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Out of sight but not gone: Sero-surveillance for cattle grubs, Hypoderma spp., in western Canada between 2008 and 2010
Veterinary Parasitology 197 (2013) 297–303
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Out of sight but not gone: Sero-surveillance for cattle grubs, Hypoderma spp., in western Canada between 2008 and 2010 Douglas D. Colwell ∗ Agriculture and Agri-Food Canada, Lethbridge Research Centre, 5403 1st Ave. S., Lethbridge, AB, Canada
a r t i c l e
i n f o
Article history: Received 24 May 2013 Received in revised form 3 July 2013 Accepted 5 July 2013 Keywords: ELISA Sero-surveillance Warbles Western Canada
a b s t r a c t Despite the lack of evidence of clinically infected cattle in western Canada since the late 1990s there is persistent evidence from serological sampling that warble fly infections remain endemic. As part of a general survey of cattle parasites conducted on calves entering a feedlot in southern Alberta sera were tested for the presence of anti-Hypoderma antibodies using an established ELISA. Calves for the study were drawn from auction markets in Manitoba, Saskatchewan, Alberta and British Columbia and serum samples were collected, according to a randomization scheme, as the calves entered a single feedlot in October through December (Survey) of three consecutive years (2008–2010). In addition, a subsample of calves originating on a ranch in southeastern Alberta (Ranch 1) that had been the subject of previous surveillance was tested. Sero-prevalence of anti-Hypoderma antibodies of the Survey calves ranged from 0.0% to 100.0% depending on origin and overall varied among the three years with the sero-prevalence being 45.6%, 26.9%, 28.5% in 2008, 2009 and 2010 respectively. Sero-prevalence in Ranch 1 calves varied among years, with one year being much higher and was different from overall Survey sero-prevalence in two of three years (31.7%, 64.1%, 38.5% in 2008, 2009, 2010 respectively). In the Survey population there was a trend to lower sero-prevalence over the three years while on Ranch 1 there was no clear trend. In the samples with ELISA adjusted absorbance values above the positive cut-off an examination of the cumulative relative frequency showed that in all years approximately 90% of the samples were in the lower 3 of 14 bins in both populations and in two of three years in Ranch 1 population. The clustering of the positive ELISA data at the lower end of the range is suggestive of low level grub infestations. The increased frequency of high positive ELISA results from Ranch 1 in 2009 potentially indicate an ‘outbreak year’ in which pupal and adult fly survival was higher than for other years of this study resulting in more infested animals with higher burdens. Examination of precipitation and temperature profiles from the subject ranch support this suggestion. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Long an important economic burden on the North American cattle industry the cattle grubs (Hypoderma spp.) have become clinically almost non-existent, particularly in western Canada, since the advent of macrocyclic lactone, broad-spectrum parasiticides. The Canadian
∗ Tel.: +1 4033172254. E-mail address: [email protected]
Beef Quality Audit reported a scant 0.1% of carcasses with evidence of grubs in 1996 (Van Donkersgoed et al., 1997) with a further decline in the subsequent audit (in 1998) to 0.008% (Van Donkersgoed et al., 2001). Since that time there has been only one small scale serological survey (Colwell, 2000) which suggested that these parasites are maintaining populations in spite of consistent use of treatments, in particular the macrocyclic lactone based compounds, to which the grubs are exquisitely sensitive (Vercruysse and Rew, 2002).
0304-4017/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.07.009
298
D.D. Colwell / Veterinary Parasitology 197 (2013) 297–303
The implication of this difference between clinical observations and serological observation is that, while a very high proportion of slaughter cattle are successfully treated for parasite control, there are residual populations of grubs in untreated cattle on farms and ranches that are capable of infesting a portion of the cattle while they are on pasture. That cattle grubs appear to be capable of persisting, even when their populations have become exceedingly small, is evidenced by experimental work of Minar (1980) and Minar and Breev (1981, 1983) and the experience in Great Britain where a well-orchestrated eradication campaign began in 1978 yet was not successful until 1991 (Tarry, 1992; Tarry et al., 1992; Webster et al., 1997). France has also had a nationally coordinated program aimed at eradication (Boulard et al., 2008) but residual populations persist, in part because of immigration from neighbouring countries that have not had such success at control. To determine if cattle grubs in western Canada remain resilient the presence of antibodies in sera were assayed. Sera, collected as part of a cattle parasite survey conducted over three consecutive years, commencing in late 2008, were tested for the presence of anti-Hypoderma antibodies in an ELISA. The survey utilized calves originating from the four western provinces of Canada as they entered a single feedlot, prior to parasiticide application. In addition, samples were obtained from a subsample of calves maintained on the ranch in southeastern Alberta that had been the subject of previous Hypoderma sero-surveillance (Colwell, 2000).
