# Patent

## Title
Modified live (JMSO strain) Haemophilus parasuis vaccine

## Abstract

The present invention is a live vaccine from a culture of cells of Haemophilus parasuis exhibiting attenuated pathogenicity and capable of triggering a protective immune response when administered to pigs. The cell culture was modified from a pathogenic parent strain by MNNG mutagenesis and was selected by complete dependence on streptomycin for growth. Several SNPs have been identified as associated with specific proteins that are associated with virulence as seen in the literature in H. parasuis or related bacterial species.

## Background
This patent application is a continuation-in-part of U.S. patent application Ser. No. 11/648,390, filed Dec. 28, 2006, now U.S. Pat. No. 8,404,253 with the title “Modified Live (JMSO Strain) Haemophilus parasuis Vaccine.”


BACKGROUND OF THE INVENTION
Haemophilus parasuis is an early colonizer of the upper respiratory tract of pigs. Non-virulent strains of this organism can be normally isolated from the nasal cavity, tonsil, and trachea of healthy pigs. Virulent strains of H. parasuis can also invade the host and cause systemic lesions characterized by fibrinous polyserositis, arthritis and meningitis.
Recent studies have demonstrated that non-virulent strains are more prevalent in the upper respiratory tract than virulent strains. However, even though the majority of pigs are colonized by non-virulent H. parasuis strains, they are not protected against systemic infection by virulent strains. It is widely recognized in the vaccine field that live vaccines may sometimes provide a higher degree and broader range of protective immunity than killed vaccines.
Controlled exposure to a low dose of live, virulent H. parasuis may reduce nursery mortality more efficiently than vaccination using commercial or autogenous killed products. However, exposure to live virulent bacteria poses significant risk of the very disease one is trying to protect against. Attenuation of the virulence of the vaccine strain in a way that is stable and also retains the immunogenicity of the live organism is therefore highly desirable to vaccine development.
Although neither the mechanisms involved in the protective immunity following controlled exposure, nor the identity of antigens which provide protective immunity, are clear at this time, it is known that protective immunity can be induced by such exposure. Moreover, pigs with controlled exposure to live H. parasuis are protected against homologous challenge through intratracheal and intraperitoneal routes, suggesting that exposed pigs develop local immune response (upper respiratory tract) as well as a systemic response. Live organisms undergo limited replication in the host at sites of infection and may thereby elicit additional arms of the immune response (e.g., local immunity) and/or augment the level of the response.
H. parasuis killed vaccines (commercial or autogenous) are known to generate poor cross-protection against different serovars. This may be due to incomplete expression of potential virulence factors when this organism is grown in vitro. Live organisms are more likely to express a wide variety of proteins and factors required for growth in vivo. Iron-regulated outer membrane proteins (IROMPS) are an example of potential virulence factors that may be expressed in vivo, but are rarely expressed in vitro. A modified live vaccine would be capable of expressing virulence factors when administered to a host. Most likely this product would induce a more involved immune response creating the potential for a safe, more efficacious vaccine.
The pork industry is in need of a safe and efficacious product capable of aiding the prevention of disease due to multiple serotypes of H. parasuis. 
SUMMARY OF THE INVENTION
The present invention provides a vaccine which provides cross-serovar protection for pigs against infection by H. parasuis, comprising an immunologically effective amount of live cells of a strain derived from a pathogenic parent strain of the organism, which are dependent upon streptomycin for growth, and which cells exhibit attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immune response that protects the animals against infection when administered as a live vaccine, and a veterinarily acceptable carrier. The present invention also provides identification of certain genes that have undergone single nucleotide polymorphisms (SNPs) in the attenuated strain. The present invention also conceptually identifies the protein products that are produced by the genes which show these SNPs, based on correlation of polynucleotide sequence with the complete genome of a standard strain (SH0165) of serovar 5 of H. parasuis and analysis of SNPs that resulted in amino acid changes. Vaccines of the invention may further comprise one or more other components including, for example, an adjuvant.
A principle object and advantage of the present invention is that it is a vaccine that provides cross-serovar protection for pigs against infection by H. parasuis. 
Another principle object and advantage of the present invention is that several SNPs in genes that encode known proteins and result in amino acid changes in the mutant strain as compared with a standard serovar 5 H. parasuis strain have been identified. The changes in the amino acids in these proteins may be responsible for the decreased virulence of the mutant strain as compared with the parent strain.


DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Applicants have discovered that cells of a pathogenic strain of H. parasuis may be attenuated, and that the resulting attenuated cells are capable of triggering an immune response that protects pigs against H. parasuis infection when administered as a live vaccine.
The term “attenuated” as used herein describes a cell, culture, or strain of H. parasuis exhibiting a detectable reduction in infectivity or virulence in vivo as compared to that of the parent strain of H. parasuis from which the attenuated cell, culture, or strain is derived. Reduction in virulence encompasses any detectable decrease in any attribute of virulence, including infectivity in vivo, or any decrease in the severity or rate of progression of any clinical symptom or condition associated with infection.
The present invention further encompasses preparation and use in a vaccine of cells of a strain of H. parasuis derived from a pathogenic parent strain of H. parasuis and which exhibit attenuated pathogenicity compared to cells of the parent strain and which are capable of triggering an immune response that protects pigs against H. parasuis infection.
The live attenuated cells of the invention are capable of triggering an immune response that protects pigs against H. parasuis infection after one or more administrations as a live vaccine. A “protective immune response” is defined as any immunological response, either antibody or cell mediated immunity, or both, occurring in the pig that either prevents or detectably reduces subsequent infection, or eliminates or detectably reduces the severity, or detectably slows the rate of progression, of one or more clinical symptoms or conditions associated with H. parasuis infection. The term “immunologically effective amount” refers to that amount or dose of vaccine or antigen that triggers a protective immune response when administered to the pig.
Example 1
Identification of the Parent Strain
The parental strain has been identified as serovar 5, H. parasuis; Newport Laboratories reference no. 3-0930-1.
The isolate was received by Newport Laboratories from Iowa State University Veterinary Diagnostic Laboratory as an agar slant labeled H. parasuis 1888 on May 2, 2003. The isolate was identified as H. parasuis based on biochemical reactions (non-hemolytic, v-factor dependent, urea, and arabinose negative). The isolate was identified as a serovar 5, H. parasuis by the Faculty of the Veterinary Medicine Department of the University of Montreal.
Example 2
Development of the Vaccine Strain
(a) Procedures used to attenuate the parental strain: Attenuation of the parent strain was accomplished by chemical mutagenesis with N-methyl-N′-nitroso-N-nitrosoguanidine (MNNG) and selection for growth dependence on media containing 400 micrograms per mL streptomycin sulfate.
The protocol for MNNG mutagenesis was adapted from Lopes et al., 2001. The parent strain was initially checked for purity by inoculating the culture on 5% sheep blood agar containing a nurse Staphylococcus sp. streak and incubating for 24 h at 37° C.+−2° C. in a candle jar.
The culture was pure, based on the absence of extraneous growth outside the Staphylococcus sp. streak and growth of the H. parasuis satellite colonies in the area adjacent to and just outside the streak. After purity was confirmed, the parent H. parasuis strain was swabbed on chocolate agar plates. Plates were incubated as indicated above.
The resulting bacterial cells were harvested by adding 1 mL of PBS to the chocolate agar plate and scraping colonies using a sterile, modified Pasteur pipet (heated and bent to the shape of a hockey stick).
Bacterial cells were pooled to one side of the plate, and an automated PIPET-AID® and a 5 mL pipet were used to collect the cell suspension. The cell suspension was then diluted in pre-warmed PPLO media (BD) containing MNNG (Sigma-Aldrich Cp., St. Louis, Mo., 30 micrograms/mL). The mixture of cells and MNNG was then incubated at 37° C.+/−2° C. for 30 minutes without agitation. Following incubation, bacteria were collected by centrifugation and washed with cold phosphate-buffered saline (PBS), pH 7.4, to eliminate residual mutagen. The washed cells were resuspended in pre-warmed PPLO media and incubated at 37° C.+/−2° C. for 40 min.
The MNNG-exposed bacteria were plated onto PPLO agar plates containing 400 mcg/mL streptomycin sulfate and onto PPLO without streptomycin sulfate and both sets of plates were incubated at 37° C.+/−2° C. for 24 hours in a candle jar. PPLO agar plates without streptomycin sulfate were used to ensure the MMNG treatment did not inactivate all bacterial cells.
Following incubation, the plates without streptomycin showed bacterial growth, confirming that the MNNG treatment did not inactivate all bacterial cells. A single colony was observed and harvested from a single PPLO plate containing streptomycin sulfate. This colony was passed to a PPLO agar plate and PPLO broth, both supplemented with 400 mcg/mL streptomycin sulfate (permissive media), and to PPLO broth and agar plate containing no streptomycin sulfate (non-permissive). Cultures were incubated as above. Growth on permissive medium and lack of growth on non-permissive medium was seen. This was the basis for selection of the modified live, streptomycin-dependent, H. parasuis vaccine strain. The attenuated H. parasuis strain and proposed Master Seed has been identified, “Haemophilus parasuis streptomycin dependent Master Seed 101304”.
The selected Master Seed has proven to be avirulent for the natural host of H. parasuis (pigs) in preliminary research. A backpassage study of 4 passes with two pigs per pass was completed.
Two pigs were inoculated intraperitoneally with 3 cc of 109 cells of the streptomycin dependent strain. Two days following inoculation, pigs were euthanized and intraperitoneal fluids were collected and passed to 2 additional pigs. This was done for a total of 4 passes. Animals were observed for clinical signs following inoculation and for clinical lesions following euthanasia. Tissues and swabs were taken from euthanized pigs and isolation of the vaccine strain and potential revertant strain was attempted. Approximately 1 mL of the subcultured peritoneal fluid was tested at Newport Laboratories for H. parasuis isolation (as above) and by H. parasuis detection PCR.
Following the administration of the vaccine strain in the backpassage and safety studies, no clinical signs associated with disease caused by H. parasuis were noticed in any pigs. All isolation attempts for the vaccine strain and the streptomycin-independent revertant strain were negative. H. parasuis detection PCR from peritoneal fluid from the preliminary backpassage study was negative.
(b) Screening methods and protocols for the identification and purification of the vaccine microorganism: The vaccine microorganism was selected based on its complete dependence on streptomycin. Purity of the vaccine strain was done according to the guidelines set forth in 9 CFR 113.27(d) and reported in the Master Seed report submitted Aug. 31, 2005.
Identification of the vaccine strain was confirmed based on biochemical reactions, dependency on streptomycin sulfate, H. parasuis detection PCR, H. parasuis Genotyping PCR, gram staining, and colony morphology. Summary for all testing was reported in the Master Seed Report submitted to USDA-CVB Mar. 23, 2006.
Both the parent and vaccine have been shown to be viable by the American Type Culture Collection (ATCC) and the following ATCC accession numbers have been assigned:
















