Feasibility of genus-specific real-time PCR for the differentiation of larvae from gastrointestinal nematodes of naturally infected sheep

Results of real-time PCR analysis of coproculture third stage larvae (L3) using genus specific TaqMan minor groove binder probes were compared with the results of morphological differentiation of L3 after coprocultured and direct morphological worm differentiation from gastrointestinal samples of eight sheep with naturally acquired nematodes infections. Faecal egg counts prior to postmortem confirmed infections with trichostrongyles with a geometric mean count of 4828 eggs per gram for all sheep. Individual egg counts correlated positively with total worm counts (correlation coefficient 0.794). Five different nematode species and one genus were found in the abomasi and small intestines: Cooperia curticei, Haemonchus contortus, Nematodirus spp., Teladorsagia (Ostertagia) circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Coproculture of faecal eggs yielded five of these, Cooperia spp., Haemonchus spp., Ostertagia/Teladorsagia spp. and Trichostrongylus spp. Comparison between morphological L3 and worm differentiation data showed high congruence (94%). The agreement between PCR analysis of L3 after coproculture and direct morphological worm differentiation was 84%. Thus, real-time PCR was found to be suitable as a speedy and reliable diagnostic tool for the assessment of gastrointestinal nematode infections of ruminants in the field.     

The epidemiology of nematode and fluke infections in cattle in the Red River Delta in Vietnam

Over a period of 13 months, faecal samples were collected monthly from approximately 45 cattle over 3 months of age. Additionally, 74 calves of 1-2 months were sampled to determine the presence of Toxocara vitulorum eggs. Individual egg counts and infective strongyle larvae from pooled faecal samples were examined. Post-mortem worm counts were carried out on six groups of tracer calves (n=12) that had been kept for 4 weeks on pasture in and around the village studied. The following helminths were identified: T. vitulorum, Cooperia punctata, C. pectinata, C. oncophora, Oesophagostomum radiatum, Trichostrongylus axei, T. colubriformis, Haemonchus spp., Fasciola spp. and Paramphistomum spp. In 8% of the samples collected from young calves, individual egg counts for T. vitulorum were found indicative for pathogenic worm burdens. Strongyle egg counts and worm counts indicated that transmission is low without a distinct seasonality. In animals of 3-9 months old, a strongyle egg count peak can be demonstrated which at a higher age steadily and significantly decreased. In faecal cultures Cooperia spp. were most prominent in all age groups throughout the year with the exception of the period September-November when Haemonchus spp. and Oesophagostomum spp. were most prevalent. Fasciola spp. eggs were found in 22% of the collected faecal samples and the egg counts were low indicating that the intensity of Fasciola spp. infection is mild. Based on the present data, regular anthelmintic treatments seem not to be justified, except for a single treatment at the age of 2 weeks against toxocariosis.     

An epidemiological study of trichostrongylidiasis in dairy cattle grazing irrigated pastures

Trichostrongylid burdens in 1 to 2-year-old dairy heifers were estimated after they grazed with a dairy herd for up to 12 months. Ostertagia ostertagi, Trichostrongylus axei and Cooperia oncophora represented 79.5, 12.7 and 7.8% respectively of all trichostrongylids recovered. The largest burden was 97,000 trichostrongyles with less than 20,000 being the typical burden. Clinical disease was not observed. The strain of O. ostertagi present was capable of inhibition and was not necessarily controlled by anthelmintics. Availability of infective larvae increased with the onset of spring when "modified tracer" calves accumulated up to 30,000 trichostrongyles over intervals of one month. From July to September each year, up to 80% of the Ostertagia burden in these animals were inhibited larvae. Faecal strongyloid egg counts fell from less than an average of 60 eggs per gram to 10 eggs per gram when the heifers were one and 2 years old, respectively. Mature cows continually passed few eggs in their faeces.     

Nematode control practices and anthelmintic resistance in dairy calves in the south west of England.

