Veterinary Parasitology 197 (2013) 487–497 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Transfer of Cystoisospora suis-specific colostral antibodies and their correlation with the course of neonatal porcine cystoisosporosis Lukas Schwarz 1 , Anja Joachim, Hanna Lucia Worliczek ∗ Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria a r t i c l e i n f o Article history: Received 22 May 2013 Received in revised form 8 July 2013 Accepted 9 July 2013 Keywords: Coccidiosis Antibody development Immunoglobulins Pig Swine a b s t r a c t Cystoisospora suis is the most pathogenic species of coccidia in suckling piglets, affecting them predominantly within their first three weeks of life. The clinical signs of neonatal cystoisosporosis include watery diarrhea and wasting, leading to significant economic losses for the farmer. Since neonatal piglets have an immature immune system, colostral transfer of maternal factors such as immune cells or antibodies is essential for controlling infections at that age. However, the role of C. suis-specific antibodies transferred from the sow to the piglets and possible correlations between antibody levels in the piglets acquired from colostrum with the clinical outcome of disease are currently not understood. To address this issue, 12 non-infected piglets and 14 piglets experimentally infected with C. suis on the third day of life were examined during their first four weeks of life. IgG, IgA, and IgM titers in the blood serum specific for sporozoites and merozoites of C. suis were evaluated, along with oocyst excretion and fecal consistency. Additionally, the antibody content in the colostrum and milk of three mother sows was determined. A transfer of naturally acquired C. suis-specific antibodies from sows to piglets with the colostrum could be demonstrated. Maternal antibodies in piglets’ blood sera did not persist for longer than 14–21 days except for IgG which was present in high titers until the end of the study. Within 2–3 weeks after birth the onset of endogenous antibody production was noticed. Titers in blood serum showed a correlation with the severity of diarrhea which was positive for IgG and IgM (possibly due to increased consumption or loss of these antibodies) and negative for IgA. C. suis-specific mucus antibodies isolated from infected and non-infected piglets (n = 6/group) on the 28th day of life were present in both groups, showing significantly higher titers of IgA and IgM in infected piglets. Maternally transferred antibodies acquired by natural infections of sows as observed in this study did not provide protection against the clinical manifestation of disease. The level and effect of transferrable maternal factors necessary for protection still need to be elucidated. However, correlations between antibody titers and fecal consistency in the piglets indicate that C. suis-specific antibodies might be useful markers for the expectable clinical severity of cystoisosporosis. © 2013 Elsevier B.V. All rights reserved. 1. Introduction ∗ Corresponding author at: Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, A-1210 Vienna, Austria. Tel.: +43 1 25077 2227; fax: +43 1 25077 2290. E-mail address: Hanna.Worliczek@vetmeduni.ac.at (H.L. Worliczek). 1 Current address: Clinic for Swine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria. 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.07.007 Cystoisospora suis is the most pathogenic species of coccidia in suckling piglets affecting the animals most severely within their first three weeks of life (Harleman and Meyer, 1983; Stuart and Lindsay, 1986; Meyer et al., 1999; Daugschies et al., 2004). Pathological changes include 488 L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 crypt hyperplasia and atrophy and necrosis of the villi in the small intestine, which cause diarrhea and decreased absorption of nutrients. These changes in turn lead to reduced and uneven weaning weights with subsequent economic losses for the farmer as a result from reduced weight gain and poor performance in the suckling and postweaning phase (Stuart et al., 1982a; Lindsay et al., 1985a,b; Mundt et al., 2005, 2006; Scala et al., 2009; Aliaga-Leyton et al., 2011a,b; Kreiner et al., 2011). At this stage of life immunity against pathogens, either passively transferred via colostrum or actively acquired, is generally considered to be of major relevance for survival and later performance of piglets (Wagstrom et al., 2000; Salmon et al., 2009; Jourquin et al., 2010). In the case of C. suis little information regarding the development of protective immunity is available, and virtually nothing is known about the role of the humoral immune system (Taylor, 1984; Jarvinen et al., 1988; Baekbo et al., 1994; Koudela and Kucerova, 1999, 2000; Worliczek et al., 2009a,b, 2010a). Piglets acquire resistance to reinfection after infections with C. suis, and naïve older piglets seem to be rather unsusceptible to clinical coccidiosis, reflecting a natural age resistance (Stuart et al., 1982b; Stuart and Lindsay, 1986; Koudela and Kucerova, 1999, 2000). Several studies have shown that cellular components are involved in the development of immunity against coccidia including C. suis (Rommel and Heydorn, 1971; Taylor, 1984; Wakelin and Rose, 1990; Yun et al., 2000; Buggelsheim, 2008; Worliczek et al., 2010a,b). However, at the time when the infection with C. suis causes the most severe clinical and pathological changes, i.e. in the first three weeks of life, the porcine immune system is functionally immature and therefore most likely unable to control the infection (Becker and Misfeldt, 1993). In general, most authors do not consider passive immunity against C. suis to be transferrable from the mother sow to the piglets (Stuart et al., 1982b; Taylor, 1984; Stuart and Lindsay, 1986; Baekbo et al., 1994); however, some proposed an involvement of colostral antibodies in resistance against natural infections with C. suis (O’Neill and Parfitt, 1976; Greve, 1985). Several studies demonstrated the protective effects of antibodies on invasive stages of poultry and bovine coccidia both in vitro and in vivo (Long and Rose, 1972; Davis and Porter, 1979; Witlock and Danforth, 1982; Crane et al., 1986a,b; Whitmire et al., 1988; Trees et al., 1989; Wallach, 2010) and passive immunity in the offspring (Wakelin and Rose, 1990; Jenkins et al., 1999; Perryman et al., 1999; Sharman et al., 2010). Taylor (1984) could not determine a correlation between piglet serum IgG