DIGESTIVE PROTOZOOSIS IN PIGS- ETHIOPATHOGENICAL, EPIDEMIOLOGICAL AND THERAPEUTICAL RESEARCHES

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1 UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE OF CLUJ-NAPOCA FACULTY OF VETERINARY MEDICINE DOCTORAL SCHOOL OF VETERINARY MEDICINE DIGESTIVE PROTOZOOSIS IN PIGS- ETHIOPATHOGENICAL, EPIDEMIOLOGICAL AND THERAPEUTICAL RESEARCHES SUMMARY OF PhD THESIS SCIENTIFIC COORDONATOR: Prof. Univ. Dr. VASILE COZMA Cluj Napoca 2015 XX

2 Introduction The thesis was divided into two parts. Part I bibliographic study includes a main chapter with six subsections, and Part II own research with three chapters and five subsections. In the researches was followed the evolution of protozoan infections in some farms in the NW region of the country in animals reared in extensive and intensive systems. Also, were carried out in laboratory experiments regarding antiparasitic effect of plants extracts. Protozoans often develop subacute and chronic forms or subclinical with other viral or bacterial infections, resulting in the application of measures often ineffective and increasing the costs for obtaining a kilogram of meat. CHAPTER 1. DIGESTIVE PROTOZOOSIS IN SWINE Some testimonies regarding the study of parasites remained in the ancient world, proving that humans and animals were sometimes victims of these creatures. Rich material accumulated from experimental data allowed, in the beginning of the twentieth century, to define some areas of parasitology including phyto-parasitology, moving towards new dimensions of modern parasitology science integrated in tasks and aspirations of human and animal medicine. After 2001 our country has recorded a significant increase in pork consumption among the population. According to operational data of the Ministry of Agriculture pork consumption increased by 8% in 2004, compared to 2003, and in 2005 was higher by 13.1% over the previous year, increasing each year on average 10%. Satisfactory results in pig husbandries- a large number of piglets and achieving large increases weight gain depends on the livestock. Parasitic diseases, acts disturbing the entire process, are seriously affecting production levels. That of the overall population of over 5 million pigs, only 1.6 million are found in industrial complexes and the rest of the households, is one of the reasons that explain the XXI

3 high frequency of nematodes in our country. Parasitic diseases of swine are important in several respects: veterinary, epidemiological, zoonotic, ecological, economic and social. Parasitic diseases cause significant economic losses in swine by reducing production (growth modification in youth, fattening reduction, decreased milk production, confiscation of parasitized slaughterhouse organs, forced slaughter), morbidity and mortality in livestock. Social importance of parasitic diseases lies in the impact on human health through direct implications transmission of diseases from animals to humans which are parasitic zoonoses. Fundamental dimensions of parasitology are based on knowledge of phenomena adaptive evolutionary essential parasite-host biosystem and applied the knowledge sphere etiologic agents, pathogenic mechanisms and means of combat. In the context of modernization and diversification of farming system - intensive, semi-intensive and extensive - won an important managerial concerns regarding minimizing losses caused by parasites and the poliparazitism, opposite the general steps in the world, protection and restoration of the balance of ecosystems. The livestock are infected subjects with a small number of parasites that have low pathogenic so that their health is not affected. These animals are considered carriers of parasites and go unnoticed, but disseminates infective elements in the environment and contaminate the rest of the herd. CHAPTER 2. RESEARCH ON INFECTION WITH CRYPTOSPORIDIUM SPP. INPIGLETS AND ASSOCIATIONS WITH OTHER ENTEROPATHOGENS Research on infection with Cryptosporidium spp. in piglets were held from March to May Biological samples were collected from pig farm in northwestern Romania, and their processing was performed in the Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine Cluj-Napoca, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Were collected from pig farm in northwest Romania, from piglets, 50 fecal samples and XXII

