Tapeworm control practices by sheep farmers in Northern Ireland.

A questionnaire to obtain information on tapeworm control practices was sent to 252 sheep farmers in Northern Ireland (NI) in 2012. Replies were received from 228 flock owners. Most farmers considered that tapeworm infections had less impact on productivity than gastrointestinal nematodes, flukes and ectoparasites. The majority of respondents (61.8%) did not treat for tapeworms. Of those that did, the average number of treatments given per year was 2.3, with some owners treating up to 6 times a year. The highest percentages of treatments were given over the period May-July. Benzimidazole compounds were the predominant class of drugs used (48.2%), followed by macrocyclic lactones (MLs) (31.2%). Levamisole, oxyclozanide, closantel and Monepantel were also used; together with MLs, their combined use accounted for 51.9% of all treatments given, and represents inappropriate product choice. Diagnostic data for tapeworm infections in NI over the period 2007-2014 was retrieved from the database held by the Veterinary Sciences Division at Stormont. Positive diagnoses remained low throughout this period: the highest recorded figure was 3.1%, in 2007. Despite there being little-to-no justification for treating sheep for M. expansa on the basis of any likely benefit to the health or production of the animals, many farmers in NI do treat for tapeworm and often with ineffective products. This is of concern, in that it could lead to the inadvertent development of anthelmintic resistance in nematode and trematode parasites.

Liver fluke control on sheep farms in Northern Ireland: A survey of changing management practices in relation to disease prevalence and perceived triclabendazole resistance.

Reports of resistance to triclabendazole (TCBZ) among fluke populations have increased in recent years. Allied to this, there has been a rise in the prevalence of the disease, which has been linked to climate change. Results from questionnaire surveys conducted in Northern Ireland (NI) in 2005 (covering the years 1999-2004) and 2011 (covering the years 2008-2011) have provided an opportunity to examine the extent to which fluke control practices have changed over a prolonged time-frame, in light of these changes. A number of differences were highlighted. There was a significant shift away from the use of TCBZ over time, with it being replaced largely by closantel. The timing of treatments had moved earlier in the year, perhaps in response to climate change (and an altered pattern of disease). In relation to the frequency of drug treatments, there were no major changes in the overall pattern of drug treatments between the two survey points, although on both occasions approximately one-third of flock owners gave more than 3 treatments per year to ewes. In lowland areas in 2011, flock owners were rotating drug classes more often (each year and at each treatment) than in 2005, whereas in upland areas, flock owners were rotating less often and more were not rotating at all. Between 2005 and 2011, the percentage of flock owners giving quarantine treatments to bought-in stock had halved, to a very low level (approximately 10%). Using data from a complementary TCBZ resistance survey (Hanna et al., 2015), it has been shown that the way in which data are selected and which efficacy formula is applied can influence the calculation of drug efficiency and impact on diagnosis of resistance.

Anthelmintic resistance in Northern Ireland (I): prevalence of resistance in ovine gastrointestinal nematodes, as determined through faecal egg count reduction testing.

The prevalence of anthelmintic resistance in Northern Ireland sheep flocks was evaluated between July and October 2011. Sampling kits were sent to 172 flock owners and returns were received from 91. Within this survey population, 27 flock owners used benzimidazole products, 10 used levamisole products, 15 used avermectin products, 26 used milbemycin products and 4 flock owners used the amino acetonitrile derivative, Monepantel. The remaining 9 flock owners used combination drenches (broad spectrum wormer plus fasciolicide). However, 15 sets of samples were ineligible for faecal egg count reduction testing due to either too low an egg count or insufficient faecal volume. Treatment efficacy below 95%, indicating significant resistance, was detected in 81% (n=24) of flocks tested for benzimidazole resistance; in 14% (n=1) of flocks tested for levamisole resistance; and in 50% (n=7) and 62% (n=13) of flocks tested for avermectin and milbemycin resistance, respectively. Monepantel resistance was absent in all (n=3) flocks tested. Combination products (broad spectrum nematocide plus flukicide) containing levamisole were entirely effective, while treatment efficacy below 95% was detected in 60% (n=3) of flocks where the nematocide in the combination product was a benzimidazole. Where parasite identification based on coproculture was completed, Trichostrongylus was the dominant genus detected in all cases post-treatment, indicating the occurrence of anthelmintic-resistant Trichostrongylus spp. populations. Benzimidazole efficacy was highest in treating Trichostrongylus spp. (51%) and lowest when treating Teladorsagia spp. Levamisole was 100% effective in treating Cooperia, but ineffective (0%) in treating Trichostrongylus spp. Avermectin efficacy was highest when treating Haemonchus contortus (100%) and Teladorsagia spp. (73%), with a marginally lower efficacy against Trichostrongylus spp. (71%). Moxidectin efficacy was 33% against Trichostrongylus spp., 68% against Teladorsagia spp., 97% against Cooperia spp. and 100% against Haemonchus contortus infections.

