Leishmania mexicana and Leishmania tropica: Cross immunity in C57BL/6 mice

Leishmania mexicana and Leishmania tropica infection were comparatively studied in C57BL/6 mice. Infection with 10⁴ amastigotes of L. mexicana was followed by the appearance of a single lesion which ulcerated in 8 weeks and healed in 24 weeks. Mice infected with 10⁴ amastigotes of L. tropica developed less severe lesions which healed in 18 weeks. In both cases healing was accompanied by a delayed hypersensitivity response and an in vitro lymphocyte reactivity to leishmanial antigens. Mice recovered from a primary infection with L. mexicana or L. tropica were resistant to both homologous and heterologous challenge. In vitro and in vivo immunological tests indicated that L. mexicana and L. tropica share antigenic determinants which are involved in cell-mediated immune responses to these parasites.     

Immunoglobulin isotype distribution of malaria-specific antibodies produced during infection with Plasmodium chabaudi adami and Plasmodium yoelii

The antibody response of mice to Plasmodium chabaudi adami and Plasmodium yoelii has been compared using a solid phase isotype-specific radioimmunoassay and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Serological cross-reactivity between these parasites was substantial. Studies using a radioimmunoassay detecting all classes of malaria-specific antibody demonstrated that during the early part of infection it was not possible to distinguish between homologous and heterologous reactions. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 50% or more of the protein antigens detected were apparently shared by both parasites although the intensity of bands was always greater with homologous reactions. However, the distribution of isotypes in the antibody (Ab) response differed in the two infections. P. chabaudi infections were characterized by a predominant and persistent IgM response, moderate IgG₂ and IgG₃ and little significant IgG₁ response during a primary infection. By contrast, IgM antibodies were transient in P. yoelii infection, IgG₂ was the predominant isotype, and both IgG₁ and IgG₃ antibodies were present during a primary infection. These differences in isotypes were also detected when sera were tested on the heterologous antigen extracts suggesting that antigens shared by P. chabaudi and P. yoelii do not necessarily induce similar antibody responses in the two infections.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

Immunology of histoplasmosis: Humoral and cellular activity from a polysacchari-de-protein complex and its deproteinized fraction in experimentally immunized mice

In a previous publication it was reported that a polysaccharide-protein complex (PPC), sensitive to -glucosidase, was isolated from Histoplasma capsulatum. This complex was strongly reactive in an agar gel diffusion assay with sera from patients with histoplasmosis, but was unreactive with sera from patients with coccidioidomycosis. Here, the studies with human sera have been expanded and attempts were made to determine the response of mice immunized with nonviable H. capsulatum or Cocccidioides immitis to PPC or its deproteinized fraction (D-PPC) using more sensitive tests for antibody and including also test for cell-mediated immunity. Histoplasmin and coccidioidin were compared with PPC or its deproteinized fraction (D-PPC) in all assays. In a counterimmunoelectrophoresis (CIE) assay, PPC and D-PPC reacted only with sera from patients with histoplasmosis, whereas cross reactions were noted with histoplasmin and coccidioidin using heterologous sera. Cross-reaction were observed with all four antigen preparations and both types of antisera using a micro complement fixation assay. The assay for macrophage migration inhibitory factor (MIF) was also relatively nonspecific, in that inhibition occurred with cells from animals sensitized with Histoplasma or Coccidioides using both homologous and heterologous antigens. In the footpad assay, histoplasmin and coccidioidin were highly cross-reactive in animals sensitized with the heterologous fungus, but the PPC and D-PPC from H. capsulatum elicited significant reactions only in animals sensitized with Histoplasma.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

Contact sensitivity in rabbits sensitized with dinitrophenylated Mycobacterium tuberculosis

The conjugation of Mycobacterium tuberculosis with DNFB results in the formation of a haptenated preparation that induces the formation of contact sensitivity when administered subcutaneously. This contact sensitivity can be measured in vivo by topical application of the free chemical and in vitro by lymphocyte transformation. The antigens suitable for the in vitro detection are those preparations obtained by the haptenation of cell membranes. Haptenation of serum proteins, of homologous and heterologous origin, does not produce antigens suitable for in vitro assay. The antigen requirements for the in vitro transformation assay of contact sensitivity are similar for adjuvant induced sensitivity as well as for free chemical induced sensitivity.     

