- Email: [email protected]
Prevalence of coccidian infection in suckling piglets in China
Veterinary Parasitology 190 (2012) 51–55
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Prevalence of coccidian infection in suckling piglets in China Wen-Jie Zhang a,b , Lin-Hai Xu a,b , Ying-Ying Liu a,b , Bing-Qing Xiong a,b , Qing-Li Zhang a,b , Fa-Cai Li a,b , Qi-Qi Song a,b , Muhanmad Kasib Khan a,b , Yan-Qin Zhou b , Min Hu a , Junlong Zhao a,b,∗ a
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China b
a r t i c l e
i n f o
Article history: Received 2 November 2011 Received in revised form 10 May 2012 Accepted 15 May 2012 Keywords: Coccidian infection Isoopora Eimeria China Suckling piglets
a b s t r a c t To determine the prevalence of coccidian infection in suckling piglets in China, fecal samples from 779 litters of suckling piglets were collected on 80 different farms in 17 provinces from September 2009 to December 2010. These samples were examined through saturated saline flotation technique. The prevalences of coccidian infection ranged from 0 to 32.5% among different provinces and the average was 16.7% (130/779). The highest prevalence of 19.9% (69/346) was found in 8–14 day-old litters of suckling piglets. Seven coccidian species were detected in the positive litters of suckling piglets, including Isospora suis (63.9%), Eimeria debliecki (46.9%), Eimeria polita (19.2%), Eimeria suis (20.8%), Eimeria perminuta (13.9%), Eimeria scabra (4.6%), and Eimeria yanglingensis (1.5%). 55.4% of the positive litters of suckling piglet infected more than one coccidian species. The results of this investigation will provide the relevant basic data for control strategies against porcine coccidiosis on pig farms in China. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Scours in the pre-weaning period is a major threat to pig industry. The main cause is assumed to be viral or bacterial pathogens. During the past decades, porcine coccidia considered as one of the most prevalent etiologic agents of diarrhea in suckling piglets has been widely accepted and well documented in a number of publications in different countries (Holm, 2001; Jacek et al., 2007; Martineau and Castillo, 2000; Meyer et al., 1999; Mundt et al., 2006; Niestrath et al., 2002; Weng et al., 2005). The clinical coccidiosis in suckling piglets is apparently manifested by diarrhea with watery or pasty yellowish feces. A detailed
∗ Corresponding author at: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Shizishan Street, Wuhan 430070, Hubei, China. Tel.: +86 27 87281810. E-mail address: [email protected] (J. Zhao). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.05.015
enteric examination revealed the damage to the mucosal surface of the jejunum and ileum, which frequently leads to secondary infections with other enteric pathogens and may also lead to mortality. Therefore, high morbility with this non-hemorrhagic diarrhea and reduced weight gain lead to the economic losses (Driesen et al., 1993; Gualdi et al., 2003; Mundt et al., 2006; Niestrath et al., 2002). It has been shown that porcine coccidian infection in piglets was common in some provinces in China (Qian and Xu, 2003; Weng et al., 2005; Wu, 2006; Yang et al., 2004; Zhou et al., 1996). However, most of the researchers focused on older animals (weaners, fatteners, and the sows), a little was known about prevalence of coccidian infection in suckling piglets, which is also considered to be susceptible to coccidia. The aim of this study was to determine the prevalence of coccidian infection and identify coccidian species in suckling piglets in China. The results would provide a fundamental data for the control of coccidian infection in suckling piglets.
52
W.-J. Zhang et al. / Veterinary Parasitology 190 (2012) 51–55
2. Materials and methods 2.1. The study area The research was conducted in 17 out of 34 provinces or autonomous regions in China from September 2009 to December 2010. The investigated provinces are the main pig industry areas in the country and located in eastern and southeastern part of China within the northern latitudes from 3◦ 30 to 53◦ 33 and eastern longitudes from 97◦ 21 to 135◦ 05 . 2.2. Sample collection A total of 779 fecal samples from suckling piglets suffering from diarrhea (age 1–4 weeks) were collected randomly on 80 pig farms from 17 provinces. The samples were taken from 3 to 5 suckling piglets per litter and were pooled. In general, 5–10 litters were selected from each farm for parasitological screening. All the fecal samples were collected directly from the rectum or off the ground within 5–10 min of excretion in a wide-mouth plastic bottle using plastic gloves and stored at 4 ◦ C until being examined. 2.3. Parasitological examination Saturated saline flotation technique was used to detect fecal sample for the presence of coccidian oocysts. This procedure was adopted as previously described by Zajac and Conboy, 2006. Coccidian oocsyts per gram (OPG) of feces was quantified using McMaster technique by Ministry of Agriculture, Fisheries and Food (MAFF, 1986). Each fecal sample was examined for three times and the mean value of three examinations was calculated for further analysis. 2.4. Species identification Fecal flotation solution positive for oocysts was taken and passed through a sieve into a glass cylinder to remove the residue, followed by centrifugation for 15 min at 200 × g. The filtered fluid was collected and transferred into a beaker containing 250 ml water. After 1 h of sedimentation, the supernatant was carefully discarded. The remaining sediment was centrifuged for 10 min at 810 × g. At last, the pure sediment containing unsporulated coccidian oocysts was transferred into a plate containing 2.5% (w/v) aqueous potassium dichromate solution at 25 ◦ C for several days, until the oocysts were sporulated. Coccidian species were identified by peculiar species morphological characteristic as described by Daugschies et al., 1999, Zhou et al. (1996) and Wu (2006). The color, shape, roughness of the outer layer of the oocyst wall and the presence of a micropyle were recorded, while length and width of oocysts and sporocysts were measured with a computer-assisted image analysis system (Nikon Eclipse 80i, NIS-Element-F3.0, Japan). All morphological data were stored and analyzed for definition of coccidian species. For each sample, at least three slides were examined under 400× magnification to identify the species.