2. Materials and methods 2.1. Animals Calves were selected as they entered a feedlot located south of Calgary, Alberta, Canada (50.3461◦ N, 113.7751◦ W) during October through December of 2008–2010. Groups of calves were identified as to the auction market from which they originated and any loads of calves that had multiple origins were removed from the data set. The auction markets from which the calves were acquired were located in four western Canadian provinces (Fig. 1). Faecal samples for gastrointestinal parasite egg counts and blood samples were collected from randomly selected individuals representing 10% of each lot of calves (Waldner et al., 2004). All calves were from six to eight months of age. Data from these samples are referred to as “Survey”. Cattle entered the study from 23, 37 and 31 auction markets located in British Columbia, Alberta, Saskatchewan and Manitoba in 2008, 2009, 2010, respectively. Distribution and approximate location of the auction markets from which the calves originated are in Fig. 1. The same sites were not always repeated in all years, but 10 sites were represented in all three years. The total number of calves tested was 500, 528 and 399 in those years. In addition, faecal and blood samples were collected from a proportion of calves from the herd of cross-bred cattle maintained at the Agriculture and Agri-Food Canada, Lethbridge Research Centre field station at One Four, Alberta (49.4000◦ N, 110.7016◦ W). All calves were from
Fig. 1. Map of the four western Canadian provinces from which samples for the Survey were drawn. Numbers indicate approximate location of the auction markets from which calves originated.
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Table 1 Sero-prevalence of anti-Hypoderma antibodies in calves sourced from auction markets located across the four western provinces of Canada in three consecutive years. The market locations are as noted on Fig. 1. Sites at which <10 animals were tested have been removed. Year
Location
# tested
# pos
# neg
%pos
Year
Location
# tested
# pos
# neg
%pos
2008 2009 2010 2008 2008 2008 2009 2010 2008 2009 2010 2009 2010 2008 2009 2010 2008 2008 2009 2009 2008 2009
1 1 1 4 5 6 6 7 9 9 9 10 10 12 12 12 13 14 14 15 16 16
45 45 31 39 47 56 12 15 75 18 17 30 10 12 32 29 50 15 18 10 20 32
12 21 13 25 26 45 1 7 56 8 5 6 3 9 8 7 31 10 13 4 14 9
33 24 18 14 21 11 11 8 19 10 12 24 8 3 24 22 19 5 5 6 6 23
26.7 46.7 41.9 64.1 55.3 80.4 8.3 46.7 74.7 44.4 29.4 20.0 30.0 75.0 25.0 24.1 62.0 66.7 72.2 40.0 70.0 28.1
2008 2009 2010 2010 2008 2008 2009 2009 2009 2009 2009 2010 2009 2010 2009 2009 2009 2010 2010 2010 2010 2010
17 17 17 18 19 20 25 31 33 35 36 38 41 42 47 49 50 54 56 57 58 59
41 27 10 16 22 23 72 27 16 10 32 26 30 36 12 14 34 22 78 38 10 22
19 11 1 13 18 10 59 8 4 2 12 12 11 11 1 4 15 6 36 13 10 6
22 16 9 3 4 13 13 19 12 8 20 14 19 25 11 10 19 16 42 25 0 16
46.3 40.7 10.0 81.3 81.8 43.5 81.9 29.6 25.0 20.0 37.5 46.2 36.7 30.6 8.3 28.6 44.1 27.3 46.2 34.2 100.0 27.3
six to seven months of age. Data from these samples are referred to as “Ranch 1”. The numbers of samples from Ranch 1 were 123, 156, and 78 for 2008, 2009 and 2010, respectively. 2.2. ELISA Sera were tested for presence and quantity of antiHypoderma antibodies using an ELISA as described by Colwell et al. (1997). Each sample was tested in duplicate on one plate and each plate had eight wells with a pooled negative serum and a titrated row of wells with a pooled positive serum. Adjusted absorbance values were calculated by subtracting the mean negative value (±2× SD) on each plate from each mean test serum value. 2.3. Weather data Daily average temperature and daily precipitation data were obtained from an Alberta Agriculture and Rural Development website (http://www.agriculture. alberta.ca/acis/alberta-weather-data-viewer.jsp). There are approximately 200 data collection points distributed throughout the province of Alberta with one located on Ranch 1. 2.4. Analysis Sero-prevalence values (i.e. proportion of those tested that were considered positive for anti-Hypoderma antibodies) were compared using the Equality of Proportions Test in SYSTAT v.13. Frequency analysis of the adjusted absorbance data above the established cut-off value was conducted with 14 bins established at 0.070–0.100, 0.101–0.200, 0.201–0.300, 0.301–0.400 and in equal sized bins through an adjusted absorbance of 1.400.
3. Results Sero-prevalence of anti-Hypoderma antibodies is presented for each Survey site in Table 1. The majority of samples originated from Alberta and Saskatchewan which together have approximately 70% of the national beef herd (http://www.statcan.gc.ca/tables-tableaux/sum-som/l01/ cst01/prim50a-eng.htm). Sero-prevalence was variable among sites in all years, ranging from 0.0 to 100.0% although many of the sample sizes at individual sites were small. Those sites that were sampled in all three years exhibited substantial variation in sero-prevalence among the years and consistent trends were not evident. Overall sero-prevalence for the entire Survey population and for Ranch 1 in the three years is presented in Fig. 2. Analysis of the Survey data showed a significant difference in sero-prevalence between 2008 and 2009 (Z = 6.213, df = 1, p > 0.001); 2008 and 2010 (Z = 5.279, df = 1, p > 0.001); but not for 2009 and 2010 (Z = −0.545, df = 1, ns). Similarly, analysis of the data from Ranch 1 showed a significant difference in sero-prevalence between 2008 and 2009 (Z = 5.373, df = 1, p > 0.001); 2009 and 2010 (Z = 3.741, df = 1, p > 0.001); but the difference in sero-prevalence between 2008 and 2010 was not significant (Z = −0.979, df = 1, ns). Differences in sero-prevalence between the Survey data and the Ranch 1 were significant for two of the three years, 2008 and 2009 (2008 Z = −2.789, df = 1, p > 0.005; 2009 Z = 8.514, df = 1, p > 0.001). The sero- prevalence was not different between the Survey and Ranch 1 in 2010 (Z = 1.757, df = 1, ns). The cumulative relative frequency of adjusted absorbance values in the 14 bins for the Survey and for Ranch 1 over the three years is presented in Fig. 3. The Survey data analysis (Fig. 3a) shows that 82.5%, 84.2% and 93.1% of the adjusted absorbance values are in the first three bins in 2008, 2009 and 2010 respectively. The analysis of Ranch 1 (Fig. 3b) data show that a lower
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Fig. 2. (a) Sero-prevalence of Hypoderma spp. in calves entering an Alberta feedlot in three consecutive years (2008–2010). The Survey calves were sourced from locations throughout western Canada. (b) Sero-prevalence of Hypoderma spp. in calves from the Lethbridge Research Centre ranch at One Four Alberta in three consecutive years (2008–2010).
