PCR COMPARISON OF TRICHOSTRONGYLE GENERA PRESENT IN SOUTH DAKOTA CATTLE WITH AND WITHOUT SPRINGTIME DEWORMING

Proceedings of the South Dakota Academy of Science, Vol. 88 (2009) 147 PCR COMPARISON OF TRICHOSTRONGYLE GENERA PRESENT IN SOUTH DAKOTA CATTLE WITH AND WITHOUT SPRINGTIME DEWORMING A.F. Harmon 1, B. C. Lucas 1 and M. B. Hildreth 1,2 Departments of Biology & Micrbiology 1 and Veterinary Sciences 2 South Dakota State University Brookings, SD 57007 ABSTRACT Recent advances in multiplex PCR technologies have made it possible to differentiate (at the genus level) trichostrongyle nematode eggs from naturally infected cattle. Because species of Cooperia in cattle are more resistant to macrocyclic lactones (ML) than Ostertagia and Haemonchus, resistance tends to first appear among Cooperia, enabling it to out-compete other genera. Therefore, a stable population of mixed trichostrongyle genera provides evidence that ML resistance is not appearing in cattle herds. The purpose of this study was to use standard multiplex PCR and real-time multiplex PCR to compare the distribution of 3 trichostrongyle genera in 8 herds that had been participating in a spring-time deworming program to another 5 herds that did not. All herds were from eastern South Dakota. Cooperia and Ostertagia were the predominate genera indentified, but Haemonchus was also frequently present. Of the 239 samples from all herds, trichostrongyle DNA was detected in 187 samples. Among these positive samples, Cooperia was found in 64%, Ostertagia in 59%, and Haemonchus in 26%. A third of the positive samples (32%) contained only 1 genus, whereas combinations of Cooperia and Ostertagia (37%) or of all 3 genera (23%) were detected. Samples containing only Cooperia (the more ML-resistant genera) were found in only 17% of the total samples. Cooperia-only samples within each herd from the 8 spring-dewormed herds ranged from 5% to 50% of the animals, but were not significantly different from the 5 herds using no spring deworming program. These data provide no evidence for ML resistance developing within the treated herds in eastern South Dakota. Keywords Trichostrongyle nematode, cattle parasites, multiplex PCR, macrocyclic lactone resistance, Haemonchus, Ostertagia, Trichostrongylus, Cooperia, Oesophagostomum, South Dakota INTRODUCTION Throughout the United States, the 5 main genera of trichostrongyle nematodes infecting cattle include Haemonchus, Ostertagia, Trichostrongylus, Cooperia

148 Proceedings of the South Dakota Academy of Science, Vol. 88 (2009) and Oesophagostomum. These nematodes cause an estimated $2 billion in economic losses to U.S cattle producers each year (Zarlenga et al. 2001). Strategic deworming programs have helped minimize these losses; the most popular programs typically involve the use of macrocyclic lactones (ML), including ivermectin, doramectin, eprinomectin, and moxidectin. The efficacy of these compounds has contributed to the popularity of these products, but this efficacy has also placed high selection pressures on cattle nematodes to develop resistance to these compounds. In cattle trichostrongyles, resistance to ivermectin has been reported throughout Europe and New Zealand (West et al. 1994; Vermunt et al. 1995; Vermunt et al. 1996; Coles et al. 1998; Loveridge et al. 2003), and in the United States (Gasbarre et al. 2005). Thus far, most reports of resistance have involved Cooperia oncophora, which is not surprising because this species is one of the dose-limiting species for ML in cattle (Njue and Prichard 2004). Due to the lack of any molecular assays for detecting ML resistant worms, the only accepted method for identifying resistance is to perform a fecal egg count reduction test (FECRT), which is reasonably useful for documenting resistance once resistance is suspected, but using FECRT to monitor the development of resistance on a regional scale is inconvenient