Setaria cervi (Filarioidea, Onchocercidae) undressing in ungulates: altered morphology of developmental stages, their molecular detection and complete sequence cox1 gene.

This work introduces new morphological and molecular information on the filaroid nematode Setaria cervi (Rudolphi, 1819) obtained from 13 infected game ungulates out of 96 dissected. The hosts comprised the following: a single moose (Alces alces), ten red deer (Cervus elaphus) and two sika deer (Cervus nippon) originating from the western and northern regions of the Czech Republic. Based on the complete sequences of the gene encoding mitochondrial cytochrome c oxidase subunit 1 (cox1), all 20 females and four males belonged to the species S. cervi. We detected three developmental female stages (adult fertile females, juvenile L5 females and L4 female larvae) differing in size and some morphological traits as the subtle structure of peribuccal crown and shape and features of tail knob. Such differences were described in detail for the first time. The phylogenetic relationships within the family Onchocercidae have been evaluated using new information on the cox1 sequence of S. cervi (maximum likelihood method, GTR + I + G model). In accordance with the latest phylogenetic studies, the present analysis confirmed the ancient separation of the subclass Setariinae from the remaining two onchocercid lineages Dirofilariinae and Onchocerinae.

Resistance of strongylid nematodes to anthelmintic drugs and driving factors at Czech goat farms.

BACKGROUND: Strongylid nematode infections may negatively affect both animal health and welfare, with deleterious consequences for livestock productivity. Many farmers in recent decades have relied on anthelmintics as the sole strategy of control, but the intensive use of these chemotherapeutics has led to the development of anthelmintic resistance (AR). Knowledge of both the efficacy of anthelmintics and factors promoting AR are essential to effectively control nematode infections, but no information on these topics for goats in the Czech Republic (CR) is available. This survey aimed to determine the occurrence of AR at Czech goat farms and to identify risk factors for the development of AR. A total of 24 herds of dairy goats across the CR were evaluated using in vitro tests for detecting AR, and a questionnaire survey was carried out to evaluate factors associated with AR. RESULTS: Resistance against benzimidazoles was confirmed at 18 (75%) farms, and the level of resistance was high in four (22%) of the affected herds based on the egg hatch test. Ivermectin-resistant nematodes were detected in 13 (54%) herds using the larval development test; Teladorsagia/Trichostrongylus and Haemonchus were the predominant types of resistant larvae. Eight (62%) of the affected herds were evaluated as highly resistant to ivermectin. Eleven (46%) of the herds were resistant to both benzimidazoles and ivermectin. This report is the first on dual AR in the CR. A univariate logistic regression analysis indicated that a high stocking rate and farmer inexperience were significantly associated with ivermectin and benzimidazole resistance, respectively. CONCLUSIONS: The results of our survey suggest that AR is widespread amongst herds of dairy goats in the CR, likely due to inappropriate practices of pasture and health management. AR may be an issue for expanding dairy-goat production in the CR in the near future unless both veterinary practitioners and farmers widely adopt strategies to prevent the development of AR.

Natural chemotherapeutic alternatives for controlling of haemonchosis in sheep.