titers against C. suis sporozoites and the course of clinical disease; however, other antibody subclasses and parasitological parameters were not investigated in her work. On the other hand, suckling piglets experimentally infected with C. suis showed an increase of B cells in mesenteric lymph nodes. This possibly reflects an activation of the humoral immune system (Worliczek et al., 2010a), although it must be assumed that piglets in the first three weeks of life are not capable of an immune response to C. suis in the same way as older pigs due to the immaturity of the immune system, and that their ability to control the parasite must therefore be limited (Becker and Misfeldt, 1993; Sinkora and Butler, 2009). It is only at an age of two weeks of life that the gut of piglets becomes colonized with lymphocytes, and complete colonization is achieved only at an age of about seven weeks (Stokes et al., 2004). Due to the inability of the neonatal immune system of piglets to react in an efficient way to C. suis infections, maternal factors transferred to the piglets via colostrum may contribute to the control of the disease. The main site for invasion and replication of C. suis is the jejunal epithelium (Stuart et al., 1980; Harleman and Meyer, 1983; Koudela and Kucerova, 2000). In this compartment, antibodies – either maternally derived from colostrum/milk or self-produced – could directly interact with stages in the gut lumen and might therefore be of major importance for immune protection against invasive sporozoites or merozoites, similar to the protection against Cryptosporidium parvum infections (Martin-Gomez et al., 2005a,b). Thus, investigations on the presence and role of C. suis-specific antibodies in the jejunal mucus might contribute to a better understanding of protective immune mechanisms. The present study was based on the hypothesis that antibodies specific against invasive stages of C. suis naturally acquired by mother sows are transferred to their piglets via colostrum uptake. Furthermore, we expected a correlation between higher maternal antibody titers in suckling piglets and a milder course of disease after experimental infections with C. suis. To address these points, we examined sow colostrum/milk as well as blood serum and jejunal mucus from infected and non-infected piglets for the presence of C. suis-specific antibodies against sporozoites and merozoites. In parallel, oocyst excretion and fecal consistency were investigated to analyze the correlation between antibody titers and the outcome of disease. 2. Materials and methods 2.1. Study animals A total of 26 conventionally raised piglets from six crossbred sows were used for this study. Four sows were housed on straw in farrowing crates in the local animal husbandry facilities at the Institute of Parasitology, University of Veterinary Medicine Vienna, Austria. Two sows (nos. 4 and 5) were kept in a biosafety unit of the University of Veterinary Medicine Vienna, Austria, to prevent infections with coccidia. These sows were housed under the same conditions as those in the non-biosafety facility but without straw. Sows arrived two weeks prior to parturition and their feces were examined for parasite shedding at this time. If fecal samples tested positive for parasites, sows were treated with fenbendazole (PANACUR® Pulver 4% für Schweine, Intervet GmbH, Vienna, Austria, 5 mg/kg body weight (BW) p.o.), ivermectin (Noromectin® , Norbrook Laboratories, Newry, Northern Ireland, 1.5 ml/50 kg BW i.m.) or sulphonamides (200 g sulfadimidine sodium ad 1333 g glucose monohydrate, hospital pharmacy of the University of Veterinary Medicine Vienna, Vienna, Austria, 50 mg/kg BW for 7 days p.o.). After treatment, a final fecal examination was carried out and sows tested negative for parasite excretion. L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 489 Table 1 Composition of litters and blood/milk sampling schedule. Piglets no. 1–8 were investigated during a pilot study (Schlepers, 2009). Study day (SD) 0 was the day of birth. Piglet number Litter Infection Blood samples on SD Milk samples on SD 1–5 6 7–8 9–14 15–20 21–26 1 2 3 4 5 6 + + + − − + 7, 14, 21, 28 7, 14, 21, 28 7, 14, 21, 28 0, 1, 7, 14, 21, 28 0, 1, 7, 14, 21 0, 1, 7, 14, 21, 28 0, 1, 7, 14, 21, 28 0, 1, 7, 14, 21 0, 1, 7, 14, 21, 28 In order to prevent C. suis introduction into the two noninfected control litters sows housed in the biosafety unit were introduced two weeks prior to the farrowing date and washed ante partum using commercial detergents. Sows were kept in a parasite-free environment and introduction of parasites was prevented by standard hygiene measures. All sows were fed once daily with a commercial diet. Municipal drinking water was available for the sows and their piglets ad libitum. Commercial piglet starter feed was offered ad libitum to the piglets from the second week of life on. The day of birth was defined as study day (SD) 0. Clinically healthy piglets with a birth weight > 900 g were included in the study. Immediately after birth, each piglet was marked individually and weighed. Body weights were measured weekly until SD 28. On SD 3, all piglets were vaccinated against Mycoplasma hyopneumoniae (Stellamun® Mycoplasma; Pfizer Pharma GmbH, Karlsruhe, Germany; 2 ml i.m.) and received iron dextran (Vanafer® , Vana, Vienna, Austria; 1 ml s.c.). Piglets from litter 4, 5 and 6 were additionally supplemented with iron dextran a second time on SD 10 because they underwent more frequent blood sampling than the other litters (see Table 1 for sampling procedures). All procedures involving animals were approved by the Animal Ethics Committee of the University of Veterinary Medicine Vienna and the Austrian Federal Ministry of Science and Research according to the Austrian Animal Protection law (BMWF-68.205/0167-II/3b/2010). 2.2. Litters Based on a pilot study which investigated eight piglets (litters 1–3) (Schlepers, 2009), three additional litters (litter 4–6) were examined in this study (Table 1). Antibody titers in blood serum samples from piglets of the pilot study were re-evaluated for the present study; data of fecal consistency and oocyst excretion were extracted from the original records. 2.3. Parasites and experimental infection The oocysts were stored at 10–12 ◦ C in aqueous suspension with 2% potassium dichromate for a maximum of six months prior to infection. Before infection oocysts were washed in tap water. Piglets were infected orally on SD 3 with 1000 sporulated oocysts of C. suis (strain Wien I) in 1 ml tap water isolated from previously experimentally infected piglets (Ruttkowski et al., 2001; Worliczek et al., 2009a). 