Copyright Blackwell Munksgaard, 2004 AJRI 2004; 52: 197–203
American Journal of Reproductive Immunology
Effect of Peptidoglycan–Polysaccharide Complex on Reproductive Efficiency in Sheep Hola´skova´ I, Lewis GS, Elliott M, Blemings KP, Dailey RA. Effect of peptidoglycan-polysaccharide complex on reproductive efficiency in sheep. AJRI 2004; 52:197–203 Blackwell Munksgaard, 2004 PROBLEM: Spontaneous mastitis or induced infections mimicking mastitis reduce pregnancy rates in ruminants. The effect of immunization with either a mastitis-related pathogen component, peptidoglycan–polysaccharide (PG–PS), or killed Streptococcus pyogenes on pregnancy outcome was investigated. METHOD OF STUDY: Ewe lambs were immunized with PG–PS (n ¼ 50) or killed bacteria (n ¼ 50) or were not immunized (control, n ¼ 100). Titers of PG–PS immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA). Ewes were bred by rams at synchronized estrus. All immunized ewes and half of the ewes not immunized were challenged with PG–PS on day 5 after breeding. Pregnancy maintenance was evaluated. RESULTS: Although the proportion of ewes pregnant at day 42 after breeding did not differ among treatments, the probability of pregnancy decreased with total dose of PG–PS (P < 0.05). CONCLUSIONS: Immunization of ewe lambs with PG–PS or killed S. pyogenes did not improve pregnancy maintenance. Furthermore, the toxic streptococcal component decreased pregnancy rate in immunized sheep in a dose-dependent manner.
INTRODUCTION Early embryonic mortality accounts for most pregnancy losses in many mammalian species (20–30% in cows,1–3 sheep,4,5 and goats,5 5–40% in rodents,6 10– 20% in primates7–9). For enhanced profitability, dairy cows must be pregnant within 90 days after parturition,10 which means they must be inseminated during peak lactation when metabolic demand is high. Maternal disease during early embryonic development can adversely affect survival of the embryo. Contraction of mastitis, an inflammation of the mammary gland, shortly after artificial insemination, but not before insemination or later in pregnancy, decreased conception rate in Jersey cows.11 Hence, infection with mastitis-causing pathogens between fertilization and placentation might be a critical factor for loss of pregnancy. Two toxic components of bacteria, lipopolysaccharide (LPS) and peptidoglycan (PG; synonyms: glycopeptide, mucopeptide, or murein), have been used to
Ida Holskov1, Gregory S. Lewis2, Meenal Elliott3, Kenneth P. Blemings1, Robert A. Dailey1 1 Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, WV, USA; 2USDA Sheep Experiment Station at Dubois, ID, USA; 3Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV, USA
Key words: Fever, gram-positive bacteria, immunization, inflammation, mastitis, pregnancy Address reprint requests to Robert A. Dailey, Division of Animal and Veterinary Sciences, POB 6108, West Virginia University, Morgantown, WV 26506-6108, USA. E-mail:
[email protected] Submitted December 8, 2003; revised June 10, 2004; accepted June 22, 2004.
delineate the link between infection, such as mastitis, and early embryonic failure. Pregnancy rate in sheep was reduced after administration of 30 or 60 lg/kg of PG on day 5 after breeding.12 The biologic attributes of PG are: it is a main immunogenic component of the cell wall of gram-positive bacteria,13–15 a macrophage and complement activator,14 as well as a B-cell mitogen.16 For these and additional reasons, PG is responsible for many clinical manifestations including inflammation, fever, leukocytosis, hypotension, decreased peripheral perfusion, malaise, sleepiness, and arthritis.16,17 These physiologic effects are brought about by inflammatory mediators17 produced by antigen presenting cells (APC) after PG binds to the Toll-like receptor-2 (TLR-2).18 PG and LPS have similar biologic actions19,20 and evoke common components of the NF-jB intracellular signaling pathway leading to secretion of inflammatory cytokines, such as tumor necrosis factor a (TNFa) in mononuclear phagocytes.21 However, the PG-TLR-2 binding, with possible cooperation of TLR-2 and TLR-6,18,22
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induces gene products leading to lower toxicity and a Th2-like response. This is in contrast to a Th1-like response and toxicity during sepsis initiated by LPS that binds to TLR-4. Antisera to group A-variant streptococci were found to be a rich source of antibodies to PG.23–27 Further, daily pretreatment of rabbits,28 guinea-pigs,29 and rats30 with repeated doses of PG28 or muramyl dipeptide (MDP), the water soluble component of PG,29,30 induced tolerance to its pyrogenic effect. The goal of this study was to investigate if induction of a humoral response prior to post-breeding exposure would block the deleterious effect of exposure on pregnancy. Ewe lambs were repeatedly inoculated with PG–polysaccharide (PS) or whole killed Streptococcus pyogenes before breeding and the effect of this immunization on early pregnancy continuation was evaluated.