Comparative Study of 14C-Labeled Purified Protein Derivative from Various Mycobacteria: II. Skin Cross-Reactivity in Sensitized Guinea Pigs

A study on skin cross-reactivity between stabilized ¹⁴C-labeled mycobacterial antigens, namely tuberculin purified protein derivative (PPD; from Mycobacterium tuberculosis), PPD-A (M. avium), PPD-Y (M. kansasii), PPD-G (M. scrofulaceum), PPD-B (M. intracellulare), and PPD-F (M. fortuitum), has been carried out in groups of guinea pigs sensitized with one of the following heat-killed mycobacteria: M. tuberculosis, M. avium, M. kansasii, M. scrofulaceum, M. intracellulare, or M. fortuitum. For each type of sensitization, the average response for the corresponding PPD antigen was higher than the average response for any of the other antigens. However, the responses to the heterologous PPD antigens were not necessarily significantly different among themselves, and the significant differences of the heterologous PPD antigens were distributed differently according to the type of sensitization. Therefore, ¹⁴C-PPD antigens skin cross-reacted in guinea pigs essentially in the same manner as reported by others for nonradioactive PPD antigens.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

Production and properties of reaginic antibodies in rabbits infected with Clonorchis sinensis or Schistosoma japonicum

The production of reaginic antibodies detected by homologous passive cutaneous anaphylaxis (PCA) was demonstrated in all rabbits experimentally infected with either Clonorchis sinensis or Schistosoma japonicum. The antibodies appeared in the sera as early as 3 weeks after exposure and persisted with relatively high titers for at least 7 weeks in some animals. The antisera of rabbits infected with C. sinensis were found to be cross reactive against heterologous trematode antigens, although PCA titers were less than 3% of the titer by the homologous antigen; no cross reaction was observed between S. japonicum antiserum and the heterologous antigens. PCA activity of the antisera was completely destroyed in some samples by heat treatment at 56 C for 2 hr, but partially in the others even after heating for 6 hr. However, the physicochemical properties of these antibodies were analogous to human IgE; the PCA activity was eluted with 0.035 M phosphate buffer from a DEAE-cellulose column and recovered in the ascending portion of the IgG peak by Sephadex G-200 gel filtration. PCA activity was found in a β region in preparative agar electrophoresis.     

Gestation and breastfeeding in schistosomotic mothers differently modulate the immune response of adult offspring to postnatal Schistosoma mansoni infection

Schistosoma mansoni antigens in the early life alter homologous and heterologous immunity during postnatal infections. We evaluate the immunity to parasite antigens and ovalbumin (OA) in adult mice born/suckled by schistosomotic mothers. Newborns were divided into: born (BIM), suckled (SIM) or born/suckled (BSIM) in schistosomotic mothers, and animals from noninfected mothers (control). When adults, the mice were infected and compared the hepatic granuloma size and cellularity. Some animals were OA + adjuvant immunised. We evaluated hypersensitivity reactions (HR), antibodies levels (IgG1/IgG2a) anti-soluble egg antigen and anti-soluble worm antigen preparation, and anti-OA, cytokine production, and CD4⁺FoxP3⁺T-cells by splenocytes. Compared to control group, BIM mice showed a greater quantity of granulomas and collagen deposition, whereas SIM and BSIM presented smaller granulomas. BSIM group exhibited the lowest levels of anti-parasite antibodies. For anti-OA immunity, immediate HR was suppressed in all groups, with greater intensity in SIM mice accompanied of the remarkable level of basal CD4⁺FoxP3⁺T-cells. BIM and SIM groups produced less interleukin (IL)-4 and interferon (IFN)-g. In BSIM, there was higher production of IL-10 and IFN-g, but lower levels of IL-4 and CD4⁺FoxP3⁺T-cells. Thus, pregnancy in schistosomotic mothers intensified hepatic fibrosis, whereas breastfeeding diminished granulomas in descendants. Separately, pregnancy and breastfeeding could suppress heterologous immunity; however, when combined, the responses could be partially restored in infected descendants.     

Transfection of Eimeria and Toxoplasma using heterologous regulatory sequences

Eimeriatenella and Toxoplasmagondii are Apicomplexan protozoa and share many similarities in biology and genomics. While the latter parasites are easily cultured in vitro and genetically manipulated, many Eimeria species are difficult to grow in vitro. We hypothesised that molecular tools for the genetic manipulation of T. gondii could be applied to the study of Eimeria parasites. Here we show that three different promoter sequences originating from E. tenella could function effectively not only in other species of the Eimeria genus (histone H4) but also in T. gondii (histone H4, actin and tubulin). Similarly, promoters of the “housekeeping” gene (tubulin) and differentially regulated gene (surface antigen gene, sag1) of T. gondii were effective in driving the expression of the yellow fluorescent protein (YFP) maker gene in E. tenella. The transfection efficiency with heterologous regulatory sequences was similar to that with homologous promoters; while the promoter strength of heterologous vectors is slightly weaker than the homologous vectors in both E. tenella and T. gondii. The results suggest that 5′ regulatory sequences are functionally conserved not only among the Eimeria species, but also between T. gondii and E. tenella, and that T. gondii could be used as a novel transfection check system for Eimeria-rooted vectors, accelerating the development of reverse genetics in Eimeria spp.     

