilaria spp. are vector-borne filarial nematodes that affect a variety of animal species, including humans. Dirofilaria immitis and Dirofilaria repens are the two main zoonotic species, but also other wildlife-associated Dirofilaria species are occasionally reported as causative agents of human dirofilariasis, including Dirofilaria striata, Dirofilaria tenuis, Dirofilaria ursi, Dirofilaria spectans, and Dirofilaria magnilarvata. Since the etiological identity of most of the species mentioned here is arguable, we summarized and critically discussed data concerning infections in humans, focusing on the reliability of Dirofilaria species identification. We advocate the importance of combined morphological and genomic approaches to provide unequivocal evidence for their zoonotic potential and pathogenicity.

Myth or truth: investigation of the jumping ability of Tunga penetrans (Siphonaptera: Tungidae).

Female sand fleas (Tunga penetrans Linnaeus, 1758, Siphonaptera: Tungidae) cause a severe parasitic skin disease known as tungiasis. T. penetrans is a small flea, measuring less than 1 mm in length. The females of this species burrow into the skin of human and animal hosts and mostly affect the feet. This has led to the anecdotal assumption that T. penetrans, unlike its relatives in the Siphonaptera family, would have a limited jumping ability potentially not reaching higher body parts. However, there is no data supporting this. This study evaluated the jumping capabilities of T. penetrans for height and distance using sticky tapes. The vertical jump of the female T. penetrans ranged from 4.5 to 100 mm with a mean of 40 mm whereas the vertical jump of the male T. penetrans ranged from 1.2 to 138 mm with a mean of 46 mm. The horizontal jump of the female T. penetrans ranged from 18 to 138 mm with a mean of 64 mm and that of the male ranged from 9 to 251 mm with a mean of 80 mm. Based on the literature, fleas of various species have been described as jumping vertically 50-100 times their size and horizontally 5-100 times their size. In this respect, sand fleas appear to have equal expert jumping abilities to their relatives. Their aggregation on people's feet is not likely a result of their poor jumping ability but might be an adaptation to the host's behavior which would require further investigations.

Validation of a serum ELISA test for cyathostomin infection in equines.

Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific 'serum score' for each horse. Validation dataset results were then used to assess the optimised test's performance and select serum score cut-off values for diagnosis of burdens above 1000, 5000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection.

Neurocognitive and mental health outcomes in children with tungiasis: a cross-sectional study in rural Kenya and Uganda.

BACKGROUND: Tungiasis, a neglected tropical parasitosis, disproportionately affects children. Few empirical studies have reported neurocognitive and mental health outcomes of children with ectoparasitic skin diseases like tungiasis. Pathophysiology of tungiasis suggests it could detrimentally affect cognition and behaviour. This study pioneered the investigation of neurocognitive and mental health outcomes in children with tungiasis. METHODS: This was a multi-site cross-sectional study including 454 quasi-randomly sampled school-children aged 8-14 from 48 randomly selected schools in two counties in Kenya and a district in Uganda. The participants were stratified into infected and uninfected based on the presence of tungiasis. The infected were further classified into mild and severe infection groups based on the intensity of the infection. Adapted, validated, and standardized measures of cognition and mental health such as Raven Matrices and Child Behaviour Checklist were used to collect data. Statistical tests including a multilevel, generalized mixed-effects linear models with family link set to identity were used to compare the scores of uninfected and infected children and to identify other potential risk factors for neurocognitive and behavioural outcomes. RESULTS: When adjusted for covariates, mild infection was associated with lower scores in literacy [adjusted ß(aß) = - 8.9; 95% confidence interval (CI) - 17.2, - 0.6], language (aß = - 1.7; 95% CI - 3.2, - 0.3), cognitive flexibility (aß = - 6.1; 95% CI - 10.4, - 1.7) and working memory (aß = - 0.3; 95% CI - 0.6, - 0.1). Severe infection was associated with lower scores in literacy (aß = - 11.0; 95% CI - 19.3, - 2.8), response inhibition, (aß = - 2.2; 95% CI - 4.2, - 0.2), fine motor control (aß = - 0.7; 95% CI - 1.1, - 0.4) and numeracy (aß = - 3; 95% CI - 5.5, - 0.4). CONCLUSIONS: This study provides first evidence that tungiasis is associated with poor neurocognitive functioning in children. Since tungiasis is a chronic disease with frequent reinfections, such negative effects may potentially impair their development and life achievements.

Efficacy of flukicides against Fasciola hepatica and first report of triclabendazole resistance on German sheep farms.