Anthelmintic resistance in Northern Ireland (III): uptake of 'SCOPS' (Sustainable Control of Parasites in Sheep) recommendations by sheep farmers.

Reports of anthelmintic resistance to multiple drugs in individual parasite species, and in multiple parasite species across virtually all livestock hosts, are increasingly common. A working group of UK researchers and practitioners devised a set of guidelines in 2003 (Sustainable Control of Parasites in Sheep, 'SCOPS') aimed at maintaining anthelmintic efficacy on farms. Over the years that followed, these guidelines were promoted through meetings, promotional literature and the agricultural press. Results from questionnaires conducted in Northern Ireland (NI) in 2005 (covering 1999-2004) and 2011 (covering 2008-2011) have provided an opportunity to examine the extent to which these campaigns have influenced parasite control on sheep farms. The percentage of flocks at risk of under-dosing through inaccurate weight estimation in NI has increased by 15.9% since 2005. The number of flocks at risk of under-dosing through non-calibrated equipment has increased by 14.3% since 2005. The size of the in refugia population may have potentially doubled, as indicated by an increased compliance with the recommendation (wherever possible) to leave a portion of the flock untreated. However, whether this is indeed the case cannot be explicitly determined without a measure of the impact of various factors, including host immunity, environment/climate, previous anthelmintic treatment and the species of parasite present.

Anthelmintic resistance in Northern Ireland. II: Variations in nematode control practices between lowland and upland sheep flocks.

A questionnaire to obtain information on nematode control practices and sheep management was sent to over 1000 farmers in Northern Ireland. Replies were received from 305 flock owners, and data from 252 of them were analysed. Farms were divided into lowland and upland areas. Sizes of pasture and stocking rates on lowland and upland farms were 59.5 hectares, 6.99 sheep/hectare and 62.9 hectares and 10.01 sheep/hectare, respectively. Mean drenching rates for lambs and adults were 2.33 and 2.44, respectively, in lowland flocks and 2.73 and 2.71, respectively, in upland flocks. Between 2008 and 2011, the most frequently identified compounds in use were benzimidazoles and moxidectin in lowland flocks, and benzimidazoles and avermectins in upland flocks. Over the same period the most frequently identified commercial formulations were Tramazole(®), Panacur(®) and Allverm(®) (white drench), Levacide(®) (yellow drench), Oramec(®) (clear drench; avermectin), Cydectin(®) (clear drench; moxidectin) and Monepantel(®) (orange drench). Most respondents (56.35%) treated their lambs at weaning and the most common time to treat ewes was identified to be pre-mating (67.86% of respondents). The results of the questionnaire survey revealed that lowland annual drench frequency was 2.33 and 2.44 in lambs and ewes, respectively, although drench frequencies were higher in upland flocks: 2.73 and 2.71 for lambs and ewes, respectively. Annual drench rotation was practiced by 43.96% of flock owners, but whether this was true rotation or pseudo-rotation (i.e., substitution of one anthelmintic product by another product belonging to the same chemical group of anthelmintics) could not be explicitly determined.

The effects of climate change on ovine parasitic gastroenteritis determined using veterinary surveillance and meteorological data for Northern Ireland over the period 1999-2009.