Trypanosoma cruzi: immunosuppressed response to different antigens in the infected mouse.

Trypanosoma cruzi: Immunosuppressed response to different antigens in the infected mouse. Experimental Parasitology45, 190–199. Trypanosoma cruzi infection in mice results in functional changes in the normal immunological responses to heterologous antigens. An immunosuppression of the 19 and 7S antibody response is observed in infected animals against both a particulate antigen and against soluble antigens. Furthermore, the immune response to the soluble T-independent antigens, DNP-Ficoll and LPS, was also similarly impaired when antigen was administered to trypanosome-infected animals. The suppression of the immune response to these antigens does not seem to involve an alteration in the macrophage, as evidenced by a normal uptake and handling of soluble ¹³¹I-labeled HSA and by a normal immune response when antigen-exposed peritoneal macrophages from trypanosome-infected mice were transferred to normal mice. These data support the concept that T. cruzi induces an immunosuppression to both T-dependent and T-independent antigens and that the depression observed is not due to an alteration in macrophage function.     

A PHYSICAL-CHEMICAL DIFFERENCE IN ANTIBODIES AGAINST THE S AND R VARIANTS OF A SINGLE BACTERIAL STRAIN

1. Rabbits were immunized with Bact. typhosum 0 901 S and 0 901 R, over a long period. Homologous and heterologous strains were sensitized with sera obtained from weekly bleedings. Agglutination titer was recorded, and the isoelectric points of the bacteria maximally sensitized were determined. 2. 0 901 S maximally sensitized with homologous immune serum had isoelectric points which became more alkaline as immunization progressed, covering a range of pH 4.8 to 5.5. 3. Strain 0 901 R maximally sensitized with homologous immune serum had isoelectric points which became more alkaline as immunization progressed, covering the range of pH 5.0 to 5.9. 4. Both 0 901 S and 0 901 R maximally sensitized with heterologous serum had isoelectric points lower than when sensitized with homologous serum. 5. The isoelectric points of both forms sensitized with increasing concentrations of homologous immune serum were determined. Increasing concentrations of homologous immune serum shifted the isoelectric point of 0 901 R from less than 2.2 for the unsensitized bacteria progressively to the alkaline side until the maximum values previously mentioned were reached. Increasing concentrations of homologous immune serum conferred upon 0 901 S isoelectric points which became only slightly more alkaline in maximal sensitization. 6. The electrophoretic mobilities of 0 901 S and 0 901 R, in each case maximally sensitized with homologous hyperimmune serum, were found to differ significantly over the whole range of pH studied.     

In vitro studies of cell-mediated immunity in rabbits sensitized withMycobacterium kansasii andMycobacterium tuberculosis

Rabbits, sensitized withM. kansasii, responded by a profound inhibition of migration of macrophages elicited by both antigens: the migration index for homologous antigen was 0.51 in the direct test and 0.65 in the indirect test; for heterologous antigen the indexes were 0.53 and 0.67. However, significant differences in reactivity were found in rabbits sensitized withM. tuberculosis. In the homologous system, high reactivity was maintained and the migration index reached the value of 0.53 in the direct and 0.63 in the indirect test. On the other hand, the heterologous antigenM. kansasii influenced the migration in both direct and indirect assays significantly less, the migration indexes being 0.62 and 0.72. The differences were statistically significant at 1 % and 5 % levels.     

Studies on the specific immunodiagnosis of larval cestode infections of cattle and sheep using antigens purified by affinity chromatography in an enzyme-linked immunosorbent assay (Elisa)

Craig P.S. and Rickard M.D. 1981. Studies on the specific immunodiagnosis of larval cestode infections of cattle and sheep using antigens purified by affinity chromatography in an enzyme-linked immunosorbent assay (ELISA). International Journal for Parasitology11: 441–449. Crude somatic or cyst fluid extracts prepared from Taenia saginata, T. hydatigena or Echinococcus granulosus were partially purified by absorption against homologous and heterologous bovine or ovine antisera on immunoabsorbent affinity columns. Antigens in parasite extracts which were eluted after binding to the homologous anti-parasite antisera (bovine or ovine) coupled to CNBr-activated Sepharose were then passed sequentially through affinity columns containing heterologous anti-parasite Ig and the ‘run-through’ antigens collected. The level of cross reactions to these absorbed antigens, in an enzyme-linked immunosorbent assay (ELISA) using sera from cattle or sheep given heterologous parasite infections (including Fasciola hepatica), were significantly decreased. Absolute specificity was not achieved, and some loss in sensitivity occurred. The absorption of cross-reactive antigen(s) using affinity Chromatographie techniques may be a useful first step in the production of species-specific immunodiagnostic antigens for larval cestode infections.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.     