Fasciola hepatica infections lead to severe health problems and production losses in sheep farming, if not treated effectively. Triclabendazole has been used extensively over decades due to its unique efficacy range against all definitive hostfluke stages but published data about the susceptibility of F. hepatica to anthelmintics in Germany are lacking. This study aimed to identify current F. hepatica infections in German sheep flocks by coproscopic examinations and to evaluate the efficacy of anthelmintics with a focus on triclabendazole in a field study conducted from 2020 to 2022. Initial screening included 71 sheep farms, many of them with known history of fasciolosis. In this highly biased sample set, the frequency of F. hepatica infection at individual sheep and farm level were 12.8% and 35.2%, respectively. Additionally, eggs of Paramphistominae were found at frequencies of 4.8% and 15.5% at individual sheep and farm level, respectively. Due to low egg shedding intensity, faecal egg count reduction (FECR) tests could only be conducted on a few farms. The efficacy of triclabendazole was tested on 11 farms and albendazole on one farm, including 3-53 sheep/farm. Individual faecal samples were collected before and two weeks after treatment to evaluate the FECR using the sedimentation or FLUKEFINDER® or a modified FLUKEFINDER® method. On all farms a coproantigen reduction test was conducted in parallel. Lacking efficacy of triclabendazole even at double dosage was shown on one farm associated with a high number of animal losses due to acute fasciolosis. On this farm, the Fasciola miracidium development test was additionally performed, revealing a high in vitro ovicidal activity of albendazole while closantel was effective in vivo. On all other farms, sufficient efficacy of triclabendazole was observed. In conclusion, triclabendazole resistance appears not to be widespread on German sheep farms but, when present, can have serious effects on animal health.

Molecular detection of Babesia spp. in dogs in Germany (2007-2020) and identification of potential risk factors for infection.

BACKGROUND: In Europe, canine babesiosis is most frequently caused by Babesia canis and Babesia vogeli, and occasionally by Babesia gibsoni.. In Germany, B. canis is recognized as endemic. The aims of this study were to assess how often Babesia spp. infections were diagnosed in a commercial laboratory in samples from dogs from Germany, and to evaluate potential risk factors for infection. METHODS: The database of the LABOKLIN laboratory was screened for Babesia spp.-positive polymerase chain reaction (PCR) tests for dogs for the period January 2007-December 2020. Sequencing was performed for positive tests from 2018 and 2019. Binary logistic regression analysis was performed to determine the effects of sex, season, and year of testing. Questionnaires were sent to the submitting veterinarians to obtain information on travel abroad, tick infestation, and ectoparasite prophylaxis of the respective dogs. Fisher's exact test was used to calculate statistical significance and P < 0.05 was considered statistically significant. RESULTS: In total, 659 out of 20,914 dogs (3.2%) tested positive for Babesia spp. by PCR. Of 172 sequenced samples, B. canis was identified in 156, B. vogeli in nine, B. gibsoni in five, and B. vulpes in two. Season had a statistically significant impact on test results when summer/winter (1.6% tested positive) was compared to spring/autumn (4.7%), with peaks in April (5.2%) and October (7.4%) [P < 0.001, odds ratio (OR) = 3.16]. Sex (male 3.5%, female 2.8%; P = 0.012, OR = 1.49) and age (< 7 years old 4.0%, ≥ 7 years old 2.3%; P < 0.001, OR = 1.76) of the tested dogs also had a statistically significant effect. A statistically significant impact was demonstrated for observed tick attachment (P < 0.001, OR = 7.62) and lack of ectoparasite prophylaxis (P = 0.001, OR = 3.03). The frequency of positive Babesia spp. tests did not significantly differ between the 659 dogs that had never left Germany and the 1506 dogs with known stays abroad (P = 0.088). CONCLUSIONS: The possibility of canine infection with B. canis needs to be especially taken into consideration in spring and autumn in Germany as the activity of the tick Dermacentor reticulatus, a potential vector for canine babesiosis, is highest in these seasons. Travel and importation of dogs are considered major factors associated with canine babesiosis in Germany. However, autochthonous Babesia spp. infections also occur in a considerable number of dogs in Germany.

Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic.

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria (Clostridium perfringens and Clostridioides difficile) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation.

Quantifying metabolic activity of Ascaris suum L3 using resazurin reduction.

BACKGROUND: Helminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine. METHODS: Ascaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity. RESULTS: We show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100-1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO2 for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively. CONCLUSIONS: Together, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro.

Development of a three-colour digital PCR for early and quantitative detection of benzimidazole resistance-associated single nucleotide polymorphisms in Haemonchus contortus.

