Efficacy of a novel intradermal Lawsonia intracellularis vaccine in pigs against experimental infection and under field conditions.

BACKGROUND: The efficacy of a novel inactivated intradermal Lawsonia intracellularis vaccine, Porcilis® Lawsonia ID, was evaluated in two experimental vaccination-challenge studies and under field conditions on a farm with a history of recurrent acute ileitis. In addition, the efficacy of the vaccine was compared to that of a commercially available live attenuated vaccine. The novel inactivated vaccine consists of a freeze-dried antigen fraction that is dissolved just prior to use in either the adjuvant or in Porcilis® PCV ID; an existing intradermal vaccine against porcine Circovirus type 2. In the two experimental vaccination-challenge studies, groups of 25 piglets were vaccinated once at 3 weeks of age or left unvaccinated as challenge control. Vaccines tested were Porcilis® Lawsonia ID as standalone (study 1) or in associated mixed use with Porcilis® PCV ID (study 2) and an orally administered commercially available live vaccine (study 1). The pigs were challenged with virulent L. intracellularis at 4 weeks (study 1) or 21 weeks (study 2) after vaccination. Post-challenge, the pigs were evaluated for clinical signs, average daily weight gain, shedding and macroscopic as well as microscopic immuno-histological ileum lesion scores. In the field study, the mortality and key performance parameters were evaluated over a period of 8 months. RESULTS: The results of the two experimental vaccination-challenge studies showed that Porcilis® Lawsonia ID as single vaccine or in associated mixed use with Porcilis® PCV ID, induced statistically significant protection against experimental L. intracellularis infection, 4 weeks or 21 weeks after vaccination. This was demonstrated by lower clinical scores, improved weight gain, reduction of L. intracellularis shedding and reduction of macroscopic as well as microscopic ileum lesion scores when compared to the controls. The protection induced was superior to that of the commercially available live vaccine. In the field study Porcilis® Lawsonia ID was highly efficacious in reducing L. intracellularis associated mortality and improving key production parameters. CONCLUSION: The results support that this new intradermal vaccine is efficacious against L. intracellularis and may be used in associated mixed use with Porcilis® PCV ID.

Efficacy of a novel inactivated Lawsonia intracellularis vaccine in pigs against experimental infection and under field conditions.

The efficacy of a novel inactivated Lawsonia intracellularis vaccine, Porcilis® Lawsonia, was compared to that of a commercially available live attenuated vaccine in three experimental vaccination-challenge studies in pigs. The efficacy of the new vaccine was further tested under field conditions on a farm with a history of acute ileitis. The novel inactivated vaccine consists of a freeze-dried antigen fraction that is dissolved just prior to use in either the adjuvant or in Porcilis® PCV M Hyo; an existing combination vaccine against porcine circovirus type 2 and Mycoplasma hyopneumoniae. The three experimental vaccination-challenge trials had a similar design and for each trial 75 piglets were used, randomly allotted to three groups of 25 piglets. The pigs were vaccinated at 4 or 5 weeks of age with either Porcilis® Lawsonia in adjuvant or in associated mixed use with Porcilis® PCV M Hyo (group 1), with the live vaccine (group 2), or left as unvaccinated controls (group 3). The pigs were challenged with virulent Lawsonia intracellularis 3, 4 or 17 weeks after vaccination. Post-challenge the pigs were evaluated for clinical signs, average daily weight gain, shedding and macroscopic as well as microscopic immuno-histological ileum lesion scores. In the field study, the mortality and key performance parameters were evaluated over a period of 8 months. The results of all three experimental vaccination-challenge trials showed that Porcilis® Lawsonia induced statistically significant protection against experimental Lawsonia intracellularis infection. This was demonstrated by lower clinical scores, improved weight gain, reduction of Lawsonia intracellularis shedding and reduction of macroscopic as well as microscopic ileum lesion scores when compared to the controls. The protection induced was superior to that of the commercially available live vaccine. In the field study, Porcilis® Lawsonia proved to be highly efficacious; reducing Lawsonia associated mortality to zero and improving key production parameters.

Safety and efficacy of a new octavalent combined Erysipelas, Parvo and Leptospira vaccine in gilts against Leptospira interrogans serovar Pomona associated disease and foetal death.