Haemophilus parasuis: Parent JMSO 50703
PTA-7710



Haemophilus parasuis: JMSO 10134
PTA-7711










Example 3
Vaccine Production
The vaccine culture was started from a working seed. The working seed was subcultured to X+5 by using PPLO media supplemented with 400 mcg/mL streptomycin, 2% dextrose, 10% horse serum, and 0.2% NAD was used. The final subculture was incubated at approximately 37° C. overnight with agitation. Following incubation, the culture was centrifuged for 20 minutes at 3,000 rpms. The cell pellet was resuspended in fresh PPLO media with 1/10 of the original volume. The vaccine was stored on ice until used. Immediately prior to vaccination, Trigen was added to the vaccine at 10% of the resuspended volume. Prior to vaccination, the vaccine was titrated and cell concentrations were determined. Cell concentration for the first vaccination was 107 to 109.60 CFUs per mL. The vaccine used for animals receiving 2 doses was prepared in the same manner. Cell concentration for the 2nd vaccination was 109.55 CFU per mL.
Example 4
Efficacy of the Vaccine
(a) Methods
At arrival, all pigs were injected with 0.3 mL of Excede IM and housed in the same room at the Research Farm (RF) for 7 days. To assure equal representation in treatment groups, pigs were randomly allotted based on litters. Prior to vaccination, non-vaccinated pigs, pigs receiving the vaccine orally, and pigs receiving the vaccine IM were housed in three separate rooms. Pigs receiving a single dose were separated from pigs receiving a 2nd dose by a solid partition. Pigs were vaccinated at approximately 4 weeks of age; pigs receiving a second dose were revaccinated 2 weeks following the first vaccination. Three weeks after the 2nd vaccination, all pigs were challenged.
To challenge the pigs, a single ampoule of the H. parasuis parent strain, Newport Laboratories, was removed from frozen storage and streaked on 5 chocolate agar plates. Plates were incubated at 37° C. in candle jars for approximately 48 hours. Using a sterile cotton swab, growth was harvested and used to inoculate 400 mL of PPLO media containing the following supplements: 10% horse serum, 2% dextrose (50%), and 0.2% NAD per mL of PPLO. Cultures were incubated at 37° C. on a shaker cart for approximately 6 hours. Following incubation, % transmittance was recorded, and cell titrations were made. All pigs were challenged intravenously and intraperitoneally with 1 mL per route.
All vaccinations were given at a rate of 0.2 mL per pig IM or orally as appropriate.
(b) Results
Rectal temperatures were taken prior to challenge and for 4 days following challenge. A temperature at or above 40.5° C. was considered a fever. Pigs were weighed prior to challenge, at the time of death or at the end of the study, as appropriate. Average Daily Gain (ADG) was determined for each pig and daily observations were made once daily by a blind observer.
Pigs were observed daily for clinical signs and were scored accordingly. A daily observation chart was constructed using the following scoring system.











