Role of adjuvants in inhibitory influence of immunization with porcine zona pellucida antigen (ZP-3) on ovarian folliculogenesis in bonnet monkeys: a morphological study

Female bonnet monkeys were immunized with 55 kDa porcine zona antigen (ZP-3), with either complete Freund's adjuvant (CFA) or sodium phthalylated lipopolysaccharide (SPLPS) as adjuvant. Anti-ZP-3 antibody titers were monitored and the effect of immunization on the ovarian morphology was assessed by high-resolution light microscopy. The study demonstrated that both adjuvants used were equally potent in eliciting antibody response against ZP-3. Although no morphological damage to ovarian components was observed in animals immunized with SPLPS as adjuvant, immunization using CFA resulted in profound ovarian follicular atrophy, sparing only the primordial follicles. The atrophic phenomenon involved those follicles that either already had or were in the process of forming zona pellucida. The results of this study indicate that choice of adjuvant may be an important consideration for immunization against zona antigens. These findings encourage further investigations for developing better immunization regimen aimed at using zona antigens for immunocontraception.     

Homologous prostatic fluid added to frozen-thawed dog spermatozoa prior to intravaginal insemination of bitches resulted in better fertility than albumin-free TALP

The aim of this study was to determine whether canine prostatic fluid has intrinsic effects resulting in higher fertility than albumin-free Tyrode's albumin lactate pyruvate (afTALP) when added to thawed semen prior to intravaginal insemination. Twenty-four German shepherd bitches were inseminated intravaginally with frozen-thawed spermatozoa to which either homologous prostatic fluid (Group P; 12 bitches) or afTALP (Group T; 12 bitches) was added to give a final insemination volume of 7mL. Each bitch was inseminated daily starting when the vaginal folds first became angular and continuing until the day before a diestrus vaginal smear was first seen. Bitches were spayed about 3 weeks after the onset of diestrus and the number of corpora lutea and the number of conceptuses counted. Group P and Group T bitches were, respectively, inseminated 5.3+/-1.0 and 5.8+/-2.1 times with 48.9+/-8.6 and 50.4+/-8.3 million progressively motile spermatozoa per insemination. Eight Group P bitches and 10 Group T bitches conceived with totals of 76 and 45 conceptuses and 126 and 117 corpora lutea, respectively. Odds of conception were taken as the number of conceptuses divided by (the number of corpora lutea minus the number of conceptuses). After adjustment for the number of progressively motile spermatozoa per day and the random effect of bitch, the addition of prostatic fluid resulted in an increased odds of conception compared to afTALP. This effect decreased as the number of progressively motile spermatozoa per day increased.     

An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits.

Emulsion adjuvants have been used for production of polyclonal antisera in rabbits (Oryctolagus cunniculi) for decades. Complete Freund's adjuvant has a reputation as a very effective immunoenhancer, but adverse physiological effects, including fever, inflammation and sterile abscess formation, have prompted a search for alternatives to complete Freund's. In this study, we quantitatively compared five adjuvant regimens: (a) a primary inoculation with complete Freund's followed by three boosts with incomplete Freund's; (b) four serial inoculations of incomplete Freund's adjuvant augmented with 6-bromoguanisine; (c) four serial inoculations with RIBI's MPL + TDM + CWS adjuvant emulsion; (d) four serial inoculations with Montanide ISA 50 emulsion; and (e) four serial inoculations with Montanide ISA 70 emulsion. We chose a small (12 amino acid) chain polypeptide coupled to bovine serum albumin as our test antigen. When compared, no system could be seen to be significantly better than a regimen of a primary immunization with complete Freund's adjuvant followed by serial reimmunization with incomplete Freund's adjuvant. The commercially available RIBI adjuvant produced significantly lower antibody levels, while other systems produced essentially equivalent levels. With all five adjuvants, antibody quantities plateaued after the second injection and further immunization did not increase titers significantly. Boost injections did yield greater intradermal tissue reaction than primary inoculations, and intramuscular inoculum volumes of 0.4 cc caused chronic lesions still detectable by the gross necropsy 2 weeks after the final injection.     

