Validation of deep amplicon sequencing of Dicrocoelium in small ruminants from Northern regions of Pakistan.

Dicrocoelium lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple Dicrocoelium species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe Dicrocoelium species in sheep and goats. First, rDNA ITS-2 sequences of four Dicrocoelium species (Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium orientalis, and Dicrocoelium chinensis) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of Dicrocoelium species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the Dicrocoelium genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as D. dendriticum and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the Dicrocoelium species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from D. orientalis and D. chinensis, which sit very close to each other in a single large clade whereas D. hospes and D. dendriticum are separated into their own clade. In conclusion each sample was identified as D. dendriticum based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of Dicrocoelium in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance.

Case report: Infection with Dicrocoelium dendriticum in a Japanese Chin dog.

Dicrocoelium dendriticum is a trematode colonising the bile ducts of herbivores. Coproscopic findings in dogs are usually considered gastrointestinal passages of eggs after ingestion of unheated liver tissue or infected ruminant faeces. Here, a Japanese Chin presented with diarrhoea and weight loss. Eggs comparable to D. dendriticum were detected in faeces and infection was confirmed via PCR and by ruling out differential diagnoses. Egg excretion continued for a period of 10 months. Praziquantel (50 mg/kg body weight [BW]) was administered orally for four consecutive days. Egg excretion 10 days after treatment entailed further treatments with 100 mg/kg BW, again for four days. Faecal samples were negative ten days and four weeks afterwards, diarrhoea resolved, and the dog gained weight. In cases of repeated coproscopic positivity for D. dendriticum, an infection with dogs acting as definitive hosts should be considered. Treatment with praziquantel at a higher dosage may be required.

Histopathology and antibody responses describe the seasonal pattern of dicrocoeliosis in small ruminants in the Himalayan ranges of Pakistan.

In some parts of the world, Dicrocoelium spp. lancet flukes cause significant production loss in pastoral livestock, and accurate diagnosis of infection is important. The aims of the present study were to describe the histopathology and to investigate the transmission patterns of Dicrocoelium amongst ten sheep and goat farms in Khyber Pakhtunkhwa and Gilgit Baltistan, Pakistan. The liver histology and indirect enzyme-linked immunosorbent assay (ELISA) analyses followed standard procedures. The liver histopathology showed intensive tissue destruction and biliary hyperplasia associated with presence of adult flukes, severe inflammatory cell infiltration, congestion of blood vessels, damaged hepatocytes, and sinusoids in the infected areas. The time of onset of infection was investigated by ELISA detection of antibodies in sheep (n = 164) and goats (n = 152). Colostral transfer of Dicrocoelium antibodies from seropositive mothers was detected in sheep and goats up to 16 weeks of age. In both sheep and goats, the estimated time of infection differed between farms and years. Infection was seen in both sheep flocks and goat herds, with high variation between flocks and herds, and the highest infection rate in lambs. Dicrocoelium infection was most prevalent in sheep and goats in September (n = 84) and August (n = 63) respectively. This study concluded Dicrocoelium causes severe inflammation and necrosis of liver tissues in sheep and goats. Colostral transfer of antibodies can be detected up to about ten weeks of age. Higher infection rates are observed during August and September in sheep than in goats, putatively due to effects of different grazing and browsing behaviors on the ingestion of ants. The results will aid in the development of effective disease control strategies to ensure optimal growth and productivity of sheep and goats.

Spatial Distribution of Dicrocoelium in the Himalayan Ranges: Potential Impacts of Ecological Niches and Climatic Variables.

PURPOSE: Dicrocoeliosis can be an important cause of production loss in ruminants due to the cost of liver condemnation at slaughter. The aim of the present study was to determine the prevalence of Dicrocoelium infection and to predict the ecological niches and climatic variables that support dicrocoeliosis in the Himalayan ranges of Pakistan. METHODS AND RESULTS: Dicrocoelium was detected in 33 of 381 liver samples and 238 of 6060 blood samples taken from sheep and goat herds in the area. The prevalence of dicrocoeliosis was higher in sheep than in goats and highest in females aged more than 3 years. An environmental risk map was created to predict active zones of transmission and showed the highest probability values in central parts of the Chitral district in the northwest of Pakistan. Climatic variables of the mean monthly diurnal temperature range (Bio2), annual precipitation (Bio12), and normalised difference vegetation index (NDVI) were found to be significantly (p < 0.05) associated with the presence of Dicrocoelium infection. CONCLUSION: Together, the findings of this study demonstrate the most suitable ecological niches and climatic variables influencing the risk of dicrocoeliosis in the Himalayan ranges of Pakistan. The methods and results could be used as a reference to inform the control of dicrocoeliosis in the region.