Table 1 Prevalence of coccidian infection in litters of suckling piglets in 17 provinces in China. Province
Examined no.
Positive no.
Prevalence (%)
Chongqing Fujian Guangdong Guangxi Hainan Heilongjiang Henan Hubei Hunan Jiangsu Jiangxi Jilin Liaoning Shandong Shanghai Sichuan Zhejiang Total
35 30 40 30 20 40 151 38 20 40 20 20 50 135 20 50 40 779
6 7 6 7 2 6 29 2 1 6 1 0 11 22 2 9 13 130
17.1 23.3 15 23.3 10 15 19.2 5.3 5 15 5 0 22 16.3 10 18 32.5 16.7
2.5. Statistical analysis Statistical analysis was performed by SAS version 8.0. The difference in prevalences of coccidia between different provinces and between different species, as well as between different age groups were determined statistically significant when P-value was <0.05 using chi-square tests. Linear regression analysis was used to show difference of variations between OPG ranges. 3. Results Coccidian oocysts were observed in 130 out of 779 litters of suckling piglets (16.7%). Out of the 80 pig farms, 66.3% (53/80) were positive for coccidian oocysts and the prevalence of coccidian oocysts on each farm ranged from 0 to 60%. The prevalences of coccidian oocysts in 17 provinces ranged from 0 to 32.5% (Table 1). Chi-square test revealed that the prevalences were significantly different (P < 0.05) between the 17 provinces. Zhejiang province showed the highest prevalence of coccidian oocysts (32.5%), followed by Guangdong and Fujian provinces with the prevalence of 23.3%, respectively. No coccidian oocysts were detected in Jilin province. Seven coccidian species of two different genus including Isospora and Eimeria were identified in the positive litters of suckling piglets. The prevalence of Eimeria species was 77.7% (101/130) on 60% (48/80) farms, and the prevalence of Isospora with only one species was 63.9% (83/130) on 52.5% (42/80) farms. A significant difference in the prevalence was observed (P < 0.05) between Isospora and Eimeria. As shown in Fig. 1, seven coccidian species were identified in positive litters including Isospora suis, Eimeria debliecki, Eimeria polita, Eimeria suis, Eimeria perminuta, Eimeria scabra, and Eimeria yanglingensis, and the prevalence were 63.9%, 46.9%, 20.8%, 19.2%, 13.9%, 4.6%, 5%, respectively. There was a highly significant difference (P < 0.01) between the prevalences of seven coccidian species. Most litters of the suckling piglets showed mixed infection. Out of 130 positive litters, 72 (55.4%) contained more
W.-J. Zhang et al. / Veterinary Parasitology 190 (2012) 51–55
53
Fig. 1. Species of coccidia identified and their prevalences in litters of suckling piglets in China.
than one coccidian species and a significant difference was observed between the single and mixed infection (P < 0.05). The percentages of single and mixed infections with different coccidian species in positive litters were shown in Table 2. Coccidian oocysts were found in four age groups in litters of suckling piglets (Table 3). 8–14 day-old litters were the highest infected (19.9%; 69/364), followed by 15–21 day-old (16.7%; 49/293), 22–28 day-old (11.7%; 7/60) and 1–7 day-old litters (6.3%; 5/80). Chi-square test showed that there are significant difference (P < 0.05) in prevalences between the four age groups. OPG values ranged from 0 to 11,346 in 0–7 day-old litters of suckling piglets, from 0 to 442,222 in 8–14 day-old litters, from 0 to 287,222 in 15-21day-old litters, and from 0 to 94,815 in 22–28 dayold litters. Linear regression analysis showed a significant reduction (P < 0.05) in the OPG values of 1–7 day-old and 22–28 day-old litters, compared with that of 8–14 day-old and 15–21 day-old litters, respectively (Table 3). 4. Discussion The prevalences of coccidian infection detected in pigs varied widely among different provinces in China (Qian and Xu, 2003; Weng et al., 2005; Wu, 2006; Yang et al., 2004; Zhou et al., 1996). This is the first extensive study on the
Table 2 Percentage with single or mixed infections of different coccidian species in litters of suckling piglets in China. Present species no.
Positive no.