proportion of values are in the first three bins in 2008 (70.0%) and 2010 (69.2%), but in 2009 that proportion was extremely low at 13.0% Average daily temperature and total daily precipitation data collected at Ranch 1 between 1 March and 30 May each year are presented in Fig. 4. In both 2008 and 2010 there were significant rainfall events toward the end of May coupled with relatively low temperatures. In contrast, 2009 was a comparatively dry year with slightly elevated temperatures during the same period. 4. Discussion The variation in sero-prevalence among years in the Survey populations, where multiple years of data were obtained, was likely a result of the calves not being sourced from the same herds, but only the same auction market.
As a consequence individual ranch management likely was variable and as a result would have affected the outcome. The increase in sero-prevalence on Ranch 1 in 2009 was substantial and unanticipated because there were no management changes to which this might be attributed. This result then suggested that environmental parameters could have been a factor. Further, analysis of the frequency of adjusted absorbance values indicated that a lower proportion of the values were in the low range in 2009 as compared to 2008 and 2010. This is suggestive of an increase in heavier infections. Although there is no direct evidence, the increased sero-prevalence and decreased frequency of low adjusted absorbance values in 2009 could be the result of the combined effects of low precipitation and warm temperatures at the end of May. Table 2 presents the total May precipitation recorded in each year with the associated sero-prevalence values showing that
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Fig. 3. (a) Cumulative relative frequency distribution of Hypoderma spp., ELISA results from calves entering an Alberta feedlot in three consecutive years. The calves were sourced from locations throughout western Canada. The ELISA results are binned into adjusted absorbance categories above the cut-off value of 0.07. (b) Cumulative relative frequency distribution of Hypoderma spp., ELISA results from calves from the Lethbridge Research Centre ranch at One Four Alberta. (c) Cumulative relative frequency distribution of Hypoderma spp., ELISA results from a previously published study (Colwell, 2000). The data are from two separate herds (herd 1 and herd 2) on Ranch 1 with a long-standing cattle grub treatment program and a second ranch (Ranch 2) where no routine treatment for grubs was practiced and infection prevalence was high (>80%).
precipitation differences were large. These conditions could have allowed increased survival of pupal and adult stages whereas the high precipitation and cool temperatures in 2008 and 2010 would likely have been detrimental to those free-living stages (Nilssen, 2006).
The weather conditions, in particular the precipitation events, at the end of May in 2008 and 2010 could have contributed to pupal or adult fly death being higher in those years as compared to 2009 (Nilssen, 1997).
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Fig. 4. (a) Daily mean temperature (◦ C) recorded at Ranch 1 between 1 March and 30 May for each of three consecutive years (2008–2010). (b) Total daily precipitation (mm) recorded at Ranch 1 between 1 March and 30 May for each of three consecutive years (2008–2010). Table 2 Total May precipitation and Ranch 1 sero-prevalence over three years 2008–2010.
May precipitation (mm) Ranch 1 prevalence
2008
2009
2010
76.3 31.7
21.0 64.1
68.8 38.5
Among year variation in overall sero-prevalence in the Survey samples suggests that across the broad region there may be environmental variables that are affecting survival of pupal and adult stages which would result in more or less infections taking place. However, unlike on Ranch 1, trends in sero-prevalence would be obscured in the larger data set because of variability in environmental variables across the entire region from which the calves originated. Comparison of Ranch 1 sero-prevalence in the current study with the previous study conducted at the same site (Colwell, 2000) showed that the sero-prevalence remained within the range reported for the period 1992–1999. Data from ELISAs conducted in 1992 were available for reexamination and frequency analysis showed a different distribution of adjusted absorbance values than in the current study. Data from separate herds maintained at the
time on Ranch 1 were compared with what was referred to as ‘Ranch 2 (Colwell, 2000) (Fig. 3c). Sero-prevalence was extremely high on Ranch 2 and the distribution of adjusted absorbance values was broad with only 10.0% of values in the lowest three bins. The Ranch 1 herds, managed and pastured separately had 35.0% and 40.0% of the values in the lowest three bins. Ranch 2 did not have a treatment program for grub control and palpation confirmed infestation levels were high (Colwell, 2000). This data supports the contention that distribution of adjusted absorbance values is reflective of increased infestation intensity. There is no correlation between level of antibodies and the intensity of cattle grub infection (Boulard, 1985; Colwell and Baron, 1990). However, no surveys have examined the distribution of ELISA results relative to known infestation levels. Examining the past survey of cattle sero-prevalence there was a strong suggestion that where sero-prevalence was high (and grub intensity confirmed); e.g., Ranch 2, 1992, there was a broader distribution of adjusted absorbance values with a much lower proportion at the lower antibody levels. From the data presented in the current study there is a relationship between seroprevalence and distribution of ELISA values which may reflect low level infestations.