Score
Clinical Signs







 4*
Moribund or dead, unable to rise, lateral recumbancy, CNS



signs.


3
Severely lame and or lethargic. Total or complete lameness on



one or more limbs, cannot support weight. Extreme difficulty in



rising, can stand when prompted, quick to lie down.


2
Moderately lame and/or lethargic. Lame but will support



weight on lame leg, must be prompted to move


1
Mildly lethargic. Ears are down, not active, slow to rise and



move.


0
Normal





*Dead pigs are given 4 points for each experimental day following death.






A treatment was considered satisfactory, if, in a valid assay, 80% or more of the vaccinated pigs in the group survived challenge and showed no lesions upon necropsy and at least 80% of the non-vaccinated control pigs do not survive challenge and/or are positive for 1 or more lesions at necropsy. To confirm infection from H. parasuis following challenge, lung and heart samples, and peritoneal, joint, and pleural swabs from each necropsied pig were collected and sent to Newport Laboratories (NPL) diagnostic lab for H. parasuis isolation.
The test was valid as 100% of non-vaccinated pigs had lesions or did not survive challenge.
Experiment #1
Homologous Protection (Serovar 5 Vaccination Serovar 5 Challenge)
Table 1 shows the mortality rate after inoculation with the vaccine and subsequent challenge with the parent strain.














Treatment Group
Mortality



















Non-vaccinates
100%



1 dose IM vaccinates
10%



2 dose IM vaccinates
0%



1 dose oral vaccinates
80%



2 dose oral vaccinates
100%










Table 2 shows the percentage of pigs in each treatment group with a fever.














Treatment Group
% of animals with a fever



















Non-vaccinates
90%



1 dose IM vaccinates
10%



2 dose IM vaccinates
0%



1 dose oral vaccinates
50%



2 dose oral vaccinates
70%



Non-vaccinated/non-challenged
0%










Table 3 shows the Average Daily Gain for each treatment group following challenge:














Treatment Group
Average daily gain (loss)









Non-vaccinates
−2.88 lbs/day



1 dose IM vaccinates
  1.65 lbs/day



2 dose IM vaccinates
  1.71 lbs/day



1 dose oral vaccinates
−2.85 lbs/day



2 does oral vaccinates
−5.51 lbs/day



Non-vaccinated/non-challenged
  2.21 lbs/day










Table 4 shows the total points (scores) registered from daily observations.














Treatment Group
Total Points



















Non-vaccinates
116



1 dose IM vaccinates
15



2 dose IM vaccinates
13



1 dose oral vaccinates
112



2 dose oral vaccinates
137



Non-vaccinated/non-challenged
0










Experiment 2
Heterologous Protection (Serovar 5 Vaccination, Heterologous Challenge)
Table 5 shows the mortality % for each treatment group when challenged with a serovar 4 strain.














Treatment group
Mortality



















Non-vaccinates
78%



1 dose vaccinates
0%



2 dose vaccinates
0%










    
    
        89% of non-vaccinated pigs were positive for one or more lesions commonly associated with H. parasuis infection. No vaccinated pigs were necropsied as no vaccinated pig met euthanasia criteria.
    
    


Table 6 shows the mortality % for each treatment group when challenged with a serovar 13 strain.














Treatment group
Mortality



















Non-vaccinates
100%



1 dose vaccinates
13%



2 dose vaccinates
0%










    
    
        All non-vaccinated pigs were positive for 1 or more lesions. Two of 8 pigs vaccinated with a single dose were positive for one or more lesions. One of 8 pigs vaccinated with 2 doses was positive for 1 or more lesions.
    
    


All non-vaccinated pigs had fevers following challenge with the parent strain, while all vaccinated pigs had normal temperatures following challenge.
Table 7 shows the average daily gain for each treatment group when challenged by the serovar 5 parent strain, 3-09302-1.














Treatment Group
Average Daily Gain (loss)









Non-vaccinates
−3.84 lbs/day  



1 dose vaccinates
1.65 lbs/day



2 dose vaccinates
1.71 lbs/day










Table 8 shows the total points (scores) for each treatment group when challenged by the serovar 5 parent strain, 3-09302-1.














Treatment Group
Total Points



















Non-vaccinates
139



1 dose vaccinates
0



2 dose vaccinates
0










All non-vaccinated pigs had fevers following challenge with the parent strain, while all vaccinated pigs had normal temperatures following challenge.
Challenge with Serotype 4 Strain
All non-vaccinated pigs challenged with strain 4-1230-2 (type 4) had a fever for at least one day. No vaccinated pigs had fevers following challenge.
Table 9 shows the average daily gain for each treatment group following challenge with the serotype 4, 4-1230-2 strain.














