Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep

Systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep

Background Anthelmintic drugs have been widely used in sheep as a cost-effective means for gastro-intestinal nematode (GIN) control. However, growing anthelmintic resistance (AHR) has created a compelling need to identify evidence-based management recommendations that reduce the risk of further development and impact of AHR. Objective To identify, critically assess, and synthesize available data from primary research on factors associated with AHR in sheep. Methods Publications reporting original observational or experimental research on selected factors associated with AHR in sheep GINs and published after 1974, were identified through two processes. Three electronic databases (PubMed, Agricola, CAB) and Web of Science (a collection of databases) were searched for potentially relevant publications. Additional publications were identified through consultation with experts, manual search of references of included publications and conference proceedings, and information solicited from small ruminant practitioner list-serves. Two independent investigators screened abstracts for relevance. Relevant publications were assessed for risk of systematic bias. Where sufficient data were available, random-effects Meta-Analyses (MAs) were performed to estimate the pooled Odds Ratio (OR) and 95% Confidence Intervals (CIs) of AHR for factors reported in ≥2 publications. Results Of the 1712 abstracts screened for eligibility, 131 were deemed relevant for full publication review. Thirty publications describing 25 individual studies (15 observational studies, 7 challenge trials, and 3 controlled trials) were included in the qualitative synthesis and assessed for systematic bias. Unclear (i.e. not reported, or unable to assess) or high risk of selection bias and confounding bias was found in 93% (14/15) and 60% (9/15) of the observational studies, respectively, while unclear risk of selection bias was identified in all of the trials. Ten independent studies were included in the quantitative synthesis, and MAs were performed for five factors. Only high frequency of treatment was a significant risk factor (OR = 4.39; 95% CI = 1.59, 12.14), while the remaining 4 variables were marginally significant: mixed-species grazing (OR = 1.63; 95% CI = 0.66, 4.07); flock size (OR = 1.02; 95% CI = 0.97, 1.07); use of long-acting drug formulations (OR = 2.85; 95% CI = 0.79, 10.24); and drench-and-shift pasture management (OR = 4.08; 95% CI = 0.75, 22.16). Conclusions While there is abundant literature on the topic of AHR in sheep GINs, few studies have explicitly investigated the association between putative risk or protective factors and AHR. Consequently, several of the current recommendations on parasite management are not evidence-based. Moreover, many of the studies included in this review had a high or unclear risk of systematic bias, highlighting the need to improve study design and/or reporting of future research carried out in this field. </p

Efficacy of targeted anthelmintic treatment for suppression of the peri-parturient egg rise in ewes and impact on 50-day lamb weights

This study was conducted to determine whether targeted anthelmintic treatment of peri-parturient ewes lambing in the winter, spring and/or autumn would suppress the peri-parturient egg rise (PPER) and improve 50-day lamb weights. Three farms in Ontario, Canada, that practiced out-of-season lambing were enrolled in 2010 and sampled for three consecutive lambing seasons (winter, spring and autumn). For each lambing season, all farms were visited three times. On the first visit, all ewes due to lamb that season were randomly allocated to treatment with ivermectin, fenbendazole or levamisole at the recommended dosage, or left untreated. Among these treated ewes, 40–60 animals (10–15 ewes per treatment group) were randomly selected for fecal sampling during the 3 sampling visits and processed individually to measure gastro-intestinal nematode (GIN) fecal egg counts (FECs). Ewe and lamb productivity data, including approximate 50-day lamb weights, were collected for all ewes lambing in each season, where available. A Fecal Egg Count Reduction Test was performed on all three farms to determine the ivermectin, fenbendazole and levamisole resistance status. Both farms A and B had fenbendazole resistance, while farm C had ivermectin and fenbendazole resistance; levamisole was effective on all three farms. The effect of targeted treatment on the subsequent PPER depended on the farm, possibly a partial surrogate variable for the different anthelmintic resistance levels on each farm, lambing season and sampling time-point. On farm A, during the winter and autumn lambing seasons, ivermectin and levamisole were more effective at reducing the FECs, compared to fenbendazole. In contrast, during the spring lambing season, treatment of ewes with ivermectin, fenbendazole or levamisole had no effect on the FECs. On farm B, all anthelmintic treatments were associated with a reduction in the FECs during the spring lambing season, while no reduction was observed during the winter and autumn lambing seasons. On farm C, the FECs decreased in ewes treated with levamisole in both the winter and spring lambing seasons, while ivermectin only reduced the FECs in ewes treated in the winter lambing season. Litter size was positively associated with FECs. Anthelmintic treatment was not associated with approximate 50-day lamb weights, although the power to detect significant difference was lower than anticipated due to only having relevant weight data from farm A. These results suggest that the efficacy of targeted treatment for the suppression of the PPER depends on the anthelmintics’ efficacy and time of treatment in relation to the grazing period

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 [<120d and ≥120d, respectively], and early or late lactation [<40d and ≥40d, 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

A survey of farm management practices and their associations with anthelmintic resistance in sheep flocks in Ontario, Canada