4 24 blood samples (Table 2.1). Depending on age piglets were divided into two groups: piglets (Fig. 2.1) and weaners (Fig. 2.2). From the category piglets were collected 28 fecal samples and 12 blood samples from piglets weaned and 22 fecal samples and 12 blood samples. Age of the piglets was between 12 and 60 days with an average of 34 days. The average age of piglets infants was 21 days and 51 days weaned piglets. Number and type of samples collected from piglets in farm N Table 2.1 Faeces samples Blood samples Average age (days) Suckling piglets Weaned piglets Total Faecal samples were collected over the floor in separate vials and were processed in the first 72 hours after collection. The blood samples were collected through puncture of jugular vein, on anticoagulant (EDTA) and were processed in the first 24 hours after collection. The faecal samples were processed through coproparasitological methods: Willis (flotation with oversaturated sodium chloride), sedimentation, faeces smears collored through Henriksen method and coproelisa technique in view of detecting the coproantigens of Cryptosporidium spp., corona and rotaviruses. The blood samples were processed for determination of: erythrocyte numbers, haemoglobin concentration, hematocrit, glucose, total proteins, albumins and gammaglobulins. By Henriksen method Cryptosporidium spp. oocysts were identified in one samples of 50 examined (2%), which originate from a suckling pig, with age of 21 days. Through ELISA, Cryptosporidium antigens have been identified in 19 samples (38%) (Table 2.9, Fig. 2.6). Following calculation of concordance index k (0,064), no correlation between the two tests was noticed. Nevertheless, the sample positive for Henriksen was also positive in coproelisa (Table 2.10) XXIII

5 Table 2.9 Situation of Cryptosporidium spp. in piglets from N farm (n=50) Method Frequency Prevalence 95% confidence interval p Henriksen 1 2 0,10-10,6 0,2 copro-elisa ,70-52,8 Level of agreement (k) between Henriksen staining and coproelisa Table 2.10 Henriksen Pozitiv Positive Negativ Negative Pozitiv Positive coproelisa Negativ Negative k (- 0,033-0,162) Fig. 2.6: Prevalence of Cryptosporidium spp. in piglets from N farm The number of positive cases in coproelisa method was higher in piglets (12/28; 42.9%) compared with weaned piglets (7/22; 31.8%) (Fig. 2.7), but this difference was not statistically significant (p = 0.31) (Table 2.11). XXIV

6 Table 2.11 Situation of Cryptosporidium spp. in piglets from N farm according with age category by coproelisa Category of piglets Frequency Prevalence 95% confidence interval p (n=28) 12 42,9 24,5-62,8 Suckling piglets 0,31 (n=22) 7 31,8 13,9-54,9 Weaned piglets Total ,70-52,8 Fig. 2.7: Prevalence of Cryptosporidium spp. in piglets from N farm according with age category by coproelisa On examination, fecal flotation and sedimentation method were identified also other parasitic infestations (Table 2.12) from 20 piglets (40%) of the 50 examined. Parasites were identified: Eimeria / Isospora (1/50, 2%); Balantidium coli (15/50; 30%); Strongyloides ransomi (5/50, 5%); strongyl type eggs (1/50, 2%) and Trichocephalus suis (1/50, 2%). The most prevalent parasite was B. coli (Fig. 2.8). XXV

7 Fig Prevalence of parasitic infection in piglets from N farm Eimeria oocysts / Isospora, eggs of T. suis and strongyl type were identified only in weaned piglets (Table 2.14). While S. ransomi was more prevalent in piglets (10.7%) than in weaned piglets (9.1%) (Table 2.13, Table 2.14), B. coli was more prevalent weaned piglets (31 8% piglets 28.6%) (Fig. 2.9). There were no statistically significant differences. Situation of parasitic infection in suckling piglets from N farm Table 2.13 Frequency Prevalence 95% confidence interval Cryptosporidium spp. 1 3,6 0,1-18,3 Eimeria/Isospora ,3 Balantidium coli 8 28,6 13,2-48,7 Strongyloides ransomi 3 10,7 2,3-28,2 Strongyl type egg ,3 Trichocephalus suis ,3 Total 11 39,3 21,5-59,4 XXVI