and thereby impractical. For this reason, it is difficult for cattle producers to get science-based information about the presence of anthelmintic resistance within a region, and so producers are prone to develop perceptions based upon rumors and misinformation. Within herds, trichostrongyle populations are typically composed of combined species, generally from 2 or more genera. As the selective pressures from repeated ML treatments build in these populations, the species with lower susceptibility to the drug will out-compete those species with higher susceptibility. Limited studies in Minnesota and Wyoming (Stromberg et al. 1991; Malczewski et al. 1996) suggest that Ostertagia tended to be the most prevalent genus of trichostrongyle, followed by Cooperia and then Haemonchus and Trichostrongylus. If ML-resistant trichostrongyle populations begin to emerge in the Northern Great Plains, the more resistant Cooperia and Trichostrongylus species should become much more prevalent than Ostertagia. Therefore, a stable population of mixed trichostrongyle genera provides evidence that selective pressures are not yet sufficient to drive the population toward ML resistance. In regions such as the Northern Great Plains, where deworming programs typically involve a single treatment and herds tend to be closed, it s possible that ML resistance may not develop to any significant extent; however, the only way to determine this is to monitor changes in the genera composition of these populations. Unfortunately, there are virtually no data on population composition for cattle strongyles from this region; as such, there is an immediate need to establish this baseline for the U.S.A. Northern Great Plains. In 2001, Zarlenga et al. (2001) described a multiplex PCR assay that can be used with DNA extracted from trichostrongyle eggs to differentiate the five most common genera of cattle trichostrongyles generally found in the United States (i.e. Cooperia, Ostertagia, Haemonchus, Trichostrongylus, and Oesophagostomum). Recent studies by Harmon et al. (2006, 2007a, 2007b) optimized a trichostrongyle-egg DNA-extraction protocol that can be used with the multiplex PCR assay, making the protocol efficient for large field studies. With these tools, it

Proceedings of the South Dakota Academy of Science, Vol. 88 (2009) 149 is now feasible to determine the genus-level population composition of cattle trichostrongyles within a commercial herd simply by collecting fecal samples from representative calves of the herd. The purpose of the present study was to use standard multiplex PCR and real-time multiplex PCR to determine which genera were dominant in the region, as well as the typical proportions of Cooperia, Ostertagia, and Haemonchus present in cattle herds from eastern South Dakota, and compare these proportions in 8 herds that had been participating in a spring-time deworming program to 5 herds that had not. METHODS At the end of the 2005 grazing season (September through November, depending on the herd), rectal fecal samples were collected from spring-born calves in 13 herds from eastern South Dakota. Attempts were made to collect at least 20 samples from each herd, because Gasbarre et al. (1996) demonstrated that this sample size was statistically representative of herd parasite loads. Pertinent information such as anthelmintic use, date of treatment, and pasture rotation scheme were collected for each herd. Only 5 of the 13 herds had not been using any avermectin-type dewormer during the preceding 5 springs. The remaining 8 herds had been treated with either a pour-on or injectable avermectin-type product during that spring (typically June), and generally at least during the preceding 5 springs. Collected fecal samples were stored at 4 ºC until eggs were isolated and counted from 254 samples using a modified Wisconsin protocol (Cox and Todd 1962). To recover eggs from each slide