Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana

G. Reiner¹, J. Eckert¹, T. Peischl¹, S. Bochert², T. Jäkel², U. Mackenstedt², A. Joachim³, A. Daugschies³ and H. Geldermann¹

Journal of Veterinary Parasitology, in Druck

¹ Department of Animal Breeding and Biotechnology, University of Hohenheim, D-70593 Stuttgart (Germany)

² Department of Parasitology, University of Hohenheim, D-70593 Stuttgart (Germany)

³ Institute for Parasitology, University of Leipzig, D-04103 Leipzig (Germany)

Abstract

Future prophylaxis needs new concepts, including natural disease resistance of hosts against infectious agents. Genomic approaches to detect and improve disease resistance in farm animals and the molecular mechanisms involved in host-parasite-interactions depends to a high degree on the trait differences between founder breeds, i.e. on the animal model. The present study evaluates differences in susceptibility/resistance against Sarcocystis miescheriana in the European Pietrain (PI) and the Chinese Meishan (ME) pig breeds, based on 25 individuals, infected orally with 5*10⁴ sporocysts of S. miescheriana. Significant differences appeared in clinical, serological, haematological and parasitological findings. The major discriminating period post infection (p.i.) was between days 42 and 45. Severity of signs was negatively correlated with specific immunoglobulin titres during the first 3 weeks p.i. and positively with the load of bradyzoites in muscle tissues of the pigs. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME. Sarcocystis-specific differences between the two breeds were in the range of 1 to 2 standard deviations. The study lays the fundamentals for further experiments to analyse chromosomal regions, candidate genes, and thus the molecular basis of Sarcocystis susceptibility/resistance as a model for host-parasite-interaction in protozoan infectious disease.

Keywords: Pig-protozoa, Sarcocystis miescheriana, disease resistance, genetics

1. Introduction

Differences in genetic susceptibility/resistance of animals, populations or breeds against viruses (Bumstead, 1998), bacteria (Adams and Templeton, 1998) and parasites (Stear and Wakelin, 1998) are well known. The development of highly informative genetic maps in species of main farm animals permits mapping of disease resistance loci using F₂-families, generated with founder animals, which are genetically different in resistance/susceptibility against a certain disease. Among the parasitoses in swine, Sarcocystis miescheriana offers a number of advantages for genetic analysis. The coccidia of the genus Sarcocystis are common parasites of ruminants, pigs and horses, which are intermediate hosts. The definite hosts are carnivores and humans. Sarcocystis miescheriana is a widespread parasite of the muscles of pigs (Barrows et al., 1982; Dubey et al., 1989). There are two generations of schizogony, forming the acute phase of infection. The first generation matures until days 8 to 9, the second generation until days 13 to 14 post infection (p.i.). The chronic phase starts with the formation of sarcocysts around day 27 p.i. (Dubey et al., 1989). Mild infections do usually not cause clinical signs in naturally infected animals, but weight gain and meat quality of infected pigs is reduced over the whole fattening period, even at subclinical doses. This results in economic losses for the producers (Daugschies et al., 1988). The aim of the present study was to analyse breed differences in susceptibility/resistance against S. miescheriana in the breeds Pietrain and Meishan, in order to prove these breeds for subsequent quantitative trait loci (QTL) mapping on traits of protozoan host-parasite-interaction.

2. Materials and methods

2.1 Animals

Pigs of both sexes of the Chinese breed Meishan (ME, n=11) and the European breed Pietrain (PI, n=14) were used. They were serologically negative for Sarcocystis miescheriana as determined with ELISA (see 2.4) and clinical healthy prior to use. The pigs were housed before infection up to an age of 83 days in two separated barns of the experimental station "Unterer Lindenhof" of the University of Hohenheim. Five ME and 6 PI were housed in stable 1 in pens of one pig each, 6 ME and 8 PI were housed in two groups in stable 2. Sex relation was balanced in the groups. The pigs were monitored over a period of 7 days prior to infection, and thus, each pig served as its own control. Throughout the period of observation, pigs were fed ad libitum with a customary cereal diet of 11.5 MJ energy, with no food additive.