8 Situation of parasitic infection in weaned piglets from N farm Table 2.14 Frequency Prevalence 95% confidence interval Cryptosporidium spp. 0 0,0 0,0-15,4 Eimeria/Isospora 1 4,5 0,1-22,8 Balantidium coli 7 31,8 13,9-54,9 Strongyloides ransomi 2 9,1 1,1-29,2 Strongyl type egg 1 4,5 0,1-22,8 Trichocephalus suis 1 4,5 0,1-22,8 Total 9 40,9 20,7-63,6 The most common association was observed between Cryptosporidium spp., and Balantidium coli (6/12, 31.58%), association seen in both piglets (4/12, 21.05%) and in weaned piglets (2 / 12; 10.53%) (Table 2.17, Fig. 2.12). Table 2.17 Identified associations between Cryptosporidium and other enteropathogens in piglets from N farm Association Frequency Prevalence Category of piglets Cryptosporidium+Rotavirus 2 10,53 Suckling piglets 6 31,58 Cryptosporidium+ 4 21,05 Suckling piglets Balantidium coli 2 10,53 Weaned piglets Cryptosporidium+ Strongyloides ransomi 2 10,53 Suckling piglets Cryptosporidium+ Balantidium coli + 1 5,26 Weaned piglets Strongyloides ransomi Cryptosporidium+ Trichocephalus suis 1 5,26 Weaned piglets +strongyl type egg Total 12 63,16 XXVII

9 Fig. 2.12: Prevalence of associations between Cryptosporidium and other enteropathogens in piglets from N farm Both in piglets and weaned infants a small number of red blood cells was observed, the values being below the lower limit of the species and age (Table 2.18). If haemoglobin and hematocrit values obtained were within physiological limits of species and age, hemoglobin concentration was slightly below the lower limit in piglets (Table 2.18). All 3 blood constant determined were lower in piglets than those in weaned piglets, but without such differences to be statistically significant. Table 2.18 Blood constants obtained in piglets from N farm Red blood cells (RBC) (mil/ml) Haemoglobin (g/dl) Total 2,88±0,53 9,5±3,36 28,58±6,64 Suckling piglets 2,8±0,53 8,65±2,98 28±7,37 Weaned piglets 2,95±0,54 10,27±3,64 29,1±6,26 p 0,54 0,31 0,73 Normal values in piglets of 2-9 weeks age Packed cell volume (PCV) (%) 5,0-7,0 9,0-11,0 27,0-31,0 Piglets glucose level in the study was above the maximum physiological value, being significantly higher in piglets (Fig. 2.15). XXVIII

10 Fig. 2.15:Histogram of glucose in piglets from N farm CHAPTER 3. A COMPARATIVE EPIDEMIOLOGICAL PROTOZOAS PIGS IN BIHOR EXTENSIVE AND INTENSIVE SYSTEMS The study of livestock in the two systems, extensive and intensive in was performed on a total of 5287 ends, stratified by age. Sampling was performed in plastic bags, labeled and sealed. Faecal samples were processed by flotation coproparasitological technique (Willis method) direct examination with saline, McMaster method and quantitative method for detecting antigens of Cryptosporidium spp in coproelisa. Analysis of data obtained show different aspects of development in pigs of digestive protozoosis in extensive and intensive systems, depending on the season, the age and zoo-hygiene conditions and technological characteristic situations of each locality studied and operating farms. XXIX

