Epidemiology of rumen fluke infection in selected buffalo farms in perak, malaysia: prevalence, molecular species identification, and associated risk factors.

Rumen flukes cause heavy economic losses in the ruminant industry worldwide, especially in tropical and subtropical countries. This study estimated the prevalence of rumen flukes in buffaloes, identified the species diversity, and determined risk factors associated with rumen fluke prevalence in Perak, Peninsular Malaysia. A cross-sectional study was conducted, and 321 faecal samples were collected from six buffalo farms. A structured questionnaire was developed, and farmers were interviewed to obtain information regarding risk factors associated with rumen fluke infection. The faecal samples were examined using sedimentation and Flukefinder® techniques. Genomic DNA was extracted from the fluke eggs recovered using the Flukefinder® method, and the internal transcribed spacer 2 (ITS2) fragment was amplified and sequenced to facilitate species identification. The results showed that the overall prevalence of rumen fluke across the sampled farms was 40.2% (129/321). Three rumen fluke species were identified, namely, Fischoederius elongatus, F. cobboldi, and Orthocoelium streptocoelium. Several management factors had a significant association (P < 0.05) with rumen fluke prevalence, including production type, cleaning of the stable, drinking water system, flooding around the farm, grazing system, pasture sharing with other livestock, and deworming program. This work constitutes the first attempt to understand the epidemiology of rumen fluke infection in the region and suggests that good farm management, pasture management, choosing appropriate drugs, and proper husbandry practices may improve buffalo health and production in areas where rumen flukes are prevalent.

Visualization and development of colorimetric loop-mediated isothermal amplification for the rapid detection and diagnosis of paramphistome infection: colorimetric PAR-LAMP.

Colorimetric detection can be applied to differentiate between positive and negative conditions. It can be coupled with loop-mediated isothermal amplification to diagnose rumen fluke or paramphistome infection, also called colorimetric PAR-LAMP. This study conducted LAMP using three candidate indicator dyes, namely malachite green (MLG), methyl green (MTG), and neutral red (NTR), and the results were observed by the naked eye. The dye concentration was optimized to obtain the most pronounced positive-negative result discrimination. Subsequently, we conducted target sensitivity tests using the DNA of Fischoederius elongatus at different concentrations. To validate the detection accuracy, the result was confirmed by gel electrophoresis. The sensitivity test presented the lowest detectable DNA concentration or limit of detection (LOD), with 1 pg for MLG, 0.5 ng for MTG, and 50 pg for NTR. Different LODs revealed inhibition of LAMP reaction and reduced efficiency of result presentation for colorimetric-based detection, particularly NTR and MTG. For MLG-LAMP, we observed no cross-reaction of non-target DNA and improved reaction with the DNA of Fischoederius cobboldi and Calicophoron sp., with multi-detection. In addition, naked eye observation and agarose gel electrophoresis (AGE) evaluation of the MLG-LAMP results showed a moderate and strong agreement with LAMP-AGE and microscopic examinations. Based on our results, colorimetric PAR-LAMP is a rapid, comfortable, and point-of-care procedure for the diagnosis of paramphistome infection.

Transcriptome and Secretome Analysis of Intra-Mammalian Life-Stages of Calicophoron daubneyi Reveals Adaptation to a Unique Host Environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock, which has seen a rapid rise in prevalence throughout Western Europe in recent years. After ingestion of metacercariae (parasite cysts) by the mammalian host, newly excysted juveniles (NEJs) emerge and invade the duodenal submucosa, which causes significant pathology in heavy infections. The immature flukes then migrate upward, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients, and avoid the host immune response. Here, transcriptome analysis of four intramammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic diseases, respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that while a family of cathepsins B with varying S2 subsite residues (indicating distinct substrate specificities) is differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is upregulated in adult worms, although they are under-represented in the secretome. The most abundant proteins in adult fluke secretions were helminth defense molecules that likely establish an immune environment permissive to fluke survival and/or neutralize pathogen-associated molecular patterns such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognize antigens from other helminths commonly found as coinfections with rumen fluke.