2.4. Evaluation of fecal samples Individual fecal samples were taken from SD 7–21 daily from infected piglets and every other day from noninfected piglets. Fecal consistency was scored according to the following key: fecal score (FS) 1 = normal, FS 2 = pasty, FS 3 = semi-liquid and FS 4 = liquid, with FS 3 and 4 considered as diarrhea (Mundt et al., 2006). Fecal samples were screened for oocysts by autofluorescence detection under UV light (Daugschies et al., 2001). Positive samples were further quantified with a modified McMaster technique (Meyer et al., 1999) and oocysts per gram of feces (OPG) were calculated. 2.5. Blood sampling Blood samples were collected by puncture of the jugular vein with a syringe and a needle (Primavette® , KabeLabortechnik GmbH, Nümbrecht-Elsenroth, Germany) according to the frequency specified in Table 1. Blood samples for serum production were centrifuged for 10 min at 1500 × g after coagulation occurred. The serum was transferred into 1.5 ml conical tubes and stored at −20 ◦ C until further use. 2.6. Colostrum/milk samples Colostrum (SD 0) and milk samples were collected from sow no. 4, 5, and 6 by manual milking (for sampled animals and dates see Table 1). Samples were centrifuged at 8400 × g for 30 min at 10 ◦ C. After centrifugation the cream phase was removed and the liquid whey was stored at −20 ◦ C until further use. 2.7. Isolation of mucus antibodies Six infected and 6 non-infected piglets were sacrificed for isolation of mucus antibodies at 28 days of age. Piglets were sacrificed using 1.2 mg/kg BW of azaperone and 10 mg/kg BW ketamine for anesthesia followed by exsanguination. Different isolation methods described for mice and pigs (Parr et al., 1998; Fang et al., 2000) were adapted for the piglet gut. A piece of 10 cm of jejunum of each sacrificed piglet was washed with PBS (PAA, Pasching, Austria) to remove fecal contents. The cleaned gut was then opened longitudinally, cut in pieces of 1 cm length, and 10 ml PBS were added. The mucus antibodies were allowed to dissolve into the PBS using a magnetic stirrer for 15 min. The tissue pieces were removed and the mucus-PBS-suspension was collected in a 15 ml tube. The 490 L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 suspension was centrifuged (1500 × g, 10 min) three times, the pellet was resuspended after each centrifugation step to dissolve as much antibodies as possible from the debris. After a final centrifugation (8000 × g, 30 min) the supernatant containing the mucus antibodies was collected and stored at −20 ◦ C until further use. 2.8. Antigen production The sporozoites used as antigen in the immunofluorescence antibody tests (IFAT) were gained by excystation (Worliczek et al., 2013) and were washed twice with PBS (1000 × g, 10 min). Merozoites were produced in cell culture (Worliczek et al., 2013) using intestinal porcine epithelial cells (IPEC-J2, ACC 701, Leibniz Institute DSMZ GmbH, Braunschweig, Germany). Briefly, IPEC-J2 were maintained in DMEM/Ham’s F12 supplemented with 5% fetal calf serum (FCS), 2 mM l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (PAA, Pasching, Austria) (Worliczek et al., 2013). An infection dose of 1:10 (sporozoites:host cells) was used. For harvesting of parasites, a calcium ionophore (calcimycin – A23187, Sigma, Vienna, Austria) was used in a final concentration of 5 ␮M in culture medium (DMEM/Ham’s F12 + 5% FCS, PAA, Pasching, Austria) on day 5 until day 7 post infectionem (Behrendt et al., 2008). After incubation of infected cells with A23187 for 20 min at 37 ◦ C supernatant containing free merozoites was collected and the flask was washed once with PBS to retrieve the remaining merozoites. Harvested merozoites were centrifuged (1000 × g, 10 min) and washed two times with PBS (1000 × g, 10 min). 2.8.1. Preparation of 10-dot slides For the IFAT 10-dot slides (Medco, Munich, Germany) were used. Parasite cells were diluted in PBS to reach a density of approximately 30 sporozoites or merozoites per dot. Coated slides were dried in a heating cabinet at 40 ◦ C overnight and stored at −80 ◦ C until further use. Fig. 1. Prevalences (in percent) of excretion and diarrhea in infected piglets (n = 14) from SD 7 to SD 28. The gray frame indicates the phase of acute infection of piglets (SD 10–19). a fluorescence microscope. Titer ≥1:40 were considered as positive. Titrations were performed to a negative titer. For statistical analysis antibody titers were transformed to numerics: 0 = neg. (< 1 : 40), 1 = 1 : 40, 2 = 1 : 80, . . ., max. : 10 = 1 : 20480. 2.10. Statistical analyses Statistical evaluation was performed using PASW Statistics 17.0 (SPSS Inc., Chicago, IL, USA). Data were split to SD and analyzed with Mann–Whitney (Wilcoxon) two-sample tests comparing infected and non-infected animals. Spearman’s rank correlation coefficients were determined for IFAT results, and the area under the curve (AUC) of OPG and FS from SD 7-21-21. Significance was assumed for p ≤ 0.05. 2.9. Immunofluorescence antibody test – IFAT 3. Results Each blood/milk serum and mucus sample was tested with sporozoites and merozoites as antigen. Slides were thawed, dried at room temperature and fixed for 10 min in acetone (−20 ◦ C) which was subsequently allowed to evaporate from the slides. Serum was added to the slides in serial dilutions starting with 1:40 and slides were incubated for 30 min at 37 ◦ C. Positive and negative references (colostral serum from a highly positive sow and precolostral blood serum, respectively) obtained from a previous trial were included in 1:40 dilutions. After two washing steps in PBS, samples were incubated (30 min, 37 ◦ C) with fluorescein-labeled conjugates [goat anti-pig IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) 1:150 in PBS/0.05% (w/v) Evans blue (Merck, Darmstadt, Germany), goat anti-pig IgA and goat anti-pig IgM (Bethyl Laboratories, Montgomery, TX, USA), both 1:1000 in PBS/0.05% (w/v) Evans blue]. After two washing steps in PBS the slides were covered with a cover slip using PBS/glycerin (1:10) and evaluated under 3.1. Fecal examination and body weights With the exception of one piglet of litter 5, non-infected piglets did not excrete oocysts during the examination period. This animal showed oocyst shedding on SD 23. Therefore, data of litter 5 were only used until SD 21 for analysis. This decision was