wet-mount, the slidecoverslip combination was placed in a 50ml centrifuge tube containing 40ml of water, and the tube was vortexed for approximately 30 seconds. Each tube was then centrifuged at 2,000g to concentrate the eggs into a pellet that was stored at -20 ºC until DNA was isolated. Egg DNA was extracted from 239 egg samples as described by Harmon et al. (2006), and analyzed with a traditional multiplex assay (MPCR) PCR assay and a real-time PCR assay (QPCR) to determine which genera of trichostrongyles were present. The MPCR assay used was described by Zarlenga et al. (2001) and Harmon et al. (2006), and was able to identify the following trichostrongyle genera: Haemonchus, Ostertagia, Trichostrongylus, Cooperia, and Oesophagostomum. The PCR reaction took place in an PE 2400 thermocycler (Applied Biosystems Inc, Foster City, CA 94404), and PCR products were visualized with a Bio-Rad ChemiDoc XRS gel imaging system (Hercules, CA). The QPCR assay was modified from an assay described by von Samson-Himmelstjerna et al. (2002) for sheep trichostrongyles, but modified for cattle trichostrongyles by Harmon et al. (2007b). This QPCR assay detected Ostertagia ostertagi and Cooperia oncophora, C. surnabada, and C. puncta (Table 1). Additionally, a primer and probe set specific for H. contortus, from a recent study (Harmon et al. 2007a), was used to determine if samples positive for Haemonchus with the MPCR assay were H. contortus. If not H. contortus, then the assumption was made that the sample contained H. placei. The real-time assay was performed on a Stratagene MX3000P

150 Proceedings of the South Dakota Academy of Science, Vol. 88 (2009) Table 1. Fecal egg output (in eggs/gram) and prevalence of selected parasites in calves from treated and untreated herds in eastern South Dakota Herd (sample number) Untreated 1 (n= 18) Untreated 2 (n= 9) Untreated 3 (n= 18) Untreated 4 (n= 22) Untreated 5 Mean Treated 1 Treated 2 Treated 3 Treated 4 (n= 36) Treated 5 (n= 21) Treated 6 (n= 21) Treated 7 Treated 8 Mean Strongyles EPG (prevalence) Nematodirus EPG (prevalence) Trichuris EPG (prevalence) Moniezia EPG (prevalence) 6.44 (66.7%) 0.22 (16.7%) 0 0 1.89 (44.4%) 0.78 (44.4%) 0 0.33 (11.1%) 47.61 (100.0%) 2.00 (33.3%) 0.28 (16.7%) 30.78+ (72.2%) 10.05 (100.0% 6.05 (81.8%) 0.32 (13.6%) 1.05 (4.5%) 1.20 (35.0%) 0.80 (30.0%) 0 8.20 (20.0%) 13.44 SEM= 8.69 1.97 SEM= 1.06 0.12 SEM= 0.07 8.07 SEM= 5.87 4.05 (75.0%) 2.70 (75.0%) 0.25 (15.0%) 22.55 (55.0%) 3.00 (80.0% 2.35 (90.0%) 0 0.05 (5.0%) 0.10 (5.0%) 0.10 (5.0%) 0 4.10 (20.0%) 23.73 (100.0%) 3.38 (57.7%) 0.08 (7.7%) 0.75 1.57 (61.9%) 0.43 (23.8%) 0 14.77 (42.9%) 25.20 (100.0%) 9.20 (50.0%) 0.35 (25.0%) 5.05+ (10.0%) 0.75 (40.0%) 1.00 (60.0%) 0.05 (5.0) 10.00+ (10.0%) 7.40 (100.0%) 2.15 (55.0%) 0 32.65+ (35.0%) 8.23 SEM= 3.64 2.66 SEM= 1.01 0.09 SEM= 0.05 11.24 SEM= 4.06 machine (La Jolla, CA). When amplification did not occur (no statistical increase in fluorescence), samples were assigned a CT value of 40. Statistical analysis was performed with GraphPad Instat software (http://www.graphpad.com). RESULTS Results from the 254 fecal egg counts are shown in Table 1. Trichostrongyle eggs were found in 71.65% of the fecal samples. There was a large amount of herd-to-herd variability among the 13 herds for both prevalence and trichostrongyle egg output, and both values among the treated herds were statistically

