Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada: comparison of detection methods for drug resistance.

In 2011, a field study was conducted to assess drug resistance of gastro-intestinal nematodes in sheep flocks in Ontario, Canada. Benzimidazole resistance in Haemonchus contortus was assessed by genetic analysis of eggs; measurement of resistant allele percentages at codons 167, 198 and 200 in the ß-tubulin gene was determined on pools of H. contortus eggs using pyrosequencing. Susceptibility to benzimidazoles in gastro-intestinal nematodes was also determined using a Faecal Egg Count Reduction Test (FECRT) and a Larval Development Assay (LDA). In total, 16 farms were assessed with the genetic test. Based on resistant allele frequencies, all of the farms (16/16) tested had benzimidazole resistance in H. contortus; the overall percentage of benzimidazole-resistant H. contortus (estimated prior to treatment using the Hardy-Weinberg formula) was 68.5%. The FECRT and LDA were performed on 11 and 13 farms, respectively. Resistance to fenbendazole was detected on 100% (11/11) of the farms where the FECRT was performed. The LDA revealed the presence of thiabendazole resistance in H. contortus in 92% (12/13) of the farms. Estimated percentages of resistant parasites in H. contortus populations obtained with the two biological tests and the genetic test were compared. The results of the genetic test were in agreement with the biological tests and confirmed that benzimidazole resistance in H. contortus is present in Ontario sheep flocks. Differences between the different methods of drug resistance detection are discussed in terms of cost, time and sampling.

A longitudinal study on the effect of lambing season on the periparturient egg rise in Ontario sheep flocks.

The epidemiology of the periparturient egg rise (PPER) of gastrointestinal nematodes (GINs) in sheep remains unclear, and may be influenced by the lambing season. This longitudinal study was performed to determine the effect of out-of-season lambing on the PPER in ewes in Ontario, and whether total plasma protein (TPP) and packed cell volume (PCV) were associated with the PPER. Six farms that practiced out-of-season lambing were enrolled, and sampled for three consecutive lambing seasons (winter, spring and autumn). For each lambing season, all farms were visited five times. On the first visit for each lambing season, 15-20 pregnant ewes and 15-20 non-pregnant/early gestation ewes were randomly selected. At each visit, fecal samples were collected from all selected animals and processed individually to measure GIN fecal egg counts (FECs). Blood samples were collected on three visits in each lambing period and processed to measure TPP and PCV. The ewes were classified into one of five production stages (maintenance [i.e. not pregnant], early or late gestation [<120 d and ≥ 120 d, respectively], and early or late lactation [<40 d and ≥ 40 d, respectively]) based on information collected during farm visits. Linear mixed models were developed for the TPP, PCV and logarithmic-transformed FEC (lnFEC). During the winter and spring lambing season, the FECs increased gradually over the gestation period and peaked during lactation, with these increases being larger in ewes with a low PCV (three-way interaction in the final model). In the autumn lambing season, the FECs started off higher in early gestation, and increased rapidly to peak in late gestation, particularly for animals with low PCV levels. In the TPP model, PCV and lnFEC were positively associated with TPP. During both autumn and winter lambing seasons, the TPP decreased from maintenance throughout gestation and early lactation, followed by an increase in late lactation, except for when there were high FECs. During the spring lambing season, TPP peaked at early gestation, and then decreased in late gestation, to increase more gradually over lactation. In the PCV model, PCV increased with TPP and decreased exponentially with increases in lnFEC. The PPER occurred during all three lambing seasons, and its magnitude and distribution varied with the lambing season, suggesting that the PPER in ewes depends on both environmental and animal physiological factors, an important consideration when implementing preventive parasite control strategies on sheep farms that practice out-of-season lambing.

Anthelmintic resistance in sheep flocks in Ontario, Canada.

Gastrointestinal nematodes (GIN) are a significant constraint to pasture-based sheep production worldwide. Anthelmintic resistance (AR) has been reported in most sheep-raising areas in the world, yet little is known about the AR status in Canada. This study was conducted to determine the frequency of AR in GIN in sheep flocks in Ontario, Canada. Forty-seven sheep flocks were enrolled in the study, and their level of parasitism was monitored monthly throughout a grazing season by analyzing owner-acquired fecal samples from 15 grazing lambs per flock. When the mean GIN fecal egg count (FEC) reached a threshold of 200 eggs per gram (epg), oral ivermectin was supplied to producers to check ivermectin efficacy; the reduction in mean FEC 14 days after ivermectin treatment was calculated. 'Drench failure' was defined as a reduction in mean FEC of <95%. In those flocks with apparent drench failure, researchers performed a Fecal Egg Count Reduction Test (FECRT), dividing sheep into 4 treatment groups (n=10-15): control (i.e. untreated), ivermectin, and, if sufficient numbers of animals - fenbendazole and levamisole. AR was defined as a reduction in mean FEC <95% and a lower 95% confidence interval <90%. Larval cultures were performed on pooled post-treatment FECRT samples. Larval Development Assays (LDAs) to detect the presence of resistance to thiabendazole and levamisole were performed prior to the ivermectin drench check on pooled owner-acquired fecal samples that reached the 200 epg threshold. Approximately 89% (42/47) of the farms reached the FEC threshold of 200 epg; 93% (39/42) of these farms performed an ivermectin drench check, and 88% (34/39) of these farms had drench failure. The FECRT was performed on 29 of the 34 farms. Resistance to ivermectin, fenbendazole and levamisole was demonstrated on 97% (28/29), 95% (19/20) and 6% (1/17) of the farms tested, respectively, with considerable variability in resistance levels among farms. Haemonchus sp. was the most commonly cultured parasite from post-treatment fecal samples. LDA results for 21 farms were available; of these, 14% (3/21) and 62% (13/21) had low and high levels of thiabendazole resistance, respectively, while none of the farms exhibited resistance to levamisole. Amongst these tested farms, resistance to both ivermectin and benzimidazoles was very common. These findings strongly suggest that AR, particularly in Haemonchus sp., is a serious problem in these sheep flocks. Thus, marked changes in GIN management need to be instituted immediately to mitigate a worsening situation.

