Selection for immune response in goats: the effect of immunization procedure on antibody response to diphtheria toxoid and human serum albumin.

The serum antibody titers to diphtheria toxoid and human serum albumin were determined in 103 goat kids from lines selected for 12 yr for high or low antibody response to diphtheria toxoid. In the 12th yr, six groups of kids were immunized with different preparations of the antigens. In all groups but one, the antigens were emulsified in Freund's incomplete adjuvant with added sonicated Mycobacterium paratuberculosis. The groups received the following treatments: Group 1 was immunized with both antigens mixed in the same syringe, Group 2 got both antigens injected separately, Group 3 got both antigens injected separately, but with a lower concentration of M. paratuberculosis, Group 4 was immunized with diphtheria toxoid only, Group 5 was immunized with human serum albumin only, and Group 6 was immunized with both antigens mixed, but without any M. paratuberculosis. The animals were immunized at 4 wk of age, and the antibody titers were determined 3 wk later by ELISA and passive hemagglutination. The mean antibody titers to both antigens were different between the selected lines (P less than .03). There was no effect of separate vs combined injections of antigens. However, there were indications of antigen suppression or competition between the antigens. Animals receiving only one antigen seemed to mount a higher antibody response to that antigen than did animals immunized with two antigens.     

Immune response of the llama (Lama glama) to tetanus toxoid vaccination

An ELISA was developed to measure serum concentrations of tetanus toxoid-specific immunoglobulins. The titers obtained with this assay were compatible with those obtained by the standard mouse toxin-neutralization test. Serum samples from 123 llamas were analyzed for ELISA titers to tetanus toxoid. Of the 82 vaccinated adults, 75 (91%) had titers greater than or equal to 1:50. The vaccination status and titers of weanlings and juveniles (3 to 12 months old) varied; of the 21 vaccinated, 17 (81%) had titers greater than or equal to 1:50 and 7 of 9 (78%) unvaccinated llamas had titers less than 1:50. The ELISA titers of unvaccinated llamas less than 8 weeks old (crias) were matched with the maternal titers. All crias with titers less than 1:50 had dams with titers greater than or equal to 1:50.     

Selection for immune response in goats: the antibody response to diphtheria toxoid after 12 years of selection.

A herd of Norwegian dairy goats was subject to selection for high and low antibody response to diphtheria toxoid for 12 yr, or approximately 5.5 generations because sires were used for one mating season, whereas dams were used several years. The herd comprised approximately 100 milking goats. Only sires were tested and selected, five to seven sires were used in each line each year, and the percentage of male kids used for breeding varied between 15 and 50%. The mean phenotypic values of the lines diverged until the 4th yr, but the lines did not diverge any further. The means of both lines decreased during the experimental period. The restricted maximum likelihood (REML) estimate of the heritability of the trait in the base population was .19, whereas the realized heritability approached zero in the last six cycles of selection. Using the REML-estimate of heritability in an individual animal model, the mean breeding values (BLUP) of the lines were significantly different. The difference in mean BLUP values between the lines increased throughout the study. Yet, this increase was only 40% from the 2nd to the 12th yr. No effect of a major gene was observed in a test based on individual BLUP.     

Possible association of antibody responses to human serum albumin and (T,G)-A--L with the bovine major histocompatibility complex (BoLA)

Antibody responses to human serum albumin (HSA) and (T,G)-A--L were determined in 130 young bulls in Norway and the BoLA types of the bulls were defined. Significant associations of some BoLA antigens with immune responsiveness were shown, indicating the likely existence of an immune response (Ir) region linked to the BoLA class I antigens. High response to HSA seems to be a dominant trait. BoLA w2 showed an association with low response to HSA. This may reflect the effect of a specific MHC-associated immune suppressor gene.     

Influence of orally administered CpG-ODNs on the humoral response to bovine serum albumin (BSA) in chickens

Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have been reported to be effective mucosal adjuvants in mice when given orally. Studies on their effectiveness in chickens are currently very limited. This study investigated whether CpG-ODNs could adjuvant the immune response to BSA when given orally to a commercial line of SCWL chickens. In two experiments, performed over time, chickens were given selected concentrations of CpG-ODNs with BSA followed by 6 consecutive days of ad libitum access to drinking water containing 1.4 mg/ml BSA. Serum responses, and in some cases intestinal specific antibodies, were measured out to 33 days post-immunization. Birds receiving a single dose of CpG-ODN had consistently higher IgG, IgM, and IgA titers in the serum, dependent upon dose, and in specific areas of the intestine when compared to the non-immunized and BSA only groups. These findings suggest that a single oral CpG-ODN administration can accelerate the kinetics of antigen specific antibodies of all three isotypes in commercial-strain chickens immunized via the drinking water using common protein antigen.     

