Iowa Livestock Health Advisory Council

FINAL REPORT

Title:Further characterization of Erysipelothrixrhusiopathiae isolates associated with slaughter house condemnations and development of a multiplex PCR panel for bacterial pathogens associated with septicemia in swine

Principle Investigator: / Tanja Opriessnig, Dr. med. vet., Ph.D.
Assistant Professor
Veterinary Diagnostic and Production Animal Medicine
College of Veterinary Medicine
Phone: 294-1137
E-mail:
Co-Investigators: / Kent J. Schwartz, DVM, MS
Clinician/Pathologist, VDPAM
College of Veterinary Medicine
Veterinary Diagnostic and Production Animal Medicine
Phone: 294-1177
SubhashinieKariyawasam, BVSc, PhD
Clinician/Molecular Diagnostician, VDPAM
College of Veterinary Medicine
Veterinary Diagnostic and Production Animal Medicine

Date of Completion of the Project:June 30, 2008

Industrial Summary

1. Statement of the problem

Condemnations at slaughter due to superficial skin lesions continue to be a cause of economic loss to swine producers in the United States and also worldwide. These skin lesions are often associated with septicemia with several bacterial pathogens being potential causes, including Erysipelothrixspp., Actinobacillussuis, Salmonella choleraesuis, Streptococcus suis, or Haemophilusparasuis. Case submissions to the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) have demonstrated that Erysipelothrixspp. continues to be a reason for slaughterhouse condemnations despite improving management practices and available vaccines. Investigation of few recent cases suggested that isolates associated with condemnations may be unique and different from those available in commercial vaccines.

2. Objective

  1. To develop a multiplex PCR that detects Erysipelothrix spp. and to determine the presence of this pathogen in slaughter house condemnations.
  2. To further characterize the Erysipelothrix spp. isolates found in slaughter house samples and to investigate the source of infection of pigs with these isolates.

3. Results

We developed a multiplex real-time PCR assay which is able to differentiate between Erysipelothrixrhusiopathiae and Erysipelothrixtonsillarum without cross-reacting with Erysipelothrixspecies group 2. This assay is a very useful addition to our Erysipelothrix toolbox and once fully validated will be used as routine-assay in the ISU-VDL. In addition, the results from our study indicated that enrichment culture for Erysipelothrix spp. improved the number of successful isolations and the enrichment culture has now been re-adapted by the ISU-VDL bacteriology section as part of their routine work-up.

Forty-eight cases of condemned samples have been received from a regional slaughterhouse facility and 37 have been diagnostically examined for the presence of Erysipelothrixspp. (the additional cases have been received in the past month and have not been fully characterized as of the submission deadline). Of the 37 cases, Erysipelothrix spp. was successfully isolated from 32cases. Therefore we were able to confirm the preliminary slaughterhouse diagnosis and proved that Erysipelothrix spp. was the cause of condemnationin these cases. The main species of Erysipelothrixidentified in slaughter house investigations wereErysipelothrixrhusiopathiaeserotypes 1a and 2.

4. Impact for the Industry

Although no new pathogenic strains of Erysipelothrix spp. have been identified in this work, the serotypes of isolates that have been recovered appear to be important. Data from investigations in herds by our group have demonstrated that seroptye 2 is associated more commonly with acute swine erysipelas outbreaks than is serotype 1. Several of these herds have been vaccinated for swine erysipelas; the vaccine strain has been serotyped as strain 1a; however the majority of the tissue isolates have been determined to be serotype 2. Additional work is warranted as vaccines currently available to United States swine producers may not be fully efficacious in protection against erysipelas. This work has shown a change in serotypes traditionally thought offas being associated with acute erysipelas and further investigation and evaluation of vaccine efficacy is needed.

Scientific Report

a. Materials and Methods

PCR development.For the development of the multiplex real-time PCR published sequences of Erysipelothrixrhusiopathiae, Erysipelothrixtonsillarum, Erysipelothrixspecies strain 1 and Erysipelothrixspecies strain 2 were aligned. Primer design focused on a non-conserved sequence between genotypes. After identification of such a sequence we designed two primer-probe combinations specific for Erysipelothrixrhusiopathiae and Erysipelothrixtonsillarum. All archived known serotypes were tested with the multiplex real-time PCR to determine specificity of the primer-probe combinations and the multiplex PCR was found to be 100% specific on these isolates. Further evaluation of the sensitivity of the assay on tissue samples from experimentally infected animals and field cases in pending.

Slaughterhouse condemnations. Tissue specimens with lesions representative of swine erysipelas responsible for carcass condemnations were identified and collected by the veterinary inspector-in-charge (IIC) at a regional slaughter facility. Tissues from the slaughter facility were collected and placed into individual specimen bags, labeled, and then frozen at -20ºC prior to transfer to our laboratory. Tissues that were collected included: tonsil, lung, heart, spleen, liver, kidney, skin, and lymph nodes.Received tissue specimens were refrigerated at a temperature of 4ºC to allow for bacterial culture. All specimens were saved at -80ºC for possible future use.