A postal survey of worming practices on West Country dairy farms was undertaken and farmers were requested to send faecal samples for nematode egg counts. The majority of the farmers who responded had a nematode control policy which was based on a mixture of anthelmintics and pasture rotation. Sixty-five per cent turned out calves on to the same paddock each year and 57 per cent treated their stock with anthelmintics during the second year. Ninety farms submitted samples for analysis but only 16 samples contained sufficient eggs to justify repeat egg counts and only eight of these produced enough eggs for in vitro tests. The small number of positive samples, even into the latter part of the year suggests a heavy use of anthelmintics with relatively clean pasture. One Somerset farm had larvae which developed in high concentrations of ivermectin, and eggs were still being passed after two treatments with ivermectin at the manufacturer's recommended dose seven days apart. Of 100 male nematodes isolated from two of the calves, 88 were Cooperia species and 12 were Nematodirus species. A controlled trial with this isolate in eight Friesian male calves showed a 44 per cent reduction in egg counts at day 7 but no significant reduction in worm burden postmortem. This is the first reported case of ivermectin-resistant Cooperia species in cattle in the United Kingdom.     

Clinical pathology of experimental Schistosoma curassoni infections in sheep and goats

The clinical pathology of Schistosoma curassoni infection in sheep and goats was studied for 22 weeks following experimental infection with 7000 and 4000 cercariae, respectively. Excretion of eggs began at week 7 after infection: in goats the numbers increased to 30 to 50 eggs per gram faeces (epg) at weeks 8 to 18, followed by a reduction. In a pregnant goat, epg values increased markedly before and after parturition. The mean faecal egg counts in sheep were lower than in goats, increasing to a maximum level of 30 epg at weeks 16 and 17 after infection. Infected sheep maintained growth rates roughly comparable with controls, whereas infected goats failed to gain as much weight as the controls. Infected goats and sheep produced eosinophil counts of about 3 x 10(3) mm-3, five and eight weeks after infection, respectively. Sheep developed a progressive anaemia from week 11 after infection, in goats blood values remained within normal limits. Differences in serum protein concentration were observed between infected and uninfected goats about nine weeks after infection, but not in sheep. Increased total protein values, hyperglobulinaemia and lowered albumin to globulin ratios were features of infected goats. Serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, gamma-glutamyl transferase, total lactate dehydrogenase and bilirubin were not significantly changed. The mean recovery in sheep was 608 worms, in goats 428 worms, but the total tissue egg counts were higher in the latter. Of the total eggs deposited in the goats 92 per cent were found in the liver with 51.5 per cent in the ovine liver. The histopathological changes were studied.     

Epidemiological studies of parasitic gastrointestinal nematodes, cestodes and coccidia infections in cattle in the highveld and lowveld communal grazing areas of Zimbabwe

Between January 1999 and December 2000 faecal samples from 16264 cattle at 12 dipping sites in the highveld and nine in the lowveld communal grazing areas of Zimbabwe were examined for gastrointestinal (GI) nematode and cestodes eggs, and coccidia oocysts. Strongyle larvae were identified following culture of pooled faecal samples collected at monthly intervals. The effects of region, age, sex and season on the prevalence of GI nematodes, cestodes and coccidia were determined. Faecal egg and oocyst counts showed an overall prevalence of GI nematodes of 43%, coccidia 19.8% and cestodes 4.8%. A significantly higher prevalence of infection with GI nematodes, cestodes and coccidia was recorded in calves (P < 0.01) than in adults. Pregnant and lactating cows had significantly higher prevalences than bulls, oxen and non-lactating (dry cows) (P < 0.01). The general trend of eggs per gram (epg) of faeces and oocysts per gram (opg) of faeces was associated with the rainfall pattern in the two regions, with high epg and opg being recorded during the wet months. The most prevalent genera of GI nematodes were Cooperia, Haemonchus and Trichostrongylus in that order. Strongyloides papillosus was found exclusively in calves. Haemonchus was significantly more prevalent during the wet season than the dry season (P < 0.01). In contrast, Trichostrongylus was present in significantly (P < 0.01) higher numbers during the dry months than the wet months, while Cooperia and Oesophagostomum revealed no significant differences between the wet and dry season. These findings are discussed with reference to their relevance for strategic control of GI parasites in cattle in communal grazing areas of Zimbabwe.     