11 The average values of sheddings of the digestive protozooses diagnosed in pigs less than 4 months from extensive and intensive systems studied are presented in Table and Figure Table Values of extensivity and intensivity in swine under 4 months intensive and extensive systems Harvesting System Species October January April Legend: E-extensivity(%); I intensivity; INT- intensive system; Balantidium coli Eimeria spp./ Isospora suis Trichomonas spp INT E(%) I EXT E(%) I Average E(%) I INT E(%) I EXT E(%) I Average E(%) I INT E(%) I EXT E(%) I Average E(%) I EXT extensive system In October, there was a much higher percentage of infected animals in the youth under 4 months in the intensive versus extensive system at all protozooan species studied. Regarding the degree of infestation the results were similar for Balantidium coli, Eimeria XXX

12 spp. / Isospora suis and Trichomonas spp. in the case of extensive system to intensive system. Balantidium coli infection prevalence in young swine (up to 5-7 weeks) of holdings in southern Germany was 80% of the total number of units under study. Cysts sheddings were maximum at the age of 5-7 weeks, 60% of animals were infected (Damriyasa and Bauer, 2006). The study carried out by us yielded similar values in extensive systems, but with the mention that in the intensive systems levels were lower (10%). Coccidiosis evolution was similar to that of balantidiosis ascertaining the differences between the two farming systems. Extensiveness maximum values were 10% in the intensive systems and 60% in the extensive system. Trichomonas spp. showed a high variability in the intensive 15 to 75% from one season to another compared to the extensive system where values were similar (Fig ). Fig : Dynamics of extensivity values in case of Balantidium coli,eimeria spp/isospora suis and Trichomonas spp. in swine reared under four months, intensive and extensive systems XXXI

13 Extensiveness values followed an upward curve for coccidiosis in intensive system (reaching 20%), and in the intensive systems the maximum values were in autumn and subsequently decrease (Fig ) Exogenous development of oocysts in favorable conditions under wet layer, airy and warm (20-28 C) -it made with some specific variations, within 4-12 days or even 22 days, and the prepatent period takes 4-9 days. In the intensive system eimeriosis extensivity varied from 5 to 13% in sows with access to pasture. In piglets in some rearing systems it reaches about 76% (Yatuse, 1983). Oocysts elimination in apparently healthy animals are reduced, of about 100 OPG and in those with symptoms OPG reach over 50,000 (Jones and McTaggart, 1985). Values close to those described by Yatuse in piglets have reached this high only in extensive system, and the values were higher in intensity in all samples collected compared to those presented by Jones and McTaggart, about 250 OPG without clinical manifestation of the disease. Table Values of extensivity and intensivity in swine over 4 months intensive and extensive systems Harvestin g System Balantidium coli Eimeria spp./isospora suis INT. E(%) I (OPG) EXT. E(%) January October Ap ril I Average E(%) I INT. E(%) I EXT. E(%) I Average E(%) I INT. E(%) XXXII

14 I EXT. E(%) I Average E(%) I Legend: E-extensivity(%); I intensivity; INT- intensive system; EXT extensive system Although the incidence of trichomoniasis decreases in April compared to January, is still higher than in October in intensive system. In contrast, in the extensive system, the highest values were obtained in October and lowest in January (Fig ). The average values of sheddings of the digestive protozoosis diagnosed in pigs over four months in extensive and intensive systems investigated are shown in Table and Figure Comparing the incidence of balantidiosis and coccidiosis in young pigs and over 4 months and their incidence in breeders we found that: In October, in the intensive system, extensivity was higher for both protozoosis in youth over 4 months In the extensive system incidence was higher in fall in breeders for both protozoosis In the winter season extensity in the case of Balantidium coli was the same in both groups of animals, being higher for coccidiosis in young pigs over 4 months in the farms studied In the extensive system the results were similar with the intensive system for coccidiosis both for extensivity and for intensivity. XXXIII

15 Fig : Dynamics of extensivity values in case of Balantidium coli and Eimeria spp/isospora suis in swine over four months reared in intensive and extensive systems Regardless of the incidence of parasitosis present in both systems studied, the degree of parasitic infestation in breeders was comparable to that of youth over 4 months, we observed an increased intensity in the extensive system than the intensive system Table Values of extensivity and intensivity in the intensive and extensive breeding systems Harvesting System Crt. October INT EXT Average Balantidium coli Eimeria spp. / Isospora suis E(%) I (CPG/OPG) E(%) I (CPG/OPG) E(%) I (CPG/OPG) XXXIV