While the influence of temperature and moisture on the free-living stages of gastrointestinal nematodes have been described in detail, and evidence for global climate change is mounting, there have been only a few attempts to relate altered incidence or seasonal patterns of disease to climate change. Studies of this type have been completed for England Scotland and Wales, but not for Northern Ireland (NI). Here we present an analysis of veterinary diagnostic data that relates three categories of gastrointestinal nematode infection in sheep to historical meteorological data for NI. The infections are: trichostrongylosis/teladorsagiosis (Teladorsagia/Trichostrongylus), strongyloidosis and nematodirosis. This study aims to provide a baseline for future climate change analyses and to provide basic information for the development of nematode control programmes. After identifying and evaluating possible sources of bias, climate change was found to be the most likely explanation for the observed patterns of change in parasite epidemiology, although other hypotheses could not be refuted. Seasonal rates of diagnosis showed a uniform year-round distribution for Teladorsagia and Trichostrongylus infections, suggesting consistent levels of larval survival throughout the year and extension of the traditionally expected seasonal transmission windows. Nematodirosis showed a higher level of autumn than Spring infection, suggesting that suitable conditions for egg and larval development occurred after the Spring infection period. Differences between regions within the Province were shown for strongyloidosis, with peaks of infection falling in the period September-November. For all three-infection categories (trichostrongylosis/teladorsagiosis, strongyloidosis and nematodirosis), significant differences in the rates of diagnosis, and in the seasonality of disease, were identified between regions.

Impact of compound alpha treatment in vivo on egg production by the liver fluke, Fasciola hepatica.

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were treated with compound alpha at a dosage of 15 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24h, 48 h and 72 h post-treatment (pt). They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system: specifically, the uterus, Mehlis' gland, ovary and vitellaria. As judged by the appearance of the uterus, normal egg production ceased within 24h of treatment; this phenomenon preceded significant changes to the other reproductive organs. Over the 3-day pt period, there was a progressive decline in the number of oogonia in the ovary, together with an increase in the number of eosinophilic and apoptotic oocytes and vacuolation and shrinkage of the ovarian tubules. There was a shift in the cell population within the vitelline follicles at 48 h pt, with relatively greater numbers of mature vitelline cells and fewer immature cells. The follicles were vacuolated and the shell globule clusters in the mature cells were disorganised. Greater disruption was seen at 72 h pt, with a reduction in the size of the follicles and rupture of cells, releasing their content into the lumen of the follicles. These histological observations were confirmed and extended at the TEM level. Thus, examination of electron micrographs showed that disruption of the shell globule clusters was evident at 48 h pt, which coincided with the start of the breakdown of the mature cells and of the nurse cell network. These degenerative changes were more conspicuous at 72 h pt. In the Mehlis' gland, shrinkage and vacuolation of the cells and their cytoplasmic extensions became progressively greater from 48 h to 72 h pt, and secretory activity declined. The changes in the reproductive organs and inhibition of egg production are put in context of the overall time-course of drug action.

Fasciola hepatica: comparative effects of host resistance and parasite intra-specific interactions on size and reproductive histology in flukes from rats infected with isolates differing in triclabendazole sensitivity.

The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study, F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke size was positively related to testis score for both isolates, and a significant negative relationship was found between percentage success of infection and metacercarial dose. The results are interpreted in terms of differing interactions between various numbers of young flukes and host immunity during invasion of and migration in the hepatic parenchyma, and of fluke intra-specific (possibly pheromonal) stimulatory effects in the final stages of development, within the host bile ducts. No significant relationships were found between host antibody levels and fluke size or testis score. False positive serological reactions were found in some rats that had been infected, but found to harbour no flukes at autopsy. Clearly the act of eliminating the flukes involved generation of an immune response.

Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola hepatica.

A sheep trial was performed to standardise a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole (TCBZ) in Fasciola hepatica). The CRT employs the BIO K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) to test for the presence of F. hepatica coproantigens in a faecal sample. If it is coproantigen-positive, the CRT protocol recommends that faecal samples are re-tested for coproantigens at 14 days post-treatment (dpt), with negative testing at this point indicating TCBZ success. Initial work aimed to confirm the sensitivity of the BIO K201 ELISA for Fasciola infection and investigate whether coproantigens represent a robust reduction marker of TCBZ efficacy. Thirty-eight, indoor-reared sheep were artificially infected with F. hepatica isolates known to be susceptible (Cullompton) and resistant (Sligo) to TCBZ action, respectively. Treatment was administered at 12 weeks post-infection (wpi), with 2 sheep groups, infected with each isolate, culled at 2 and 4 weeks post-treatment (wpt), respectively. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period. Additional work focused on the effect of temperature on faecal sample collection and storage. Faecal samples collected from sheep positive for F. hepatica infection were sub-sampled and left at room temperature. Individual sub-samples were tested by ELISA on consecutive days and these readings compared to the original test result on the day of collection. In addition, ELISA values were compared between faecal sub-samples prepared on the day of sampling and post storage at -20°C. Also, an immunocytochemical study was performed to determine the tissue site of origin of the coproantigen protein in the fluke. Results showed that the BIO K201 ELISA was sensitive for Fasciola coproantigens, with coproantigens detectable from 5 wpi onwards. The suitability of coproantigens as a diagnostic marker of TCBZ efficacy was supported by the absence and presence of coproantigens in TCBZ-treated Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) F. hepatica infections at 2 and 4 wpt, respectively. Study results suggest that low to moderate temperature has little, if any, impact on coproantigen stability in faecal samples, but that higher temperatures may have. Immunolabelling for the coproantigen showed that it was specific to the gastrodermal cells of both adult and juvenile flukes.