Neuromuscular system of the causative agent of dicrocoeliosis, Dicrocoelium lanceatum. I. 5-Hydroxytryptamine in the nervous system.

The trematode Dicrocoelium lanceatum known as lancet fluke, is a causative agent of dicrocoeliosis, a widespread parasitic disease of the grazing ruminants. The investigation of the major neurotransmitters and their functions are an important step in the development of a new pharmacological strategy of the struggle against the dicrocoeliosis affecting the neuronal signal substances and the functions of its nervous system. The aim of this work was to study the presence and localization of the neurotransmitter serotonin (5-HT, 5-Hydroxytryptamine) in the nervous system of D. lanceatum using immunocytochemical technique and confocal laser scanning microscopy. For the first time the data on the presence and distribution of serotonin-immunopositive components in the central and peripheral compartments of the nervous system of D. lanceatum has been obtained. Serotonin-immunopositive neurons and neurites were identified in paired brain ganglia, in the brain commissure, longitudinal nerve cords and connective nerve commissures. The innervation of the oral and ventral suckers by serotonergic nerve structures was revealed. The distal part of the reproductive system and the region of the reproductive pore were intensively innervated by serotonergic neurites. Serotonin-immunopositive neurons and neurites were also revealed in the proximal region of the reproductive system. The data obtained suggest that the serotonergic nervous system is involved in the regulation of the attachment organs and the reproductive system functions in D. lanceatum. The new results on the morphological and functional organization of the D. lanceatum nervous system increase our knowledge of the structure and function of nervous system of trematodes of various taxonomic groups and support the possibility of the exploitation of the serotonergic system of the parasite as a target for anthelmintic drugs.

A survey of sheep dicrocoeliosis in Sulaymaniyah slaughterhouse, northern Iraq in 2013-2014.

Dicrocoelium dendriticum is a helminth which lives in the bile ducts and gall bladder of mammalian definitive hosts. Dicrocoeliosis is thought to be endemic in some countries and it has an increasing prevalence in Northern Iraq, potentially due to increased importation of infected animals, particularly sheep, in high numbers from neighboring countries. The parasite's ability to infect rodents, wild animals, livestock and humans means this parasite is of significant interest in veterinary and human medicine. While D. dendriticum causes relatively mild clinical disease in animals, infection leads to liver condemnation at slaughter and subsequent economic losses to farmers. In this study, the livers of 91,486 sheep slaughtered at Sulaymaniyah New Slaughterhouse (Northern Iraq) were visually inspected for D. dendriticum infection between November 2013 and March 2014, with 1269 livers rejected due to D. dendriticum in this period, representing 29.4% of all condemned livers. The highest rate of rejection due to D. dendriticum infection was seen in December, possibly linked to increased numbers of intermediate host snails during the wet season. Routine inspection of condemned livers revealed adult flukes of D. dendriticum and eggs were seen in the faeces of imported sheep, indicating these animals may be a vector for introduction of D. dendriticum to this region. Due to the complex life cycle and wide range of animals which it infects, we cannot establish the exact route of introduction into Iraq; however, our study suggests that slaughterhouse workers, farmers and local health authorities should be aware of the presence of D. dendriticum, and the potential risks it represents to both human and animal health. Our data also suggest that some level of quarantine or border checks may be useful to prevent further introduction of D. dendriticum or other pathogens into Iraq, although this may prove difficult until accurate diagnostic assays are developed.

Evaluation of zoonotic platyhelminthe infections identified in slaughtered livestock in Iran, 2015-2019.

