The induction and kinetics of an anti-DNP IgE response in dogs

Twenty-eight dogs immunized to dinitrophenol-ascaris (DNP-ASC) at birth and then three times at two week intervals produced serum anti-DNP antibody. The IgM response was detected one week after primary immunization and lasted for up to five weeks. The IgE and IgG antibody response in general was not present until week three but persisted through the immunization schedule. Although variation in the level and duration of the antibody response was detected between individual dogs, each dog did have a response that included all three isotypes examined.     

Immunomodulatory effects of C3bgp on the antibody response to hemocyanin in outbred rabbits and the F1 generation of breeding with siblings

Dynamics and quantitative analyses of monospecific antibody during the primary and secondary humoral responses were determined in outbred rabbits and in the F1 generation of breeding with siblings. The antibody response in rabbits immunized with Keyhole Limpet Hemocyanin (KLH) was studied during a 4-month immunization period. ELISA determination of anti-KLH Ig and anti-KLH IgG alone, in preimmune and immune rabbit sera, was performed. Antibody response in both groups of rabbits was similar when assessed by anti-rabbit Ig but displayed differences when assessed by anti-rabbit IgG. A statistically significant increase in anti-KLH IgG was observed in the F1 inbred rabbits compared to the control group after primary immunization from days 14 to 35. Immunomodulation also elicited differences in the antibody response in the two groups of animals. C3-binding glycoprotein isolated from Cuscuta europea (C3bgp), applied simultaneously with antigen (KLH), produced a much stronger secondary immune response than the antigen alone, in both experimental groups. The enhancement of anti-KLH Ig in C3bgp-treated inbred rabbits was statistically significant in comparison with nontreated inbred rabbits. A significant increase in anti-KLH IgG was observed only for the inbred group after treatment with C3bgp. The results demonstrate that the F1 generation of breeding with sibling leads to significant differences in antibody responses to immunization compared with outbred rabbits, as well as to immunomodulation with C3bgp.     

猪囊虫病基因工程疫苗的体液与细胞免疫反应

为了探讨以 Ｉ Ｓ Ｃ Ｏ Ｍ 作佐剂的猪囊虫病基因工程疫苗的免疫机理,对其诱导的体液免疫与细胞免疫反应进行了测定。用上述疫苗免疫 ９ 头试验猪,采用间接 Ｅ Ｌ Ｉ Ｓ Ａ 检测体液免疫反应及通过淋巴细胞转化试验、 Ａ Ｎ Ａ Ｅ染色试验、 Ｅ玫瑰花环形成试验等检测细胞免疫反应;用该疫苗和铝胶苗分别免疫昆明小鼠各 ２０ 只,分别检测体液免疫反应和 Ｔ 淋巴细胞抑制／杀伤亚群的动态变化。体液免疫的检测结果显示,免疫后 ７ 天即出现抗体,２１ 天后抗体全部转阳,持续的时间不少于 １９３ 天,效价明显高于铝胶苗;细胞免疫检测结果显示,免疫猪外周血 Ｔ淋巴细胞转化率、 Ａ Ｎ Ａ Ｅ＋ 细胞和粗粒型 Ａ Ｎ Ａ Ｅ＋ 细胞、 Ｅ Ｒ Ｆ Ｃ和 Ｅａ Ｒ Ｆ Ｃ细胞显著升高,免疫小鼠 Ｔ淋巴细胞抑制／杀伤亚群显著升高;与铝胶苗及对照组比较,差异极显著。以上结果表明猪囊虫病基因工程疫苗可同时激发动物的体液免疫和细胞免疫反应,增强了机体的免疫调节功能及杀伤性 Ｔ 淋巴细胞功能。     

A preliminary study to evaluate the immune responses induced by immunization of dogs with inactivated Ehrlichia canis organisms