Cross-reactivity between human and feline Ia antigens, using a monoclonal antibody HLA-D.m1

A murine monoclonal antibody HLA-D.m1 produced against human Ia antigens which react with 10% to 30% of human peripheral-blood mononuclear cells was found to react with greater than 90% of feline mononuclear cells. The antigen was not expressed on granulocytes. By direct immunofluorescence, 7% to 41% of feline lymphocytes expressed surface immunoglobulins. In mixed-lymphocyte reaction, the addition of antibody HLA-D.m1 to cultures abolished proliferation. These studies indicate that most feline peripheral-blood mononuclear cells express Ia-like antigens.     

Characteristics of the response of ovine granulocytes (PMNs) to zymosan-activated serum (ZAS) and to recombinant human interleukin-8 (IL-8)

The chemotactic activity of zymosan-activated serum (ZAS) and of two concentrations of recombinant human IL-8 (IL-8(25), 25 ng/ml; IL-8(50), 50 ng/ml) for ovine polymorphonuclear granulocytes (PMNs) was tested in a modified Boyden chamber. Thick cellulose acetate filters and the leading front method were used to quantify the movements of the cells. Both ZAS and IL-8(25) exerted a chemotactic effect on ovine PMNs (P < 0.01): IL-8(50) induced a more homogeneous response (P < 0.001). To verify the characteristics of the responsiveness to the chemokines after short-term (st) or long-term (lt) repeated samplings, chemotaxis was investigated 1 (T1st), 2 (T2st), 24 (T3st) and 48 h (T4st) after the basal sampling (T0st) and 15 days (T1lt) after the basal sampling (T0lt). No differences in chemotaxis were found in long-term repeated samplings. In contrast an increase in the responsiveness to IL-8(25) and to IL-8(50) (P < 0.05) was detected at T2st in comparison with T0st. Furthermore, the significance of the distance run by activated PMNs compared with the controls, increased from T0st to T2st, as a sign of a more homogeneous response to the chemokines. In the absence of evident changes in circulating leucocyte numbers and in serum cortisol concentrations, these findings could be interpreted as a consequence of a different expression of chemoattractant receptors on the membrane of PMNs collected at different times.     

Oral bioavailability of sulphonamides in ruminants: A comparison between sulphamethoxazole,sulphatroxazole, and sulphamerazine,using the dwarf goat as animal model

Summary

The various sulphonamides show marked differences in disposition characteristics after administration to ruminants. For use in combination with a diaminopyrimidine derivative such as trimethoprim or baquiloprim, it is essential that a sulphonamide has similar pharmacokinetic properties in order to obtain optimal synergy. In the present study the pharmacokinetics of sulphamethoxazole, sulphatroxazole, and sulphamerazine were investigated in dwarf goats (n=6) after IV and intraruminal administration at a dose of 30 mg/kg bodyweight. In addition, the in vitro binding of sulphamerazine to ruminal contents was studied as a possible explanation for a reduced absorption rate. Sulphamethoxazole showed the most rapid absorption after intraruminal administration (mean t_max ± SD : 0.8 ± 0.2h). However, the drug was rapidly eliminated from the plasma (t_1/2ß : 2.4 ± 1.5h) and the bioavailability was only 12.4 ± 4.7 %, most likely due to an extensive ‘first‐pass’ effect. The bioavailability of orally administered sulphamerazine and sulphatroxazole was much higher (67.6 ± 13.5 % and 70.2 ± 32.3 %, respectively). After intraruminal administration, sulphatroxazole showed the highest plasma peak concentration (26.1 ± 6.3 mg/I) and the longest plasma half‐life (4.7 ± 1.8h) and mean residence time (13.9 ± 4.5 h). Sulphamerazine showed considerable binding to rumen contents in vitro. Based on its pharmacokinetic properties sulphatroxazole appears to be a suitable candidate to be used in combination with the more recently developed diaminopyrimidines such as baquiloprim.     

Studies on adjuvant-induced arthritis, tumor transplantability, and serologic response to bovine serum albumin in Sendai virus-infected rats.

Sendai virus, one of the most prevalent of the murine viruses, was studied in regard to its capability to alter various functions of rats given a second, unrelated antigenic challenge. Rats given a single foot-pad injection of Freund's complete adjuvant (FCA) had an 85% incidence of adjuvant arthritis. The adjuvant disease was significantly less severe (P less than 0.01) in those rats given 0.05 ml of 10(5.5) median egg infective doses of egg-propagated Sendai virus intranasally 7 days before injection of adjuvant. Rats given Sendai virus concurrently with the FCA, or any time after FCA was injected, did not have a lessened severity of the arthritic reaction, as compared with that in control animals. Sendai virus infection had no detectable effect on median tumor dose requirement for Walker carcinosarcoma cells in rats or on the antibody response to bovine serum albumin.     