BACKGROUND: Parallel in vitro and in vivo experiments were designed to evaluate promising chemotherapeutic alternatives for controlling haemonchosis in ruminants. In vitro anthelmintic activities (egg hatch test - EHT; larval development test - LDT) of aqueous and methanolic herbal extracts Mix1 and Mix2 were investigated. The in vivo effects of dietary supplementation with Mix1 and Mix2 on the parasitological status, inflammatory response, antioxidant parameters and microbial community of the lambs infected experimentally with Haemonchus contortus were investigated. Lambs were divided into four groups for the in vivo study: uninfected control lambs (C), infected lambs (I), infected lambs supplemented with Mix1 (I + Mix1) and infected lambs supplemented with Mix2 (I + Mix2). The experimental period was 70 days. RESULTS: The number of eggs per gram (EPG) of feces was quantified 22, 30, 37, 44, 51, 58, 65 and 70 days post-infection, and mean abomasal worm counts were assessed 70 days post-infection. Quantitative analyses identified 57.3 and 22.2 mg/g phenolic acids, 41.5 and 29.5 mg/g flavonoids and 1.4 and 1.33 mg/g protoberberine-type alkaloids in Mix1 and Mix2, respectively. The methanolic extracts of the herbal mixtures in both in vitro tests had higher anthelmintic effects (P < 0.01) than the aqueous extracts, but the effects did not differ significantly between Mix1 and Mix2 (P > 0.05). I + Mix1 and I + Mix2 lowered mean EPGs between 44 and 70 d by 58.1 and 51.6%, respectively. The level of IgG antibodies against H. contortus increased significantly after infection in each infected group. CONCLUSION: These results represent the first monitoring of the in vitro anthelmintic effects of herbal mixtures on H. contortus. The in vivo experiment indicated that the anthelmintic effect was not sufficient for the elimination of parasites, but this herbal treatment may affect the host over a longer term, reducing the parasitic infection in the host.

Ovicidal and larvicidal activity of extracts from medicinal-plants against Haemonchus contortus.

The use of medicinal plants (MP) containing bioactive compounds is an alternative strategy to control of parasitic nematode of small ruminants Haemonchus contortus at various stages of their life cycle. The aims of this study were to determine the in vitro anthelmintic activity of both aqueous and methanolic extracts from 13 medicinal plants typical for Central Europe, and to determine quantity of selected plant secondary metabolites (PSMs) in the methanolic extracts. In vitro egg hatch test and larval development tests were conducted to determine the possible anthelmintic effects of methanolic and aqueous extracts of the roots of Althaea officinalis L., Petasites hybridus L. and Inula helenium L.; flowers of Malva sylvestris L. and Chamomilla recutita L.; leaves of Plantago lanceolata L. and Rosmarinus officinalis L.; seeds of Foeniculum vulgare Mill. and stems of Solidago virgaurea L., Fumaria officinalis L., Hyssopus officinalis L., Melisa officinalis L. and Artemisia absinthium L. on eggs and larvae of H. contortus. Ultra-performance liquid chromatography and tandem mass spectroscopy was used for quantifying six PSMs: gallic acid (GA), rutin (RU), diosmin (DI), hesperidin (HE), quercetin (QU) and kaempferol (KA). RU content of the most effective methanolic extracts was in the order: M. sylvestris (9.33 mg/g DM) > A. absinthium (6.10 mg/g DM) > C. recutita (0.42 mg/g DM). The highest concentration of QU (44.8 mg/g DM) and KA (6.59 mg/g DM) were detected in stems of F. officinalis comparing to the other evaluated plants. The most significant (p < 0.05) anthelmintic effects exhibited methanolic extracts of A. absinthium in both in vitro tests (i.e., egg hatch test and larval development test). Additionally, only two methanolic extracts of C. recutita and M. sylvestris were comparable to activity of A. absinthium using the larval development test. Wider spectrum of aqueous extracts exhibited stronger ovicidal activity in comparison to methanolic extracts. The similar trend was observed in evaluating of larvicidal activity of aqueous and methanolic plant extracts.

Long-term occurrence of Trichuris species in wild ruminants in the Czech Republic.

The aim of this study was to identify Trichuris species in wild ruminants from 32 localities in the Czech Republic using morphological and molecular methods (ITS1-5.8S RNA-ITS2 region polymorphisms). Trichurids were obtained from 176 wild ruminants (roe deer, sika deer, red deer, fallow deer and mouflons) that were culled between 2009 and 2017. Trichuris discolor is the predominant trichurid of all of the above-mentioned wild ruminants, whereas Trichuris ovis was identified less frequently in roe deer, fallow deer, sika deer and mouflons. Red deer were parasitised exclusively by T. discolor. Young hosts under 1 year of age were more intensively infected by trichurids than were adults (χ2 = 32.02, p = 0.00). Trichurid prevalence results obtained through coprological methods and those based on parasitological dissections differed significantly (χ2 = 16.26, p = 0.00). The regression analysis indicated that the eggs per gram (EPG) threshold (20 EPG) was exceeded only if the host was parasitised by more than 7 trichurid females. Full concordance between the positive results obtained by the coprological methods and those obtained via direct dissections was achieved when the number of trichurid females per host exceeded 51.