2.2 Infection and clinical examination

Pigs were infected orally at an age of 97±0.5 days (d0) with a dose of 50.000 sporocysts per animal. Sporocysts were collected from the intestine of a dog previously fed cyst-harbouring pig meat as described in detail by Rommel et al. (1995) and were stored thereafter at 4°C until use two weeks later.

Clinical examination (rectal temperature, heart rates) took place daily from day 7 before infection (day -7) to day 21 after infection, and weekly from day 28 until day 77.

On days 0, 7, 14, 21, 28, 35, 42, and 49, blood samples were obtained from cranial vena cava of each pig.

2.3 Haematology and blood levels of tissue enzymes

Differential leucocyte counts were made by distinguishing 100 leucocytes on Giemsa-stained thin blood films. Activities of creatin kinase (CK) and aspartate aminotransferase (AST) in the plasma were measured using UV-tests (Boehringer, Mannheim).

2.4 Antibody titers

Sarcocystis-specific antibody titers were determined by ELISA. Antigen was prepared from a muscle sample obtained from a pig slaughtered 73 days after infection with 50.000 sporocysts and positioned in a 96-well microtiter plate at a concentration of 1.6 µg antigen per well (IgG), 1.6 µg/well (IgA), and 0.5 µg/well (IgM). Sera were diluted with PBS-BSA (1%) 1:200 (IgG), 1:100 (IgA), and 1:400 (IgM). The Konjugat was horseraddish peroxidase-coupled goat anti-porcine IgG diluted with PBS-BSA 1:30.000 (IgG), 1: 20.000 (IgA), and 1: 20.000 (IgM).

2.5 Determination of sarcocyst density in muscles

Pigs were slaughtered after 80±3 days p.i. Numbers of brachyzoites were counted after trypsin-digestion of 12 g muscle tissue for 45 minutes at 37°C, according to the method described by Jäkel et al. (1999) in heart, Musculus longissimus dorsi (M.l.d.), M. semimembranosus (M.sm.), diaphragm and Musculus masseter (M.m.). Bradyzoites were counted in a Neubacher counter.

2.6 Statistical analysis

Traits were mainly calculated as deviations from values reached before infection (days -7 to 0). Statistical analysis was done with the procedure GLM of the SAS-program-package. Analyses of variance were used including the effects of breed (PI/ME), sex (male,female) and group (single pen/group) were considered. Sums of squares (type III) were attributed to each factor, given all other terms were in the model.

3. Results

3.1 Clinical findings

Body temperature was 0.37°C higher in PI than in ME during the whole period of examination (Fig. 1A). Mean temperature prior to inoculation was 39.49°C in PI and 39.12°C in ME. Deviations from base-line temperature prior to infection are shown in Figure 1B. A first fever peak was seen in PI (39.51°C), including days 9 and 10 (ME: 39.05°C). A second fever peak, in average 1.3°C above baseline in both breeds, with mean body temperatures of 40.8 in PI and 40.4 in ME was found at day 13 p.i. Base line temperatures were reached again on day 17 and stayed stabil in ME, until the end of the investigation (day 77). In PI, a third fever peak with temperatures about 40°C was observed between days 42 and 45. Individual body temperature varied considerably. Maximal temperatures on days 45 were 41.0°C in PI and 39.7 in ME. Temperature peaks were not influenced by single/group-housing nor by the sex (data not shown). Breed differences were found on days 8 to 9 and 35 to 49 p.i. Pietrain had always the higher increase in temperature.

Heart rates of PI were significantly higher than those of ME (Fig. 2A). Deviations from base-line values of the period before infection (Fig. 2B), were small in ME. Heart rates of the Pietrain pigs were seen to decrease during the periods day 12 to day 16 and day 35 to the end of the investigation.

