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Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection
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ARTICLE IN PRESS Veterinary Parasitology xxx (2013) xxx–xxx
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Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection Scott Bowdridge a,1 , Kathryn MacKinnon a,2 , Joshua C. McCann a,3 , Anne M. Zajac b , David R. Notter a,∗ a b
Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA 24061, USA
a r t i c l e
i n f o
Article history: Received 11 May 2011 Received in revised form 28 November 2012 Accepted 14 January 2013 Keywords: Hair sheep Colostrum IgA Haemonchus
a b s t r a c t Antibody levels produced in response to gastro-intestinal nematode (GIN) parasite infection are typically higher in GIN-resistant breeds than susceptible breeds. Consequently, GINresistant ewes should generate greater parasite-specific antibody in colostrum and milk, potentially providing greater passive immunity to young lambs. To test this hypothesis, we monitored immunoglobulin levels in wool and hair-type sheep infected with Haemonchus contortus for 35 days following the end of a 45-day autumn breeding season and subsequently for 6 weeks around the time of parturition. Ten, first-parity ewes of each type were infected with 12,000 H. contortus L3 larvae following the end of breeding. In response to infection, hair ewes generated greater serum IgA (P < 0.05), although the pattern of IgA production was similar between the types. Following experimental infection, wool ewes were incapable of clearing the parasite infection well in advance of parturition. Prior to parturition, hair ewes had lower FEC and greater circulating H. contortus-specific IgA. However, no difference was seen in total or antigen-specific IgA production in the colostrum and milk of either breed. These data further demonstrate that hair-type sheep can rapidly reduce fecal egg output and generate greater humoral immunity as evidenced by higher levels of circulating antigen-specific antibody, but there is no evidence to suggest GIN-resistant sheep preferentially mobilize antigen-specific IgA to colostrum or milk. Thus, no clear difference exists between types of sheep in ability to deliver parasite-specific IgA to their offspring. © 2013 Published by Elsevier B.V.
1. Introduction Hair-type sheep are well known for their resistance to gastro-intestinal trichostrongylid parasites, however the
∗ Corresponding author. Tel.: +1 5402315135. E-mail addresses: [email protected] (S. Bowdridge), [email protected] (K. MacKinnon), [email protected] (J.C. McCann), [email protected] (D.R. Notter). 1 Present address: Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506, USA. 2 Present address: World Wide Life Sciences, 8810 Westgate Park Dr. Suite #108, Raleigh, NC 27617, USA. 3 Present address: Department of Animal Science, Texas A&M University, College Station, TX 77843, USA.
biological mechanism of resistance has yet to be fully characterized. St. Croix sheep, originating in the Caribbean, have developed a natural resistance to parasites that is at least partly mediated by their increased humoral immune response to helminth parasites (Gamble and Zajac, 1992). Parasite-resistant sheep have a distinct advantage in developing a more potent acquired immune response as evidenced by an increase in circulating and local antibodies (MacKinnon et al., 2010). In utero antibody transfer generally does not occur in livestock (Butler, 1999), but amounts of IgG and IgA in colostrum are substantial. In neonatal lambs, pinocytotic absorption of antibodies allows for passive immunity to occur, but this ability declines rapidly, as do antibody concentrations in milk (Butler, 1999). Measurable levels of
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Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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Fig. 1. Schematic timeline of experimental events. The first trial was conducted from day 0 to day 35 after infection with H. contortus. The second trial was initiated at least 3 weeks prior to parturition, which began on December 22, 2006. Milk samples were collected at 3 and 6 weeks following parturition.
the antibodies IgG and IgA remain in the milk of sheep for at least 30 days after parturition and may provide protection to newborn offspring (Pfeffer et al., 2005). IgA is typically found in surface secretions and in high levels in saliva, intestinal fluid, milk, colostrum and urine (Tizard, 2004). The major role of IgA is immune exclusion of pathogens, as it is secreted in the mucus of the small intestine and protects the lumen from invasion by viruses, bacteria or parasites (Tizard, 2004). Increased production of IgA is also an indicator of immune responsiveness to gastro-intestinal nematode infection (Amarante et al., 2005; Martinez-Valladares et al., 2005; Pfeffer et al., 2005), especially during challenge helminth infection. Plasma levels of parasite-specific antibodies in neonatal lambs have been reported to be directly proportional to the levels of parasite-specific antibody in the colostrum or milk of the ewe (Pfeffer et al., 2005). However, there is a lack of information as to the perceived ability of parasite-resistant sheep to have greater colostrum or milk antibody levels or whether immune clearance of parasite infection during gestation can enhance levels of antigen-specific antibodies passively transferred to the neonate. Thus the goal of this experiment was to compare acute and long-term periparturient immune responses of hair and wool-type ewes challenged with H. contortus immediately following breeding. 2. Materials and methods 2.1. Sheep and experimental design Ten St. Croix hair sheep ewe lambs and 10 ewe lambs of a composite line of wool sheep developed at Virginia Tech were used for this study. Animals from the composite line were made up of 50% Dorset, 25% Finnsheep and 25% Rambouillet breeding and have been shown to be significantly more susceptible than St. Croix to infection by H. contortus (Notter et al., 2003; Vanimisetti et al., 2004). Animals were born in January 2006 and exposed to rams for 45 days beginning at approximately 7 months of age in early August. All ewes were housed indoors from the end of breeding in September 2006. Animals and procedures used in this study were approved by the Virginia Tech IACUC Committee. Following breeding and natural parasite exposure on pastures known to be contaminated with H. contortus
and other trichostrongylid nematodes, ewes were treated with levamisole (8 mg/kg) (Agri-lab, St. Joseph, MO), which reduced individual fecal egg counts (FEC) to ≤ 50 eggs/g, and kept in drylot for 2 weeks (Fig. 1). Animals were then orally administered 12,000 H. contortus L3 larvae and this challenge infection was allowed to persist through the periparturient period. Fifty days after ram exposure, transabdominal ultrasound was used to determine pregnancy and thereby identify 10 pregnant ewe lambs of each type to be included in the study. Additionally three pregnant ewe lambs of each type were maintained during the periparturient period as uninfected controls to evaluate control IgA levels. Pregnant ewes were housed indoors in drylot throughout lambing and early lactation in order to avoid environmental exposure to infective larvae. A 16% CP cornsoybean ration, grass hay, and water were provided ad libitum. 2.2. Parasitological techniques Adult H. contortus were collected from euthanized sheep and pulverized in an ice-cold glass tissue homogenizer to release developing eggs. Homogenate was mixed with egg-free feces to obtain a mono-specific larval culture. The Baermann technique (Zajac and Conboy, 2006) was used to collect H. contortus L3 larvae which were then used to infect two feedlot-raised donor wethers that were dewormed to a FEC of 0, as determined by McMaster. At least 21 days after infection, feces were collected from donor lambs and cultured at 30 ◦ C for 7–8 days. Collected larvae were stored at 4 ◦ C in deionized water, and used within 1 month to orally infect experimental animals. Fecal egg counts were determined using the modified McMaster technique (Whitlock, 1948), and PCV were determined by the micro-hematocrit centrifuge method (NCCLS, 2000). 2.3. Blood collection and extraction of milk immunoglobulins Blood was collected via jugular venipuncture into a 10 ml vacutainer tube (BD) for serum antibody analysis and into a 4 ml EDTA-treated vacutainer tube (BD) for measurement of PCV. Since ewes did not lamb on the same day blood samples were taken twice weekly and the sample taken closest (±2 days) to the predefined time-point was used
Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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7000 (A)
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Day after infection Fig. 2. Response to H. contortus infection after breeding. Ten ewe lambs of each type were infected with 12,000 H. contortus L3 larvae shortly following a 45-day breeding season. Response to infection was measured by (a) fecal egg count (FEC), (b) packed cell volume (PCV) and (c) total serum IgA. Means differ between types by *P < .05, **P < 0.01, ***P < 0.001. Means for FEC and PCV with different letters indicate a significant effect of day (P < 0.05). Error bars represent standard error of means.