Comparison of two assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance to triclabendazole in Fasciola hepatica in sheep.

A sheep trial was performed to evaluate two diagnostic assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance of Fasciola hepatica to triclabendazole (TCBZ). The FECRT defines successful TCBZ treatment as a 95% or greater reduction in fluke faecal egg counts (FECs) at 14 days post-treatment (dpt). The CRT defines effective TCBZ treatment as faeces negative for Fasciola coproantigens at 14dpt, as measured by the commercial BIO K201 coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium). Forty-nine indoor-reared sheep were split into four trial groups and each sheep was infected with 200 metacercariae of 1 of 4 F. hepatica isolates, previously described as susceptible (Cullompton and Fairhurst) and resistant (Leon and Oberon) to TCBZ action, respectively. TCBZ treatment was administered at 12 weeks post-infection (wpi) to one sub-group in each infected sheep group, and these sheep were culled at 4 weeks post-treatment (wpt). Untreated sheep sub-groups, were culled at a parallel time-point, that is, at 16wpi. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period, sub-sampled and examined by a standardised egg sedimentation protocol and by the BIO K201 ELISA. Results supported the use of both the FECRT and the CRT for the diagnosis of resistance of F. hepatica to TCBZ. In addition, the study confirmed the TCBZ susceptibility of the Cullompton and Fairhurst F. hepatica isolates and the TCBZ resistance of the Oberon F. hepatica isolate. However, the Leon F. hepatica isolate was found to be susceptible, rather than resistant, to TCBZ action.

Disruption of egg formation by Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host.

Eight indoor-reared cross-bred sheep with no prior exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Twelve weeks after infection, sheep were treated with 10mg/kg triclabendazole. Two sheep were euthanised per time period; at 48 h, 72 h and 96 h post-treatment (pt). Two untreated control sheep were euthanised at 96 h pt. Flukes were recovered from the liver and, if present, from the gall bladder of the sheep. They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system; specifically, the uterus, vitelline follicles, Mehlis' gland and ovary. Over the 4-day post-treatment period, there was a progressive reduction in the number of oogonia and oocytes in the ovary and evidence of apoptosis. Vacuolation and a decrease in the number of Mehlis' gland cells were observed from 48 h pt onwards and disruption of the normal role of the gland in egg formation was evident. The vitelline follicles showed a gradual decrease in size and became vacuolated; the population structure in each follicle changed to be one consisting mainly of mature cells and the production of shell protein material declined. The follicle became disorganised as the cells broke down and released their contents into the lumen of the follicle. While the uterus appeared to contain eggs at 48 h pt in whole-mount specimens, no properly-formed eggs were observed in histological sections. By 96 h pt, the uterus was completely devoid of eggs. Overall, egg production was seen to be severely affected by TCBZ treatment and flukes were incapable of producing normal eggs within 2 days of treatment. The implications of this in terms of the epidemiology of the disease are discussed.

Fasciola hepatica: histological changes in the reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes after treatment in vivo with TCBZ and the related benzimidazole derivative, Compound Alpha.

