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Gastrointestinal nematodes in grazing dairy cattle from small and medium-sized farms in southern Poland
Veterinary Parasitology 198 (2013) 250–253
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Gastrointestinal nematodes in grazing dairy cattle from small and medium-sized farms in southern Poland J. Piekarska a,∗ , K. Płoneczka-Janeczko b , M. Kantyka a , M. Kuczaj c , M. Gorczykowski a , K. Janeczko d a Department of Internal Medicine and Clinic of Diseases of Horses, Dogs and Cats, Division of Parasitology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Norwida 31, 50-375 Wroclaw, Poland b Department of Epizootiology with Clinic of Birds and Exotic Animals, Wroclaw University of Environmental and Life Sciences, Pl. Grunwaldzki 45, 50-366 Wroclaw, Poland c Institute of Animal Breeding, Faculty of Biology and Animal Breeding, Wroclaw University of Environmental and Life Sciences, Chełmo´ nskiego 38C, Wroclaw, Poland d Department of Reproduction and Clinic for Farm Animals, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Pl. Grunwaldzki 49, 50-366 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Received 9 May 2013 Received in revised form 25 July 2013 Accepted 26 July 2013 Keywords: Gastrointestinal nematode eggs Dairy cattle BTM Multiplex PCR O. ostertagi
a b s t r a c t This study aimed to estimate the prevalence of gastrointestinal nematodes and the intensity of infection in grazing dairy cattle from small and medium-sized farms in southern Poland. The level of antibodies against Ostertagia ostertagi in the bulk tank milk (BTM) from the animals was also assessed. Rectal fecal samples collected from 361 cows on 20 farms were examined using Willis-Schlaaf flotation and the McMaster method. BTM samples were tested for the presence of O. ostertagi antibodies using ELISA. Multiplex PCR was used to identify the third-stage larvae (L3) of gastrointestinal nematodes derived from the culture of pooled fecal samples from sampled farms. Gastrointestinal nematode eggs were found in the samples from 18 of the 20 herds with a prevalence range from 20.4 to 94.5%. The average number of eggs excreted in the feces of the herds was 200 eggs per gram (EPG). Antibodies to O. ostertagi were found in 20 of the examined herds (100%), of which 6 had optical density ratios (ODR) greater than 0.5. PCR results showed the presence of three nematode species: Ostertagia ostertagi, Cooperia oncophora and Oesophagostomum radiatum. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Gastrointestinal (GI) nematodes that cause substantial losses in dairy cattle breeding include members of the Trichostrongylidae (Trichostrongylus, Haemonchus, Ostertagia, Cooperia) and Molineidae (Nematodirus) families and Oesophagostomum species. Previous research confirmed that the negative effects of infection in dairy cows with subclinical symptoms included a decrease in milk yield of
∗ Corresponding author. Tel.: +48 713205420. E-mail address: [email protected] (J. Piekarska). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.07.039
up to and even over 11% (Gross et al., 1999; Charlier et al., 2009). The interpretation of fecal examination results has certain limitations. In order to distinguish gastrointestinal parasite species, it is necessary to conduct larval culture, perform morphometric analysis of the third larvae stage, or use PCR. The Ostertagia ostertagi antibody levels in bulk tank milk samples (BTM) provide an important indicator of potential production losses in dairy herds. Potential loss of milk yield is already observed at antibody concentrations of 0.5 ODR (Charlier et al., 2007). The greatest possibilities of infection with GI nematodes in Poland fall in late May and June and late August and
J. Piekarska et al. / Veterinary Parasitology 198 (2013) 250–253
September, as those are the periods with the most favorable weather conditions for infection. Small and medium-sized dairy farms have a significant share in the total milk production in Poland, but their parasitological status is unknown, so each examination is very important to fill this gap in our knowledge. Testing also has a huge impact on farmers’ awareness. For them, the investment into deworming seems high, especially with preparations that have no withdrawal period, and there are no data for veterinarians to support the recommended prevention programs. The main objective of this study was to investigate the prevalence and intensity of GI nematode invasion in dairy herds from small and medium-sized farms in southern Poland, to determine the O. ostertagi-specific antibody levels in BTM samples from the examined herds to estimate potential losses in production, and to identify the GI nematode species occurring in the studied herds. 