for analysis, prior to parturition. Milk (10 ml from each half of the udder) and blood samples were collected from each ewe within 12 h after parturition. Colostrum and milk samples were pooled by individual, diluted 1:1 in PBS (pH 7.4), and stored at -20 ◦ C. Diluted milk samples were precipitated in 36% ammonium sulfate and centrifuged at 3000 × g for 20 min at 4 ◦ C, and the whey portion was removed. Ammonium sulfate, in the whey portion, was exchanged with PBS (pH7.4) using a 3000 MW membrane dialysis cassette (Pierce, Rockford, IL) in 1 L of PBS (pH 7.4) at 4 ◦ C overnight (Pfeffer et al., 2005). 2.4. Determination of total and antigen-specific IgA Crude worm antigen (CWA) was extracted from adult H. contortus collected from the abomasum of a previously infected donor. Worms were washed twice in PBS (pH 7.4), diluted to a final volume of 7 ml, and homogenized in an ice-cold glass homogenizer. Homogenate was centrifuged at 15,000 × g at 4 ◦ C for 1 h, protein content of the supernatant was measured using the BCA assay (Pierce, Rockford, IL), and samples were stored at −80 ◦ C. To determine the appropriate concentration of CWA in this assay a checkerboard titration was performed. Antigen-specific and total IgA were assayed using 96-well plates (Nunc, Rochester, NY) coated with either 0.4 g/ml of crude worm antigen
(CWA) or polyclonal anti-sheep IgA (1:100) (Bethyl, Montgomery, TX) diluted in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 ◦ C. Plates were washed 3 times in PBS with 0.05% Tween-20, blocked using PBS with 0.1%BSA (PBS-B), and allowed to incubate for 1 h at room temperature (RT). After another wash step, serum and milk samples were diluted (1:1000 and 1:20, respectively) in PBS-B, added to duplicate wells, and samples were allowed to incubate for 2 h at RT. To confirm antigen specificity PBS-B was added to two wells on each plate in place of sample. Subsequent blank absorbance was subtracted from sample absorbance to correct for non-specific binding. Polyclonal anti-sheep IgA conjugated with HRP (Bethyl Laboratories Inc., Montgomery, TX.) was diluted 1:1500 and incubated at RT for 1 h. After a final wash step, TMB substrate (Pierce, Rockford, IL) was added and plates were read at a wavelength of 450 nm, on a Biotek EL-311 spectrophotometer. Intra-sample, intergroup variation was accepted when the CV was less than 10%. Inter-plate variation was evaluated by comparing the absorbance of the standards and which had a CV of less than 10%. Total IgA values were compared to a sheep IgA standard curve that was run in duplicate and 2-fold serially diluted eight places with a starting concentration of 6 g/ml (Accurate Chemical Co., Westbury, NY).
Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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Fecal egg counts and immunoglobulin data were not normally distributed and were transformed as log10 (FEC + 25) (LFEC) or log10 (ABS + 1), respectively, and analyzed using the general lineal model of SAS (SAS Institute, Cary, NC). Fixed effects in the model included breed, time and two-way interactions. Significant differences in LS means analysis were compared using t-tests with critical probability values calculated using the Bonferroni adjustment for comparisons among more than two means. Means of transformed FEC and absorbance values were backtransformed for reporting.
3000 FEC (eggs/g)
2.5. Statistical analysis
(A) Hair Wool
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3. Results
Ewe lambs were infected after breeding to evaluate differences in response to infection. By this age, and with a history of grazing pastures known to be contaminated with H. contortus (Fig. 1), both types of ewes have had opportunity to develop sufficient immunological memory to effectively respond to parasite infection. After challenge infection there was a significant increase in FEC, but no difference was observed between breed types (Fig. 2a). Concomitant reductions in PCV were observed, beginning on day 21 and continuing to day 35 (Fig. 2b), thereby confirming successful H. contortus infection. Changes in total serum IgA levels (T-IgA) in both types revealed a two-stage antibody production pattern (Fig. 2c). Average T-IgA through 35 days post-infection was consistently higher in hair sheep (P < 0.05) and peaked twice; once at day 5 (13.2 mg/ml) and again at day 28 (17.3 mg/ml). The initial pattern of increase in T-IgA production in wool ewes was similar to that observed in hair ewes, although of lesser magnitude. However, the pronounced IgA increase observed in hair sheep at day 28 was not mirrored in wool ewes. Hair sheep appeared to have higher initial levels of circulating antibody and were able to rapidly increase antibody production following an additional acute parasite challenge.