Differentiating paramphistome species in cattle using DNA barcoding coupled with high-resolution melting analysis (Bar-HRM).

Paramphistomosis is caused by paramphistome or amphistome parasites, including Fischoederius elongatus, Gastrothylax crumenifer, Orthocoelium parvipapillatum, and Paramphistomum epiclitum. The control and prevention of these parasite outbreaks are difficult because of the wide occurrence of these species. Besides, the clinical manifestations and their egg characteristics are similar to those of other intestinal flukes in the paramphistome group, leading to misdiagnosis. Here, we employed DNA barcoding using NADH dehydrogenase (ubiquinone, alpha 1) (ND1) and cytochrome c oxidase subunit I (COI), coupled with high-resolution melting analysis (Bar-HRM), for species differentiation. As a result, ParND1_3 and ParCOI4 resulted in positive amplification in the paramphistomes and Fasciola gigantica, with significantly different melting curves for each species. The melting temperatures of each species obtained clearly differed. Regarding sensitivity, the limit of detection (LoD) for all species of paramphistomes was 1 pg/µl. Our findings suggest that Bar-HRM using ParND1_3 is highly suitable for the differentiation of paramphistome species. This approach can be used in parasite detection and epidemiological studies in cattle.

Exploration of factors associated with spatial-temporal veterinary surveillance diagnoses of rumen fluke (Calicophoron daubneyi) infections in ruminants using zero-inflated mixed modelling.

Rumen fluke (Calicophoron daubneyi) has emerged as a prominent parasite of ruminants in Europe over the past decades. Epidemiological questions remain regarding this observed increase in prevalence as well as the prospect for future paramphistomosis risk. This study aimed to identify factors associated with the temporal−spatial prevalence of rumen fluke as measured by veterinary surveillance in a temperate region using zero-inflated negative binomial mixed modelling. Modelling revealed that summer rainfall, raindays and sunshine hours and mean winter temperature as significant positively associated climate variables for rumen fluke prevalence over space and time (P < 0.05). Rumen fluke prevalence was also higher in counties with higher cattle/sheep densities and was positively associated with rumen fluke case rates in the previous years (P < 0.05). Equivalent models for fasciolosis prevalence revealed no significant association with winter temperature and sunshine hours, (P > 0.05). These results confirm a strong association between rainfall and the prevalence of both fluke species in a temperate environment, likely due to the role of Galba truncatula as their intermediate snail host. It also highlights the potential added importance of winter temperature and sunshine hours in rumen fluke epidemiology when compared to liver fluke.

DNA barcoding of rumen flukes (Paramphistomidae) from bovines in Germany and Austria.

Rumen flukes have received growing veterinary attention in western and central Europe during the past two decades because of an increase in prevalence of infection in cattle and sheep, including cases of severe clinical disease. Historically, rumen fluke infections in Europe were assumed to be caused mainly by Paramphistomum cervi (or species, which were later considered to be synonymous with P. cervi), but more recently molecular studies demonstrated Calicophoron daubneyi to be the predominating species. For the present investigation, adult rumen flukes isolated from 23 cattle originating from ten farms in Germany (Saxony [1], Baden-Württemberg [4], Bavaria [5]) and one farm in Austria (Tyrol) were analyzed to establish partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) and the complete sequence of the nuclear internal transcribed spacer 2 (ITS2). Flukes of five animals (dairy cows from three farms in Bavaria) were determined as P. leydeni, and flukes of 18 animals (dairy cows or cattle from cow-calf operations from eight farms in Saxony [1], Baden-Württemberg [4], Bavaria [2], and Tyrol [1]) were identified as C. daubneyi. Based on the molecular analysis of adult rumen flukes collected from cattle, the results of this investigation confirm the common occurrence of C. daubneyi in Germany and reveal the first definitive findings of P. leydeni in Germany and C. daubneyi in Austria.

