Comment on: Occurrence of Hookworm and the First Molecular and Morphometric Identification of Uncinaria stenocephala in Dogs in Central Europe.

BACKGROUND: Hookworms are blood-feeding nematodes that infect millions of people and animals worldwide. The most common species infecting dogs in Europe are representatives of the family Ancylostomatidae, which differ in invasiology, biology and morphological features. However, the differentiation of invasions of species such as Uncinaria stenocephala and Ancylostoma caninum based on a coproscopic examination is problematic. For this reason, it is recommended to use molecular diagnostics for this purpose. The authors of the article investigated the prevalence of U. stenocephala in dogs kept in various living conditions in Slovakia and developed a two-step morphology-molecular analysis-based strategy to identify the genus and the species of eggs and larvae of the Ancylostomatidae family in dogs. CONCLUSIONS: In our opinion, this work is very much needed as it shows how to effectively diagnose hookworm infestations. However, we do not agree with the information in the title of the article because such studies have already been carried out in Central Europe by other authors.

Synthesis and anthelmintic activity of novel thiosemicarbazide and 1,2,4-triazole derivatives: In vitro, in vivo, and in silico study.

INTRODUCTION: Intestinal parasitic infections are neglected diseases and, due to the increasing resistance of parasites to available drugs, they pose an increasing therapeutic challenge. Therefore, there is a great need for finding new compounds with antiparasitic activity. OBJECTIVES: In this work, new thiosemicarbazide and 1,2,4-triazole derivatives were synthesized and tested for their anthelmintic activity. METHODS: The synthesis was carried out by classical methods of organic chemistry. Anthelmintic activity tests were carried out in vitro (Rhabditis sp., Haemonchus contortus, Strongylidae sp.) in vitro (Heligmosomoides polygyrus/bakeri), and in silico analysis was performed. RESULTS: Quinoline-6-carboxylic acid derivative compounds were designed and synthesized. The highest activity in the screening tests in the Rhabditis model was demonstrated by compound II-1 with a methoxyphenyl substituent LC50 = 0.3 mg/mL. In the next stage of the research, compound II-1 was analyzed in the H. contortus model. The results showed that compound II-1 was active and had ovicidal (percentage of dead eggs > 45 %) and larvicidal (percentage of dead larvae > 75 %) properties. Studies in the Strongylidae sp. model confirmed the ovicidal activity of compound II-1 (percentage of dead eggs ≥ 55 %). In vivo studies conducted in the H. polygyrus/bakeri nematode model showed that the number of nematodes decreased by an average of 30 % under the influence of compound II-1. In silico studies have shown two possible modes of action of compound II-1, i.e. inhibition of tubulin polymerization and SDH. The test compound did not show any systemic toxic effects. Its influence on drug metabolism related to the activity of cytochrome CYP450 enzymes was also investigated. CONCLUSION: The results obtained in the in vitro, in vivo, and in silico studies indicate that the test compound can be described as a HIT, which in the future may be used in the treatment of parasitic diseases in humans and animals.

The first case of Spiroxys contortus in European pond turtle (Emys orbicularis) in the wild in Poland.

During standard parasitological dissection of 4 individuals of European pond turtle (E. orbicularis) (Linneaus, 1758) one nematode was found. In a morphological examination the parasite was identified as Spiroxys contortus (Rudolphi, 1819) and confirmed by DNA analysis based on sequencing of the small subunit ribosomal RNA gene. The partial 18S rDNA gene was deposited to NCBI GenBank with the accession number MN629917. This is the first molecular evidence of S. contortus in E. orbicularis from wild from Poland. The analyzed sample genotype sequence shows 100% similarity to the reference specimen from Argentina.

Very low intraspecific sequence variation in selected nuclear and mitochondrial Parascaris univalens genes.