2. Materials and methods 2.1. Study area and farms Fecal examinations were conducted in October and November 2011, in 20 grazing dairy cattle herds: 12 from Lower Silesia and 8 from Lesser Poland (Galicia). The herd size ranged from 10 to 200 animals and their average age was about 5.5 years. The mean number of lactation periods across all the herds was 3.51 ± 0.124. The material consisted of 361 rectal fecal samples from lowland black-and-white and red Polish cows. The percentage of examined fecal samples from a given herd was between 19 and 73, depending on herd size. The grazing period had started at the end of April and ended in November 2011. The last anti-parasitic compounds had been given to cattle on 2 of the 20 farms 6 months prior to the study. The products used as the last treatment included ivermectin (Farm No. 19) and oxfendazole (Farm No. 20) (Table 1). The cattle on the other 18 farms had never been treated before sampling. The herds originated in areas characterized by moderate climates with maritime and continental features. The annual rainfall ranges from 550 to 700 mm, and July is the wettest month. The average temperature in Lower Silesia is −0.6 ◦ C (30.92 ◦ F) in January and 17.8 ◦ C (64.04 ◦ F) in July. The warmest month in Lesser Poland is July, with an average temperature of 14.5 ◦ C (58.1 ◦ F). January is the coldest month, with an average temperature of −9.4 ◦ C (15.08 ◦ F). The snow cover lasts 50 days on average. 2.2. Parasitological techniques (fecal egg counts, larval culture) The fecal samples were examined using Willis-Schlaaf flotation with saturated NaCl solution (Pilarczyk et al., 2009). A modified version of the McMaster technique using saturated NaCl solution as the flotation fluid and a detection level of 50 gastrointestinal nematode eggs per gram of feces was performed on all individual fecal samples (Vadlejch et al., 2011). Pooled coprocultures obtained from the positive fecal samples from each herd were prepared and incubated at 24 ◦ C in moist conditions for 14 days to obtain
251
the infective third stage larvae (L3) that were harvested using the Baermann technique (Pilarczyk et al., 2009). 2.3. Molecular identification of gastrointestinal nematodes The DNA of third-stage infective GI larvae collected from coprocultures was isolated using the Genomic Set AX ´ Genotyping was conducted STOOL Mini Kit (DNA-Gdansk). using multiplex PCR. The parameters of the reaction and peculiar starters were planned based on Zarlenga et al. (2001). Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats and from the 3 -end of the small subunit rDNA and 5 -end of the large subunit rDNA were used to generate five primer sets that, when used simultaneously in multiplex PCR, produce a unique electrophoretic DNA banding pattern characterized by single DNA fragments for: Ostertagia ostertagi (257 bp) primer sequence forward 5 -TAAAAGTCGTAACAAGGTATCTGTGGT reverse 5 -GTCTCAAGCTCAACCATAACCAACATGG; Haemonchus placei (176 bp) primer sequence forward 5 -CATTTTCGTCTTGGGCGATAT reverse 5 -TGAGACCGCACGCGTTGATTCGAA; Oesophagostomum radiatum (329 bp) primer sequence forward 5 -GCAGAACCGTGACTATGGTC reverse 5 -GACAAGGAGATCACGACATCAGCAT; Trichostrongylus colubriformis (243 bp) primer sequence forward 5 -CAGGGTCAGTGTCGAATGGTCATTGTCAAATA reverse 5 -CAGGGTCAGTGGTTGCAATACAAATGATAATT; Cooperia oncophora (151 bp) primer sequence forward 5 -TCGATGAAGAGTTTTCGGTGTTC reverse 5 -TTCACGCTCGCTCGTGACTTCA.
The PCR products (10 l) were separated via 5% agarose gel electrophoresis. 2.4. Bulk-tank milk ELISAs Bulk tank milk (BTM) samples of 30 ml were taken from the milk tank of each of the 20 herds after the morning milking, according to Polish standard PN-A-86002:1999: Raw milk. Milk samples were centrifuged at 16,000 × g for 4 min and the fat fraction was removed. The BTM skim milk samples were tested for the presence of O. ostertagi antibodies using SVANOVIR O. ostertagi-Ab ELISA test (Svanova Biotech Ab, Uppsala, Sweden) according to the manufacturer’s instructions. The optical density was measured at 492 nm and the test results were expressed as the optical density ratio (ODR). The ODR value was calculated for each herd according to the formula: (ODsample − ODnegative control )/(ODpositive control − ODnegative control ) 2.5. Statistical analysis The confidence limits (confidence level: 95%, p < 0.05) of infection incidence (%) were calculated according to the modified Wald method (Sauro, 2005). All of the statistical
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J. Piekarska et al. / Veterinary Parasitology 198 (2013) 250–253
Table 1 Results of examination of cattle herds in Lower Silesia (herd no. marked with *) and Lesser Poland for gastrointestinal nematodes (2011). Herd no.