Treatment Group
Average Daily Gain (loss)









Non-vaccinates
−2.43 lbs/day  



1 dose vaccinates
1.27 lbs/day



2 dose vaccinates
1.86 lbs/day










Table 10 shows total points (scores) registered from daily observations were.














Treatment Groups
Total Points



















Non-vaccinates
99



1 dose vaccinates
0



2 dose vaccinates
0










Challenge with Serotype 13 Strain
All non-vaccinated pigs challenged with strain 5-1124-23 (type 13) had a fever for at least one day following challenge. Four pigs from the 1 dose vaccinates had a fever for one or more days following challenge and 1 pig from the 2 dose vaccinates had a fever for one or more days.
Table 11 shows the average daily gain for each treatment group following challenge with the serotype 13, 5-1124-23 strain.














Treatment Groups
Average Daily Gain (loss)









Non-vaccinates
−4.12 lbs/day  



1 dose vaccinates
0.99 lbs/day



2 dose vaccinates
0.79 lbs/day










Table 12 shows the total points (scores) registered from daily observations.














Treatment Groups
Total Points



















Non-vaccinates
120



1 dose vaccinates
15



2 dose vaccinates
6










Discussion/Conclusions: The H. parasuis ML (modified live) vaccine had clear protection against all 3 challenge strains. Mortality, average daily gain, daily observations, and body temperature data all support the efficacy of and the potential for the ML vaccine to be used as a tool for controlling disease caused by H. parasuis. Results support the efficacy of the vaccine against heterologous challenge strains.
Example 5
Mutant Characterization Using DNA Sequencing
Materials and Methods:
PCR/Sequencing Primer Development:
The Haemophilus influenzae streptomycin resistance (strA) gene was used as a model for primer development as there was little published sequence data for H. parasuis. A freeware primer selection software was used to select primers for this gene.









strA Forward



(SEQ ID NO: 13)



TAC GCA AAC CGC GTG TGA



 



strA Reverse



(SEQ ID NO: 14)