Haemonchus contortus is the most pathogenic nematode in small ruminants and anthelmintic resistance (AR) hampers its efficient control. Early detection of AR status is required to reduce selection for AR and cannot be achieved using phenotypic tests. For benzimidazoles (BZs), the detection of AR-associated alleles characterised by single nucleotide polymorphisms (SNPs) in the isotype 1 ß-tubulin gene allows early AR detection in strongyles. The F200Y, F167Y, E198A and E198L polymorphisms have been described in BZ-resistant populations with a clear variation in frequencies between regions. A novel digital PCR (dPCR) enables the detection of all of the above-described polymorphisms in H. contortus. Assays were validated using synthetic DNA fragments containing these SNPs. Then, larvae obtained and pooled at farm level from 26 Austrian and 10 Italian sheep farms were analysed. For all assays a detection limit of 15 copies/µl of resistance alleles and a high level of accuracy were demonstrated, allowing to detect allele frequencies of 1% in most samples. In Austrian samples, elevated frequencies of F200Y resistance alleles were detected on all farms. Polymorphisms in codon 167 and codon 198 were identified in H. contortus from Austria for the first time. In Italian samples, the frequency of resistance alleles was still comparatively low, but F200Y resistance alleles were traceable. In conclusion we developed for the first time dPCR assays that target all SNPs of relevance associated with BZ-resistance in H. contortus. Future research on AR development could benefit from an early onset of SNP-based surveillance that would include the developed assays for all SNPs of relevance. Improved surveillance in the long term will include other important, though less pathogenic, nematode genera in the analyses.

Coproscopical diagnosis of patent Fasciola hepatica infections in sheep - A comparison between standard sedimentation, FLUKEFINDER® and a combination of both.

The liver fluke Fasciola hepatica is a highly pathogenic and zoonotic trematode with a cosmopolitan distribution. In livestock, infections may lead to significant economic losses if not diagnosed promptly and treated effectively. Particularly for small ruminants, the standard method for the detection of fluke infection is based on coproscopical methods such as the sedimentation method, which detects F. hepatica eggs in faecal samples. In this respect a recent innovative coproscopical approach to diagnose patent infections is the FLUKEFINDER® method, which relies on differential sieving before sedimentation. These two methods and a combination of both methods that allows larger amounts of faeces to be processed with the FLUKEFINDER® apparatus were compared, to assess which method is most appropriate to determine the prevalence and intensity of F. hepatica egg shedding. The methods were compared for their ability to recover eggs from ovine faecal samples containing different numbers of fluke eggs per gram (EPG) of faeces and diluting the samples further by mixing with faeces from uninfected sheep. To compare the specificity of the test procedures, positive and negative samples with a low EPG were analysed in parallel by an investigator blinded to the nature of the samples. Significant differences concerning the EPG outcome were found: The FLUKEFINDER® method demonstrated the highest EPG values (p < 0.001) in the undiluted samples as well as in all mixing levels, followed by the modified FLUKEFINDER® method. The standard sedimentation showed the lowest EPG values and the highest variability between technical replicates. The precision of the FLUKEFINDER® method and the modified FLUKEFINDER® method were significantly higher than the precision of the standard sedimentation as determined by comparison of variability between technical replicates. The highest raw egg counts were detected using the modified FLUKEFINDER® method. The FLUKEFINDER® method and the combined method showed a sensitivity of 100 % even at the lowest egg concentrations, whereas the sensitivity of the standard sedimentation was 98.1 % for the same set of samples (i.e. one false negative sample). In a separate investigation aiming to estimate the specificity no differences were found between the three methods: all protocols showed 100 % specificity and were able to correctly distinguish between truly positive and truly negative samples without any evidence of cross-contamination between positive and negative samples processed in parallel.

Comparison of two molecular barcodes for the study of equine strongylid communities with amplicon sequencing.

Basic knowledge on the biology and epidemiology of equine strongylid species still needs to be improved to contribute to the design of better parasite control strategies. Nemabiome metabarcoding is a convenient tool to quantify and identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA gene, with a limited investigation of its predictive performance for cyathostomin communities. Using DNA pools of single cyathostomin worms, this study aimed to provide the first elements to compare performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. Barcode predictive abilities were compared across various mock community compositions of two, five and 11 individuals from distinct species. The amplification bias of each barcode was estimated. Results were also compared between various types of biological samples, i.e., eggs, infective larvae or adults. Bioinformatic parameters were chosen to yield the closest representation of the cyathostomin community for each barcode, underscoring the need for communities of known composition for metabarcoding purposes. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types. However, imperfect correlations were found between relative abundances from infective larvae and other life-stages for Cylicostephanus species using the ITS-2 barcode. While the results remain limited by the considered biological material, they suggest that additional improvements are needed for both the ITS-2 and COI barcodes.

