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.

Fasciola hepatica: Specificity of a coproantigen ELISA test for diagnosis of fasciolosis in faecal samples from cattle and sheep concurrently infected with gastrointestinal nematodes, coccidians and/or rumen flukes (paramphistomes), under field conditions.

Chronic fasciolosis is often diagnosed by faecal egg counting (FEC), following concentration of the eggs in the sample by a zinc sulphate floatation method. However, concentration by a sedimentation technique gives improved sensitivity. Interpretation of FEC results for fasciolosis is complicated by factors such as the long pre-patent period and irregular egg shedding. Thus, FEC reduction tests (FECRT), when used alone, are not completely reliable for diagnosis of anthelmintic susceptibility or resistance in local fluke populations, especially when parasite burdens are small. A Fasciola hepatica coproantigen ELISA test has been introduced which more accurately reflects the presence of flukes in the host bile ducts in late pre-patent infections, and absence of flukes following successful chemotherapeutic intervention. The aim of the present study was to elucidate the specificity of the F. hepatica coproantigen ELISA technique, particularly regarding potential cross-reactivity with rumen fluke (paramphistome), gastrointestinal nematode and coccidian infections. The method involved parallel testing of a large battery of faecal samples from field-infected cattle and sheep using floatation and sedimentation FECs and coproantigen analysis. No evidence was found for significant false positivity in the F. hepatica coproantigen ELISA due to paramphistome, coccidian and/or gastrointestinal nematode co-infections. With sedimentation FECs less than 10 F. hepatica eggs per gram (epg), the likelihood of a positive coproantigen result for the sample progressively decreased. Diagnosis of fasciolosis should be based on consideration of both FEC and coproantigen ELISA findings, to ensure optimum sensitivity for pre-patent and low-level infections.

Fasciola hepatica: a comparative survey of adult fluke resistance to triclabendazole, nitroxynil and closantel on selected upland and lowland sheep farms in Northern Ireland using faecal egg counting, coproantigen ELISA testing and fluke histology.

In order to investigate the incidence and distribution of adult fluke resistance to the fasciolicide tricalbendazole (TCBZ) amongst populations of Fasciola hepatica in sheep flocks in Northern Ireland (NI), individual rectal faeces samples were collected from 3 groups of 20 sheep, before (pre-dose), and 21 days after (post-dose) treatment of the animals with TCBZ, nitroxynil or closantel, on each of 13 well-managed sheep farms distributed across the province. The efficacy of each flukicide was determined for each farm, using faecal egg count reduction (FECRT) and F. hepatica coproantigen ELISA testing. In certain flocks, 2 sheep with high pre-dose faecal egg counts (FEC) were killed 3 days and 21 days respectively after TCBZ treatment, and the histology of the fluke reproductive organs was compared with that of flukes from untreated sheep, and from sheep treated with nitroxynil or closantel 2 days prior to death, using haematoxylin and eosin (H&E) staining and an in situ hybridisation method (TdT-mediated dUDP nick end labelling [TUNEL]) to demonstrate apoptosis. Results from FECRT revealed that in all flocks with a high fluke burden, TCBZ was ineffective in treating chronic fasciolosis, and this finding was generally supported by the results of the coproantigen reduction test (CRT). The histology of reproductive organs of flukes from TCBZ-treated sheep in these flocks was normal, when compared with untreated flukes, and this, together with the FECRT and CRT findings, indicated a likely diagnosis of TCBZ resistance in all the flocks with a high fluke burden. In contrast, nitroxynil and closantel were found to be fully effective against TCBZ-resistant flukes in each of the flocks bearing a high chronic fluke burden. All of the flocks with a high fluke burden and TCBZ resistance were managed on lowland in the South and East of NI. Upland flocks, in the North and West, had low fluke burdens, or were clear of infection; and FECs were too low to allow valid resistance testing. The study highlights the high level of penetration of TCBZ resistance throughout F. hepatica populations in areas of intensively managed sheep production with a high level of fluke challenge. Further, it emphasises the importance of pre-emptive chemotherapeutic action against chronic fasciolosis, using flukicides effective against the egg-producing adult flukes to minimise pasture contamination for the next season's lamb crop. This study also exemplifies the use of several complementary methods (FECRT; CRT; fluke histology; comparative anthelmintic efficacy testing) for confirmation of a diagnosis of fluke drug 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.

Specificity of a coproantigen ELISA test for fasciolosis: lack of cross-reactivity with Paramphistomum cervi and Taenia hydatigena.