1 2 3 4 Total
58 50 20 2 130
Percentage (%) 44.6 38.5 15.4 1.5 100
prevalence of coccidian infection in suckling piglets carried out in China. In the present study, we investigated 779 litters of suckling piglets on 80 different farms in 17 provinces of China. The overall prevalence of coccidian infection in litters of suckling piglets was 16.7% (130/779), ranging from 0 to 32.5% among different provinces. The overall prevalence of coccidian infection was lower than previous reports in some European countries that the prevalences were from 20% to 68% (Eysker et al., 1994; Jacek et al., 2007; Roepstorff et al., 1998; Wieler et al., 2001). However, it was a little higher than the prevalence of 10.4% reported in Australia (Johnson et al., 2008). There are 15 Eimeria and 2 Isospora species reported all over the world (Pellerdy, 1974). In the present study, seven species were detected in litters of suckling piglets (Fig. 1). During the past decade, PCR methods based on conserved 18S rRNA and ITS-1 rDNA regions were developed for differentiation of porcine coccidian species (Johnson et al., 2008; Ruttkowski et al., 2001). PCR methods were used for coccidian species determination because of its sensitivity and specificity. Until now, as there are only four porcine coccidian species whose target genes are available (I. suis, E. polita, E. scabra, E. porci), the traditional method based on morphological characteristic is still an effective method for differentiation of porcine coccidian species. Due to some differences in the developmental cycles of two genus of coccidian and changes of microclimate, the time period needed for I. suis oocysts to turn to infective stage is shorter than that of Eimeria spp. (Jacek et al., 2007; Lindsay et al., 1984). This situation enabled a quick spreading of the I. suis infection in piglets. I. suis has also been reported in many countries as a major coccidian pathogen, leading to the piglet diarrhea (Driesen et al., 1993; Eysker et al., 1994; Farkas et al., 2004; Jacek et al., 2007; Koudela and Vitovec, 1986; Mundt et al., 2005). Similar results were observed in our study, where I. suis was detected as the highest coccidian pathogen in the piglet farms. Multiple
54
W.-J. Zhang et al. / Veterinary Parasitology 190 (2012) 51–55
Table 3 Prevalence and intensity of coccidian infection in litters of suckling piglets at different age in China. Animal group (days)
Examine no.
Positive no.
Prevalence (%)
Mean OPG (range)
1–7 8–14 15–21 22–28
80 346 293 60
5 69 49 7
6.3 19.9 16.7 11.7
5162 (0–11,346) 25,730 (0–442,222) 23,655 (0–287,222) 12,432 (0–94,815)
OPG: oocysts per gram of feces.
infections of Eimeria species were very common, which was also reported in other studies in China (Zhou et al., 1996; Wu, 2006). Single and mixed infections with two species were more commonly observed than the multiple infections with three or four species. I. suis was the most frequently detected as a sole infecting agent. Of the mixed infections, I. suis, E. debliecki, E. polita were frequently present. The age related prevalences of isosporosis varied widely among different countries. It was maximum at 7–10 dayold piglets in Canada and Great Britain (Robinson et al., 1983; Sanford, 1983). Henriksen et al. (1989) detected the highest prevalence of isosporosis in 7–14 day-old piglets in Denmark. Sayd and Kawazoe (1996) reported the highest prevalence of isosporosis in 10–19 day-old piglets in Brazil. Yang et al. (2004) reported the highest prevalence of coccidian infection at the third week of piglet age in Sichuan, China. In the present study, it was highest at the second week of age (19.9%), where it almost sustained up to third week (16.7%). In conclusion, the present study provided evidence that porcine coccidia are widely prevalent in pig farms in China. The high prevalences of coccidian infection in piglet farms are meaningful that it is necessary to carry out suitable control programs. Furthermore, it is needed to pay attention on highly prevalent coccidian species (I. suis and E. debliecki) to control piglet diarrhea in China. Integrated strategies should be taken to eliminate risk factors in pig farms.
Acknowledgements We are grateful to Bayer (Sichuan) Animal Health Co., Ltd. for the help in the collection of fecal samples and part of financial support.
References Daugschies, A., Imarom, S., Bollwahn, W., 1999. Differentiation of porcine Eimeria by morphologic algorithms. Vet. Parasitol. 81, 201–210. Driesen, S.J., Carland, P.G., Fahy, V.A., 1993. Studies on preweaning piglet diarrhoea. Aust. Vet. J. 70, 259–263. Eysker, M., Boerdam, G.A., Hollanders, W., Verheijden, J.H., 1994. The prevalence of Isospora suis and Strongyloides ransomi in suckling piglets in The Netherlands. Vet. Q. 16, 203–205. Farkas, R., Szeidemann, Z., Majoros, G., 2004. Prevalence and geographical distribution of isosporosis in swine farms of Hungary. In: Proceedings of the 18th IPVS, Hamburg, Germany, p. 314. Gualdi, V., Vezzoli, F., Luini, M., Nisoli, L., 2003. The role of Isospora suis in the ethiology of diarrhoea in suckling piglets. Parasitol. Res. 90 (Suppl. 3), S163–S165.