made since no pathological changes are expected before the second day post infection and C. suis has a prepatent period of 4–5 days (Stuart et al., 1982a; Lindsay et al., 1985a; Mundt et al., 2006). No diarrhea occurred in the non-infected piglets over the whole study period. The acute phase of infection was characterized by diarrhea and excretion of oocysts in the infected piglets (SD 10–19). Oocysts in feces of piglets infected on SD 3 were detected from SD 8 until SD 21. Maximum prevalences of 85.7% and 78.6% were seen on SD 10 (1st excretion peak) and SD 16 (2nd excretion peak; Fig. 1). Thereafter, oocyst L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 491 Table 2 Mean body weights in gram (in brackets: standard deviation) and body weight gain of infected (+) and non-infected (−) piglets over the study period. SD = study day. Infection SD 0 SD 7 SD 14 SD 21 SD 28 Body weight gain in % + − 1922.86 (495.77) 1462.50 (113.07) 3032.86 (376.67) 2641.67 (287.49) 4297.86 (858.99) 4466.67 (809.98) 6062.14 (1271.92) 6508.33 (988.30) 8291.43 (1500.88) 7683.33 (549.24) 341.88 (60.11) 416.92 (44.99) excretion declined until the end of fecal examination. During the examination period, oocyst excretion occurred at least once in each infected piglet for an average of 7.1 days. Diarrhea was first observed on SD 10 in 57.1% of the infected piglets (Fig. 1) and persisted for a maximum of four days. Altogether, FS > 2 occurred in 10/14 (71.4%) of the infected piglets. Body weight gain was higher in the non-infected group but without significant differences between infected and non-infected piglets (Table 2). 3.2. C. suis-specific antibodies in the colostrum/milk of sows C. suis-specific antibodies were detected in colostrum and milk from three sows (two with non-infected, one with infected piglets). Titers of all antibody classes declined from the day of farrowing until SD 7 (Fig. 2). Afterwards, no clear pattern was seen for individual sows. 3.3. C. suis-specific antibodies in the blood serum of piglets IgG, IgA and IgM specific for merozoite and sporozoite antigen of C. suis were detected in both groups, showing significant differences between infected and non-infected animals (Table 3). Precolostral sera were observed to be negative for all piglets and all antibody classes (Fig. 3). On SD 1 significant differences for IgA against merozoites (Fig. 3C) and IgG, IgA, and IgM against sporozoites (Fig. 3B, D, F) between infected and non-infected piglets were found (Table 3). No correlations between colostral titers and piglet serum titers were found on SD 1 (data not shown). The initial differences in antibody titers after colostrum uptake equalized until SD 7, on this study day no significant differences between groups were detectable. In the first 24 h of life (SD 0 to SD 1) antibody titers of all classes increased strongly (Fig. 3A–F). With the exception of IgG against merozoites, all antibody titers declined rapidly after SD 7 in both infected and non-infected piglets. During the acute phase of infection (SD 10–19) no increase in antibody titers was seen in infected piglets. Only on SD 21 and 28 IgG and IgM against sporozoites increased significantly in infected piglets (Fig. 3B, F). The decrease of IgG against merozoites was only moderate in non-infected piglets. In infected animals this decrease, reflected by significantly lower IgG titers on SD 14, 21 and 28 was considerably stronger (Table 3 and Fig. 3A). IgA titers against merozoites on SD 1 were significantly correlated with the AUC of OPG values (Table 4 and Fig. 4). Fig. 2. Antibody titers of IgG, IgA and IgM against merozoites and sporozoites of C. suis in colostrum/milk of three sows. Sows no. 4 and 5 suckled noninfected (−) piglets. Sow no. 6 suckled infected (+) piglets. The gray frame indicates the phase of acute infection (SD 10–19). Study day 0 = day of parturition (colostral sampling). 492 L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 Fig. 3. Mean blood serum antibody titers (IgG, IgA, IgM) of infected and non-infected piglets against merozoites and sporozoites of C. suis. Significant differences between groups are marked with an asterisk. The gray frame indicates the phase of acute infection (SD 10–19). Study day 0 = day of birth (precolostral sampling). Significant correlations with the AUC of the FS were detected for IgM titers against merozoites on SD 1 (Table 4 and Fig. 5A), for IgG against merozoites on SD 21 and 28 (Fig. 5B, C) and for IgA titers against merozoites on SD 14, 21 and 28 (Fig. 5D–F). For antibodies against sporozoites significant positive correlations with the AUC of the FS were found for IgM on SD 7 and 14 (Table 4 and Fig. 5G, H). 3.4. Mucus samples In all investigated mucus samples (6 from infected, 6 from non-infected animals from SD 28), antibodies against Fig. 4. Correlation between IgA titers against merozoites from infected piglets with the area under the curve (AUC) of oocysts per gram of feces (OPG) on study day (SD) 1. Dots represent the data of individual piglets, the line represents the regression. The according formula and the coefficient of determination (R2 ) can be found in the respective graph. stages of C. suis from at least one class were present. Significantly higher titers were found in infected piglets for IgA and IgM against merozoites and for IgM against sporozoites. In contrast, significantly lower titers were detected in infected piglets for IgG against merozoites (Table 5). 4. Discussion C. suis infections are most severe in neonatal piglets which are not only agammaglobulinemic at birth but also show limited cellular immune responses (Rothkötter and Pabst, 1989; Bailey et al., 2001). In a previous study an increased frequency of B-cells was detected in the mesenteric lymph nodes (MLN) of piglets infected with C. suis during the acute phase of infection (Worliczek et al., 2010a), suggesting an active humoral immune response to the parasite. However, the role of maternal as well as piglet derived antibodies during piglet cystoisosporosis is still an unresolved issue (O’Neill and Parfitt, 1976; Stuart et al., 1982b; Taylor, 1984; Greve, 1985; Stuart and Lindsay, 1986; Baekbo et al., 1994). In the present study the transfer of maternal IgG, IgA and IgM against two different invasive stages of C. suis, sporozoites and merozoites, from sows to piglets could be demonstrated. The three sows under investigation regarding antibody content in colostrum and milk were non-naïve as reflected by the presence of