D.D. Colwell / Veterinary Parasitology 197 (2013) 297–303
The UK experience at the end of their eradication campaign suggests that sero-surveillance is a good alternative for detection of low level, persistent cattle grub populations (Webster et al., 1997). Similar observations on the utility of sero-surveillance have been made following control campaigns in China (Guan et al., 2005). A recent study demonstrates the persistence of clinical cattle grub infection in two locations in USA (Rehbein et al., 2013). The number of larvae manually detected was low compared to levels reported from the late 1990s in Canada (Colwell, 2000) and would likely have escaped detection had the hosts not been part of a parasiticide efficacy study. ELISA, employing either sera or milk, for determination of Hypoderma prevalence is routine (Otranto et al., 2001; Haine et al., 2004; Guan et al., 2005; Otranto et al., 2005; Simsek et al., 2008; Balkaya et al., 2010) and is often used as a surveillance tool in place of palpation or examination of carcasses at slaughter. However, currently available AbELISAs over-estimate the proportion of cattle that exhibit clinical hypodermosis as shown in Rehbein et al. (2013). Serology however provides a sensitive indication of how many individuals actually are exposed to infestation as a number of animals will harbour larvae that die in the host prior to reaching the back where they are clinically detectable. This study indicates that cattle grubs remain well entrenched in western Canada despite the extensive use of macrocyclic lactone parasiticides. Use of these products has increased with the appearance of generic products and the subsequent decline in cost. The study also provides a reminder that while these parasites are no longer a cost burden to producers they lurk in the background and could threaten a return to clinical relevance should treatment programs move away from macrocyclic lactone products. There is currently no documented evidence of a reduction in efficacy of these products for control of nematodes in western Canada, but should the evidence become clear and producers react by changing anthelminthic compounds not effective against arthropods there is potential for a resurgence of cattle grub populations. Acknowledgements I would like to thank the staff and management of Feedlot Health Management Services for their help organising and supervising sample collections. The help of staff at the Lethbridge Research Centre ranch at One Four Alberta is also greatly appreciated. I also thank Dawn Gray for her excellent technical help. This is contribution #38713016 from the Lethbridge Research Centre. References Balkaya, I., Simsek, S., Saki, C.E., 2010. A serological and molecular survey of cattle hypodermosis in east-Turkey. Vet. Parasitol. 173, 287–291. Boulard, C., 1985. Avantages de l’immunodiagnostic de l’hypodermose bovine établi par hémagglutination passive et par ELISA à partir du sérum et du lactosérum, sur la numération des varons. Ann. Rech. Vét. 16, 335–343.
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Boulard, C., Argenté, M., Argenté, G., Languille, J., Paget, L., Petit, E., 2008. A successful, sustainable and low cost control-programme for bovine hypodermosis in France. Vet. Parasitol. 158, 1–10. Colwell, D.D., 2000. Persistence of cattle grubs (Diptera: Oestridae) on a Canadian ranch with long-term, continuous therapeutic control. Vet. Parasitol. 94, 127–132. Colwell, D.D., Baron, R.W., 1990. Early detection of cattle grub infestation (Hypoderma lineatum DeVill. and H. bovis L.) (Diptera: Oestridae) using ELISA. Med. Vet. Entomol. 4, 35–42. Colwell, D.D., Baron, R.W., Lysyk, T.J., 1997. Influence of parasiticide treatment on kinetics of antigen specific antibody responses in cattle infested with Hypoderma lineatum (Diptera: Oestridae). Vet. Parasitol. 68, 175–186. Guan, G., Luo, J., Ma, M., Yang, D., Wang, Y., Gao, J., Sun, H., Liu, Z., Liu, A., Dang, Z., Boulard, C., Yin, H., 2005. Sero-epidemiological surveillance of hypodermosis in yaks and cattle in north China by ELISA. Vet. Parasitol. 129, 133–137. Haine, D., Boelaert, F., Pfeiffer, D.U., Saegerman, C., Lonneux, J.F., Losson, B., Mintiens, K., 2004. Herd-level seroprevalence and risk-mapping of bovine hypodermosis in Belgian cattle herds. Prev. Vet. Med. 65, 93–104. Minar, J.K., Breev, K.A., 1981. Investigations on low and basic populations of the warble fly Hypoderma bovis (Hypodermatidae) and its significance for the persistence of hypodermatosis. Parazitologia 15, 163–167. Minar, J., 1980. Studies of the fundamental populations of the warble fly Hypoderma bovis (De Geer). Acta Univ. Carol., Biol. 12, 353–359. Minar, J., Breev, K.A., 1983. Studies on the low and fundamental populations of the warble fly Hypoderma bovis. Folia Parasitol. 29, 351–360. Nilssen, A.C., 2006. Pupal biology and metamorphosis behaviour. In: Colwell, D.D., Hall, M., Scholl, P.J. (Eds.), The Oestrid Flies: Biology, Host–Parasite Relationships, Impact and Management. CAB International, pp. 124–139. Nilssen, A.C., 1997. Effect of temperature on pupal development and eclosion dates in the reindeer oestrids Hypoderma tarandi and Cephenemyia trompe (Diptera: Oestridae). Environ. Entomol. 26, 296–306. Otranto, D., Testini, G., Sottili, R., Capelli, G., Puccini, V., 2001. Screening of commercial milk samples using ELISA for immuno-epidemiological evidence of infection by the cattle grub (Diptera: Oestridae). Vet. Parasitol. 