Packed Cell Volume (%)
40 3.1. Initial response to challenge infection following breeding
(B)
35
30
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20 2 2 3 1 3 1 Week before Week after Parturition Fig. 3. Evaluation of FEC and PCV during periparturient period. An autumn artificial infection of H. contortus was allowed to persist in pregnant ewe lambs and evaluated for 3 weeks before and after parturition. (a) Fecal egg count (FEC) was measured to evaluate parasite load, and (b) packed cell volume (PCV) was used to measure the impact and magnitude of infection. Error bars represent standard error of means.
PCV (31.6%) than wool ewes (28.5%) over the periparturient period (P < 0.05; Fig. 2b), and the breed type by time interaction was again not significant. 3.3. Serum and milk antibody levels in periparturient yearling ewes
3.2. FEC and PCV levels in periparturient yearling ewes Data collection during the periparturient period (Fig. 1) began on December 22, 2006 approximately 1 month after the end of the data collection period shown in Fig. 3. Although there was no significant time by type interaction, wool ewes had higher average FEC than hair ewes during the periparturient period (1124 versus 205 eggs/g of feces; P < 0.001). By approximately 3 weeks before parturition, hair ewes had nearly cleared their previous parasite infection, with FEC of <500 eggs/g (Fig. 3a). In contrast, FEC remained high in wool ewes until 1 week prior to parturition, after which time FEC declined. Fecal egg counts in hair sheep also declined immediately before parturition, albeit from a much lower baseline, and the analysis of transformed FEC revealed no significant breed type by time interaction. Hair ewes likewise had a significantly higher
Among infected ewes, wool ewes had much higher serum T-IgA levels at, and 3 weeks after, parturition compared to infected hair ewes or to control ewes of either type (P < 0.01) (Fig. 4a). Alternatively, CWA-IgA levels in infected hair ewes were much higher (P < 0.05) (Fig. 4b) than those observed in wool ewes or uninfected controls of either type. These data indicate that hair ewes generate and maintain a potent antigen-specific, humoral response to infection with H. contortus. Neither, the greater circulating T-IgA at and immediately following parturition in wool ewes, nor the elevated CWA-IgA observed in hair ewes were accompanied by higher concentrations of T-IgA in colostrum or milk (Fig. 4c and d). T-IgA or CWA-IgA levels in milk were not significantly different than uninfected controls when analyzed by breed. When total IgA was analyzed on the basis of
Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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CWA - IgA
Total IgA 0.4
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Fig. 4. Antibody response in serum and milk after lambing. Total IgA (T–IgA) in (A) serum and (C) milk and antigen–specific IgA (CWA–IgA) in serum (B) and (D) milk were measured at parturition, 3 and 6 weeks after parturition Uninfected animals did not receive an inoculation after the autumn breeding season. Means differ between types by *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent standard error of means.
4. Discussion During the acute response to experimental infection we found no differences between breeds in FEC up to 35 days following experimental infection. This result is surprising, as St. Croix sheep have been shown to have markedly lower FEC than sheep of this composite line following infection with H. contortus (Notter et al., 2003). Ewe lambs in this trial were bred to lamb at 1 year of age and were therefore maintained on a diet of concentrates and highquality forages. The effect of nutritional supplementation on reducing effects of parasitism are well-known (Eady et al., 2003), and in particular effects of protein supplementation on FEC reduction (Fox, 1997; Kahn et al., 2003). An increased nutritional plane may have improved resistance in wool ewes during the acute phase of infection accounting for the absence of differences in FEC and PCV at this time. When infected with identical doses of H. contortus during challenge infection a transient T-IgA response was observed in both types and may reflect changes in H. contortus life stages. Ultimately, wool ewes failed to generate T-IgA concentration equivalent to hair ewes within the first 35 days following challenge infection. Early elevated IgA concentration in hair ewes may have reflected
residual IgA production from previous natural infection, indicating that the rest period following anthelmintic treatment was insufficient to allow for antibody levels to regress. Nonetheless, circulating IgA concentration has been shown to be correlated with IgA activity at the local site of infection (Stear et al., 1995) and associated with FEC reduction in GIN-resistant sheep (MacKinnon et al., 6
**
Uninfected Infected
5 4
IgA (mg/ml)
infection we saw a significant difference between uninfected controls and the average of all infected animals at all time points (Fig. 5).
3 2 1 0
Fig. 5. Total IgA in milk of uninfected and infected ewes. Milk samples from uninfected ewes were only collected at parturition whereas samples from infected ewes were collected at parturition and 3 and 6 weeks after parturition. Data presented in this graph represent the average Total IgA in milk of all uninfected ewes at parturition and of all infected ewes across all time points. Means differ between types by **P < 0.01. Error bars represent standard deviation of means.
Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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2010). IgA may provide protection against gastro-intestinal trichostrongylids through multiple mechanisms including larval immobilization (Harrison et al., 2003), complement activation (McCoy et al., 2008), suppressing egg production in the adult female (Stear et al., 1995) or aid in adult killing via Fc receptor binding (Kooyman et al., 1997). Although IgA has been shown to be an important component of protective immunity (McCoy et al., 2008), the IgA response is driven by cellular immunity and T-helper cell-2 activation. Therefore elevated IgA levels observed may be more indicative of enhanced immunity in hair sheep rather than inferring a specific mechanism of GINresistance. Differences in the mean FEC between breed types were observed during the periparturient period. Hair ewes had a marked reduction when compared to their FEC at day 35, while wool ewes maintained an elevated fecal egg output. A pattern of FEC reduction was observed in wool ewes from week 3 to week 1 prior to parturition, but whether that reduction was due to immunity or parasite senescence in the host is not known based on the data we have collected. At parturition and at week 3 of lactation, wool ewes had more T-IgA in their serum than hair ewes, presumably an artifact of elevated FEC prior to parturition. An increase in serum concentration of IgA has been reported to be associated with concentrations of IgA in colostrum and milk (Jertborn et al., 1986). However, no differences existed in concentrations of T-IgA in the milk or colostrum of hair and wool ewes. Thus, higher concentration of T-IgA in the serum at parturition may be more reflective of recovery from infection than an ability to mobilize IgA to milk. Mean T-IgA levels for all ewes in the periparturient period were much lower than those observed immediately following challenge and the lower level of T-IgA observed in hair sheep after parturition is consistent with normal IgA serum concentration (Tizard, 2004), supporting the conclusion that hair sheep had a low periparturient FEC and thus did not require elevated antibody production. Infection status impacted total IgA levels observed in milk among both types when compared to uninfected controls, indicating that prior infection did have an effect on antibody level available in milk. Ultimately, no differences were observed in T-IgA of colostrum or milk, and in our study circulating antibody concentration was not reflective of antibody concentration in milk. Parasite-specific antibody levels of hair sheep were significantly higher than those of wool sheep at parturition and through 6 weeks after parturition. Higher CWA-IgA levels in hair sheep presumably represent greater immunological memory because, unlike wool ewes, hair ewes did not show evidence of substantial infection shortly before parturition. Lower serum CWA-IgA levels of wool ewes may be accounted for by infection; due to antibody sequestration at the site of infection. Colostrum and milk levels of CWA-IgA did not differ between breeds, indicating no elevated ability of hair-type sheep in mobilizing antigen-specific antibody to colostrum or milk. This study documents differences in IgA production of parasite-resistant hair and parasite-susceptible wool
sheep infected with H. contortus. Upon challenge infection, hair ewes had a greater and more rapid humoral immune response, and were able to both expeditiously reduce FEC levels and also maintain significantly higher circulating levels of antigen-specific IgA. Higher levels of circulating antibodies in hair sheep were not, however, reflective of antibody levels in colostrum or milk, suggesting that ewes of the two types possess comparable capacities to provide potential IgA-mediated protection to the neonate. Future studies utilizing a cross-fostering approach may be required to elucidate the effects of colostral antibodies on parasitism in lambs. References Amarante, A.F.T., Bricarello, P.A., Huntley, J.F., Mazzolin, L.P., Gomes, J.C., 2005. Relationship of abomasal histology and parasite-specific immunoglobulin A with the resistance to Haemonchus contortus infection in three breeds of sheep. Vet. Parasitol. 128, 99–107. Butler, J.E., 1999. Immunoglobulins and immunocytes in animal milks. In: Ogra, P.L. (Ed.), Mucosal Immunology. Academic Press, New York, pp. 1531–1554. Eady, S.J., Woolaston, R.R., Barger, I.A., 2003. Comparison of genetic and nongenetic strategies for control of gastrointestinal nematodes of sheep. Livest Prod. Sci. 81, 11–23. Fox, M.T., 1997. Pathophysiology of infection with gastrointestinal nematodes in domestic ruminants: recent developments. Vet. Parasitol. 72, 285–308. Gamble, H.R., Zajac, A.M., 1992. Resistance of St. Croix lambs to Haemonchus contortus in experimentally and naturally acquired infections. Vet. Parasitol. 41, 211–225. Harrison, G.B.L., Pulford, H.D., Hein, W.R., Barber, T.K., Shaw, R.J., McNeill, M.M., Wakefield, S.J., Shoemaker, C.B., 2003. Immune rejection of Trichostrongylus colubriformis in sheep; a possible role for intestinal mucus antibody against an L3-specific surface antigen. Parasit. Immunol. 25, 45–53. Jertborn, M., Svennerholm, A.M., Holmgren, J., 1986. Saliva, breast milk, and serum antibody responses as indirect measures of intestinal immunity after oral cholera vaccination or natural disease. J. Clin. Microbiol. 24, 203–209. Kahn, L.P., Knox, M.R., Walkden-Brown, S.W., Lea, J.M., 2003. Regulation of the resistance to nematode parasites of single- and twin-bearing Merino ewes through nutrition and genetic selection. Vet. Parasitol. 114, 15–31. Kooyman, F.N.J., Van Kooten, P.J.S., Huntley, J.F., MacKellar, A., Cornelissen, A.W.C.A., Schallig, H.D.F.H., 1997. Production of a monoclonal antibody specific for ovine immunoglobulin E and its application to monitor serum IgE responses to Haemonchus contortus infection. Parasitology 114, 395–406. MacKinnon, K.M., Zajac, A.M., Kooyman, F.N.J., Notter, D.R., 2010. Differences in immune parameters are associated with resistance to Haemonchus contortus in Caribbean hair sheep. Parasit. Immunol. 32, 484–493. Martinez-Valladares, M., Vara-Del Rio, M.P., Cruz-Rojo, M.A., RojoVazquez, F.A., 2005. Genetic resistance to Teladorsagia circumcinta: IgA and parameters at slaughter in Churra sheep. Parasit. Immunol. 27, 213–218. McCoy, K.D., Stoel, M., Stettler, R., Merky, P., Fink, K., Senn, B.M., Schaer, C., Massacand, J., Odermatt, B., Oettgen, H.C., Zinkernagel, R.M., Bos, N.A., Hengartner, H., Macpherson, A.J., Harris, N.L., 2008. Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection. Cell Host Microbe 4, 362–373. NCCLS, Procedure for Determining Packed Cell Volume by the Microhematocrit method; Approved Standard-Thrid Edition. NCCLS document H7-A3 (ISBN 1-56238-413-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA 2000. Notter, D.R., Andrew, S.A., Zajac, A.M., 2003. Responses of hair and wool sheep to a single fixed dose of infective larvae of Haemonchus contortus. Small Rumin. Res. 47, 221–225. Pfeffer, A., Shaw, R.J., Green, R., Phegan, M.D., 2005. The transfer of maternal IgE and other immunoglobulins specific for Trichostrongylus colubriformis larval excretory/secretory product to the neonatal lamb. Vet. Immunol. Immunopath. 108, 315–323.
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Stear, M.J., Bishop, S.C., Doligalska, M., Duncan, J.L., Holmes, P.H., Irvine, J., McCririe, L., McKellar, Q.A., Sinski, E., Murray, M., 1995. Regulation of egg production, worm burden, worm length and worm fecundity by host responses in sheep infected with Ostertagia circumcincta. Parasit. Immunol. 17, 643–652. Tizard, I.R., 2004. Veterinary Immunology: An Introduction, 7th edition. Saunders, Philadelphia, PA.
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Vanimisetti, H.B., Greiner, S.P., Zajac, A.M., Notter, D.R., 2004. Performance of hair sheep composite breeds: resistance of lambs to Haemonchus contortus. J. Anim. Sci. 82, 595–604. Whitlock, H.V., 1948. Some modifications of the McMaster helminth eggcounting technique apparatus. J. Council Sci. In. Res. 21, 177–180. Zajac, A.M., Conboy, G.A., 2006. Veterinary Clinical Parasitology, 7th edition. Wiley-Blackwell, Hoboken, NJ.