Enhancement of delayed-type hypersensitivity and induction of interferon by the lipophilic agents DDA and CP-20,961

The lipophilic amines dimethyl dioctadecyl ammonium bromide (DDA) and N,N-dioctadecyl-N′, N′-bis(2-hydroxyethyl)propanediamine (CP-20,961) are compared on their capacities to induce interferon, nonspecific protection to viral infection, and enhancement of delayed-type hypersensitivity (DH). DDA, a well-known adjuvant for the induction of DH is a moderate interferon inducer like CP-20,961. On the other hand, CP-20,961, a known interferon inducer and resembling in structure DDA, is shown to enhance DH to inactivated Semliki Forest virus (SFV). Nonspecific protection to challenge with a lethal dose of either SFV or encephalomyocarditis (EMC) virus was induced on injection of both compounds.     

Active immunization against riboflavin carrier protein results in peri-implantation embryonic loss leading to pregnancy termination in rats: use of alternate adjuvants

To investigate the mechanism of pregnancy termination following immuno-neutralization of riboflavin carrier protein (RCP) and to use acceptable adjuvants, we actively immunized female rats with reduced and carboxymethylated RCP (RCM-RCP) using various adjuvants (during primary immunization) such as sodium phthalylated lipopolysaccharide (SPLPS), purified S. typhi outer membrane proteins (porins) and a combination of them. Rats (5-14 per group) were immunized with alugel adsorbed RCM-RCP (100 microg/dose) either alone or with SPLPS or porins or SPLPS+porins. Control animals received RCM-RCP emulsified with Fruend's completelincomplete adjuvants (FCA/FIA). All animals received five boosters at intervals of 21 days. The lowest (4 X 10(-3)) and the highest (> 70 X 10(-3)) anti-RCM-RCP antibody titers were observed in alugel adsorbed-RCM-RCP group and control groups, respectively. Immunized animals showed reduced fertility following 3rd, 4th and 5th boosters. Reduction in fertility was 30-60% in alugel adsorbed RCM-RCP group, 90-100% in FCA-RCM-RCP group and 80-90% in SPLPS+porins group. Fertility reduction was not strictly correlatable with the serum antibody titers. RCP-specific IgG could be localized in the uterine endometrial glands and luminal epithelial cells in the immunized animals. Animals in the FCA/FIA group showed abnormal implantation/resorption sites and their histological sections showed degenerated embryos. But, day 5 preimplantation embryos were normal. These results show that (a) SPLPS+porins can be used as adjuvants in place of FCA/FIA for active immunization against RCM-RCP and (b) early termination of pregnancy in the immunized animals is due largely to the failure of normal embryo implantation.     

Immunogenic Properties of Colloidal Gold

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or its combination with Freund's adjuvant). Application of colloidal gold also enhanced nonspecific immune responses, such as lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens or complete antigens (without other adjuvants) was shown to induce the production of highly active antibodies. In addition, the amount of antigen used for animal immunization with colloidal gold was an order of magnitude lower, compared to immunization with complete Freund's adjuvant. This fact can be evidence for adjuvant properties of colloidal gold proper.     

Luteal function of induced corpora lutea in the bitch

Nineteen anestrous bitches with a mean of 22 kg body weight and ranging from 2 to 4 years of age were induced to exhibit estrus and ovulate using PMSG and HCG. Twelve days after the first day of estrus, bitches were assigned to four treatment groups. Group (A) consisted of six bitches, Group (B) of five bitches and Groups (C) and (D) of four bitches each. At this time, bitches in Groups (A), (B) and (C) were laparotomized and those assigned to Groups (A) and (B) were bilaterally hysterectomized leaving the cervix and oviducts intact. Although bitches in Group (C) were laparotomized, they were not hysterectomized. Group (D) bitches were not subjected to any surgical procedures. Homologous uterine extract was prepared from each bitch in Group (A) and administered intramuscularly beginning on day 25 (day 0 = first day of estrus) and continued every other day for 61 days post-estrus. Bitches in Group (B) were similarly injected with equal volumes of 0.9% saline. Blood samples, obtained prior to laparotomy and every other day for 85 days thereafter, were assayed for plasma progesterone concentrations using radioimmunoassay. One bitch in each of Groups (A) and (D) did not form luteal tissue following treatment with PMSG and HCG although both bitches exhibited estrus following treatment. All other bitches showed an increase in progesterone levels (4 to 19 ng/ml) between the first day of estrus and 10 days post-estrus. Thereafter, progesterone levels progressively declined in all groups with levels below 1 ng/ml between 38 to 40 days post-estrus. Results of this study suggested that CL formed in the bitch following PMSG and HCG treatment have a reduced function compared to non-induced CL of a normal, non-fertile estrous cycle. Such premature CL regression appears to be independent of the presence or absence of the uterus.     