Clinical signs of disease such as weakness, anorexia and reduced water intake, were seen in all infected pigs, in particular at days 12 and 42, but not measured quantitatively. None of the ME, but two PI died (days 47 and 63), with no specific necropsy findings.

3.2 Serum enzyme activities

Creatine kinase (CK) values of the pigs were statistically significant different in both breeds, before and after infection with Sarcocystis (Fig. 3A). CK-values rose linearly from day 0 to day 42. In Meishan, CK-values did not change until day 14, and then started to rise too. CK-values relatively to the base-line situation ante infectionem were different in both breeds on day 14 p.i. (Fig. 3B). Basal values (a.i.) differed between breeds also for AST-values (Fig. 3C). After infection with Sarcocystis, AST-values reached 213% and 148% of the basal levels in ME, but 313% and 2036% in PI at days 14 and 42, respectively (Fig. 3D). Breed differences were significant at day 42 (p<0.0001).

3.3 Leucocyte count

As documented in Fig. 4, relative numbers of circulating leucocytes varied with time after infection with Sarcocystis. Lymphocyte numbers decreased during the first 14 days after infection and were elevated from days 21 to 42 (Fig. 4A). The degree of decrease was higher in PI than in ME (p<0.05). Monocyte numbers increased until day 21 and decreased to base-line values until day 42. The rate of decrease was faster in PI than in ME (Fig. 4B). Relative numbers of neutrophils stayed more or less base-line in PI, except of a decrease at day 21 p.i. An increase at day 14 in ME lead to distinct breed differences on that day (Fig. 4C). Relative amounts of basophils varied only slightly, with no significant differences between breeds (Fig. 4D). Course of eosinophils numbers were parallel in both breeds, with no significant breed differences (Fig. 4E).

3.4 Serology

The IgM anti Sarcocystis response increased up to day 21 in both breeds (Fig. 5A). Antibody titers were significantly higher in ME at days 21 and 28. A further increase was found in PI up to day 42, but not in ME. A specific IgG response was recognised from day 21, with a larger increase in PI than in ME (Fig. 5B). Breed differences in IgG-titers were increasing from day 21 to the end of the investigation (day 82). IgA-levels increased slightly two weeks after infection with Sarcocystis, with almost no differences between breeds (Fig. 5C).

3.5 Bradyzoites in muscle tissues

At day 82 post infection, several muscles were analysed for numbers of bradyzoites. The numbers varied clearly with the muscle, animal and breed (Fig. 6). Muscles of PI had always higher numbers of bradyzoites. Breeds differed significantly in all muscles, except for M. masseter (M.m.). Highest differences occured in M. longissimus dorsi (M.l.d.; p<0.001) and in the heart (p<0.01).

Logarithms of ME bradycoite counts varied between 3.0 and 4.6, i.e. 20.000 bradyzoites per g in M.l.d. and 140.000 bradyzoites per g in M.m. Corresponding logarithms of numbers in PI were 5.0 to 5.6 (330.000 bradycoits per g M.l.d. and 6.3 Mio bradyzoites per g M.m.) - average counts were 130.000/g in ME and 2.71 Mio/g in PI.

3.6 Trait-differences between breeds

Differences between PI and ME regarding Sarcocystis resistance/susceptibility, are shown in Table 1 in units of their standard deviations (SD). Major differences were seen for clinical traits at day 42 p.i., and for the numbers of sarcocysts counted at day 82. Some differences were also found at days 14 (relative numbers of leucocytes) and 28 (immunoglobulines). Differences in IgG-titers were significant at any time set.