The safety and protective efficacy of a new octavalent combination vaccine containing inactivated Erysipelothrix rhusiopathiae, Parvovirus, and Leptospira interrogans (sensu lato) serogroups Canicola, Icterohaemorrhagiae, Australis (Bratislava), Grippotyphosa, Pomona and Tarassovi - Porcilis(®) Ery+Parvo+Lepto - was evaluated in laboratory studies and under field conditions. The safety (2× overdose and repeated dose) was tested in 26 gilts. In this study, neither vaccine related temperature increase nor other systemic reactions were observed after intramuscular vaccination. No local reactions were observed except for one animal that had a small local reaction (2cm diameter) that lasted for 5 days after the third vaccination. Efficacy was tested in 40 gilts. A group of 20 gilts was vaccinated at 20 and 24 weeks of age with Porcilis(®) Ery+Parvo+Lepto and a group of 20 age- and source-matched animals served as the control group. The gilts were inseminated at 41 weeks or 66 weeks of age and were challenged with serovar Pomona 10 weeks after insemination, corresponding to 6 months (n=2×10) and 12 months (n=2×10) after the last vaccination. After both the 6- and 12-month challenges the control animals developed clinical signs (fever, lethargy and anorexia) and leptospiraemia as determined by positive blood culture. In addition, both the 6- and 12-month challenges resulted in death of 21% and 27% of the total number of foetuses in the control groups, respectively. Clinical signs and leptospiraemia were statistically significantly lower in vaccinated gilts after both the 6- and 12-month challenges. In addition, foetal death was statistically significantly lower (3% and 2%, respectively) in vaccinated gilts after both the 6- and 12 month challenges. The vaccine was tested further under field conditions on a Portuguese farm with a history of an increasing abortion rate associated with a Leptospira serovar Pomona infection (confirmed by PCR and serology). This study was designed as an observational-longitudinal field study. At the start of the study, all breeding sows and replacement gilts on the farm were vaccinated twice with Porcilis(®) Ery+Parvo+Lepto at an interval of 4 weeks. Starting six months after the primary vaccination schedule, the animals were re-vaccinated during the second week of every subsequent lactation. New replacement gilts were vaccinated using the same schedule. After vaccination, the abortion rate reduced rapidly from 12.6% in winter months of 2012 (December 2011 to March 2012) to 0.5% in winter months of 2013, a statistical significant decrease of 96%. The total number of abortions on the farm decreased from 55 in 2012 to 6 in 2013. Thereafter, the abortion rate remained stable and in the period December 2013 to April 2014 was still low (0.6%). In conclusion, the present studies demonstrate that the octavalent Porcilis(®) Ery+Parvo+Lepto vaccine can be safely used in gilts and sows and induces significant protection, for the duration of at least one year, against serovar Pomona induced clinical signs, leptospiraemia and foetal death. Protection against Pomona associated reproductive failure was confirmed under field conditions where a significant reduction in abortion rate was observed.

Veterinary vaccines for public health and prevention of viral and bacterial zoonotic diseases.

To meet with the increasing demand for food, the scale of world food production is increasing, as is the transport of animals and food products. At the same time, the contact of animals with the environment remains unchanged or, in the case of free-ranging animals, is even increasing. A number of microorganisms have established themselves in farmed animals, which although relatively harmless to animals are pathogenic to man. In this article, the options for reducing the risk of transferring zoonotic agents from animals (particularly farm animals) to man using veterinary vaccines against viral and bacterial diseases are described.

Vaccine potential of recombinant Ornithobacterium rhinotracheale antigens.

Ornithobacterium rhinotracheale is a pathogen involved in respiratory infection and systemic disease in poultry. Previously, eight potential vaccine candidates were identified that induced cross-protective immunity when administered to chickens as a multi-component vaccine. In this study, we analyzed the immunogenicity of these eight recombinant proteins by subunit vaccination, and characterized the different proteins and corresponding genes more thoroughly by sequencing, in vitro expression analysis, and cellular localization experiments. We found, that all genes encoding the eight antigens were highly conserved among different O. rhinotracheale serotypes, but the different antigens were not expressed by all serotypes. Cellular fractionation experiments indicated that the majority of the antigens are predominantly located in the outer membrane fraction. Vaccination of chickens with single-antigen vaccines demonstrated that the Or77 antigen was protective against serotypes that expressed Or77 in vitro, suggesting that the protein has strong potential as a vaccine antigen. Furthermore, immunization with four-component subunit vaccines indicated the existence of immunogenic synergism between the candidate vaccine antigens.

Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes worldwide severe losses in pig farming. The virulence of the 15 serotypes of A. pleuropneumoniae is mainly determined by the three major RTX toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX toxin, ApxIV, is produced by all 15 serotypes only during infection of pigs, but not under in vitro conditions. Pigs infected with A. pleuropneumoniae show specific antibodies directed against ApxIV. In contrast, antibodies against the other three toxins ApxI, ApxII and ApxIII are also found in pigs free of A. pleuropneumoniae. The antibodies to the three latter might result from other, less pathogenic Actinobacillus species such as A. rossii and A. suis. We used a recombinant protein based on the N'-terminal part of ApxIV to serologically detect A. pleuropneumoniae infections in pigs by immunoblot analysis. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected A. pleuropneumoniae infections in the second to third week post infection. We developed an indirect ELISA based on the purified recombinant N'-terminal moiety of ApxIV. The analysis of sera from pigs that were experimentally or naturally infected by A. pleuropneumoniae, and of sera of pigs that were free of A. pleuropneumoniae, revealed that the ELISA had a specificity of 100% and a sensitivity of 93.8%. The pre-validation study of the ApxIV-ELISA revealed that the latter was able to detect A. pleuropneumoniae-positive herds, even when clinical and pathological signs of porcine pleuropneumonia were not evident. Pigs vaccinated with a subunit vaccine Porcilis App were serologically negative in the ApxIV-ELISA.