The generation of monoclonal antibodies in mice: influence of adjuvants on the immune response, fusion efficiency and distress

The objective of this study was to find a reliable alternative to Freund's adjuvant in order to reduce the distress imposed on the animals without impairing the fusion efficiency for immune-positive clones. For this purpose several commercially available adjuvants and adjuvant formulations representing different classes of molecules were compared. Humoral responses and animals' distress evaluated by clinical assessment and histopathological examinations were investigated and compared to fusion efficiencies. In a first set of experiments seven adjuvants were tested essentially to determine their potential to induce distress. Poly(A).poly(U) and GERBU were selected for further investigations due to their low overall toxicity. They were combined with five different antigens and compared to the classic Freund's adjuvant system (CFA/IFA) and to control immunizations without adjuvant. The results showed that adjuvants of very low toxicity could induce a high fusion efficiency. According to a standardized immunization protocol, GERBU induced polyclonal titres similar to Freund's whereas animals treated with poly(A).poly(U) did not attain titres higher than mice immunized with antigen in saline. Poly(A).poly(U) however, exhibited the best fusion efficiency, Freund and GERBU were slightly less efficient. Therefore poly(A).poly(U) and GERBU may serve as valuable alternatives to Freund's adjuvant for generating monoclonal antibodies. Furthermore, these two adjuvants are very easy to use.     

Evaluation of ethylene-vinyl acetate copolymer as a non-inflammatory alternative to Freund's complete adjuvant in rabbits

Ethylene-vinyl acetate copolymer (EVAc) was evaluated as an antigen delivery device in laboratory rabbits. Bovine serum albumin (BSA) was incorporated with EVAc in a pellet, which was implanted subcutaneously. Serum antibody titers to BSA in four implanted rabbits were equal to titers in four rabbits injected twice with BSA in complete Freund's adjuvant. Three of four rabbits implanted with EVAc displayed no inflammation or systemic illness in response to the pellet. The fourth rabbit repeatedly developed a small abscess at the implantation site, but the lesions were less severe than complete Freund's adjuvant injection sites. The EVAc pellet is recommended as a non-inflammatory alternative method to Freund's adjuvants for producing serum antibody in rabbits.     

Differentiation between pyometra and cystic endometrial hyperplasia/mucometra in bitches by prostaglandin F2alpha metabolite analysis

Bitches with pyometra are potential emergency cases which may be clinically difficult to differentiate from cases of cystic endometrial hyperplasia (CEH) in combination with mucometra. In the present study plasma prostaglandin F(2alpha), as measured by its main metabolite 15-keto-13,14-dihydro-PGF(2alpha) (PG-metabolite) concentrations, blood biochemical and hematological parameters were measured in 59 bitches with pyometra, 10 bitches with CEH and nine controls to determine if PG-metabolite could differentiate between the three uterine conditions. Bitches with pyometra had significantly higher plasma levels of PG-metabolite than bitches with CEH (P=0.002) and the controls (P=0.002). PG-metabolite analysis alone had a high sensitivity (98.3%) and a high specificity (80.0%) for the differentiation of pyometra versus CEH in bitches where fluid in the uterus was diagnosed. When a combination of PG-metabolite and percentage band neutrophils (PBN) was used for differentiation of the two diagnoses, a sensitivity of 100% and specificity of 90.0% was obtained. This means that the combination of PG-metabolite and PBN analysis allows for differentiation between cases of pyometra and CEH. If the PG-metabolite level in a bitch is >or=4,524 pmol l(-1), there is a 99% probability of the diagnosis pyometra versus CEH. Levels of PG-metabolite >or=3,054 pmol l(-1), >or=2,388 pmol l(-1) or>or=1,666 pmol l(-1) indicates a 95%, 90% or 80% probability of pyometra, respectively. At high PG-metabolite levels (above about 3,000 pmol l(-1)), PG-metabolite alone is enough for differentiation of pyometra versus CEH. The results of the present study showed that PG-metabolite analysis is valuable in the diagnosis and prediction of severity of uterine diseases.     

