Inverse relationship between IgE and worm burdens in cattle infected with Ostertagia ostertagi

l'eterinao' Parasitology, 47 ( 1993 ) 87-97 Elsevier Science Publishers B.V., Amsterdam

87

Inverse relationship between IgE and worm burdens in cattle infected with Ostertagia

ostertagi D.G. Baker ~ a n d L.J. G e r s h w i n Department ~[ l "eterinarv Mtcrohioh~gy and lmmunoh~gy, School ~?[ | ~'terinao' .~ledicme, Umversity of ('ahJbrnia, Davis, ()! 95616. US.I (Accepted 20 September 1992 )

ABSTRACT Baker, D.G. and Gershwin, L.J., 1993. Inverse relationship between lgE and worm burdens in cattle infected with Ostertagta ostertagi. Vet. I'arasitol., 47: 87-97. Changes in serum total and Ostertagia-specific IgE levels, and pepsinogen concentrations were evaluated in 28 Holstein calves naturally or experimentally infected with Ostertagia ostertagi. In addition, IgE and pepsinogen concentrations were determined in abomasal lymph. Results showed that ( 1 ) 13mph lgE responses were inversely correlated v,ith worm burdens, and (2) serum lgE levcls wcrc unreliable for predicting worm burdens.

INTRODUCTION

The abomasal nematode Ostertagia ostertagi is an important parasite of cattle in North America (Gibbs and Herd, 1986). Type I ostertagiosis results when calves, during their first grazing season, are exposed to large numbers of infective third-stage larvae (L3), which mature within their gastric glands. Type 2 disease occurs primarily in yearling animals after their first grazing season. Disease results when large numbers of fourth-stage larvae (L4), previously inhibited in their development (hypobiosis), resume development to adults. The rate of hypobiosis increases as seasonal environmental conditions worsen. Development resumes about the time that climatic conditions improve. Immunoglobulin E (IgE) is well recognized as an antibody isotype imporCorrespondence to: L.J. Gershwin, Department of VeterinaD' Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. *Present address: Zoonotic Diseases Laboratory, Livestock and Poultry Sciences Institute, United States Department of Agriculture, Bldg. 1040, BARC-East, 10300 Baltimore Avenue, Behsville, MD 20705, USA.

f~ 1993 F.lscvicr Science Publishers B.V. All rights reserved 0304-4017/93/$06.00

88

I).(i. BAKER AND I,.J. (iERSHWIN

tant in the host response to parasite infection (Jarrett and Miller, 1982). In cattle, IgE responses have been detected in animals infected with Fasciola hepatica (Doyle, 1973 ) and Sarcocystis cruzi (Granstrom et al., 1990 ). Two recent studies in this laboratory have documented lgE responses in cattle infected with O. ostertagi. However, results from the two studies appeared to conflict. In the first report, serum total lgE levels werc inversely related to worm burdens in calves grazed for l-month periods (Thatcher et al., 1989). In a subsequent study, continuously infected calves were found to have serum total and Ostertagia-specific IgE levels that increased during the season of the year when adult worm burdens were also thought to be increasing ( Baker and Gershwin, 1992 ). It has been postulated that the pathology observed in ostertagiosis may be due in part to an IgE-mediated Type I hypersensitivity response, since infection is associated with apparent changes in abomasal permeability and local increases in numbers of mast cells and eosinophils (Armour et al., 1979; Klesius, 1988; Wiggin and Gibbs, 1990). Additional studies were thercfore needed to evaluate the IgE response of cattle to O. ostertagi. The objectives of the present study were to determine ( 1 ) if there was a total or Ostertagia-specific IgE response, and (2) if serum or lymph lgE levels were related to infection status and therefore might be useful predictors of infection. MATERIALS AND METHOI)S

Location o.fstudy This study was conducted at the University of California Sierra Foothill Range Field Station, Browns Valley, California. The topographical features of the station have been reported previously (Baker and Fisk, 1986). The climate at the station is Mediterranean, characterized by hot, dry summers and mild, rainy winters.

