Cloning, sequencing and expression of white rhinoceros (Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of rhinoceros IFN-gamma specific antibodies

Bovine tuberculosis (BTB) is endemic in African buffalo (Syncerus caffer) in the Kruger National Park (KNP). In addition to buffalo, Mycobacterium bovis has been found in at least 14 other mammalian species in South Africa, including kudu (Tragelaphus strepsiceros), Chacma baboon (Papio ursinus) and lion (Panthera leo). This has raised concern about the spillover into other potentially susceptible species like rhinoceros, thus jeopardising breeding and relocation projects aiming at the conservation of biodiversity. Hence, procedures to screen for and diagnose BTB in black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum) need to be in place. The Interferon-gamma (IFN-gamma) assay is used as a routine diagnostic tool to determine infection of cattle and recently African buffalo, with M. bovis and other mycobacteria. The aim of the present work was to develop reagents to set up a rhinoceros IFN-gamma (RhIFN-gamma) assay. The white rhinoceros IFN-gamma gene was cloned, sequenced and expressed as a mature protein. Amino acid (aa) sequence analysis revealed that RhIFN-gamma shares a homology of 90% with equine IFN-gamma. Monoclonal antibodies, as well as polyclonal chicken antibodies (Yolk Immunoglobulin-IgY) with specificity for recombinant RhIFN-gamma were produced. Using the monoclonals as capture antibodies and the polyclonal IgY for detection, it was shown that recombinant as well as native white rhinoceros IFN-gamma was recognised. This preliminary IFN-gamma enzyme-linked immunosorbent assay (ELISA), has the potential to be developed into a diagnostic assay for M. bovis infection in rhinoceros.     

Analysis of repeated tests for interferon-gamma (IFN-gamma) response and faecal excretion for diagnosis of subclinical paratuberculosis in Danish cattle

A total of 315 cattle were tested for infection with Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) at three consecutive samplings, using the interferon-gamma (IFN-gamma) test on whole blood and bacteriological culture of faecal samples. Of 205 cattle from 10 infected herds 99 (48%) were positive in the IFN-gamma test on at least one sampling using "IDEXX-criteria" for interpretation, and of 110 cattle from five non-infected herds three (3%) were positive. Forty-four animals from infected and one from non-infected herds tested positive at all three samplings. Although support for the specificity of the IFN-gamma test was provided by these results, they also indicate problems with false positives. Approximately half of the positive animals did not give the same result at all three samplings, indicating that repeated testing increases the chance of detecting reactors. Changing, or fluctuating, IFN-gamma test results occurred most frequently in animals younger than 1 year, indicating that the IFN-gamma test should be applied only to animals 1 year and older. M. paratuberculosis was isolated from 16 (4%) of 371 cattle, all from infected herds. Fifteen culture-positive cattle tested positive at least once in the IFN-gamma test. It was not possible to predict from the IFN-gamma test result the number of animals that would eventually develop disease. However, the test may be useful to detect animals that have been exposed to M. paratuberculosis earlier in their lives, and the testing of young cattle could be included in a control program to check for the effectiveness of preventing transmission of infection to calves and to identify animals at risk of developing disease later in their lives.     

Effects of supplemental chromium on interferon-gamma (IFN-gamma) mRNA expression in response to Newcastle disease vaccine in broiler chicken