3.7 Correlations between traits

Correlations between selected traits from different days after inoculation with S. miescheriana are given in Table 2. For this purpose, the traits were selected at days after inoculation, for which the largest differences between breeds have been observed. Body temperature at day 42 (TD42) was closely related with AST (AST42) and CK (CK42). The traits were significantly correlated with each other, with immunoglobulin levels at days 14 and 21, and with bradyzoite numbers in muscle tissues at slaughter (e.g. logM.l.d.). TD14 was positively correlated with numbers of bradyzoites in muscle tissues at slaughter (logAV). Variance in body temperature at day 42 can be explained by a model including IgM titers at days 14 and 21 and bradycoite numbers in M.l.d. to an extent of 70.8% (p<0.0001), with a multiple correlation-coefficient of 0.84. Relations between clinical findings, immunoglobuline levels and bradyzoite counts are summarized in Fig. 7. In five groups, formed on the basis of rectal temperatures at day 42 p.i., temperature increases from group 1 to group5 (p<0.0001). A parallel course could be found for the logarithm of bradyzoite counts (e.g. in M.l.d.; p<0.05), and an antiparallel course for the logarithm of IgM titers at day 21 (IgM21; p<0.05). Pigs of the breed PI are concentrated within the severly affected groups 4 and 5. They are missing in group 1. Meishan pigs on the other hand are mainly within groups 1 to 3 and are not represented within group 5.

Discussion

The results of this study show variation in susceptibility/resistance to infection with S. miescheriana between genetically diverse pigs. The Chinese Meishan breed is characterised as a hyperprolific line with high fat deposition and low muscularity in carcass (Ford, 1997). The European Pietrain breed is meat producing with inferiour reproductive performance and often accompanied by the problem of stress susceptibility.

The oral dose of 50.000 sporocysts was high enough to induce clinical signs in all pigs at distinct stages p.i., and produced a breed-specific course of disease. Statistically significant differences were seen in clinical findings, serology and haematology at different periods of time p.i., and in sarcocyst numbers of different muscle tissues. All the symptoms showed one major peak at the time of the second schizogony in Meishan, but two peaks in Pietrain, one at the second schizogony and a second at the time of sarcocyst formation, starting from day 28. The maximum of this peak was reached around days 42 to 45. A third peak in PI, representing the first schizogony was seen on days 9 and 10, but was not significant. Correlations indicate, that the severity of clinical, serological and haematological signs during the period of sarcocysts formation is negatively correlated with specific immunoglobuline titers during the first 3 weeks p.i., i.e., the rapid onset of immunity in Meishan eliminates merozoites during acute phase of Sarcocystis infection. The number of merozoites is decreased, leading to lower loads of sarcocysts in muscle tissues. One of the main factors forming these differences is the formation of IgM. It is quicker in ME than in PI. A statistical model regarding IgM titers at day 14 and 21 and bradyzoite numbers in M.l.d. explained 70.8% of the total variance in body temperature at day 42. PI-pigs showed similar clinical signs as ME during the phase of the second schizogony (day 14), when only small differences were seen. Larger differences between breeds were visible in the logCK14 level and in haematological parameters at day 14 p.i. Differences in immunoglobuline titres (IgM and IgG) rose from day 14, until day 49, respectively the end of the study (day 82). The number of sarcocysts in muscle tissues, mainly in the M.l.d, were more than 20 times higher in PI than in ME. Taking into account a more than 4-fold higher M.l.d.-mass in PI, the load that formed from the inoculation of 5*10⁴ sarcocyst load can be expected to be 80 times higher in Pietrain than in Meishan.

The employment of disease resistance in farm animals as a measure of prophylaxis and disease prevention needs genetic markers, linked to disease resistance genes. Genome-wide scans of F₂-families, derived from founder breeds differing in resistance/susceptibility against a certain pathogene or a group of causative agents, are valuable starting points to identify gene-loci involved (Reiner et al. 2002). As resistance/susceptibility most often is a quantitative problem, gene-loci involved are often called Quantitative Trait Loci (QTLs). Success of QTL-experiments depend on the trait differences between founder breeds relative to the within breed variation (Haley and Andersson, 1994). With differences in the range of 1 to 6 standard deviations between Meishan and Pietrain pigs, for parameters of susceptibility/resistance after infection with S. miescheriana, one of the major requirements for QTL-analysis in F₂-individuals formed on the basis of Pietrain and Meishan pigs as founder breeds is given.