BACKGROUND: Platyhelminth infections of livestock can result in considerable economic losses. This study aims to evaluate the spatial frequency of cystic echinococcosis (CE), dicrocoeliasis, and fascioliasis in livestock slaughtered in Iran during the years 2015-2019 and estimate direct costs associated with organ condemnation due to these parasites. METHODS: Abattoir data from 413 abattoirs representing all 31 Iranian provinces were collected from the Iran Veterinary Organization. Infection prevalence was calculated per year at the province level. The Local Moran's I statistic was performed to evaluate spatial autocorrelation of animals positive at slaughter for the years 2015-2019. Direct costs associated with condemned livers were calculated for each parasitic condition, with costs associated with condemned lungs also included for CE. RESULTS: Overall prevalence values for the study timeframe were as follows: sheep and goat fascioliasis 1.56% (95% CI: 1.56-1.56%), cattle fascioliasis 3.86% (95% CI: 3.85-3.88%), sheep and goat dicrocoeliasis 4.63% (95% CI: 4.62-4.63%), cattle dicrocoeliasis 3.08% (95% CI: 3.07-3.09%), sheep and goat CE 5.32% (95% CI: 5.32-5.33%), and cattle CE 7.26% (95% CI: 7.24-7.28%). Northwest Iran had the highest prevalence of CE and fascioliasis. High infection areas for Dicrocoelium spp. included the provinces of Zanjan, Gilan, Qazvin, and Tehran, which are located in northern Iran. Direct economic losses for sheep and goat fascioliasis, dicrocoeliasis, and CE for the study period were US$13,842,759, US$41,771,377, and US$22,801,054, respectively. Direct economic losses for cattle fascioliasis, dicrocoeliasis, and CE for the study period were US$1,989,200, US$1,668,986, and US$2,656,568, respectively. CONCLUSION: Our findings provide valuable data for future monitoring of these important parasitic diseases in Iranian livestock. Disease control strategies are required to reduce the economic and public health impact of these platyhelminths.

Parasitic causes of meat and organs in cattle at four slaughterhouses in Sistan-Baluchestan Province, Southeastern Iran between 2008 and 2016.

This 8-year (from 2008 to 2016) retrospective study calculated the percentage of carcass and organ (lung and liver) condemnations and estimated the direct financial costs at four slaughterhouses in Sistan-Baluchestan Province, Southeastern Iran. Each carcass and organ (lung and liver) was thoroughly examined through inspection, palpation and incision following the standard protocol. Identification of the parasites was performed macroscopically. The total direct economic loss due to meat's condemnation was estimated by adding weights of each organ or carcass part and multiplying individual organ totals by their 2016 market unit price. A total of 857,039 cattle were slaughtered during this period, 64,497 livers (7.5%), 31,401 lungs (3.6%) and the carcasses of 1,171 cattle (0.1%) were condemned due to lesions caused by parasites. The main parasitic lesions in the condemned livers were attributed to Echinococcus granulosus (4.2%), Fasciola spp. (3.1%) and Dicrocoelium dendriticum (0.1%). All the condemned lungs were due to E. granulosus (3.6%). Taenia saginata cysticerci were detected in 0.1% of inspected animals. Liver condemnation due to cystic echinococcosis was the highest in fall (4.7%, p < 0.001); while lung condemnation was the highest during spring (3.98%, p < 0.001). Liver condemnation due to Fasciola spp. was the lowest in winter (2.99%, p < 0.001). Carcass condemnation as a result of cysticercosis was the highest in summer (p < 0.001). Considering the 2016 market prices, condemnations due to the studied parasites caused direct costs estimated U.S. $ 3,191,879. To the best of our knowledge, this is the first report estimating the monetary losses due to parasitic infections in the slaughterhouses of this province. Due to the high financial impact of the studied parasites, a control programme should be implemented to decrease this impact.

Dicrocoelium spp. in cattle from Wa, Ghana: prevalence and phylogeny based on 28S rRNA.