Characterization of tungiasis infection and morbidity using thermography in Kenya revealed higher disease burden during COVID-19 school closures.

BACKGROUND: Tungiasis is a neglected tropical skin disease caused by the sand flea Tunga penetrans. Female fleas penetrate the skin, particularly at the feet, and cause severe inflammation. This study aimed to characterize disease burden in two highly affected regions in Kenya, to test the use of thermography to detect tungiasis-associated inflammation and to create a new two-level classification of disease severity suitable for mapping, targeting, and monitoring interventions. METHODS: From February 2020 to April 2021, 3532 pupils age 8-14 years were quasi-randomly selected in 35 public primary schools and examined for tungiasis and associated symptoms. Of the infected pupils, 266 were quasi-randomly selected and their households visited, where an additional 1138 family members were examined. Inflammation was assessed using infra-red thermography. A Clinical score was created combining the number of locations on the feet with acute and chronic symptoms and infra-red hotspots. RESULTS: The overall prevalence of tungiasis among all the school pupils who were randomly selected during survey rounds 1 and 3 was 9.3% [95% confidence interval (CI): 8.4-10.3]. Based on mixed effects logistic models, the odds of infection with tungiasis among school pupils was three times higher in Kwale (coastal Kenya) than in Siaya [western Kenya; adjusted odds ratio (aOR) = 0.36, 95% CI: 0.18-0.74]; three times higher in males than in females (aOR = 3.0, 95% CI: 2.32-3.91) and three times lower among pupils sleeping in a house with a concrete floor (aOR = 0.32, 95% CI: 0.24-0.44). The odds of finding an infected person among the household population during surveys before the COVID-19 pandemic was a third (aOR = 0.32, 95% CI: 0.19-0.53) of that when schools were closed due to COVID-19 restrictions and approximately half (aOR = 0.44, 95% CI: 0.29-0.68) in surveys done after school re-opening (round 3). Infection intensity was positively correlated with inflammation as measured by thermography (Spearman's rho = 0.68, P < 0.001) and with the clinical score (rho = 0.86, P < 0.001). Based on the two-level classification, severe cases were associated with a threefold higher level of pain (OR = 2.99, 95% CI: 2.02-4.43) and itching (OR = 3.31, 95% CI: 2.24-4.89) than mild cases. CONCLUSIONS: Thermography was a valuable addition for assessing morbidity and the proposed two-level classification of disease severity clearly separated patients with mild and severe impacts. The burden of tungiasis was considerably higher in households surveyed during COVID-19 restrictions suggesting underlying risks are found in the home environment more than in school.

Autochthonous Babesia canis infections in 49 dogs in Germany.

BACKGROUND: Vector-borne diseases are of increasing importance in Germany. Since 2015, autochthonous cases have been increasingly documented in Berlin/Brandenburg. OBJECTIVES: Describe autochthonous Babesia canis infection in the Berlin/Brandenburg region. ANIMALS: Forty-nine dogs with autochthonous B. canis infection. METHODS: Evaluation of history, clinical signs, laboratory abnormalities, treatment, and outcome. RESULTS: Dogs were presented between March and August (9) and September and January (40) in the years 2015-2021. Historical and clinical findings were lethargy (100%), pale mucous membranes (63%), fever (50%), and pigmenturia (52%). Common clinicopathological findings were thrombocytopenia (100%), anemia (85%), intravascular hemolysis (52%), pancytopenia (41%), and systemic inflammatory response syndrome (SIRS; 37%). Babesia detection was based on blood smear evaluation (n = 40) and PCR targeting the 18S rRNA gene of piroplasms (n = 49). Sequencing indicated 99.47% to 100% identity to B. canis sequences from GenBank. All dogs were treated with imidocarb (2.4-6.3 mg/kg; median, 5 mg/kg); 8 dogs received 1, 35 received 2, and 1 dog each received 3, 4, or 5 injections, respectively. Continued PCR-positive results were detected in 7 dogs after the 1st, in 5 after the 2nd, in 2 after the 3rd, and in 1 28 days after the 4th injection. Four dogs were euthanized and 3 dogs died. CONCLUSIONS AND CLINICAL IMPORTANCE: Autochthonous B. canis infections in Berlin/Brandenburg were associated with severe clinicopathological changes, SIRS, and multiorgan involvement. Testing by PCR during and after treatment is advisable to monitor treatment success. Screening of blood donors in high-risk areas and year-round tick protection is strongly recommended.

