Mitogen stimulation of canine normal and myasthenia gravis lymphocytes

Responses of canine lymphoid tissues to mitogens were studied in five normal dogs and in two dogs with acquired myasthenia gravis (MG). In the normal dogs, lymph-node-derived lymphocytes gave the most consistent proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM), as determined by thymidine incorporation; and in most cases PHA, lipopolysaccharide (LPS), and PWM stimulated total IgG production, as determined by ELISA. Splenic lymphocytes had the greatest capacity for increased total IgG production. In the myasthenic dogs total IgG production by unstimulated lymph-node-derived lymphocytes was 88 micrograms/ml and 153 micrograms/ml, much higher than that of unstimulated normal dog lymphocytes (mean less than 1.0 microgram/ml). All mitogens resulted in suppression rather than stimulation of IgG production by lymphocytes from dogs with MG. Production of antibodies to acetylcholine receptors (AChRs) was detected in the supernatants of lymphocyte cultures from one of the dogs with MG at a rate of 78 fmol/5 x 10(5) cells per week and was not detected in culture supernatants of control dogs. This study demonstrates that lymph nodes may be an important site of antibody production in myasthenic dogs and provides the necessary groundwork for future studies of the cellular immunology of canine MG.     

Peripheral lymphocyte function in dogs with Brucella canis infection

Peripheral lymphocyte function in dogs with Brucella canis infection was evaluated using in vitro lymphocyte stimulation with the mitogens PHA, Con A and PWM, and killed Brucella canis organisms. Bitches with naturally occurring Brucella canis infection were compared to negative controls. There was no difference in the response to Con A and PWM between these two groups. Lymphocytes from infected dogs were less responsive (p less than .05) to PHA than were lymphocytes from controls. There was a significant (p less than .005) difference in response to Brucella canis antigen between the two groups. Lymphocytes from infected dogs were stimulated by Brucella canis antigen, whereas those from controls did not respond.     

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.     

Cell-mediated immunity in the prepatent primary infection of dogs with Echinococcus granulosus

Cell-mediated immunity was studied in 21 parasite-naive dogs by transformation of peripheral lymphocytes with phytohemagglutinin (PHA), concanavalin A (Con A), or hydatid fluid protein (HFP) at 29 days of a primary infection with Echinococcus granulosus, and by delayed cutaneous hypersensitivity to HFP at 36 days of the infection. Infection depressed significantly the ability of unstimulated cells to proliferate but enhanced the response to Con A. The lymphocytes of four dogs reacted to HFP significantly above the average, and one dog exhibited cutaneous reactivity to 0.2 micrograms of HFP. Five or 1 but not 0.2 micrograms of HFP produced non-specific skin reactions in parasite-naive dogs. The 7 dogs with the highest reactivity to PHA or Con A had significantly fewer parasites than the 7 less reactive dogs. The differences in other parasite characteristics were not significant in the 7 dogs with the greatest or smallest reactivities. Correlation tests showed an inverse correlation of PHA reactivity with establishment and maturation of parasites, of Con A reactivity with growth and maturation, and of HFP with maturation.     

Changes in lymphocyte blastogenic response of mares during the perinatal period

A fluorometric assay was applied to evaluate blastogenesis of equine lymphocytes. Optimal culture conditions were as follows; concentrations of phytohaemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were 1 microgram/ml, 40 micrograms/ml and 10 micrograms/ml, respectively, when 5 X 10(5) lymphocytes were incubated with culture medium containing 20% pooled horse serum (PHS) for 120 hours. The relative mean stimulation index of healthy non-pregnant mares were 5.107 +/- 0.323 (M +/- SE) with PHA, 4.019 +/- 0.183 with Con A and 3.610 +/- 0.131 with PWM. Sequentially the blastogenic responses of lymphocytes from twenty mares were observed during various stages of the perinatal period. Response decreased gradually before parturition was lowest at the time of parturition (PHA: 1.923 +/- 0.174, Con A: 1.698 +/- 0.206 and PWM: 1.706 +/- 0.177), and then increased gradually after parturition towards non-pregnant levels.     