A commercial coproantigen ELISA test for fasciolosis, based on the use of MM3 monoclonal antibody for antigen capture, was investigated for possible cross-reactivity with Paramphistomum cervi, a trematode that commonly infects cattle and sheep grazing in fluke-infested pasture in Ireland. Histological sections of adult and immature Fasciola hepatica and P cervi were incubated with MM3 monoclonal antibody, and its binding to tissue-localised coproantigen was subsequently visualised by immunocytochemistry. In a related study, the soluble antigenic fractions derived from homogenates of P cervi adults and Taenia hydatigena metacestodes were tested for cross-reactivity with MM3 monoclonal antibody in an antigen-capture ELISA, using known F hepatica-positive and F hepatica-negative ovine faecal samples as natural controls. It was found that, while intense immunocytochemical labelling was located over the gastrodermis and gut contents of adult and immature F hepatica, sections of adult and immature P cervi were unlabelled. In the ELISA tests, the soluble fractions of F hepatica reacted strongly with MM3 monoclonal antibody, but those of P cervi and T hydatigena gave negative results. These findings support the specificity of the coproantigen ELISA test for fasciolosis in areas where paramphistomosis and cysticercosis are liable to occur singly or as coinfections with F hepatica.

Control of caseous lymphadenitis in six sheep flocks using clinical examination and regular ELISA testing.

In an effort to control the spread of caseous lymphadenitis (CLA) infection, flocks of affected sheep on six holdings were tested serologically at regular intervals using an antibody ELISA with a mean (sd) specificity of 99 (1) per cent and a sensitivity of 79 (5) per cent. Western blot assays to detect antibodies to the phospholipase D (PLD) exotoxin of Corynebacterium pseudotuberculosis were used as a further test when ELISA results were inconclusive. Owners were advised to remove from the flock any sheep that demonstrated clinical signs of CLA or tested positive for PLD by ELISA or western blot. Of the six trial flocks, one was dispersed after only two blood tests, and in another the recommendations for CLA control were not followed and infected animals were retained within the flock. In the remaining four flocks, the testing regimen and other advice enabled the disease to be controlled to such an extent that the appearance of new clinical cases of CLA was effectively halted. This remained the case for up to five years after the end of the trial. In two of these flocks, a small number of seropositive animals were detected at the last flock test). However, on the other two holdings all sheep were seronegative in the final two flock tests, consistent with the complete eradication of infection.

Parameter estimation and simulations of a mathematical model of Corynebacterium pseudotuberculosis transmission in sheep.

Caseous lymphadenitis (CLA) is an infectious disease of sheep caused by Corynebacterium pseudotuberculosis. It is prevalent in most sheep producing countries and was introduced into the UK sheep population in 1991. The pathogen invades the host through epithelium and forms an abscess in the local draining lymph node. Typically, disease presents as clinical, with overt (externally visible) swollen lymph nodes (the parotid, submandibular, prefemoral, prescapular, popliteal or mammary) or sub-clinical, with abscesses in the lungs and associated thoracic (bronchial and mediastinal) lymph nodes. We present a mathematical model in which disease is categorised as overt and/or respiratory (sub-clinical), using the above groupings. In both situations sheep may be infected and may or may not be infectious. In the model, overt abscesses may resolve and respiratory abscesses are considered to be present for life. Using the location of the abscesses, three routes of transmission are postulated: overt to overt, respiratory to overt and respiratory to respiratory. Data from four naturally infected flocks were used to describe populations of sheep with epidemic CLA and to estimate transmission coefficients for each of the postulated transmission routes. The infection process parameters were derived from literature where possible. Parameters were estimated using maximum likelihood methods and compared to the data using a multinomial distribution. The distribution of abscesses in the flocks was similar to endemic data reported in other studies. In the model most infected sheep developed abscesses, and approximately 36% of sheep with overt abscesses recovered from infection. The average time for respiratory abscesses to become infectious was 41 days. In these data, overt to overt transmission was the most frequent route of transmission since it had the highest coefficient in the model compared with respiratory to overt and respiratory to respiratory transmission. Transmission coefficients specific for each flock significantly (P<0.05) improved the model fit to the data. In simulations using values of best-fitting parameter combinations, the proportion of sheep infected was between 0.39 and 0.60 at equilibrium. This is the first mathematical model of C. pseudotuberculosis infection, the parameter estimates indicate that aspects of the infection process could be utilised to design control strategies.

Investigations into an outbreak of tuberculosis in a flock of sheep in contact with tuberculous cattle.