Seasonal dynamics of endoparasitic infections at an organic goat farm and the impact of detected infections on milk production.

This study evaluated patterns and species composition of parasitic infections detected over a 1-year period at an organic goat farm. As a result of coprological examination, the overall prevalence of observed strongylids (99%), coccidia of the genus Eimeria (98%), and Muellerius capillaris lungworms (93%) was calculated. The most prevalent strongylids recovered from incubated fecal samples were Haemonchus contortus (42%), genera Trichostrongylus (23%), Oesophagostomum columbianum (13%), and Teladorsagia circumcincta (11%). A maximum intensity of coccidia infection 5150 oocysts per gram, strongylids infection 9900 eggs per gram and lungworm infection 867.26 larvae per gram were detected. The various effects (including environment, host, and parasites) on milk yield, lactose, protein, and fat were evaluated using generalized linear mixed models. Milk yield (P < 0.0001), milk fat (P < 0.01), and lactose (P < 0.0001) were affected by month, i.e., these parameters were influenced by the month of the year, regardless of the individual goat. With the intensity of infection detected in our study, only protein content was affected (P < 0.01) by parasitic infection (exclusively caused by strongylids). Correlation between measurements from one individual revealed that the goat itself can substantially decrease protein content but has much less of an effect on fat, milk yield, and lactose. Based on our results, we can conclude that a low intensity of parasitic infections does not significantly affect milk yield and the qualitative parameters of milk.

Reliable molecular differentiation of Trichuris ovis and Trichuris discolor from sheep (Ovis orientalis aries) and roe deer (Capreolus capreolus) and morphological characterisation of their females: morphology does not work sufficiently.

The main aim of the study was to evaluate associations between morphological variability of Trichuris females from sheep and roe deer and their rDNA polymorphism in whipworm populations from the Czech Republic. The results introduced the use of new molecular markers based on the internal transcribed spacer (ITS)1-5.8S RNA-ITS2 region polymorphisms, as useful tools for the unambiguous differentiation of congeners Trichuris ovis and Trichuris discolor. These markers revealed both parasites in roe deer and in sheep; however, T. ovis females predominated in sheep while T. discolor females occurred mostly in roe deer. Additional analysis of ITS1-5.8 rRNA-ITS2 discovered the genetic uniformity of the analysed T. discolor but high haplotype variation of T. ovis. Simultaneously, molecularly designated female individuals of both species were categorised into four morphotypes (MT) on the basis of morphology of genital pore area. MT1 and MT4 (vulvar opening on everted vaginal appendage/on visible cuticular bulge) occurred only in T. ovis, MT2 (uneverted vagina-vulvar opening without any elevation) was identified only in T. discolor and MT3 (transient type of vulvar opening on a small swelling) was observed in both species. Statistical analysis of biometric data confirmed that morphology of vulva is not a reliable marker for the species determination. On the basis of the ITS1-5.8S RNA-ITS2 region variability, we carried out a phylogenetic analysis (maximum likelihood method, Hasegawa-Kishino-Yano model) which showed that T. ovis haplotypes from the Czech Republic and Ireland and T. discolor haplotypes from the Czech Republic, Spain, Iran and Japan are sister OTUs.

Faecal egg count reduction test in goats: Zooming in on the genus level.