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Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection Scott Bowdridge a,1 , Kathryn MacKinnon a,2 , Joshua C. McCann a,3 , Anne M. Zajac b , David R. Notter a,∗ a b
Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA 24061, USA
a r t i c l e
i n f o
Article history: Received 11 May 2011 Received in revised form 28 November 2012 Accepted 14 January 2013 Keywords: Hair sheep Colostrum IgA Haemonchus
a b s t r a c t Antibody levels produced in response to gastro-intestinal nematode (GIN) parasite infection are typically higher in GIN-resistant breeds than susceptible breeds. Consequently, GINresistant ewes should generate greater parasite-specific antibody in colostrum and milk, potentially providing greater passive immunity to young lambs. To test this hypothesis, we monitored immunoglobulin levels in wool and hair-type sheep infected with Haemonchus contortus for 35 days following the end of a 45-day autumn breeding season and subsequently for 6 weeks around the time of parturition. Ten, first-parity ewes of each type were infected with 12,000 H. contortus L3 larvae following the end of breeding. In response to infection, hair ewes generated greater serum IgA (P < 0.05), although the pattern of IgA production was similar between the types. Following experimental infection, wool ewes were incapable of clearing the parasite infection well in advance of parturition. Prior to parturition, hair ewes had lower FEC and greater circulating H. contortus-specific IgA. However, no difference was seen in total or antigen-specific IgA production in the colostrum and milk of either breed. These data further demonstrate that hair-type sheep can rapidly reduce fecal egg output and generate greater humoral immunity as evidenced by higher levels of circulating antigen-specific antibody, but there is no evidence to suggest GIN-resistant sheep preferentially mobilize antigen-specific IgA to colostrum or milk. Thus, no clear difference exists between types of sheep in ability to deliver parasite-specific IgA to their offspring. © 2013 Published by Elsevier B.V.
1. Introduction Hair-type sheep are well known for their resistance to gastro-intestinal trichostrongylid parasites, however the
∗ Corresponding author. Tel.: +1 5402315135. E-mail addresses: [email protected] (S. Bowdridge), [email protected] (K. MacKinnon), [email protected] (J.C. McCann), [email protected] (D.R. Notter). 1 Present address: Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506, USA. 2 Present address: World Wide Life Sciences, 8810 Westgate Park Dr. Suite #108, Raleigh, NC 27617, USA. 3 Present address: Department of Animal Science, Texas A&M University, College Station, TX 77843, USA.
biological mechanism of resistance has yet to be fully characterized. St. Croix sheep, originating in the Caribbean, have developed a natural resistance to parasites that is at least partly mediated by their increased humoral immune response to helminth parasites (Gamble and Zajac, 1992). Parasite-resistant sheep have a distinct advantage in developing a more potent acquired immune response as evidenced by an increase in circulating and local antibodies (MacKinnon et al., 2010). In utero antibody transfer generally does not occur in livestock (Butler, 1999), but amounts of IgG and IgA in colostrum are substantial. In neonatal lambs, pinocytotic absorption of antibodies allows for passive immunity to occur, but this ability declines rapidly, as do antibody concentrations in milk (Butler, 1999). Measurable levels of
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Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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Fig. 1. Schematic timeline of experimental events. The first trial was conducted from day 0 to day 35 after infection with H. contortus. The second trial was initiated at least 3 weeks prior to parturition, which began on December 22, 2006. Milk samples were collected at 3 and 6 weeks following parturition.
the antibodies IgG and IgA remain in the milk of sheep for at least 30 days after parturition and may provide protection to newborn offspring (Pfeffer et al., 2005). IgA is typically found in surface secretions and in high levels in saliva, intestinal fluid, milk, colostrum and urine (Tizard, 2004). The major role of IgA is immune exclusion of pathogens, as it is secreted in the mucus of the small intestine and protects the lumen from invasion by viruses, bacteria or parasites (Tizard, 2004). Increased production of IgA is also an indicator of immune responsiveness to gastro-intestinal nematode infection (Amarante et al., 2005; Martinez-Valladares et al., 2005; Pfeffer et al., 2005), especially during challenge helminth infection. Plasma levels of parasite-specific antibodies in neonatal lambs have been reported to be directly proportional to the levels of parasite-specific antibody in the colostrum or milk of the ewe (Pfeffer et al., 2005). However, there is a lack of information as to the perceived ability of parasite-resistant sheep to have greater colostrum or milk antibody levels or whether immune clearance of parasite infection during gestation can enhance levels of antigen-specific antibodies passively transferred to the neonate. Thus the goal of this experiment was to compare acute and long-term periparturient immune responses of hair and wool-type ewes challenged with H. contortus immediately following breeding. 2. Materials and methods 2.1. Sheep and experimental design Ten St. Croix hair sheep ewe lambs and 10 ewe lambs of a composite line of wool sheep developed at Virginia Tech were used for this study. Animals from the composite line were made up of 50% Dorset, 25% Finnsheep and 25% Rambouillet breeding and have been shown to be significantly more susceptible than St. Croix to infection by H. contortus (Notter et al., 2003; Vanimisetti et al., 2004). Animals were born in January 2006 and exposed to rams for 45 days beginning at approximately 7 months of age in early August. All ewes were housed indoors from the end of breeding in September 2006. Animals and procedures used in this study were approved by the Virginia Tech IACUC Committee. Following breeding and natural parasite exposure on pastures known to be contaminated with H. contortus
and other trichostrongylid nematodes, ewes were treated with levamisole (8 mg/kg) (Agri-lab, St. Joseph, MO), which reduced individual fecal egg counts (FEC) to ≤ 50 eggs/g, and kept in drylot for 2 weeks (Fig. 1). Animals were then orally administered 12,000 H. contortus L3 larvae and this challenge infection was allowed to persist through the periparturient period. Fifty days after ram exposure, transabdominal ultrasound was used to determine pregnancy and thereby identify 10 pregnant ewe lambs of each type to be included in the study. Additionally three pregnant ewe lambs of each type were maintained during the periparturient period as uninfected controls to evaluate control IgA levels. Pregnant ewes were housed indoors in drylot throughout lambing and early lactation in order to avoid environmental exposure to infective larvae. A 16% CP cornsoybean ration, grass hay, and water were provided ad libitum. 2.2. Parasitological techniques Adult H. contortus were collected from euthanized sheep and pulverized in an ice-cold glass tissue homogenizer to release developing eggs. Homogenate was mixed with egg-free feces to obtain a mono-specific larval culture. The Baermann technique (Zajac and Conboy, 2006) was used to collect H. contortus L3 larvae which were then used to infect two feedlot-raised donor wethers that were dewormed to a FEC of 0, as determined by McMaster. At least 21 days after infection, feces were collected from donor lambs and cultured at 30 ◦ C for 7–8 days. Collected larvae were stored at 4 ◦ C in deionized water, and used within 1 month to orally infect experimental animals. Fecal egg counts were determined using the modified McMaster technique (Whitlock, 1948), and PCV were determined by the micro-hematocrit centrifuge method (NCCLS, 2000). 2.3. Blood collection and extraction of milk immunoglobulins Blood was collected via jugular venipuncture into a 10 ml vacutainer tube (BD) for serum antibody analysis and into a 4 ml EDTA-treated vacutainer tube (BD) for measurement of PCV. Since ewes did not lamb on the same day blood samples were taken twice weekly and the sample taken closest (±2 days) to the predefined time-point was used
Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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7000 (A)
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Day after infection Fig. 2. Response to H. contortus infection after breeding. Ten ewe lambs of each type were infected with 12,000 H. contortus L3 larvae shortly following a 45-day breeding season. Response to infection was measured by (a) fecal egg count (FEC), (b) packed cell volume (PCV) and (c) total serum IgA. Means differ between types by *P < .05, **P < 0.01, ***P < 0.001. Means for FEC and PCV with different letters indicate a significant effect of day (P < 0.05). Error bars represent standard error of means.