Broth Enrichment Technique. The Wood’s method consisting of an Erysipelothrixselective broth (ESB) was used to test all specimens for this investigation. ESB was prepared as indicated by Dr. Wood and dispensed aseptically into sterile tubes, stored at 4ºC, and used within the two-week time period.4 Tissue specimens were cut into sections to a size of 2 x 3cm using a sterile scalpel blade for each specimen. Sections were then placed into a sterile whirl-pak bag and 2mL of sterile 0.85% physiologic saline solution was added. The bag containing the tissue and saline was then placed into a Seward Stomacher® 80 Biomaster and stomached on the normal setting for three-minutes. Following removal from the stomacher, 300µm of fluid was removed via sterile pipette and added to the tube containing ESB. ESB test medium tubes were then incubated at 37ºC. At both 24 and 48 hours a subculture from the test medium was made onto a CNA (Gram-positive selective) and sodium azide-crystal violet agar as described by Packer (Packers medium).2 The inoculum was spread over a 1mm square area with a sterile loop, and then using a sterile wooden applicator was streaked for isolation. Inoculated plates were then incubated at 37ºC and observed for colonies with characteristics resembling Erysipelothrixspp. Suspect colonies were sub cultured to 5% sheep blood agar plates, incubated at 37ºC, and biochemically confirmed as Erysipelothrixspp.

b. Results and Discussion

The PCR assay was found to differentiate between all Erysipelothrixrhusipathiae and Erysipelothrixtonsillarum serotypes without cross-reacting with Erysipelothrixspecies type 2. The modification of the currently used gel-based differential PCR assay into a real-time multiplex PCR assay will be very useful for future diagnostic investigations as the assay sensitivity and specificity markedly improved.

Forty-eight cases of condemned samples have been received from a regional slaughterhouse facility and 37 of which have been diagnostically examined for the presence of Erysipelothrixspp. (the additional cases have been received in the past month and have not been fully characterized as of the submission deadline). Of these 37 cases, 32 have been successful in the isolation of Erysipelothrixspp. and have thus been determined to have been condemned for swine erysipelas. Table 1 provides the number of cases submitted, stage of lesion as reported by the veterinary inspector-in-charge at the slaughter facility, and the corresponding serotype isolated from the lesion. Lesion duration was determined by the veterinarian inspector-in-charge at the slaughter facility. Subacute/acute lesions were identified by superficial skin lesions, and chronic lesions were characterized by arthritic changes present in joints.

Table 1. Cases submitted and their corresponding lesion and serotype.

Case / Lesion / Isolate
Number / Duration / Serotype
12 / Subacute/Acute / 1a
17 / Subacute/Acute / 2
2 / Chronic / 2
1 / Chronic / 11

It has been reported that the serotypes most frequently found in disease outbreaks are of the species Erysipelothrixrhusiopathiae serotypes 1a, 1b, and 2. Serotypes 1a and 1b have been reported as being associated with acute erysipelas, whereas serotype 2 is often associated with the chronic form of the disease.5During the summer of 2001 an increase in the frequency of swine erysipelas occurred in the United States, and as reported by Opriessnig et al. the primary serotype involved (79.6%) was 1a.3 The majority of serotype prevalence investigations for swine erysipelas have been conducted outside of the United States, but it has been reported to involve all three major serotypes, 1a, 1b, and 2. In 2006,Eamens et al. investigated vaccine breakdowns and acute cases of erysipelas in Australia. Their work concluded that a shift occurred in the past 15-20 years implicating that serotype 1a was not the major cause of acute swine erysipelas, that in fact serotype 2 is the predominant factor.1 The serotypes from condemned tissues that have been collected in this investigation seem to support these observations. Initial data from other investigations in herds by our group have demonstrated that pigs diagnosed with acute swine erysipelas seem to support this conclusion as well. Serotype 2 is involved in acute swine erysipelas more often and not as previously reported serotype 1a. Several of these herds have been vaccinated for swine erysipelas and the vaccine has been serotyped as type 1a; however,the majority of our tissue isolates have been determined to be serotype 2. Additional work is warranted as vaccines currently available to United States swine producers may not be fully efficacious in protection against erysipelas. This work has shown thus far that a change in serotypes traditionally thought to be the cause of acute erysipelas may be occurring and further investigation is needed.

c. Tables and Figures

This has already been covered in section “b”.

d. References

1.Eamens GJ, Forbes WA, Djordjevic SP: 2006, Characterisation of Erysipelothrix rhusiopathiae isolates from pigs associated with vaccine breakdowns. Vet Microbiol 115:329-338.

2.Fidalgo SG, Riley TV: 2004, Detection of Erysipelothrix rhusiopathiae in clinical and environmental samples. Methods Mol Biol 268:199-205.

3.Opriessnig T, Hoffman LJ, Harris DL, et al.: 2004, Erysipelothrix rhusiopathiae: genetic characterization of midwest US isolates and live commercial vaccines using pulsed-field gel electrophoresis. J Vet Diagn Invest 16:101-107.

4.Wood RL: 1965, A selective liquid medium utilizing antibiotics for isolation of Erysipelothrix insidiosa. Am J Vet Res 26:1303-1308.

5. Yong-Jian S, Wei H: 1990, Proc Int Pig Vet Soc: 193

List of Presentations and Publications

Opriessnig T, Bender J, Irwin C, Halbur PG. Erysipelothrixrhusiopathiae: Characterization of recent outbreaks by using advanced diagnostic tools. Volume 1:137. 20th International Pig Veterinary Society Congress, Durban, South Africa, June 22-26, 2008.

Bender J, Kinyon J, Irwin C, Opriessnig T. Erysipelothrixrhusiopathiae: Comparison of diagnostic tools and characterization of recent outbreaks. 47th North Central Conference of Veterinary Laboratory Diagnosticians. Madison, Wisconsin, pp. 40-42, June 5-6. 2008.Award winning presentation:Veterinary Laboratory Diagnosticians Graduate Student Award 2008