Herd size
No. of tested feces samples/no. of positive samples
Mean no. of lactation periods ± SEMa
Infection incidence (CIb )
Mean egg count EPG
BTM ODR
Larvoscopy/ Baermann
Multiplex PCR
Ostertagia ostertagi NR Oesophagostomum radiatum, Cooperia oncophora NR NR NR NR Ostertagia ostertagi Ostertagia ostertagi Cooperia oncophora NR Ostertagia ostertagi NR Cooperia oncophora NR Ostertagia ostertagi NR Oesophagostomum radiatum, Cooperia oncophora NR NR
1* 2 3
30 11 13
11/10 8/3 9/6
3.73 ± 0.65 3.88 ± 0.81 5.11 ± 1.24
80.3% (60.0–99.9) 41.5% (13.4–69.6) 61.6% (35.0–88.2)
350 115 425
0.612 0.054 0.073
+ + +
4 5* 6* 7 8* 9* 10 11* 12 13* 14* 15* 16 17* 18
14 20 25 40 54 30 10 35 28 50 50 150 76 90 30
9/4 6/5 15/6 21/15 30/5 12/8 7/5 15/9 15/12 15/8 15/9 29/17 22/22 30/7 22/17
2.78 3.33 2.93 2.95 2.67 3.58 4.29 3.33 5.67 4.67 2.87 2.24 3.68 3.10 4.09
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
46.1% (18.8–73.3) 70.3% (41.7–98.8) 42.0% (19.7–64.3) 68.1% (49.7–86.4) 20.4% (6.8–34.0) 62.6% (38.8–86.4) 63.8% (35.2–92.4) 57.9% (35.6–80.2) 73.8% (54.0–93.7) 52.6% (30.1–75.2) 57.9% (35.6–80.2) 57.6% (40.7–74.5) 94.5% (87–100) 26.3% (11.5–41.2) 73.2% (56.1–90.2)
200 160 125 215 110 300 70 113 288 200 80 189 250 150 265
0.070 0.263 0.427 0.567 0.654 0.778 0.177 0.254 0.723 0.340 0.145 0.112 0.764 0.363 0.119
+ + + + + + + + + + + + + + +
19* 20*
150 200
31/0 39/0
4.13 ± 0.40 3.56 ± 0.32
0.311 0.101
− −
0.74 0.80 0.32 0.44 0.29 0.48 0.97 0.41 0.94 0.89 0.41 0.23 0.63 0.35 0.68
– –
EPG = eggs per gram of feces; BTM = bulk tank milk; NR = not recorded. a Mean age of tested cows in herd was expressed as a mean number of lactation periods per herd. b CI = 95% confidence limits according to the modified (adjusted) Wald method.
analyses, including descriptive statistics and the Kendall Tau correlation were performed using the STATISTICA ver. 10.0 software package. 3. Results and discussion The results of this study demonstrated that GI nematode infections occur frequently in dairy cattle in Lower Silesia and Lesser Poland. The eggs of GI nematodes were found in 168 out of 361 stool specimens tested (46.5%). The infected animals came from 18 of the 20 examined dairy herds. The prevalence of infection averaged 58.4%, while the average intensity of egg excretion of GI nematodes in the infected herds was 200 (range from 70 to 425) per gram of feces. Specific antibodies for O. ostertagi were found in the BTM samples in all of the examined dairy herds. The mean ODR for all of the examined BTM samples was 0.345 (range from 0.07 to 0.778). In studies using multiplex PCR, three nematode species were found in 9 of the 18 infected herds. O. ostertagi was confirmed in five examined herds and Cooperia oncophora in four, with Oesophagostomum radiatum present in two where Cooperia oncophora was also detected (Table 1). There was a significant positive correlation between the presence of O. ostertagi DNA in the pooled fecal samples and the mean value of the BTM ODR being greater than 0.612 (Kendall r = 0.628, p ≤ 0.05). In a previous study of selected farms in Poland, the prevalence of GI nematodes in cows was found to range between 12.7 and 32.1%. A prevalence of 42.6% was detected on a large farm (Pilarczyk et al., 2009). A high incidence of dairy cattle infection was observed in the
Netherlands (88.5%) and Belgium (94%) in other studies (Borgsteede et al., 2000; Agneessens et al., 2000). O. ostertagi is one of the most important factors limiting milk production in grazing cattle because of the prolonged susceptibility to reinfection (Rinaldi and Geldhof, 2012). O. ostertagi ELISA can be used as an indicator to evaluate whether GI nematode infections are impairing productivity (Charlier et al., 2009). In dairy herds, a negative relationship was established between the anti O. ostertagi antibody level in BTM and the annual average milk yield (Charlier et al., 2005). In addition, herds with a high BTM antibody level had a higher milk-yield response to anthelmintic treatment than those with a low level (Charlier et al., 2007). In Europe in 2005–2007, the average BTM ODR value for grazing dairy herds was 0.3 in Italy, 0.5 in Sweden, 0.6 in Portugal, and 0.8 in Ireland and the UK (Forbes et al., 2008; Bennema et al., 2010). The seroprevalence of O. ostertagi in dairy cattle herds in Poland was examined for the first time in 2009 with the use of the ELISA test. The mean BTM ODR was 0.53 (range 0.43–0.63; p < 0.05) (Płoneczka-Janeczko et al., 2011). In this study, the mean ODR in the examined region of Poland was generally lower (0.345) than that found in the 2009 study. However, in 6 of the 20 examined BTM samples, the ODR calculated for stocks based on the concentration of antibodies was >0.5, which may be correlated with a decrease in milk yield ranging from 0.5 to over 2.5 kg of milk per cow per day (Forbes et al., 2008). In addition, molecular studies confirmed the presence of O. ostertagi mainly in herds where the ODR value was >0.6 (Table 1). A comprehensive and integrated program for parasite eradication could effectively reduce the level of occurrence and increase the safety of grazing animals (Mercier et al.,