MATERIALS AND METHODS Animals and Treatment Groups The study was conducted at the USDA, ARS, US Sheep Experiment Station (Dubois, ID) using 8-month-old Rambouillet ewe lambs with approximate body weights (bw) of 50 kg. Ewes were on the ranch from early spring to late summer and then housed in outdoor pens (approximately 50 ewes/pen) and fed a standard balanced diet. During the breeding season ewes were assigned to blocks of four randomized treatments (n ¼ 50/group). Sheep in the first group (control) were injected with 3 mL saline on days 0, 22 and 5 days after breeding (this corresponded to 47 days from first injection). Sheep in the second group were inoculated with 3 mL saline on days 0 and 22, and with 3 mL PG–PS (60 lg PG/kg bw, without adjuvant) 5 days after breeding to mimic bacterial infection without immunization. Sheep in the third group were immunized on days 0 and 22 with 3 mL PG–PS (30 lg PG/kg bw, without adjuvant) and injected with 3 mL PG–PS (60 lg PG/kg bw, without adjuvant) 5 days after breeding. Sheep in the fourth group were immunized with heat-killed S. pyogenes (0.23 g wet cells/ewe, approximately equivalent to 30 lg PG/kg bw) on days 0 and 22, and were injected with PG–PS 5 days after breeding (60 lg PG/kg bw). Injections on days 0 and 22 were administered s.c. in the neck. Injections given 5 day after breeding were administered i.v. in the jugular. The estrous cycles of all ewes were synchronized 9 days after the second injection using progesterone pessaries (MAP, Canada) for 10 days31,32 with injection of prostaglandin F2a (15 mg/ewe i.m.; Lutalyse; Pharmacia and Upjohn, Inc., Kalamazoo, MI, USA) 5 days before progester BLACKWELL MUNKSGAARD, 2004
one withdrawal. At removal of pessaries, ewes received 400 IU of eCG and 200 IU of hCG (PG 600; 5 mL/ sheep, i.m.; Intervet, Millsboro, DE, USA) and were mated with fertile rams within 36 hr of gonadotropin injection (male to female ratio 1:10). About 160 ewes responded to estrous synchronization and only those that were bred remained in the experiment (n ¼ 36–41 per group).
Blood Samples and Data Collection Jugular blood samples were collected on days 0, 13, 35, 47, (day of challenge) and 84. Pregnancy status was examined on day 42 after breeding by transrectal ultrasonography33 using an Aloka 500 console with a 7.5 MHz linear transducer (Corometrics Medical Systems, Wallingford, CT, USA). Pregnancy data were confirmed at day 60 of gestation by abdominal sector ultrasonography. Peptidoglycan–Polysaccharide Isolation and Whole Killed Cells Preparation The PG–PS was prepared34 from S. pyogenes group A, type 3 (ATCC 10389). The teichoic acid and carbohydrates were not removed so that the final product had the PS attached to PG. Content of PG in PG–PS was 51% as determined by a rhamnose assay35 that measured the amount of methylpentoses, which is a function of the PG content.36 The PG–PS was suspended in 0.9% sodium chloride solution (1 mg PG–PS/1 mL) and sonicated for 90 min to ensure solubility and prevent aggregation of PG–PS molecules during storage. For preparation of whole killed cells, S. pyogenes were grown as described,34 killed by 1 hr pasteurization (60C), filtered, washed, suspended in sterile saline, and stored frozen. Assay of Antibodies Activation of humoral immunity was determined by measuring the immunoglobulin G (IgG) antibodies to PG–PS from 10 randomly selected serum samples from each treatment group using an enzyme-linked immunosorbent assay (ELISA).37 Since standard sheep PG– PS anti-sera are not commercially available, the ELISA assay was modified to include a titer-dilution protocol to determine the relative concentrations of IgG antibodies in the serum samples. One serum sample with an intermediate response (based on preliminary ELISA assays) relative to the other serum samples was selected as the standard. This standard was included on all ELISA plates analyzed. Flat bottom ELISA plates (96WL Easywash, HB, Fisher Scientific, Pittsburgh, PA, USA) were coated with 100 lL/well of sonicated PG–PS solution [0.002 lg PG/lL phosphate-buffered saline (PBS) with 0.04% sodium azide] and blocked with PBSTG