Equines were over decades considered to be infected by two morphologically virtually indistinguishable ascarid species, Parascaris univalens and Parascaris equorum. Reliable species discrimination is only possible using enzyme isoelectric focussing and karyotyping with P. univalens having one and P. equorum two chromosome pairs. However, presumably the complexity of both methods prevented their routine use in nearly all previous studies about prevalence and drug resistance of Parascaris spp. These have barely been performed on the species level although most studies stated presence of one or the other species. Recently, only P. univalens has been identified by karyotyping and the last published study identifying P. equorum dates back to 1989. In order to improve species-specific detection, molecular markers are required. Here, partial 12S rRNA, cytochrome oxidase I (COI) and complete internal transcribed spacer (ITS)-1 and - 2 sequences were obtained from 24 karyotyped Parascaris specimens from Poland and 6 German specimens (not karyotyped) and used in phylogenetic analyses with orthologous sequences from GenBank. All karyotyped specimens were identified as P. univalens. In the phylogenetic analysis, they formed very homogenous clusters for all target genes and in a multi-locus analysis. Within this cluster, almost all sequences from GenBank were also included, no matter if they had been assigned to P. univalens or P. equorum. However, a small number of P. univalens ITS and COI sequences originating from donkeys from a single farm in China formed a highly supported sister cluster suggesting that they might represent another Parascaris genotype or species. Our data also strongly suggest that nearly all ITS and COI sequences previously deposited in GenBank and assigned to P. equorum actually represent P. univalens. The fact that significantly different sequences can be found in Parascaris spp. suggests that PCR-based species diagnosis will be possible once molecular markers have been identified for P. equorum from karyotyped specimens.

Echinococcus multilocularis infection in horses in Poland.

A cross-sectional survey was performed to obtain first information on the prevalence of Echinococcus multilocularis infection in slaughter horses from central, eastern and southern Poland, a country with a highly endemic occurrence of this tapeworm in red foxes. White tough nodular lesions being 3-10 mm in size, sharply demarcated and spherically or irregularly shaped were found in 54 of 365 livers examined. Histologically, focal granulomatous necrotizing inflammations and sometimes PAS-positive acellular lamellar structures being characteristic of the E. multilocularis metacestode stage were visible; protoscoleces were not found. E. multilocularis DNA was detected in six of 19 hepatic lesions examined by nested PCR. Our results from molecular and morphological diagnostics suggest an overall prevalence of alveolar echinococcosis between 4.7% and 14.8% in the horse population studied. Horses as dead-end hosts do not play any role in the life cycle of E. multilocularis but may serve as additional sentinel animals in monitoring the environmental contamination with tapeworm eggs.

A survey of ivermectin resistance in Parascaris species infected foals in south-eastern Poland.

Parascaris spp. are major gastro-intestinal nematodes that infect foals and can lead to respiratory symptoms, poor growth, and in some cases obstruction of the small intestine and death. Ivermectin resistance has been reported for Parascaris spp. in many countries. In Poland, the knowledge of the level of resistance against ivermectin in Parascaris spp. is limited. The aim of this study was to examine the efficacy of ivermectin against Parascaris spp. in foals from south-eastern Poland. Foals (n = 225 = reared in 7 stud farms) were treated orally with ivermectin paste. Faecal samples were collected from the rectum of each foal or from the environment straight after defaecation on 1 day prior and 2 weeks after deworming. A faecal egg count (FEC) was performed using the McMaster method with a minimum detection limit of 50 eggs/g. FEC reduction (FECR) was calculated using the Faecal Egg Count Reduction Test. The statistical analysis was limited to foals excreting more than 150 eggs/g before treatment and to stud farms with at least 6 foals excreting at or above this level. Confidence intervals were determined by 1000 bootstraps at farm level and the contribution of sex and age to FECR was quantified using a generalized equation estimation procedure. Parascaris spp. eggs were found in 40% of the foals. Following ivermectin treatment, Parascaris spp. eggs were identified in 28.4% of the foals. The mean estimated FECR ranged from 44% to 97% and average efficacy was 49.3%. FECR was more pronounced in older foals (P-values = 0. 003). The FECR was more pronounced in males than in females (P value = 0.028). This study is the first to indicate a reduced efficacy of ivermectin against Parascaris spp. in foals in Poland.

The Heat Shock Protein HSP70 Promotes Th17 Genes' Expression via Specific Regulation of microRNA.