TGC GTG CTT CAA CAC TAC GA






Genomic DNA Extraction:
Cultures of the parent and the JMSO strain were swabbed onto multiple agar media (Chocolate agar for parent strain, PPLO agar with 400 mg/mL of streptomycin for the JMSO strain). Plates were incubated at 37° C.+/−3 in a candle jar until sufficient lawn of growth was obtained (approx. 48 hrs).
The Qiagen Genomic DNA extraction kit was used to obtain the DNA for sequencing. The method was as follows:
Aseptically, 5 mL of 0.25% Tryptic Soy Broth (TSB) was added to each plate. With a sterile loop, the growth was scraped off the agar surface, the plate was tipped to the side and the bacterial suspension was collected with a pipet and pooled into a tared centrifuge tube. The suspensions were centrifuged at 4,500 rpm for 10 min. The supernatant was decanted and the remaining pellet was weighed (for calculation of extraction lysis steps). Pelleted cells were frozen until lysis could be conducted.
Buffer B1 was prepared by adding 100 mg/mL solution of RNase A to obtain a final concentration of 200 g/mL. Eleven mL of Buffer B1 was added to the frozen pellets and the tubes vortexed until a uniform suspension was obtained. To the suspension, 300 ul of a lysozyme stock (100 mg/ml) and 500 ul of Proteinase K were added. The tubes were incubated at 37° C.+/−3 for 1 hr. The sample was deproteinized with Buffer B2, by adding 4 ml to the suspension. The tube was vortexed for a few seconds and then was placed in a water bath at 50° C. for 30 min.
A 500/G Genomic Tip (provided in the kit) was set up for each of the parent and DMSO digested pellets. The tips were equilibrated with 10 ml of Buffer QBT. The tip was emptied by gravity flow. The digested pellet was vortexed for 10 sec at maximum speed and diluted 1:1 with Buffer QBT. One half of the total volume of the digested pellet was poured into the Genomic Tip. A syringe plunger was used to apply positive pressure to help the eluation (20-40 drops/min maximum flow rate). The Genomic Tip was then washed twice with 15 ml of Buffer QC. The genomic DNA was then eluted into a sterile tube with 15 ml of Buffer QF. The DNA was then precipitated with 10.5 ml of room temperature isopropanol by mixing and centrifuging at >5000×g for 15 min. The supernatant was removed and the DNA pellet washed with 4 ml of cold 70% ethanol. The pellets were vortexed and the samples centrifuged briefly. The supernatant was carefully removed and the remaining DNA pellet was allowed to air dry for 5-10 minutes. The DNA pellet was resuspended in TE buffer (pH 8.0) and the DNA allowed to dissolve overnight at 37° C.+/−3.
The resuspended DNA was then evaluated using a 2% agarose gel. The evaluation revealed high molecular weight DNA. The DNA was then quantified using a UV Spectrophotometer for the PCR amplification.
Amplification of Target Gene:
Amplification of the strA gene was conducted on the genomic DNA samples. Approximately 20 ng of DNA was used in this reaction. The “per reaction mix” consisted of the following: 37.8 μl of sterile molecular grade water, 5.0 μl of Taq DNA polymerase buffer (provided with Taq), 1.0 μl of dNTP mix (100 μM each), 10 μM of each forward and reverse primers, and 0.2 μl of Taq DNA polymerase. The samples were amplified at the following conditions: 94° C. for 5 min., followed by 30 cycles of 94° C. for 30 sec., 54° C. for 20 sec., and 68° C. for 30 sec.
DNA Sequencing:
The PCR product was purified of residual dNTPs and Taq DNA polymerase in preparation for sequencing using the QIAQUICK® PCR Purification Kit (Qiagen). The purified PCR product was resolved on a 2% agarose gel, to estimate quantity for sequencing step.
A Dye Terminator Cycle Sequencing (DTCS) and a Beckman Coulter CEQ 8000 (Beckman Coulter) were used to sequence this product. The reaction was set up per the recommendations in the DTCS Quick Start Master Mix kit provided by Beckman Coulter. Forward and reverse sequence reactions were set up using 0.15 M of primer.
Results:
The sequencing parameters resulted in the complete strA gene. A BLAST search revealed that the sequence obtained was an exact match to the strA gene of H. influenzae. Both forward and reverse sequences were obtained, thru multiple repetitions. The sequences were analyzed using MegAlign (DNASTAR Inc, Madison, Wis.) software and a consensus sequence was obtained. The alignment and comparison of the parent and JMSO consensus sequences revealed two nucleotide changes, coding for two amino acid differences. Nucleotide 68: GCA(Alanine) parent to GAA(Glutamic Acid) JMSO strain. Nucleotide 272: CCG(Proline) parent to CTG(Leucine) JMSO.
The nucleotide and sequence data (SEQ ID NOS: 1-4) are identified in a sequence listing document filed with this application.
Example 6
Construction of Genomic Libraries and Characterization of SNPs
Log phase cultures of H. parasuis parent and mutant strains were pelleted separately by centrifuging at 10,000×g for 5 min. The pellet was used for genomic DNA isolation using Qiagen DNA mini kit as per manufacturer's instructions (Qiagen, Valencia, Calif.). The purified genomic DNA was visualized by using DNA electrophoresis technique and quantified in a spectrophotometer (Maniatis et al, Cold spring harbor laboratory). The genomic DNA was used in the preparation of a library for sequencing as per manufacturer's protocols (Illumina Genomic DNA Prep Kit, Illumina, San Diego, Calif.). The genomic DNA libraries of both parent and mutant strains of H. parasuis were denatured and subjected to cluster amplification on a Single End Flow Cell v 4 with a Cluster Generation Station instrument (Illumina) to generate raw cluster intensity of  ̃600,000 mm2. Sequencing was performed on a Genome Analyzer GAII for 56 cycles using Sequencing Kit reagents (Illumina). The generation of sequence and downstream analysis were performed at the University of Nebraska, Lincoln genomics facility and at the University of Washington.
Comparison of Sequences:
The sequences were compared against the reported GenBank genome sequence of H. parasuis SH0165 (Xu et. al. “Genomic Characterization of H. parasuis SH0165, a Highly Virulent Strain of Serovar 5 Prevalent in China,” PLoS ONE, Vol. 6, Issue 5, May 2011, www.plosone.org) and the homology was identified with known defined genes. The sequences of both the parent and mutant were compared with each other to look for any single nucleotide polymorphisms (SNPs) that occur in the coding regions of the genes. This analysis revealed several SNPs and those that resulted in the amino acid changes were considered for further verification. The SNPs that resulted in the amino acid change were verified using Sanger Sequencing technique according to established protocols.
The gsMapper software was used to analyze the SNPs between the parent vs. mutant and mutant vs. SH0165. The softwares gsMapper and newbler assembler (Genome Sequencer 20, Version 1.0.53) are proprietary softwares developed by Hoffmann-La Roche Ltd. Given two sets of raw reads (eg., readA and readB) from different genomes, the newbler assembler was used to create two independent sets of contigs, named as contigA and contigB. The contig sequence from one genome was used as a reference while the reads from the other genome were aligned based on them using gsMapper software. Specifically, two gsMapper runs were carried out, where readA was mapped to contigB in one run and readB to contigA in another run. The gsMapper software utilizes a reference sequence (SH0165) to aid the assembly of raw read data. In addition to sequence assembly, this software gives a “high confidence difference (HCD)” file that summarizes all regions, where the sequence alignment shows differences between reference and multiple read sequences spanning that region. From “readA mapped to contigB” gsMapper run, readA sequences spanning a HCD region (e.g., HCD1) were checked to identify regions within contigA sequence, where they were assembled by the previous newbler run. If this particular region also appears in the HCD output file from the “readB mapped to contigA” gsMapper run, where readB sequences spanning this HCD have been assembled to the region where the previous HCD1 is found, this HCD was considered to be an SNP. For the comparison of genomes between different isolates, each SNP analysis was supported by at least 10 reads from each genome, where at least 80% of the reads spanning that region show the difference. Finally, the SNP data is filtered based on contig base quality score (at least 60) as well as whether or not the SNP is found on a homopolymer region of 3 bases or more to reduce false positive error.
Results: Single Nucleotide Polymorphisms (SNP) Identified in the Vaccine Strain of H. parasuis. 
The SNPs that resulted in the change of amino acid are discussed below regarding their association with virulence as seen in the literature on H. parasuis or related bacterial species.
The complete ORF of the gene is shown in sequence listings filed with this patent application.