for analysis, prior to parturition. Milk (10 ml from each half of the udder) and blood samples were collected from each ewe within 12 h after parturition. Colostrum and milk samples were pooled by individual, diluted 1:1 in PBS (pH 7.4), and stored at -20 ◦ C. Diluted milk samples were precipitated in 36% ammonium sulfate and centrifuged at 3000 × g for 20 min at 4 ◦ C, and the whey portion was removed. Ammonium sulfate, in the whey portion, was exchanged with PBS (pH7.4) using a 3000 MW membrane dialysis cassette (Pierce, Rockford, IL) in 1 L of PBS (pH 7.4) at 4 ◦ C overnight (Pfeffer et al., 2005). 2.4. Determination of total and antigen-specific IgA Crude worm antigen (CWA) was extracted from adult H. contortus collected from the abomasum of a previously infected donor. Worms were washed twice in PBS (pH 7.4), diluted to a final volume of 7 ml, and homogenized in an ice-cold glass homogenizer. Homogenate was centrifuged at 15,000 × g at 4 ◦ C for 1 h, protein content of the supernatant was measured using the BCA assay (Pierce, Rockford, IL), and samples were stored at −80 ◦ C. To determine the appropriate concentration of CWA in this assay a checkerboard titration was performed. Antigen-specific and total IgA were assayed using 96-well plates (Nunc, Rochester, NY) coated with either 0.4 g/ml of crude worm antigen
(CWA) or polyclonal anti-sheep IgA (1:100) (Bethyl, Montgomery, TX) diluted in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 ◦ C. Plates were washed 3 times in PBS with 0.05% Tween-20, blocked using PBS with 0.1%BSA (PBS-B), and allowed to incubate for 1 h at room temperature (RT). After another wash step, serum and milk samples were diluted (1:1000 and 1:20, respectively) in PBS-B, added to duplicate wells, and samples were allowed to incubate for 2 h at RT. To confirm antigen specificity PBS-B was added to two wells on each plate in place of sample. Subsequent blank absorbance was subtracted from sample absorbance to correct for non-specific binding. Polyclonal anti-sheep IgA conjugated with HRP (Bethyl Laboratories Inc., Montgomery, TX.) was diluted 1:1500 and incubated at RT for 1 h. After a final wash step, TMB substrate (Pierce, Rockford, IL) was added and plates were read at a wavelength of 450 nm, on a Biotek EL-311 spectrophotometer. Intra-sample, intergroup variation was accepted when the CV was less than 10%. Inter-plate variation was evaluated by comparing the absorbance of the standards and which had a CV of less than 10%. Total IgA values were compared to a sheep IgA standard curve that was run in duplicate and 2-fold serially diluted eight places with a starting concentration of 6 g/ml (Accurate Chemical Co., Westbury, NY).
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Fecal egg counts and immunoglobulin data were not normally distributed and were transformed as log10 (FEC + 25) (LFEC) or log10 (ABS + 1), respectively, and analyzed using the general lineal model of SAS (SAS Institute, Cary, NC). Fixed effects in the model included breed, time and two-way interactions. Significant differences in LS means analysis were compared using t-tests with critical probability values calculated using the Bonferroni adjustment for comparisons among more than two means. Means of transformed FEC and absorbance values were backtransformed for reporting.
3000 FEC (eggs/g)
2.5. Statistical analysis
(A) Hair Wool
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3. Results
Ewe lambs were infected after breeding to evaluate differences in response to infection. By this age, and with a history of grazing pastures known to be contaminated with H. contortus (Fig. 1), both types of ewes have had opportunity to develop sufficient immunological memory to effectively respond to parasite infection. After challenge infection there was a significant increase in FEC, but no difference was observed between breed types (Fig. 2a). Concomitant reductions in PCV were observed, beginning on day 21 and continuing to day 35 (Fig. 2b), thereby confirming successful H. contortus infection. Changes in total serum IgA levels (T-IgA) in both types revealed a two-stage antibody production pattern (Fig. 2c). Average T-IgA through 35 days post-infection was consistently higher in hair sheep (P < 0.05) and peaked twice; once at day 5 (13.2 mg/ml) and again at day 28 (17.3 mg/ml). The initial pattern of increase in T-IgA production in wool ewes was similar to that observed in hair ewes, although of lesser magnitude. However, the pronounced IgA increase observed in hair sheep at day 28 was not mirrored in wool ewes. Hair sheep appeared to have higher initial levels of circulating antibody and were able to rapidly increase antibody production following an additional acute parasite challenge.
Packed Cell Volume (%)
40 3.1. Initial response to challenge infection following breeding
(B)
35
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20 2 2 3 1 3 1 Week before Week after Parturition Fig. 3. Evaluation of FEC and PCV during periparturient period. An autumn artificial infection of H. contortus was allowed to persist in pregnant ewe lambs and evaluated for 3 weeks before and after parturition. (a) Fecal egg count (FEC) was measured to evaluate parasite load, and (b) packed cell volume (PCV) was used to measure the impact and magnitude of infection. Error bars represent standard error of means.