Observations on the egg output resulting from continuous low level infections with Ostertagia circumcincta in lambs

Faecal egg counts were determined twice weekly for two groups of four-month-old worm free cross Suffolk lambs experimentally infected with 100 and 320 Ostertagia circumcincta larvae, five times per week for 20 weeks. The group mean egg count in both groups rose to a maximum of approximately 500 eggs per gram during weeks 12 to 14 and had fallen to less than half of that value at slaughter. There was no statistical difference in egg output between these two groups or between them and a comparable group of lambs receiving 4000 Ostertagia circumcincta larvae per day. No significant changes in live weight gain or serum pepsinogen levels were observed in the lambs given 100 and 320 larvae per day.     

Strongyle egg shedding consistency in horses on farms using selective therapy in Denmark

Knowledge of horses that shed the same number of strongyle eggs over time can lead to the optimization of parasite control strategies. This study evaluated shedding of strongyle eggs in 424 horses on 10 farms when a selective anthelmintic treatment regime was used over a 3-year period. Faecal egg counts were performed twice yearly, and horses exceeding 200 eggs per gram (EPG) of faeces were treated. The results are presented as probabilities of the egg count outcome, when two previous egg counts are known. A horse with no strongyle eggs detected in the two previous faecal examinations had an 82% probability of a zero, and a 91% of being below 200 eggs per gram in the third examination. A horse with the two previous egg counts below 200 EPG had an 84% probability of being below 200 EPG the third time as well. When faecal egg counts exceeded 200 EPG on the previous two counts, the probability for a horse exceeding 200 EPG the third time was 59%. In conclusion, these data demonstrate consistent shedding from one grazing season to another in a majority of horses despite treatment of horses exceeding 200 EPG.     

Transfer of adult Strongylus vulgaris via stomach tube

Patent infections with Strongylus vulgaris were established in 6 of 8 helminth-free ponies given 41 to 101 adult worms via nasogastric tube. The parasites were removed from the cecum and ventral colon and transferred within 1 to 2 hours of the death of the donor horses. Eggs were found in the feces of the recipients in 2 or 3 days; egg counts reached maximum, 28 eggs per gram of feces, at 4 weeks after ponies were inoculated. In 6 ponies euthanatized 3 to 7 weeks after parasitic transfers were done, 28% of the inoculated worms were found alive at necropsy. A 7th pony was maintained as a donor for establishing infections for chemotherapy trials and, although never passing more than 6 eggs per gram of feces, shed infective larvae over a period of 2 years.     

Control of endoparasites in horses with a gel containing moxidectin and praziquantel

A gel formulation containing moxidectin (20 g/kg) and praziquantel (125 g/kg) reduced the geometric mean faecal strongyle egg count in horses to below 100 eggs per gram of faeces (epg) for at least 12 weeks despite their being exposed continuously to reinfection from pasture grazed by treated and untreated horses. The geometric mean egg count of horses treated with a proprietary paste containing abamectin (3.7 g/kg) and praziquantel (46.2 g/kg) increased steadily from six weeks after the treatment, peaking at over 820 epg after 12 weeks. Relative to the efficacy of the abamectin/praziquantel treatment, the reduction in mean faecal egg count compared with the pretreatment counts was significantly (P<0.05) better in the horses treated with moxidectin and praziquantel from eight weeks after the treatment. Both products eliminated tapeworms from horses in a non-invasive modified critical trial.     

Prevalence of cattle herds with ivermectin resistant nematodes in the hot sub-humid tropics of Mexico