Dicrocoeliosis is a trematode infection in cattle, sheep and goats caused by the small liver fluke, Dicrocoelium spp. Though endemic in Ghana, its disease situation is poorly understood. In the present study, the prevalence, distribution and worm load of Dicrocoelium spp. in cattle at slaughter in Wa were determined. A total of 389 cattle were screened during meat inspection for liver flukes, and polymerase chain reaction accompanied by DNA sequencing of the 28S rRNA gene was used to identify Dicrocoelium spp. Generally, prevalence of bovine dicrocoeliosis (small liver fluke) stood at 19.54 % with prevalence in males and females being 17.62 % and 21.43 %, respectively. Animals under 2 years suffered more infection than older ones (23.08 % vs. 16.80 %). Dicrocoelium infection was recorded in animals from all the communities where slaughtered cattle came from. On average, 31 flukes per infected animal were recorded. A molecular confirmatory test on seven flukes identified them as D. hospes. This preliminary study highlights the importance of bovine dicrocoeliosis in Ghana and has identified D. hospes as a causal agent. The data provides basis for further studies to appraise the trematode disease situation in animals and phylogeny of Dicrocoelium spp. circulating in Ghana.

Treatment of sarcoptic and chorioptic mange in an alpaca (Vicugna pacos) herd with a combination of topical amitraz and subcutaneous ivermectin.

Clinical history: An outbreak of intense pruritus and weight loss in a herd of 40 alpacas (Vicugna pacos) in the south-west of France was investigated after the death of 14 adults. One alpaca was referred to a veterinary teaching hospital for diagnosis and treatment but died soon after and one of the dead alpacas was submitted for necropsy. Clinical findings: The remaining alpacas were intensely pruritic with variably severe and extensive alopecia, erythema, lichenification and crusting on the face, ventral abdomen and distal limbs. Superficial skin scrapes from five animals revealed large numbers of Sarcoptes scabiei mites, and less frequent and numerous Chorioptes bovis mites. Coproscopic examinations revealed a median of 1,350 (min 500, max 8800) strongyle epg. The alpaca admitted for treatment was anaemic and hypoalbuminaemic. Skin scrapes revealed copious S. scabiei and C. bovis mites. The two alpacas examined post-mortem had similar skin lesions to those examined on-farm and were cachexic. One had lung lesions attributed to protostrongylid infestation and its liver contained numerous Dicrocoelium spp. adults. Diagnosis: Sarcoptic and chorioptic mange with secondary superficial bacterial skin infection, associated with severe internal parasitism and underfeeding. Treatment and outcome: All 25 alpacas were treated topically with a 3% chlorhexidine shampoo followed by a 0.025% amitraz wash at the initial visit and then 1, 2, 3, 7 and 9 weeks later. A systemic treatment with S/C 500 µg/kg ivermectin was administered at the initial visit and then 2, 7 and 9 weeks later. The alpacas were treated orally with 50 mg/kg praziquantel to control dicrocoeliosis. Nutritional measures, including increased pasture area and supplemental feeding were simultaneously implemented. Pruritus was reduced 1 week after the start of treatment and had resolved after 2 weeks. After 9 weeks, skin lesions were markedly improved. Six months after the initial visit, skin lesions entirely resolved and superficial skin scrapes, taken from half of the animals, were negative for mites. Clinical relevance: This is the first report of the use of two acaricides combined with a chlorhexidine shampoo to successfully treat simultaneous sarcoptic and chorioptic mange in alpacas.

Molecular confirmation of Dicrocoelium dendriticum in the Himalayan ranges of Pakistan.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to confirm the species identity of Dicrocoeliid flukes collected from the Chitral valley in the Himalayan ranges of Pakistan. The morphology of 48 flukes belonging to eight host populations was examined; but overlapping traits prevented accurate species designation. Phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess within and between species variation and re-affirm the use of species-specific single nucleotide polymorphism markers. PCR and sequencing of 34 corresponding fragments of ribosomal DNA and 14 corresponding fragments of mitochondrial DNA from the Chitral valley flukes, revealed 10 and 4 unique haplotypes, respectively. These confirmed for the first time the molecular species identity of Pakistani lancet liver flukes as D. dendriticum. This work provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings.