TABLE 13







30S ribosomal protein S12













SNP
NUCLEOTIDE
AMINO ACID



SEQ ID
POSITION
CHANGE
CHANGE
















1-4
68
C > A
A > E



1-4
272
C > T
P > L










The 30S ribosomal protein S12 plays a role in the susceptibility/resistance/dependence for streptomycin antibiotic. The above two mutations were reported to be responsible for the streptomycin dependence seen in our vaccine strain in the parent patent application.







TABLE 14







N-acetyl-D-glucosamine kinase













SNP
NUCLEOTIDE
AMINO ACID



SEQ ID
POSITION
CHANGE
CHANGE







5-8
456
G > A
M > I










N-Acetyl-L-glutamate kinase (NAGK) is involved in the metabolism of the bacteria. It plays a role in the arginine biosynthesis and also responsible for ornithine synthesis. The amino acid arginine is required for the survival of the bacteria. Previous studies have shown that mutations in nagK gene resulted in decreased arginine synthesis in Pseudomonas aeruginosa. 

    J Mol Biol. 2009 Jun. 19; 389(4):748-58. N-acetyl-L-glutamate kinase (NAGK) from oxygenic phototrophs: P(II) signal transduction across domains of life reveals novel insights in NAGK control. Beez S, Fokina O, Herrmann C, Forchhammer K.
    J Bacteriol. 2004 November; 186(21):7273-9. The N-acetyl-D-glucosamine kinase of Escherichia coli and its role in murein recycling. Uehara T, Park JT.
    J Bacteriol. 2008 April; 190(8):3018-25. Basis of arginine sensitivity of microbial N-acetyl-L-glutamate kinases: mutagenesis and protein engineering study with the Pseudomonas aeruginosa and Escherichia coli enzymes. Fernández-Murga M L, Rubio V.









TABLE 15







F0F1 ATP synthase subunit C













SNP
NUCLEOTIDE
AMINO ACID



SEQ ID
POSITION
CHANGE
CHANGE







9-12
188
C > T
A > V










ATP synthase is an enzyme involved in the synthesis of ATP, an energy molecule that is required for the metabolic activities of the bacteria. This protein is also reported to be playing a role in the antibiotic resistance of the bacteria. The new generation antibiotics are:

    Nat Chem Biol. 2007 June; 3(6):323-4. Diarylquinolines target subunit c of mycobacterial ATP synthase. Koul A, Dendouga N, Vergauwen K, Molenberghs B, Vranckx L, Willebrords R, Ristic Z, Lill H, Dorange I, Guillemont J, Bald D, Andries K.


Because of the above determination of polynucleotide sequences in the mutant strain with the polynucleotide sequences of a known H. parasuis genome, and thus the conceptual determination of the protein products, Applicant expects that further experimentation will show the absence or altered function of the protein products in the mutant as compared to the parent strain. Such experiments can be carried out by methods known in the art such as enzyme assays, Western blots, immunohistochemical assays and ELISA assays.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety to the extent allowed by applicable law and regulations. In case of conflict, the present specification, including definitions, will control.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore desired that the present embodiment be considered in all respects as illustrative and not restrictive, reference being made to the appended claims rather than to the foregoing description to indicate the scope of the invention.

