Characterization of immunostimulatory components of orf virus (parapoxvirus ovis).

Inactivated orf virus (ORFV, parapoxvirus ovis) induces antiviral activity in animal models of acute and chronic viral infections and exerts strong effects on human immune cells. ORFV activates antigen presenting cells (APC) via CD14 and, probably, Toll-like receptor signalling, and triggers the release of IFN-γ that has been identified as the key mediator of the antiviral activity. After delineating virus proteins as being the most likely active constituent, we aimed to characterize the ORFV proteins responsible for the therapeutic effect. By using a vaccinia virus/ORFV expression library we identified several multi-gene DNA fragments with strong immunomodulatory activity. Together these fragments contain 27 ORFs. The encoded proteins are related to virion structure and transcription but are otherwise unrelated. Two proteins were separately expressed and purified, and demonstrated immunostimulatory activity. Gene expression profiles induced by ORFV and the identified fragments were investigated by microarray analysis. Interestingly, all active fragments induced a similar gene-expression pattern, differing only in quantitative aspects. Obviously, several proteins of ORFV activate similar cellular pathways, modulating APC to generate a strong T-helper 1-dominated immune response. This was balanced by additional induction of immune dampening mechanisms, suggesting regulatory differences compared to single cytokine therapies. We conclude that ORFV may have the potential to enrich the armamentarium of antiviral therapies.

Longitudinal prevalence study of diarrheagenic Escherichia coli in dairy calves.

A longitudinal study (cohort study) elaborating 1,224 rectal swabs from 221 calves aging between 1 and 12 weeks was conducted on 11 dairy farms (i) to ascertain associations between diarrhea and shedding of diarrheagenic E. coli and (ii) to facilitate the zoonotic potential assessment of E. coli strains shed by young calves. Calves were screened weekly by PCR of swab cultures for shedding of enterotoxigenic E coli [ETEC; by detection of heat stable (est) and heat labile enterotoxin genes (elt)], diffusely adhering E. coli [DAEC; diffuse adhesion (daa)], typical enteropathogenic E. coli [EPEC; bundle-forming pili (bfpA) and intimin (eae)] as well as enterohemorrhagic E. coli [EHEC, intimin (eae) and Shiga toxin (stx)]. In addition, EHEC-hemolysin- (Hly(EHEC)) and alpha-hemolysin- (alpha-Hly) producing E. coli were detected by inoculation of blood agar plates. Within the 221 calves, prevalences were 69.7% (25.2% of the 1,224 samples) for Hly(EHEC)-producing E. coli, 55.3% (19.3%) for eae, and 18.2% (4.5%) for stx. E. coli strains exhibiting an alpha-Hly phenotype were detected in 66.5% of the calves and 21.9% of fecal samples. The est gene was detectable in 31.7% of the calves from only 9 of 11 herds and in 7.8% of the samples. Calves shedding DAEC or typical EPEC were not identified. The detection frequency of virulence traits significantly depended on the calves' age and shedding dynamics differed between the traits. A significant correlation between calf diarrhea and shedding of EHEC virulence traits was determined for several postnatal periods (1 week: Hly(EHEC); 1st & 10th week: eae; 4th week stx). Shedding of ETEC (est) was associated with diarrhea in newborn calves (1st week) only. Hly(EHEC)- and alpha-Hly-producing E. coli were shed significantly more frequently by diarrheic calves in 1st and 8th week of life, respectively. The knowledge gained in this study highlights the high prevalence of zoonotic E. coli already in calves. The age-dependent shedding dynamic of the various E. coli pathovars has to be considered regarding prophylaxis as well as planning intervention studies, both for calves and humans.

Immunization with an alphatoxin variant 121A/91-R212H protects mice against Clostridium perfringens alphatoxin.

As shown previously, a recombinant alphatoxin variant (rAT121A/91) constructed from the naturally occurring Clostridium perfringens mutant strain 121A/91, was devoid of enzymatic (PLC), hemolytic and lethal activity (18). In the present study, the recombinant variant was altered by an oligonucleotide-directed reversion of an arginine in position 212 for a histidine residue, corresponding to the sequence of the wild-type alphatoxin. The new variant rAT121A/91R212H proved to be negative in enzymatic, hemolytic and lethal activity as well. RAT121A/91 as well as rAT121A/91R212H was used for i.p. immunization of balb/c mice. The immune response was studied in ELISA as well as in the mouse neutralization test. Furthermore, immunized mice were challenged by i.p. application of active C. perfringens alphatoxin. In all immunized groups, mice developed high anti-alphatoxin titers (up to 1:128000). Antisera of both groups were able to reduce the hemolytic effect of native alphatoxin with predominance of anti-rAT121A/91R212H sera. During neutralization experiments, mice receiving a mixture of anti-rAT121A/91R212H and wild-type toxin were protected completely, whereas an anti-rAT121A/91/toxin mixture prolonged time until death but failed in protection. I.p immunization with rAT121A/91R212H yielded a significant protection rate (76%) when mice were challenged intraperitoneal with wild-type toxin. Our cumulative data indicates that the reversion of arginine in position 212 to histidine for rAT121A/91R212H was necessary to induce production of protective antibodies against wild-type alphatoxin of C. perfringens.