Twenty-four shed-reared lambs were each infected orally with 250 metacercariae of Fasciola hepatica, using either the triclabendazole (TCBZ)-sensitive Cullompton isolate or the TCBZ-resistant Sligo isolate. Twelve weeks after infection the lambs were treated with TCBZ (10mg/kg) or with the experimental fasciolicide, Compound Alpha (Cpd alpha), a benzimidazole derivative of TCBZ (15mg/kg). The lambs were euthanised 48, 72 and 96h after TCBZ treatment, or 24, 48 and 72h after Cpd alpha treatment, and flukes were collected from the liver and/or gall bladder of each animal. Untreated animals harbouring 12-week infections were euthanized 24h after administration of anthelmintic to the treatment groups, and the untreated flukes provided control material. A semi-quantitative assessment of the degree of histological change induced by the two drugs after different times of exposure was achieved by scoring the intensity of three well-defined lesions that developed in the testes and uteri of a representative sample of flukes from each lamb. In general, it was found that in those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), there was progressive loss of cell content due to apparent failure of cell division to keep pace with expulsion of the mature or effete products. Further, actively dividing cell types tended to become individualised, rounded and condensed, characteristic of apoptotic cell death. Protein synthetic activity was apparently inhibited in the Mehlis' secretory cells. In the uterus, where successful formation of shelled eggs represents the culmination of a complex sequence of cytokinetic, cytological and synthetic activity involving the vitelline follicles, the ovary and the Mehlis' gland, histological evidence indicating failure of ovigenesis was evident from 24h post-treatment onwards. The development of these lesions may be related to the known anti-tubulin activity of the benzimidazole class of anthelmintics, to the induction of apoptosis in cells where mitosis or meiosis has aborted due to failure of spindle formation, and to drug-induced inhibition of protein synthesis. The semi-quantitative findings indicated that Cpd alpha is slightly less efficacious than TCBZ itself in causing histological damage to the reproductive structures of TCBZ-sensitive flukes, and that, like TCBZ, it caused no histological damage in flukes of the TCBZ-resistant isolate. This study illustrates the potential utility of histological techniques for conveniently screening representative samples of flukes in field trials designed to validate instances of drug resistance or to test the efficacy of new products against known drug-resistant and drug-susceptible fluke isolates. It also provides reference criteria for drug-induced histopathological changes in fluke reproductive structures which may aid interpretation of TEM findings.

Surface changes in adult Fasciola hepatica following treatment in vivo with the experimental fasciolicide, compound alpha.

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks postinfection. Adult flukes were recovered from the bile ducts at 24, 48, and 72 h post-treatment (p.t.). Changes to the surface morphology of the flukes were assessed using scanning electron microscopy (SEM). Flukes were still active at 24 h p.t. and displayed limited areas of disruption, which were restricted to the oral cone region. At 48 h p.t., a reduced level of motility was observed in approximately 50% of the flukes recovered. Swelling of the tegument was more widespread and was accompanied by blebbing and partial loss of the tegumental covering of the spines. By 72 h p.t., the reduction in motility was greater, and approximately one quarter of the flukes recovered were inactive. In the majority of the flukes examined, the midbody region was marked by a discoloration of the flukes' tissues. This was seen to be due to the loss of the tegumental syncytium. Sloughing extended into the tail region in some specimens and, in the more badly-affected specimens, the basal lamina was breached to expose the underlying musculature. Elsewhere on the body, the tegument that remained was relatively normal, although areas of swelling and blebbing were present. Overall, the results provided information on the time-scale of changes to the surface morphology of the fluke that underpin the efficacy of compound alpha.

Ultrastructural changes to the tegumental system and the gastrodermal cells in adult Fasciola hepatica following in vivo treatment with the experimental fasciolicide, compound alpha.

Sheep infected with the triclabendazole-susceptible, Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24, 48 and 72 h post-treatment (p.t.). Ultrastructural changes to the flukes were assessed using transmission electron microscopy (TEM), with a view to gathering information on the mechanism(s) of action for compound alpha and on the possible route of its entry into F. hepatica. The tegumental syncytium was more severely affected than the gut at all time-points p.t. with compound alpha, suggesting a predominantly trans-tegumental route of uptake. Disruption to the tegumental system became increasingly severe over time. A stress response was observed at 24 h p.t. and took the form of blebbing and increases in the production and transport of secretory bodies. By 72 h p.t., extensive tegumental loss and degeneration of the tegumental cell bodies had occurred. Degeneration of subtegumental tissues and internal flooding were also observed. Changes in the gastrodermal cells were slow to develop: reduced secretory activity was evident at 72 h p.t.. There was progressive disruption to the somatic muscle layers, with disorganization of the muscle blocks and loss of muscle fibres.

An evaluation of the efficacy of compound alpha and triclabendazole against two isolates of Fasciola hepatica.