A comparative analysis of sphenoid bone between domestic sheep (ovis aries) and goat (capra hircus) using geometric morphometrics

The sphenoid bone forms the rostral part of the base of the neurocranium and is composed of two segments, the presphenoid [os praesphenoidale] and the basisphenoid [os basisphenoidale]. Rarely studied in osteology, we tested whether it can provide distinctive features between domestic sheep (Ovis aries L., 1758) and goat (Capra hircus L., 1758). For this goal, we studied a sample comprised by 53 dry modern skulls of adult sheep (n = 36) and goat (n = 17) subjects from a modern comparative collection by means of geometric morphometric techniques using a total of 26 anatomical points (2 saggital landmarks and 24 semilandmarks). Results showed that form (size + shape) differences appear between both species: sphenoid among sheep tends to be bigger, longer and wider than in goats, differences of width being mainly located on basisphenoid width.     

Detection of serum adiponectin in the cat, bear, and horse using antibody from a commercial ELISA kit

Adiponectin is a hormone secreted from adipose tissue that closely associates with insulin resistance. In contrast to other adipokines, adiponectin levels decrease as fat mass increases. The ability to measure adiponectin in veterinary species will allow researchers to assess hormone function and may lead to improved screening tests and treatments for insulin resistance and type II diabetes. A commercial ELISA kit (B-bridge International) is available for adiponectin quantification in mice and rats. To test this assay's ability to bind adiponectin in several veterinary species known to develop insulin resistance with obesity, we conducted a Western blot analysis comparing mouse, cat, bear, and horse serum using the antibody provided with the assay. The proteins were subjected to denaturing SDS-PAGE and transferred to a nitrocellulose membrane. Blocking was performed in 5% milk solution. The membrane was then incubated with the rabbit anti-mouse adiponectin antibody provided with the ELISA kit, followed by incubation with peroxidase-labeled goat anti-rabbit IgG. Detection of bound antibody was performed using enhanced chemoluminescence. Bands corresponding to the adiponectin monomer (30 kDa) were apparent for each sample tested. Therefore, the antibody provided in the ELISA kit appears to bind with adiponectin in all species tested. Our laboratory has previously validated the ELISA mouse/rat adiponectin kit for use in cats. High DNA homology across species and strong antibody binding at the 30 kDa site warrant validation of the kit's accuracy in other species at risk for insulin resistance (eg. horses and bears).     

Characterization of the interferon gamma response to Lawsonia intracellularis using an equine proliferative enteropathy challenge (EPE) model

Lawsonia intracellularis is the etiological agent of infectious intestinal hyperplasia for which several clinical diseases have been described including proliferative enteropathy (PE), intestinal adenomatosis, and ileitis. While initially recognized as the causative agent of PE in pigs, L. intracellularis is now viewed as an emerging cause of intestinal hyperplasia in a wide range of mammalian species, including horses. Equine proliferative enteropathy (EPE) has been reported worldwide though definitive diagnosis is difficult and the epidemiology of the disease remains poorly understood. Weanlings, in particular, appear to be most at risk for infection, though the reasons for their particular susceptibility is unknown. Using an infectious challenge model for EPE, we demonstrate that EPE, like porcine proliferative enteropathy, can exhibit three clinical forms: classical, subclinical and acute. Out of six pony weanlings, one developed signs of classic EPE, one developed acute EPE, and two developed subclinical EPE. Attempts to induce pharmacological stress through the use of dexamethasone failed to have any effect on outcome. Peripheral blood cells collected from those weanlings that developed clinical EPE exhibited decreased expression of interferon-gamma (IFN-γ) following in vitro stimulation with L. intracellularis. By contrast, those weanlings that did not develop clinical disease generated a robust IFN-γ response. These results indicate IFN-γ likely plays a significant role in protection from disease caused by L. intracellularis in the equid.     

Elimination of homologous and heterologous (bovine) serum albumin from the blood circulation in the dog

The elimination of intravenously administered I¹³¹-labelled bovine serum albumin has been compared to the elimination rate of relabelled homologous serum albumin in normal and bled dogs, which had lost considerable blood volumes. The investigation shows that during the first four to five days after the administration the elimination is similar of heterologous and homologous serum albumin. This proves that bovine serum albumin can be regarded to be an equivalent plasma expander to homologous serum albumin in the dog. Elimination of homologous as well as heterologous serum albumin follows a simple exponential curve during four to five days after administration. The intravascular half-lives for homologous serum albumin were 6.4 ±1.5 days and 6.4 ± 0.6 days respectively in control and bled dogs. Corresponding values for heterologous (bovine) serum albumin were 5.0 ± 0.3 and 4.8 ± 0.4 days respectively.The quote for cencentrations of homologous and heterologous serum albumin in different tissues was found to be relatively constant approximately 1.4. An exception was the stomach wall in bled dogs which had a quote of 1.1 only.     