99, 241–248. Otranto, D., Zalla, P., Testini, G., Zanaj, S., 2005. Cattle grub infestation by Hypoderma sp. in Albania and risks for European countries. Vet. Parasitol. 128, 157–162. Rehbein, S., Holste, J.E., Smith, L.L., Lloyd, J.L., 2013. The efficacy of eprinomectin extended-release injection against Hypoderma spp. (Diptera: Oestridae) in cattle. Vet. Parasitol. 192, 353–358. Simsek, S., Utuk, A.E., Koroglu, E., Dumanli, N., 2008. Seroprevalence of hypodermosis in cattle in some provinces of Turkey. Res. Vet. Sci. 84, 246–249. Tarry, D.W.,1992. The effects of UK eradication measures on residual warble fly populations. In: COST 811. Improvements in the Control Methods for Warble-Fly in Cattle and Goats. Office for Official Publications of the European Commission, Brussels, pp. 21–27. Tarry, D.W., Sinclair, I.J., Wassal, D.A., 1992. Progress in the British hypodermosis eradication programme: the role of serological surveillance. Vet. Rec. 131, 310–312. Van Donkersgoed, J., Jewison, G., Mann, M., Cherry, B., Altwasser, B., Lower, R., Wiggins, K., Dejonge, R., Thorlakson, B., Moss, E., Mills, C., Grogan, H., 1997. Canadian beef quality audit. Can. Vet. J. 38, 217–225. Van Donkersgoed, J., Jewison, G., Bygrove, S., Gillis, K., Malchow, D., McLeod, G., 2001. Canadian beef quality audit 1998–1999. Can. Vet. J. 42, 121–126. Vercruysse, J., Rew, R., 2002. General efficacy of the macrocyclic lactones to control parasites of cattle. In: Vercruysse, J., Rew, R. (Eds.), Macrocyclic Lactones in Antiparasitic Therapy. CABI Publishing, Oxon, UK, pp. 185–232. Waldner, C., Wildman, B.K., Hill, B.W., Fenton, R.K., Pittman, T.J., Schunicht, O.C., Jim, G.K., Guichon, P.T., Booker, C.W., 2004. Determination of the seroprevalence of Neospora caninum in feedlot steers in Alberta. Can. Vet. J. 45, 218–224. Webster, K.A., Dawson, C., Flowers, M., Richards, M.S., 1997. Serological prevalence of Hypoderma species in cattle in Great Britain (1995/96) and the relative value of serological surveillance over clinical observation. Vet. Rec. 141, 261–263.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Out of sight but not gone: Sero-surveillance for cattle grubs, Hypoderma spp., in western Canada between 2008 and 2010 Douglas D. Colwell ∗ Agriculture and Agri-Food Canada, Lethbridge Research Centre, 5403 1st Ave. S., Lethbridge, AB, Canada
a r t i c l e
i n f o
Article history: Received 24 May 2013 Received in revised form 3 July 2013 Accepted 5 July 2013 Keywords: ELISA Sero-surveillance Warbles Western Canada
a b s t r a c t Despite the lack of evidence of clinically infected cattle in western Canada since the late 1990s there is persistent evidence from serological sampling that warble fly infections remain endemic. As part of a general survey of cattle parasites conducted on calves entering a feedlot in southern Alberta sera were tested for the presence of anti-Hypoderma antibodies using an established ELISA. Calves for the study were drawn from auction markets in Manitoba, Saskatchewan, Alberta and British Columbia and serum samples were collected, according to a randomization scheme, as the calves entered a single feedlot in October through December (Survey) of three consecutive years (2008–2010). In addition, a subsample of calves originating on a ranch in southeastern Alberta (Ranch 1) that had been the subject of previous surveillance was tested. Sero-prevalence of anti-Hypoderma antibodies of the Survey calves ranged from 0.0% to 100.0% depending on origin and overall varied among the three years with the sero-prevalence being 45.6%, 26.9%, 28.5% in 2008, 2009 and 2010 respectively. Sero-prevalence in Ranch 1 calves varied among years, with one year being much higher and was different from overall Survey sero-prevalence in two of three years (31.7%, 64.1%, 38.5% in 2008, 2009, 2010 respectively). In the Survey population there was a trend to lower sero-prevalence over the three years while on Ranch 1 there was no clear trend. In the samples with ELISA adjusted absorbance values above the positive cut-off an examination of the cumulative relative frequency showed that in all years approximately 90% of the samples were in the lower 3 of 14 bins in both populations and in two of three years in Ranch 1 population. The clustering of the positive ELISA data at the lower end of the range is suggestive of low level grub infestations. The increased frequency of high positive ELISA results from Ranch 1 in 2009 potentially indicate an ‘outbreak year’ in which pupal and adult fly survival was higher than for other years of this study resulting in more infested animals with higher burdens. Examination of precipitation and temperature profiles from the subject ranch support this suggestion. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Long an important economic burden on the North American cattle industry the cattle grubs (Hypoderma spp.) have become clinically almost non-existent, particularly in western Canada, since the advent of macrocyclic lactone, broad-spectrum parasiticides. The Canadian
∗ Tel.: +1 4033172254. E-mail address: [email protected]
Beef Quality Audit reported a scant 0.1% of carcasses with evidence of grubs in 1996 (Van Donkersgoed et al., 1997) with a further decline in the subsequent audit (in 1998) to 0.008% (Van Donkersgoed et al., 2001). Since that time there has been only one small scale serological survey (Colwell, 2000) which suggested that these parasites are maintaining populations in spite of consistent use of treatments, in particular the macrocyclic lactone based compounds, to which the grubs are exquisitely sensitive (Vercruysse and Rew, 2002).