PCV (31.6%) than wool ewes (28.5%) over the periparturient period (P < 0.05; Fig. 2b), and the breed type by time interaction was again not significant. 3.3. Serum and milk antibody levels in periparturient yearling ewes
3.2. FEC and PCV levels in periparturient yearling ewes Data collection during the periparturient period (Fig. 1) began on December 22, 2006 approximately 1 month after the end of the data collection period shown in Fig. 3. Although there was no significant time by type interaction, wool ewes had higher average FEC than hair ewes during the periparturient period (1124 versus 205 eggs/g of feces; P < 0.001). By approximately 3 weeks before parturition, hair ewes had nearly cleared their previous parasite infection, with FEC of <500 eggs/g (Fig. 3a). In contrast, FEC remained high in wool ewes until 1 week prior to parturition, after which time FEC declined. Fecal egg counts in hair sheep also declined immediately before parturition, albeit from a much lower baseline, and the analysis of transformed FEC revealed no significant breed type by time interaction. Hair ewes likewise had a significantly higher
Among infected ewes, wool ewes had much higher serum T-IgA levels at, and 3 weeks after, parturition compared to infected hair ewes or to control ewes of either type (P < 0.01) (Fig. 4a). Alternatively, CWA-IgA levels in infected hair ewes were much higher (P < 0.05) (Fig. 4b) than those observed in wool ewes or uninfected controls of either type. These data indicate that hair ewes generate and maintain a potent antigen-specific, humoral response to infection with H. contortus. Neither, the greater circulating T-IgA at and immediately following parturition in wool ewes, nor the elevated CWA-IgA observed in hair ewes were accompanied by higher concentrations of T-IgA in colostrum or milk (Fig. 4c and d). T-IgA or CWA-IgA levels in milk were not significantly different than uninfected controls when analyzed by breed. When total IgA was analyzed on the basis of
Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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CWA - IgA
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Fig. 4. Antibody response in serum and milk after lambing. Total IgA (T–IgA) in (A) serum and (C) milk and antigen–specific IgA (CWA–IgA) in serum (B) and (D) milk were measured at parturition, 3 and 6 weeks after parturition Uninfected animals did not receive an inoculation after the autumn breeding season. Means differ between types by *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent standard error of means.
4. Discussion During the acute response to experimental infection we found no differences between breeds in FEC up to 35 days following experimental infection. This result is surprising, as St. Croix sheep have been shown to have markedly lower FEC than sheep of this composite line following infection with H. contortus (Notter et al., 2003). Ewe lambs in this trial were bred to lamb at 1 year of age and were therefore maintained on a diet of concentrates and highquality forages. The effect of nutritional supplementation on reducing effects of parasitism are well-known (Eady et al., 2003), and in particular effects of protein supplementation on FEC reduction (Fox, 1997; Kahn et al., 2003). An increased nutritional plane may have improved resistance in wool ewes during the acute phase of infection accounting for the absence of differences in FEC and PCV at this time. When infected with identical doses of H. contortus during challenge infection a transient T-IgA response was observed in both types and may reflect changes in H. contortus life stages. Ultimately, wool ewes failed to generate T-IgA concentration equivalent to hair ewes within the first 35 days following challenge infection. Early elevated IgA concentration in hair ewes may have reflected
residual IgA production from previous natural infection, indicating that the rest period following anthelmintic treatment was insufficient to allow for antibody levels to regress. Nonetheless, circulating IgA concentration has been shown to be correlated with IgA activity at the local site of infection (Stear et al., 1995) and associated with FEC reduction in GIN-resistant sheep (MacKinnon et al., 6
**
Uninfected Infected
5 4
IgA (mg/ml)
infection we saw a significant difference between uninfected controls and the average of all infected animals at all time points (Fig. 5).
3 2 1 0
Fig. 5. Total IgA in milk of uninfected and infected ewes. Milk samples from uninfected ewes were only collected at parturition whereas samples from infected ewes were collected at parturition and 3 and 6 weeks after parturition. Data presented in this graph represent the average Total IgA in milk of all uninfected ewes at parturition and of all infected ewes across all time points. Means differ between types by **P < 0.01. Error bars represent standard deviation of means.
Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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2010). IgA may provide protection against gastro-intestinal trichostrongylids through multiple mechanisms including larval immobilization (Harrison et al., 2003), complement activation (McCoy et al., 2008), suppressing egg production in the adult female (Stear et al., 1995) or aid in adult killing via Fc receptor binding (Kooyman et al., 1997). Although IgA has been shown to be an important component of protective immunity (McCoy et al., 2008), the IgA response is driven by cellular immunity and T-helper cell-2 activation. Therefore elevated IgA levels observed may be more indicative of enhanced immunity in hair sheep rather than inferring a specific mechanism of GINresistance. Differences in the mean FEC between breed types were observed during the periparturient period. Hair ewes had a marked reduction when compared to their FEC at day 35, while wool ewes maintained an elevated fecal egg output. A pattern of FEC reduction was observed in wool ewes from week 3 to week 1 prior to parturition, but whether that reduction was due to immunity or parasite senescence in the host is not known based on the data we have collected. At parturition and at week 3 of lactation, wool ewes had more T-IgA in their serum than hair ewes, presumably an artifact of elevated FEC prior to parturition. An increase in serum concentration of IgA has been reported to be associated with concentrations of IgA in colostrum and milk (Jertborn et al., 1986). However, no differences existed in concentrations of T-IgA in the milk or colostrum of hair and wool ewes. Thus, higher concentration of T-IgA in the serum at parturition may be more reflective of recovery from infection than an ability to mobilize IgA to