Prevalence of Liver Trematode Infections in Cattle in the Province of Agri in Turkey

Objective: This study was conducted to determine the prevalence of distomatosis in cattle in the Agri province in Turkey. Methods: The livers and bile ducts of 200 slaughtered cattle were examined macroscopically, and the gall bladders were examined by sedimentation. The presence of Fasciola hepatica eggs and antigens in 188 cattle were investigated using sedimentation and coproantigen ELISA, respectively. Egg counts per gram of faeces (EPG) was determined using the modified McMaster sedimentation method. Results: Adult Fasciola hepatica and Dicrocoelium dendriticum were found in the bile ducts of 47 and 25 cattle, respectively. Examination of the gall bladders of cattle revealed the presence of eggs of Fasciola hepatica and D. dendriticum in 63 and 48 cattle, respectively. The coproantigens of Fasciola hepatica were found in 148 (78.7%) cattle using ELISA. Additionally, the eggs of F. hepatica were found in 63 (33.5%) cattle by sedimentation, with the value of EPG between 17 and 83. The prevalence of D. dendriticum was determined as 25.5%, with the value of EPG between 17 and 67. Conclusion: In this study, distomatosis was detected in cattle in Agri province using coproantigen ELISA and sedimentation methods along with macroscopic examination. The findings necessitate an urgent implementation of an effective prevention and control program, which can increase the awareness of cattle breeders regarding these diseases. Furthermore, there is also a need for more detailed studies on these diseases.

Molecular characterization and immunodiagnostics of Dicrocoelium dendriticum species isolated from sheep of north-west Himalayan region.

Despite its extensive presence among grazing ruminants, dicrocoeliosis, also known as 'small liver fluke' disease, is poorly known and often underestimated by researchers and practitioners in many countries. The accurate identification and prepatent diagnosis of Dicrocoelium dendriticum infection is an essential prerequisite for its prevention and control. In the present study, the morphologically identified specimens isolated from the bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of the second internal transcribed spacer (ITS-2) of our isolates showed a high degree of similarity with D. dendriticum using the BLAST function of the National Center for Biotechnology Information (NCBI). The phylogenetic analysis of our isolates showed a close relationship with previously described D. dendriticum isolates from different countries. The antigenic profiles of somatic and excretory/secretory (E/S) antigens of D. dendriticum were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from sheep naturally infected with D. dendriticum. By SDS-PAGE, 16 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited six seroreactive bands ranging from 27 to 130 kDa. Among these, the 84 and 130 kDa bands were quite specific, with high diagnostic specificity and sensitivity. The E/S fraction comprised nine distinct bands, as revealed by SDS-PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited five antigenic bands ranging from 27 to 130 kDa. Among these, the 130 kDa band was found to be quite specific, with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 84 and 130 kDa in somatic fraction and 130 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also encourage further studies regarding their vaccine potential.

Prevalence of hydatid cyst, Fasciola spp. and Dicrocoelium dendriticum in cattle and sheep slaughtered in Sabzevar abattoir, Iran.

Hydatid cyst, Dicrocoelium dendriticum, and Fasciola spp. are common parasites among ruminants. The current study was conducted to assess the prevalence of these parasites in cattle and sheep slaughtered in Sabzevar abattoir, from April 2014 to March 2016. Total of 1653 cattle and 26720 sheep were inspected for helminthic infections of liver and lung in different seasons. The prevalence rates were found to be 684 (2.55%) and 235 (14.21%) in sheep and cattle, respectively. The infection rate of liver in cattle was estimated to be 5.86%, 0.84% and 5.68% for hydatid cysts, Fasciola spp. and D. dendriticum, respectively and lung infection with hydatid cyst in cattle was 1.81%. Moreover, the liver infection rate of sheep was estimated to be 0.87%, 0.06% and 2.13% with hydatid cysts, Fasciola spp. and D. dendriticum, respectively and lung infection with hydatid cyst was 0.35%. The rate of infection with D. dendriticum was higher in the both animals than the other parasites. The results of this study indicated that the prevalence of the aforementioned parasites was high, which caused high economic losses. Therefore, sanitary and preventive measures should be taken.

Predicting environmental suitability and geographical distribution of Dicrocoelium dendriticum at littoral of Caspian Sea: An ecological niche-based modeling.