Animals Twenty-eight Holstein steers, 2.8-4.8 months of age ( m e a n = 3 . 9 ) and weighing 108.9-186.0 kg ( m e a n = 150.2 kg) at the start of the study, were used. Animals had been raised under conditions precluding infection with gastrointestinal heiminths and were deemed free of worms by fecal flotation examination (Foreyt, 1986). Calves were inoculated with bacterins of common clostridial (Coopers Animal Health, Kansas City, KS) and leptospiral (Norden Laboratories, Lincoln, NE) agents, as well as modified live IBR, PI3 and BVD vaccines (Norden). Two experimentally infected and two non-infected calves were not vaccinated. This omission was apparently of no con-

89

IgE AND WORM BURDENS IN CATTLE INFECTED WITH OSTI-RII4(;I,4 OS]'t:RI~-IGI TABLE 1 Infection a n d s a m p l i n g schedule Group No.

Weeks post-pasture t u r n o u t 0

1 2 3 4 5 6

19 D e c ? 8 Feb. ~ 10 Mar. ~ 10 Mar. t X3 Xa

1

X X

2

X X

3

4

5

6

7

8

X X X

S

N S S

N N

X

18 Jan. 2 10 Mar.: 10 Apr. 2 X S S

25

27

28

29

25 Aug. z

X

S

N

N N

~Placed on pasture a n d sampled. -~Removed from pasture a n d sampled. ~Observation period began. 4Experimcntally infected with O. ostertagt. X = o t h e r s a m p l e s collected: S-cannulation surgeD'; N = necropsy.

sequence. All calves received levamisole (Pitman-Moore, Mundelein, IN) (8 mg per kg body weight) subcutaneously on the day of purchase. Calves were assigned as purchased into four groups of five steers each. Three groups were pastured for 1 month each, while one group was pastured for 5.5 months (Table 1 ). Placement of calves on pasture was timed to allow for the development of specific patterns of ostertagism (Baker and Fisk, 1986 ). The pastures had been grazed the previous year by animals from another study that had not been treated with anthelmintics. Following pasture exposure, steers were housed under conditions precluding further infection. Alfalfa hay was provided twice daily and water was available ad libitum. In addition, four steers remained as uninfected controls (Group 5 ) and four steers were experimentally infected with 200 000 viable O. ostertagi L 3 (Group 6). Infective larvae had been harvested from fecal-vermiculite cultures (Louisiana isolate kindly provided by Dr. J.C. Williams, Louisiana State University). These eight control steers were similarly housed and fed.

Surgery Abomasal lymphatic cannulation surgery was performed on naturally infected steers between 2 and 3 weeks following removal from pasture, and 1 month after experimental infection or the start of the observation period in control groups. The surgical procedure has been described previously (Baker et al., 1991 ).

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l).(i. BAKER A N D L.J. ( I E R S | t W I N

Sample collection Samples were collected before natural or experimental infection with O.

ostertagi, and at intervals thereafter (Table 1 ). On each sampling date, 20 ml blood were collected by jugular venipuncture and allowed to clot. Serum was harvested following centrifugation and was stored at - 2 0 ° C until used. The last serum sample included in the data analysis was that which had been collected 1-2 weeks before surgery. Following surgery, 25 ml abomasai lymph was collected into plastic vials containing 100 U heparin.

Parasite enumeration Numbers ofabomasal parasites were estimated using the method described by Charles and Baker (1988). Briefly, 10% aliquots of abomasal and small intestinal contents were fixed in neutral buffered formalin (NBF) and examined for nematode parasites. The abomasa and small intestines were soaked separately overnight in tap water. After soaking, the tissues were washed and the mucosal washings were fixed in NBF and similarly examined. Worm data were transformed log~o ( x + 1 ) before analysis.

Total and Ostertagia-spec~[ic IgE ELISA Details of the total and Ostertagia-specific IgE ELISA have been reported previously ( Baker and Gershwin, 1992 ). Briefly, for total lgE, microtitcr plates were sensitized with mouse anti-bovine epsilon chain monoclonai antibody and incubated. Following blocking and washing, test samples, standards, PBS and fetal bovine serum (FBS) (negative control ) samples were added to wells and incubated. Horseradish peroxidase-conjugated rabbit anti-bovine immunoglobulin followed. Finally, O-phenylenediamine served as substrate. Results were expressed as lgE units per mi serum or lymph. For Ostertagia-specific IgE, soluble somatic extract of third-stage larval ( L~ ) O. ostertagi was prepared and used to sensitize microtiter plates. Following incubation,wells were blocked and washed. Test serum samples werc precipitated with 27.5% saturated ammonium sulfate and the supernatants were added to the wells. Subsequent incubations included mouse anti-bovine cpsilon chain monoclonai antibody, followed by peroxidase conjugated goat antimouse immunoglobulin. Finally, O-phenylenediamine again served as substrate and results were expressed as a positive/negative ratio ( P / N ) with FBS serving as negative.