Epidemiological study on factors influencing the occurrence of helminth eggs in horses in Germany based on sent-in diagnostic samples.

Gastrointestinal nematodes are ubiquitous parasites of grazing equines with Parascaris spp., and strongyles being the most relevant ones regarding the prevalence and potential disease severity. Despite their importance, epidemiological data regarding the presence and egg-shedding intensities of these parasites are scarce. Data from 1067 horse samples collected on German horse farms initially to compare diagnostic methods were used for epidemiological analyses. Due to its higher sensitivity, presence/absence data were based on a combined sedimentation/flotation technique while faecal egg counts were based on Mini-FLOTAC. For strongyles, 46.5% of the samples were positive and the median egg-shedding intensity was 40 (range 5-2590). In multivariate analyses, prevalence and egg-shedding intensity were significantly influenced by season, age group and sample type. The drug used for the last treatment and the number of foals on the yard only affected prevalence while the number of horses on the yard and sex were only significant for egg-shedding intensity. For Parascaris spp., a prevalence of 4.6% and a median egg-shedding intensity of 0 (range 5-905) were observed. In multivariate analyses, the age group, the time since the last anthelmintic treatment, presence and number of foals had significant effects on ascarid prevalence whereas egg-shedding intensity was significantly influenced by age group and season only. Parascaris occurred only on yards with foals, but with an increasing number of foals, Parascaris egg-shedding intensity decreased. Prevalence and egg-shedding intensity were influenced by different but partially overlapping variables for Parascaris and strongyles.

Prevalence, Infection Intensity and Molecular Diagnosis of Mixed Infections with Metastrongylus spp. (Metastrongylidae) in Wild Boars in Uzbekistan.

The aim of the present study was to characterize the diversity of Metastrongylus spp. in wild boars and the earthworm intermediate host species contributing to the maintenance of the life cycle. Here, wild boars were subjected to parasitological necropsies, and lungworm species were identified morphologically, followed by confirmation using ITS-2 sequencing and a phylogenetic analysis. Earthworms were collected from wild boar habitats and investigated for the presence of larvae. The prevalence of Metastrongylus spp. in wild boars was 78.8%, and many individuals were positive for all three detected species, Metastrongylus pudendotectus, Metastrongylus salmi and Metastrongylus elongatus. The phylogenetic analysis did not clearly resolve all species, except for M. pudendotectus. Age group and season had no influence on prevalence, while intensity was significantly higher in autumn than in spring and summer (Kruskal-Wallis followed by Dunn's test). Three out of six investigated earthworm species were positive for metastrongyloid larvae (prevalence of 10.4-16.7%), but neither their phylogenetic relationship nor ecological microhabitats were able to explain these differences. Further sequence data should be used to improve the resolution in phylogenetic trees to determine potential cryptic species in the genus, while the application of deep sequencing approaches might provide insights into species-specific epidemiology and pathology.

Transgenic Expression of Haemonchus contortus Cytochrome P450 Hco-cyp-13A11 Decreases Susceptibility to Particular but Not All Macrocyclic Lactones in the Model Organism Caenorhabditis elegans.

The number of reported macrocyclic lactones (ML) resistance cases across all livestock hosts is steadily increasing. Different studies in the parasitic nematode Haemonchus contortus assume the participation of cytochrome P450s (Cyps) enzymes in ML resistance. Still, functional data about their individual contribution to resistance or substrate specificity is missing. Via microinjection, transgenic Caenorhabditis elegans expressing HCON_00141052 (transgene-Hco-cyp-13A11) from extrachromosomal arrays were generated. After 24 h of exposure to different concentrations of ivermectin (IVM), ivermectin aglycone (IVMa), selamectin (SEL), doramectin (DRM), eprinomectin (EPR), and moxidectin (MOX), motility assays were performed to determine the impact of the H. contortus Cyp to the susceptibility of the worms against each ML. While transgene-Hco-cyp-13A11 significantly decreased susceptibility to IVM (four-fold), IVMa (2-fold), and SEL (3-fold), a slight effect for DRM and no effect for MOX, and EPR was observed. This substrate specificity of Hco-cyp-13A11 could not be explained by molecular modeling and docking studies. Hco-Cyp-13A11 molecular models were obtained for alleles from isolates with different resistance statuses. Although 14 amino acid polymorphisms were detected, none was resistance specific. In conclusion, Hco-cyp-13A11 decreased IVM, IVMa, and SEL susceptibility to a different extent, but its potential impact on ML resistance is not driven by polymorphisms.