Lymphocyte transformation test in veterinary clinical immunology

Lymphocyte transformation test is a powerful tool in laboratory testing of immunologic competence of animals. The impaired function of the lymphocytes or presence of mitogenesis suppressing factors in the patient serum were detected by comparing lymphocyte transformation (expressed as thymidine incorporation) obtained in media containing either autologous, homologous, or fetal calf serum additions. Most valuable results were obtained by using at least two, preferably three, different phytomitogens: concanavlin A (Con A), pokeweed mitogen (PWM), and pl ytohemagglutinin (PHA) at optimal concentrations (Con A, 15 μg/ml, PWM and PHA, 5 μg/ml) and decreased concentrations (Con A, 5 μg/ml, PWM and PHA, 1 μg/ml). Mitogenesis induced by lipopolysaccharide was considerably smaller and not used routinely. With 2 × 10⁵ lymphocytes/well, the background count of unstimulated lymphocytes in autologous serum in healthy dogs was usually between 100 and 400 counts/min (CPM), in clinically healthy cattle and horses from 200 to over 2000 CPM. Higher CPM were rarely detected without clinical disease. Increased background counts were often associated with viral infections, leukemias and lymphoreticular hyperplasias, decreased background counts were associated with various diseases. The stimulation indexes (SI) of healthy animals in autologous serum with Con A, (5 μg/ml) or PWM or PHA (1 μg/ml) were in the range from 100 to 1000 in the dogs, in the tens for Con A and in hundreds for PWM and PHA in horses and cattle. Increased SI were present during the incubation period of various diseases. Decreased SI were associated with numerous infectious and lymphoreticular diseases and were caused by any of the following: (1) the presence of serum immunosuppressive factor(s) in the patient serum, (2) the decreased response of lymphocytes to mitogens, or (3) increased mitogenicity of lymphocytes due to unidentified serum factors in absence of phytomitogens.     

In vitro lymphocyte stimulation by concanavalin A and with histamine as a co-mitogen in dogs with atopic dermatitis.

Cell-mediated immune function was assessed in a group of dogs with atopic dermatitis by measuring the responses of peripheral-blood lymphocytes (PBL) to various concentrations of Concanavalin A (Con A) and comparing them to those of normal dogs. No difference from normal was found in any of the stimulation indices neither was spontaneous tritium uptake of unstimulated cells different between the groups. We also measured the response to Con A stimulation in vitro of PBL preincubated for 24 h, either in cell-culture medium at 37 degrees C, or in whole blood containing EDTA at room temperature, as an indirect measure of function of a subgroup of suppressor cells. Preincubation caused enhancement of mitogenesis for normal dog lymphocytes but not for the atopic dog cells, particularly for suboptimal concentrations of Con A. No differences were found in the responsiveness following incubation in cell-culture medium between normal and atopic dog cells but for both groups the cells preincubated in whole blood were generally more responsive. Histamine, which is one of the mediators of type 1 hypersensitivities such as atopy, can modulate lymphocyte function. At 10(-4) and 10(-8) M histamine, when added simultaneously with Con A, enhanced mitogenesis of normal dog PBL but suppressed mitogenesis of atopic dog PBL. By using histamine H1 and H2 antagonists, we concluded that histamine enhanced mitogenesis via H1, receptors and suppressed it via H2 receptors. Our results suggest that there are abnormalities in lymphocyte function in dogs with atopic dermatitis which may be important in the pathogenesis of the disease.     

Duck lymphocytes--III. Transformation responses to some common mitogens

The lymphocyte transformation (LT) test was performed using duck blood lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A), lentil lectin (LC), Roman snail lectin (HP), peanut agglutinin (PNA), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), horseshoe crab lectin (HSC), pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS). Cells were cultured in microtitre trays, at 41.6 degrees C, 8 x 10(5) cells in 200 microliters medium (= 4 x 10(6) cells/ml) supplemented with 10% pooled duck serum. Mitogens were added at final concentrations of 0.1-100 micrograms/ml and triplicate cultures at each concentration were harvested daily for scintillation counting 6 hr after addition of 1 microCi [3H]thymidine. Three patterns of response were observed. The responses to Con A, LC, HP and HSC were greatest at high mitogen concentrations (40-100 micrograms/ml) throughout the 7 days of culture. With PHA, PNA, WGA and LPS maximum stimulation was obtained at 3-5 days, at which time the cells were responding to lower concentrations of mitogen than were required at other times during the experiment. The response to BSS and PWM showed increasing sensitivity to lower concentrations of mitogen during the first 3 days of culture and then stimulated most strongly at 2-10 micrograms/ml in cultures harvested after 4-7 days. Cells from two ducks were cultured for 3 and 5 days with selected concentrations of these mitogens; the results confirmed the variation in response to different mitogens. It is possible that these patterns of response are the outcome of stimulating different populations of duck lymphocytes.     