0304-4017/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.07.009
298
D.D. Colwell / Veterinary Parasitology 197 (2013) 297–303
The implication of this difference between clinical observations and serological observation is that, while a very high proportion of slaughter cattle are successfully treated for parasite control, there are residual populations of grubs in untreated cattle on farms and ranches that are capable of infesting a portion of the cattle while they are on pasture. That cattle grubs appear to be capable of persisting, even when their populations have become exceedingly small, is evidenced by experimental work of Minar (1980) and Minar and Breev (1981, 1983) and the experience in Great Britain where a well-orchestrated eradication campaign began in 1978 yet was not successful until 1991 (Tarry, 1992; Tarry et al., 1992; Webster et al., 1997). France has also had a nationally coordinated program aimed at eradication (Boulard et al., 2008) but residual populations persist, in part because of immigration from neighbouring countries that have not had such success at control. To determine if cattle grubs in western Canada remain resilient the presence of antibodies in sera were assayed. Sera, collected as part of a cattle parasite survey conducted over three consecutive years, commencing in late 2008, were tested for the presence of anti-Hypoderma antibodies in an ELISA. The survey utilized calves originating from the four western provinces of Canada as they entered a single feedlot, prior to parasiticide application. In addition, samples were obtained from a subsample of calves maintained on the ranch in southeastern Alberta that had been the subject of previous Hypoderma sero-surveillance (Colwell, 2000).
2. Materials and methods 2.1. Animals Calves were selected as they entered a feedlot located south of Calgary, Alberta, Canada (50.3461◦ N, 113.7751◦ W) during October through December of 2008–2010. Groups of calves were identified as to the auction market from which they originated and any loads of calves that had multiple origins were removed from the data set. The auction markets from which the calves were acquired were located in four western Canadian provinces (Fig. 1). Faecal samples for gastrointestinal parasite egg counts and blood samples were collected from randomly selected individuals representing 10% of each lot of calves (Waldner et al., 2004). All calves were from six to eight months of age. Data from these samples are referred to as “Survey”. Cattle entered the study from 23, 37 and 31 auction markets located in British Columbia, Alberta, Saskatchewan and Manitoba in 2008, 2009, 2010, respectively. Distribution and approximate location of the auction markets from which the calves originated are in Fig. 1. The same sites were not always repeated in all years, but 10 sites were represented in all three years. The total number of calves tested was 500, 528 and 399 in those years. In addition, faecal and blood samples were collected from a proportion of calves from the herd of cross-bred cattle maintained at the Agriculture and Agri-Food Canada, Lethbridge Research Centre field station at One Four, Alberta (49.4000◦ N, 110.7016◦ W). All calves were from
Fig. 1. Map of the four western Canadian provinces from which samples for the Survey were drawn. Numbers indicate approximate location of the auction markets from which calves originated.
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Table 1 Sero-prevalence of anti-Hypoderma antibodies in calves sourced from auction markets located across the four western provinces of Canada in three consecutive years. The market locations are as noted on Fig. 1. Sites at which <10 animals were tested have been removed. Year
Location
# tested
# pos
# neg
%pos
Year
Location
# tested
# pos
# neg
%pos
2008 2009 2010 2008 2008 2008 2009 2010 2008 2009 2010 2009 2010 2008 2009 2010 2008 2008 2009 2009 2008 2009
1 1 1 4 5 6 6 7 9 9 9 10 10 12 12 12 13 14 14 15 16 16
45 45 31 39 47 56 12 15 75 18 17 30 10 12 32 29 50 15 18 10 20 32
12 21 13 25 26 45 1 7 56 8 5 6 3 9 8 7 31 10 13 4 14 9
33 24 18 14 21 11 11 8 19 10 12 24 8 3 24 22 19 5 5 6 6 23
26.7 46.7 41.9 64.1 55.3 80.4 8.3 46.7 74.7 44.4 29.4 20.0 30.0 75.0 25.0 24.1 62.0 66.7 72.2 40.0 70.0 28.1
2008 2009 2010 2010 2008 2008 2009 2009 2009 2009 2009 2010 2009 2010 2009 2009 2009 2010 2010 2010 2010 2010
17 17 17 18 19 20 25 31 33 35 36 38 41 42 47 49 50 54 56 57 58 59
41 27 10 16 22 23 72 27 16 10 32 26 30 36 12 14 34 22 78 38 10 22
19 11 1 13 18 10 59 8 4 2 12 12 11 11 1 4 15 6 36 13 10 6
22 16 9 3 4 13 13 19 12 8 20 14 19 25 11 10 19 16 42 25 0 16
46.3 40.7 10.0 81.3 81.8 43.5 81.9 29.6 25.0 20.0 37.5 46.2 36.7 30.6 8.3 28.6 44.1 27.3 46.2 34.2 100.0 27.3
six to seven months of age. Data from these samples are referred to as “Ranch 1”. The numbers of samples from Ranch 1 were 123, 156, and 78 for 2008, 2009 and 2010, respectively. 2.2. ELISA Sera were tested for presence and quantity of antiHypoderma antibodies using an ELISA as described by Colwell et al. (1997). Each sample was tested in duplicate on one plate and each plate had eight wells with a pooled negative serum and a titrated row of wells with a pooled positive serum. Adjusted absorbance values were calculated by subtracting the mean negative value (±2× SD) on each plate from each mean test serum value. 2.3. Weather data Daily average temperature and daily precipitation data were obtained from an Alberta Agriculture and Rural Development website (http://www.agriculture. alberta.ca/acis/alberta-weather-data-viewer.jsp). There are approximately 200 data collection points distributed throughout the province of Alberta with one located on Ranch 1. 2.4. Analysis Sero-prevalence values (i.e. proportion of those tested that were considered positive for anti-Hypoderma antibodies) were compared using the Equality of Proportions Test in SYSTAT v.13. Frequency analysis of the adjusted absorbance data above the established cut-off value was conducted with 14 bins established at 0.070–0.100, 0.101–0.200, 0.201–0.300, 0.301–0.400 and in equal sized bins through an adjusted absorbance of 1.400.