Characterisation of the gene encoding suilysin from Streptococcus suis and expression in field strains.

The gene encoding suilysin was cloned from Streptococcus suis serotype 2 strain P1/7. Analysis of the nucleotide and translated amino acid sequence confirmed suilysin to be a member of the thiol activated cytolysin group (TACY). The pneumolysin from Streptococcus pneumoniae is the most closely related orthologous gene known. Suilysin was overexpressed in E. coli in an active haemolytic form. A strong correlation between the presence of the sly gene and haemolytic activity in the supernatant of S. suis field strains was found. Of 158 strains tested, 63% contained the gene. Within the (most prevalent) serotype 2, the sly gene was demonstrated in 95% of the strains isolated in Eurasia, but only in 7% of the strains from North America.

Molecular analysis of the dnaK locus of Leptospira interrogans serovar Copenhageni.

Analysis of the dnaK locus of Leptospira interrogans serovar Copenhageni identified four genes in the order hrcA, grpE, dnaK and dnaJ. This is the first time a homologue of hrcA has been identified in a spirochete. The hrcA gene and a regulatory sequence, designated CIRCE, play a significant role in the regulation of the dnaK locus of several Gram+ organisms. Their presence upstream of dnaK in Leptospira suggested a similiar regulatory mechanism. Transcriptional analysis using reverse transcriptase-PCR demonstrated transcription of all four genes and indicated that hrcA and grpE were co-transcribed, as were grpE and dnaK. Whilst hrcA, grpE and dnaK were closely linked on the chromosome, transcription terminators between dnaK and dnaJ and downstream of dnaJ suggested that this latter gene exists in its own operon. Primer extension analysis located functional promoters upstream of hrcA and grpE; however, no evidence of a functional promoter could be found for dnaJ. Moreover, transcripts encompassing the first three genes or the entire locus could not be demonstrated, suggesting that the four genes are regulated independently at the transcriptional level. These results indicate that the regulation of the dnaK locus of Leptospira differs somewhat from that observed in other organisms.

Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains.

A method has been developed which allows the determination of the activator, the structural and the secretion genes of the three toxins ApxI, ApxII and ApxIII in Actinobacillus pleuropneumoniae in only two PCR reactions. The oligonucleotide primers were designed to amplify a significant part of the activator and structural genes apxICA, apxIICA and apxIIICA together in a single PCR reaction giving amplification products which differ in length, in order to be clearly separated by agarose gel electrophoresis. Variations in the apxIA and apxIIIA genes which were found in different serotypes were taken into account in the design of the primers to give a uniform amplification product for both variants of the apxIA and the apxIIIA genes. The secretion genes apxIBD and apxIIIBD are also detected in a single PCR reaction containing two pairs of oligonucleotide primers which yield two differently sized fragments to differentiate between apxIBD and apxIIIBD genes. The reference strains of A. pleuropneumoniae serotypes 1-12 and 104 field strains representing all serotypes obtained from various laboratories worldwide were analysed for their content of apx genes. The two PCR reactions give toxin gene patterns which are characteristic for different groups of serotypes in A. pleuropneumoniae and allow the rapid differentiation of five toxin type groups, group 1 including serotypes 1, 5a, 5b, 9 and 11, group 2 including serotypes 2, 4, 6, 8, group 3 with serotype 3, group 4 with serotype 7 and 12 and group 5 with serotype 10. The method enhances and facilitates differentiation of A. pleuropneumoniae strains for diagnostics and epidemiology and allows the detection of serotypes with atypical toxin patterns.

Rapid and specific detection of pathogenic Leptospira species by amplification of ribosomal sequences.

We have developed an assay for the detection of pathogenic Leptospira that is based on the polymerase chain reaction. With the combination of agarose gel electrophoresis and blotting, pathogenic Leptospira can be discriminated specifically from nonpathogenic Leptospira and other bacterial species. This method, based on the amplification of 16S ribosomal RNA sequences, is able to detect 10 leptospiral cells/mL in cattle urine samples and 100 leptospiral cells/mL in pig urine samples. Using this assay leptospires were detected in urine samples from cattle that were experimentally infected with Leptospira interrogans serovar hardjo type hardjobovis.

Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni.

