Description of the Problem and Recent Related Research:
A marked increase in the number of dirty eggs has been reported among layers in Pennsylvania. Examination of several birds from some of these flocks has revealed a high prevalence of cecal spirochetes on Victoria Blue 4R stained cecal mucosal scrapings. The Serpulina-like spirochete has been recovered from cecal scrapings in relatively pure anaerobic culture on SBJ or CVS differential/selective media as shown from VB4R and Gram stained smears prepared from the primary culture (Figures 1 & 2). However, the organism will not subculture easily, making biochemical characterization of the organism difficult. Determining the species if Serpulina recovered is important and there are both pathogenic and commensal strains. Several Serpulina species-specific primers, including S. pilosicoli have been published and these organisms are described as the most likely agents of dirty egg syndrome. A new species of Serpulina, S. alvinipulli, pathogenic for chickens, has recently been described. Use of PCR to differentiate between potentially virulent Serpulina species and spirochetes that may be part of the normal cecal flora would enhance our ability to study this syndrome.
An Australian group of investigators has utilized sequences of the NADH oxidase gene (nox) to compare isolates. The Serpulina have been grouped into 4 subsets using primers NOX1 = S. hyodysenteriae, a pathogen of swine, NOX2 = S. intermedia and NOX3 = S. pilosicoli, a pathogen of both chickens and humans. The non-pathogenic groups, S. innocens and S. murdochii belong to group NOX4 (Atyeo et al.). A newly describe species of Serpulina that is pathogenic for chickens, S. alvinipulli, has been described by Stanton et al. If these primer sets are not able to differential between the species of Serpulina recovered, attempts will be made to compare the strains by restriction fragment length polymorphism (RFLP) or ribotyping. Once we are able to correctly identify the etiological agent, we can begin to address possible predisposing epidemiological factors such as diet, potential mammalian reservoirs such as swine, introduction by pests, and potential therapies.
A. Prepare primers and a suitable PCR protocol allowing us to identify the Serpulina species recovered from diagnostic submissions of birds from flocks experiencing dirty eggs.
B. Develop a standardized method for determining the antimicrobial susceptibility of the spirochete. In vivo testing may be employed if a suitable test flock can be identified.
C. Correlate the management practices, nutritional information and other elements of the case history that might indicate predisposing factors.
Development of PCR primers or a probe specific for the Serpulina species associated with dirty egg syndrome would allow us to study the prevalence of this difficult to culture microorganism.
Multiple samples of cecal contents, possible even manure, could be evaluated by PCR and enable us to examine specimens from flocks on different diets, with different genetic backgrounds, and confirm these or discover other predisposing factors.
A. Prepare primers and a suitable PCR protocol allowing us to identify the Serpulina species recovered from diagnostic submissions of birds from flocks experiencing dirty eggs. The NOX primers (1-4) will be synthesized at the PSU Biotechnology Institute based upon the published sequences. The newly defined S. alvinipulli species will be screened to determine if the NOX primers can differentiate the new species. If not, degenerate primers will be designed based upon conserved sequences, the S. alvinipulli NADH oxidase region of the genome will be sequenced, and new S. alvinipulli specific primer pairs will be selected.
B. Develop a standardized method for determining the antimicrobial susceptibility of the spirochete. In vivo testing of the antimibrobial agents may be employed if a suitable test flock can be identified. Brain Heat Infusion Broth supplemented with 7-10% fetal calf serum has been reported to support the growth of several Serpulina species. If this media will successfully support the growth of the avian cecal spirochetes, the quality control and quality assurance organisms must be tested to determine if this is an acceptable means of antimicrobial susceptibility testing. A minimal inhibitory concentration broth dilution format will be used, performed under anaerobic conditions. If a suitable test flock can be identified in the field, hens will be brought to Wiley Biocontainment facility and housed in battery cages. Different antibiotic treatment groups will be established and the efficacy measured by cecal moisture content as well as the microbiology parameters listed below.
C. A complete history including ration formulation, genetic line of birds, recent history of stresses such as H2O changes, treatments for coccidia, antibiotic therapy, feed changes, or any other predisposing factors will be examined.
Laboratory Testing: 6-10 birds will be necropsied and ceca removed for examination. Sections will be taken and fixed for histological examination following hematoxalin and eosin staining. Another ceca will be submitted to the bacteriology laboratory at PSU/ADL for culture and microscopic examination following Victoria Blue 4 R (VB4R) staining of cecal mucosal scrapings. The cecal mucosal scrapings will also be plated in CVS and SBJ media incubated anaerobically. The number of culture positive specimens and a quantitative assessment of the anaerobic growth will be recorded. Also, a quantitative assessment of the number of spirochetes observed in the VB4R stained slides will also be recorded. Tabulative statistics, regression analysis and analysis of variance will be performed using Mini-Tab to determine if there is a correlation between the histopathology, microbiology and management factors.
Brief Description of the Roles of Individual Investigators:
Dr. Patty Dunn will act as the lead diagnostician, evaluating gross pathology and histopathology. She will conduct the in vivo antimicrobial susceptibility study in collaboration with Dr. David Kradel.
Dr. Chobie DebRoy will oversee the anaerobic culture, susceptibility testing and PCR analysis.
Dr. David Kradel will gather the history, ration, and other management data from the producers, and assist with the in vivo antimicrobial susceptibility testing.
The first six months of the study will be needed to prepare and evaluate the PCR primers and antimicrobial susceptibility techniques. The next 12 months we will examine birds from affected flocks, record and evaluate management data. The final 6 months will be devoted to evaluation of the data and the in vivo antimicrobial susceptibility testing.
Atyeo, R. F., T. B. Stanton, N. S. Jensen, D. S. Suriyaarachichi, and D. J. Hampson. 1999. Differentiation of Serpulina species by NADH oxidase gene (nox) sequence comparisons and nox-based polymerase chain reaction tests. Vet. Microbiol. 67(1):47-60).
Stanton, T. B., D. Postic, and N. S. Jensen. 1998. Serpulina alvinipulli sp. nov., a new Serpulina species that is enteropathogenic for chickens. Inst. J. Syst. Bacteriolo. 49(pt 3):669-676.
Budget: $33,244 24 months
Budget Justification:
The greatest expenditures will be associated with the time required to develop and evaluate the primer pairs for PCR and to develop an assay for antimicrobial susceptibility testing. A skilled technician will be required to assist with these efforts. No additional equipment will be required for this effort.