Evaluation of anthelmintic drugs against egg development of rumen flukes recovered from cattle raised in the humid tropics of Mexico.

Paramphistomosis is a parasitic disease endemic in ruminants nearly worldwide. In the present study, an in vitro screening of the main anthelmintics used in Mexico was carried out to determine the mean lethal dose for rumen fluke eggs from cattle in a humid, warm region. Rumen flukes were obtained from cattle slaughtered in the states of Tabasco and Chiapas in Mexico. Eggs were collected using a 37-µm sieve and quantified. Then, an in vitro incubation study was performed: 100 eggs were placed into the wells of polystyrene microtiter plates. Anthelmintic products were tested on the eggs at concentrations ranging from 0.0015 to 3.0 mg/ml for rafoxanide, 0.0025 to 10.20 mg/ml for nitroxinil and 0.0015 to 3 mg/ml for closantel to determine the median lethal dose (LD50) and maximum lethal dose (LD99). A control group (water) was included in each plate. Three different species of rumen flukes (Calicophoron brothriophoron, Calicophoron clavula and Paramphistomum cervi) belonging to five isolates were identified. Nitroxinil had the highest efficacy against rumen fluke eggs, with an LD50 of 0.11 to 65 µg/ml, whereas rafoxanide showed the lowest efficacy with an LD50 ranging from 500 to 1713 µg/ml. Closantel showed high variability in the LD50 among the different analysed isolates (17 to 122 µg/ml). The evaluated flukicidal drugs presented differential efficacy against the development of rumen fluke eggs. The efficacy of the drugs will vary depending on the geographical area of origin of the animals.

Rumen fluke in Irish sheep: prevalence, risk factors and molecular identification of two paramphistome species.

BACKGROUND: Rumen flukes are trematode parasites found globally; in tropical and sub-tropical climates, infection can result in paramphistomosis, which can have a deleterious impact on livestock. In Europe, rumen fluke is not regarded as a clinically significant parasite, recently however, the prevalence of rumen fluke has sharply increased and several outbreaks of clinical paramphistomosis have been reported. Gaining a better understanding of rumen fluke transmission and identification of risk factors is crucial to improve the control of this parasitic disease. In this regard, a national prevalence study of rumen fluke infection and an investigation of associated risk factors were conducted in Irish sheep flocks between November 2014 and January 2015. In addition, a molecular identification of the rumen fluke species present in Ireland was carried out using an isolation method of individual eggs from faecal material coupled with a PCR. After the DNA extraction of 54 individual eggs, the nuclear fragment ITS-2 was amplified and sequenced using the same primers. RESULTS: An apparent herd prevalence of 77.3 % was determined. Several risk factors were identified including type of pasture grazed, regional variation, and sharing of the paddocks with other livestock species. A novel relationship between the Suffolk breed and higher FEC was reported for the first time. The predominant rumen fluke species found was C. daubneyi. Nevertheless, P. leydeni was unexpectedly identified infecting sheep in Ireland for the first time. CONCLUSIONS: An exceptionally high prevalence of rumen fluke among Irish sheep flocks has been highlighted in this study and a more thorough investigation is necessary to analyse its economic impact. The isolation of individual eggs coupled with the PCR technique used here has proven a reliable tool for discrimination of Paramphistomum spp. This technique may facilitate forthcoming studies of the effects of paramphistomosis on livestock production. The most noteworthy finding was the identification of P. leydeni affecting sheep in Ireland, however further studies are required to clarify its implications. Also, a significant relationship between Suffolk breed and a heavier infection was found, which can be used as a starting point for future research on control strategies of rumen fluke infection.

Disease screening profiles and colostrum management practices on 16 Irish suckler beef farms.