A chromosomal gene library of Leptospira interrogans serovar copenhageni strain Wijnberg was constructed in phage lambda gt11. Plaque immunoassay with R alpha P64 antiserum identified one clone expressing a putative groEL homologue. DNA sequence analysis of the 2.4 kb EcoRI-Bam HI cloned fragment from strain Wijnberg revealed two open reading frames encoding polypeptides of 10.5 kDa (Hsp10) and 58 kDa (Hsp58). Sequence comparison of the deduced amino acid sequences of these ORFs confirmed the operon as the groE equivalent of Leptospira. Transcriptional analysis suggested that this operon is primarily under the control of an E sigma 70 promoter element. However, both Hsp10 and Hsp58 were overexpressed under heat-shock conditions as determined by [35S]-methionine pulse labelling experiments. As no functional heat-shock promoter could be identified, a 9bp inverted repeat, located between the transcription and translation start sites, may play a role in the upregulation of this operon under heat-shock conditions, similar to mechanisms described for several Gram-positive organisms.

Presence of putative sphingomyelinase genes among members of the family Leptospiraceae.

The presence of multiple DNA elements in pathogenic members of the family Leptospiraceae, similar to the sphA sphingomyelinase gene from Leptospira borgpetersenii, was demonstrated by low-stringency hybridization experiments. These DNA elements were designated putative sphingomyelinase genes. Grouping of strains by similarity of hybridization patterns corresponds to the species subdivision of the family Leptospiraceae on the basis of genetic characteristics. Therefore, hybridization with the sphA gene can be used as a taxonomic tool. These hybridization experiments indicate the presence of two groups of genetically related pathogenic Leptospira species.

Molecular analysis of a sphingomyelinase C gene from Leptospira interrogans serovar hardjo.

A thermolabile hemolysin from Leptospira interrogans serovar hardjo, strain Sponselee, was shown to specifically degrade sphingomyelin. Nucleotide sequence determination revealed that sphingomyelinase activity was encoded by an open reading frame of 1,668 nucleotides. Although a putative signal sequence could be identified, no evidence for protein export in either L. interrogans or Escherichia coli was obtained. The apparent molecular mass of the expression product in E. coli minicells was 41.2 kilodaltons, whereas open reading frame 1 encoded a protein of 63,268 daltons. The observed difference may be explained by processing at the carboxy-terminal part of the hemolysin in E. coli. A high degree of similarity on the DNA and protein levels with Staphylococcus aureus beta-hemolysin and sphingomyelinase C from three Bacillus cereus strains was observed. The presence of various sphingomyelinase genes within the L. interrogans species is demonstrated.

The use of immunodeficient male (CBA/N x BALB/c) F1 mice to produce monoclonal antibodies directed to proteins of Leptospira interrogans rather than to immunodominant lipopolysaccharides.

A method, using an immunodeficient mouse strain, for the production of monoclonal antibodies directed exclusively against the proteins in an antigen mixture also containing immunodominant LPS, is described. Male (CBA/N x BALB/c) F1 mice were immunized with an outer envelope antigen mixture from Leptospira interrogans strain Wijnberg containing both lipopolysaccharides and proteins. The immune response in these mice was shown to be predominantly directed against protein antigens. Hybridoma cell lines were generated by fusing spleen cells from a (CBA/N x BALB/c) F1 mouse with BALB/c Sp2/0 plasmacytoma cells. Hybridoma cell lines producing monoclonal antibodies reacting with the outer envelope preparation were identified by ELISA. All epitopes recognized by the monoclonal antibodies are sensitive to proteinase K degradation and resistant to oxidation by periodate indicating that they are located on proteins. All epitopes are located on a 35 kDa protein and specific for the pathogenic L. interrogans species.

Cloning of a hemolysin gene from Leptospira interrogans serovar hardjo.

A DNA fragment encoding both hemolysin and sphingomyelinase C activity was cloned from the pathogenic bacterium Leptospira interrogans serovar hardjo. Initial clones were obtained by screening a genomic library in EMBL3 for hemolytic activity. Both hemolytic and sphingomyelinase C activities were coded for by a 3.9-kilobase BamHI fragment. The hemolysin was expressed from its own promoter in Escherichia coli K-12. Similar DNA sequences were also present in the serovars tarassovi and ballum.

RNA and protein analysis of an X-ray-induced hereditary cataract in rat.

We have analyzed the consequences of a mutation causing a recessive hereditary cataract in the rat. The lenses of these rats were examined both by histological and molecular biological methods. In the mutant rat lens development starts normally during early fetal life, but becomes progressively disrupted. The structure of the lens is clearly abnormal around the time of birth. Surprisingly, changes at the molecular level occur much later than the histological effects can be observed. Lens epithelial and cortex fiber cells apparently continue to synthesize the lens-specific crystallin mRNAs and proteins in spite of the damaged fiber cells.