We report the effects of supplemental chromium (Cr) on interferon-gamma (IFN-gamma) expression in response to Newcastle disease virus (NDV) vaccine in broiler chicken. Two hundred and eighty day old straight run broiler chicks were randomly selected and allocated at random to one of the seven treatment groups replicated four times having 10 chicks in each replication. Treatment 1 (C) was a standard control diet in which no Cr was offered to birds either through feed or drinking water. In treatments 2-4 (F500, F1000 and F1500), Cr in the form of chromium picolinate (CrPic) was added at rates of 500, 1000 and 1500 ppb in diets, respectively. In treatments 5-7 (W250, W500 and W750), Cr as added to the drinking water at rates of 250, 500 and 750 ppb, respectively. The feeding was continued from 1 to 49 d of age. On 49 d, three birds from each replicate, i.e. 12 birds/treatment were inoculated intramuscularly with R(2)B strain of NDV. Twelve unvaccinated birds along with 12 vaccinated birds from each treatment (four birds each day) were then sacrificed on 1, 3 and 7 days post-immunization to study the IFN-gamma expression using quantitative real time PCR. Following the immunization, the IFN-gamma mRNA expression in spleen was significantly (P<0.01) either up-regulated or down-regulated at day 1 and day 3 post-stimulation. Whereas, IFN-gamma mRNA expression reached basal level in all the vaccinated groups at day 7 post-stimulation. IFN-gamma mRNA expression on day 1 was approximately two and four times higher than the control (C) levels in F500 and W500 groups, respectively. On 3 day post-immunization, IFN-gamma mRNA expression in spleen was about 40 and 27 times higher than controls (C) in F500 and W500 groups, respectively. Other groups (F1000, F1500, W250 and W750) showed down-regulation of IFN-gamma mRNA expression. The results suggested that the dosage of chromium modulates the expression of IFN-gamma and route has effect on the onset and duration of the response. Thus, the supplementation of chromium at appropriate dose might be helpful to enhance the IFN-gamma mRNA expression in response to NDV.     

Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus

Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-gamma response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-alpha was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-gamma response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-alpha expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-alpha or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-gamma response to the PRRSV vaccine could be achieved by substituting polyinosinic-polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines.     

Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals

Cytokines produced by T helper (Th) cells are important in orchestrating the immune response during health and disease. Recent reports indicated that cytokine mRNA expression in foals is often quantitatively lower than that of adult horses suggesting that foal T cells are not fully mature. Here, peripheral blood mononuclear cells from foals and adult horses were stimulated with phorbol 12-myristate 13-acetate and analyzed for intracellular interferon-gamma (IFN-γ), interleukin-4 (IL-4) and IL-10 production, representing the Th1, Th2 and regulatory T_R1 cell phenotypes respectively, by flow cytometry. In agreement with previous reports, all three cytokines were quantitatively reduced in foals compared to adults. However, the balance between Th1 and Th2 cytokines (IFN-γ/IL-4 ratio) showed a clear Th1-biased response in foals by 6 and 12 weeks of life, while similar IFN-γ/IL-10 ratios were found in foals and adult horses. By day 5 after birth, intracellular IFN-γ production by foal CD4⁺ and CD8⁺ T cells resembled that in adults. Overall, IL-4 production was low in foals. IL-4⁺ cells peaked at day 5 of age when IL-4 was mainly produced by IgE⁺ cells. Relative percentages of IL-4⁺ Th2 cells were significantly lower in foals at all time points. The data suggested that equine neonates and young foals have an impaired Th2 response, that the immune response of foals is Th1 biased, that IFN-γ production by Th and cytotoxic T cells is qualitatively similar to adult horses, and regulatory IL-10 production by T cells is developmentally mature in foals during the first three months of life.     

Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease.     

Increased Th1/Th2 (IFN-gamma/IL-4) Cytokine mRNA ratio of rat embryos in the pregnant mouse uterus

Somatic cell nuclei can be dedifferentiated in ooplasm from another species, and interspecies cloned embryos can be implanted into the uteri of surrogates. However, no full pregnancies have been achieved through interspecific mammalian cloning. Rat blastocysts transferred into mouse uteri provide a unique model for studying the causes of interspecific pregnancy failure. In this study, intraspecific pregnancy (mouse-mouse) and interspecific pregnancy (rat-mouse) models were established. On Day 9 of pregnancy, the fetoplacental units were separated from the uterine implantation sites and the expression of messenger (m)RNA was quantitated by real-time PCR. We compared the mRNA expression levels of type-1 T helper (Th1) and type-2 T helper (Th2) cytokines, interferon-gamma (IFN-gamma), and interleukin-4 (IL-4) in fetoplacental units between intraspecific and interspecific pregnancy groups. The mRNA expression of IFN-gamma in the fetoplacental units of the interspecific pregnancy group was significantly higher than that of the intraspecific pregnancy group (P<0.05). The mRNA expression of IL-4 in the interspecific pregnancy group was significantly lower than that in the intraspecific pregnancy group (P<0.05). We also analyzed the ratio of IFN-gamma/IL-4 mRNA, and an increased IFN-gamma/IL-4 mRNA ratio was observed in the interspecific pregnancy compared with that in the intraspecific pregnancy group. The IFN-gamma and IL-4 mRNA expressions indicate that there is a Th1/Th2 imbalance in the feto-maternal interface of interspecific pregnancies. Bias of Th1 cytokine dominance may be a barrier to reproductive success between species.     

