Flow Cytometric Analysis of Cellular Changes in Mice after Intradermal Inoculation with a Liposome–Iscom Adjuvanted Vaccine

As it is not known what changes to leucocyte homeostasis are mandatory for effective adjuvant action, the biological relevance of systemic changes elicited by different vaccine formulations can only be interpreted in the context of the immunological outcomes. We used flow cytometry to quantify the changes in leucocyte subsets induced in mice intradermally immunized with SAMA4 (adjuvant group), outer membrane proteins (OMP) purified from Actinobacillus pleuropneumoniae (OMP antigen group), SAMA4 adjuvanted OMP (OMP vaccine group), or phosphate-buffered saline (PBS: control group). This approach allowed direct comparisons to be made between the effects of antigen, adjuvant or antigen–adjuvant complexes on immune effector cell populations. Antigens complexed with the liposome–iscom hybrid adjuvant, SAMA4, generated strong antibody responses and cytotoxic T-cell activity in animals immunized intradermally, reflecting remobilization and recruitment of specific cell populations. Splenomegaly, due to granulocytosis, monocytosis and megakaryocytosis, was most prominent in the OMP vaccine group. Histological examination of spleen sections confirmed that these changes were due primarily to splenic haematopoiesis. Circulating numbers of granulocytes and monocytes increased significantly (P < 0.05) in the blood of the OMP vaccine group, as did granulocyte numbers in the lungs (P < 0.05). No changes in T- and B-cell numbers were detected by flow cytometry in the spleens, lungs or blood over the 28-day period in any treatment group. Thymocyte numbers (predominantly CD4⁺CD8⁺ cells) in the OMP vaccine group fell by 95% within 3 days of immunization. Identical cellular responses were obtained when an innocuous antigen, ovalbumin, was complexed with SAMA4 instead of OMP, thus demonstrating that the adjuvant effects of SAMA4 were due to synergistic interaction between antigen and adjuvant and not due to the presence of toxic components. The association of strong adaptive immune responses with such complex changes in leucocyte homeostasis induced by complexing adjuvant and antigen suggested that the changes were important for effective vaccination and were not purely circumstantial.     

Oral immunization of mice with attenuated Salmonella typhimurium aroA expressing a recombinant Mycoplasma hyopneumoniae antigen (NrdF).

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia, a commercially expensive respiratory disease of swine. Salmonella typhimurium SL3261 was used as a live carrier of plasmid pKF1, which encodes a 15-kDa recombinant M. hyopneumoniae protein. This expressed recombinant protein consists of the carboxy-terminal 11 kDa of a 42-kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein. Rabbit anti-15-kDa serum was able to inhibit the growth of viable M. hyopneumoniae J in vitro. When used as a live oral vaccine, S. typhimurium SL3261(pKF1) induced a significant secretory immunoglobulin A immune response in the lungs of mice orally immunized against the M. hyopneumoniae antigen. Utilization of live oral vaccines expressing potentially protective M. hyopneumoniae proteins, such as the NrdF antigen, which can stimulate a lung mucosal response against surface-accessible proteins may provide a cost-effective alternative to the present control strategies used for porcine enzootic pneumonia.     

Molecular characterization of a ribonucleotide reductase (nrdF) gene fragment of Mycoplasma hyopneumoniae and assessment of the recombinant product as an experimental vaccine for enzootic pneumonia.

A Mycoplasma hyopneumoniae clone bank was screened with hyperimmune pig serum. One clone exhibited sequence homology to the prokaryotic R2 subunit of ribonucleotide reductase and was expressed as an 11-kDa protein fused to beta-galactosidase. The vaccine potential of the fusion protein was assessed in pig trials. Following experimental challenge with a virulent isolate of M. hyopneumoniae, gross lung pathology (mean Goodwin lung score) of vaccinated animals, irrespective of adjuvant treatment, was significantly reduced compared with that of control unvaccinated pigs (P < 0.05).     

Oral immunization of swine with attenuated Salmonella typhimurium aroA SL3261 expressing a recombinant antigen of Mycoplasma hyopneumoniae (NrdF) primes the immune system for a NrdF specific secretory IgA response in the lungs

Salmonella typhimurium SL3261 (aroA mutant) expressing a recombinant Mycoplasma hyopneumoniae antigen was used to orally immunize swine against porcine enzootic pneumonia. This construct, designated S. typhimurium aro A SL3261 (pKF1), expressed a recombinant protein containing the carboxy-terminal 11 kDa of a 42 kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein. Here we demonstrate that this antigen is present in all seven geographically diverse strains of M. hyopneumoniae tested, and is recognized by the swine immune system after experimental infection with the virulent M. hyopneumoniae Beaufort strain. The immune response of swine orally immunized twice with S. typhimurium SL3261 (pKF1) on day 0 and day 14 was evaluated. Oral immunization with S. typhimurium SL3261 (pKF1) primed the immune system to elicit a significant (P<0.05) secretory IgA response against the 15 kDa NrdF antigen in the respiratory tract of swine, post-challenge, compared to control groups. Blood lymphocytes from swine immunized with S. typhimurium SL3261 (pKF1) proliferated significantly (P<0.05) following stimulation with M. hyopneumoniae whole-cell extracts compared to control groups 14 days post-vaccination. Following challenge with virulent M. hyopneumoniae, swine immunized with S. typhimurium SL3261 (pKF1) showed higher average daily weight gains and reduced lung pathology compared to control groups.     