To describe parasite control and farm management practices commonly used by Ontario sheep farmers, and to determine whether any of these practices were associated with the level of anthelmintic resistance (AR) to ivermectin, fenbendazole or levamisole, we conducted fecal egg count reduction (FECR) tests in Ontario sheep flocks, and administered a questionnaire pertaining to farm practices that were considered putative risk factors for AR. In the previous 5 years, most of the producers had used ivermectin and fenbendazole drenches (95% and 68%, respectively), while only 11% had used levamisole drench. Producers treated their animals a mean of 2.6 times per year. Routine treatment was practiced by 82% of the producers; most ewes were treated either at lambing (55%) and/or at the beginning of winter housing (48%). The majority of the producers (82%) also used targeted or targeted selective treatment; however, it was often in addition to, rather than in lieu of, routine treatment. Twenty-five producers (66%) brought in new animals in the previous year. Many producers (45%) did not calibrate the drench gun before use. The mean FECR percentages following treatment with ivermectin, fenbendazole, and levamisole were 23.7%, 28.6% and 99.1%, respectively. Although univariable analyses identified several marginally significant risk factors (0.10> p >0.05), none were significant in the final model for ivermectin FECR percentage. In contrast, use of benzimidazoles in the previous 5 years was associated (p =0.01) with increased resistance (lower mean FECR percentage) to fenbendazole. Levamisole resistance could not be modeled due to the very low levels of resistance on the farms surveyed. This study: (1) provided a picture of management practices employed by Ontario sheep producers who were experiencing AR to one or more anthelmintic drugs on their farms; and (2) allowed us to identify areas for further AR risk factor research

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 200epg threshold. Approximately 89% (42/47) of the farms reached the FEC threshold of 200epg; 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 situatio

The effect of estus synchronization treatment on somatic cell count of transitional-anestrus local-Damascus cross breed goats' milk

An experiment was conducted to examine the effect of estrus synchronization protocols and steroid hormones concentrations on somatic cell count (SCC) of transitional-anestrus local-Damascus cross goats’ milk. Fifty-six goats (2–4-year old) were randomly assigned to three groups: fluorogestone acetate (FGA, n = 19), FGA-Prostaglandin (FGA-PGF, n = 19) and control (n = 18) groups. Intravaginal sponge containing 40 mg FGA was inserted for 13 days and an injection of 600 IU equine chorionic gonadotropin (eCG) was administered for goats of FGA and FGA-PGF groups at the time of sponge removal (day 0). In addition, goats of FGA-PGF group were injected with 10 mg dinoprost tromethamine (PGF₂α) on day 0. Five fertile local-Damascus cross bucks were turned-in with all goats on day 0. Blood and milk samples were collected from all goats on days -13 (beginning of experiment), -6, 0, 1, 2, 7, 13 and 20 (end of the experiment). Four-year old and second-parity goats had significantly higher (p < 0.05) SCC of both udder halves than 2- and 3-year old and first-parity goats, respectively. There was a significant effect (p < 0.05) for treatment and number of kids born in the last kidding season on SCC of both udder halves. Neither estradiol nor progesterone concentrations were correlated with SCC in goats in this experiment. The SCC of both udder halves and left udder halves in goats of the control and FGA groups, respectively, increased significantly (p < 0.05) after sponge removal and buck introduction when compared with day 0, with no differences in the FGA-PGF group. This increase in SCC of the control and FGA groups coincided with peak estrus behavior. However, SCC was far below the upper limit of the current standard for normal milk. In conclusion, induction of estrus with progestagen based programs and buck introduction may cause temporary significant increase in SCC. However, the SCC values during this period of temporary increase were still in the range of acceptable values for normal milk. With the current standards for SCC of 1,000,000/ml as legal limit for abnormal milk control programs in goats, estrus synchronization programs and the estrus status should not be considered when bulk-tank milk SCC is being investigated.A. Q. Talafha, S. Q. Lafi, M. M. Ababne

The effect of estrus synchronization treatments on somatic cell count of transitional-anestrus Awassi ewes' milk

Fifty-three transitional-anestrus Awassi ewes, randomly assigned to three groups: fluorogestone acetate (FGA, n = 18), FGA-Prostaglandin (FGA-PGF, n = 18) and control (n = 17), were used to examine the effect of estrus synchronization protocols and steroid hormones concentrations on milk somatic cell count (SCC). Intravaginal FGA sponge was inserted for 13 days and 600 IU equine chorionic gonadotropin was administered for ewes of FGA and FGA-PGF groups at the time of sponge removal (day 0). In addition, 10 mg was administered to ewes of FGA-PGF group on day 0. Blood and milk samples were collected from all ewes on days -13, -6, 0, 1, 2, 7 and 14. Estradiol had significant positive correlation with the SCC during the periods of sponge insertion (P = 0.015, r = 0.235) and within two days (P = 0.063 r = 0.23) after sponge removal with no correlation with SCC of both udder halves during the luteal phase. Progesterone concentrations, on the other hand, had a significant positive correlation (P < 0.001; r = 0.420) with the SCC of both udder halves during the luteal phase of the experiment, but not during the periods of sponge insertion and expected estrus. SCC returned under the influence of endogenous progesterone on days 7 and 14 to pre-synchronization values. In conclusion, sheep milk SCC is affected significantly with induction of estrus and steroid hormones concentrations. However, peak SCC recorded during estrus was far below the upper limit of the current standard for normal milk. With the current standards for SCC of 1,000,000/ml as legal limit for abnormal milk control programs in sheep, estrus synchronization programs and the estrus status should not be considered when bulk-tank milk SCC is being investigated, but should be considered during the process of setting new standards.A. Q. Talafha, S. Q. Lafi, M. M. Ababne