The faecal egg count reduction test (FECRT) is the most widely used method to assess treatment efficacy against gastrointestinal nematodes (GIN). Information on genera composition of the GIN community is not available with this test and it is commonly obtained by identifying cultured third-stage larvae (L3) or through molecular assays in the post-treatment survey, but results provided are usually only qualitative or semi-quantitative. The updated WAAVP guidelines now recommend assessing anthelmintic efficacy for each GIN genus/species separately (genus-specific FECRT), but this approach is poorly employed in Europe and in goats especially. For this reason, four FECRT trials were conducted using oxfendazole and eprinomectin in two Italian goat farms. Samples were processed individually using the McMaster technique and then pooled to create two samples from faeces of 5 animals each. Pooled samples were analysed using the McMaster and cultured for seven days at 26°C to obtain L3s. The genus-specific FECRT was based on larval identification, integrating coproculture and FEC results. Larvae were identified as Haemonchus, Trichostrongylus, Teladorsagia, Oesophagostomum / Chabertia and Bunostomum. Molecular assays (a multiplex real-time PCR and two end-point PCRs) were also implemented on pooled samples to support the morphological identification. The Spearmann Rho test confirmed a high correlation between the two approaches (Rho = 0.941 and Rho = 0.914 respectively for Haemonchus and Trichostrongylus, the two most common genera). Both oxfendazole and eprinomectin were effective in one farm, while none in the other farm (FECR = 75.9% and 73.3% respectively). In the second farm, the genus-specific FECRT highlighted a different response to treatment among genera: oxfendazole lacked efficacy against both Haemonchus and Trichostrongylus spp., eprinomectin only against Haemonchus, while all other genera were susceptible to both drugs. This study brings new attention on the importance of adopting a genus-specific approach to identify and quantify differences in susceptibility to anthelmintics among genera in goats, providing support for FECRT interpretation, anthelmintic resistance evaluation and evidence-based GIN control.

Cephenemyia stimulator and Hypoderma diana infection of roe deer in the Czech Republic over an 8-year period.

A survey of naso-pharyngeal and subcutaneous myiasis affecting roe deer (Capreolus capreolus) was conducted in the Czech Republic over an 8-year period (1999-2006). A total of 503 bucks and 264 does from six hunting localities were examined. The sampling area comprised predominantly agricultural lowlands and a mountain range primarily covered by forest. Since 1997, the deer have been treated each winter across the board with ivermectin (150 mg/kg, CERMIX® pulvis, Biopharm, CZ). Parasites found were the larvae of Hypoderma diana and Cephenemyia stimulator. There were no significant differences in warble fly infection among captured animals in the individual hunting localities. Overall, 146 (28.8%) of 503 animals (bucks) were infected with Cephenemyia stimulator larvae; body size of the second instar larva reached 13-18 mm. The prevalence ranged from 16.1 to 42.9% per year, and the mean intensity from 6 to 11 larvae per animal. Additionally, a total of 264 roe deer (does) were examined for H. diana larvae, and 77 (29.1%) were found to be positive; body size of the second instar larva reached 17 mm. The prevalence ranged from 18.8 to 50.0% per year, and the mean intensity from 13 to 22 larvae per animal. The results showed that the bot flies, Cephenemyia stimulator as well as H. diana, are common parasites in roe deer in the Czech Republic, and that through the help of treatment (ivermectin), it is possible to keep parasite levels low. The body weights of infected and non-infected H. diana deer did not differ significantly.

An alien parasite in a changing world - Ashworthius sidemi has lost its traditional seasonal dynamics.

A non-native nematode Ashworthius sidemi has emerged in captive fallow deer in Central and Eastern Europe over the last decade. Although this parasite has been spreading in the wild outside it's native distributional range and colonising local European host species since the middle of the last century, limited information has been published on the seasonality of A. sidemi and its susceptibility to anthelmintics. To address this knowledge gap, we conducted a study to investigate seasonal dynamics of the non-native parasite in the current Central European climate conditions. We collected freshly voided faecal pellets at four-week intervals from February 2018 to February 2020 at a fallow deer reserve with a known history of A. sidemi presence. The faecal pellets obtained were pooled after each site visit (n = 25) and coprocultured to obtain the third stage larvae of trichostrongylid nematodes at monthly intervals. Total genomic DNA was extracted from the recovered larvae. Using real-time multiplex PCR, A. sidemi DNA was detected in 17 out of 25 larval samples (68% prevalence). During the monitoring period, the annual administration of ivermectin based premix (Cermix) took place in January 2018, 2019, and 2020, and additionally a mixture of rafoxanide and mebendazole (Rafendazol) was administered once in spring 2019. The probability of parasite presence was significantly influenced by the time since the drug administration (p = 0.048) and the mean temperature at the location (p = 0.013). Larval samples negative for A. sidemi were always identified shortly after the drug administration. However, rapid pasture contamination by the parasite eggs from two to three months after Cermix administration and within one month after Rafendazol administration suggest only a short-lived efficacy of both administered drugs. The abundance of A. sidemi DNA was positively affected by mean temperature (p = 0.044) and remained relatively stable throughout the monitoring period, with the highest peak in August 2018 and 2019. Pasture contamination with A. sidemi eggs occurred almost all year round, with the exception of the beginning of 2018, 2019, and 2020. These findings indicate adaptation of a non-native parasite to the current climatic conditions of the Czech Republic resulted in negligible seasonal patterns of parasite egg shedding.