Optimal conditions for in vitro mitogen-induced proliferation of peripheral blood lymphocytes in breeding foxes

The proliferative response of fox peripheral blood lymphocytes to nonspecific mitogens: leucoagglutinin (LA), concanavalin A (Con A) and pokeweed mitogen (PWM) was studied. Microcultures were kept at 39 degrees C in a humidified atmosphere containing 5% CO2. The highest 3H-thymidine incorporation was observed, when Con A was used, while LA and PWM showed weaker but significant stimulatory action. Optimal doses of mitogens were: 5 micrograms/ml for Con A, 5 micrograms/ml for LA and a dilution of 1:100 for PWM. The maximal stimulation index for Con A was about 240 and up to 100 for LA or PWM. The maximal lymphocyte proliferation was observed when culture media were supplemented with 10% serum. When proliferation kinetics were studied, the peak response was observed on Day 2.     

Effect of ammonia on viability and blastogenesis of bovine lymphocytes

The effect of addition of ammonia into the tissue culture on viability and functions of bovine lymphocytes was studied. The concentrations of ammonia in the tissue cultures represented toxic, subtoxic, and normal concentrations of ammonia in the bovine blood during clinical and subclinical urea toxicosis. Lymphocytes separated from peripheral bovine blood were incubated in control medium and test medium with various concentrations of ammonia and/or PHA or Con A. Viability of the lymphocytes was measured by trypan blue exclusion test and their mitogenic reactivity by incorporation of ³H thymidine into DNA of lymphocytes. Approximately 30% bovine lymphocytes were killed by ammonia in medium during 72 hours of incubation. Ammonia also affected the response of lymphocytes to stimulation with PHA or Con A as well as mixed lymphocyte culture reaction. The mitogenic response of lymphocytes was also reduced when lymphocytes were preincubated with ammonia for even 1 hour. The mitogenic response was not restored when the number of lymphocytes preincubated with ammonia was reconstituted to the initial concentration to compensate for the killed lymphocytes before stimulation with PHA. Therefore, addition of ammonia to the culture either killed lymphocytes or permanently impaired their functions.     

Optimum conditions for the turkey lymphocyte transformation test.

Optimum conditions for turkey lymphocyte transformation tests were determined. Thrice-washed turkey buffy-coat cells obtained after slow centrifugation (40 x g, 10 minutes) responded well to mitogenic stimulation. Turkey lymphocytes isolated on Ficoll-containing separation media largely lost their ability to respond to mitogens. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when bovine fetal serum was used at a 2.5% concentration or pooled turkey serum and autologous plasma were used at a 1.25% concentration. Higher concentrations of turkey serum or plasma decreased the responses when sub-optimum doses of concanavalin-A (Con A) or phytohemagglutinin-P (PHA-P) were used. Serum-free cultures gave higher stimulation indices than cultures with serum only when sub-optimum doses of Con A or PHA-P were used. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. Responses were usually greatest with final concentrations of 5 micrograms Con A/ml, 10 micrograms PHA-P/ml, and 20 micrograms pokeweed mitogen (PWM)/ml and when the cultures were incubated in 96-well microplates at 40 C in humidified air with 5% CO2 for 40-42 hours with pulsing with 3H-thymidine during the final 16 hours of incubation.     

In vitro stimulation of pig lymphocytes after infection and vaccination with Aujeszky's disease virus

Mitogenic and antigenic lymphocyte stimulation were examined in Aujeszky's disease virus (ADV) infected pigs and in pigs vaccinated with modified live ADV. Neither infection nor vaccination had any effect on lymphocyte responsiveness to phytohaemagglutinin (PHA), pokeweed mitogen (PWM) or concavalin A (Con A). ADV antigen-responsive lymphocytes began to appear in the peripheral blood between 7 and 14 days after inoculation and could still be demonstrated in blood and spleen of infected pigs at 174 days after infection. In vaccinated pigs, sensitized peripheral blood lymphocytes could be detected up to at least 35 days after revaccination. Pre-incubation of ADV antigen with specific antibody markedly reduced lymphocyte stimulation. Non-immunized pigs showed no lymphocyte response to ADV antigen. Infected pigs exhibited no lymphocyte reactivity against antigens of non-infected cells.     

Effect of sodium diethyldithiocarbamate, Corynebacterium parvum and mycobacterium cell wall extract on in vitro blastogenic responses of bovine blood lymphocytes

In vivo inoculation of three-month-old calves with sodium diethyldithiocarbamate (DTC), killed Corynebacterium parvum or mycobacterium cell wall extract (MCWE) resulted in an enhancement of in vitro peripheral blood lymphocyte blastogenic responses to mitogens phytohemagglutinin (PHA) and Concanavalin A (Con A) in the first three days after treatment. In a separate experiment, blood lymphocytes isolated from a healthy nontreated calf were incubated in vitro in presence of each of the same immunostimulating agents and tested for their blastogenic responses to PHA and Con A. The results showed that all immunostimulants, excepting DTC, enhanced the in vitro blastogenic responses of lymphocytes to PHA and Con A. Finally, addition of MCWE to cultures of blood lymphocytes isolated from calves vaccinated intramuscularly with bovine rotavirus and adjuvant resulted in an enhancement of the in vitro lymphocyte transformation to rotavirus. Our study demonstrated that DTC, killed Corynebacterium parvum and mycobacterium cell wall extract were able to enhance bovine T cell proliferation in vitro.     