T helper cells type 17 (Th17) are orchestrators of autoimmune conditions, including multiple sclerosis (MS), but mechanisms of Th17 pathogenicity remain unknown. MicroRNAs (miRNA) are known to control T cells. To understand the function of miRNA in Th17, we have established a T cell line, EL4-TCR+, that resembles the expression pattern of the Th17 cells. Subsequently, we have evaluated the crosstalk between miRNA and Th17 genes' expression using a combination of gene expression profiling, gene expression manipulation, RNA and protein immunoprecipitation, as well as confocal microscopy. We have found that Th17-related miRNA were strongly expressed in EL4-TCR+ cells following the binding of the cluster of differentiation 3 (CD3) component of the T cell receptor (TCR). Furthermore, a specific inhibition of these miRNA resulted in downregulation of the critical Th17 genes' expression. Surprisingly, this mechanism relied on the function of the stress signal regulator heat shock protein 70 (HSP70). Upon activation, HSP70 co-localized intracellularly with miRNA processing proteins. Precipitation of HSP70 resulted in enrichment of the Th17-associated miRNA. Finally, HSP70 inhibition led to downregulation of the Th17 genes' expression and ameliorated development of autoimmune demyelination. Our study demonstrated that HSP70 facilitates specific miRNA function leading to Th17 genes' expression, a mechanism linking stress and autoimmunity.

Potential sources of infection with selected zoonotic agents in the veterinary work environment - pilot studies.

INTRODUCTION AND OBJECTIVE: The problem of occupational biohazards is very important, especially in the field of agriculture and in human and veterinary medicine. The aim of the study was to determine the potential sources of infection in veterinary professionals with selected zoonotic agents, including: Toxoplasma gondii, Giardia duodenalis, Leptospira spp., Cryptosporidium spp. and Coxiella burnetii. MATERIAL AND METHODS: A total of 50 air samples from barns, piggeries and veterinary surgeries were examined for the presence of Leptospira spp. and C. burnetii DNA. Serum samples of 86 pigs and 80 cows were tested for the presence of antibodies to Leptospira spp. and to phase I and II C. burnetii antigens. Serum of 70 cats were tested for the presence of antibodies to T. gondii and 65 samples of cat faeces for the presence of T. gondii oocysts. The presence of G. duodenalis and Cryptosporidium spp. were examined in 50 of dog faeces and 50 of bovine faeces samples. RESULTS: DNA of Leptospira spp. was detected in 2 air samples from the piggeries (4%). C. burnetii DNA was not found in any sample. Anti-Leptospira spp. antibodies were detected in 51 (59.3%) of examined pigs. Neither anti-Leptospira spp. nor anti-C. burnetii antibodies were found among samples of bovine serum. Anti-T. gondii antibodies was found in 52 cat serum samples (74.3%). Among samples of cat faeces, no T. gondii oocysts were detected. In one sample of cattle stool (2%), G. duodenalis was detected and in another (2%) - Cryptosporidium spp. G. duodenalis was detected in 7 samples (14%) and Cryptosporidium spp. in 2 samples (2%) of dog faeces. CONCLUSIONS: The results of this study demonstrate the potential risk of infection with Leptospira spp. in veterinarians working with pigs. Veterinarians could be also be at risk of infection with T. gondii and G. duodenalis.

Echinococcus multilocuraris -first recorded case of Norway rat (Rattus norvegicus) in Poland.

INTRODUCTION: Echinococcus multilocularis is a very dangerous zoonotic parasite threatening human health. The red fox is the main definitive host, and cats and dogs less commonly. Rats can be intermediate hosts. OBJECTIVE: The aim of the study was to determine the parasitofauna of Norway rats and some cats and dogs living on a farm near a forest. MATERIAL AND METHODS: A parasitological section on 15 Norway rats was conducted. The internal organs were examined by means of macroscopic and microscopic methods. For molecular examination, a QIAmp DNA Mini Kit (Qiagen) was used. RESULTS: Based on necropsy, parasitological and molecular examinations, of the 15 examined rats, 1 was found to have larvae of E. multilocularis, while 3 others had eggs of Hymenolepis diminuta, H. nana and Syphacia obvelata. The faeces of the pets did not contain any developmental forms of parasites. CONCLUSIONS: This is the first case of Echinococcus multilocularis infestation in a rat in Poland.

Factors affecting prevalence and abundance of A.perfoliata infections in horses from south-eastern Poland.