Concentrations of Zn, Mn, Cu and Cd in different tissues of perch (Perca fluviatilis) and in perch intestinal parasite (Acanthocephalus lucii) from the stream near Prague (Czech Republic).

We monitored concentrations of Cd, Cu, Mn and Zn in acantocephalan parasites (Acanthocephalus lucii) and its final host (Perca fluviatilis). The concentrations in parasites were found to be significantly higher than those found in the muscle, gonads and liver of fish host. The bioaccumulation factor values (BF=C(parasite)/C(host muscle)) were 194, 24.4, 2.2 and 4.7 for Cd, Cu, Mn and Zn, respectively. This suggests a benefit for the host due to the high accumulation of toxic cadmium.

Essential oils in the ranunculaceae family: chemical composition of hydrodistilled oils from Consolida regalis, Delphinium elatum, Nigella hispanica, and N. nigellastrum seeds.

In this study, we analyzed the chemical composition of volatile oils hydrodistilled from seeds of Consolida regalis, Delphinium elatum, Nigella hispanica, and N. nigellastrum using GC and GC/MS. In C. regalis, octadecenoic (77.79%) and hexadecanoic acid (8.34%) were the main constituents. Similarly, the oils from D. elatum and N. hispanica seeds consisted chiefly of octadecadienoic (42.83 and 35.58%, resp.), hexadecanoic (23.87 and 28.59%, resp.), and octadecenoic acid (21.67 and 19.76%, resp.). Contrastingly, the monoterpene hydrocarbons α-pinene (34.67%) and ß-pinene (36.42%) were the main components of N. nigellastrum essential oil. Our results confirm the presence of essential oils in the family Ranunculaceae and suggest chemotaxonomical relationships within the representatives of the genera Consolida, Delphinium, and Nigella. In addition, the presence of various bioactive constituents such as linoleic acid, (-)-ß-pinene, squalene, or carotol in seeds of D. elatum, N. hispanica, and N. nigellastrum indicates a possible industrial use of these plants.

Effect of Acanthocephalus lucii infection on total mercury concentrations in muscle and gonads of fish host (Perca fluviatilis).

The samples from 13 perches (Perca fluviatilis) - muscle with skin and bones; fish gonads; and acanthocephalan parasites were analysed for mercury (Hg). Hg concentrations were present in all analysed samples. There were found no statistically significant difference in Hg concentration in fish tissues between perches either with or without infection by the acanthocephalan parasite, Acanthocephalus lucii). In this study there was no evidence that acanthocephalan worms accumulate mercury from hosts. For this reason, A. lucii is not a suitable bioindicator for mercury pollution.

Non-native Nematode Ashworthius sidemi Currently Dominates the Abomasal Parasite Community of Cervid Hosts in the Czech Republic.