A study of porcine lymphocyte populations. II. Characterization of porcine lymphocyte populations

The percentage of E rosette forming cells amounted to 26% of the blood lymphocytes and 34% of the spleen cells in German Landrace pigs. 10% of the live lymphocytes in the peripheral blood and 22% of the spleen cells were EAC rosette forming cells. The number of E rosettes could be increased by treatment of sheep erythrocytes with neuraminidase. The number of lymphoid cells reacting with protein A in the peripheral blood and in the spleen of pigs correlated well with the number of EAC rosette forming cells. The mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) are potent stimulators of pig lymphoid cells. The mitogenic stimulation of pig lymphocytes could not be influenced significantly by the removal of phagocytic cells. By neuraminidase treatment the mitogen induced stimulation rate was decreased. For the mitogenic stimulation of porcine lymphoid cells in the presence of PHA, Con A and PWM T cells were required. Bacterial lipopolysaccharides (LPS) stimulated only B cells to a small degree.     

Lymphocyte blastogenic responses of calves experimentally infected with Ostertagia ostertagi

Twelve 9-week-old calves were divided into four groups; two groups were maintained helminth-free as controls and the other groups were given Ostertagia ostertagi infective-stage larvae (L3) orally. One group received 100,000 L3 as a single inoculum and the other group received L3 in increasing dosages at weekly intervals for 8 consecutive weeks. The blastogenic responses to concanavalin A (Con A), phytohemagglutinin (PHA), and a soluble larval antigen from O. ostertagi (SLA) of peripheral blood lymphocytes were evaluated using tritiated thymidine incorporation into DNA as a measure of blastogenesis. The responses to Con A of all infected calves were significantly depressed while the responses to PHA were not. SLA, at concentrations of 4 micrograms ml-1 and above, caused blastogenic activity in lymphocytes from uninfected calves. Using SLA at 1 microgram ml-1 in lymphocyte cultures supplemented with autologous serum, an antigen-induced blastogenic response was detected in calves receiving serial inoculations of L3.     

Immunomodulatory properties of a strain of Mycobacterium chelonae. I. Mouse lymphocyte responses in vitro

Immunomodulatory properties of a strain of live Mycobacterium chelonae (Mch) was investigated in an in vitro lymphocyte transformation system. Murine splenocyte activation by this bacterium was characterized by polyclonal lymphoproliferative responses in a dose dependent fashion. Optimal doses ranging from 20 to 80 micrograms of Mch (wet weight) per ml of cell suspension induced a very significant mitogenic effect. Higher doses (100 micrograms) of Mch manifested a decreased rate of tritiated thymidine ([3H] TdR) uptake whereas responsiveness of splenic lymphocytes to lower doses (0.156 microgram) was not modified. Contrary to the splenocyte responses activation of murine thymocytes by this mycobacterium is characterised by a decreased proliferation as compared to the background count of unstimulated cells. Simultaneous addition of Mch with optimal doses of Concanavalin A (Con A) and Phytohemaglutinin (PHA) potentiated polyclonal mitogenic responses of murine splenocytes to these two lectins. However, proliferation of these lymphocytes to Lypopolysaccharide (LPS) induction was not modified. BALB/C and DBA/2 spenocytes were found to be more responsive to stimulation by this Mycobacterium as compared to those of C3H/Ou and to a lesser degree to those of C57BL/6 mice.     