Henriksen, S.V.A., Christensen, J.P.B., Yvoré, P., 1989. Coccidiosis in piglets in Denmark. Shedding of oocyst of Isospora suis in relation to age of the host. Coccidia and intestinal ciccidiomorphs. In: Yvoré, P. (Ed.), 5th International Coccidiosis Conference. Tours, France, 17–20 October, 1989, Ed. INRA Publ. 489–492. Holm, A., 2001. Coccidiosis in piglets seen from the point of view of the practising veterinarian. Parasitol. Res. 87, 357–359. Jacek, K., Irena, Z., Tomasz, C., 2007. Prevalence of Isospora suis and Eimeria spp. in suckling piglets and sows in Poland. Vet. Parasitol. 142, 171–175. Johnson, J., Samarasinghe, B., Buddle, R., Armson, A., Ryan, U., 2008. Molecular identification and prevalence of Isospora sp. in pigs in Western Australia using a PCR–RFLP assay. Exp. Parasitol. 120, 191–193. Koudela, B., Vitovec, J., 1986. Coccidiosis of suckling piglets. Veterinarstvi 36, 23–24. Lindsay, D.S., Ernst, J.V., Current, W.L., Stuart, B.P., Stewart, T.B., 1984. Prevalence of oocysts of Isospora suis and Eimeria spp. from sows on farms with and without a history of neonatal coccidiosis. J. Am. Vet. Med. Assoc. 185, 419–421. MAFF, 1986. Manual of Veterinary Parasitological Laboratory Techniques. Ministry of Agriculture, Fisheries and Food, HMSO, London, pp. 78–90. Martineau, G.P., Castillo, J.D., 2000. Epidemiological, clinical and control investigations on field porcine coccidiosis: clinical, epidemiological and parasitological paradigms? Parasitol. Res. 86, 834–837. Meyer, C., Joachim, A., Daugschies, A., 1999. Occurrence of Isospora suis in larger piglet production units and on specialized piglet rearing farms. Vet. Parasitol. 82, 277–284. Mundt, H.C., Cohnen, A., Daugschies, A., Joachim, A., Prosl, H., Schmaschke, R., Westphal, B., 2005. Occurrence of Isospora suis in Germany, Switzerland and Austria. J. Vet. Med. B 52, 93–97. Mundt, H.C., Joachim, A., Becka, M., Daugschies, A., 2006. Isospora suis: an experimental model for mammalian intestinal coccidiosis. Parasitol. Res. 98, 167–175. Niestrath, M., Takla, M., Joachim, A., Daugschies, A., 2002. The role of Isospora suis as a pathogen in conventional piglet production in Germany. J. Vet. Med. B 49, 176–180. Pellerdy, L.P., 1974. Coccidia and Coccidiosis [M], 2nd ed. Verlag Peal Parey, Berlin. Qian, X.Z., Xu, Q.Q., 2003. Investigation on the prevalence of coccidia in pigs in the City of Taizhou, Jiangsu Province, People’s Republic of China. Chin. J. Vet. Parasitol. 11 (1), 31–32 (in Chinese). Robinson, Y., Morin, M., Higins, R., 1983. Experimental transmission of intestinal coccidiosis to piglets: clinical, parasitological and pathological findings. Can. J. Comp. Med. 47, 401–407. Roepstorff, A., Nilsson, O., Oksanen, A., Gierde, B., Richter, S.H., Ortenberg, E., Christensson, D., Martinsson, B.K., Bartlett, P.C., Nansen, P., Eriksen, L., Helle, O., Nikander, S., Larsen, K., 1998. Intestinal parasites in swine in the Nordic countries: prevalence and geographical distribution. Vet. Parasitol. 76, 305–319. Ruttkowski, B., Joachim, A., Daugschies, A., 2001. PCR-based differentiation of three porcine Eimeria species and Isospora suis. Vet. Parasitol. 95, 17–23. Sanford, S.E., 1983. Porcine neonatal coccidiosis: clinical, pathological, epidemiological and diagnostic features. Calif. Vet. 37, 26–30. Sayd, S.M.O., Kawazoe, U., 1996. Prevalence of porcine neonatal isosporosis in Brazil. Vet. Parasitol. 67, 169–174. Weng, Y.B., Hu, Y.J., Li, Y., Li, B.S., Lin, R.Q., Xie, D.H., Gasser, R.B., Zhu, X.Q., 2005. Survey of intestinal parasites in pigs from intensive farms in Guangdong Province, People’s Republic of China. Vet. Parasitol. 127, 333–336. Wieler, L.H., Ilieff, A., Herbat, W., Bajer, C., Vieler, E., Bauerfeind, R., Failing, K., Klos, H., Wengert, A., Baljer, G., Zahner, H., 2001. Prevalence of enteropathogens in suckling and weaned piglets with diarrhea in South Germany. J. Vet. Med. B 48, 151–159.
W.-J. Zhang et al. / Veterinary Parasitology 190 (2012) 51–55 Wu, J.B., 2006. Investigation on the prevalence of coccidia in pigs in the district of Nanchang, Jiangxi province, People’s Republic of China. Chin. J. Vet. Parasitol. 2 (14.) (in Chinese). Yang, G.Y., Zeng, Z.Y., Guo, L., Xiu, Y.H., Wang, S.X., Cheng, Y., Lan, H., Li, Q.H., 2004. Epidemiological investigation of coccidiasis on piglets in some farms in Sichuan Province, People’s Republic of China. Chin. J. Vet. Sci. Technol. 34 (3), 31–33 (in Chinese).
55
Zajac, A.M., Conboy, G.A., 2006. Veterinary Clinical Parasitology. Blackwell Publishing, USA. Zhou, J.X., Hou, A.Z., Zhang, L., Wang, S.R., Wang, T.Q., Feng, X.R., 1996. Investigation on the prevalence of coccidia in pigs in Henan Province, People’s Republic of China. Chin. J. Vet. Parasitol. 4 (2), 41–42 (in Chinese).