J. Piekarska et al. / Veterinary Parasitology 198 (2013) 250–253
2001). The lack of eggs found in the fecal samples from two of the examined herds can be explained by the animals having been dewormed before the grazing season. This led to a low level of contamination of their pastures during this period. Knowledge of the epidemiology of particular species of parasites allows very good results to be achieved in reducing the level of infection. The methodology is based on: continuous monitoring of the degree of infestation; deworming prior to putting the animals out to pasture and at the end of the grazing season; rational grazing, preferably through a quartered system, maintaining the required occupancy and pasture load as well as the rotation time, thus potentially reducing egg and larva consumption by the grazing animals. Providing dairy cows with favorable grazing conditions is one of the main factors determining farm profitability (Mejía et al., 2011). In conclusion, this study indicated that the prevalence of GI parasites was relatively high in small dairy cattle farms in the southern Poland. In 30% of the examined herds, the concentration of O. ostertagi antibodies in the BTM exceeded 0.5 ODR, i.e. the level above which the loss of milk yield may be significant. The fecal and serological research and the molecular studies on the nematode larvae (L3) confirmed that the lack of anti-parasitic prevention combined with the grazing model reinforced the occurrence of parasitic invasions in small-sized cattle farms in southern Poland. Integrated grazing strategies and prophylaxis measures should be adopted to control intestinal parasite infection in dairy cattle in this region of Poland. References Agneessens, J., Claerebout, E., Dorny, P., Borgsteede, F.H., Vercruysse, J., 2000. Nematode parasitism in adult dairy cows in Belgium. Vet. Parasitol. 90, 83–92. Bennema, S.C., Vercruysse, J., Morgan, E., Stafford, K., Höglund, J., Demeler, J., von Samson-Himmelstjerna, G., Charlier, J., 2010. Epidemiology and risk factors for exposure to gastrointestinal nematodes in dairy herds in northwestern Europe. Vet. Parasitol. 173, 247–254.
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Borgsteede, F.H., Tibben, J., Cornelissen, J.B., Agneessens, J., Gaasenbeek, C.P., 2000. Nematode parasites of adult dairy cattle in the Netherlands. Vet. Parasitol. 89, 287–296. Charlier, J., Claerebout, E., Duchateau, L., Vercruysse, J., 2005. A survey to determine relationships between bulk tank milk antibodies against Ostertagia ostertagi and milk production parameters. Vet. Parasitol. 129, 67–75. Charlier, J., Duchateau, L., Claerebout, E., Vercruysse, J., 2007. Predicting milk-production responses after an autumn treatment of pastured dairy herds with eprinomectin. Vet. Parasitol. 143, 197–390. Charlier, J., Höglund, J., von Samson-Himmelstjerna, G., Dorny, P., Vercruysse, J., 2009. Gastrointestinal nematode infections in adult dairy cattle: impact on production, diagnosis and control. Vet. Parasitol. 164, 70–79. Forbes, A.B., Vercruysse, J., Charlier, J., 2008. A survey of the exposure to Ostertagia ostertagi in dairy cow herds in Europe through the measurement of antibodies in milk samples from the bulk tank. Vet. Parasitol. 157, 100–107. Gross, S.J., Ryan, W.G., Ploeger, H.W., 1999. Antihelmintic treatment of adult dairy cows and its effect on milk production. Vet. Rec. 144, 581–587. Mejía, M.E., Perri, A.F., Licoff, N., Miglierina, M.M., Cseh, S., Ornstein, A.M., Becu-Villalobos, D., Lacau-Mengido, I.M., 2011. Comparison of three methods for gastrointestinal nematode diagnosis determination in grazing dairy cattle in relation to milk production. Vet. Parasitol. 183, 74–177. Mercier, P., Steffan, P.E., White, C.R., 2001. Effects of treatments with endectocide on the weight gain of grazing cattle in a warm temperate climate. Vet. Rec. 149, 266–269. Pilarczyk, B., Balicka-Ramisz, A., Kozak, W., Ramisz, A., 2009. Occurrence of endoparasites in heifers imported to Poland from the Netherlands. Arch. Tierz. 