3. Results Sero-prevalence of anti-Hypoderma antibodies is presented for each Survey site in Table 1. The majority of samples originated from Alberta and Saskatchewan which together have approximately 70% of the national beef herd (http://www.statcan.gc.ca/tables-tableaux/sum-som/l01/ cst01/prim50a-eng.htm). Sero-prevalence was variable among sites in all years, ranging from 0.0 to 100.0% although many of the sample sizes at individual sites were small. Those sites that were sampled in all three years exhibited substantial variation in sero-prevalence among the years and consistent trends were not evident. Overall sero-prevalence for the entire Survey population and for Ranch 1 in the three years is presented in Fig. 2. Analysis of the Survey data showed a significant difference in sero-prevalence between 2008 and 2009 (Z = 6.213, df = 1, p > 0.001); 2008 and 2010 (Z = 5.279, df = 1, p > 0.001); but not for 2009 and 2010 (Z = −0.545, df = 1, ns). Similarly, analysis of the data from Ranch 1 showed a significant difference in sero-prevalence between 2008 and 2009 (Z = 5.373, df = 1, p > 0.001); 2009 and 2010 (Z = 3.741, df = 1, p > 0.001); but the difference in sero-prevalence between 2008 and 2010 was not significant (Z = −0.979, df = 1, ns). Differences in sero-prevalence between the Survey data and the Ranch 1 were significant for two of the three years, 2008 and 2009 (2008 Z = −2.789, df = 1, p > 0.005; 2009 Z = 8.514, df = 1, p > 0.001). The sero- prevalence was not different between the Survey and Ranch 1 in 2010 (Z = 1.757, df = 1, ns). The cumulative relative frequency of adjusted absorbance values in the 14 bins for the Survey and for Ranch 1 over the three years is presented in Fig. 3. The Survey data analysis (Fig. 3a) shows that 82.5%, 84.2% and 93.1% of the adjusted absorbance values are in the first three bins in 2008, 2009 and 2010 respectively. The analysis of Ranch 1 (Fig. 3b) data show that a lower
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Fig. 2. (a) Sero-prevalence of Hypoderma spp. in calves entering an Alberta feedlot in three consecutive years (2008–2010). The Survey calves were sourced from locations throughout western Canada. (b) Sero-prevalence of Hypoderma spp. in calves from the Lethbridge Research Centre ranch at One Four Alberta in three consecutive years (2008–2010).
proportion of values are in the first three bins in 2008 (70.0%) and 2010 (69.2%), but in 2009 that proportion was extremely low at 13.0% Average daily temperature and total daily precipitation data collected at Ranch 1 between 1 March and 30 May each year are presented in Fig. 4. In both 2008 and 2010 there were significant rainfall events toward the end of May coupled with relatively low temperatures. In contrast, 2009 was a comparatively dry year with slightly elevated temperatures during the same period. 4. Discussion The variation in sero-prevalence among years in the Survey populations, where multiple years of data were obtained, was likely a result of the calves not being sourced from the same herds, but only the same auction market.
As a consequence individual ranch management likely was variable and as a result would have affected the outcome. The increase in sero-prevalence on Ranch 1 in 2009 was substantial and unanticipated because there were no management changes to which this might be attributed. This result then suggested that environmental parameters could have been a factor. Further, analysis of the frequency of adjusted absorbance values indicated that a lower proportion of the values were in the low range in 2009 as compared to 2008 and 2010. This is suggestive of an increase in heavier infections. Although there is no direct evidence, the increased sero-prevalence and decreased frequency of low adjusted absorbance values in 2009 could be the result of the combined effects of low precipitation and warm temperatures at the end of May. Table 2 presents the total May precipitation recorded in each year with the associated sero-prevalence values showing that
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Fig. 3. (a) Cumulative relative frequency distribution of Hypoderma spp., ELISA results from calves entering an Alberta feedlot in three consecutive years. The calves were sourced from locations throughout western Canada. The ELISA results are binned into adjusted absorbance categories above the cut-off value of 0.07. (b) Cumulative relative frequency distribution of Hypoderma spp., ELISA results from calves from the Lethbridge Research Centre ranch at One Four Alberta. (c) Cumulative relative frequency distribution of Hypoderma spp., ELISA results from a previously published study (Colwell, 2000). The data are from two separate herds (herd 1 and herd 2) on Ranch 1 with a long-standing cattle grub treatment program and a second ranch (Ranch 2) where no routine treatment for grubs was practiced and infection prevalence was high (>80%).
precipitation differences were large. These conditions could have allowed increased survival of pupal and adult stages whereas the high precipitation and cool temperatures in 2008 and 2010 would likely have been detrimental to those free-living stages (Nilssen, 2006).