DNA-loaded bacterial ghosts efficiently mediate reporter gene transfer and expression in macrophages.

There is a demand for efficient and safe DNA delivery vehicles mediating gene transfer and expression. We present bacterial ghosts as a novel platform technology for DNA delivery and targeting of macrophages. Bacterial ghosts are cell envelopes of gram-negative bacteria that are devoid of the cytoplasmic content. Escherichia coli ghosts were loaded with plasmid DNA and linear double-stranded DNA. Confocal laser scanning microscopy and flow cytometry confirmed that the DNA localized to the inner lumen of bacterial ghosts and was not associated with the outer surface of the bacteria. Up to approximately 6000 plasmids could be loaded per single ghost and the amount of loaded DNA correlated with the DNA concentration used for loading. E. coli ghosts loaded with plasmids encoding the enhanced green fluorescent protein (EGFP) targeted efficiently murine macrophages (RAW264.7) and mediated effective gene transfer. The EGFP was expressed by more than 60% of the macrophages as measured by flow cytometry detecting the green fluorescence and immunocytochemical staining with antibodies specific for EGFP.

Reinfection with Chlamydophila abortus by uterine and indirect cohort routes reduces fertility in cattle preexposed to Chlamydophila.

This study investigated the effects of controlled reinfection on fertility of cattle naturally preexposed to Chlamydophila abortus. All animals had high prechallenge levels of immunoglobulin M (IgM), IgG, IgG1, and IgG2 serum antibodies against ruminant C. abortus in a chemiluminescent enzyme-linked immunosorbent assay. Twenty virgin heifers were estrus synchronized with prostaglandin F2, artificially inseminated 2 to 3 days later, and challenged immediately by intrauterine administration of 0, 10(4), 10(5), 10(6), or 10(8) inclusion-forming units (IFU) of C. abortus. Ten heifers were estrus synchronized, inseminated, and uterine challenged 2 weeks later. These animals were also indirectly exposed to C. abortus infection (cohort challenged) by contact with their previously challenged cohorts. Pregnancy was determined by rectal palpation 42 days after insemination. All anti-C. abortus antibody isotypes increased in heifers following uterine challenge with 10(8) IFU. A total of 11, 83, 50, 66, and 0% of heifers were pregnant after uterine challenge with 0, 10(4), 10(5), 10(6), and 10(8) IFU of C. abortus, respectively. A total of 50 and 65% of heifers were pregnant with and without cohort challenge, respectively. Uterine inoculum dose and cohort challenge (or, alternatively, a negative pregnancy outcome [infertility]) correlated highly significantly with a rise in postchallenge anti-C. abortus IgM levels over prechallenge levels. Logistic regression modeled fertility, with uterine challenge dose and cohort challenge or prechallenge IgM as predictors (P < 0.05). The models predict that the uterine C. abortus inoculum causing infertility is 8.5-fold higher for heifers without cohort exposure and 17-fold higher for heifers with high IgM levels than for heifers with cohort exposure or with low IgM levels.

Induction of an antigen-specific immune response and partial protection of cattle against challenge infection with foot-and-mouth disease virus (FMDV) after lipopeptide vaccination with FMDV-specific B-cell epitopes.

To evaluate the potential of chemically synthesized lipopeptides for vaccination against foot-and-mouth disease (FMD), seven lipopeptides containing the immunostimulating principle of bacterial lipoproteins and linear B-cell epitopes of FMDV strain O(1)Kaufbeuren (O(1)K) were used to immunize cattle (n=7). Animals were vaccinated once and 21 days after immunization animals were infected with the homologous virus. Four animals were protected. After vaccination, as well as after challenge infection, B- and T-cell responses were examined. Sera were tested for virus- and peptide-specific antibodies and showed after vaccination only a weak antibody response. After challenge infection, an increase in antibody titre was obvious but there was no correlation between antibody titre and protection. The reactivity of the cellular immune system was detected by analyses of PBMCs for virus- and peptide-specific T-lymphocytes. With regard to the virus-specific T-lymphocytes, a heterogeneous reactivity could be shown. No correlation between virus-specific T-cell proliferation and protection was found. Obvious was the fact that all protected animals showed after vaccination a strong T-cell response against at least one of the peptides used for immunization. These results suggest a correlation between the onset of an antigen-specific T-cell reaction and protection.