52, 265–271. Płoneczka-Janeczko, K., Piekarska, J., Rypuła, K., Mazurkiewicz, M., 2011. A survey of anti-Ostertagia ostertagi antibody levels in bulk tank milk samples (BTM) in dairy herds in Lower Silesia Region (Poland). Pol. J. Vet. Sci. 14, 135–136. Rinaldi, M., Geldhof, P., 2012. Immunologically based control strategies for ostertagiosis in cattle: where do we stand? Parasite Immunol. 34, 254–264. Sauro, J., 2005. Confidence interval calculator for a completion rate. Measuring usability, http://www.measuringusability.com/ wald.htm#marg (accessed: 30.12.2010). ´ M., Zaichenko, I., Cadková, Z., Jankovská, I., Langrová, Vadlejch, J., Petrtyl, I., Moravec, M., 2011. Which McMaster egg counting technique is the most reliable? Parasitol. Res. 109, 1387–1394. Zarlenga, D.S., Chute, M.B., Gasbarre, L.C., Boyd, P.C., 2001. A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle. Vet. Parasitol. 97, 201–211.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Gastrointestinal nematodes in grazing dairy cattle from small and medium-sized farms in southern Poland J. Piekarska a,∗ , K. Płoneczka-Janeczko b , M. Kantyka a , M. Kuczaj c , M. Gorczykowski a , K. Janeczko d a Department of Internal Medicine and Clinic of Diseases of Horses, Dogs and Cats, Division of Parasitology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Norwida 31, 50-375 Wroclaw, Poland b Department of Epizootiology with Clinic of Birds and Exotic Animals, Wroclaw University of Environmental and Life Sciences, Pl. Grunwaldzki 45, 50-366 Wroclaw, Poland c Institute of Animal Breeding, Faculty of Biology and Animal Breeding, Wroclaw University of Environmental and Life Sciences, Chełmo´ nskiego 38C, Wroclaw, Poland d Department of Reproduction and Clinic for Farm Animals, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Pl. Grunwaldzki 49, 50-366 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Received 9 May 2013 Received in revised form 25 July 2013 Accepted 26 July 2013 Keywords: Gastrointestinal nematode eggs Dairy cattle BTM Multiplex PCR O. ostertagi
a b s t r a c t This study aimed to estimate the prevalence of gastrointestinal nematodes and the intensity of infection in grazing dairy cattle from small and medium-sized farms in southern Poland. The level of antibodies against Ostertagia ostertagi in the bulk tank milk (BTM) from the animals was also assessed. Rectal fecal samples collected from 361 cows on 20 farms were examined using Willis-Schlaaf flotation and the McMaster method. BTM samples were tested for the presence of O. ostertagi antibodies using ELISA. Multiplex PCR was used to identify the third-stage larvae (L3) of gastrointestinal nematodes derived from the culture of pooled fecal samples from sampled farms. Gastrointestinal nematode eggs were found in the samples from 18 of the 20 herds with a prevalence range from 20.4 to 94.5%. The average number of eggs excreted in the feces of the herds was 200 eggs per gram (EPG). Antibodies to O. ostertagi were found in 20 of the examined herds (100%), of which 6 had optical density ratios (ODR) greater than 0.5. PCR results showed the presence of three nematode species: Ostertagia ostertagi, Cooperia oncophora and Oesophagostomum radiatum. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Gastrointestinal (GI) nematodes that cause substantial losses in dairy cattle breeding include members of the Trichostrongylidae (Trichostrongylus, Haemonchus, Ostertagia, Cooperia) and Molineidae (Nematodirus) families and Oesophagostomum species. Previous research confirmed that the negative effects of infection in dairy cows with subclinical symptoms included a decrease in milk yield of
∗ Corresponding author. Tel.: +48 713205420. E-mail address: [email protected] (J. Piekarska). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.07.039
up to and even over 11% (Gross et al., 1999; Charlier et al., 2009). The interpretation of fecal examination results has certain limitations. In order to distinguish gastrointestinal parasite species, it is necessary to conduct larval culture, perform morphometric analysis of the third larvae stage, or use PCR. The Ostertagia ostertagi antibody levels in bulk tank milk samples (BTM) provide an important indicator of potential production losses in dairy herds. Potential loss of milk yield is already observed at antibody concentrations of 0.5 ODR (Charlier et al., 2007). The greatest possibilities of infection with GI nematodes in Poland fall in late May and June and late August and