Equine Anoplocephalosis constitute a significant problem in horses worldwide. The aim of this study was to analyse intrinsic (host age and sex) and extrinsic (management type, pasture type and moisture) factors that influence the prevalence and FEC of A. perfoliata infections. Faecal samples were collected from 994 horses managed in studs or individually between 2012 and 2014. The Sedimentation-flotation method was applied for coproscopic analysis, and faecal egg counts were calculated. The overall prevalence was 25.1% (21.4-29.0) with the highest prevalence (36.1% [28.1-44.8]) found in horses 10-20 years old. The individuals kept in studs showed three times higher A. perfoliata prevalence compared to the ones managed individually. The prevalence significantly differed between pasture types, with individuals kept in studs (37.6% [34.3-40.9]) showing four times higher prevalence than horses kept individually (9.2% [4.8-16.5]). More horses kept on watery (42.0% [36.6-47.6]) and semi-watery (35.9% [31.3-40.7]) pastures were infected than those on dry (6.6% [4.6-9.2]) pastures. The overall A. perfoliata FEC in all examined individual was 2.67 and differed within sex, with mares showing 4.3 - times higher FEC of infection than stallions. Horses bred in studs (3.65±0.289) showed higher FEC than these bred individually (1.28±0.198). There was the effect of pasture type on A. perfoliata FEC, with horses kept on joint pastures (4.06±0.29) showing higher FEC than individuals kept individually (0.88±0.23). Pasture moisture significantly affected A. perfoliata FEC with the highest FECs in horses from watery pastures. Horses bred on dry pastures showed 16 times lower FEC than horses bred on watery pastures. Host age also significantly affected A. perfoliata FEC, with the oldest individuals showing the highest mean FEC. The presented analysis of intrinsic and extrinsic factors may help to overcome A. perfoliata infections in horses in different breeding systems. Understanding the role of management and pasture type risk factors that influence this parasitosis may benefit both breeders and veterinary surgeons.

Variations in the Rate of Infestations of Dogs with Zoonotic Nematodes and the Contamination of Soil in Different Environments.

Companion animals are an important aspect in human life. However, they may also be considered a source of pathogens. An example of zoonotic parasitoses is toxocarosis or cutaneous larva migrans (CLM). The aim of the study was to detect zoonotic nematodes of dogs living in different areas and the intensity of contamination in parasite polluted environments that are hazardous to human health. The fecal samples were examined using standard flotation and decantation methods as well as McMaster's quantitative technique. The soil samples in urban and rural areas were examined using a modified flotation method as described by Quinn et al. Statistical analyses were performed by IBM SPSS Statistics Version 23. The overall prevalence of parasites in dogs was 38%, 17.02% and 56.60% from urban and rural areas, respectively. The percentage values of nematodes important for human health (Toxocaracanis, Ancylostomatidae, Trichurisvulpis) remained at the same level (16%). The infected dogs were dominated by a single parasite species, the main was T.canis (28.95%). In total, 54.30% of the soil samples were contaminated with parasite eggs. The contamination of urban and rural sandpits was 40% and 60%, respectively. The molecular examinations of soil samples using LAMP (loop-mediated isothermal amplification) confirmed the presence of nematode eggs of the species T.canis in all samples previously classified as positive.

Analysis of intrinsic and extrinsic factors influencing the dynamics of bovine Eimeria spp. from central-eastern Poland.

Eimeria infections are common in cattle worldwide, however, little is known about the invasion dynamics of this unicellular parasite. Therefore, the aim of this study was to analyze intrinsic (host age) and extrinsic (herd size and management system) factors influencing the dynamics of Eimeria spp. found in calves from CE Poland. Fecal samples were collected from 356 calves from different types of management systems and from different herd sizes. Flotation and McMaster method were used for parasitological investigation. Oocysts were differentiated on the basis of morphological criteria. Eight Eimeria species were identified and mean species richness (MSR) was significantly affected by host age. The highest MSR was noted for middle age animals. There was an association between species, with a highly significant co-occurrence of Eimeria bovis with Eimeria zuernii. The presence of E. bovis significantly increased the percentage of individuals carrying E. zuernii. The presence of E. bovis significantly increased the percentage of individuals carrying Eimeria canadensis. The overall prevalence of Eimeria spp. reached 52.8% and was significantly affected by the age of cows, with the highest prevalence in animals between 5-10 months old. The most prevalent species were E. bovis (37.4%), E. zuernii (19.9%) and E. canadensis (12.1%). The prevalence of E. bovis was affected by host age (the highest prevalence in age class 2 animals) and management type (the highest prevalence in individuals raised in groups). The prevalence of E. zuernii was affected by age (the lowest prevalence was noted in the oldest individuals) and herd size (individuals infected were present only in the middle and large size herds), whereas the prevalence of E. canadensis was affected by all three factors. Overall, mean OPG of the combined Eimeria spp. was 458.84 (37.93) and differed significantly between age classes. Mean OPGs were generally low for young and mature animals but high for middle age class animals. There was no significant influence of extrinsic factors on OPG. Taking into consideration the presented results, it is necessary to underline the role of intrinsic and extrinsic factors affecting the prevalence and abundance of Eimeria spp. in calves. Understanding the dynamics of Eimeria infections in calves may help to overcome this most common bovine parasite.