ELISA and direct immunofluorescence test to detect equine arteritis virus (EAV) using a monoclonal antibody directed to the EAV-N protein

A monoclonal antibody (mAb) directed against the equine arteritis virus (EAV) nucleocapsid (N) protein was used for indirect enzyme-linked immunosorbent assays (ELISAs) using viral antigen from different sources. The same mAb was labelled with fluorescein isothiocyanate for direct immunofluorescence tests (DIFTs). The N-specific mAb appeared to be suitable for the detection in both ELISA and DIFT of different EAV strains and field isolates from semen and tissue samples after passage in lines of RK-13, Vero and fetal equine kidney cells. The ELISA described is an easy and fast method which can be used in most cases to replace the microneutralization test to prove the EAV specificity of the cytopathic effect of cell cultures. The DIFT, however, is more sensitive than both the ELISA and the microneutralization test because EAV antigen can be detected even in cell cultures without or with very weak cytopathic effect.     

Evaluation of serum and secretory antibody responses to an immunodominant recombinant merozoite surface antigen, p150, using a sensitive enzyme-linked immunosorbent assay

An equation obtained from linear-regression analysis of positive/negative ratios and log of enzyme-linked immunosorbent assay (ELISA) titers of coccidia-immune serum samples was used to accurately predict the antibody titers of chickens immunized with a recombinant merozoite surface protein (p150). Chickens immunized twice intramuscularly with the recombinant p150 antigen emulsified in complete Freund's adjuvant developed a dose-dependent anti-p150 antibody response 14 days after primary immunization. Serum IgG and IgM and secretory biliary IgA antibodies were detected 2 months after primary immunization. Oral challenge with live Eimeria parasites significantly enhanced both the serum and secretory anti-p150 antibody titers. These results indicate that vaccination of chickens with the p150 recombinant merozoite antigen can induce a parasite-specific host immune response.     

The seasonal antibody response in juvenile summer flounder (Paralichthys dentatus) to the hemoflagellate Trypanoplasma bullocki

An immuno-blot assay was used to investigate the serum antibody response in flounder injected with formalin-killed flagellates (immunized) and those injected with saline (control) and challenged with live T. bullocki after 21 days. Fish were held at 20 degrees C and at ambient temperature from October through June. At 20 degrees C immunized fish had significantly higher antibody titers than control fish, but immunized fish were not protected from infection with T. bullocki. At ambient temperature, after initial flagellate growth phase, antibody titer varied directly with temperature (2-25 degrees C) and T. bullocki intensity varied inversely with titer. Flagellates were eliminated from the peripheral circulation in both immunized and control fish when antibody titer peaked in May. Recovered fish were immune to homologous challenge for at least one year.     

Immunohistochemical identification of amyloid, using an anti-human serum amyloid P component (SAP) antibody, is possible in ruminants but not in dogs and cats

Amyloidosis represents a heterogenous group of diseases that have in common the deposition of fibrils composed of proteins of beta-pleated sheet structure, a structure which can be specifically identified by histochemistry using the Congo red or similar stains. Amyloid consists primarily of the amyloid fibrils but also of the amyloid P component (AP). This component, which is identical with the serum counterpart (SAP), is found in all types of human amyloid, and immunohistochemical identification of AP has been proposed as an adjunct to the universal, type-independent diagnosis of human amyloidosis. In the present study of animal amyloidosis, we compared the amyloid-specific Congo red stain with an immunohistochemical protocol using an anti-human SAP antibody for the identification of amyloid in formalin fixed tissue samples. The species and types of amyloidoses investigated were: (i) seven cows, one yak (Bos grunniens), and one sheep affected with amyloidosis of presumed AA type, (ii) one dog with a pancreatic endocrine tumour producing amyloid of presumed AIAPP type, (iii) two cats with presumed AIAPP-amyloidosis of the islets of Langerhans, one cat with presumed AA-amyloidosis, and one cat with an amyloid-producing odontogenic tumour. Intense immunostaining co-localized with amyloid, identified by its congophilia and green birefringence, using a protocol without any antigen retrieval in each of the seven cows, the yak and the sheep. The method seemed more sensitive in the ruminants than the Congo red stain, but was unable to detect amyloid in the dog and the cats regardless of the application of various antigen retrieval protocols. However, specific identification of amyloid still rests on the Congo red method or similar histochemical techniques.     

Urinary oestrogen excretion in the goat during the oestrous cycle and after administration of pregnant mare serum gonadotrophin (PMSG)

Two goats were placed in cages constructed for urine collection. Samples were taken daily and stored in the frozen state until analysis. The urine was processed according to a method designed for determination of oestrone and oestradiol-17 α in bovine urine (Lunaas unpublished).