milk. Mean T-IgA levels for all ewes in the periparturient period were much lower than those observed immediately following challenge and the lower level of T-IgA observed in hair sheep after parturition is consistent with normal IgA serum concentration (Tizard, 2004), supporting the conclusion that hair sheep had a low periparturient FEC and thus did not require elevated antibody production. Infection status impacted total IgA levels observed in milk among both types when compared to uninfected controls, indicating that prior infection did have an effect on antibody level available in milk. Ultimately, no differences were observed in T-IgA of colostrum or milk, and in our study circulating antibody concentration was not reflective of antibody concentration in milk. Parasite-specific antibody levels of hair sheep were significantly higher than those of wool sheep at parturition and through 6 weeks after parturition. Higher CWA-IgA levels in hair sheep presumably represent greater immunological memory because, unlike wool ewes, hair ewes did not show evidence of substantial infection shortly before parturition. Lower serum CWA-IgA levels of wool ewes may be accounted for by infection; due to antibody sequestration at the site of infection. Colostrum and milk levels of CWA-IgA did not differ between breeds, indicating no elevated ability of hair-type sheep in mobilizing antigen-specific antibody to colostrum or milk. This study documents differences in IgA production of parasite-resistant hair and parasite-susceptible wool
sheep infected with H. contortus. Upon challenge infection, hair ewes had a greater and more rapid humoral immune response, and were able to both expeditiously reduce FEC levels and also maintain significantly higher circulating levels of antigen-specific IgA. Higher levels of circulating antibodies in hair sheep were not, however, reflective of antibody levels in colostrum or milk, suggesting that ewes of the two types possess comparable capacities to provide potential IgA-mediated protection to the neonate. Future studies utilizing a cross-fostering approach may be required to elucidate the effects of colostral antibodies on parasitism in lambs. References Amarante, A.F.T., Bricarello, P.A., Huntley, J.F., Mazzolin, L.P., Gomes, J.C., 2005. Relationship of abomasal histology and parasite-specific immunoglobulin A with the resistance to Haemonchus contortus infection in three breeds of sheep. Vet. Parasitol. 128, 99–107. Butler, J.E., 1999. Immunoglobulins and immunocytes in animal milks. In: Ogra, P.L. (Ed.), Mucosal Immunology. Academic Press, New York, pp. 1531–1554. Eady, S.J., Woolaston, R.R., Barger, I.A., 2003. Comparison of genetic and nongenetic strategies for control of gastrointestinal nematodes of sheep. Livest Prod. Sci. 81, 11–23. Fox, M.T., 1997. Pathophysiology of infection with gastrointestinal nematodes in domestic ruminants: recent developments. Vet. Parasitol. 72, 285–308. Gamble, H.R., Zajac, A.M., 1992. Resistance of St. Croix lambs to Haemonchus contortus in experimentally and naturally acquired infections. Vet. Parasitol. 41, 211–225. Harrison, G.B.L., Pulford, H.D., Hein, W.R., Barber, T.K., Shaw, R.J., McNeill, M.M., Wakefield, S.J., Shoemaker, C.B., 2003. Immune rejection of Trichostrongylus colubriformis in sheep; a possible role for intestinal mucus antibody against an L3-specific surface antigen. Parasit. Immunol. 25, 45–53. Jertborn, M., Svennerholm, A.M., Holmgren, J., 1986. Saliva, breast milk, and serum antibody responses as indirect measures of intestinal immunity after oral cholera vaccination or natural disease. J. Clin. Microbiol. 24, 203–209. Kahn, L.P., Knox, M.R., Walkden-Brown, S.W., Lea, J.M., 2003. Regulation of the resistance to nematode parasites of single- and twin-bearing Merino ewes through nutrition and genetic selection. Vet. Parasitol. 114, 15–31. Kooyman, F.N.J., Van Kooten, P.J.S., Huntley, J.F., MacKellar, A., Cornelissen, A.W.C.A., Schallig, H.D.F.H., 1997. Production of a monoclonal antibody specific for ovine immunoglobulin E and its application to monitor serum IgE responses to Haemonchus contortus infection. Parasitology 114, 395–406. MacKinnon, K.M., Zajac, A.M., Kooyman, F.N.J., Notter, D.R., 2010. Differences in immune parameters are associated with resistance to Haemonchus contortus in Caribbean hair sheep. Parasit. Immunol. 32, 484–493. Martinez-Valladares, M., Vara-Del Rio, M.P., Cruz-Rojo, M.A., RojoVazquez, F.A., 2005. Genetic resistance to Teladorsagia circumcinta: IgA and parameters at slaughter in Churra sheep. Parasit. Immunol. 27, 213–218. McCoy, K.D., Stoel, M., Stettler, R., Merky, P., Fink, K., Senn, B.M., Schaer, C., Massacand, J., Odermatt, B., Oettgen, H.C., Zinkernagel, R.M., Bos, N.A., Hengartner, H., Macpherson, A.J., Harris, N.L., 2008. Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection. Cell Host Microbe 4, 362–373. NCCLS, Procedure for Determining Packed Cell Volume by the Microhematocrit method; Approved Standard-Thrid Edition. NCCLS document H7-A3 (ISBN 1-56238-413-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA 2000. Notter, D.R., Andrew, S.A., Zajac, A.M., 2003. Responses of hair and wool sheep to a single fixed dose of infective larvae of Haemonchus contortus. Small Rumin. Res. 47, 221–225. Pfeffer, A., Shaw, R.J., Green, R., Phegan, M.D., 2005. The transfer of maternal IgE and other immunoglobulins specific for Trichostrongylus colubriformis larval excretory/secretory product to the neonatal lamb. Vet. Immunol. Immunopath. 108, 315–323.
Please cite this article in press as: Bowdridge, S., et al., Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Vet. Parasitol. (2013), http://dx.doi.org/10.1016/j.vetpar.2013.01.008
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Stear, M.J., Bishop, S.C., Doligalska, M., Duncan, J.L., Holmes, P.H., Irvine, J., McCririe, L., McKellar, Q.A., Sinski, E., Murray, M., 1995. Regulation of egg production, worm burden, worm length and worm fecundity by host responses in sheep infected with Ostertagia circumcincta. Parasit. Immunol. 17, 643–652. Tizard, I.R., 2004. Veterinary Immunology: An Introduction, 7th edition. Saunders, Philadelphia, PA.
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Vanimisetti, H.B., Greiner, S.P., Zajac, A.M., Notter, D.R., 2004. Performance of hair sheep composite breeds: resistance of lambs to Haemonchus contortus. J. Anim. Sci. 82, 595–604. Whitlock, H.V., 1948. Some modifications of the McMaster helminth eggcounting technique apparatus. J. Council Sci. In. Res. 21, 177–180. Zajac, A.M., Conboy, G.A., 2006. Veterinary Clinical Parasitology, 7th edition. Wiley-Blackwell, Hoboken, NJ.
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