Development of specific diagnostic test for small ruminant lentivirus genotype E

Small ruminant lentivirus (SRLV) belonging to the highly divergent genotype E has recently been identified in the Italian goat breed Roccaverano. In this report we have developed a specific serological test based on recombinant matrix/capsid antigen fusion protein. Performance has been evaluated and compared with a similar test based on genotype B antigen. Herds under study were selected according to the infectious status characterized by blood PCR and sequencing. Results clearly showed that B and E based recombinant ELISA only detected homologous infection and an apparent cross-reactivity was recorded in a herd in which co-infection was present. Three commercially available ELISAs showed different abilities in detecting genotype E infection, being the whole virus-based immunoassay the best choice. Genotype E-recombinant antigen was not detected in ELISA by three commercially available Mabs known to be cross-reactive among CAEV and MVV capsid antigens, further supporting the high divergence of the E genotype from others. Finally, a SRLV-free herd according to commercial ELISA testing, was analysed in the same area where genotype E was identified and few animals belonging to Roccaverano breed were found slightly reactive with the E antigens. Our results suggest that the prevalence of genotype E in other small ruminant populations may be conveniently estimated using a comparative assay based on a combination of genotype specific recombinant antigens and may highlight a wider space in which SRLVs evolve.     

Serological characterization of the new genotype E of small ruminant lentivirus in roccaverano goat flocks

Maedi visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are a heterogeneous group of infectious agents affecting sheep and goats. Due to their natural cross-species infection they are referred to as small-ruminant lentiviruses (SRLV). Recently a new genetic cluster, highly divergent from MVV and CAEV was identified in the north-west part of Italy. A panel of genotype E specific antigens was developed and evaluated in flocks infected with B1 and E strains. The results clearly indicate that a strain specific antigen is required to correctly identify animals infected with different genotypes.     

Serological characterization of the new genotype E of small ruminant lentivirus in roccaverano goat flocks

Maedi visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are a heterogeneous group of infectious agents affecting sheep and goats. Due to their natural cross-species infection they are referred to as small-ruminant lentiviruses (SRLV). Recently a new genetic cluster, highly divergent from MVV and CAEV was identified in the north-west part of Italy. A panel of genotype E specific antigens was developed and evaluated in flocks infected with B1 and E strains. The results clearly indicate that a strain specific antigen is required to correctly identify animals infected with different genotypes.

     

Immune response during coccidiosis in SC and FP chickens. I. In vitro assessment of T cell proliferation response to stage-specific parasite antigens

Development of cell-mediated immunity (CMI) and comparative effectiveness of different stage-specific coccidia antigens in T cell activation during avian coccidiosis were evaluated in two inbred strains of chickens using a specific in vitro T cell proliferation assay. Lymphocytes from chickens infected with different Eimeria spp. showed proliferative response to sporozoites, merozoites or Eimeria soluble antigen (Esa) excreted by cultured parasites. Detectable CMI response was observed at 21 day P.I. in chickens infected with E. tenella and E. maxima. Generally lower T cell response was observed in chickens infected with E. acervulina. Merozoites were highly immunogenic compared to sporozoites. Esa prepared from cultured parasites was as effective as whole parasites in evoking a T cell response. Although strain variation in T cell response to parasites or Esa was observed during a primary infection, substantially enhanced T cell response was observed 3 days after a secondary infection in both strains of chickens. The results of the present investigation suggest that Esa may be a major parasite antigen released to the immune system during early stages of infection and relevant to the development of protective immunity.     

Experimental infection with Tritrichomonas suis in heifers.

Nine heifers were intravaginally challenged with 9.3x10(6) Tritrichomonas suis reference strains. Vaginal mucus and serum samples were collected weekly 4 weeks post-inoculation. Vaginal mucus was cultured for T. suis and sera was tested by ELISA against whole cell antigens for T. suis and Tritrichomonas foetus. All vaginal mucus cultures were T. suis-negative during the experiment. ELISA values for both antigens were similar and differences were not significant (P>0.05). Positive control serum samples from one heifer vaccinated against T. foetus showed anti-T. suis ELISA values. We concluded that T. suis intravaginal inoculation induced a low level of serum immune response in heifers measured by ELISA and both protozoa probably share a common antigen. However, under the experimental conditions of this trial, colonization of the heifers' genital tract was not possible in any of the nine animals.     

Countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats

The procedure of countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats was carried out for antemortem diagnosis of T. hydatigena cysticercosis in experimentally and naturally infected goats. The antigens of cyst fluid, scolex and membrane of T. hydatigena metacestodes were purified and compared. The sensitivity of the test in experimentally and naturally infected goats was 57.1 and 52.5%, respectively, whereas its specificity using antisera raised against T. solium cysticercosis, hydatid cyst and Fasciola gigantica was 66.7 and 83.4% with partially purified and fractionated antigens, respectively. Of all three antigens, the cyst fluid antigen was found to be most reactive. The test could be employed for antemortem diagnosis of T. hydatigena cysticercosis using purified antigen.     

Relationships between infection with caprine arthritis encephalitis virus, intramammary bacterial infection and somatic cell counts in dairy goats.

Somatic cell counts, the bacteriological condition of the milk and antibodies against caprine arthritis encephalitis virus (CAEV) were measured monthly throughout lactation in 121 lactating goats of the Murcia-Granada breed in four commercial dairy goat herds. The prevalence of bacterial intramammary infection was 5.6 per cent and the prevalence of CAEV infection was 20.6 per cent. An analysis of variance revealed a significant effect of herd, intramammary infection and the interaction between intramammary infection and CAEV on the somatic cell count. In udder halves free of intramammary infection, the somatic cell counts were significantly lower in seronegative goats than in seropositive goats (P<0.05), but the difference was not significant in udder halves persistently infected by bacteria. There was a significant increase in somatic cell counts due to bacterial intramammary infection (P<0.01) in the seronegative goats, but this effect was not present in the seropositive animals.     

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.     

Augmented T lymphocyte responses and abnormal B lymphocyte numbers in goats chronically infected with the retrovirus causing caprine arthritis-encephalitis

Caprine arthritis-encephalitis is a retrovirus-induced disease resulting in lymphoproliferative lesions of the CNS and joints. Peripheral blood leukocytes of chronically infected goats were analyzed for the types of cells present and for their reactivity to viral antigen and polyclonal stimulants. Two of 9 infected goats had abnormal numbers of B lymphocytes--one elevated and the other deficient. Lymphocyte reactivity to viral antigens was transiently detectable by a lymphoblastogenic assay in 5 of the 9 goats. The reactive cells were peanut agglutinin-negative T lymphocytes. Concanavalin A induced more division in T lymphocytes of infected goats than in lymphocytes of noninfected goats, whereas the reactions to phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide were no different in the 2 goat groups. It is concluded that goats infected by the caprine arthritis-encephalitis virus have antigen-reactive T lymphocytes and that infection promotes the response to a nonspecific T-cell stimulant.     

Orientation of bovine CTL responses towards PIM, an antibody-inducing surface molecule of Theileria parva, by DNA subunit immunization

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8(+) cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.     

Modified protection against Toxoplasma gondii lethal infection and brain cyst formation by vaccination with SAG2 and SRS1

Numerous studies have supported the importance of immunity to SAG1, the most predominant antigen of Toxoplasma tachyzoite, in protection against Toxoplasma gondii infection. Nevertheless, vaccination with SAGI provides insufficient protection when compared with that of Toxoplasma lysate (TL). In order to screen the Toxoplasma antigens for immunogenic potential shown by modified protection or induction of specific immune response after infection, recombinant antigens were prepared in Eschericha coli using DNA fragments corresponding to SAG1, SAG2, SAG3, SRS1 and P54 of T. gondii RH strain maintained in our laboratory. Each of the recombinant antigen products or a mixture of the five antigens (Mix) was used to vaccinate mice. Mice then received a lethal dose of T. gondii. Up to 25% of the mice vaccinated with SAG2, SRS1, P54 and Mix survived, whereas there were no survivors in gene 10- (negative control), SAG1- and SAG3- vaccinated groups. In all the survivors, brain cysts were not observed. Conversely, vaccination with TL almost completely protected mice in the acute phase but permitted brain cyst formation and resulted in gradual decrease of survivors to 33% during 4 months of experiments. Western blot analysis on convalescent sera showed an extensive IgG induction to a 30 kDa antigen in TL-vaccinated mice, a 22 kDa in SAG2-vaccinated mice and a 55 kDa in P54-vaccinated mice. The protection modified by boost in specific antibody is suggestive of the immunogenic potential of SAG2, SRS1 and possibly P54 against T. gondii infection.     