Seventy indoor-reared sheep were divided into 10 groups to test the efficacy of the experimental fasciolicide, compound alpha (15mg/kg) against triclabendazole (TCBZ)-resistant and TCBZ-susceptible F. hepatica infections. Activity against the Sligo TCBZ-resistant isolate was tested at three time points post-infection (p.i.): 3 days, 4 weeks and 12 weeks (Groups 1-3, respectively). A parallel trial was carried out using TCBZ (10mg/kg) (Groups 5-7): this provided a direct comparison between the efficacies of the two drugs. Group 4 served as an untreated Sligo control. Groups 8 and 9 were setup to test the efficacy of TCBZ and compound alpha against 12-week-old and 4-week-old TCBZ-susceptible, Cullompton infections, respectively. Group 10 served as an untreated Cullompton control. Sheep were sacrificed at 16 weeks p.i. and efficacies were determined. All remaining flukes were collected and measured, before being processed for whole-mount staining to assess the condition of their reproductive structures (testis, vitellaria, ovary and uterus). A second study was carried out to test the activity of compound alpha (15mg/kg) against mature 12-week-old TCBZ-susceptible F. hepatica infections in sheep. Eighteen sheep were divided into two groups, A and B. Group A was treated and Group B served as an untreated control group. Efficacy was determined by reduction in faecal egg counts. The results showed that, whilst compound alpha was very active against adult TCBZ-susceptible flukes, producing a 100% reduction in faecal egg counts, it only caused a 62.5% reduction in fluke burden against juvenile flukes. Moreover, compound alpha was not effective against any stage of infection with TCBZ-resistant F. hepatica in sheep. Data from the trial also revealed biological differences between the two isolates. Thus, Sligo flukes were smaller in size and produced fewer eggs than the Cullompton flukes and their cysts were less infective to sheep. However, they reached the bile ducts more quickly and their eggs appeared in the faeces >2 weeks earlier.

Surface and internal tegumental changes in juvenile Fasciola hepatica following treatment in vivo with the experimental fasciolicide, compound alpha.

Eight indoor-reared, crossbred sheep with no pre-exposure to Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole-susceptible, Cullompton isolate of F. hepatica. Anthelmintic dosing occurred at 4 weeks post-infection using 15mg/kg compound alpha. Two treated sheep per time period were euthanized at 24h, 48h and 72h post-treatment with compound alpha. The two sheep from the control group were euthanized alongside the 24h alpha-treated sheep. Juvenile flukes were recovered from each of the sheeps' liver and processed for examination by electron microscopy. The surface morphology of the flukes' tegument was assessed using scanning electron microscopy (SEM). The ultrastructure of the tegumental syncytium and underlying tegumental cells and connections and somatic musculature were investigated using transmission electron microscopy (TEM). Both the SEM and TEM results revealed a level of disruption that increased with time, culminating at 72h with extensive tegumental loss and substantial degeneration of the cell bodies. The effects of compound alpha on the surface morphology were not particularly apparent until 48h post-treatment, when disruption included swelling and blebbing of the tegument. At 72h post-treatment, SEM revealed loss of the entire syncytial layer over large areas of the flukes. In the areas where the syncytium was lost and the basal lamina exposed, lesions of varying sizes had developed, revealing underlying tissues. Though minor forms of disruption to the ultrastructure of the syncytium were observed using TEM 24h post-treatment, it was at 48h post-treatment that substantial stress responses occurred. They included the presence of autophagic vacuoles and 'open' bodies at the apex of the syncytium and swelling of the basal infolds. The mitochondria within the syncytium and tegumental cells became progressively more disrupted over the three time periods and, by 72h post-treatment, they were frequently distorted and swollen in appearance, and contained severely swollen cristae. By 72h, the number of secretory bodies, particularly T1 bodies, had become significantly depleted in their respective cell bodies, cytoplasmic processes and in the tegumental syncytium. Both the circular and longitudinal muscle bundles were severely disrupted 72h post-treatment. They frequently contained a reduced number of muscle fibres and, in more severe instances, there was an absence of fibres altogether.

Biochemical changes induced by hypomagnesaemia in lactating cows and ewes.

Severe hypomagnesaemia was induced in lactating cows and lactating sheep by feeding them magnesium-deficient diets for 17 and 14 days, respectively. Hypomagnesaemia in cows was associated with abnormally high rates of change in the numbers of leucocytes, neutrophils, monocytes and platelets. There were increases in the concentration of iron in the liver of the hypomagnesaemic ewes and in the heart of the hypomagnesaemic cows, which were not associated with a haemolytic process. The percentage of some of the peroxidisable fatty acids was lower in the heart tissue of hypomagnesaemic cows, but the reduction was not associated with significant lipid peroxidation.