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Prevalence of coccidian infection in suckling piglets in China Wen-Jie Zhang a,b , Lin-Hai Xu a,b , Ying-Ying Liu a,b , Bing-Qing Xiong a,b , Qing-Li Zhang a,b , Fa-Cai Li a,b , Qi-Qi Song a,b , Muhanmad Kasib Khan a,b , Yan-Qin Zhou b , Min Hu a , Junlong Zhao a,b,∗ a
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China Key Laboratory of Animal Epidemical Diseases and Infectious Zoonoses, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, Hubei, China b
a r t i c l e
i n f o
Article history: Received 2 November 2011 Received in revised form 10 May 2012 Accepted 15 May 2012 Keywords: Coccidian infection Isoopora Eimeria China Suckling piglets
a b s t r a c t To determine the prevalence of coccidian infection in suckling piglets in China, fecal samples from 779 litters of suckling piglets were collected on 80 different farms in 17 provinces from September 2009 to December 2010. These samples were examined through saturated saline flotation technique. The prevalences of coccidian infection ranged from 0 to 32.5% among different provinces and the average was 16.7% (130/779). The highest prevalence of 19.9% (69/346) was found in 8–14 day-old litters of suckling piglets. Seven coccidian species were detected in the positive litters of suckling piglets, including Isospora suis (63.9%), Eimeria debliecki (46.9%), Eimeria polita (19.2%), Eimeria suis (20.8%), Eimeria perminuta (13.9%), Eimeria scabra (4.6%), and Eimeria yanglingensis (1.5%). 55.4% of the positive litters of suckling piglet infected more than one coccidian species. The results of this investigation will provide the relevant basic data for control strategies against porcine coccidiosis on pig farms in China. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Scours in the pre-weaning period is a major threat to pig industry. The main cause is assumed to be viral or bacterial pathogens. During the past decades, porcine coccidia considered as one of the most prevalent etiologic agents of diarrhea in suckling piglets has been widely accepted and well documented in a number of publications in different countries (Holm, 2001; Jacek et al., 2007; Martineau and Castillo, 2000; Meyer et al., 1999; Mundt et al., 2006; Niestrath et al., 2002; Weng et al., 2005). The clinical coccidiosis in suckling piglets is apparently manifested by diarrhea with watery or pasty yellowish feces. A detailed
∗ Corresponding author at: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Shizishan Street, Wuhan 430070, Hubei, China. Tel.: +86 27 87281810. E-mail address: [email protected] (J. Zhao). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.05.015
enteric examination revealed the damage to the mucosal surface of the jejunum and ileum, which frequently leads to secondary infections with other enteric pathogens and may also lead to mortality. Therefore, high morbility with this non-hemorrhagic diarrhea and reduced weight gain lead to the economic losses (Driesen et al., 1993; Gualdi et al., 2003; Mundt et al., 2006; Niestrath et al., 2002). It has been shown that porcine coccidian infection in piglets was common in some provinces in China (Qian and Xu, 2003; Weng et al., 2005; Wu, 2006; Yang et al., 2004; Zhou et al., 1996). However, most of the researchers focused on older animals (weaners, fatteners, and the sows), a little was known about prevalence of coccidian infection in suckling piglets, which is also considered to be susceptible to coccidia. The aim of this study was to determine the prevalence of coccidian infection and identify coccidian species in suckling piglets in China. The results would provide a fundamental data for the control of coccidian infection in suckling piglets.
52
W.-J. Zhang et al. / Veterinary Parasitology 190 (2012) 51–55
2. Materials and methods 2.1. The study area The research was conducted in 17 out of 34 provinces or autonomous regions in China from September 2009 to December 2010. The investigated provinces are the main pig industry areas in the country and located in eastern and southeastern part of China within the northern latitudes from 3◦ 30 to 53◦ 33 and eastern longitudes from 97◦ 21 to 135◦ 05 . 2.2. Sample collection A total of 779 fecal samples from suckling piglets suffering from diarrhea (age 1–4 weeks) were collected randomly on 80 pig farms from 17 provinces. The samples were taken from 3 to 5 suckling piglets per litter and were pooled. In general, 5–10 litters were selected from each farm for parasitological screening. All the fecal samples were collected directly from the rectum or off the ground within 5–10 min of excretion in a wide-mouth plastic bottle using plastic gloves and stored at 4 ◦ C until being examined. 2.3. Parasitological examination Saturated saline flotation technique was used to detect fecal sample for the presence of coccidian oocysts. This procedure was adopted as previously described by Zajac and Conboy, 2006. Coccidian oocsyts per gram (OPG) of feces was quantified using McMaster technique by Ministry of Agriculture, Fisheries and Food (MAFF, 1986). Each fecal sample was examined for three times and the mean value of three examinations was calculated for further analysis. 2.4. Species identification Fecal flotation solution positive for oocysts was taken and passed through a sieve into a glass cylinder to remove the residue, followed by centrifugation for 15 min at 200 × g. The filtered fluid was collected and transferred into a beaker containing 250 ml water. After 1 h of sedimentation, the supernatant was carefully discarded. The remaining sediment was centrifuged for 10 min at 810 × g. At last, the pure sediment containing unsporulated coccidian oocysts was transferred into a plate containing 2.5% (w/v) aqueous potassium dichromate solution at 25 ◦ C for several days, until the oocysts were sporulated. Coccidian species were identified by peculiar species morphological characteristic as described by Daugschies et al., 1999, Zhou et al. (1996) and Wu (2006). The color, shape, roughness of the outer layer of the oocyst wall and the presence of a micropyle were recorded, while length and width of oocysts and sporocysts were measured with a computer-assisted image analysis system (Nikon Eclipse 80i, NIS-Element-F3.0, Japan). All morphological data were stored and analyzed for definition of coccidian species. For each sample, at least three slides were examined under 400× magnification to identify the species.