Ashworthius sidemi is an abomasal nematode typical for Asiatic cervids such as sambar (Rusa unicolor) or sika deer (Cervus nippon). This non-native parasite was introduced into Europe via sika deer in the late 19th and early 20th centuries. The current dynamic spread of this parasite amongst autochthonous wild cervids occurs independently of human activities, and A. sidemi has a negative impact on the health of wild ruminants and may pose a threat to the conservation of endangered wild ungulates and to livestock. This invasive parasite has been previously detected in the Czech Republic, but more accurate information on A. sidemi is required. Only limited information is generally available on the factors influencing the spread of abomasal nematodes in wild ruminants, so more information is necessary for planning effective strategies of parasite control. We therefore conducted a survey on the abomasal nematodes in cervids in both game reserves and hunting grounds across the Czech Republic, taking into account the hosts (species, age, sex) and environmental factors (monthly average temperature). The abomasa of 104 animals belonging to five cervid species originating from various locations of the country were collected. Data on host (species, sex, and age group) and the monthly average temperature in the region were obtained for each animal. The parasitological analyses indicated that 92% of the abomasa were infected by nematodes. Ashworthius sidemi was the most prevalent (72%) and abundant (80% of the total recovered individuals) nematode species and was detected in all cervid species except white-tailed deer. The intensity of A. sidemi was highest in roe deer (Capreolus capreolus) and fallow deer (Dama dama), but A. sidemi abundance did not depend substantially on the host or environmental factors. In contrast, the abundance of nematodes from the subfamily Ostertagiinae was influenced by the host species and temperature. Parasitic load was significantly higher in roe deer and during the warmer periods of the survey. We also detected another non-native nematode species, Spiculopteragia houdemeri. The results of our study suggest that the non-native nematode A. sidemi is now widespread amongst cervid hosts in the Czech Republic, probably due to the high sensitivity of autochthonous cervids to A. sidemi infections as well as adaptation of this parasite to the current climatic conditions of this country.

Total Failure of Fenbendazole to Control Strongylid Infections in Czech Horse Operations.

The control of strongylid infections has become challenging globally for equine practitioners due to the development of anthelmintic resistance. Comprehensive information on anthelmintic resistance in the Czech Republic, however, is still lacking. This study monitored the current efficacy of fenbendazole, pyrantel embonate, ivermectin and moxidectin. Forty-eight of 71 operations met the criteria (≥6 horses with ≥200 eggs per gram), with 969 fecal egg count reduction tests performed. Anthelmintic resistance was evaluated on an operation level based on fecal egg count reduction (FECR) and the lower limit of the 95% credible interval (LLCI) using Bayesian hierarchical models. General anthelmintic efficacy across all operations was assessed by posterior FECRs and the occurrence of sub-zero efficacies. Ivermectin and moxidectin demonstrated excellent efficacy (FECR 99.8-100%; 99.4-100 LLCI) in 45 and 23 operations, respectively, pyrantel embonate demonstrated sufficient efficacy in 15 operations and resistance was suspected in seven operations (FECR 88.1-99.1%; 72.5-98.5 LLCI). Fenbendazole, however, was not effective in a single operation (FECR 19.1-77.8%; 8.1-50.1 LLCI) out of 18. Fenbendazole had the highest probability of sub-zero efficacy (29.1%), i.e., post-treatment fecal egg counts exceeded the pre-treatment counts. Our data indicate an increase in the development of anthelmintic resistance, resulting in total failure of fenbendazole and a reduced efficacy of pyrantel embonate. Introducing advanced approaches of parasite control in the Czech Republic to slow the spread of anthelmintic resistance is thus needed.

In vitro synergistic effects of baicalin with oxytetracycline and tetracycline against Staphylococcus aureus.

OBJECTIVES: In this study we examined the in vitro synergistic effect of baicalin, the flavone constituent of Scutellaria spp., in combinations with oxytetracycline and tetracycline on the growth of Staphylococcus aureus. METHODS: The MICs were determined by the broth microdilution method and the effect of combinations was evaluated according to the sum of fractional inhibitory concentration indices (FICIs). RESULTS: Synergistic activity (FICI  ≤  0.5) was observed for combinations of baicalin with oxytetracycline or tetracycline against 4 of 10 strains tested, whereas the baicalin/oxytetracycline combination possessed the strongest synergistic effect (FICI = 0.418). CONCLUSIONS: Baicalin acts synergistically with oxytetracycline and tetracycline, enhancing its antimicrobial activity against S. aureus, including methicillin- and tetracycline-resistant strains.