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Statistics The proportion of ewes pregnant on day 42 after breeding was the primary variable measured to indicate treatment response. Data were evaluated using Pearson’s chi-square test of homogeneity. The preplanned comparisons of percentage of ewes pregnant were: (i) each of the immunized groups (PG–PS, killed cells), and the non-immunized PG–PS challenged group was compared with the control; (ii) all sheep exposed to PG–PS compared with the control; (iii) all immunized sheep (immunized with PG–PS and immunized with killed cells) compared to non-immunized PG–PS challenged group; and (iv) sheep immunized with PG–PS compared to sheep immunized with killed cells. These comparisons using Pearson’s chisquare tests, allowed tests for the overall effect of bacterial component challenge, the protective effect of each immunization and the relative efficiency of each type of immunization. Logistic regression analysis (JMP statistical software Version 5.0, 2002; SAS Institute, Cary, NC, USA) was used for prediction of pregnancy from the cumulative dose of PG–PS (days 0 + 22 + 47 doses in all treatment groups
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except for sheep immunized with killed cells). Antibody response analysis was performed on the logarithm of titer using repeated measures anova (SAS GLM Procedure, 1999; SAS Institute). Significance level was set at a ¼ 0.05.
RESULTS Humoral Immune System Activation Ewes were immunized with PG–PS (30 lg PG/kg), or heat-killed S. pyogenes, or saline on day 0, and day 22. Ewes were mated with fertile rams 20 days after the second injection, and then challenged with 60 lg PG/ kg or saline 5 days after breeding. High concentrations of IgG antibodies to PG–PS were detected in immunized ewes (Fig. 1). Titers increased after immunization and the booster injection as detected by a treatment · day interaction (P < 0.05). Titers in all immunized animals (groups 3 and 4) were greater than in non-immunized ewes (groups 1 and 2) by day 13 (P < 0.05). Titers of PG–PS antibodies in sheep immunized with killed cells (group 4) were greater (P < 0.05) than in PG–PS-immunized ewes (group 3) on days 35, 47, and 82.
Control Non-immunized and challenged with PG-PS Immunized with PG-PS and challenged with PG-PS Immunized with killed cells and challenged with PG-PS 100,000 IgG titer (Log scale)
(50 lg Tween 20 and 0.5 g porcine gelatin in 100 mL PBS). For each plate, seven serum samples and the standard were transferred to the first column of the plate (100 lL sample/well). Next, samples were serially diluted using PBSTG in the remaining wells (one sample per row of wells). The plates were then incubated overnight at 4C. After incubation, plates were washed, alkaline phosphatase conjugated rabbit anti-sheep IgG was added (RBT anti-sheep IgG, Fisher Scientific; 100 lL/well diluted 1:3000 in PBSTG), and the plates were incubated at 37C for 1 hr. Substrate (100 lL/well, p-nitrophenyl phosphate in 1 m diethanolamine with 0.5 mm MgCl2, pH 9.8; Fisher Scientific) was applied, and plates were read in 10 min at a wavelength of 405 nm (Universal Microplate Spectrophotometer; lQuant; BIO-TECH Instruments, Inc., Winooski, VT, USA). Optical densities (OD) for each plate were standardized by (i) plotting the ODs of the standard, (ii) determining the mid-point (or one of two maximum) OD of the standard, (iii) determining the location (well or dilution point) of ODs in the remaining samples that is most similar to the standard’s mid-point OD, and (iv) recording the corresponding dilution point. This method allowed differences in anti-PG–PS IgG concentrations to be evaluated, based on the number of dilutions required for each sample to reach a standard optical density. To minimize error, only the linear portion of the standard curve was included in determination of the OD mid-point of the standard sample.