Table 1 Prevalence of coccidian infection in litters of suckling piglets in 17 provinces in China. Province
Examined no.
Positive no.
Prevalence (%)
Chongqing Fujian Guangdong Guangxi Hainan Heilongjiang Henan Hubei Hunan Jiangsu Jiangxi Jilin Liaoning Shandong Shanghai Sichuan Zhejiang Total
35 30 40 30 20 40 151 38 20 40 20 20 50 135 20 50 40 779
6 7 6 7 2 6 29 2 1 6 1 0 11 22 2 9 13 130
17.1 23.3 15 23.3 10 15 19.2 5.3 5 15 5 0 22 16.3 10 18 32.5 16.7
2.5. Statistical analysis Statistical analysis was performed by SAS version 8.0. The difference in prevalences of coccidia between different provinces and between different species, as well as between different age groups were determined statistically significant when P-value was <0.05 using chi-square tests. Linear regression analysis was used to show difference of variations between OPG ranges. 3. Results Coccidian oocysts were observed in 130 out of 779 litters of suckling piglets (16.7%). Out of the 80 pig farms, 66.3% (53/80) were positive for coccidian oocysts and the prevalence of coccidian oocysts on each farm ranged from 0 to 60%. The prevalences of coccidian oocysts in 17 provinces ranged from 0 to 32.5% (Table 1). Chi-square test revealed that the prevalences were significantly different (P < 0.05) between the 17 provinces. Zhejiang province showed the highest prevalence of coccidian oocysts (32.5%), followed by Guangdong and Fujian provinces with the prevalence of 23.3%, respectively. No coccidian oocysts were detected in Jilin province. Seven coccidian species of two different genus including Isospora and Eimeria were identified in the positive litters of suckling piglets. The prevalence of Eimeria species was 77.7% (101/130) on 60% (48/80) farms, and the prevalence of Isospora with only one species was 63.9% (83/130) on 52.5% (42/80) farms. A significant difference in the prevalence was observed (P < 0.05) between Isospora and Eimeria. As shown in Fig. 1, seven coccidian species were identified in positive litters including Isospora suis, Eimeria debliecki, Eimeria polita, Eimeria suis, Eimeria perminuta, Eimeria scabra, and Eimeria yanglingensis, and the prevalence were 63.9%, 46.9%, 20.8%, 19.2%, 13.9%, 4.6%, 5%, respectively. There was a highly significant difference (P < 0.01) between the prevalences of seven coccidian species. Most litters of the suckling piglets showed mixed infection. Out of 130 positive litters, 72 (55.4%) contained more
W.-J. Zhang et al. / Veterinary Parasitology 190 (2012) 51–55
53
Fig. 1. Species of coccidia identified and their prevalences in litters of suckling piglets in China.
than one coccidian species and a significant difference was observed between the single and mixed infection (P < 0.05). The percentages of single and mixed infections with different coccidian species in positive litters were shown in Table 2. Coccidian oocysts were found in four age groups in litters of suckling piglets (Table 3). 8–14 day-old litters were the highest infected (19.9%; 69/364), followed by 15–21 day-old (16.7%; 49/293), 22–28 day-old (11.7%; 7/60) and 1–7 day-old litters (6.3%; 5/80). Chi-square test showed that there are significant difference (P < 0.05) in prevalences between the four age groups. OPG values ranged from 0 to 11,346 in 0–7 day-old litters of suckling piglets, from 0 to 442,222 in 8–14 day-old litters, from 0 to 287,222 in 15-21day-old litters, and from 0 to 94,815 in 22–28 dayold litters. Linear regression analysis showed a significant reduction (P < 0.05) in the OPG values of 1–7 day-old and 22–28 day-old litters, compared with that of 8–14 day-old and 15–21 day-old litters, respectively (Table 3). 4. Discussion The prevalences of coccidian infection detected in pigs varied widely among different provinces in China (Qian and Xu, 2003; Weng et al., 2005; Wu, 2006; Yang et al., 2004; Zhou et al., 1996). This is the first extensive study on the
Table 2 Percentage with single or mixed infections of different coccidian species in litters of suckling piglets in China. Present species no.
Positive no.