Proceedings of the South Dakota Academy of Science, Vol. 88 (2009) 151 similar to that of the untreated. A little more than half of the calves (51.2%) showed evidence of Nematodirus infections; however, egg output was relatively low and no differences existed between the treated and untreated herds. Trichuris eggs were found in very few calves from any of the herds. The only tapeworm, Moniezia, was found in 21.2% of the total number of calves. Egg output was highly variable in these calves, but showed no treatment effects. Among the trichostrongyles, Cooperia and Ostertagia were the predominate genera indentified through PCR analysis, but Haemonchus was also present in 9 of the herds (Table 2). Of the 239 samples further analyzed with PCR, trichostrongyle DNA was detected in 187 samples. Among these positive samples, Table 2. Fecal egg output for trichostrongyle eggs and the genera identified from those eggs (expressed as prevalence from calves within each treated and untreated herd) Herd (sample number) Untreated 1 (n= 18) Untreated 2 (n= 9) Untreated 3 (n= 18) Untreated 4 (n= 22) Untreated 5 Mean (SEM) Treated 1 Treated 2 Treated 3 Treated 4 (n= 26) Treated 5 (n= 21) Treated 6 (n= 21) Treated 7 Treated 8 Mean (SEM) Strongyles EPG (Identified) 6.44 (ID= 13) 1.89 (ID= 6) 47.61 (ID= 18) 10.05 (ID= 22) 1.20 (ID= 4) 13.44 (8.69) 4.05 ID= 11 3.00 ID= 18 0.10 ID= 6 17.33 ID= 23 1.57 ID= 20 25.20 ID= 20 0.75 ID= 9 7.40 ID= 17 8.23 (3.64) Cooperia (C) Percent Ostergia (O) Percent Haemonchus (H) Percent C & O Percent C & H Percent H & O Percent C & H & O Percent 23.08% 38.46% 0% 15.38% 0% 0% 23.08% 33.33% 16.67% 16.67% 0% 0% 16.67% 16.67% 0% 0% 0% 5.56% 5.56% 0% 88.89% 4.55% 0% 0% 59.09% 0% 0% 36.36% 0% 75.00% 0% 25.00% 0% 0% 0% 12.19% (6.79) 26.03% (14.13) 3.33% (3.33) 21.01% (10.44) 1.11% (1.11) 3.33% (1.11) 33.87% (15.15) 18.18% 36.36% 0% 36.36% 9.09% 0% 0% 5.56% 22.22% 0% 61.11% 5.56% 0% 5.56% 50.00% 50.00% 0% 0% 0% 0% 0% 17.39% 0% 13.04% 4.35% 21.74% 8.70% 34.78% 50.00% 5.00% 5.00% 30.00% 10.00% 0% 0% 5.00% 0% 0% 85.00% 0% 5.00% 5.00% 44.44% 11.11% 0% 44.44% 0% 0% 0% 0% 11.76% 0% 58.82% 0% 0% 29.41% 23.82% (7.47) 17.06% (6.37) a 2.255% (1.66) Values with similar subscripted letters were statistically different P < 0.01. abc 40.01% (10.20) c 5.80% (2.73) b 1.71% (11.17) 9.35% (5.06)

152 Proceedings of the South Dakota Academy of Science, Vol. 88 (2009) Cooperia was found in 64.4%, Ostertagia in 59.0%, and Haemonchus in 26.8%. One calf from one herd tested positive for Oesophagostomum, but Trichostrongylus was not detected in any of the calves. One-third of the positive samples (32.1%) contained only 1 genus, whereas combinations of Cooperia and Ostertagia (36.4%) or of all 3 genera (24.1%) were detected in the majority of the samples (60.4%). As expected, herds with very low egg counts tended to have a higher predominance of calves with only 1 trichostrongyle genus. Calves infected only with Cooperia (the more ML-resistant genera) were found in only 16.6% of the total samples. The percentage of calves infected only with Cooperia varied considerably within each herd, ranging from 5% to 50% of the animals, but the prevalence of calves infected only with Cooperia in the treated herds was not significantly different from the 5 herds using no spring deworming program. DISCUSSION In a 1993 survey of 98 cow-calf herds from South Dakota, 94.0% of the calves showed evidence of trichostrongyle infections (i.e., at least 1 trichostrongyle egg), and the mean egg output from these worms in these calves was 33.79 EPG (Hildreth et al. 2007). The lower prevalence and egg output demonstrated in the present study is not surprising because numerous studies have shown that the intensity of trichostrongyle infections varies considerably from one herd to another, and even from year to year within the same herds (Stromberg and Corwin 1993). Prevalence and egg output values from Nematodirus and Trichuris were very similar to those described by Hildreth et al. (2007), but Moniezia was also slightly lower. The mean egg output values for the trichostrongyles and Trichuris were numerically lower in the treated herds than in the untreated, but these values were numerically higher for Nematodirus and Moniezia in the treated herds. None of these numerical differences, however, were statistically significant. Because avermectin treatments had been performed 3 to5 months prior to sample collection, it is not