Ehrlichia canis is an intracellular pathogen that causes canine monocytic ehrlichiosis. Although the role of antibody responses cannot be discounted, control of this intracellular pathogen is expected to be by cell mediated immune responses. The immune responses in dogs immunized with inactivated E. canis organisms in combination with Quil A were evaluated. Immunization provoked strong humoral and cellular immune responses, which were demonstrable by Western blotting and lymphocyte proliferation assays. By Western blotting antibodies to several immunodominant E. canis proteins were detected in serum from immunized dogs and antibody titres increased after each immunization. The complement of immunogenic proteins recognized by the antisera were similar to those recognized in serum from infected dogs. Upon challenge with live E. canis, rapid anamnestic humoral responses were detected in the serum of immunized dogs and primary antibody responses were detected in the serum from control dogs. Following immunization, a lymphocyte proliferative response (cellular immunity) was detected in peripheral blood mononuclear cells (PBMNs) of immunized dogs upon stimulation with E. canis antigens. These responses were absent from non-immunized control dogs until after infection with live E. canis, when antigen specific-lymphocyte proliferation responses were also detected in the PBMNs of the control dogs. It can be thus concluded that immunization against canine monocytic ehrlichiosis may be feasible. However, the immunization regimen needs to be optimized and a detailed investigation needs to be done to determine if this regimen can prevent development of acute and chronic disease.     

Modulation of Ovarian Function in Female Dogs Immunized with Bovine Luteinizing Hormone Receptor

Adult female dogs were immunized with 0.5 mg bovine luteinizing hormone receptor (LH-R) encapsulated in a silastic subdermal implant and subsequently with four intramuscular booster injections of 0.1 mg LH-R each. Circulating LH-R antibody was detected in the sera 3 weeks post-implant. The appearance of LH-R antibody was associated with a decline in the serum progesterone concentrations to a range of 0–0.5 ng/ml until day 365 in the immunized dogs in comparison with a range of 5–10 ng in the control animals, suggesting a lack of ovulation and corpus luteum function in immunized dogs. The immunized dogs did not show signs of `standing heat' and failed to ovulate when induced by LH-RH challenge. Serum oestradiol levels, however, remained in the range of 30–40 pg/ml in both the immunized and the control dogs. With the decline in the antibody titres, the hormonal profile and vaginal cytology returned to a fertile state and the dogs exhibited signs of `standing heat', as well as vaginal bleeding. Dogs immunized with LH-R did not show any serious metabolic, local or systemic adverse effects. The hypothalamic–pituitary gonadal axis remained intact as indicated by little difference in pituitary LH levels between control and immunized animals, and by the release of LH by LH-RH challenge. These studies demonstrate that active immunization of female dogs with LH-R could immunomodulate ovarian function to cause a reversible state of infertility. It may be postulated that, due to extensive interspecies homology, a recombinant LH receptor-based immunocontraceptive vaccine may also be effective in other vertebrates.     

Improvement of the immune response to foot and mouth disease virus vaccine in calves by using Avridine as adjuvant.