BACKGROUND: Calf output is a key element in determining the profitability of a suckler beef enterprise. Infectious agents such as Bovine Virus Diarrhoea (BVD) virus, colostrum management and parasitic challenge can all affect calf output. Prior to the national BVD eradication programme, there was little published information on either the prevalence or effect of BVD in Irish beef herds. There is little published information on colostrum management practices in Irish commercial beef herds and there have also been few studies published on the prevalence of liver fluke or rumen fluke infection in Irish beef herds. Sixteen farms participating in the Teagasc/Farmers Journal BETTER farm beef programme were used in this study. Fourteen herds were screened for the presence of BVD virus in 2010 using RT-PCR. In 13 herds, blood samples were collected from calves (2-14 days of age) in November 2011 - April 2012 to determine their passive immune status using the zinc sulphate turbidity (ZST) test, while in 12 herds, blood and faecal samples were taken in order to determine the level of exposure to gastrointestinal and hepatic helminths. RESULTS: The overall prevalence of BVD virus-positive cattle was 0.98% (range 0 - 3% per herd, range 0.6 - 3.0% per positive herd). Eighteen of the 82 calves (22%) sampled had ZST values less than 20 units (herd mean range 17.0 - 38.5 units) indicating a failure of passive transfer. The overall animal-level (herd-level) prevalence of liver fluke and rumen fluke infection in these herds was 40.5% (100%) and 20.8% (75%), respectively. CONCLUSIONS: The potential costs associated with the presence of animals persistently infected with BVD virus through the increased use of antibiotics; the rate of failure of passive transfer of colostral immunoglobulins and the high prevalence of liver fluke infection in these herds highlight that some Irish suckler beef farms may not be realizing their economic potential due to a range of herd health issues. The use of farm-specific herd health plans should be further encouraged on Irish suckler beef farms.

Calicophoron daubneyi (Paramphistomidae) in deer of the Sumava National Park, Czech Republic - Consequence of prevalent rumen fluke infection in cattle.

A substantial parallel increase in prevalence and geographical spread of the rumen fluke, Calicophoron daubneyi, in livestock in western and central Europe has been recognized in the recent past. In the course of the examination of rectum feces of 471 red deer (Cervus elaphus) and one sika deer (Cervus nippon) from the Fascioloides magna endemic Sumava National Park in the years 2021 and 2022, rumen fluke eggs were detected in four red deer (0.8%) and the sika deer and identified as eggs of C. daubneyi by molecular analysis. Subsequent examination of rectal fecal samples of 247 beef cattle from 22 herds of 14 farms located in or nearby the national park revealed rumen fluke eggs in 53 samples (21.5%) originating from 16 herds of 11 farms, molecularly identified as C. daubneyi eggs as well. One C. daubneyi egg positive red deer and three C. daubneyi egg positive cattle samples also contained fasciolid eggs, respectively, which were detected in 9.5% or 3.6% of the total samples from red deer or cattle, respectively. Results of this investigation reveal the first finding of C. daubneyi in sika deer worldwide and in red deer in mainland Europe and add to the growing number of reports on C. daubneyi in livestock in Europe. Considering that the ratio of cattle excreting rumen fluke eggs exceeded that of deer substantially, it can reasonably be assumed that the C. daubneyi infections in deer are a consequence of the prevalent infection in cattle, illustrating a pathogen spillover event from livestock into wildlife.

Identification of Cotylophoron cotylophorum (Fischoeder, 1901) in cattle on St. Kitts, West Indies and its relationship with African and Asian populations.

There is limited information about the species of rumen fluke (Family Paramphistomidae) in the Caribbean. However, knowledge of species distribution is needed to better understand disease risk and epidemiology. Morphological identification is challenging with more recent DNA sequencing enabling a better understanding of rumen fluke distribution. In this study, rumen fluke specimens, collected between 2015 and 2016 from cattle on the island of St. Kitts, West Indies, were analysed. The ribosomal internal transcribed spacer 2 (ITS-2) region of rDNA was amplified using generic trematode primers. Results from Sanger sequencing were compared to reference sequences in GenBank and indicated the species was Cotylophoron cotylophorum with 100% sequence identity and 91% query cover. The ITS2 sequences were then compared to previously published ITS2 sequences for the Cotylophoron genus. When all the St. Kitts C. cotylophorum ITS2 sequences were compared with all other Cotylophoron sequences from India, Kenya, and Zimbabwe, three variable nucleotide sites, resulting in five unique haplotypes, were identified. Nine ITS2 sequences shared haplotype 1, which included all those from St. Kitts and single representatives from India and Kenya, potentially indicating global movement of this species.