Diagnosis and therapy of Capillaria plica infection: report and literature review.

Worldwide, data on Capillaria (Pearsonema) plica infections of the urinary tract in domestic carnivores are limited. Nevertheless, cystitis with hematuria, dysuria or pollakiuria may suggest a C. plica infection. A three-year old dog from Poland showing pollakiuria and hematuria was presented. At urine analysis, C. plica eggs were found in the urine sediment. The dog was unsuccessfully treated with a daily oral dose of fenbendazole 50 mg/kg BW for 5 days, while a single subcutaneous administration of ivermectin 0.2 mg/kg BW once effectively eliminated C. plica infection.

Seasonal changes of diagnostic potential in the detection of Anoplocephala perfoliata equine infections in the climate of Central Europe.

For this study, 724 gastrointestinal tracts of slaughter horses were investigated to determine the prevalence, intensity of Anoplocephala perfoliata and tapeworm development stages over the second, third and fourth quarter of 2012 and the first quarter of 2013. For each positive horse, faecal samples were collected from the rectum or small colon for coproscopic examinations. The samples were analysed using dedicated modified sedimentation-flotation methods. In total, 52 horses were infected with A. perfoliata in the course of the study, with an overall prevalence of 7.2 %. The prevalence changed over the study period; however, not markedly. The overall mean of A. perfoliata abundance was 12.3 (3.23) and did not differ significantly between the quarters. Mean invasion intensity did not differ significantly between the quarters. The quantity of mature tapeworms did not differ significantly over the study period; however, there was a significant difference in the number of immature tapeworms. The highest number of mature tapeworms was found in the first quarter of 2013. The number of detected tapeworm eggs rose significantly over the study period. The total number of tapeworms did not have a significant influence on the presence/absence of detected eggs. However, there was a noticeable difference between the number of mature tapeworms and presence/absence of eggs in faeces. This clearly indicates that the efficacy of the modified sedimentation-flotation method is influenced by seasonality, and therefore the most effective and reliable time for detection of A. perfoliata in equines is the first quarter of the year.

Comparison of the sensitivity of coprological methods in detecting Anoplocephala perfoliata invasions.

The autopsy of 487 slaughter horses revealed the presence of Anoplocephala perfoliata in 36 animals. The invasions varied in the intensity (3 to 2,069 tapeworms) and in the level of tapeworms' proglottid maturity. Twenty nine horses were found to contain tapeworms with gravid proglottid. Fecal samples collected from the rectum were tested using following techniques: flotation with solution-saturated NaCl, decantation, McMaster's, and modified sedimentation-flotation methods (50 g feces samples, flotation solution-saturated NaCl and sucrose, specific gravity 1.25 g/ml). The number of A. perfoliata positive fecal samples was significantly higher using the sedimentation-flotation methods 21 (58.33%) than flotation 6 (16.66%), decantation 3 (8.33%), and McMaster's 1 (2.77%) techniques. The sensitivities of the coprological methods during the patent period were 20.69, 10.34, 3.45, and 72.41% for the flotation, decantation, McMaster's, and sedimentation-flotation method, respectively. Sedimentation-flotation techniques proved to be more sensitive than other one. The lowest intensity of invasion possible to detect using this method was nine tapeworms with gravid proglottid.

Cutaneus myxosporidiasis in the Australian green tree frog (Litoria caerulea).

This case is reported with the intention of highlighting the presentation of cutaneous myxosporidiasis in Australian tree frog (Litoria caerulea) caused by genus Myxobolus. The morphology and morphometric characteristic of the spores were determined using light microscopy and differential interference contrast microscopy. Spores were pyriform in shape in frontal view and oval in lateral view, and the average size was respectively 11.4 × 6.0 × 4.5 µm (12.1 - 9.5 × 6.3 - 5.4 × 5.0 - 4.1 µm). To the best of our knowledge, this is the second case of skin invasion caused by myxosporeans in amphibians.