J. Piekarska et al. / Veterinary Parasitology 198 (2013) 250–253
September, as those are the periods with the most favorable weather conditions for infection. Small and medium-sized dairy farms have a significant share in the total milk production in Poland, but their parasitological status is unknown, so each examination is very important to fill this gap in our knowledge. Testing also has a huge impact on farmers’ awareness. For them, the investment into deworming seems high, especially with preparations that have no withdrawal period, and there are no data for veterinarians to support the recommended prevention programs. The main objective of this study was to investigate the prevalence and intensity of GI nematode invasion in dairy herds from small and medium-sized farms in southern Poland, to determine the O. ostertagi-specific antibody levels in BTM samples from the examined herds to estimate potential losses in production, and to identify the GI nematode species occurring in the studied herds. 2. Materials and methods 2.1. Study area and farms Fecal examinations were conducted in October and November 2011, in 20 grazing dairy cattle herds: 12 from Lower Silesia and 8 from Lesser Poland (Galicia). The herd size ranged from 10 to 200 animals and their average age was about 5.5 years. The mean number of lactation periods across all the herds was 3.51 ± 0.124. The material consisted of 361 rectal fecal samples from lowland black-and-white and red Polish cows. The percentage of examined fecal samples from a given herd was between 19 and 73, depending on herd size. The grazing period had started at the end of April and ended in November 2011. The last anti-parasitic compounds had been given to cattle on 2 of the 20 farms 6 months prior to the study. The products used as the last treatment included ivermectin (Farm No. 19) and oxfendazole (Farm No. 20) (Table 1). The cattle on the other 18 farms had never been treated before sampling. The herds originated in areas characterized by moderate climates with maritime and continental features. The annual rainfall ranges from 550 to 700 mm, and July is the wettest month. The average temperature in Lower Silesia is −0.6 ◦ C (30.92 ◦ F) in January and 17.8 ◦ C (64.04 ◦ F) in July. The warmest month in Lesser Poland is July, with an average temperature of 14.5 ◦ C (58.1 ◦ F). January is the coldest month, with an average temperature of −9.4 ◦ C (15.08 ◦ F). The snow cover lasts 50 days on average. 2.2. Parasitological techniques (fecal egg counts, larval culture) The fecal samples were examined using Willis-Schlaaf flotation with saturated NaCl solution (Pilarczyk et al., 2009). A modified version of the McMaster technique using saturated NaCl solution as the flotation fluid and a detection level of 50 gastrointestinal nematode eggs per gram of feces was performed on all individual fecal samples (Vadlejch et al., 2011). Pooled coprocultures obtained from the positive fecal samples from each herd were prepared and incubated at 24 ◦ C in moist conditions for 14 days to obtain
251
the infective third stage larvae (L3) that were harvested using the Baermann technique (Pilarczyk et al., 2009). 2.3. Molecular identification of gastrointestinal nematodes The DNA of third-stage infective GI larvae collected from coprocultures was isolated using the Genomic Set AX ´ Genotyping was conducted STOOL Mini Kit (DNA-Gdansk). using multiplex PCR. The parameters of the reaction and peculiar starters were planned based on Zarlenga et al. (2001). Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats and from the 3 -end of the small subunit rDNA and 5 -end of the large subunit rDNA were used to generate five primer sets that, when used simultaneously in multiplex PCR, produce a unique electrophoretic DNA banding pattern characterized by single DNA fragments for: Ostertagia ostertagi (257 bp) primer sequence forward 5 -TAAAAGTCGTAACAAGGTATCTGTGGT reverse 5 -GTCTCAAGCTCAACCATAACCAACATGG; Haemonchus placei (176 bp) primer sequence forward 5 -CATTTTCGTCTTGGGCGATAT reverse 5 -TGAGACCGCACGCGTTGATTCGAA; Oesophagostomum radiatum (329 bp) primer sequence forward 5 -GCAGAACCGTGACTATGGTC reverse 5 -GACAAGGAGATCACGACATCAGCAT; Trichostrongylus colubriformis (243 bp) primer sequence forward 5 -CAGGGTCAGTGTCGAATGGTCATTGTCAAATA reverse 5 -CAGGGTCAGTGGTTGCAATACAAATGATAATT; Cooperia oncophora (151 bp) primer sequence forward 5 -TCGATGAAGAGTTTTCGGTGTTC reverse 5 -TTCACGCTCGCTCGTGACTTCA.