The epidemiological analysis of the cattle population during the eradication plan of foot and mouth disease (FMD) in Argentina clearly indicated a higher incidence of the disease in animals within their first year of age. It is important to improve the efficacy of the vaccination in those animals. In a previous report, we have shown the effect of an immunomodulator, Avridine (Avr), in the enhancement of the immune response elicited by FMD virus (FMDV) vaccines in experimental hosts [Berinstein, A., Pérez Filgueira, M., Schudel, A., Zamorano, P., Borca, M., Sadir, A.M., 1993. Avridine and LPS from Brucella ovis: effect on the memory induced by foot-and-mouth disease virus vaccination in mice. Vaccine 11, 1295-1301]. In this report, we analyze the effect of Avr in the improvement of the anti-FMDV immune response elicited in young animals immunized with a tetravalent vaccine. The anti-FMDV antibody response was evaluated using a liquid-phase blocking sandwich ELISA (LPBE) [Smitsaart, E.N., Zanelli, M., Rivera, I., Fondevila, N., Compaired, D., Maradei, E., Bianchi, T., O'Donnell, V., Schudel, A.A., 1998. Assessment using ELISA of the herd immunity levels induced in cattle by foot and mouth disease oil vaccines. Prev. Vet. Med 33, 283-296] while the cellular response was detected using an antigen specific lymphoproliferative test [Zamorano, P., Wigdorovitz, A., Chaher, M., Fernández, F., Sadir, A., Borca, M., 1994. Localization of B and T cell epitopes on a synthetic peptide containing the major immunogenic site of FMDV O1 Campos. Virology 201, 383-387]. The results show that, while no differences were detected in the cellular response, the anti-FMDV antibody reaction was significantly (<0.05) higher in animals immunized with the immunogen containing Avr. At 90 days post vaccination, 89-100% of the animals immunized with Avr presented predicted protection (PP) higher than 82% while just 50-61% of the animals immunized with vaccine without immunomodulator presented that characteristic. Also, it is shown that the increase in the anti-FMDV antibody titre in animals immunized with the vaccine containing Avr was mediated by an increase in the levels of both IgG1 and IgG2 which presented a significative correlation with LPELISA antibodies titres. It is concluded that the addition of Avr in the FMDV vaccines improve the immune status of the calves, the cattle population that suffers the highest epidemiological risk.     

Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.     

A modified live canine parvovirus vaccine. II. Immune response

The safety and efficacy of an attenuated canine parvovirus (A-CPV) vaccine was evaluated in both experimental and in field dogs. After parenteral vaccination, seronegative dogs developed hemagglutination-inhibition (HI) antibody titers as early as postvaccination (PV) day 2. Maximal titers occurred within 1 week. Immunity was associated with the persistence of HI antibody titers (titers greater than 80) that endured at least 2 years. Immune dogs challenged with virulent CPV did not shed virus in their feces. The A-CPV vaccine did not cause illness alone or in combination with living canine distemper (CD) and canine adenovirus type-2 (CAV-2) vaccines, nor did it interfere with the immune response to the other viruses. A high rate (greater than 98%) of immunity was engendered in seronegative pups. In contrast, maternal antibody interfered with the active immune response to the A-CPV. More than 95% of the dogs with HI titers less than 10 responded to the vaccine, but only 50% responded when titers were approximately 20. No animal with a titer greater than 80 at the time of vaccination became actively immunized. Susceptibility to virulent CPV during that period when maternal antibody no longer protects against infection, but still prevents active immunization, is the principal cause of vaccinal failure in breeding kennels where CPV is present. Reduction, but not complete elimination, of CPV disease in large breeding kennels occurred within 1-2 months of instituting an A-CPV vaccination program.     

A Leishmania infantum multi-component antigenic protein mixed with live BCG confers protection to dogs experimentally infected with L. infantum

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.     

犬细小病毒基因疫苗对幼貉的免疫试验

应用已构建的pcDCPV和pIRCPVIL2重组质粒配制成疫苗,对幼貉进行免疫,并对其免疫反应进行观察。应用夹心ELISA方法检测外周血中特异性的抗细小病毒血清抗体免疫球蛋白(IgG)的水平,MTT比色法检测外周血中淋巴细胞的转化率,对基因疫苗的免疫保护进行观察。结果表明,用pcDCPV和pIRCPVIL2重组质粒免疫后的幼貉对细小病毒的攻击具有一定的保护性,为pcDCPV和pIRCPVIL2重组质粒的应用奠定了基础。     

轮状病毒和大肠杆菌不同免疫方式免疫奶牛乳中抗体消长规律的研究

以引起婴儿腹泻的轮状病毒RV和大肠杆菌E.coli免疫妊娠后期的奶牛，使之产生抗这两种病原的抗体。免疫方式分为两种：一种是分别用E.coli和RV作免疫原免疫奶牛，另一种以两者同时免疫奶牛。然后定期采乳，分别以试管凝集反应和反应间接血凝抑制试验检测大肠杆菌抗体和轮状病毒抗体，并比较这两种不同免疫方式免疫奶牛乳中抗体的消长规律。试验结果表明：单独以大肠杆菌或轮状病毒作免疫原与这两者同时作免疫原，免疫奶牛乳中抗体消长规律相似，抗体效价也相近，且均可维持近两个月。     