The objective was to determine the prevalence of semi-intensive Bos indicus and Bos indicus × Bos taurus cattle herds with ivermectin (IVM) resistant nematodes in a sub-humid tropical zone of Mexico using the faecal egg count reduction test (FECRT). Thirty-three herds (28 beef and 5 dual purpose herds) were monitored in a period of 6 months (September 2008 to February, 2009). Only 14 of the 33 herds were included in the trial. The other herds had not enough animals with sufficient nematode eggs per gram of faeces (EPG) to be included in a FECRT. Some farms were visited twice trying to find more animals with egg counts higher than 150 EPG. In the 14 surveyed herds the calves were randomly distributed into two groups: (a) treatment group received 0.2mg of IVM/kg BW sc on day 0, and (b) control group without treatment. Faecal samples were obtained from each animal on days 0 and 14 post-treatment. Reduction percentages (% R) and 95% CI were calculated. The prevalence of cattle herds with IVM resistant nematodes was 78.6%. Those suspected of IVM resistance were 21.4%. All surveyed herds used IVM from two to three times a year (mainly beginning and end of the wet season) during 1-11 consecutive years. The farm with stronger resistance used IVM for 11 consecutive years (% R=0%; 95% CI=0-47%). Genera of nematodes resistant to IVM were: Ostertagia, Haemonchus, Cooperia and Trichostrongylus. A considerable effort is needed to perform FECRT in cattle herds under hot sub-humid tropical conditions.     

An evaluation of a modified preventive drenching programme on commercial sheep farms

The effectiveness of a commonly advocated five drench preventive drenching programme in lambs was assessed on nine farms in the Gisborne, Hawkes Bay and Wairarapa districts between December 1983 and March 1985. The productive performance of two groups of preventively drenched lambs was compared with lambs in which parasitism was suppressed by fortnightly drenching from weaning until mid-August 1984. One of the groups received the standard five drench programme commencing at weaning and finishing in March 1984. The other group received six additional 4-weekly drenches, with the last drench in mid-August 1984. All the other lambs on the farms were drenched according to the five drench programme. The availability of infective larvae on pasture and the build-up of parasitic infection in the lambs was monitored. The effects of the different drenching programmes on liveweight and faecal egg counts was followed until March 1985, when the animals were 18 months old. Between March and July 1984 on all nine farms there was a significant depression of growth rates of the standard five drench group when compared with the suppression-drenched group (P<0.01). The mean liveweight difference from December 1983 to July 1984 for all farms was 4.0 kg (P<0.05). Hogget wool weights were significantly depressed (mean difference 0.37 kg, P<0.05). Faecal egg counts rose steadily after the completion of the five drench programme in March, exceeding 1000 eggs per gram on seven farms and 4000 eggs per gram on three of these farms. Trichostrongylus spp. was the major constituent of the winter parasite increase except on one farm when Cooperia spp. succeeded a previous Haemonchus dominance. Two farms had major build-ups of Haemonchus spp., one in early May, the other in June. Six farms salvage-drenched the standard five drench groups. The lambs in the standard drench groups on these farms recovered about half the bodyweight difference between August and March. The mean weight gains to July and wool weights of the modified drench programme groups were reduced slightly compared with the suppression-drenched group (mean liveweight difference 1.4 kg; mean wool weight difference 0.15 kg). No major rises in faecal egg counts occurred. Herbage larval counts exceeded 1000 larvae/kg at some stage on seven farms and, of these, the counts exceeded 4000 larvae/kg on three farms.     

Equine helminth infections: control by selective chemotherapy

A programme of selective anthelmintic therapy was used in a herd of 31 horses. Faecal egg counts were done during the months of September, November, January, March, May and the following September. Horses with greater than or equal to 100 eggs per gram (epg) were treated with ivermectin, and those with less than 100 epg were not treated. The criteria for adequate internal parasite control in the herd was a median herd faecal egg count of less than or equal to 100 epg. Effectiveness of selective therapy was assessed by faecal egg count after nine months of treatment and was determined to be adequate when a median herd egg count of 0 epg was obtained. However, on returning from pasture the following September, median herd egg count had risen to 325 epg. A statistically significant correlation was seen in the paired September faecal egg counts of the horses in that initial September faecal egg count was predictive for the following September. Initial September faecal egg count was related to the number of anthelmintic treatments required during the period of selective therapy, whereas age of horse was not. We propose that faecal egg counts be incorporated into strategic anthelmintic programmes as an economical tool for identifying and targeting herd members predisposed to shedding elevated numbers of helminth eggs.     