The PCR products (10 l) were separated via 5% agarose gel electrophoresis. 2.4. Bulk-tank milk ELISAs Bulk tank milk (BTM) samples of 30 ml were taken from the milk tank of each of the 20 herds after the morning milking, according to Polish standard PN-A-86002:1999: Raw milk. Milk samples were centrifuged at 16,000 × g for 4 min and the fat fraction was removed. The BTM skim milk samples were tested for the presence of O. ostertagi antibodies using SVANOVIR O. ostertagi-Ab ELISA test (Svanova Biotech Ab, Uppsala, Sweden) according to the manufacturer’s instructions. The optical density was measured at 492 nm and the test results were expressed as the optical density ratio (ODR). The ODR value was calculated for each herd according to the formula: (ODsample − ODnegative control )/(ODpositive control − ODnegative control ) 2.5. Statistical analysis The confidence limits (confidence level: 95%, p < 0.05) of infection incidence (%) were calculated according to the modified Wald method (Sauro, 2005). All of the statistical
252
J. Piekarska et al. / Veterinary Parasitology 198 (2013) 250–253
Table 1 Results of examination of cattle herds in Lower Silesia (herd no. marked with *) and Lesser Poland for gastrointestinal nematodes (2011). Herd no.
Herd size
No. of tested feces samples/no. of positive samples
Mean no. of lactation periods ± SEMa
Infection incidence (CIb )
Mean egg count EPG
BTM ODR
Larvoscopy/ Baermann
Multiplex PCR
Ostertagia ostertagi NR Oesophagostomum radiatum, Cooperia oncophora NR NR NR NR Ostertagia ostertagi Ostertagia ostertagi Cooperia oncophora NR Ostertagia ostertagi NR Cooperia oncophora NR Ostertagia ostertagi NR Oesophagostomum radiatum, Cooperia oncophora NR NR
1* 2 3
30 11 13
11/10 8/3 9/6
3.73 ± 0.65 3.88 ± 0.81 5.11 ± 1.24
80.3% (60.0–99.9) 41.5% (13.4–69.6) 61.6% (35.0–88.2)
350 115 425
0.612 0.054 0.073
+ + +
4 5* 6* 7 8* 9* 10 11* 12 13* 14* 15* 16 17* 18
14 20 25 40 54 30 10 35 28 50 50 150 76 90 30
9/4 6/5 15/6 21/15 30/5 12/8 7/5 15/9 15/12 15/8 15/9 29/17 22/22 30/7 22/17
2.78 3.33 2.93 2.95 2.67 3.58 4.29 3.33 5.67 4.67 2.87 2.24 3.68 3.10 4.09
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
46.1% (18.8–73.3) 70.3% (41.7–98.8) 42.0% (19.7–64.3) 68.1% (49.7–86.4) 20.4% (6.8–34.0) 62.6% (38.8–86.4) 63.8% (35.2–92.4) 57.9% (35.6–80.2) 73.8% (54.0–93.7) 52.6% (30.1–75.2) 57.9% (35.6–80.2) 57.6% (40.7–74.5) 94.5% (87–100) 26.3% (11.5–41.2) 73.2% (56.1–90.2)
200 160 125 215 110 300 70 113 288 200 80 189 250 150 265
0.070 0.263 0.427 0.567 0.654 0.778 0.177 0.254 0.723 0.340 0.145 0.112 0.764 0.363 0.119
+ + + + + + + + + + + + + + +
19* 20*
150 200
31/0 39/0
4.13 ± 0.40 3.56 ± 0.32
0.311 0.101
− −
0.74 0.80 0.32 0.44 0.29 0.48 0.97 0.41 0.94 0.89 0.41 0.23 0.63 0.35 0.68
– –
EPG = eggs per gram of feces; BTM = bulk tank milk; NR = not recorded. a Mean age of tested cows in herd was expressed as a mean number of lactation periods per herd. b CI = 95% confidence limits according to the modified (adjusted) Wald method.