Dicrocoeliasisis caused by the small liver fluke (Dicrocoelium spp.), mainly Dicrocoelium dendriticum in domestic and wild ruminants. The small liver fluke is the probable predisposing cause of economic burden. The impact of geographic and climatic factors on the incidence of dicrocoeliasis has been severely ignored in different geographical areas. Due to the lack of data regarding dicrocoeliasis in Iran, this study was aimed to investigate the prevalence and intensity of ovine and bovine Dicrocoelium infection in the coastal strip south of the Caspian Sea. Fecal samples were obtained from the cattle and sheep in three provinces of Guilan, Mazandaran and Golestan at the littoral of the Caspian Sea. All collected samples were then tested by flotation methods for determining the number of eggs per gram of feces (EPG). Moreover, we applied maximum entropy niche-based modeling (MaxEnt), coupled with remote sensing and the Geographical Information System (GIS) to visualize the spatial distribution and risk factors of Dicrocoelium dendriticum at the littoral of Caspian Sea. A total of 2688 stool samples were collected from cattle (n = 1344) and sheep (n = 1344) in coastal provinces of the Caspian Sea including Guilan (n = 1280), Mazandaran (n = 768) and Golestan (n = 640) provinces. Based on the data presented here, the highest rate of infection was observed in Guilan and Mazandaran provinces. The results revealed the prevalence rates of 36.72% and 6.09% for sheep and cattle in Guilan province, respectively. This rate was 22.4% for sheep and 3.91% for cattle in Mazandaran province. However, the rate of sheep infection was 90% in some point locations. Dicrocoelium infection was found to be significantly different between three provinces in sheep (P < 0.00001, Chi = 111.633). Our findings exhibited a high reliability of the MaxEnt model, and area under the curve (AUC) values of the training and test data sets were determined to be 0.852 and 0.818, respectively. Jackknife analysis showed the relative variable contribution to the model performance, where four variables were found as key influential factors that highly affected the habitat suitability of the presence of the lancet fluke including the precipitation of driest quarter (Bio17), altitude, temperature seasonality (Bio4), and precipitation of driest month (Bio14). The findings of this study demonstrated a high presence rate of Dicrocoelium infection at the littoral of Caspian Sea, Iran. Moreover, climatic variables can be considered as important predictive factors affecting the distribution of infection in the studied areas. Further studies based on the findings of the GIS are also very important in the country for planning control programs.

Dicrocoeliosis in extensive sheep farms: a survey.

BACKGROUND: This study investigated the epidemiological and molecular aspects of dicrocoeliosis in extensive sheep farms. METHODS: From 2013 to 2014, copromicroscopical analyses in 190 dairy sheep farms and anatomo-pathological inspections in six slaughterhouses were carried in Sardinia, Italy. Rectal faecal samples were analyzed using the FLOTAC® method, and anatomo-pathological examinations were based on detecting thickened terminal bile ducts (TTBDs). In addition, genetic analyses were conducted on representative DNA samples of adult Dicrocoelium spp. RESULTS: Ninety-seven (51.1%) out of 190 sheep farms were coprologically positive for Dicrocoelium spp. In the liver, on the surface and cut surface, TTBDs were reported in 40.1% (309/770) and 15.3% (118/770) of the animals examined, respectively, with an overall prevalence of 25.5% (196/770). No intraspecific genetic variation was observed among the Dicrocoelium dendriticum isolates. CONCLUSIONS: Our survey reveals the widespread presence of D. dendriticum in Sardinia, although seasonal, geographical and climatic conditions might be key factors in modulating the infection prevalence. Examining typical lesions due to D. dendriticum in the liver in abattoirs can be used as a marker for tracking chronic dicrocoeliosis infection.

Hepatic Dicrocoelium dendriticum infection in a miniature horse.

A miniature horse which died following humane seizure from an Ontario farm was emaciated with serous atrophy of fat. Autopsy revealed hepatic atrophy and moderate periportal fibrosis. Eggs and trematodes seen in the liver were identified as Dicrocoelium dendriticum. This appears to be the first reported case of infection of equids in North America with Dicrocoelium dendriticum.