1 2 3 4 Total
58 50 20 2 130
Percentage (%) 44.6 38.5 15.4 1.5 100
prevalence of coccidian infection in suckling piglets carried out in China. In the present study, we investigated 779 litters of suckling piglets on 80 different farms in 17 provinces of China. The overall prevalence of coccidian infection in litters of suckling piglets was 16.7% (130/779), ranging from 0 to 32.5% among different provinces. The overall prevalence of coccidian infection was lower than previous reports in some European countries that the prevalences were from 20% to 68% (Eysker et al., 1994; Jacek et al., 2007; Roepstorff et al., 1998; Wieler et al., 2001). However, it was a little higher than the prevalence of 10.4% reported in Australia (Johnson et al., 2008). There are 15 Eimeria and 2 Isospora species reported all over the world (Pellerdy, 1974). In the present study, seven species were detected in litters of suckling piglets (Fig. 1). During the past decade, PCR methods based on conserved 18S rRNA and ITS-1 rDNA regions were developed for differentiation of porcine coccidian species (Johnson et al., 2008; Ruttkowski et al., 2001). PCR methods were used for coccidian species determination because of its sensitivity and specificity. Until now, as there are only four porcine coccidian species whose target genes are available (I. suis, E. polita, E. scabra, E. porci), the traditional method based on morphological characteristic is still an effective method for differentiation of porcine coccidian species. Due to some differences in the developmental cycles of two genus of coccidian and changes of microclimate, the time period needed for I. suis oocysts to turn to infective stage is shorter than that of Eimeria spp. (Jacek et al., 2007; Lindsay et al., 1984). This situation enabled a quick spreading of the I. suis infection in piglets. I. suis has also been reported in many countries as a major coccidian pathogen, leading to the piglet diarrhea (Driesen et al., 1993; Eysker et al., 1994; Farkas et al., 2004; Jacek et al., 2007; Koudela and Vitovec, 1986; Mundt et al., 2005). Similar results were observed in our study, where I. suis was detected as the highest coccidian pathogen in the piglet farms. Multiple
54
W.-J. Zhang et al. / Veterinary Parasitology 190 (2012) 51–55
Table 3 Prevalence and intensity of coccidian infection in litters of suckling piglets at different age in China. Animal group (days)
Examine no.
Positive no.
Prevalence (%)
Mean OPG (range)
1–7 8–14 15–21 22–28
80 346 293 60
5 69 49 7
6.3 19.9 16.7 11.7
5162 (0–11,346) 25,730 (0–442,222) 23,655 (0–287,222) 12,432 (0–94,815)
OPG: oocysts per gram of feces.
infections of Eimeria species were very common, which was also reported in other studies in China (Zhou et al., 1996; Wu, 2006). Single and mixed infections with two species were more commonly observed than the multiple infections with three or four species. I. suis was the most frequently detected as a sole infecting agent. Of the mixed infections, I. suis, E. debliecki, E. polita were frequently present. The age related prevalences of isosporosis varied widely among different countries. It was maximum at 7–10 dayold piglets in Canada and Great Britain (Robinson et al., 1983; Sanford, 1983). Henriksen et al. (1989) detected the highest prevalence of isosporosis in 7–14 day-old piglets in Denmark. Sayd and Kawazoe (1996) reported the highest prevalence of isosporosis in 10–19 day-old piglets in Brazil. Yang et al. (2004) reported the highest prevalence of coccidian infection at the third week of piglet age in Sichuan, China. In the present study, it was highest at the second week of age (19.9%), where it almost sustained up to third week (16.7%). In conclusion, the present study provided evidence that porcine coccidia are widely prevalent in pig farms in China. The high prevalences of coccidian infection in piglet farms are meaningful that it is necessary to carry out suitable control programs. Furthermore, it is needed to pay attention on highly prevalent coccidian species (I. suis and E. debliecki) to control piglet diarrhea in China. Integrated strategies should be taken to eliminate risk factors in pig farms.
Acknowledgements We are grateful to Bayer (Sichuan) Animal Health Co., Ltd. for the help in the collection of fecal samples and part of financial support.
References Daugschies, A., Imarom, S., Bollwahn, W., 1999. Differentiation of porcine Eimeria by morphologic algorithms. Vet. Parasitol. 81, 201–210. Driesen, S.J., Carland, P.G., Fahy, V.A., 1993. Studies on preweaning piglet diarrhoea. Aust. Vet. J. 70, 259–263. Eysker, M., Boerdam, G.A., Hollanders, W., Verheijden, J.H., 1994. The prevalence of Isospora suis and Strongyloides ransomi in suckling piglets in The Netherlands. Vet. Q. 16, 203–205. Farkas, R., Szeidemann, Z., Majoros, G., 2004. Prevalence and geographical distribution of isosporosis in swine farms of Hungary. In: Proceedings of the 18th IPVS, Hamburg, Germany, p. 314. Gualdi, V., Vezzoli, F., Luini, M., Nisoli, L., 2003. The role of Isospora suis in the ethiology of diarrhoea in suckling piglets. Parasitol. Res. 90 (Suppl. 3), S163–S165.