Identification of foot-and-mouth disease virus-specific linear B-cell epitopes to differentiate between infected and vaccinated cattle.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. For several years, vaccination of animals, which had proven to be successful for the eradication of the disease, has been forbidden in the United States and the European Community because of the difficulty of differentiating between vaccinated and infected animals. In this study, detailed investigations of the bovine humoral immune response against FMD virus (FMDV) were performed with the aim of identifying viral epitopes recognized specifically by sera derived from FMDV-infected animals. The use of overlapping 15-mer synthetic peptides, covering the whole open reading frame of FMDV strain O(1)K in a peptide enzyme-linked immunosorbent assay, allowed the identification of 12 FMDV strain O(1)K-specific linear B-cell epitopes. Six of these linear B-cell epitopes, located in the nonstructural proteins, were used in further assays to compare the reactivities of sera from vaccinated and infected cattle. Antibodies recognizing these peptides could be detected only in sera derived from infected cattle. In further experiments, the reactivity of the six peptides with sera from animals infected with different strains of FMDV was tested, and strain-independent infection-specific epitopes were identified. Thus, these results clearly demonstrate the ability of a simple peptide-based assay to discriminate between infected and conventionally FMD-vaccinated animals.

Inactivated parapoxvirus ovis (Orf virus) has antiviral activity against hepatitis B virus and herpes simplex virus.

It is known that some viruses are able to induce vigorous immune reactions. This study shows that inactivated parapoxvirus ovis (Orf virus), strain D1701 (PPVO), induces an autoregulatory cytokine response that involves the upregulation of IL-12, IL-18, IFN-gamma and other T helper 1-type cytokines and their subsequent downregulation, which is accompanied by induction of IL-4. An increase in IL-10 expression was also found in the livers of PPVO-treated mice. PPVO protects mice from lethal herpes simplex virus type 1 infection and guinea pigs from recurrent genital herpes disease. With dosages as low as 500 000 virus particles, PPVO is more potent than the current standard 3TC therapy in hepatitis B virus transgenic mice. No signs of inflammation or any other side effects were observed. PPVO induces IL-12, TNF-alpha and, together with a suboptimal concentration of Concanavalin A, IFN-gamma in human peripheral blood leukocytes as well. The principle of an autoregulatory cytokine induction by an inactivated virus might have advantages over existing immune therapies and it is concluded that inactivated PPVO should be investigated further for its potential use in antiviral therapy.

Quantitative detection of Chlamydia psittaci and C. pecorum by high-sensitivity real-time PCR reveals high prevalence of vaginal infection in cattle.

Bovine vaginal cytobrush specimens were analyzed for the presence of Chlamydia spp. by a high-sensitivity, high-specificity quantitative PCR. The 53% prevalence of low-level Chlamydia psittaci and C. pecorum genital infection detected in virgin heifers suggests predominantely extragenital transmission of Chlamydia in cattle and conforms to the high seroprevalence of anti-Chlamydia antibodies.

The quantity of nitric oxide released by macrophages regulates Chlamydia-induced disease.

Intracellular bacteria of the genus Chlamydia cause numerous typically chronic diseases, frequently with debilitating sequelae. Genetic determinants of disease susceptibility after infection with Chlamydia bacteria are unknown. C57BL/6 mice develop severe pneumonia and poor immunity against Chlamydia after moderate respiratory infection whereas BALB/c mice are protected from disease and develop vigorous Th1 immunity. Here we show that infected C57BL/6 macrophages release more NO synthesized by NO synthase 2 (NOS2) than BALB/c macrophages and have lower mRNA concentrations of arginase II, a competitor of NOS2 for the common substrate, l-arginine. Reduction, but not elimination, of NO production by incomplete inhibition of NOS2 abolishes susceptibility of C57BL/6 mice to Chlamydia-induced disease. Thus, the quantity of NO released by infected macrophages is the effector mechanism that regulates between pathogenic and protective responses to chlamydial infection, and genes controlling NO production determine susceptibility to chlamydial disease.