Effect of sodium cloprostenol and flunixin meglumine on luteolysis and the timing of birth in bitches.

At birth, the physiological role of prostaglandins in bitches is unclear. Bitches were treated before parturition with either saline, the prostaglandin analogue, sodium cloprostenol, or the prostaglandin synthetase inhibitor, flunixin meglumine. The animals were examined regularly to determine the onset of parturition and a series of blood samples were taken to define the hormonal profiles before, during and after birth. Animals treated with cloprostenol whelped earlier than did controls. In addition, the prostaglandin F2 alpha metabolite surge and decrease in plasma progesterone concentration and rectal temperature were earlier than in controls. Flunixin meglumine disrupted the normal 13,14-dihydro-15-keto prostaglandin F2 alpha profile but did not abolish prostaglandin synthesis completely or delay the onset of labour in treated animals. This study confirms that prostaglandins induce luteolysis and the onset of labour in the bitch. However, the partial inhibition of prostaglandin synthesis does not prevent parturition.     

Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and RIBI adjuvants in mice.

Freund's adjuvant and the RIBI adjuvant system were compared for their immune potentiating and toxic effects. Each adjuvant was administered with benzo(a)pyrene (BaP), a nonimmunogenic hapten, conjugated to a bovine gamma globulin (BGG) carrier protein to 10 mice intraperitoneally. Complete Freund's adjuvant was used at initial immunization, while incomplete Freund's was used for booster immunizations. Five mice were given the immunogen conjugate (BaP-BGG) in saline as a control. Antibody titers were determined by ELISA to both hapten and carrier after each of the two booster immunizations. Titers to BaP were 2- and 27-fold higher for RIBI than for Freund's after each of two booster immunizations. Titers to bGG were 119 and 12-fold higher for RIBI compared with Freund's. Titers to both immunogens were markedly less when administered in saline. Body weights were monitored in all three groups for the duration of the study. No differences were observed among the three groups. Mice from each group were euthanized at regular intervals to assess pathology. Splenic weight:body weight ratios were determined at the time of necropsy, and no differences were noted among the three groups. Granulomatous inflammatory lesions were most severe in the Freund's immunized mice, less severe in those immunized with RIBI, and least with saline. Results indicate that the RIBI system was more effective in potentiating an immune response and elicited less tissue reaction than did Freund's adjuvant with this particular immunogen.     

Reproductive patterns in the domestic dog--a retrospective study of the Drever breed

The aim of this study was to examine the differences, between seasons of the year, in the distribution of matings and whelpings, litter size, pup deaths, and sex ratio in domestic dogs. Furthermore, we wanted to examine the effects of age and parity of the bitch at the time of whelping on litter size, as well as the effect of litter size on gestational length. A final aim was to investigate the fertility and frequency of whelping problems in a private kennel of Drever dogs. Data from the Swedish Kennel Club (SKK) registry for the Drever breed during 1995-2006, comprising a total of 2717 litters, were analyzed together with more detailed data from a private, professional kennel of Drevers, with a total of 285 matings and 224 whelpings, during the same time period. The most matings took place during winter, and the fewest during summer; consequently, most whelpings occurred during the winter and spring seasons. Of the 285 mated bitches, 78.6% whelped, 6.25% experienced dystocia, and 5.36% underwent Cesarean section. The pup death rate was 7.6%. The largest litters were born during spring. Litter size was negatively correlated with duration of pregnancy (r=-0.18). Each pup more than average caused a shortening of the gestation by 0.25 days, and each pup less a corresponding lengthening. Bitches giving birth to their first litter after 4 years of age produced a smaller litter than younger bitches. Litter size decreased after 5 years in all bitches. The number of born pups at the private kennel increased from the first to the third parity, then decreased. The number of registered pups increased from the first to the second parity in the SKK data and from the second to the third parity in the data from the private kennel, then decreased. Mating a bitch only once resulted in a smaller litter size. None of the studied factors had any effect on the sex ratio of the pups. There were significant differences between males in whelping rate among the mated bitches, but no difference in mean litter size, which indicates a female problem rather than a male one. Available data suggest that the domestic dog is still under considerable seasonal influence, although modified by ambient and management factors.     