16 January April Legend: INT EXT Media INT EXT Media E-extensivity(%);I intensivity; E(%) I (CPG/OPG) E(%) I (CPG/OPG) E(%) I (CPG/OPG) E(%) I (CPG/OPG) E(%) I (CPG/OPG) E(%) I (CPG/OPG) C.P.G. cysts per gram faeces; O.P.G.- oocysts per gram faeces INT- intensive system; EXT extensive system Breeding pigs are responsive to both parasites, but with some differences in terms of their evolution dynamics, depending on the season, but in adult pigs subclinical disease progresses, these being carriers and disseminators of parasites. Regarding the intensity of balantidiosis in sows and boars, in winter are the smallest values of CPG. The average values were increased in the spring season ( CPG), for extensiveness.intensity of coccidiosis / isosporosis was higher in the extensive system compared to the extensive system in October and January but in spring the situation is reversed, in all cases the values are close (Fig ). Comparative research on the epidemiology of swine digestive protozoosis reared in intensive and extensive systems in Bihor county, conducted in the months of October, January and April led to the following: XXXV

17 In the herds surveyed the following species or genera were identified: Balantidium coli, Eimeria spp. / Isospora suis, Trichomonas spp. and Cryptosporidium spp. In pigs under 4 months extensivity of protozoan infections had higher mean values in the three harvests, in extensive system, except for harvesting in January, where Trichomonas spp. had a higher value in the intensive system. The fattening pigs over 4 months bred intensively developed infections with B.coli, Eimeria spp / Isospopra suis; regarding balantidiosis the average value of extensivity was of maximum 55% in the first collection; maximum intensity had the coccidiosis (275 OPG) in April. In the youth over 4 months of breeding flocks Balantidium coli showed a mean extensivity in all collections from 37.66% in the intensive versus extensive system, which was 53.33%; extensiveness value for coccidiosis was in extensive system 4 percent higher than the intensive system. Breeders showed a higher value of extensiveness in infections with B. coli and Eimeria / Isospora,in the extensive system compared to the intensive system. Maximum intensity was CPG to B. coli in pigs in extensive system. Extensivity of Cryptosporidium parvum infection was 4% in January, in intensive system, in other seasons being no longer diagnosed. Maximum intensity in coproelisa was of UDO (optical density units). CHAPTER 4. RESEARCH ON ANTIPARASITIC EFFECT OF HYDROALCOHOLIC EXTRACTS OF WORMWOOD AND MARIGOLD IN BALANTIDIUM COLI INFECTION IN SWINE Experimental research on the antiparasitic effect of hydroalcoholic extracts of wormwood (Artemisia absinthium) and tagetes (Tagetes patula) in B. coli XXXVI

18 infection in pigs, carried out during on a total of 32 pigs aged 3 to 5 months male and female, from actual maintained extensive systems have led to the following: In the case of the hydroalcoholic extract of wormwood (Artemisia absinthium), B. coli infection extensity decreased from 60% to 30% after 7 days post-treatment and 20% respectively at 14 and 28 days post-treatment, and the values of CPG dropped from 350 to 266.6; 175 and 50 after 7, 14 and 28 days post-treatment and OPG values decreased from 350 to 266.6; 175 and 50 after 7, 14 and 28 days post-treatment. In the case of the hydroalcoholic extract of tagetes (Tagetes patula) B. coli infection extensity increased from an initial value of 50% to 60% after 7 and 14 days post-treatment and 70% respectively on day 28 of the experiment, and the values of CPG dropped from 240 (pre-treatment) to 78.57, at the end of the experiment. In the control group, extensivity of B. coli infection ranged between 58.3 and 66.6% and infection intensivity ranged between and CPG. XXXVII