Efficacy of an anti-fertility vaccine based on mammalian gonadotrophin releasing hormone (GnRH-I)--a histological comparison in male animals

A N-terminal modified gonadotrophin releasing hormone (GnRH-I, tetanus toxoid-CHWSYGLRPG-NH2) conjugate was evaluated histologically in a number of male animal species (mice, dogs and sheep). The immunogen has previously been shown to be highly effective in rats, by suppressing both steroidogenesis and spermatogenesis. However, cross-species efficacy of peptide vaccines is known to be highly variable. Therefore, a comparative evaluation of reproductive tissues from animals immunized against this immunogen adsorbed onto an alum-based adjuvant was made. The sheep and dogs were chosen, as use of anti-fertility vaccines in these species is important in farming and veterinary practice. Changes in testicular size were measured during the immunization period and the greatest alteration (attributed to gonadal atrophy) was observed in the rat. Following euthanasia, the testicular tissue was evaluated for spermatogenesis. The most susceptible species to GnRH-I ablation was the rat, which showed significant (P < 0.0001) arrest in spermatogenesis compared with untreated controls. Testicular sections taken from treated animals were completely devoid of spermatozoa or spermatids, in comparison with 94% of the untreated controls showing evidence of spermatogenesis. The immunized mice and rams also showed significant arrest (P < 0.0001). There was a 30-45% decrease in spermatogenesis and total azoospermia was not apparent. However, the least responsive were the dogs, which showed little significant variation compared to untreated animals and only a 5% decrease in activity. A comparison of the specific IgG response to GnRH-I indicated that in sheep and dogs the response was not maintained, unlike in rodents, suggesting that suppression of fertility may be due to differences in immune responses in different animal species.     

A comparison of the immune responses of dogs exposed to canine distemper virus (CDV) - Differences between vaccinated and wild-type virus exposed dogs

Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals.     

Antileishmanial antibody profile in dogs naturally infected with Leishmania chagasi

Visceral leishmaniasis (VL) presents vigorous Th2 immune response, which is mainly characterized in human by augmented expression of Il-4, polyclonal B cell activation, intense hypergammaglobulinemia and production of antileishmanial IgE antibodies. However, few aspects of this type of immune response have been demonstrated in studies of canine visceral leishmaniasis (CVL). This work investigated by ELISA and western immunoblotting the production of antileishmanial IgE antibodies (IgE Ab) in symptomatic and asymptomatic dogs naturally infected by Leishmania chagasi, and also compared this IgE immune response with those of IgG, IgG1 and IgG2 antibodies. Three groups of dogs were evaluated: 12 VL dogs with positive Leishmania biopsies (GI), 44 dogs with a positive leishmanial indirect fluorescent antibody test (IFAT), 30 of them presenting clinical signs of VL and 14 asymptomatic (GII) and 21 healthy dogs living in kennels located in leishmaniasis endemic areas (GIII), which were seronegative in the IFAT. Eighteen dogs from an area free of CVL were used as controls (GIV). Antileishmanial IgE antibodies were detected in 4 of 12 VL dogs from group I (33%) and 14 of 30 symptomatic dogs from group II (47%). While all asymptomatic dogs from group II (100%) were seronegative for antileishmanial IgE Ab, 7 of 21 healthy animals from group III (33%) had these immunoglobulins. A strong correlation was verified between antileishmanial IgG and IgG2 antibody titers in all symptomatic dogs, but only 15 of these 42 animals (36%) produced simultaneously IgE, IgG, IgG1 and IgG2 antibodies to Leishmania. IgE antibodies recognized leishmanial antigens of 12, 36, 61, 81 and 118 KDD, while a more complex pattern of immunoblotting was verified mainly for IgG and IgG2 antibodies from symptomatic animals. IgG1 and IgG2 antibodies shared the recognition of L. chagasi polypeptides of 118, 81, 61, 36, 18, 14 and 12 KDD, being more intense the immune reactions between IgG1 Ab and the leishmanial polypeptides of 61 and 36 KDD, and also between IgG2 antibodies and the antigens of 26, 21, 18, 14 and 12 KDD. Our results suggest that the polyclonal production of antileishmanial antibodies that includes IgE Ab could characterize a Th2 immune response in CVL and can help the laboratory diagnosis of this disease.     