Induction of Th1 immune responses to Japanese cedar pollen allergen (Cry j 1) in mice immunized with Cry j 1 conjugated with CpG oligodeoxynucleotide

We determined whether a major Japanese cedar pollen allergen (Cry j 1) conjugated with CpG oligodeoxynucleotide would enhance allergen-specific Th1 responses in mice. Cry j 1 conjugated with CpG (Cry j 1-CpG) induced IL-12 in the spleen cells of naïve mice. Cry j 1-CpG immunization of BALB/c mice suppressed anti-Cry j 1 IgE response and enhanced anti-Cry j 1 IgG_2a to subsequent Cry j 1 and alum adjuvant injection. CD4⁺T cells isolated from the spleens in mice immunized with Cry j 1-CpG produced higher IFN-γ levels than did CD4⁺T cells obtained from mice as negative controls. Our results suggested that Cry j 1-CpG immunization can induce Cry j 1-specific Th1 immune responses, thereby inhibiting IgE response to the pollen allergen.     

Th1 and Th2 indices of the immune response in pigs vaccinated against Taenia solium cysticercosis suggest various host immune strategies against the parasite

Kinetics of the production of serum antibody levels and Th1 (IL-2, IFN-gamma) and Th2 (IL-4, IL-10) cytokines was studied in five pigs vaccinated with a synthetic tri-peptide vaccine (S3Pvac) against Taenia solium, a vaccine that has been shown protects pigs against naturally acquired infection. Healthy pigs of mixed genetic background, similar to those bred in rural villages of Mexico, were vaccinated with S3Pvac or with adjuvant alone, kept in sanitary conditions and bled at different times after vaccination to study the development of their specific immune response. Peripheral blood mononuclear cells (PBMCs) of vaccinated pigs showed a significant increment in the production of Th1 cytokines (IL-2 and IFN-gamma) but not of Th2 cytokines (IL-4 and IL-10) after specific PBLs stimulation with all the individual peptides. A Th1-inclined cytokine profile leading to an exacerbated local inflammation at the early installation stage of the cysticercus may possibly interfere with their successful establishment in the serum antibodies against total cysticercus antigens and against each of the three different peptides comprising S3Pvac were detected 7-51 days after vaccination. Antibodies against GK-1 interfered with the cysticerci development into intestinal tapeworms in prednisolone-treated hamsters. The sub-lethal crippling effect of anti-GK-1 antibodies upon cysticerci indicates to a therapeutic application of S3Pvac in infected pigs having potential epidemiological consequences, as it could aid in decreasing the number of tapeworms expected to develop from the few cysticerci that survive in the vaccinated pigs.     

Induction of immune response and protection from equine viral arteritis (EVA) by formalin inactivated-virus vaccine for EVA in horses

Thirty-nine horses included 3 pregnant mares were examined by inoculating with formalin inactivated-virus vaccine for EVA. Antibody response of horses after one dose vaccination was somewhat poor and 50% effective inoculum dose of the vaccine should be included 10(8.4) pfu of virus before inactivation. After 2 doses given at an interval of 4 weeks, the horses developed such high titer of SN antibody as up to 1:5,120. The SN titer declined rather rapidly, but supplemental administration of the vaccine at an interval of more than 2 months elicited a prompt antibody response and SN titers persisted as 1:80 to 1:320 at 6 months after the administration. Therefore, supplemental administration of the vaccine as booster every 6 months or 1 year would be capable of maintaining high titer of SN antibody. The inactivated-virus vaccine prevented horses from clinical disease of EVA and protected pregnant mares from abortion by challenge exposure with virulent virus. Fifty percent protective dose in SN titer of 1:43 was confirmed by clinical signs and viremia.     