C. suis-specific IgG, IgA and IgM antibodies in both secretions. No clear patterns were observed for the dynamics of their antibody titers. This and the small sample size do not permit conclusions on an antibody development in the milk from parturition until weaning. The level of persisting antibodies L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 493 Fig. 5. Correlations between blood serum titers from infected piglets with the area under the curve (AUC) of fecal score (FS). Only results with significant correlations are presented as scatter plots. Dots represent the data of individual piglets, the line represents the regression. The according formula and the coefficient of determination (R2 ) can be found in the respective graph. IgM against merozoites and the AUC of FS on SD 1 (A); IgG against merozoites and the AUC of FS on SD 21 (B) and 28 (C); IgA against merozoites and the AUC of FS on SD 14 (D), 21 (E) and 28 (F); IgM against sporozoites and the AUC of FS on SD 7 (G) and 14 (H). in sows might have been caused by previous transient infections or might be maintained by persisting extraintestinal stages (Harleman and Meyer, 1984). Antibodies from the sows were transferred to the piglets via colostrum within hours after birth, leading to high titers in the blood of piglets after colostrum uptake. Individual differences were most likely due to differences in colostrum consumption (Klobasa et al., 1981). The majority of maternal antibodies detected in the piglets decreased rapidly after this initial peak. At the age of one week both 494 Table 3 Results of Mann–Whitney U tests comparing antibody titers of IgG, IgA and IgM in infected (+) and non-infected (−) piglets against merozoites and sporozoites (SD 1–28). Significant results (p ≤ 0.05) are shown in bold characters. Infection SD 1 n IgG-Merozoites IgA-Merozoites IgM-Merozoites IgG-Sporozoites IgM-Sporozoites SD 14 SD 21 SD 28 X̄  p n X̄  p n X̄  p n X̄  8.58 8.33 4.08 5.00 5.67 6.00 5.67 6.50 4.25 5.17 5.83 7.00 0.79 1.21 0.79 0.63 0.49 0.63 0.49 0.55 0.62 0.75 0.58 0.00 0.678 12 14 12 14 12 14 12 14 12 14 12 14 8.67 7.71 2.17 2.21 3.67 3.29 4.00 4.00 2.75 2.14 4.08 3.64 0.49 1.68 1.19 1.12 0.49 0.83 0.60 1.18 0.75 1.41 0.67 1.50 0.374 12 14 12 14 12 14 12 14 12 14 12 14 8.67 6.79 0.50 0.79 1.67 1.57 2.42 2.79 0.83 0.43 2.25 1.71 0.49 1.37 0.67 1.12 0.65 1.09 0.67 0.89 0.72 0.65 0.75 1.33 0.002 12 14 12 14 12 14 12 14 12 14 12 14 8.08 5.64 0.00 0.29 0.08 0.29 1.25 2.64 0.17 0.43 0.67 1.57 0.67 <0.001 12 1.5 14 0.00 0.182 12 0.83 14 0.29 0.201 12 0.47 14 0.75 0.001 12 0.93 14 0.39 0.441 12 0.85 14 0.65 0.034 12 1.45 14 0.017 0.258 0.010 0.023 0.001 0.899 0.322 0.820 0.297 0.462 0.605 0.659 0.255 0.125 0.262 p n X̄  p 8.00 5.36 0.00 0.29 0.17 0.36 0.50 3.21 0.00 0.50 0.00 2.29 0.89 1.28 0.00 0.61 0.41 0.50 0.55 0.89 0.00 0.76 0.00 0.83 0.001 0.232 0.406 <0.001 0.103 <0.001 SD: study day, n: sample size, X̄: arithmetic mean, : standard deviation. Table 4 Spearman’s rank correlation coefficients (in brackets: p-value) of IFAT-results correlated with the area under the curve (AUC) of fecal scores (FS) and oocysts per gram of feces (OPG). AUC of FS and OPG of individual infected piglets were correlated with IFAT-results from SD 1–28. Significant results (p ≤ 0.05) are shown in bold characters. SD n AUC OPG AUC FS Merozoites IgG 1 7 14 21 28 6 14 14 14 14 Sporozoites IgA IgM IgG Merozoites IgA 0.270 (0.604) −0.845 (0.034) 0.338 (0.512) 0.098 (0.854) 0.772 (0.072) −0.137 (0.640) −0.205 (0.482) −0.280 (0.333) −0.302 (0.293) −0.031 (0.915) −0.323 (0.260) 0.335 (0.242) −0.099 (0.736) 0.079 (0.788) 0.055 (0.853) −0.433 (0.122) −0.36 (0.903) 0.235 (0.418) −0.161 (0.582) −0.399 (0.157) −0.449 (0.108) 0.273 (0.345) 0.129 (0.659) 0.311 (0.280) −0.289 (0.316) IgM IgG Sporozoites IgA IgM IgG C −0.412 (0.417) −.0171 (0.745) 0.857 (0.029) −0.594 (0.214) −0.257 (0.374) 0.306 (0.288) −0.422 (0.133) −0.187 (0.523) 0.249 (0.392) −0.371 (0.192) 0.403 (0.153) −0.594 (0.025) 0.318 (0.269) 0.066 (0.821) −0.401 (0.155) 0.657 (0.011) −0.607 (0.021) 0.177 (0.545) −0.012 (0.967) −0.145 (0.620) 0.824 (<0.001) −0.534 (0.049) −0.371 (0.192) 0.186 (0.525) SD: study day; n: sample size; C: not calculable, because at least one variable shows constant values. IgA IgM 0.235 (0.654) 0.166 (0.570) 0.338 (0.238) 0.018 (0.951) 0.224 (0.442) C 0.562 (0.036) 0.621 (0.018) 0.465 (0.094) 0.005 (0.987) L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 IgA-Sporozoites 12 6 12 6 12 6 12 6 12 6 12 6 − + − + − + − + − + − + SD 7 L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 495 Table 5 Results of Mann–Whitney U tests comparing jejunal mucus antibody titers of infected (+) and non-infected (−) piglets against merozoites and sporozoites on study day 28. Significant results (p ≤ 0.05) are shown in bold characters. Jejunum-IgG-Merozoites Jejunum-IgA-Merozoites Jejunum-IgM-Merozoites Jejunum-IgG-Sporozoites Jejunum-IgA-Sporozoites Jejunum-IgM-Sporozoites Infection (n) X̄  Min Max p − (6) + (6) − (6) + (6) − (6) + (6) − (6) + (6) − (6) + (6) − (6) + (6) 3.33 2.33 0.33 2.5 0.83 2.33 1.33 1.67 0.83 1.33 0.33 1.33 0.52 0.82 0.52 0.84 0.75 1.03 1.03 1.21 1.17 1.37 0.52 0.82 3 1 0 1 0 1 0 0 0 0 0 0 4 3 1 3 2 3 3 3 3 3 1 2 0.031 0.004 0.027 0.614 0.495 0.041 n: sample size, X̄: arithmetic mean, : standard deviation. groups had similar antibody titers. Therefore, the initial differences were not considered to be important for later antibody development. After the decrease of antibody titers in piglet sera upon the initial colostrum uptake, the only major increase was detected for IgG and IgM against sporozoites. The production of IgM by piglets starts around the seventh day of life (Porter and Hill, 1970) and was described to exceed the colostral IgM titers from the 14th day of life on (Habe, 1974; Klobasa et al., 1991; Rooke et al., 2003). We assume that increasing antibody titers between week three and four of life are caused by endogenous antibody production of infected piglets. This assumption is supported by the increase of B-cells in the MLN of infected piglets (Worliczek et al., 2010a). In contrast to all other titers, IgG against merozoites persisted longer and did not decrease to the same extent. The reason for this is not known. Moreover, less IgG was found in infected piglets from day 14 on compared to non-infected ones. Beside the specific antibodies detected in blood serum of piglets they were also found in the jejunal mucus. Infected animals had significantly higher IgA and IgM titers against