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Fig. 1. Anti-peptidoglycan–polysaccharide (PG–PS) immunoglobulin G (IgG) titers in Rambouillet sheep. Ewes were immunized with PG–PS (30 lg PG/kg), or heat-killed Streptococcus pyogenes, or saline on day 0, and day 22. Ewes were mated with fertile rams 20 days after second injection, and then challenged with 60 lg PG/kg or saline 5 days after breeding. Antibodies to PG–PS were measured by enzyme-linked immunosorbent assay (ELISA) in serum of 10 randomly selected sheep from each treatment group on days 0, 13, 35, 47 (day of the challenge), and 82. Bars represent mean titers (dilution rates) and the associated S.E.M. Repeated measures anova detected significant treatment by day interactions (P < 0.05) and also differences (P < 0.05) in titers of immunized compared with not immunized sheep. IgG titers of ewes immunized with whole killed bacteria were higher than in ewes immunized with PG–PS (P < 0.05).
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Pregnancy The percentages of ewes pregnant after treatment with saline, purified PG–PS, or killed S. pyogenes were 73% in control (group 1), 65% in non-immunized, PG–PS challenged (group 2), 47% in immunized with PG–PS (group 3), and 56% in sheep immunized with killed cells (group 4; P > 0.05; Fig. 2). The percentage of pregnant animals in the group immunized with PG–PS was significantly less than in the control group (P < 0.05). Control ewes had a greater pregnancy rate (73% versus 56%, P ¼ 0.08) than all PG–PSchallenged ewes (groups 2, 3, and 4). The percentage of immunized ewes that were pregnant (52%, combined groups 3 and 4), did not differ from the non-immunized, challenged group (65%, group 2, P > 0.05). Additionally, the effect of immunization with PG–PS did not differ from the effect of immunization with killed cells on pregnancy maintenance (47% versus 56%; P > 0.05). Using logistic regression, the cumulative dose of PG–PS (day 0 + day 22 + day 47) was a useful predictor of pregnancy (P < 0.05) as an increased cumulative dose of PG–PS decreased the proportion of ewes pregnant (Fig. 3).
% Pregnant
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Non-immunized Immunized with Immunized with and challenged PG-PS and killed cells and with PG-PS challenged with challenged with PG-PS PG-PS
Fig. 2. Histogram of the effect of treatment on percentage of ewes pregnant in Rambouillet sheep (N ¼ 160). Sheep were either injected with saline on days 0 and 22, or were immunized with peptidoglycan– polysaccharide (PG–PS) (30 lg PG/kg) or killed bacteria (0.23 g wet cells/sheep). Ewes were bred with rams 20 days later and injected with saline (control) or PG–PS (60 lg PG/kg body weight, all other groups) 5 days after breeding. Pregnancy was evaluated by presence of a viable embryo on day 42 after breeding using transrectal ultrasonography. Overall association of treatment and pregnancy was not detected by chi-square test (P > 0.05). After partitioning and grouping the contingency table according to pre-planned comparisons, chi-square analyses revealed that the proportion of pregnant animals in the group immunized with PG–PS was significantly lower than in the control ewes (P < 0.05). There was also a tendency for a greater pregnancy rate in control sheep (73%) than in all PG–PS-challenged ewes (56%, combined groups 2, 3, and 4, P ¼ 0.08). BLACKWELL MUNKSGAARD, 2004
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Fig. 3. Model predicting the percentage of ewes pregnant from the cumulative amount (first, plus second, plus challenge dose) of peptidoglycan–polysaccharide (PG–PS), excluding the killed cellsimmunized sheep. Logistic regression model: P(y) ¼ 1/ [1 + e ) ()1.05 + 0.009· x)], x ¼ cumulative amount of PG–PS, y ¼ predicted probability of pregnancy; N ¼ 119, P < 0.05.