The determination and relationship of four coexisting paramphistomes in perspective of integrative taxonomic investigation.

Co-infections with Orthocoelium species and other paramphistomes were found in different ruminant hosts from two provinces of Thailand. Whilst O. parvipapillatum coexisted with Paramphistomum epiclitum in the same cattle (Bos taurus) from Pathum Thani Province, Thailand, O. dicranocoelium and Fischoederius elongatus were found in water buffalo (Bubalus bubalis) from Chiang Mai Province. Morphological, histological, and tegumental surface features of both Orthocoelium species were intensively investigated for species differentiation. Statistical analysis of eight morphometric ratios presented morphological differences for three paramphistomes in the Paramphistomidae family and some relationships among paramphistomes in different definitive hosts. The genetic relationships of the co-infecting paramphistomes were investigated using p-distance and phylogenetic tree analyses. Genetic variations in the Orthocoelium co-infecting paramphistomes, P. epiclitum and F. elongatus, were calculated and compared to DNA sequence alignments based on internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit I (COI) DNA markers. In addition, the phylogenetic tree constructions from both DNA markers and their concatenated sequence (ITS2 + COI) were used for species confirmation and the presentation of genetic relationships between co-infecting paramphistomes and other paramphistomes. This study improves the basic taxonomical description and understanding of parasite-parasite and host-parasite interactions from the perspectives of morpho-histological, morphometric, and genetic variation in co-infecting paramphistomes and Orthocoelium species in different hosts.

Multispecies helminth parasitism of grazing dairy cows in Germany and Austria, examined in the housing period.

Helminth composition and burden data for dairy cows have not been reported for >40 years for Germany and even less information is available for Austria. In the context of two recent studies, helminth parasitism was studied in 32 cows (23 from six farms in Bavaria and Tyrol; 9 from one farm in Saxony) from pasture-based dairy farms necropsied during the housing period. Helminths were enumerated and identified based on morphological characters (all helminths but rumen flukes) or molecular techniques (rumen flukes). Thirteen species of gastrointestinal nematodes and two species each of liver flukes (Fasciola hepatica, Dicrocoelium dendriticum) and rumen flukes (Calicophoron daubneyi, Paramphistomum leydeni) were recorded; no lungworms were recovered from any cow. Early fourth-stage (inhibited) larval Ostertagia species nematodes (210 to 140,600) were recovered from all cows, 31 each had adult Ostertagia ostertagi/Ostertagia lyrata (40 to 2020) and Trichostrongylus axei (10 to 53,400), 23 Oesophagostomum radiatum (1 to 242) and 20 Cooperia punctata (10 to 3330). Other nematodes present in descending order of prevalence were: Cooperia oncophora/Cooperia surnabada, Ostertagia leptospicularis/Ostertagia kolchida, Oesophagostomum venulosum, Bunostomum phlebotomum, Chabertia ovina, Nematodirus helvetianus, Trichostrongylus longispicularis, Haemonchus contortus and Aonchotheca bilobata. The cows from Bavaria and Tyrol harbored more total gastrointestinal nematodes than that from Saxony (geometric mean adult plus inhibited larval nematodes, 6510 vs. 2051, respectively). However, in both cohorts of cows abomasal nematodes accounted for ∼97% of the total nematode burden with inhibited larval Ostertagia species nematodes contributing over 70% of the total gastrointestinal nematode burden and âˆ¼ 96% of the Ostertagia species burden. Approximately 44%, 37% and 19% of the cows harbored <5000, 5000 to 10,000 or > 10,000 total gastrointestinal nematodes, respectively. Fecal nematode egg and coproculture nematode larval counts significantly correlated with the cows' total adult nematode burden (rs = 0.354, p < 0.05, and rs = 0.608, p < 0.001, respectively). Although the magnitude of nematode burden to exert production effects on dairy cows is not well defined and may vary relative to several factors including nutritional supplementation, the level of mixed parasitism found in this investigation supports consideration of grazing dairy cows in helminth control measures, especially at the time of housing in autumn.