免疫佐剂对幽门螺杆菌免疫原性的影响

幽门螺杆菌是一类高传染性的致病细菌,能导致人类多种疾病。为比较不同佐剂对该致病菌的免疫原性的影响,实验中利用简单的布氏肉汤添加环糊精液体培养基体外培养幽门螺杆菌,分别与福氏佐剂、自制油佐剂、氢氧化铝佐剂混合免疫昆明种小鼠,经间接ELISA法分析抗血清效价证实,三种免疫佐剂都能有效地刺激小鼠对幽门螺杆菌产生明显的体液应答,其中福氏佐剂的效果最好,自制油佐剂略强于氢氧化铝佐剂的免疫活化作用。三者免疫的抗血清效价分别为,福氏佐剂1∶25600,自制油佐剂1∶12800,氢氧化铝佐剂1∶12800。     

The choice of adjuvants in Mycoplasma vaccines

The use of adjuvants in vaccine production is an important aspect of potent vaccines. This investigation was concerned with finding the most efficient adjuvants for use in Mycoplasma vaccines produced in Nigeria. Four different vaccines were produced from the Gladysdale strain of Mycoplasma mycoides subspecies mycoides. They differed depending on the type of adjuvants used. Each vaccine was used to vaccinate eight cattle using a dose of 1 ml. Two other groups of eight cattle were used as controls. One of the two groups received 1 ml dose of inactivated Gladysdale vaccine without adjuvant while the second group received 1 ml dose of saline. The number of cattle that had the peak complement fixing (CF) antibody titres of 1/80 in each group of cattle was four for vaccine containing aluminium hydroxide gel, eight for vaccine containing liquid paraffin, one for vaccine containing sodium alginate and one for vaccine without adjuvant. Seven cattle from the group vaccinated with vaccine containing Freund's incomplete adjuvant had peak CF antibody titres of 1/80 or higher. The two groups vaccinated with vaccine containing liquid paraffin and Freund's incomplete adjuvant survived challenge at 6 months post vaccination. Freund's incomplete adjuvant and liquid paraffin containing 10% Arlacel A are the most efficient adjuvants.     

Contraceptive potential of porcine zona pellucida in cats

The contraceptive potential of solubilized porcine zona pellucida (spZP) was studied in 2 groups of cats after active immunization using slightly different protocols. Cats from Group 1 (n = 3) were immunized with a total of 300 8g spZP divided in 4 s.c. multisite injections (each of 37.5 8g) given at 10 day intervals followed by a booster 150 days after the initial immunization. Cats from Group 2 (n = 5) were immunized with a total of 400 8g spZP divided in 4 i.m. injections (each of 50 8g) given at 2 wk intervals followed by a booster 92 days after initial immunization. Immunogen was emulsified in Complete Freund Adjuvant for the first dose and in Incomplete Freund Adjuvant for the following 3 doses. The respective controls were immunized in the same manner using only adjuvant and PBS. Immunofluorescence studies showed intense fluorescence on the zona pellucida (ZP) of the oocytes isolated from immunized cats, as well as on the ZP of porcine and cat oocytes preincubated in immune sera. Sera from cats immunized with spZP inhibited in vitro binding was demonstrated in oocytes isolated from immunized group 1 cats. In vivo fertility data in Group 2 cats revealed that only 1 of 5 cats became pregnant, the one with the lowest anti-spZP titer. The results from the experiments reported above, suggest that in this preliminary study spZP can elicit antibodies with contraceptive potential in actively immunized female cats.     