01

lgE ANI) WORM BURDENSIN CAT] LE INFI-iTED WI II.10,',TERTI.IGI.IOSIERII.IGI

Pepsinogen assays Serum pepsinogen levels were determined by a method reported previously (Baker and Gershwin, 1992). Pepsinogen levels are expressed as International Units (IU) of tyrosine (1 I U = 1 /tmole tyrosine produced per liter serum per minute at 37°C).

Statistical analysis Statistical analyses were carried out using the Number Cruncher Statistical System (NCSS) computer program (Dr. J. Hintze, Kaysville, UT). Student's t-test was used to determine whether changes in total or specific IgE and pepsinogen were different from zero. Analysis of variance (ANOVA) was performed to compare responses of groups for the variables examined. Fisher's least significant differences test was used as a post-test. Significance was evaluated at the P < 0.05 level. Regression analysis was performed to determine the value of the variables examined for predicting the number and stages of O. ostertagi present. TABLE 2 Abomasal parasites recovered from steers naturally or experimentally infected with O. o.slertagt Group No.

Total nematodes in a b o m a s u m ~

Total adult worms

Total immature worms

Immature worms EL4

L~/I.5

61728 (26472)

53376 (21962)

8032 (5150)

4200 (2492)

3832 (3777)

2

80212 (39126)

51338 (21552)

35518 (22332)

31528 (20648)

3990 (3057)

3

71725 (23787)

26605 (10249)

45020 (18873)

38460 (18926)

6560 (4714)

4

61989 (10658)

41043 (11401)

20386 (5999)

5722 (3439)

14664 (3200)

0 42325 (24153)

0 40900 (22582)

0 1425 (1915)

~Number of all nematodes in the abomasum (mean +_S.D. ).

0 838 (1156)

0 587 (762)

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D.(i. BAKER A.NI) l..J. ( i E R S H ~ ' I N

RESULTS

Results of parasite speciation and enumeration are presented in Table 2.

Ostertagia sp. accounted for a mean of 99.5% ( S . D . = 0 . 6 ) of nematodes recovered from the abomasum, with Trichostrongylus axei accounting for the remainder. In the small intestine, Cooperia sp. was the predominant nematode recovered, while small numbers o f Trichostrongylus sp. and Nematodirus sp. were also recovered. Worm genera were in agreement with the results of previous studies carried out on these pastures (Baker and Fisk, 1986 ). Only abomasai worm burdens will be considered further. Changes in serum total and Ostertagia-specific lgE levels and serum pepsinogen concentrations are presented in Table 3. For no groups were changes in serum total or specific IgE different from zero. No differences were found between groups for change in serum total IgE. However, significant differences were noted between groups for change in specific IgE. Changes in serum pepsinogen levels were different from zero for all infected groups. Concentrations of total and Ostertagia-specific IgE, and pepsinogen in TABLE 3

Changes in serum total and OsWrta,eta-spccific IgE and pcpsinogen in steers naturall,, or cxperimentall',' infected with O. osterta~t Group

Change in total

Change in

("hange in

No.

IgE ~

O.stertagta-speci fi c

peps i n o g c n '

IgE b 1

102.3" (34.111

90.6" (8.3)

1.26" 1//.731

2

86.5" (16.1)

102.Y ''b (24.4)

1.63" (0.30)

3

112.9" ( 32.3 )

117.5 ~' ( 19.4 )

1.36 J ((}.611)

4

97.4 a (124.8)

82.8:' (23.3)

1.22" (11.44)

5

114.2 ~ (69.8)

91.3 ''h (20.01

0.18 ~' 10.201

6

190.8 a (202.5)

84.3" (15.2)

2.0t,~" (I.271

tMean (S.D.) changes in serum total and Ostertagia-specific IgE expressed as percent of pro-infection values. 2Mean (S.D.) changes in serum pepsinogen expressed as I.U. tyrosine. Within a column, numbers with superscripts in common are not significantb different I P > 0.05 ).