Haemonchus contortus (Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response

Selection of resistant animals and host immunization have been proposed as alternative methods for the control of gastrointestinal nematode parasites. However, a better knowledge of the mechanisms involved in protective immunity against these parasites is required for the development of optimal strategies. In this study, 3 month old INRA 401 lambs (n = 81) were allocated into three groups: uninfected control, challenged either once (primary-infected animals) or twice (previously-infected animals) with 10,000 Haemonchus contortus L3. Uninfected control and challenged animals were sequentially sacrificed at 3, 7, 15 and 28 days post challenge. In both challenged groups, a clear Th2-oriented immune response was observed in the abomasal lymph node and mucosa. IL-4 and IL-13 mRNA over-expression, recruitment of eosinophils, mast cells and globule leukocytes and production of specific systemic IgG and mucosal IgA were observed earlier in previously-infected animals than in primary-infected ones. At 28 days post infection, no differences between intensities of these responses were observed between the challenged groups. Worm establishment rates were similar in previously-infected and primary-infected lambs. However, reductions of worm development, female fecundity and fecal egg output were observed in previously-infected sheep. We conclude that H. contortus infection in young INRA 401 lambs induced an unequivocal Th2 immune response resulting in the regulation of worm egg production without affecting their establishment.     

Induction of protective immune response against both PPRV and FMDV by a novel recombinant PPRV expressing FMDV VP1

Peste des petits ruminants (PPR) and foot-and-mouth disease (FMD) are both highly contagious diseases of small domestic and wild ruminants caused by the PPR virus (PPRV) and the FMD virus (FMDV). In this study, a recombinant PPRV expressing the FMDV VP1 gene (rPPRV/VP1) was generated and FMDV VP1 expression did not impair replication of the recombinant virus in vitro and immunogenicity in inducing neutralizing antibody against PPR in goats. Vaccination with one dose of rPPRV/VP1 induced FMDV neutralizing antibody in goats and protected them from challenge with virulent FMDV. Our results suggest that the recombinant PPRV expressing the FMDV VP1 protein is a potential dual live vectored vaccine against PPRV and FMDV.     

Cellular immune response and immunotoxicity induced by DON (deoxynivalenol) in piglets

Veterinary Research Communications -     

Administration of poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) as adjuvant activated mixed Th1/Th2 immune responses in pigs

The selection of an optimal adjuvant to enhance the potency and longevity of antigen specific immune responses has always been imperative for the development of more effective and safer vaccines. A balanced type of immune enhancing ability of a new adjuvant known as polyphosphazene (PCEP) has been demonstrated in mice. In the present study we have compared the humoral and cellular immune responses to vaccine formulations containing Actinobacillus pleuropneumoniae outer membrane antigen (OmlA) with PCEP or Emulsigen as adjuvants. Our data showed a significant improvement and a shift toward more balanced immune responses when OmlA antigen was formulated with PCEP and CpG ODN. Moreover, in contrast to Emulsigen, immunization with PCEP did not result in local injection site reactions highlighting its potential as a safe and effective adjuvant for pigs. Further, the ease of formulation, administration and relatively low per dose cost of PCEP make it a promising adjuvant for pigs.     

Detection of impaired T cell-mediated immune responses to herpesvirus (BHV-1) in cattle

Interleukin 2 (IL-2) functions in the regulation of cell-mediated immune responses both in vivo and in vitro. Therefore, IL-2 production has been studied in patients with immunological disorders to determine the level of the immune defect. However, the cause(s) of low responses to selected antigens by cells from clinically normal individuals has not been examined. The ability of cells from clinically normal individual cattle to produce and respond to IL-2 was investigated as a measure of specific cell-mediated immune response. Cells from the majority of animals (6/7 in a representative experiment) could produce IL-2 in response to mitogen or a specific antigen, Bovine Herpesvirus 1 (BHV-1). Proliferation of lymphocytes from the majority of low responding animals (2/3 and 4/4 in separate experiments) could be restored to a level similar to high responders by the addition of exogenous IL-2 after endogenous IL-2 depletion. IL-2 receptor expression was indirectly assessed by the ability of activated cells to absorb IL-2. One individual's cells were incapable of absorbing exogenous IL-2 and failed to proliferate indicating a lack of activation and expression of receptors. In addition, exogenous IL-2 was able to enhance proliferation of both high and low responders in the presence of endogenous IL-2. These results suggest that proliferation of bovine peripheral blood mononuclear cells is dependent on the presence of IL-2. In addition, IL-2 production can be used to measure specific cell-mediated immune responses.