merozoites and higher IgM titers against sporozoites in jejunal mucus than non-infected piglets. In contrast, IgG titers against merozoites were significantly higher in non-infected animals – this finding is in good agreement with reduced IgG blood serum titers against merozoites in infected piglets and possibly reflects a consumption of these antibodies due to infection. Overall, the results suggest a similar importance of IgA and IgM in the local immune response to C. suis as described for mucosal antibodies in piglets in general (Butler, 2006; Suzuki et al., 2007). Despite the high serum titers experimental infection with C. suis on SD 3 led to oocyst shedding and diarrhea in the piglets. There was no protection from a patent infection with C. suis after colostrum uptake from non-naïve sows, as described previously (Stuart and Lindsay, 1986; Baekbo et al., 1994). In general, protective effects due to maternal factors might be caused by antibodies as well as the transfer of cellular components (Williams, 1993; Wagstrom et al., 2000; Salmon et al., 2009). We assume that in the case of sows naturally infected at an unknown time point in the past, the level of C. suis-specific maternal factors in the colostrum was not high enough to provide protection. For the apicomplexan parasite C. parvum similar phenomena were described but the oral administration of hyperimmune colostrum to calves and mice protected them against experimental infections (Fayer et al., 1989; Martin-Gomez et al., 2005b). This leads to the question whether this strategy could be applied to prevent piglets from neonatal cystoisosporosis. A relationship between antibody titers, fecal consistency and oocyst shedding could be observed but not all correlations could be interpreted. The significant correlations found on SD 1 cannot be interpreted validly, since the majority of the samples showed similar titers and correlations were based on 2–3 deviating values only, indicating a spurious relationship. In contrast, reliably significant correlations between higher antibody titers and more liquid feces (i.e., diarrhea) were found for IgM against sporozoites on SD 7 and 14 and for IgG against merozoites on SD 21 and 28. The watery diarrhea caused by C. suis is due to the destruction of the epithelial lining with subsequently reduced absorption of water and ions in the affected small intestines (Mundt et al., 2006). Assuming that this is a result of high parasite reproduction in the gut, higher antibody titers might be related to a higher incidence of antigenpresenting events and subsequently a stronger onset of endogenous antibody production, as described for toxoplasmosis or trypanosomiasis (Marinho et al., 1999; Singh et al., 2010). The significant negative correlation on SD 14–28 of IgA against merozoites with the fecal score was based on data of 14 infected animals but the small variation among IgA titers affects the validity of these correlations. Nevertheless, the results provide a first indication for an involvement of IgA in the immune response to the infection, either via a direct protective effect of IgA against C. suis or indirectly, with IgA serving as a marker for an on-going protective immune response based on other mechanisms. Beside the transfer of immunoglobulins (Ig), pathogen-specific lymphocytes can also be transferred with the colostrum and might lead to protective effects (Tuboly et al., 1988; Williams, 1993; Tuboly and Bernath, 2002). However, there is no indication for a direct involvement of IgA in the immune protection against C. suis so far. 496 L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 5. Conclusion We could demonstrate that naturally acquired C. suisspecific antibodies are transferred from the sow to the piglets with the colostrum. With the exception for IgG against merozoites, antibodies do not persist for longer than 14–21 days but 2–3 weeks after birth the onset of endogenous antibody production was noticed. Antibodies acquired by previous natural infections of the sows did not provide passive protection against an experimental infection of suckling piglets with a moderate dose of oocysts. The supposed onset of antibody production by the piglets around the age of three weeks supports the hypothesis that age resistance to clinical porcine cystoisosporosis is also based on the maturation of the adaptive immune system (Worliczek et al., 2010a). The hypothesis that higher titers of C. suis-specific antibodies resulting from colostrum uptake are positively correlated with a milder course of disease has to be rejected as such. But based on all determined correlations, C. suis-specific antibodies might be useful markers for the expectable clinical severity of cystoisosporosis. Correlations for IgG and IgM indicate a relationship between higher antibody titers and a more pronounced severity of the disease. For IgA the situation was contrary with a positive correlation between higher antibody titers and less pronounced diarrhea. This might reflect a direct protective effect of this particular Ig class or could indicate that IgA responses are concomitant to protective immune responses such as T-cell dependent mechanisms, either of maternal origin or piglet derived. We assume that the level of transferrable maternal factors providing protection is not reached by naturally acquired infections of sows. Whether a protection from a clinical manifestation of porcine neonatal coccidiosis could be achieved by a boost of maternal responses, e.g. by immunization of sows ante partum, has to be elucidated. Conflict of interest statement To the author’s knowledge there are no conflicts of interest. Acknowledgements The authors are grateful to B. Ruttkowski, R. Selista, S. Rohrer, M. Lastufka and I. Raphaelis for their technical support and to Georg Duscher for proofreading the manuscript. The work was funded by the Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria. References Aliaga-Leyton, A., Friendship, R., Dewey, C.E., Todd, C., Peregrine, A.S., 2011a. Isospora suis infection and its association with postweaning performance on three southwestern Ontario swine farms. J. Swine Health Prod. 19 (2), 94–99. Aliaga-Leyton, A., Webster, E., Friendship, R., Dewey, C., Vilaca, K., Peregrine, A.S., 2011b. An observational study on the prevalence and impact of Isospora suis in suckling piglets in southwestern Ontario, and risk factors for shedding oocysts. Can. Vet. J. 52 (2), 184–188. Baekbo, P., Christensen, J., Henriksen, S.A., Nielsen, K., 1994. Attempts to induce colostral immunity against Isospora suis infections in piglets. In: Proceedings of the 13th IPVS Congress, Bangkok, Thailand, 26–30 June 13. Bailey, M., Plunkett, F.J., Rothkötter, H.J., Vega-Lopez, M.A., Haverson, K., Stokes, C.R., 2001. Regulation of mucosal immune responses in effector sites. Proc. Nutr. Soc. 60 (4), 427–435. Becker, B.A., Misfeldt, M.L., 1993. Evaluation of the mitogen-induced proliferation and cell surface differentiation antigens of lymphocytes from pigs 1 to 30 days of age. J. Anim. Sci. 71 (8), 2073–2078. Behrendt, J.H., Taubert, A., Zahner, H., Hermosilla, C., 2008. Studies on synchronous egress of coccidian parasites (Neospora caninum, Toxoplasma gondii, Eimeria bovis) from bovine endothelial host cells mediated by calcium ionophore A23187. Vet. Res. Commun. 