Henriksen, S.V.A., Christensen, J.P.B., Yvoré, P., 1989. Coccidiosis in piglets in Denmark. Shedding of oocyst of Isospora suis in relation to age of the host. Coccidia and intestinal ciccidiomorphs. In: Yvoré, P. (Ed.), 5th International Coccidiosis Conference. Tours, France, 17–20 October, 1989, Ed. INRA Publ. 489–492. Holm, A., 2001. Coccidiosis in piglets seen from the point of view of the practising veterinarian. Parasitol. Res. 87, 357–359. Jacek, K., Irena, Z., Tomasz, C., 2007. Prevalence of Isospora suis and Eimeria spp. in suckling piglets and sows in Poland. Vet. Parasitol. 142, 171–175. Johnson, J., Samarasinghe, B., Buddle, R., Armson, A., Ryan, U., 2008. Molecular identification and prevalence of Isospora sp. in pigs in Western Australia using a PCR–RFLP assay. Exp. Parasitol. 120, 191–193. Koudela, B., Vitovec, J., 1986. Coccidiosis of suckling piglets. Veterinarstvi 36, 23–24. Lindsay, D.S., Ernst, J.V., Current, W.L., Stuart, B.P., Stewart, T.B., 1984. Prevalence of oocysts of Isospora suis and Eimeria spp. from sows on farms with and without a history of neonatal coccidiosis. J. Am. Vet. Med. Assoc. 185, 419–421. MAFF, 1986. Manual of Veterinary Parasitological Laboratory Techniques. Ministry of Agriculture, Fisheries and Food, HMSO, London, pp. 78–90. Martineau, G.P., Castillo, J.D., 2000. Epidemiological, clinical and control investigations on field porcine coccidiosis: clinical, epidemiological and parasitological paradigms? Parasitol. Res. 86, 834–837. Meyer, C., Joachim, A., Daugschies, A., 1999. Occurrence of Isospora suis in larger piglet production units and on specialized piglet rearing farms. Vet. Parasitol. 82, 277–284. Mundt, H.C., Cohnen, A., Daugschies, A., Joachim, A., Prosl, H., Schmaschke, R., Westphal, B., 2005. Occurrence of Isospora suis in Germany, Switzerland and Austria. J. Vet. Med. B 52, 93–97. Mundt, H.C., Joachim, A., Becka, M., Daugschies, A., 2006. Isospora suis: an experimental model for mammalian intestinal coccidiosis. Parasitol. Res. 98, 167–175. Niestrath, M., Takla, M., Joachim, A., Daugschies, A., 2002. The role of Isospora suis as a pathogen in conventional piglet production in Germany. J. Vet. Med. B 49, 176–180. Pellerdy, L.P., 1974. Coccidia and Coccidiosis [M], 2nd ed. Verlag Peal Parey, Berlin. Qian, X.Z., Xu, Q.Q., 2003. Investigation on the prevalence of coccidia in pigs in the City of Taizhou, Jiangsu Province, People’s Republic of China. Chin. J. Vet. Parasitol. 11 (1), 31–32 (in Chinese). Robinson, Y., Morin, M., Higins, R., 1983. Experimental transmission of intestinal coccidiosis to piglets: clinical, parasitological and pathological findings. Can. J. Comp. Med. 47, 401–407. Roepstorff, A., Nilsson, O., Oksanen, A., Gierde, B., Richter, S.H., Ortenberg, E., Christensson, D., Martinsson, B.K., Bartlett, P.C., Nansen, P., Eriksen, L., Helle, O., Nikander, S., Larsen, K., 1998. Intestinal parasites in swine in the Nordic countries: prevalence and geographical distribution. Vet. Parasitol. 76, 305–319. Ruttkowski, B., Joachim, A., Daugschies, A., 2001. PCR-based differentiation of three porcine Eimeria species and Isospora suis. Vet. Parasitol. 95, 17–23. Sanford, S.E., 1983. Porcine neonatal coccidiosis: clinical, pathological, epidemiological and diagnostic features. Calif. Vet. 37, 26–30. Sayd, S.M.O., Kawazoe, U., 1996. Prevalence of porcine neonatal isosporosis in Brazil. Vet. Parasitol. 67, 169–174. Weng, Y.B., Hu, Y.J., Li, Y., Li, B.S., Lin, R.Q., Xie, D.H., Gasser, R.B., Zhu, X.Q., 2005. Survey of intestinal parasites in pigs from intensive farms in Guangdong Province, People’s Republic of China. Vet. Parasitol. 127, 333–336. Wieler, L.H., Ilieff, A., Herbat, W., Bajer, C., Vieler, E., Bauerfeind, R., Failing, K., Klos, H., Wengert, A., Baljer, G., Zahner, H., 2001. Prevalence of enteropathogens in suckling and weaned piglets with diarrhea in South Germany. J. Vet. Med. B 48, 151–159.
W.-J. Zhang et al. / Veterinary Parasitology 190 (2012) 51–55 Wu, J.B., 2006. Investigation on the prevalence of coccidia in pigs in the district of Nanchang, Jiangxi province, People’s Republic of China. Chin. J. Vet. Parasitol. 2 (14.) (in Chinese). Yang, G.Y., Zeng, Z.Y., Guo, L., Xiu, Y.H., Wang, S.X., Cheng, Y., Lan, H., Li, Q.H., 2004. Epidemiological investigation of coccidiasis on piglets in some farms in Sichuan Province, People’s Republic of China. Chin. J. Vet. Sci. Technol. 34 (3), 31–33 (in Chinese).
55
Zajac, A.M., Conboy, G.A., 2006. Veterinary Clinical Parasitology. Blackwell Publishing, USA. Zhou, J.X., Hou, A.Z., Zhang, L., Wang, S.R., Wang, T.Q., Feng, X.R., 1996. Investigation on the prevalence of coccidia in pigs in Henan Province, People’s Republic of China. Chin. J. Vet. Parasitol. 4 (2), 41–42 (in Chinese).