Which McMaster egg counting technique is the most reliable?

This study focuses on the comparison of three selected modifications of the McMaster counting technique, namely the McMaster method modified by Wetzel (W) and Zajícek (Z), as well as the concentration McMaster technique according to Roepstorff and Nansen (R&N). These modifications differ in the weights of faeces examined (W, 2 g/Z, 1 g/R&N, 4 g), flotation solutions (W, NaCl/Z, MgSO(4) + Na(2)S(2)O(3)/R&N, NaCl + glucose), centrifugation (W, none/Z, 2,000 RPM for 2 min and 2,000 RPM for 1 min/R&N, 1,200 RPM for 5 min), number of McMaster chambers investigated (W, 3/Z, 2/R&N, 2), and multiplication factors used (W, 67/Z, 33/R&N, 20). To investigate the sensitivity and reliability of these methods, nematode eggs (Teladorsagia circumcincta) were used. Parasite elements are distributed through negative binomial distribution in naturally infected host faeces, and the number of parasite elements in a given amount of faeces sample is unknown to man. Therefore, we decided to prepare the exact number of eggs which were added to the parasite negative faeces; the faecal sample was then investigated. From this perspective, this is the first time a comparison of the McMaster methods has been so accurately investigated. This approach allows us to evaluate the real sensitivity and reliability of the tested method. As the findings of this study indicate, the highest sensitivity and reliability were obtained using the Roepstorff and Nansen modification. This McMaster modification is able to detect 20 eggs per sample (in 70% of samples). Concentrations of 200 and 500 eggs can be found in almost 100% of samples. Moreover, this method is simple, cheap and fast. For these reasons, we can recommend this method for routine veterinary practice.

Intestinal parasite Acanthocephalus lucii (Acanthocephala) from European perch (Perca fluviatilis) as a bioindicator for lead pollution in the stream "Jevanský potok" near Prague, Czech Republic.

Lead concentrations in the tissues of perch and its parasites were determined as mg/kg dw. Lead was found at higher concentrations in the acanthocephalans (11.56) than in different tissues (liver, gonads and muscle with skin and bone) of perch. With respect to fish tissues, the highest concentrations of lead were present in the liver (1.24), followed by the gonads (0.57) whereas the lowest concentrations were in the muscle with skin and bone (0.21). The bioconcentration factors for lead indicated that parasites accumulate metals to a higher degree than fish tissues--lead concentrations in acanthocephalans were 9.32, 19.27 and 55.05 higher than in liver, gonads and muscles of host, respectively.

Nematode-induced pathological lesions and alterations of mucin pattern identified in abomasa of wild ruminants.

Pathological lesions as well as mucin alterations in abomasa infected by nematodes have been thoroughly studied in livestock, but such data from wild ruminants are limited or completely lacking. Pathological data for Ashworthius sidemi, an invasive nematode are particularly rare. We necropsied the abomasa of 21 wild ruminants belonging to five cervid species and detected mixed nematode infections, dominated by A. sidemi. Samples from both gross lesions and mucous membranes without macroscopically apparent pathological alterations were subjected to standard histological procedures and histochemical staining. Histological examination found chronic abomasitis, manifested by edema, and hyperemia. Various degrees of lymphoplasmacytic infiltration were observed in all samples. Initial fibrosis (8/20, 40%) was detected in samples from both gross lesions and areas without macroscopically visible changes. Tissue from hemorrhagic lesions was superficially eroded. Generalized loss of surface polysaccharides was apparent in all samples. Only residual periodic acid-Schiff and Alcian blue (pH2.5) positivity was detected in the upper abomasal pits and in mucosal neck. This study found that nematode infections, mostly by A. sidemi, caused chronic inflammation and negatively affected abomasal mucin formation in wild ruminants.

The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays.

BACKGROUND: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies. METHODS: Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC. RESULTS: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp. CONCLUSIONS: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.