Multi-detection for paramphistomes using novel manually designed PAR-LAMP primers and its application in a lateral flow dipstick (LAMP-LFD) system.

Loop-mediated isothermal amplification (LAMP) has been applied for the detection of various parasites, and its application in lateral flow dipstick (LFD) can improve the convenience of point-of-care diagnosis. A novel PAR-LAMP probe and primers were designed by manual selection from a region of low variation in the ITS-2 DNA sequence. Up to six species of rumen fluke were detected by LAMP and LAMP-LFD in this study. Target specificity and sensitivity were tested, revealing a high target specificity (accuracy) and a low limit of detection (sensitivity). Different target sensitivities of paramphistome were presented, including 5 pg for Gastrothylax crumenifer and Carmyerius sp.; 1 pg for Fischoederius elongatus, Orthocoelium parvipapillatum, and O. dicranocoelium; and 0.1 pg for Paramphistomum epiclitum. LAMP-LFD can detect a paramphistome egg even in contaminated in feces that was spiked with the egg under laboratory conditions. In addition, natural paramphistome infection in cattle from Surat Thani and Khon Kaen provinces, Thailand, was evaluated by detection of egg contamination in fecal specimens using PAR-LAMP primers. The PAR-LAMP detection result was also statistically evaluated by microscopic examination of feces. This study presents the application of novel manually designed primers in a LAMP-LFD system for improving performance in detection and diagnosis assays for paramphistomosis.

Complex morphological characterization and morphometric-molecular discrimination of two paramphistome species co-infecting cattle, Orthocoelium sp. and Paramphistomum epiclitum.

Co-infection by two paramphistome species, Orthocoelium sp. and Paramphistomum epiclitum, is found in cattle in Thailand. The morphological features of these and other paramphistomes under a light microscope are similar, resulting in misidentification and misdiagnosis. We classified these paramphistomes into three morphological variation types, namely Orthocoelium sp., P. epiclitum MV1 (immature), and P. epiclitum MV2 (matured). Ten morphological characteristics were investigated, and the values were transformed into 25 ratio criteria for statistical investigation. Morphometric analysis can classify the variation of these specimens using differences in the bifurcal level, the vitellaria starting level, the starting level of the anterior testis, and the center level of the posterior testis positions by body length ratios. These ratios can separate the samples into three morphologically different groups, whereas molecular analysis based on the nuclear internal transcribed spacer 2 (ITS2) region and the mitochondrial cytochrome c oxidase subunit I (COI) gene could only distinguish two specific groups. In addition, the Orthocoelium specimen, related to O. dicranocoelium and O. parvipapillatum according to morphological and histological analysis, was monophyletic grouped via ITS2 analysis. Our study provides a scientific basis for the taxonomic classification and clustering of morphologically varying species, improving the identification, detection, and diagnosis of co-infecting paramphistomes.

The effect of naturally acquired rumen fluke infection on animal health and production in dairy and beef cattle in the UK.