The weather conditions, in particular the precipitation events, at the end of May in 2008 and 2010 could have contributed to pupal or adult fly death being higher in those years as compared to 2009 (Nilssen, 1997).
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Fig. 4. (a) Daily mean temperature (◦ C) recorded at Ranch 1 between 1 March and 30 May for each of three consecutive years (2008–2010). (b) Total daily precipitation (mm) recorded at Ranch 1 between 1 March and 30 May for each of three consecutive years (2008–2010). Table 2 Total May precipitation and Ranch 1 sero-prevalence over three years 2008–2010.
May precipitation (mm) Ranch 1 prevalence
2008
2009
2010
76.3 31.7
21.0 64.1
68.8 38.5
Among year variation in overall sero-prevalence in the Survey samples suggests that across the broad region there may be environmental variables that are affecting survival of pupal and adult stages which would result in more or less infections taking place. However, unlike on Ranch 1, trends in sero-prevalence would be obscured in the larger data set because of variability in environmental variables across the entire region from which the calves originated. Comparison of Ranch 1 sero-prevalence in the current study with the previous study conducted at the same site (Colwell, 2000) showed that the sero-prevalence remained within the range reported for the period 1992–1999. Data from ELISAs conducted in 1992 were available for reexamination and frequency analysis showed a different distribution of adjusted absorbance values than in the current study. Data from separate herds maintained at the
time on Ranch 1 were compared with what was referred to as ‘Ranch 2 (Colwell, 2000) (Fig. 3c). Sero-prevalence was extremely high on Ranch 2 and the distribution of adjusted absorbance values was broad with only 10.0% of values in the lowest three bins. The Ranch 1 herds, managed and pastured separately had 35.0% and 40.0% of the values in the lowest three bins. Ranch 2 did not have a treatment program for grub control and palpation confirmed infestation levels were high (Colwell, 2000). This data supports the contention that distribution of adjusted absorbance values is reflective of increased infestation intensity. There is no correlation between level of antibodies and the intensity of cattle grub infection (Boulard, 1985; Colwell and Baron, 1990). However, no surveys have examined the distribution of ELISA results relative to known infestation levels. Examining the past survey of cattle sero-prevalence there was a strong suggestion that where sero-prevalence was high (and grub intensity confirmed); e.g., Ranch 2, 1992, there was a broader distribution of adjusted absorbance values with a much lower proportion at the lower antibody levels. From the data presented in the current study there is a relationship between seroprevalence and distribution of ELISA values which may reflect low level infestations.
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The UK experience at the end of their eradication campaign suggests that sero-surveillance is a good alternative for detection of low level, persistent cattle grub populations (Webster et al., 1997). Similar observations on the utility of sero-surveillance have been made following control campaigns in China (Guan et al., 2005). A recent study demonstrates the persistence of clinical cattle grub infection in two locations in USA (Rehbein et al., 2013). The number of larvae manually detected was low compared to levels reported from the late 1990s in Canada (Colwell, 2000) and would likely have escaped detection had the hosts not been part of a parasiticide efficacy study. ELISA, employing either sera or milk, for determination of Hypoderma prevalence is routine (Otranto et al., 2001; Haine et al., 2004; Guan et al., 2005; Otranto et al., 2005; Simsek et al., 2008; Balkaya et al., 2010) and is often used as a surveillance tool in place of palpation or examination of carcasses at slaughter. However, currently available AbELISAs over-estimate the proportion of cattle that exhibit clinical hypodermosis as shown in Rehbein et al. (2013). Serology however provides a sensitive indication of how many individuals actually are exposed to infestation as a number of animals will harbour larvae that die in the host prior to reaching the back where they are clinically detectable. This study indicates that cattle grubs remain well entrenched in western Canada despite the extensive use of macrocyclic lactone parasiticides. Use of these products has increased with the appearance of generic products and the subsequent decline in cost. The study also provides a reminder that while these parasites are no longer a cost burden to producers they lurk in the background and could threaten a return to clinical relevance should treatment programs move away from macrocyclic lactone products. There is currently no documented evidence of a reduction in efficacy of these products for control of nematodes in western Canada, but should the evidence become clear and producers react by changing anthelminthic compounds not effective against arthropods there is potential for a resurgence of cattle grub populations. Acknowledgements I would like to thank the staff and management of Feedlot Health Management Services for their help organising and supervising sample collections. The help of staff at the Lethbridge Research Centre ranch at One Four Alberta is also greatly appreciated. I also thank Dawn Gray for her excellent technical help. This is contribution #38713016 from the Lethbridge Research Centre. References Balkaya, I., Simsek, S., Saki, C.E., 2010. A serological and molecular survey of cattle hypodermosis in east-Turkey. Vet. Parasitol. 173, 287–291. Boulard, C., 1985. Avantages de l’immunodiagnostic de l’hypodermose bovine établi par hémagglutination passive et par ELISA à partir du sérum et du lactosérum, sur la numération des varons. Ann. Rech. Vét. 16, 335–343.
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