lgE AND WORM BURDENS IN CATTLE INFECTED WITH OSIERE.IGL,! OSTERI)!GI

93

abomasal lymph, are presented in Table 4. No differences were found between groups in levels of lymph total IgE. Significant group differences in lymph Ostertagia-specific IgE were noted. Large within-group variation prevented lymph pepsinogen levels in Groups 1, 4 and 6 from significantly exceeding those of the uninfected controls. Regression analysis revealed a direct correlation between change in serum Ostertagia-specifilc IgE and lymph Ostertagia-specific IgE levels (P=0.045, r=0.40). This relationship was particularly strong for the 3 groups (1-3) exposed to pasture for 1 month each (P=0.016, r=0.65). Regression analysis for predicting worm burdens revealed relationships between change in serum pepsinogen and numbers of all stages of Ostertagia sp., including early L4 (EL4) (P=0.012, r=0.48), late L4/L5 (P=0.010, r=0.49), adult (P<0.001, r=0.61 ) and total (P<0.001, r=0.60) worms. Lymph pepsinogen levels were also correlated with numbers of all stages of Ostertagia sp., most notably EL4 (P=0.006, r=0.57) and adult (P=0.009, r=0.53) worms. Lymph total IgE inversely correlated with numbers of EL 4 (P---0.015, r= - 0 . 4 8 ) and late La/L 5 ( e = 0.009, r= --0.51 ). Because calves of Groups 1-3 were all experiencing naturally acquired Type 1 ostertagiosis, and to comTABLE 4 Total and Ostertagia-specific IgE and pepsinogen in abomasal lymph of steers naturally or experimentally infected with O. ostertagt Group No.

Total IgE ( IgE units )

Ostertagia-specific lgE ( P / N ratio )

Pepsinogen ( I.U. tyrosinc )

1

3.29 a (l.10)

2.02 a,l' (0.32)

5.92 a.h (3.64)

2

4.25 ~ (3.96)

2.44 ".~.~ (I.02)

8.90 . (7.32)

3

5.84 a (3.24)

3.21 c (0.73)

8.50" (1.35)

4

1-55 ~ (1.17)

1.86 a (0.47)

6.14 "'~ (5.64)

5

7.97 a (6.85)

2.75 a.b.~ (0.72)

0.73 b (0.53)

6

9.1 I ~ (12.01)

2.92 bx (0.83)

5.56 a.h (2.53)

Mean (S.D.) concentrations of total and Ostertagta-specific lgE and pepsinogcn in abomasal lymph. Within a column, numbers with superscripts in c o m m o n arc not significantly diflbrcnt ( I ' > 0.05 ).

94

D.G. BAKER ANt) L.J. GERSHWIN

pare results from the current study with a previous report incorporating similar parasite exposure schedules (Thatcher et al., 1989), regression analyses of IgE results were repeated using only data from Groups 1-3. When this was done, there was an inverse correlation between lymph Ostertagia-specific IgE and numbers of adult Ostertagia sp. (P=0.037; r = - 0 . 5 8 ). DIS('USSION

Based on parasite burdens and serum pepsinogen responses, calves in each group had distinct patterns of parasitism (Anderson et al., 1966; Armour, 1970; Baker, 1988). Calves in Group l were experiencing moderate Type l ostertagiosis. Group 2 calves had moderate Type l ostertagiosis with superimposition of pre-Type 2 ostertagiasis. Group 3 calves had mild Type l ostertagiosis, as well as pre-Type 2 ostertagiasis. Group 4 calves were experiencing Type 2 ostertagiosis, while Group 6, the experimentally infected calves, were experiencing moderate Type 1 ostertagiosis. Changes in pepsinogen concentration were in agreement with numerous reports demonstrating increases in serum pepsinogen levels in cattle and sheep infected with Ostertagia sp. (Armour et al., 1979; Snider ct al., 1981; Yakoob et al., 1983). Pepsinogen concentrations in the lymph were comparable to those reported by other workers (Smith et al., 1987 ). When all groups of steers were considered together, regression analysis was of little value for revealing correlations between IgE levels and worm burdens. However, when regression analysis was repeated using data from the 15 calves with naturally acquired Type l ostertagiosis (Groups 1-3), thc predictivc value of lymph Ostertagia-specific IgE was greatly improved, and reflected an inverse relationship between IgE levels and adult parasite burden. This is in agreement with our previous report showing an inverse relationship between serum total IgE and number of O. ostertagi (Thatcher et al., 1989). In that study, mean changes of IgE levels were greater ( 31 IgE units) in animals with < 1000 total helminths than in animals with > 7500 total helminths (5 lgE units). In the present study, levels of helminth infection were considerably greater. In fact, no infected animal had < 1000 total helminths. One explanation for the apparent lack of IgE response in heavily infcctcd cattle is that mast cells and other inflammatory cells might sequester IgE on their surfaces, thereby reducing the concentration of circulating IgE (Thatcher et al., 1989). In addition, increased mast cell turnover might further decrease IgE levels in serum or lymph. If this were true, we would expect to find the smallest apparent IgE response in the calves with Type 2 ostertagiosis, since increases in mast cells, eosinophils and globule leukocytes have been demonstrated most prominently in calves with chronic infections (Wiggin and Gibbs, 1990; Snider et al., 1988). Significantly, calves in Group 4 had among the