References

Adams, L.G., Templeton, J.W., 1998. Genetic resistance to bacterial diseases of animals. Rev. Sci. Tech. Off. Int. Epiz. 17, 200-219.

Barrows, P.L., Prestwood, A.K., Green, C.E., 1982. Experimental Sarcocystis miescheriana infections: Disease in growing pigs. Am. J. Vet. Res. 43, 1409-1412.

Bumstead, N., 1998. Genetic resistance to avian viruses. Rev. Sci. Tech. Off. Int. Epiz. 17, 249-255.

Daugschies, A.T., Schnieder, T., Rommel, M., Bickardt, K., 1988. The effects of Sarcocystis miescheriana infections on blood enzymes and weight gain of stress-sensitive and stress-insensitive pigs. Vet. Parasitol. 27, 221-229.

Dubey, J.P., Speer, C.A., Fayer, R., 1989. Sarcobradysis of animals and man. CRC Press Inc. , Boca Raton, Florida, 215 p.

Ford, S.P., 1997. Embryonic and fetal development in different genotypes in pigs. J. Reprod. Fertil. Suppl. 52, 165-176.

Haley, C.S., Andersson, L., 1994. Linkage mapping of quantitative trait loci in plants and animals. In: Dear, P.H. (ed.): Genome Mapping, IRL press, Oxford, NewYork, Tokio, pp 49-71.

Jäkel, T., Archer-Baumann, C., Boehmler, A.M., Sorger, I., Henke, M., Kliemt, D., Mackenstedt, U., 1999. Identification of a subpopulation of merozoites of Sarcocystis singaporensis that invades and partially develops inside muscle cells in vitro. Parasitology 118, 235-244.

Reiner, G., Melchinger, E., Kramarova, M., Pfaff, E., Büttner, M., Saalmüller, A., Geldermann, H., 2002. Detection of quantitative trait loci for resistance/susceptibility to the pseudorabies virus in swine. J. Gen. Virol. 83, 167-172.

Rommel, M., Tenter, A.M., Horn, K., Daugschies, A., Mertens, C., Heydorn, A.O., Entzeroth, R., Eschenbacher, K.H., Klein, H., 1995. Sarcocystis. In: "Guidelines on techniques in coccidiosis research" (eds. J. Eckert, R. Braun, M.W. Shirley, P. Coudert). Office for Official Publications of the European Communities, Luxembourg, pp. 241-276.

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Table 1: Trait differences between PI and ME in standard deviations (SD).

Table 2: Correlations between selected clinical, serological and parasitological traits obtained at different days post inoculation with 5*10⁴ sporocysts of Sarcocystis miescheriana.

Fig. 1: Least Square Means ± SE of rectal temperatures (A) and deviations from base line ante infectionem of rectal temperatures (B) of Pietrain (PI) and Meishan (ME) after oral infection with 5*10⁴ sporocysts of Sarcocystis miescheriana. Significances: A: differences in rectal temperatures are statistically significant at least on the p<0.05 threshold except of days 1, 11, 12 and 15. B: *: p<0.05; ***: p<0.001.

Fig. 2: Least Square Means ± SE of heart rates (A) and deviations from base line ante infectionem of heart rates (B) of Pietrain (PI) and Meishan (ME) after oral infection with 5*10⁴ sporocysts of Sarcocystis miescheriana. Significances: A: B: *: p<0.05; **: p<0.01; all other measures were significant at p<0.001.

Fig. 3: Creatine kinase (CK, U/l, mean log) values (A), deviation of CK-values from base-line a.i. (B), Aspartate aminotransferase (AST, U/l) values (C) and deviation of AST-values from a.i. base-line (D) of Pietrain (PI) and Meishan (ME) on days 0, 14 and 42 after oral infection with 5*10⁴ sporocysts of Sarcocystis miescheriana. *: p<0.05; **: p < 0.01; ***p<0.001.