Preliminary Analysis of the Results of Selective Therapy Against Strongyles in Pasturing Horses

Control of horse parasites often omits application of measures to eradicate the free-living stages in pastures and frequently relies on chemotherapy only. Selective therapy was used for Spanish Sport horses grazing either in the same pasture (continuous) or in rotated meadows. In each group, equines exceeding a cutoff value of 300 strongyle eggs per gram of feces received ivermectin or moxidectin. Efficacy of the treatment was assessed by estimating reduction of fecal egg counts and the number of horses shedding parasite eggs (PHR). Coprocultures revealed presence of the cyathostomins Cyathostomum and Gyalocephalus spp. In all treated groups, a 100% value for both reduction of fecal egg counts and PHR against cyathostomins was obtained, and PHR values ranged from 100% to 12%. The longest strongyle egg reappearance period was observed in horses undergoing rotation grazing and receiving ivermectin (9 weeks), compared with a 6-week period recorded for the other treated equines. Our results seem to point that the efficacy of selective therapy in equine herds could be reduced if the horses with fecal egg counts below the threshold value (thus not receiving chemotherapy) remain grazing in the same pastures with the treated ones. It is strongly suggested that interested parties consider performing periodic fecal analyses to monitor fecal egg counts, together with the percentage of horses passing eggs in feces, to improve the effect of this procedure.     

Effect of fenbendazole and ivermectin on development of strongylate nematode eggs and larvae in calf feces.

Thirty-nine weaned steer calves (mean weight 284 kg) were maintained under dry-lot conditions and assigned (based on fecal nematode egg count) to one of three treatment groups of 13 animals each as follows: control (no treatment), fenbendazole (5 mg kg-1), and ivermectin (0.2 mg kg-1). Fecal samples were collected 12 h before treatment, at treatment, and 12, 24, 48 and 72 h after treatment for determination of nematode eggs per gram, and (after culture) infective larvae per gram and population distribution. The effect of treatment on egg development was observed in feces collected 12 and 24 h after treatment. There was essentially no difference in efficacy, based on egg counts, of fenbendazole and ivermectin. Egg count was reduced 100% by both anthelmintics at 72 h after treatment. Viability, based on percent of eggs reaching the infective larval stage, of developing stages at 12, 24, and 48 h after fenbendazole treatment was 0.1%, 1.1%, and 0%; after ivermectin treatment the corresponding values were 23.7%, 30.1%, and 28.6%, respectively. Fenbendazole treatment resulted in little or no development of eggs and/or larvae in feces deposited 12 and 24 h after treatment, whereas development proceeded normally (compared with the control group) in ivermectin treated feces. Population distribution of infective larvae was predominantly Haemonchus and Cooperia with some Ostertagia and Oesophagostomum.     

Seasonal variations in the numbers of trichostrongylid nematode eggs and their larvae in the faeces of farmed goats in Malaysia.

Faecal worm egg counts of goats from two farms in Penang Island, West Malaysia, were monitored over a period of 14 months. The faecal egg count pattern followed that of total rainfall. The humid tropical environment was favourable for the development of various species of trichostrongylid nematodes, namely Haemonchus contortus, Trichostrongylus spp., Oesophagostomum spp. and Cooperia spp. Generally, H. contortus was observed to be the predominant species, more so in the monsoon months of the year.     

Determining Treatment to Control Two Multidrug-Resistant Parasites on a Texas Horse Farm

A study was undertaken at the Texas A&M Horse Center to evaluate and compare the effectiveness of three anthelmintics—ivermectin, fenbendazole, and a combination of ivermectin and pyrantel pamoate—on fecal egg count reductions of cyathostomes and Parascaris equorum in 30 naturally infected foals. The foals were randomized into three treatment groups, with individuals being rerandomized after each 8-week observation period. The treatments of ivermectin and fenbendazole were given at the manufacturer's recommended doses, and the pyrantel treatment was given at two times the manufacturer's recommended dose. Fecal egg counts were performed at the time of treatment and at 2-week intervals after treatment for a total of 8 weeks. Each foal received a total of three treatments during the course of the study. Fecal egg counts were performed by a modified McMaster's test, with a sensitivity of 25 eggs per gram of feces, and by the modified Wisconsin double centrifugal flotation technique, with a sensitivity of 0.2 eggs per gram of feces. Fecal egg reduction percentages were calculated. Analysis of the results showed that ivermectin, either used alone or with pyrantel, was a more effective anthelmintic for cyathostome (small strongyle) control than fenbendazole. Fenbendazole and pyrantel showed a higher initial reduction in Parascaris egg counts when compared with the ivermectin-only-treated group, but this difference lessened over time. The use of the combination treatment showed the best results for controlling both parasites, indicating that a combination of anthelmintics may be necessary to control parasites on some equine farms.     