## Claims

1. A vaccine for protecting pigs against infection by H. parasuis, comprising a veterinarily acceptable carrier and an immunologically effective amount of live cells of an isolated strain of a serovar derived from a pathogenic parent strain of H. parasuis, wherein said cells exhibit attenuated pathogenicity in pigs as compared to those of the parent strain and wherein said cells are capable of triggering an immune response that protects the pig against H. parasuis infection or reduction in disease when administered as a live vaccine, wherein the parent strain and the isolated strain are serovar 5 of H. parasuis, wherein the vaccine provides heterologous protection against at least serovars 4 and 13; and
wherein the live cells differ from the virulent parent strain in that they have mutated versions of the genes encoding the 30S ribosomal protein S12, the N-acetyl-D-glucosamine kinase, and the F0F1 ATP synthase subunit C, with respect to the corresponding genes present in the parent strain.

2. The vaccine of claim 1, wherein the isolated strain was derived from the pathogenic parent strain by MNNG mutagenesis.

3. The vaccine of claim 2, wherein the isolated strain is dependent on streptomycin for growth, said dependency being associated with a strA gene.

4. The vaccine of claim 3, wherein the DNA sequence of the strA gene of the parent strain is SEQ ID NO: 1 and wherein the DNA sequence of the strA gene of the isolated strain is SEQ ID NO: 2.

5. The vaccine of claim 4, wherein the protein encoded by the strA gene of the parent strain is SEQ ID NO: 3 and wherein the protein encoded by the strA gene of the isolated strain is SEQ ID NO: 4.

6. The vaccine of claim 5, wherein the protein encoded by DNA sequence SEQ ID NO: 1 comprises a portion of 30S ribosomal subunit S12 and the protein encoded by DNA sequence SEQ ID NO: 2 comprises at least one amino acid change in said portion of said 30S ribosomal subunit S12.

7. The vaccine of claim 4, wherein the strA gene of the isolated strain differs from the strA gene of the parent strain and wherein the difference comprises a change at nucleotide 68 from C to A and a change at nucleotide 272 from C to T.

8. The vaccine of claim 6, wherein said amino acid change comprises a change at residue 23 from alanine to glutamic acid and a change at residue 91 from proline to leucine.

9. The vaccine of claim 2, wherein SEQ ID NO: 5 comprises an open reading frame of the parent strain and wherein SEQ ID NO: 6 comprises a protein encoded by SEQ ID NO: 5 and wherein SEQ ID NO: 7 comprises an open reading frame of the isolated strain and wherein SEQ ID NO: 8 comprises a protein encoded by SEQ ID NO:7.

10. The vaccine of claim 9, wherein the protein encoded by SEQ ID NO: 5 comprises N-acetyl-D-glucosamine kinase and wherein the protein encoded by SEQ ID NO: 7 comprises N-acetyl-D-glucosamine kinase having at least one amino acid change from said protein encoded by SEQ ID NO: 5.

11. The vaccine of claim 2, wherein SEQ ID NO: 9 comprises an open reading frame of the parent strain and wherein SEQ ID NO: 10 comprises a protein encoded by SEQ ID NO: 9 and wherein SEQ ID NO: 11 comprises an open reading frame of the isolated strain and wherein SEQ ID NO: 12 comprises a protein encoded by SEQ ID NO: 11.

12. The vaccine of claim 11, wherein the protein encoded by SEQ ID NO: 9 comprises F0F1 ATP synthase subunit C and wherein the protein encoded by SEQ ID NO: 11 comprises F0F1 ATP synthase subunit C having at least one amino acid change from said protein encoded by SEQ ID NO: 9.

13. An isolated and purified DNA from the isolated strain of claim 1, said isolated and purified DNA having the nucleotide sequence set forth in SEQ ID NO: 7.

14. The isolated and purified DNA of claim 13, wherein said isolated and purified DNA codes for a polypeptide having the amino acid sequence set forth in SEQ ID NO: 8.

15. The isolated DNA of claim 14, wherein said polypeptide comprises N-acetyl-D-glucosamine kinase.

16. An isolated DNA having at least 85% homology with the DNA of claim 15.

17. An isolated and purified DNA from the isolated strain of claim 1, said isolated and purified DNA having the nucleotide sequence set forth in SEQ ID NO: 11.

18. The isolated and purified DNA of claim 17, wherein said isolated and purified DNA codes for a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12.

19. The isolated and purified DNA of claim 18, wherein said polypeptide comprises F0F1 ATP synthase subunit C.

20. An isolated DNA having 85% homology with the DNA of claim 19.

21. An isolated and purified DNA from the isolated strain of claim 1, comprising a combination of SEQ ID NO: 7 and SEQ ID NO: 11.

22. An isolated and purified DNA from the isolated strain of claim 1, comprising a combination of SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 11.