Immunological properties of recombinant classical swine fever virus NS3 protein in vitro and in vivo

Classical swine fever (CSF) is a highly contagious and often fatal disease of pigs characterised by fever, severe leukopenia and haemorrhages. With vaccines having an importance in disease control, studies are seeking improved protein-based subunit vaccine against the virus (CSFV). In this respect, recombinant viral NS3 protein was analysed for its immunopotentiating capacity, particularly in terms of cytotoxic immune responses. NS3 was effective at inducing in vitro responses, quantified by lymphoproliferation, IFN-gamma ELISPOT, flow cytometric detection of activated T cell subsets, and cytotoxic T cell assays. Peripheral blood mononuclear cells from CSFV-immune pigs could be stimulated, but not cells from na?ve animals. In addition to the IFN-gamma responses, induction of both CD4+ T helper cell and CD8+ cytotoxic T cells (CTL) were discernible--activation of the latter was confirmed in a virus-specific cytolytic assay. Attempts were made to translate this to the in vivo situation, by vaccinating pigs with an E2/NS3-based vaccine compared with an E2 subunit vaccine. Both vaccines were similar in their abilities to stimulate specific immune responses and protect pigs against lethal CSFV infection. Although the E2/NS3 vaccine appeared to have an advantage in terms of antibody induction, this was not statistically significant when group studies were performed. It was also difficult to visualise the NS3 capacity to promote T-cell responses in vivo. These results show that NS3 has potential for promoting cytotoxic defences, but the formulation of the vaccine requires optimisation for ensuring that NS3 is correctly delivered to antigen presenting cells for efficient activation of CTL.     

Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes

Two groups of sheep were experimentally infected by intratracheal route with two small ruminant lentivirus (SRLV) isolates belonging to different genotypes (It-561 genotype A3 and It-Pi1 genotype B2). Seroconversion was evaluated using recombinant homologous and heterologous matrix protein/capsid antigen fusion protein. Results clearly indicate that seroconversion against homologous antigen was detected well in advance as regards heterologous antigen in both groups, although the advantage of using homologous antigen was less evident in detecting seroconversion against the caprine arthritis encephalitis virus (CAEV)-like strain, compared with the maedi-visna virus (MVV)-like infection. Commercially available ELISAs detect CAEV-like seroconversion earlier than MVV-like infection suggesting a closer relationship between CAEV-like isolate and the antigen used in the latter ELISA tests. Seven recombinant subunits developed from matrix protein and capsid antigen of strain K1514 (prototype A1) were used to better define the antibody response in sheep infected with It-561 isolate. Two animals clearly reacted against type specific epitopes in the early stage of infection. This study highlights the relative insensitivity of gag encoded cross-reacting epitopes during the early stage of infection and suggests the development of novel diagnostic tests based on both genotype specific antigens.     

Kinetic analysis of cattle anti-Brucella abortus S45/20 non-agglutinating antibodies in antibody-dependent cell-mediated cytotoxicity

Calves inoculated with Brucella abortus S45/20 produced, against surface antigens, non-agglutinating antibodies (NAAb) which were isolated and purified. A kinetic analysis was carried out of NAAb in antibody-dependent cell-mediated cytotoxicity (ADCC) using sheep red blood cells labelled with surface antigen from B. abortus S45/20 as target cells. Three parameters were examined: time of incubation, effector cell:target cell (E:T) ratio and NAAb dose. It was found that the NAAb were not able to mediate ADCC with bovine spleen cells.     

Comparative immunoelectrophoretic analysis of Echinococcus granulosus, Taenia hydatigena and Taenia pisiformis cyst fluid antigens by hyperimmune rabbit sera.

Cyst fluid antigens of Echinococcus granulosus, Taenia hydatigena and T pisiformis were examined by electrophoresis using homologous and heterologous hyperimmune rabbit sera to these antigens. While arc 5 forming antibodies were identified in sera from rabbits immunised with E granulosus and T hydatigena cyst fluids, antibodies responsible for forming precipitating antigen B band were detected in rabbit antisera to E granulosus, T hydatigena and T pisiformis antigens. T hydatigena cyst fluid appears to contain antigen similar to E granulosus antigen 5 and probably antigen B while T pisiformis cyst fluid has mainly an antigen close to hydatid antigen B.     