DISCUSSION The PG–PS and killed S. pyogenes were used to immunize against a bacterial challenge of gram-positive mastitis. Immunization with the isolated component, or the whole killed pathogen, was expected to neutralize or opsonize the pathogen in the event of further bacterial challenge, thus preventing or reducing the early pregnancy loss if mastitis had occurred shortly after breeding. A humoral immune response to PG–PS, or killed S. pyogenes, was induced as demonstrated by abundant IgG antibodies to PG–PS in both groups of immunized sheep. A high immunogenicity of PG, related to different antigenic epitopes of the molecule,25,27 has been shown to induce strong polyclonal B-cell activation in mice.38,39 However, a protective effect of PG–PS antibodies to the challenge of PG–PS was not demonstrated for maintenance of early pregnancy. Furthermore, the opposite trend was observed as the total quantity of PG–PS administered was inversely related to pregnancy rate. The hypothesis that immunization with PG–PS might protect against mastitis-induced pregnancy loss was based on two lines of earlier evidence. First, repeated injections of PG in rabbits,20,28 mice,20 and guinea-pigs29 induced tolerance to the pyrogenic effect of the PG, and moreover, mice became resistant to infections with group A streptococci.20 Secondly, early embryonic loss in mice induced by a bacterial infection could be prevented by prior immunization with LPS, as long as the anti-LPS antibody titers remained above 1/500.40 However, data of the present experiment did not support the hypothesis that immunization of sheep with PG–PS would lead to protection of pregnancy. Rather, early pregnancy was lost more in animals with high anti-PG–PS titers. This negative effect may be attributed to sensitizing effect of PG–PS on the
EFFECT OF PEPTIDOGLYCAN–POLYSACCHARIDE COMPLEX immune system.41 If sensitized, the immune system would react by mounting an even stronger inflammatory response to the PG–PS challenge with a subsequent negative effect on pregnancy. Perhaps 5 days after mating, corresponding to blastocyst formation in the uterus, the embryo or dam is extremely sensitive to the inflammatory cytokines or other mediators, which could be up-regulated in the immunized (sensitized) sheep. A number of different cells and molecules could participate in the early pregnancy loss. It is of note that activated non-specific maternal immune effector cells such as macrophages and natural killer cells, have been shown to result in embryonic resorption in mice.42–44 Likewise, inflammatory mediators secreted by macrophages, interleukin (IL)-1, TNFa and nitric oxide (NO), play an important role in embryonic loss.6 Inflammatory cytokines IL-1 and IL-6,45 as well as IL8 and TNFa46 were produced by human amniochorionic membranes, after an Escherichia coli challenge in vitro. A macrophage activation marker in the decidua of mice was expressed before early embryo loss.47 Increased prostaglandin F2a (PGF2a), measured by its metabolite, PGFM, was also associated with early pregnancy loss in sheep after PG challenge on day 5.12 The immunomodulatory function of progesterone in sheep changes with gestation day48 and could be affected by PGF2a. Taken together, interactions of the innate immune and endocrine systems as opposed to humoral immune function should be investigated to discern the mechanism of early pregnancy loss at the time of bacterial challenge in sheep. In earlier experiments,23–26 the anti-PG antibody response in rabbits was more marked to heat-killed cells than to PG. Likewise in this experiment, heatkilled cells were more immunogenic than PG–PS in sheep. These differences may be due to the differential processing of the antigen.49 Interestingly, sheep immunized with killed bacteria had higher PG–PS antibody titers than sheep immunized with PG–PS, but the difference was not reflected in maintenance of pregnancy (56% versus 47%, P > 0.05). This further supports the notion that presence of antibodies to PG– PS does not guarantee a protective mechanism against the pathogen. Both immunoadjuvant50,51 and immunosuppressive51–53 actions of PG antibodies on mice lymphocytes have been described. Nevertheless, immunization with PG–PS or with killed cells followed by PG–PS challenge did not seem to have any lasting negative effect on conception, because the pregnancy rate, including the second breeding service averaged 95% across all groups (data not shown). This second breeding service encompasses the ewes, which did not conceive or possibly lost their embryo at the time of the PG–PS challenge but were able to conceive at the
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next estrus (in 16–17 days). Embryo loss at the time of the PG–PS challenge is consistent with the idea that the inflammatory and/or stress mediators have an immediate negative effect on early pregnancy. In conclusion, immunization of ewes with isolated PG–PS or with heat-killed S. pyogenes did not prevent PG–PS-induced pregnancy reduction. Moreover, the total amount of PG–PS ewes received lowered the probability of successful pregnancy. Our data are consistent with the possible onset of hypersensitivity after repeated inoculation with gram-positive PG–PS, leading to decreased retention of pregnancy.
Acknowledgements Our sincere thanks go to Rosana Schafer, PhD, for excellent guidance with the ELISA; to Meghan Wulster-Radcliffe, PhD, and to Alison Brown Dixon, PhD, for kind help with sheep management, inoculations, and ultrasonography at the USDA, ARS, US Sheep Experiment Station at Dubois, Idaho.
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