A study of two protocols combining aglepristone and cloprostenol to treat open cervix pyometra in the bitch

To compare the efficacy and safety of two protocols using a combination of aglepristone and cloprostenol for the treatment of open cervix pyometra in the bitch and to describe the progesterone (P4) serum profiles before and during treatments, 15 bitches were randomly allocated into two treatment groups: I (n = 8): aglepristone was administered at 10mg/kg, s.c., on Days 1, 3, 8, and 15 (if not cured), combined with cloprostenol at the dose of 1 microg/kg, s.c., on Days 3 and 8, and II (n = 7): received the same treatment with aglepristone as Treatment I but cloprostenol on Days 3, 5, 8 10, 12, and 15 (if not cured). Before the beginning of the treatments and then on Days 8, 15, and 29 all bitches were evaluated for clinical signs, side effects, hemogram, serum P4 concentrations, and uterus diameters. Bitches in both treatment groups, with (n = 6) or without (n = 9; > or =1.2 ng/ml) initial basal P4 serum concentrations, achieved treatment success without side effects and no significant differences, either on Day 15 (6/8 for Treatment I and 4/7 for Treatment II) or on Day 29 (2/8 for Treatment I and 3/7 for Treatment II). In both treatments groups, clinical signs, blood parameters, and uterine diameters improved to normal values throughout the experiments. A significant interaction between day and treatment was found for percentage change in P4 when all bitches were considered together. Redevelopment of pyometra in the next estrous cycle occurred in 20% of the bitches. One nonrecurrent bitch was mated and whelped a normal litter. It is concluded that these two combined protocols proved to be efficient and safe in reversing clinical signs of open cervix pyometra independently of initial P4 concentrations and that the number of cloprostenol administrations seemed to have an effect on P4 serum changes throughout treatments.     

Production and preparation of hyperimmune bovine colostrum for passive immunotherapy of cryptosporidiosis.

Pregnant cows were immunized to produce hyperimmune bovine colostrum (HBC) by intramuscular injection or intramammary infusion (TI) followed by 3 successive TI boosters with Cryptosporidium parvum (Cp) oocyst antigen mixed with Freund's (F) or Ribi (R) adjuvant. Control cows received no Cp. Colostrum from all cows was skimmed of butterfat and tested for specific anti-Cp immunoglobulin isotypes by ELISA. The HBC from Cp-F and Cp-R immunized cows had IgG1 titers exceeding 1:400,000 and 1:800,000, respectively. Some HBC from Cp-F immunized cows was freeze-dried to facilitate storage and some were irradiated at 42.5 kGy to kill potentially contaminating pathogens. Freeze-drying, but not irradiation, reduced IgG1 titers by only one dilution. Neither treatment affected Western blot banding patterns.     

Active immunization of the cow against oestradiol-17beta

Six beef heifers were immunized over a 4-month period with an oestradiol-17beta-BSA conjugate in Freund's adjuvant. There was an interference with oestrus in the treated heifers; 2 ceased to exhibit oestrus, one exhibited one oestrus and three exhibited oestrus after Day 47 of treatment. The control heifers treated with Freund's adjuvant had normal oestrous cycles. The antiserum titre rose in all treated heifers and attained its highest level in the 2 animals in which oestrus did not recur. The temporal changes in plasma LH, progesterone and oestradiol were normal during the pretreatment period, but became abnormal during the 120 days after immunization. Although plasma oestradiol-17beta rose at the expected time of oestrus after treatment, it was apparently effectively neutralized by the antiserum induced by treatment as evidenced by the absence of an LH surge. Plasma progesterone levels fell to baseline and remained low, indicating lack of formation of corpora lutea.     

Comparison of immune and adverse effects induced by AdjuVac and Freund's complete adjuvant in New Zealand white rabbits (Oryctolagus cuniculus)

Though Freund's complete adjuvant effectively increases immune response to vaccines in various species, its potentially severe inflammatory effects have led many animal researchers to seek alternative immunological adjuvants. In a study of New Zealand white rabbits, the authors compared the immune and adverse effects of Freund's complete adjuvant with the effects of two formulations of AdjuVac, an immunological adjuvant previously developed by their group. All three adjuvants improved humoral immune response but also caused inflammation. Inflammatory reactions caused by AdjuVac, however, tended to be less severe than those caused by Freund's complete adjuvant.