The incidence of paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, has greatly increased within Europe in the last 15-20 years. However, the production impacts of this disease are poorly understood. This study firstly aimed to investigate the prevalence of rumen fluke in England and Northern Ireland (NI) by conducting an abattoir survey of dairy and beef cattle which also allowed the impact of rumen fluke on carcass weight, conformation and fat classification to be assessed. Secondly, an experiment aimed to assess the impact of C. daubneyi infection on diarrhea score, production loss and welfare in dairy heifers, while also evaluating the impacts of treating infected heifers with oxyclozanide. Rumen fluke prevalence was greater in NI than in England, with 53.8% (95% CI 51.9 - 55.9%) of the NI cattle carcases sampled being infected compared to 16.3% (95% CI 15.8 - 16.8%) and 17.9% (95% CI 17.4 - 18.4%) detected at the two abattoirs in England. However, there was no significant difference (P > 0.05) in the cold carcass weight between infected and non-infected cattle. Similarly, carcass conformation and fat classification were unaffected (P > 0.05) by the presence of rumen fluke. In the second experiment, daily live weight gain (DLWG), diarrhea score and welfare score were also unaffected (P > 0.05) by rumen fluke infection and by oxyclozanide treatment against rumen fluke. The farms in this experiment were managed to a high standard and animals had no intercurrent disease. Therefore, these findings suggest that on well-managed farms, production losses (growth rates) should not be compromised as a result of sub-clinical rumen fluke infection.

Rumen fluke infections (Paramphistomidae) in diarrhoeal cattle in western France and association with production parameters.

This study was conducted to assess the impact of rumen flukes (RFs) (Paramphistomatidae) on various production parameters of cattle in Normandy. Faecal and blood samples were taken between 1 January 2010 and 31 December 2019 as part of the routine diagnostic activity for diarrhoea in weaned cattle, including a quantitative parasitological analysis coupled with a search for Johne's disease (paratuberculosis). Information on slaughter and carcass weight, inter-farm movements and mortality was obtained from the French national registration database (BDNI). The study was conducted at two levels using adapted categorical RF variables: 1) at the cattle level (>12 months), an estimation of presence of adult parasites using egg count in faecal samples (negative vs. positive) and 2) at the herd level, an estimation of 6-24 months of the dairy heifers exposure to larval forms on the basis of the proportion of dairy cattle shedding eggs in the herd (three classes). At the cattle level, the outcome variables were carcass weight (model 1), case-control status for mortality within 30 days of diagnosis (model 2) and case-control status for slaughter within 365 days of diagnosis (model 3). At the herd level, the outcome variable was the mortality ratio for dairy heifers aged 6-24 months (Model 4). Other important covariates were used to improve model fit. Multivariate analyses were performed using a linear mixed model (model 1), generalised estimating equations (GEE) (models 2 and 3) and a multinomial logistic model (model 4). Overall, 1291 out of 4315 cattle (29.9%) were found to excrete RF eggs and 19.6% of the positives had an excretion score of >200 eggs/g. The prevalence increased steadily with age class and was higher in beef cattle than dairy cattle (42.7% vs. 26.9%) in the years 2015-2019 compared to 2010-2014 (33.3% vs. 26.5%) and in November-February (33.2%) compared to March-June (28.9%) and July-October (27.3%). Rumen fluke variables were not found to be explanatory factors of outcome variables at both animal and herd levels. In contrast, significant negative associations were observed between outcome variables and other health covariates, such as Johne's disease, GI nematode, bovine viral diarrhoea and coccidia statuses. In conclusion, RFs are prevalent in cattle reared in Normandy but this does not result in significant production losses. Therefore, the value to farmers of oxyclozanide treatment at an effective dose for paramphistomosis after simple identification of RF eggs in the faeces seems limited.

Paramphistome Species in Water Buffaloes and Intermediate Hosts in the Kizilirmak Delta in Samsun Province, Turkey.