Infection parDicrocoelium dendriticumhépatique chez un cheval miniature. Un cheval miniature qui est mort à la suite d'une saisie humanitaire dans une ferme de l'Ontario était émacié et avait une atrophie séreuse des tissus adipeux. L'autopsie a révélé une atrophie hépatique et une fibrose périportale modérée. Des oeufs et des trématodes observés dans le foie ont été identifiés comme Dicrocoelium dendriticum. Il semblerait qu'il s'agit du premier cas signalé d'infection des équidés par Dicrocoelium dendriticum en Amérique du Nord.(Traduit par Isabelle Vallières).

Evaluation of molecular methods for the field study of the natural history of Dicrocoelium dendriticum.

There is a need for improved methods for the study of the impacts of climatic and livestock management change on the epidemiology of production-limiting helminth parasitic diseases. In this study we report the application of molecular methods to describe the natural history of the small lancet fluke, Dicrocoelium dendriticum on Machair pastures on the Inner Hebridean Isle of Coll. Our results build upon those of the only previous historic field study of D. dendriticum in the British Isles that had been undertaken on the same study site. We demonstrate the value of combining conventional parasitological methods with PCR amplification of a mitochondrial DNA fragment for the detection of D. dendriticum in ants and snails, and PCR amplification of ITS2 and 28S ribosomal DNA fragments to support the species identity of the intermediate hosts, to improving understanding of the epidemiology of D. dendriticum. We report the presence of D. dendriticum infection in cattle, sheep and rabbits grazing on Machair pastures. D. dendriticum infection was identified in a high percentage of the snails, identified as Cochlicella acuta and Cernuella virgata, and in a high percentage of Formica fusca and Myrmica ruginoides ants that were collected from, or clinging to, the tops of flowers. We have identified the involvement of different intermediate host species and higher prevalences of snail and ant infection than previously reported, in part reflecting differences between the sensitivity and specificity of morphological and molecular speciation methods. Overall, our results highlight the complex life history of dicrocoeliosis and illustrate the parasite's generalist host strategy that confers potential to exploit new niches created by climatic change or grazing management for habitat conservation.

Study on the Relationship between Liver Parasitic Infections and Serum Vitamin A and ß-Carotene Status in Cattle.

OBJECTIVE: This study was conducted to evaluate the relationship between serum levels of vitamin A and ß-carotene in the liver of cattle. METHODS: A total number of 150 samples were selected according to the type of parasitic infections of the liver after postmortem examination and confirmation. Parasitic lesions in the liver were subdivided into three major parasites, including Fasciola hepatica, Dicrocoelium dendriticum, and hydatid cysts. Fifty samples were obtained from cattle without any parasitic infection as a control group. Serum levels of vitamin A, ß-carotene concentrations (µg/dL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity were assayed. RESULTS: Naturally infected cattle with D. dendriticum, F. hepatica and hydatid cyst showed lower vitamin A levels. Serum ß-carotene levels were significantly decreased in all groups (p<0.05). ALT and AST activities in animals with parasitic diseases were statistically higher than in control group (p<0.01). A significant negative correlation was determined between the ß-carotene, vitamin A levels and enzyme activities of the liver in all the three types of liver infections (p<0.001). CONCLUSIONS: This study indicated that serum levels of vitamin A and ß-carotene decline was present in cattle with liver parasite infection and vitamin supplements should be supplied.

Comparison of Fecal Egg Counts and ELISA for the Diagnosis of Dicrocoelium Dendriticum Infection.

From economical point of view, Dicrocoelium Dendriticum (D. dendriticum) causes a lot of damages to the livestock industry annually. So, the rapid diagnosis of infection is very important. The diagnosis is based on egg per count of feces (EPG) test because detection according to clinical symptoms is difficult. Since EPG is not accurate and sensitive, the serological methods become important for the diagnosis of this parasite as they are more accurate in comparison to EPG test and they are able to diagnose infection in a short time. In this study, somatic and Excretory-secretory antigens (EsAg) were isolated. The ELISA test was set up according to positive and negative sera and the results which were obtained compared to those obtained by the EPG test. The prevalence of infection in 550 samples by ELISA and EPG methods were 56% and 7% respectively, which shows the significant difference between these methods in examining the rate of infection. Based on the results, the specificity and sensitivity in ELISA test were 95% and 94%, respectively. The results showed that the ELISA is a more reliable test in comparison to EPG test for the rapid diagnosis of D. dendriticum infection.