Targeting to porcine sialoadhesin receptor improves antigen presentation to T cells

Antibody-mediated targeting of antigen to specific antigen presenting cells (APC) receptors is an attractive strategy to enhance T cell immune responses to weak immunogenic antigens. Here, we describe the characterization of two monoclonal antibodies (mAb) against different epitopes of porcine sialoadhesin (Sn) and evaluate in vitro the potential of targeting this receptor for delivery of antigens to APC for T cell stimulation. The specificity of these mAb was determined by amino acid sequence analysis of peptides derived from the affinity purified antigen. Porcine Sn is expressed by macrophages present in the border between white and red pulp of the spleen and in the subcapsular sinus of lymph nodes, an appropriate location for trapping blood and lymph-borne antigens. It is also expressed by alveolar macrophages and monocyte-derived dendritic cells (MoDC). Blood monocytes are negative for this molecule, but its expression can be induced by treatment with IFN-a. MAb bound to Sn is rapidly endocytosed. MAb to sialoadhesin induced in vitro T cell proliferation at concentrations 100-fold lower than the non-targeting control mAb when using T lymphocytes from pigs immunized with mouse immunoglobulins as responder cells and IFN-a treated monocytes or MoDC as APC, suggesting a role of sialoadhesin in antigen uptake and/or delivery into the presentation pathway in APC.     

Expression of recombinant Toxoplasma gondii P24

The gene encoding Toxoplasma gondii P24 has been reported previously. To determine the function of P24 against immune systems in the near future, we prepared recombinant P24 antigens using Escherichia coli, insect cells infected with recombinant baculovirus and mammalian cells infected with recombinant vaccinia virus. The P24 antigens derived from E. coli, insect cells and mammalian cells were detected with mouse immune sera against P24 or T. gondii homogenates by Western blot analysis; these corresponded to the authentic P24 and secreted into the supernatants of the insect and mammalian cell cultures. These proteins were not effected by tunicamycin treatment in cultured cells, indicating that recombinant P24 did not contain N-linked sugars. Recombinant P24 was separated by two-dimensional electrophoresis and analyzed by Western blotting. From these results, P24 was acidic protein and had identical isoelectric point with the authentic P24.     

Study of peripheral blood cell populations involved in the immune response of goats naturally infected with Mycobacterium caprae

Tuberculosis in goats caused by Mycobacterium bovis and Mycobacterium caprae has noteworthy sanitary and economic implications. Current diagnostic assays are based on cellular immunity and although they have demonstrated a high sensitivity, some animals remain undetected. In the present study, flow cytometry has been used to determine changes in CD4+, CD8+ and CD25+ T cell populations in peripheral blood from naturally infected goats. Proportion of lymphocytes producing PPD-specific interferon-gamma (IFN-γ) was calculated and an ELISA for detection of PPD-specific IFN-γ was performed to measure the cytokine in plasma. The infected goats showed percentages of CD4+ T cells between 27.31% and 47.23% and there were not significant differences (p=0.113) with the non-infected control goats although the mean percentage was lower in this group. Regarding CD8+ T cells, a higher percentage was observed in healthy goats compared to controls (p=0.081). The mean percentage of lymphocytes expressing CD25 without antigen stimulation (30.65±3.91) was higher in lesion and/or culture-positive animals than in the controls (21.84±1.21; p=0.053). The percentage of CD4+/IFN+ T cell population stimulated with bovine PPD was a reliable marker of infection, since the mean percentage in the infected goats was significantly higher than in the controls (p<0.05). Tuberculosis in goats caused by M. caprae induced changes in cellular populations similar to those described for M. bovis in cattle.     

Mitogen and antigen-specific induction of lymphoblast transformation in cats with subclinical toxoplasmosis.

Lymphoblast transformation in whole blood was assessed by 3H-thymidine incorporation after stimulation by concanavalin A and Toxoplasma gondii secretory and intracellular antigens in samples from cats with experimentally induced toxoplasmosis. Toxoplasma gondii-specific immunoglobulin M, immunoglobulin G, and circulating antigens were also measured throughout the study period. Lymphocytes from all cats were responsive to concanavalin A pre- and post-inoculation with T. gondii. Suppression of mitogen-stimulated lymphoblast transformation during the course of infection was not observed. Both the secretory and intracellular antigens stimulated lymphoblast transformation in many cats from Week 2 to Week 52 post-inoculation. Lymphoblast transformation was stimulated more frequently by intracellular antigens (66.25%) than by secretory antigens (48.75%). Lymphoblast transformation was not induced by T. gondii antigens in any cat prior to appearance of T. gondii-specific antibodies in serum or during the oocyst shedding period. Cats with persistent antigenemia had the most consistent lymphoblast transformation results induced by T. gondii-specific antigens.