32 (4), 325–332. Buggelsheim, M., 2008. Interaktionen von Isospora suis mit dem Immunsystem des Schweins: Auswirkungen der Infektion auf die lokale Immunantwort im Dünndarm. University of Veterinary Medicine Vienna, Vienna, Austria (Doctoral Thesis). Butler, J.E., 2006. Why I Agreed to Do This. Dev. Comp. Immunol. 30 (1–2), 1–17. Crane, M.S., Gnozzio, M.J., Murray, P.K., 1986a. Eimeria tenella (Eucoccidiorida): a quantitative assay for sporozoite infectivity in vivo. J. Protozool. 33 (1), 94–98. Crane, M.S., Norman, D.J., Gnozzio, M.J., Tate, A.C., Gammon, M., Murray, P.K., 1986b. Eimeria tenella: quantitative in vitro and in vivo studies on the effects of mouse polyclonal and monoclonal antibodies on sporozoites. Parasite Immunol. 8 (5), 467–480. Daugschies, A., Bialek, R., Joachim, A., Mundt, H.C., 2001. Autofluorescence microscopy for the detection of nematode eggs and protozoa, in particular Isospora suis, in swine faeces. Parasitol. Res. 87 (5), 409–412. Daugschies, A., Imarom, S., Ganter, M., Bollwahn, W., 2004. Prevalence of Eimeria spp. in sows at piglet-producing farms in Germany. J. Vet. Med. Ser. B: Infect. Dis. Vet. Public Health 51 (3), 135–139. Davis, P.J., Porter, P., 1979. A mechanism for secretory IgA-mediated inhibition of the cell penetration and intracellular development of Eimeria tenella. Immunology 36 (3), 471–477. Fang, L., Gan, Z., Marquardt, R.R., 2000. Isolation, affinity purification, and identification of piglet small intestine mucosa receptor for enterotoxigenic Escherichia coli k88ac+ fimbriae. Infect. Immun. 68 (2), 564–569. Fayer, R., Andrews, C., Ungar, B.L., Blagburn, B., 1989. Efficacy of hyperimmune bovine colostrum for prophylaxis of cryptosporidiosis in neonatal calves. J. Parasitol. 75 (3), 393–397. Greve, E., 1985. Isospora suis species in a Danish SPF-herd. Nord. Vet. Med. 37 (3), 140–144. Habe, F., 1974. Die quantitativen Veränderungen der Immunglobuline im Blutserum der Ferkel bei verschiedenen Aufzuchtverfahren. Justus Liebig University Giessen, Giessen, Germany (Doctoral Thesis). Harleman, J.H., Meyer, R.C., 1984. Life cycle of Isospora suis in gnotobiotic and conventionalized piglets. Vet. Parasitol. 17 (1), 27–39. Harleman, J.H., Meyer, R.C., 1983. Isospora suis infection in piglets. A review. Vet. Q. 5 (4), 178–185. Jarvinen, J.A., Zimmerman, G.L., Schons, D.J., Guenther, C., 1988. Serum proteins of neonatal pigs orally inoculated with Isospora suis oocysts. Am. J. Vet. Res. 49 (3), 380–385. Jenkins, M.C., O’Brien, C., Trout, J., Guidry, A., Fayer, R., 1999. Hyperimmune bovine colostrum specific for recombinant Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in immunosuppressed adult mice. Vaccine 17 (19), 2453–2460. Jourquin, J., van Gelderen, R., Goossens, L., 2010. Better colostrum distribution increases piglet survival in high prolific sows. In: 21st IPVS Congress, 18–21 July, Vancouver. Klobasa, F., Werhahn, E., Butler, J.E., 1981. Regulation of humoral immunity in the piglet by immunoglobulins of maternal origin. Res. Vet. Sci. 31 (2), 195–206. Klobasa, F., Werhahn, E., Habe, F., 1991. The absorption of colostral immunoglobulins in newborn piglets. III. Influence of the duration of colostrum administration. Berl. Munch. Tierarztl. Wochenschr. 104 (7), 223–227. Koudela, B., Kucerova, S., 2000. Immunity against Isospora suis in nursing piglets. Parasitol. Res. 86 (10), 861–863. Koudela, B., Kucerova, S., 1999. Role of acquired immunity and natural age resistance on course of Isospora suis coccidiosis in nursing piglets. Vet. Parasitol. 82 (2), 93–99. Kreiner, T., Worliczek, H.L., Tichy, A., Joachim, A., 2011. Influence of toltrazuril treatment on parasitological parameters and health performance of piglets in the field—an Austrian experience. Vet. Parasitol. 183 (1–2), 14–20. L. Schwarz et al. / Veterinary Parasitology 197 (2013) 487–497 Lindsay, D.S., Blagburn, B.L., Ernst, J.V., Current, W.L., 1985a. Experimental coccidiosis (Isospora suis) in a litter of feral piglets. J. Wildl. Dis. 21 (3), 309–310. Lindsay, D.S., Current, W.L., Taylor, J.R., 1985b. Effects of experimentally induced Isospora suis infection on morbidity, mortality, and weight gains in nursing pigs. Am. J. Vet. Res. 46 (7), 1511–1512. Long, P.L., Rose, M.E., 1972. Immunity to coccidiosis: effect of serum antibodies on cell invasion by sporozoites of Eimeria in vitro. Parasitology 65 (3), 437–445. Marinho, C.R., D’Imperio Lima, M.R., Grisotto, M.G., Alvarez, J.M., 1999. Influence of acute-phase parasite load on pathology, parasitism, and activation of the immune system at the late chronic phase of Chagas’ disease. Infect. Immun. 67 (1), 308–318. Martin-Gomez, S., Alvarez-Sanchez, M.A., Rojo-Vazquez, F.A., 2005a. Immunization protocols against Cryptosporidium parvum in ovines: protection in suckling lambs. Vet. Parasitol. 129 (1–2), 11–20. Martin-Gomez, S., Alvarez-Sanchez, M.A., Rojo-Vazquez, F.A., 2005b. Oral administration of hyperimmune anti-Cryptosporidium parvum ovine colostral whey confers a high level of protection against cryptosporidiosis in newborn NMRI mice. J. Parasitol. 91 (3), 674–678. 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 (4), 277–284. Mundt, H.C., Cohnen, A., Daugschies, A., Joachim, A., Prosl, H., Schmäschke, R., Westphal, B., 2005. Occurrence of Isospora suis in Germany, Switzerland and Austria. J. Vet. Med. B: Infect. Dis. Vet. Public Health 52 (2), 93–97. Mundt, H.C., Joachim, A., Becka, M., Daugschies, A., 2006. Isospora suis: an experimental model for mammalian intestinal coccidiosis. Parasitol. Res. 98 (2), 167–175. O’Neill, P.A., Parfitt, J.W., 1976. Observations on Isospora suis infection in a minimal disease pig herd. Vet. Rec. 98 (16), 321–323. Parr, E.L., Bozzola, J.J., Parr, M.B., 1998. Immunity to vaginal infection by herpes simplex virus type 2 in adult mice: characterization of the immunoglobulins in vaginal mucus. J. Reprod. Immunol. 