analyses, including descriptive statistics and the Kendall Tau correlation were performed using the STATISTICA ver. 10.0 software package. 3. Results and discussion The results of this study demonstrated that GI nematode infections occur frequently in dairy cattle in Lower Silesia and Lesser Poland. The eggs of GI nematodes were found in 168 out of 361 stool specimens tested (46.5%). The infected animals came from 18 of the 20 examined dairy herds. The prevalence of infection averaged 58.4%, while the average intensity of egg excretion of GI nematodes in the infected herds was 200 (range from 70 to 425) per gram of feces. Specific antibodies for O. ostertagi were found in the BTM samples in all of the examined dairy herds. The mean ODR for all of the examined BTM samples was 0.345 (range from 0.07 to 0.778). In studies using multiplex PCR, three nematode species were found in 9 of the 18 infected herds. O. ostertagi was confirmed in five examined herds and Cooperia oncophora in four, with Oesophagostomum radiatum present in two where Cooperia oncophora was also detected (Table 1). There was a significant positive correlation between the presence of O. ostertagi DNA in the pooled fecal samples and the mean value of the BTM ODR being greater than 0.612 (Kendall r = 0.628, p ≤ 0.05). In a previous study of selected farms in Poland, the prevalence of GI nematodes in cows was found to range between 12.7 and 32.1%. A prevalence of 42.6% was detected on a large farm (Pilarczyk et al., 2009). A high incidence of dairy cattle infection was observed in the
Netherlands (88.5%) and Belgium (94%) in other studies (Borgsteede et al., 2000; Agneessens et al., 2000). O. ostertagi is one of the most important factors limiting milk production in grazing cattle because of the prolonged susceptibility to reinfection (Rinaldi and Geldhof, 2012). O. ostertagi ELISA can be used as an indicator to evaluate whether GI nematode infections are impairing productivity (Charlier et al., 2009). In dairy herds, a negative relationship was established between the anti O. ostertagi antibody level in BTM and the annual average milk yield (Charlier et al., 2005). In addition, herds with a high BTM antibody level had a higher milk-yield response to anthelmintic treatment than those with a low level (Charlier et al., 2007). In Europe in 2005–2007, the average BTM ODR value for grazing dairy herds was 0.3 in Italy, 0.5 in Sweden, 0.6 in Portugal, and 0.8 in Ireland and the UK (Forbes et al., 2008; Bennema et al., 2010). The seroprevalence of O. ostertagi in dairy cattle herds in Poland was examined for the first time in 2009 with the use of the ELISA test. The mean BTM ODR was 0.53 (range 0.43–0.63; p < 0.05) (Płoneczka-Janeczko et al., 2011). In this study, the mean ODR in the examined region of Poland was generally lower (0.345) than that found in the 2009 study. However, in 6 of the 20 examined BTM samples, the ODR calculated for stocks based on the concentration of antibodies was >0.5, which may be correlated with a decrease in milk yield ranging from 0.5 to over 2.5 kg of milk per cow per day (Forbes et al., 2008). In addition, molecular studies confirmed the presence of O. ostertagi mainly in herds where the ODR value was >0.6 (Table 1). A comprehensive and integrated program for parasite eradication could effectively reduce the level of occurrence and increase the safety of grazing animals (Mercier et al.,
J. Piekarska et al. / Veterinary Parasitology 198 (2013) 250–253
2001). The lack of eggs found in the fecal samples from two of the examined herds can be explained by the animals having been dewormed before the grazing season. This led to a low level of contamination of their pastures during this period. Knowledge of the epidemiology of particular species of parasites allows very good results to be achieved in reducing the level of infection. The methodology is based on: continuous monitoring of the degree of infestation; deworming prior to putting the animals out to pasture and at the end of the grazing season; rational grazing, preferably through a quartered system, maintaining the required occupancy and pasture load as well as the rotation time, thus potentially reducing egg and larva consumption by the grazing animals. Providing dairy cows with favorable grazing conditions is one of the main factors determining farm profitability (Mejía et al., 2011). In conclusion, this study indicated that the prevalence of GI parasites was relatively high in small dairy cattle farms in the southern Poland. In 30% of the examined herds, the concentration of O. ostertagi antibodies in the BTM exceeded 0.5 ODR, i.e. the level above which the loss of milk yield may be significant. The fecal and serological research and the molecular studies on the nematode larvae (L3) confirmed that the lack of anti-parasitic prevention combined with the grazing model reinforced the occurrence of parasitic invasions in small-sized cattle farms in southern Poland. Integrated grazing strategies and prophylaxis measures should be adopted to control intestinal parasite infection in dairy cattle in this region of Poland. References Agneessens, J., Claerebout, E., Dorny, P., Borgsteede, F.H., Vercruysse, J., 2000. Nematode parasitism in adult dairy cows in Belgium. Vet. Parasitol. 90, 83–92. Bennema, S.C., Vercruysse, J., Morgan, E., Stafford, K., Höglund, J., Demeler, J., von Samson-Himmelstjerna, G., Charlier, J., 2010. Epidemiology and risk factors for exposure to gastrointestinal nematodes in dairy herds in northwestern Europe. Vet. Parasitol. 173, 247–254.
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