Analysis of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum by multilocus enzyme electrophoresis.

The genetic diversity of 74 Australian field isolates of Erysipelothrix rhusiopathiae and 22 reference strains for serovars of E. rhusiopathiae or Erysipelothrix tonsillarum was examined by multilocus enzyme electrophoresis. Four serovar reference strains of E. tonsillarum (strains KS 20 A, Wittling, Lengyel-P, and Bano 107 for serovars 25, 3, 10, and 22, respectively) were genetically distinct from E. rhusiopathiae. However, the E. tonsillarum reference strain for serovar 14 (Iszap-4) and the reference strain for serovar 13 (Pecs-56), which has been said to represent a new genomic species, were found to cluster with typical isolates and reference strains of E. rhusiopathiae. Our reference strain for serovar 7 (Rotzunge) was also genetically typical of E. rhusiopathiae, thus indicating that these serotype reactivities cannot be relied upon as a means of identifying isolates as E. tonsillarum. Australian field isolates of E. rhusiopathiae were genetically diverse. Those recovered from sheep or birds were more diverse than those isolated from pigs, and isolates of serovar 1 were more diverse than those of serovar 2. The diversity found among isolates of the same serovar and the presence of isolates of different serovars in the same electrophoretic types (ETs) indicated that serotyping of E. rhusiopathiae was unreliable for use as an epidemiological tool. Some ETs contained isolates recovered from different animal species. ET 41 contained 32.2% of the field isolates and two reference strains, indicating that this clone of E. rhusiopathiae is both widespread and commonly associated with disease in various species of animals.     

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease.     

Rapid Detection of Mycobacterium paratuberculosis in Clinical Samples from Ruminants and in Spiked Environmental Samples by Modified BACTEC 12B Radiometric Culture and Direct Confirmation by IS900 PCR

The suitability of a radiometric culture medium consisting of BACTEC 12B with PANTA PLUS, mycobactin J, and egg yolk was evaluated for detection of Mycobacterium paratuberculosis in feces, mesenteric lymph nodes, and intestinal walls from cattle, sheep, and goats. In addition, a simple method that would enable the rapid identification of Mycobacterium paratuberculosis by IS900 PCR in the primary cultures was sought so that subculture to secondary egg-free radiometric medium could be avoided. An ethanol extraction followed by differential centrifugation was used to separate M. paratuberculosis from PCR inhibitors in the primary culture. PCR was then undertaken with the pellet, after boiling to lyse the mycobacteria; if this test was negative, the DNA in the lysate was purified with guanidine thiocyanate and silica. Cultures of feces, ilea, and mesenteric lymph nodes from cattle, sheep, and goats known to have or suspected of having Johne’s disease yielded positive PCR results 1 to 7 weeks after inoculation. Similar results were obtained with soil and pasture samples that had been spiked with M. paratuberculosis. The results suggested that radiometric culture was more sensitive than histopathology in detecting M. paratuberculosis infection in sheep and goats and more sensitive than culture on Herrold’s egg yolk medium for the detection of the infection in cattle. Of 259 individual PCR tests with samples from cultures with growth indices of ≥10,237 (91.5%) were positive, with only 28 (11.8%) requiring both ethanol and silica preparation to yield a positive result. Of the 22 negative PCR results for samples from cultures with growth indices of ≥10, 18 were for samples from cultures that had only just developed evidence of growth. PCR-positive cultures tended to remain PCR positive over successive weeks. Flexibility in the timing of the sampling for PCR is thus possible, facilitating batch processing of samples in large-scale disease control programs for ruminants.     

A study of the foodborne pathogens: Campylobacter, Listeria and Yersinia, in faeces from slaughter-age cattle and sheep in Australia.

In a study of faeces from 475 slaughter-age cattle and sheep from 19 herds or flocks, Campylobacter species (C. jejuni and C. coli) were cultured from all production systems studied and from 73.7 per cent (14/19) of herds or flocks. Within individual properties there was a higher prevalence in cattle than in sheep, with Campylobacter being most commonly isolated from feedlot cattle. The median prevalences and ranges were: for dairy cattle, six per cent (0-24%), feedlot beef cattle, 58 per cent (12-92%) pasture beef cattle, two per cent (0-52%), mutton sheep, 0 per cent (0-4%) and prime lambs eight per cent. Listeria ivanovii was cultured from one dairy cow but Yersinia enterocolitica was not cultured from any animal. Campylobacter is the leading bacterial causative agent of acute diarrhoea in humans in many industrialised countries. While the role of cattle and sheep in producing human campylobacteriosis either directly or via contaminated food, remains to be epidemiologically clarified, this study suggests that the production system, particularly for cattle, may be an important consideration.     

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel^® or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n = 10 per group) or Suvaxyn^® M. hyo (n = 12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n = 12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn^® M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3_P216, but not against the remaining vaccine subunit antigens. Alhydrogel^® and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn^® M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn^® M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1β, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn^® M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.