Fig. 4: Deviations of relative numbers of leucocytes at days 0 to 42 in Pietrain (PI) and Meishan (ME) after oral infection with 5*10⁴ sporozoites of Sarcocystis miescheriana. (A) Lymphocytes, (B) Monocytes, (C) Neutrophiles, (D) Basophiles, (E) Eosinophiles. *: p<0.05; **: p<0.01.

Fig. 5: Antibody titers of IgM (A), IgG (B) and IgA (C) in Pietrain (PI) and Meishan (ME) after oral infection with 5*10⁴ sporocysts of Sarcocystis miescheriana. *: p<0.05; **:p<0.01; ***:p<0.001.

Fig. 6: Logarithm of bradyzoite numbers (Mio/g) of Pietrain (PI) and Meishan (ME) in heart, Musculus longissimus dorsi (M.l.d.), M. semimembranosus (M. sm.), diaphragm, M. masseter (M.m.) and on average after inoculation with 5*10⁴ sporocysts of Sarcocystis miescheriana. *: p < 0.05; **: p<0.01; ***: p<0.001.

Table 1

Trait	day 14	day 21	day 28	day 35	day42
Rectal¹ temperature	0.08	1.49	2.73*	1.45	5.63***
Heart-rate¹	4.72**	0.22	3.69*	1.44	6.32***
AST¹	0.39	-	-	-	1.47***
logCK¹	0.79*	-	-	-	0.29
IgM	0.34	0.96*	1.00**	0.62	1.09
IgG	0.67*	0.79**	0.33**	0.89***	2.02***
IgA	0.36	0.44	0.09	0.09	0.69
Lymphocytes¹	0.72*	0.87	0.49	-	0.47
Monocytes¹	0.24	0.29	1.10*	-	0.18
Eosinophiles¹	0.54	0.02	0.24	-	0.23
Basophiles¹	0.42	0.24	0.45	-	0.27
Neutrophiles¹	1.06**	0.56	0.56	-	0.05
LogCyst heart²	4,67**
logCyst M.l.d.²	5,38***
logCyst M.sm.²	2,67*
logCyst diaphragm²	2,62*
LogCyst M.m.²	1,80
LogCyst average²	4.57**

¹ deviations from values obtained prior to infection; ² bradyzoite counts at 82 days p.i.;

*: p<0.05; **: p<0.01; ***:p<0.001; -: not measured.

Table 2

	TD42	AST42	CK42	IgM14	IgM21	IgG14	IgG21	IgG82	logMld	LogAV
TD14	-.04	-.26	-.03	.16	-.04	.12	.07	.07	.39*	.51**
TD42	-	.70***	.67***	-.57**	-.45*	-.57**	-.48*	.26	.58**	.30
AST42		-	.80***	-.49**	-.50**	-.55**	-.50**	.53**	.47*	.24
logCK42			-	-.46*	-.58**	-.45***	-.47***	.54**	.35*	.34*
IgM14				-	-.69***	-.52**	.22	-.11	-.10	.17
IgM21					-	.62***	.30	-.17	-.35*	-.01
IgG14						-	.39*	.17	-.01	.13
IgG21							-	-.05	-.20	-.26
IgG82								-	.60**	.54**
LogM.l.d.									-	.70***

TD: deviation in temperature from values ante infectionem; LogM.l.d.: logarithm of bradyzoite numbers in M.longissimus dorsi; LogAV: average logarithm of bradyzoite numbers in the muscle tissues under investigation. *: p<0.05; **: p<0.01; ***:p<0.001;

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Fig. 7: Relations between clinical findings (TD42 in °C; deviation in temperature on day 42 from values ante infectionem), immunoglobulin levels (log IgM21; logarithm of IgM titer on day 21), bradycoite counts in M. longissimus dorsi (log M.l.d.) and breed distribution in five groups, formed on the basis of rectal temperatures on day 42 p.i..