PURPOSE: This study was carried out to determine the paramphistome species parasitizing water buffaloes (WBs) grazing in the Kizilirmak Delta in Samsun Province, Turkey, and the intermediate hosts of the parasites. METHODS: Between August 2016 and July 2018, abattoirs in Samsun Province were visited weekly and 139 slaughtered WBs were examined for paramphistome species. In the same period, 550 snails (300 Galba truncatula, 200 Physella (Physa) acuta and 50 Planorbis planorbis) were collected from pastures grazed by WBs during monthly sampling in the spring and autumn. Adult parasites were identified through the use of histological and molecular methods and larval stages were identified with a molecular method. RESULTS: Forty-five of the 139 WBs (32.4%) were infected with species of the family Paramphistomidae and a total of 4761 (mean 105.8) parasites were collected from them. The genera Paramphistomum and Calicophoron were distinguished from each other by examining the development status of the pars musculosa and the degree of lobulation of the testes in histological sections. Calicophoron daubneyi was present in all the infected animals and Paramphistomum cervi in only 3 of the same animals. There were larval forms in only 19 of the specimens identified as G. truncatula. Calicophoron daubneyi was molecularly diagnosed in 12 of the 19 infected G. truncatula and this result was confirmed by PCR and PCR-RFLP. Calicophoron daubneyi was also identified molecularly as being present in all 45 infected adult WBs and as developmental stages in 12 of 300 (4%) intermediate hosts, G. truncatula. The DNA sequences from the adult parasites in the definitive hosts and larval forms in intermediate hosts were allocated the codes MH939278 and MH939279, respectively, in GenBank. CONCLUSION: For the first time in Turkey, C. daubneyi was identified molecularly and its intermediate host was identified as G. truncatula. Calicophoron daubneyi was identified as the overwhelmingly dominant paramphistome species in WBs in the Kizilirmak Delta, with P. cervi found in mixed infections in only three animals.

Rumen Fluke in Great Britain.

Calicophoron daubneyi is the primary rumen fluke (RF) found in Europe in ruminants and infection is more common in cattle than in sheep. The incidence of RF has appeared to increase greatly throughout Europe in the last 10-15 years, with outbreaks of clinical paramphistomosis confirmed in ruminants in many countries, including Great Britain and Ireland. Clinical disease, due to immature stages developing in the small intestine, appears infrequently but can occur, usually in the autumn or winter within weeks of beginning to graze wet pasture. Although disease due to adult RF has not been proven, subclinical production losses have been attributed to adult RF infection by some researchers. As the intermediate host for RF and the liver fluke (Fasciola hepatica) is the mud snail (Galba truncatula), similar habitats and environmental conditions favour both parasites. There may, however, be differences in parasite development and interactions within both the final and intermediate hosts. No anthelminthic product is licensed for treatment of ruminants for RF in the UK. However, oxyclozanide, licensed for the treatment of adult F. hepatica infection, has been shown to have activity, but it may be more effective against the adult than the immature stages. The future prevalence of RF due to climate change and limited treatment options is unpredictable. Infection and clinical disease could become more common and RF is worthy of further research.

A Universal Approach to Molecular Identification of Rumen Fluke Species Across Hosts, Continents, and Sample Types.

Rumen fluke are parasitic trematodes that affect domestic and wild ruminants across a wide range of countries and habitats. There are 6 major genera of rumen fluke and over 70 recognized species. Accurate species identification is important to investigate the epidemiology, pathophysiology and economic impact of rumen fluke species but paramphistomes are morphologically plastic, which has resulted in numerous instances of misclassification. Here, we present a universal approach to molecular identification of rumen fluke species, including different life-cycle stages (eggs, juvenile and mature fluke) and sample preservation methods (fresh, ethanol- or formalin-fixed, and paraffin wax-embedded). Among 387 specimens from 173 animals belonging to 10 host species and originating from 14 countries on 5 continents, 10 rumen fluke species were identified based on ITS-2 intergenic spacer sequencing, including members of the genera Calicophoron, Cotylophoron, Fischeroedius, Gastrothylax, Orthocoelium, and Paramphistomum. Pairwise comparison of ITS-2 sequences from this study and GenBank showed >98.5% homology for 80% of intra-species comparisons and <98.5% homology for 97% of inter-species comparisons, suggesting that some sequence data may have been entered into public repositories with incorrect species attribution based on morphological analysis. We propose that ITS-2 sequencing could be used as a universal tool for rumen fluke identification across host and parasite species from diverse technical and geographical origins and form the basis of an international reference database for accurate species identification.