Serological investigations of cell adhesion in the slime molds,Dictyostelium discoideum,Dictyostelium purpureum,and Polysphondylium violaceum

1. Antibodies to slime molds were produced by injecting D. discoideum and D. purpureum amebas from 48 hour cultures into rabbits. 2. Anti-D. discoideum and anti-D. purpureum sera caused agglutination of homologous amebas from 24 to 26 hour cultures, agglutination of certain heterologous amebas from 30 to 36 hour cultures, and agglutination of all heterologous amebas from 43 to 48 hour cultures. 3. The data show that new surface antigens are formed in cultures after 26 hours and it is suggested that the new antigens are concerned with cell adhesion. 4. The probable role of surface antigens in the interaction of cells of different species of slime molds was discussed.     

Resistance of dogs to Echinococcus granulosus

Further studies on the immunization of dogs against E. granulosus indicated that certain dogs have a natural resistance, which is not mediated by specific antibodies or sensitized lymphocytes to tapeworm secretory antigens. In a pilot experiment with two pups, E. granulosus recovered from an immunized pup were arrested in their development and exhibited 100% inhibition of egg production compared to 100% egg production in mature worms recovered from a control pup. When the experiment was repeated with 10 pups, three of five control pups given Freud's adjuvant and Bordetella pertussis carried E. granulosus which were arrested in their development and showed 100% inhibition of egg production. Two of five immunized pups also exhibited signs of resistance. Arrested worms recovered 40 days after infection appeared to be at a stage equivalent to about 18 days of optimal development. Although immunized pups exhibiting resistance showed strong positive in vitro and in vivo immune responses, all control pups gave negative reactions to tapeworm secretory antigens.     

Ascaris suum: Immunosuppression in mice during acute infection

Mice inoculated by stomach intubation with 10,000 embryonated Ascaris suum eggs, 4, 11, or 21 days before an intraperitoneal (ip) immunization with 2 × 10⁸ sheep erythrocytes (SRBC) had reduced numbers of direct (IgM) splenic hemolytic plaques measured at 4 days after immunization and only a marginal reduction in indirect plaques (IgG) measured at 9 days after immunization. Lower dosages of Ascaris eggs or simultaneous inoculation of Ascaris eggs and SRBC did not suppress antibody responses to SRBC. No reduction in a secondary antibody response to SRBC injected 4 days after Ascaris inoculation was observed. IgM and IgG hemagglutinin titers, as distinguished by 2-mercaptoethanol sensitivity, were suppressed in mice injected ip with 10⁸ SRBC 10 days following inoculation of 10,000 Ascaris eggs, but titers in both Ig classes were similar in infected and control mice injected with 2 × 10⁹ SRBC. At Day 20, antibody titers following ip injection of 1.0 or 100 μg of ovalbumin in alum were reduced in mice infected with 10,000 Ascaris eggs 4 days before antigen injection.Contact hypersensitivity to oxazalone was not altered in mice sensitized at 5 or 14 days after inoculation of 10,000 Ascaris eggs. The delayed hypersensitivity response, measured by footpad swelling, to an optimum intravenous sensitizing dosage of SRBC was inhibited in mice sensitized 10 days after Ascaris infection, but not inhibited in mice sensitized at 21 or 32 days after infection. In contrast, the delayed hypersensitivity response to subcutaneous sensitization with SRBC 10 days after Ascaris infection was not altered.     

Ns1 Is a Key Protein in the Vaccine Composition to Protect Ifnar(?/?) Mice against Infection with Multiple Serotypes of African Horse Sickness Virus

African horse sickness virus (AHSV) belongs to the genus Orbivirus. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2 and NS1 proteins from AHSV-4. IFNAR^(−/−) mice inoculated with DNA/rMVA-VP2,-NS1 from AHSV-4 in an heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies specific of AHSV-4. In addition, vaccination stimulated specific T cell responses against the virus. The vaccine elicited partial protection against an homologous AHSV-4 infection and induced cross-protection against the heterologous AHSV-9. Similarly, IFNAR^(−/−) mice vaccinated with an homologous prime-boost strategy with rMVA-VP2-NS1 from AHSV-4 developed neutralizing antibodies and protective immunity against AHSV-4. Furthermore, the levels of immunity were very high since none of vaccinated animals presented viraemia when they were challenged against the homologous AHSV-4 and very low levels when they were challenged against the heterologous virus AHSV-9. These data suggest that the immunization with rMVA/rMVA was more efficient in protection against a virulent challenge with AHSV-4 and both strategies, DNA/rMVA and rMVA/rMVA, protected against the infection with AHSV-9. The inclusion of the protein NS1 in the vaccine formulations targeting AHSV generates promising multiserotype vaccines.