Trichostrongylid infections in sheep after rainfall during summer in southern Australia

OBJECTIVE: To relate trichostrongylid infections acquired by sheep during summer to prevailing weather conditions. PROCEDURE: Groups of worm-free 'tracer' sheep were put onto pastures, previously contaminated with trichostrongylid eggs, for successive periods of 2 weeks from December to March. After grazing the sheep were housed for 6 weeks. Weekly worm egg counts and worm counts were used to estimate the numbers of worms acquired and related to weather conditions during the grazing period. RESULTS: No worm eggs were detected in the faeces of sheep that grazed at the end of January when only 7 mm of rainfall was recorded. At other times rainfall between 12 and 24 mm occurred and strongyle egg counts were generally either < 50 or > 150 eggs per g (epg). Mean counts of 1,100 Ostertagia and Trichostrongylus adults gave rise to mean counts of about 350 epg whereas about 6,000 Nematodirus spp were associated with mean egg counts of about 200 Nematodirus spp epg. CONCLUSIONS: Rainfall events during summer determine the numbers of trichostrongylid larvae acquired by sheep in summer but further studies are necessary before the implications for strategic control programs in southern Australia can be fully assessed.     

Observations on the free-living stages of cattle gastrointestinal nematodes

A 4-year study on the free-living stages of cattle gastrointestinal nematodes was conducted to determine (a) the development time from egg to infective larvae (L3) inside the faecal pats, (b) the pasture infectivity levels over time, and (c) the survival of L3 on pasture. Naturally infected calves were allowed to contaminate 16 plots on monthly basis. Weekly monitoring of eggs per gram of faeces (epg) values and faecal cultures from these animals provided data for the contamination patterns and the relative nematode population composition. At the same time, faecal pats were shaped and deposited monthly onto herbage and sampled weekly to determine the development time from egg to L3. Herbage samples were collected fortnightly over a 16-month period after deposition to evaluate the pasture larval infectivity and survival of L3 over time. The development time from egg to L3 was 1-2 weeks in summer, 3-5 weeks in autumn, 4-6 weeks in winter, and 1-4 weeks in spring. The levels of contamination and pasture infectivity showed a clear seasonality during autumn-winter and spring, whilst a high mortality of larvae on pasture occurred in summer. Ostertagia spp., Cooperia spp. and Trichostrongylus spp. were predominant and a survival of L3 on pasture over a 1-year period was recorded in this study.     

Reduced efficacy of anthelmintics in young compared with adult horses

Studies on a Thoroughbred breeding farm in Ohio from 1982 to 1988 demonstrated the value of three anthelmintic pastes (ivermectin, oxibendazole, pyrantel pamoate) in controlling benzimidazole resistant cyathostomes (small strongyles) in adult horses. However, a comparison of drug efficacy in suppressing faecal egg counts for the full period between treatments showed a significant reduction in efficacy of all drugs in yearling horses compared with adults. Mean faecal egg counts of adult horses were generally kept below 100 eggs per gram (epg) of faeces when using oxibendazole or pyrantel pamoate at four to five week intervals and ivermectin at eight week intervals. By contrast, mean counts of young horses rose as high as 655 epg (oxibendazole), 729 epg (pyrantel pamoate) and 852 epg (ivermectin) within the same time period after treatment. Individual counts of treated yearlings sometimes exceeded 3,000 epg. Three distinct mechanisms appeared to be involved in the poor results in young horses. These were 1) anthelmintic refuge, 2) anthelmintic resistance, and 3) anthelmintic avoidance.