Evaluation of immune response to artificial infections of Haemonchus contortus in Gulf Coast Native compared with Suffolk lambs.

The Gulf Coast Native (Native) breed of sheep among many others is identified as being relatively resistant to Haemonchus contortus, an abomasal nematode parasite of small ruminants. Understanding the mode of immune response that helps these breeds of sheep control infection could help design and implement appropriate control programs. In this experiment, the components of the immune response during the early infection period in resistant Native lambs were evaluated and compared with susceptible Suffolk breed of sheep. Groups (n=5) of six month old Native and Suffolk lambs were given infective larvae as one time (single) or trickle experimental infections. Fecal, blood, and serum samples were collected on days 0, 2, 7, 14 and 21 post-infection. Abomasal mucosa and regional lymph node samples were collected at the time of necropsy on days 14 and 21. There was no significant difference in number of worms recovered at necropsy but the ratio of adult versus larvae was significantly greater in single infected Suffolk than Native lambs. Native lambs had significantly greater numbers of mast cells and eosinophils in the abomasal mucosa and serum IgG production was significantly greater compared to Suffolk lambs. Native lambs also showed a trend of increased level of serum IgA and IgE compared to Suffolk lambs.

Effect of feeding sericea lespedeza leaf meal in goats experimentally infected with Haemonchus contortus.

Effect of sericea lespedeza [SL; Lespedeza cuneata (Dum-Cours.) G. Don.] leaf meal feeding was evaluated in two experiments in indoor reared goats with experimental infection of Haemonchus contortus larvae. In the first experiment, ten 8-10 month old male Spanish and Alpine cross kids pair matched for body weight and age were fed SL or bermudagrass [BG; Cynodon dactylon (L.) Pers.] hay one week before infection and were infected with 5000 H. contortus L(3). The animals were maintained on the same diet for the remaining period and were slaughtered 28 days post-infection (DPI) to determine the establishment of incoming infective larvae. Goats fed SL had lower establishment (P<0.05) of H. contortus larvae than that of the control goats fed BG hay. In the second experiment, twenty-five 8-10 months old male Alpine cross, Saanen, Nubian×Saanen and Spanish kids reared in confinement on BG were experimentally infected with 5000 H. contortus L(3). On 35 DPI, the animals were allocated to two groups after blocking by fecal egg count (FEC), and one group was fed SL leaf meal (n=13), and another control group remained on BG (n=12). Four goats/group were slaughtered successively on days 7, 14, and 28 days post SL feeding, except on day 7, when five SL fed goats were slaughtered. Fecal egg counts and blood packed cell volume (PCV) were measured at weekly intervals and worm count, female worm fecundity, worm length and mucosal eosinophils, mast cells and globule leucocytes were measured after slaughter. Goats fed SL had a lower FEC (P<0.05) one week after feeding, as compared to those fed on BG, and the values remained at low level thereafter. Similarly, PCV was also significantly affected by feeding (P<0.01), and feeding and time interaction (P<0.05). However, worm burden, female worm fecundity, parasite length, and mucosal inflammatory cell count were similar between the groups. Feeding SL reduced the establishment of infective larvae and FEC of H. contortus in experimental studies and this plant could be used for biological control of parasite infection under field conditions to limit the harmful effects of the parasites in goats.

A Th2 type of immune response is associated with increased resistance to Haemonchus contortus in naturally infected Gulf Coast Native lambs.

Haemonchus contortus is one of the major nematode parasites causing substantial economic losses in small ruminant farming worldwide. Recently, effectiveness of anthelmintic treatment has decreased due to an increasing problem of nematode populations that have developed resistance to anthelmintics. Efforts to develop effective vaccines have had limited success. There are certain breeds of sheep that are relatively resistant to the parasite including Gulf Coast Native (Native) sheep. Understanding the protective nature of the immune response that helps these breeds of sheep control infection could enable the development of vaccines to enhance control programs. This experiment was designed to compare the immunological responses of resistant Native versus susceptible Suffolk sheep in order to identify the mechanisms responsible for this resistance. Immune responses were evaluated in naturally infected Native and Suffolk lambs that grazed pasture contaminated predominantly with H. contortus. Ten lambs of each breed grazed together for 42 days. Fecal, blood and serum samples were collected on 0, 2, 4, 7, 10, 14, 21, 28, 35 and 42 days of exposure. Five lambs of each breed were necropsied on day 35 and five on day 42 for nematode recovery and abomasal tissue sample collection. Throughout the course of infection, Native lambs had significantly lower FEC, significantly lower PCV reduction percent, and significantly higher serum IgE after day 14 and increased expression of IL-4 on day 10 post-exposure compared to Suffolk lambs. At both necropsy time points, Native lambs had significantly greater numbers of mucosal mast cells, eosinophils and globule leukocytes in abomasal mucosa than Suffolk lambs. Results indicated that Native lambs had a more pronounced immune response to infection with H. contortus than Suffolk lambs which may be responsible for the observed resistance to infection.