38 (1), 15–30. Perryman, L.E., Kapil, S.J., Jones, M.L., Hunt, E.L., 1999. Protection of calves against cryptosporidiosis with immune bovine colostrum induced by a Cryptosporidium parvum recombinant protein. Vaccine 17 (17), 2142–2149. Porter, P., Hill, I.R., 1970. Serological changes in immunoglobulins IgG, IgA and IgM and Escherichia coli antibodies in the young pig. Immunology 18 (4), 565–573. Rommel, M., Heydorn, A.O., 1971. Transfer of immunity to Eimeria infections by lymphocytes. Z. Parasitenkd. 36 (3), 242–250. Rooke, J.A., Carranca, C., Bland, I.M., Sinclair, A.G., Ewen, M., Bland, V.C., Edwards, S.A., 2003. Relationships between passive absorption of immunoglobulin G by the piglet and plasma concentrations of immunoglobulin G at weaning. Livest. Prod. Sci. 81 (2–3), 223–234. Rothkötter, H.J., Pabst, R., 1989. Lymphocyte subsets in jejunal and ileal Peyer’s patches of normal and gnotobiotic minipigs. Immunology 67 (1), 103–108. Ruttkowski, B., Joachim, A., Daugschies, A., 2001. PCR-based differentiation of three porcine Eimeria species and Isospora suis. Vet. Parasitol. 95 (1), 17–23. Salmon, H., Berri, M., Gerdts, V., Meurens, F., 2009. Humoral and cellular factors of maternal immunity in swine. Dev. Comp. Immunol. 33 (3), 384–393. Scala, A., Demontis, F., Varcasia, A., Pipia, A.P., Poglayen, G., Ferrari, N., Genchi, M., 2009. Toltrazuril and sulphonamide treatment against naturally Isospora suis infected suckling piglets: is there an actual profit? Vet. Parasitol. 163 (4), 362–365. Schlepers, M., 2009. Isospora suis: haematological parameters and antibody-development during porcine coccidiosis in suckling piglets. Utrecht University, Utrecht, The Netherlands (Doctoral Thesis). Sharman, P.A., Smith, N.C., Wallach, M.G., Katrib, M., 2010. Chasing the golden egg: vaccination against poultry coccidiosis. Parasite Immunol. 32 (8), 590–598. Singh, J., Graniello, C., Ni, Y., Payne, L., Sa, Q., Hester, J., Shelton, B.J., Suzuki, Y., 2010. Toxoplasma IgG and IgA, but not IgM, antibody titers increase in sera of immunocompetent mice in association with proliferation of 497 tachyzoites in the brain during the chronic stage of infection. Microbes Infect. 12 (14–15), 1252–1257. Sinkora, M., Butler, J.E., 2009. The ontogeny of the porcine immune system. Dev. Comp. Immunol. 33 (3), 273–283. Stokes, C.R., Bailey, M., Haverson, K., Harris, C., Jones, P., Inman, C., Pie, S., Oswald, I.P., Williams, B.A., Akkermans, A.D.L., Sowa, E., Rothkötter, H.J., Miller, B.G., 2004. Postnatal development of intestinal immune system in piglets: implications for the process of weaning. Anim. Res. 53, 325–334. Stuart, B.P., Lindsay, D.S., Ernst, J.V., Gosser, H.S., 1980. Isospora suis enteritis in piglets. Vet. Pathol. 17 (1), 84–93. Stuart, B.P., Gosser, H.S., Allen, C.B., Bedell, D.M., 1982a. Coccidiosis in swine: dose and age response to Isospora suis. Can. J. Comp. Med. 46 (3), 317–320. Stuart, B.P., Sisk, D.B., Bedell, D.M., Gosser, H.S., 1982b. Demonstration of immunity against Isospora suis in swine. Vet. Parasitol. 9 (3–4), 185–191. Stuart, B.P., Lindsay, D.S., 1986. Coccidiosis in swine. Vet. Clin. North Am. Food Anim. Pract. 2 (2), 455–468. Suzuki, K., Ha, S.A., Tsuji, M., Fagarasan, S., 2007. Intestinal IgA synthesis: a primitive form of adaptive immunity that regulates microbial communities in the gut. Semin. Immunol. 19 (2), 127–135. Taylor, J.R., 1984. Immune response of pigs to Isospora suis (Apicomplexa, Eimeriidae). Auburn University, Auburn, Alabama, United States of America (Doctoral Thesis). Trees, A.J., Karim, M.J., McKellar, S.B., Carter, S.D., 1989. Eimeria tenella: local antibodies and interactions with the sporozoite surface. J. Protozool. 36 (4), 326–333. Tuboly, S., Bernath, S., 2002. Intestinal absorption of colostral lymphoid cells in newborn animals. Adv. Exp. Med. Biol. 503, 107–114. Tuboly, S., Bernath, S., Glavits, R., Medveczky, I., 1988. Intestinal absorption of colostral lymphoid cells in newborn piglets. Vet. Immunol. Immunopathol. 20 (1), 75–85. Wagstrom, E.A., Yoon, K.J., Zimmerman, J.J., 2000. Immune components in porcine mammary secretions. Viral Immunol. 13 (3), 383–397. Wakelin, D., Rose, M.E., 1990. Immunity to coccidiosis. In: Long, P.L. (Ed.), Coccidiosis in Man and Domestic Animals. CRC Press Inc., Boca Raton, FL, pp. 281–306. Wallach, M., 2010. Role of antibody in immunity and control of chicken coccidiosis. Trends Parasitol. 26 (8), 382–387. Whitmire, W.M., Kyle, J.E., Speer, C.A., Burgess, D.E., 1988. Inhibition of penetration of cultured cells by Eimeria bovis sporozoites by monoclonal immunoglobulin G antibodies against the parasite surface protein P20. Infect. Immun. 56 (10), 2538–2543. Williams, P.P., 1993. Immunomodulating effects of intestinal absorbed maternal colostral leukocytes by neonatal pigs. Can. J. Vet. Res. 57 (1), 1–8. Witlock, D.R., Danforth, H.D., 1982. Surface changes induced by immune serum on Eimeria tenella sporozoites and merozoites. J. Protozool. 29 (3), 441–445. Worliczek, H.L., Gerner, W., Joachim, A., Mundt, H.C., Saalmüller, A., 2009a. Porcine coccidiosis – investigations on the cellular immune response against Isospora suis. Parasitol. Res. 105 (Suppl. 1), 151–155. Worliczek, H.L., Mundt, H.C., Ruttkowski, B., Joachim, A., 2009b. Age, not infection dose, determines the outcome of Isospora suis infections in suckling piglets. Parasitol. Res. 105 (Suppl. 1), 157–162. Worliczek, H.L., Buggelsheim, M., Alexandrowicz, R., Witter, K., Schmidt, P., Gerner, W., Saalmüller, A., Joachim, A., 2010a. Changes in lymphocyte populations in suckling piglets during primary infections with Isospora suis. Parasite Immunol. 32 (4), 232–244. Worliczek, H.L., Ruttkowski, B., Joachim, A., Saalmüller, A., Gerner, W., 2010b. Faeces, FACS, and functional assays – preparation of Isospora suis oocyst antigen and representative controls for immunoassays. Parasitology 137 (11), 1637–1643. Worliczek, H.L., Ruttkowski, B., Schwarz, L., Witter, K., Tschulenk, W., Joachim, A., 2013. Isospora suis in an epithelial cell culture system – an in vitro model for sexual development in coccidia. PLoS ONE 8 (7), e69797, http://dx.doi.org/10.1371/journal.pone.0069797. Yun, C.H., Lillehoj, H.S., Lillehoj, E.P., 2000. Intestinal immune responses to coccidiosis. Dev. Comp. Immunol. 24 (2–3), 303–324.