Intestinal and serum antibody response in gnotobiotic piglets to oral immunization with Escherichia coli

The local and systemic immune response to a formolized E. coli oral vaccine was investigated in 13 gnotobiotic piglets. Beginning at ten days of age animals received a daily dose of 10¹⁰ or 10¹¹ bacteria, on ten consecutive days. Intestinal loop tests with one animal of each group on day 26 showed protection which was more pronounced in the animal dosed 10¹⁰ bacteria compared with the other immunized piglet. Immunoglobulin class-specific antibodies to O and K antigens were determined by ELISA technique. In serum no IgG or IgA antibodies were found, whereas IgM-anti O149 antibodies in both immunized groups reached their highest level at day 4 of dosing and decreased thereafter. IgM-anti K88 antibodies were first detected at day 10 of dosing. Both immunized groups had comparable serum levels at days 20 and 30. Also in gut secretion the IgM antibody response was predominant, and higher levels were found in the 10¹⁰ group than in the 10¹¹ group. IgG and IgA antibody response were also detected in secretion.     

镇江市2008年兽用狂犬病疫苗免疫效果观察

为评价镇江市家犬兽用狂犬病疫苗的免疫效果,探讨影响免疫效果的因素,采集镇江市不同地区免疫犬和非免疫犬只的血清样本,ELISA法测定其抗体产生情况。结果表明,镇江市总的免疫犬只抗体阳性率为34．53％（67／194）;不同地区犬只抗体阳性率差异显著（χ2 =23．743,P=O．000）;犬只的免疫3剂次及以上与3剂次以...     

Standardization and kinetics of in vitro bovine blood lymphocyte stimulation with bovine rotavirus

Two groups of 3-month old calves were immunized intramuscularly with attenuated bovine rotavirus and boosted 21 and 42 days later. The first group of three calves were vaccinated with live virus emulsified with incomplete Freund's adjuvant (IFA) and the second group was immunized with live virus suspended in phosphate buffered saline (PBS). Three other calves, serving as controls, were inoculated with PBS emulsified with IFA. The specific cell-mediated and antibody responses of the animals were studied. Preliminary analysis of in vitro peripheral blood lymphocyte transformation to bovine rotavirus determined optimal conditions as: 96 h culture period, 5 X 10(5) cells per culture in RPMI 1640 medium containing 10% heat-inactivated bovine fetal serum and the use of inactivated virus in the cell culture at a concentration of 5 X 10(6) median tissue culture infective dose before inactivation. Specific blastic stimulation was observed on calves immunized with the rotavirus emulsified with IFA after the second and third vaccine inoculation with stimulation index values varying from 2.00 to 5.73. Serum neutralizing antibody titers of 1/25,600 were also induced in the same calves. Calves immunized with rotavirus-PBS suspension developed a mean antibody titer of 1/1,600, but showed no specific lymphocyte stimulation. No increase in specific immune responses was detected in the control animals.     

Characterization of anti-idiotype reagents to bovine herpesvirus-1 monoclonal antibody

A monoclonal antibody (MAb) to a neutralization epitope on the 97-kD glycoprotein of bovine herpesvirus-1 (BHV-1) was used to prepare an anti-idiotypic antibody in rabbits. Purified F(ab')2 fragments of the MAb were used to immunize the animals and the sera containing the greatest anti-idiotype activity were identified by ELISA. After digestion of the immunoglobulins with pepsin and purification by affinity chromatography, anti-idiotype F(ab')2 fragments reacted specifically with the MAb in ELISA. Binding of the anti-idiotypic (anti-id) antibody was inhibited by preincubation of the MAb with BHV-1. Using an ELISA inhibition assay with BHV-1, the anti-id reagent inhibited the binding of anti-BHV-1 MAb to BHV-1, suggesting that the anti-id mimics an epitope of the 97-kD glycoprotein by binding the antigen combining site of the MAb. Development and characterization of this anti-id and future studies of its immunomodulatory effects are discussed.     