Tuberculosis, associated with Mycobacterium bovis infection, occurs infrequently in sheep. A sheep flock, which was potentially exposed to a high level of infection from in-contact tuberculous cattle, was examined for evidence of infection. Six sheep that had given a positive reaction to the comparative intradermal tuberculin test were examined post mortem. Tuberculous lesions were present in four of these sheep. Lesion morphology and distribution in the sheep was similar to that in cattle. M. bovis was cultured from the lesions and the isolates were strain typed by spoligotyping and variable number of tandem repeats (VNTR) typing. Tuberculin-reacting sheep also reacted positively to an assay for in vitro release of interferon-gamma. This paper describes the first report of an outbreak of tuberculosis in sheep in either Britain or Ireland. The report describes immunology and pathology findings and, using molecular typing techniques, suggests that the sheep had been infected from in-contact cattle.

Detection of chlamydial antibody by fetal serology--an aid to the diagnosis of ovine abortion.

Two serological tests (indirect immunofluorescence and enzyme-linked immunosorbent assay) were developed for the detection of fetal antibody to Chlamydia psittaci. Fetal blood and thoracic fluid from 126 field cases of suspected ovine chlamydial abortion were examined using both tests. Placenta and fetal tissues (lung, liver, and kidney) from the same animals were also examined by the following conventional diagnostic methods: isolation in McCoy cells, detection of chlamydial lipopolysaccharide (LPS), modified Ziehl-Nielsen staining, and direct fluorescent antibody staining of chlamydia in frozen cryostat sections. Seventy cases were positive by fetal serology, and of these, 68 were also positive by isolation and/or LPS detection. The remaining 56 cases had negative fetal serology, and of these, 39 were positive by isolation and/or LPS detection. Results indicate that fetal serology, although less sensitive than either isolation in McCoy cells or detection of chlamydial LPS antigen, may be of particular use when placenta is not available.

Bog asphodel (Narthecium ossifragum) poisoning in cattle.

Fifteen cows among a herd of 50 suckler cows and calves rapidly lost body condition and became dull and anorexic after grazing pasture containing bog asphodel (Narthecium ossifragum) during the summer of 1989. The affected cows had evidence of kidney damage characterised by elevated plasma urea and creatinine concentrations. Eleven cows died and diffuse renal tubular necrosis was present in three cows which were examined post mortem. Similar renal lesions were reproduced experimentally by feeding bog asphodel to a healthy calf.

Infectious agents in respiratory disease of housed, fattening lambs in Northern Ireland.

A two-year longitudinal, microbiological and pathological survey of respiratory disease in lambs housed for fattening at three-and-a-half to four months of age was undertaken. In the first year samples of nasal mucus and blood were taken from lambs each week for the first nine weeks after entry to a fattening unit and each week one lamb was examined post mortem. In the second year two additional fattening units were included in the survey, when samples of blood and nasal mucus were taken from lambs twice weekly for three weeks after entry and two lambs from each unit were examined post mortem eight to 11 days after entry to the unit. In both years the lambs had a nasal discharge and were coughing. Mycoplasma ovipneumoniae and Pasteurella haemolytica were the organisms most consistently isolated from the lungs, trachea and nasal mucus. Mycoplasma arginini and parainfluenza-3 virus were also isolated. Post mortem examination lesions of atypical, pasteurella-type and parasitic pneumonias were seen. In the second year an abattoir survey of pneumonia lesions was undertaken. Areas of pulmonary consolidation were seen in 27.5 per cent, bands of consolidation in 47.5 per cent and muellerius-type lesions in 28 per cent of the lungs examined. No significant correlation was found between the slaughter weights of the lambs and the extent of the lung lesions at slaughter.

Pathological changes in the pericardium and meninges of cattle associated with Clostridium chauvoei.

Twenty-nine cases of Clostridium chauvoei infection in cattle were investigated over a two-year period. Fourteen had lesions of myositis only, eight had lesions of both myositis and fibrinous pericarditis, six had lesions of fibrinous pericarditis only and one had lesions of purulent meningitis only. Cl chauvoei was identified in all the lesions using the fluorescent antibody technique.

Possible involvement of leptospires in abortion, stillbirths and neonatal deaths in sheep.

Using culture, immunofluorescence and fetal serology, leptospiral infection was demonstrated in aborted, stillborn and weak lambs from nine out of 42 flocks investigated during the 1980 and 1981 lambing seasons. Three serogroups of leptospira, namely the Hebdomadis, Australis and Pomona serogroups, were implicated.