surprising that no statistical differences were detectable. As with many other treatment studies involving macrocyclic lactones that have documented diminished trichostrongyle egg output following treatment (Rew and Vercruysse 2002), it is likely that significant differences would have been detectible in herds from the present study if samples had been collected in mid-july. As with studies from states directly east (i.e., Minnesota) and west (i.e., Wyoming) of South Dakota (Stromberg et al. 1991; Malczewski et al. 1996), this project also demonstrated that Cooperia and Ostertagia were the 2 most common trichostrongyle genera detected in cattle from eastern South Dakota. Haemonchus placei is generally considered to be a more southern species (Gibbs and Herd 1986); however, H. placei was found regularly in previous necropsy studies from the Northern Plains (Stromberg et al. 1991; Malczewski et al. 1996). The demonstration of Haemonchus in calves from more than half of the herds in the present study was a little surprising. Trichostrongylus was occasionally identified in both the Minnesota study by Stromberg et al. (1991) and the Wyoming study by Malczewski et al. (1996), but was not detected in the South Dakota herds.

Proceedings of the South Dakota Academy of Science, Vol. 88 (2009) 153 Necropsy studies of area cattle are needed to confirm the PCR results, especially in relation to Trichostrongylus. Because species of Cooperia in cattle are more resistant to the MLs than Ostertagia and Haemonchus, populations of Cooperia tend to out-compete the other genera as resistance increases. Therefore, the common presence of mixed trichostrongyle genera in both the treated and the untreated herds provides good evidence that ML resistance is not a problem in these herds from eastern South Dakota. Because the eastern side of the Northern Great Plains would have a slightly higher parasite load (Hildreth et al. 2007), any ML resistance that might develop in this region would likely develop in eastern South Dakota. As such, these data provide no evidence for ML resistance developing within the Northern Great Plains. ACKNOWLEDGEMENTS This work was supported in part by the South Dakota State University Agricultural Experiment Station and Pfizer Animal Health. Special thanks to Craig Dybedahl for help with organizing fecal collections. This project also utilized the South Dakota State University Functional Genomics Core Facility supported in part by the National Science Foundation/EPSCoR Grant No. 0091948 and by the State of South Dakota. LITERATURE CITED Coles, G.C., K.A. Stafford, and P.H.S. MacKay. 1998. Ivermectin-resistant Cooperia species from calves on a farm in Somerset. Veterinary Records 142(10): 255-256. Cox, D.D., and A.C., Todd. 1962. Survey of gastrointestinal parasitism in Wisconsin dairy cattle. Journal of the American Veterinary Medical Association 141: 706-709. Gasbarre, L.C., E.A. Leighton, and D. Bryant. 1996. Reliability of a single fecal egg per gram determination as a measure of individual and herd values for trichostrongyle nematodes of cattle. American Journal of Veterinary Research 57: 168-171. Gasbarre, L.C., L.L. Smith, P.A. Pillit. 2005. Identification of cattle nematode parasites resistant to multiple classes of anthelmintics in a commercial cattle population in the U.S. Presented at American Association of Veterinary Parasitologist 50th Annual Meeting July 18 2005 in Minneapolis MN USA. Gibbs, H. C. and R.P. Herd. 1986. Nematodiasis in cattle: importance, species involved, immunity, and resistance. Veterinary Clinics of North America: Food Animal Practice 2(2), 211-224. Harmon, A.F., Z.B. Williams, L.D. Holler, and M.B. Hildreth. 2007a. Comparison of three different preservatives for morphological and Real-time PCR analyses of Haemonchus contortus eggs. Veterinary Parasitology 145(3-4): 361-365.

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