IgE AND WORM BURDENS IN CATTLE INFECTED WITtl OSTI-RT)IGIA OSTI'.'RI~-IGI

95

greatest decreases in serum total and specific IgE, and had the least amount of IgE in their lymph. As noted by Cross and Klesius (1989), there are other possible explanations for apparent decreases in antibody response, including non-specific inhibition of lymphocyte proliferation by parasite proteins, depression of lgE production by histamine or prostaglandin E, and enhancement in number or function of suppressor T cells. Lastly, quenching might have occurred due to parasite antigens binding antigen-specific IgE, although it would seem unlikely that enough antigen could be taken up by the host to detectably reduce total IgE levels. Additional studies are needed to determine which of these mechanisms might be involved. Results of the present study are in general disagreement with those of another study recently carried out in our laboratory. In that study, continuously infected calves demonstrated total and Ostertagia-specific IgE reponses, which paralleled adult worm burdens, based on fecal egg counts and historical epidemiologic patterns of parasitism on the pastures used (Baker and Gershwin, 1992 ). Calves in that study likely bore much lighter worm burdens than the animals of the present study. It may be that above a threshold level of parasitism, mast cell production and turnover increase sufficiently to quench circulating IgE. The question arises as to why our results differ from the reports of others citing measurable IgE responses in parasitized animals (Jarrett and Miller, 1982 ). Other parasites for which IgE responses have been measured undergo tissue migration during their life cycles. In contrast, O. ostertagi is only minimally invasive, occupying the lumena of gastric glands but not actively breaching the epithelium. It may be that parasites with systemic migration routes elicit qualitatively a n d / o r quantitatively different IgE responses. It was of interest that specific IgE levels in the lymph of uninfected controls (Group 5) exceeded, although not significantly, those of three of the other groups. We have noted similar results with serum. Antibodies cross-reactive with intestinal microflora may be responsible. Alternatively, there may have been non-specific binding of lymph or serum antibodies to Ostertagia antigen, as suggested by Canals and Gasbarre (1990). In conclusion, lymph and to a lesser extent serum IgE levels were inversely correlated with numbers of O. ostertagi. Serum IgE levels are therefore unreliable for predicting worm burdens in parasitized cattle. We propose that circulating IgE levels decline due to binding to an increasing population of mast cells, and possibly other cells, In addition, increases in mast cell turnover may further reduce IgE levels. ACKNOWLEDGEMENTS

This work was supported by USDA special Grant No. 88-34116-3650 and the Western Regional (W-102) Parasite Control Program. The authors wish

96

D.(;. BAKER AND I..J. GERSHWIN

to t h a n k Drs. J i e C h e n a n d C o n g f e n Li f o r t e c h n i c a l a s s i s t a n c e . T h e a u t h o r s also t h a n k the s t a f f o f t h e U n i v e r s i t y o f C a l i f o r n i a Sierra Foothill R a n g e Field S t a t i o n , B r o w n s V a l l e y , C a l i f o r n i a , for t e c h n i c a l a s s i s t a n c e .

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1988. Sequential histopathologic changes of Type 1, pre-Type II and Type II ostertagiasis in cattle. Vet. Parasitol., 27:169-179. Thatcher, E., Gershwin, L.J. and Baker, N.F., 1989. Levels of serum IgE in response to gastrointestinal nematodes in cattle. Vet. Parasitol., 32:153-161. Wiggin, C.J. and Gibbs, H.C,, 1990. Adverse immune reactions and the pathogenesis of Ostertagia ostertagi infections in calves. Am. J. Vet. Res., 51: 825-832. Yakoob, A., Holmes, P.H. and Armour, J., 1983. Pathophysiology of gastrointestinal trichostrongyles in sheep: plasma losses and changes in plasma pepsinogen levels associated with parasite challenge of immune animals. Res. Vet. Sei., 34: 305-309.