Molecular studies of T-lymphocytes from cattle infected with bovine leukemia virus

Although bovine leukemia virus (BLV) is mainly associated with infections of B-lymphocytes, we have previously reported the statistically significant increase in the T-lymphocytes obtained from BLV-infected asymptomatic aleukemic (AL) cattle. In this report the presence of BLV provirus in the DNA of immunoaffinity purified T-lymphocytes from AL animals was assessed using a highly specific radiolabelled (32P) BLV-DNA provirus probe and solid phase DNA hybridization. The BLV provirus was found in the DNA of the peripheral blood mononuclear cells of all AL animals tested and three of the four purified T-lymphocyte preparations from these animals. The purified T-lymphocyte preparations used in this study contained less than 4% detectable B-lymphocytes. One animal had no detectable B-lymphocytes in the purified T-lymphocyte preparation and the DNA from these cells also gave positive hybridization results. The lymphocyte blastogenesis assay was then used as an indicator of the functional ability of lymphocytes from these BLV-infected AL cattle to respond to mitogenic stimuli. The responsiveness of lymphocytes from these animals to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweek mitogen (PWM) was comparable to that of lymphocytes from BLV-negative animals when changes in 3H-thymidine uptake (c.p.m.) were used as measurement of mitogenic-induced blastogenesis. This indicated that infection of the T-lymphocytes by BLV does not appear to alter the overall response of the lymphocyte populations to mitogenic stimuli. High levels of spontaneous blastogenesis in the absence of mitogenic stimulation were observed for lymphocyte preparations of AL animals. The reason for this proliferation of lymphocytes is unclear; however, sera from these AL animals were found to contain a blastogenesis-augmenting factor(s) when added to lymphocytes from BLV-negative control animals in the presence of Con A, PHA and PWM.     

Mitogen stimulation of lymphocytes in pigs with hereditary vitamin C deficiency

Recently an inherited vitamin G deficiency in the pigs presumably based on an autosomal recessive gene was decribed* Homozygotes are in contrast to heterozygotes and normal pigs unable to synthesize ascorbic acid. In an experiment comprising 3 littermate pigs, 2 homozygous and 1 heterozygous for the vitamin C deficiency gene, the influence of ascorbic acid depletion, and repletion on mitogen stimulation of peripheral blood lymphocytes was studied. Ascorbic acid depletion of the vitamin C dependent pigs resulted in a rapid decline in plasma ascorbic acid. Response of lymphocytes to stimular tion with Concanavalin A (Con A) and phytohemagglutinin M (PHA) decreased more slowly reaching a minimum, which coincidedi with the occurrence of the first clinical symptoms of scurvy. Following resupplementation with vitamin C the plasma content of ascorbic acid rapidly returned to normal, while the lymphocyte response to Con A and PHA stimulation only gradually approached the initial values. The repletion with ascorbic acid caused a transitory increase in the response to pokeweed mitogen (PWM) stimulation. The significance of these findings in relation to the cellular immune system in normal pigs is discussed.     

Optimum conditions for the chicken lymphocyte transformation test.

Optimum conditions for chicken (Gallus gallus) lymphocyte transformation tests were determined. Thrice-washed chicken buffy-coat cells obtained after slow centrifugation (40 x g for 10 minutes) responded substantially better to mitogenic stimulation than lymphocytes isolated on separation media containing Ficoll. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when fetal bovine serum was used at a 5% concentration or pooled chicken serum and autologous plasma were used at a 1.25% concentration. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. When 1.25% chicken serum was used in the cultures, responses were usually greatest with final concentrations of 30-50 micrograms/ml of concanavalin A (Con A) and 30-50 micrograms/ml of phytohemagglutinin-P (PHA-P). The optimum concentration of pokeweed mitogen (PWM) varied from 1 to 40 micrograms/ml among the birds and was practically impossible to establish in general. The incubation in humidified air with 5% CO2 was significantly better at 40 C than at 37 C. The total culture time of 40 hours including pulsing with 3H-thymidine during the final 16 hours of incubation was the best for Con A- and PHA-P-stimulated cells, whereas a longer incubation of 64 hours gave the highest results with PWM stimulations.     

Evidence of immunosuppression by Demodex canis.

Three clinically normal beagles, 3 beagles with localized demodectic mange (LDM), and 3 beagles with generalized demodectic mange (GDM) were investigated simultaneously 1-3 and 4-6 weeks from the appearance of the clinical signs. Blood clinical examination and reactivity of peripheral lymphocytes to Con A and PHA were investigated in the first instance, and reactivity to Con A, PHA, and LPS in the second. Eight aliquots were used in each blastogenesis assay for each dog. All dogs were negative for rheumatoid factor. The results of blastogenesis showed that many observations were distributed non-normally, and that not all dogs in each group responded homogeneously. Comparison of blastogenesis results between dogs demands careful statistical analysis. Responses to mitogens were normal in all dogs at 1-3 weeks except for the LDM dogs that showed an increased response to PHA. Only the response to Con A was moderately inhibited in the LDM dogs at 4-6 weeks. All responses were severely depressed in the GDM dogs at 4-6 weeks. This means that immunosuppression follows rather than precedes the clinical manifestations of GDM, and implies that the phenomenon is induced by the parasite or the host's reaction to it.