Serum antibody response of Indian major carp, Labeo rohita to three species of pathogenic bacteria; Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens

The immune response to mixed whole cell antigens of Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens, the common Gram negative bacterial pathogens associated with diseases of Indian major carps were evaluated for their efficacy in triggering antibody responses in rohu, Labeo rohita (Ham.). The rohu yearlings were either immunized with antigens from single bacterial strain, A. hydrophila, E. tarda and P. fluorescens or a combination of all three. An antibody response was detected at 1st week post immunization that rose significantly (p<0.05) at 4th week post immunization in all the immunized groups. The antibody level started declining after 8th week but persisted up to 10th week post immunization in all the immunized groups. Similarly, no significant difference (p>0.05) in the antibody level was found between groups immunized with single and mixed bacterial antigens. Moreover, the use of mixed bacterial antigens did not jeopardize the specific immune response to the vaccine components. Upon challenge with single pathogen, a high relative percent survival was recorded in the group immunized with mixed bacterial antigens and was comparable to those fish immunized with the single bacteria.     

Immune response to Neospora caninum in naturally infected heifers and heifers vaccinated with inactivated antigen during the second trimester of gestation

The objective of this study was to compare the immune response to Neospora caninum in naturally infected heifers and heifers inoculated with a killed whole N. caninum tachyzoite preparation during the second trimester of gestation. Nine Holstein heifers were used in this study; three naturally infected heifers were born from seropositive dams, and six seronegative heifers were born from seronegative dams. Four seronegative heifers were subcutaneously vaccinated with a killed whole N. caninum tachyzoite preparation at weeks 13, 15 and 17 of gestation. A killed whole N. caninum tachyzoite preparation containing 45 mg of protein/5 ml dose was formulated with 70% of mineral oil adjuvant (13% consisting of Arlacel C, 85% Marcol 52 and 2% Tween-80). Similarly, two seronegative heifers (negative controls) were inoculated with mock-infected bovine monocytes in oil adjuvant. Humoral immune responses were tested by using an indirect fluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting isotype specific antibodies. Cellular immune responses were assessed by lymphocyte proliferation test (LPT) and IFN-gamma production. N. caninum-specific antibody responses increased in immunized cattle by week 15 of gestation (mean reciprocal antibody titers 450+/-252), peaked at week 23 (mean 16,000+/-6400). Maximum antibody response in naturally infected heifers was observed at week 19 of gestation (mean: 3467+/-2810). Mean serum IFAT titers were significantly higher in immunized heifers compared with those in naturally infected heifers from weeks 17 to 25 (P < 0.05). Analysis of isotype specific antibodies in naturally infected heifers revealed a predominant IgG1 response in one heifer and a predominant IgG2 response in the other two. Similar titers of IgG1 and IgG2 occurred in immunized heifers. Control heifers remained seronegative throughout the study by IFAT and ELISA. Significant antigen-specific proliferation responses were only detected in naturally infected heifers in week 19 of gestation. Peripheral mononuclear blood cells (PMBC) from immunized animals produced IFN-gamma in similar concentrations to those of infected animals (P > 0.05). No abortion was seen in any experimental group; however, one calf from a vaccinated heifer died due to dystocia. All calves from vaccinated and control heifers were seronegative by IFAT at 6 months of age; in contrast, calves born from naturally infected heifers remained seropositive with titers > or = 200. Killed vaccine induced similar immune responses to those found in chronically, naturally infected cattle which did not abort; however, different immune pathways may be followed in vaccinated and natural infected heifers with differences in degree of protective immunity.