1. Results Discussion
RAPD is a commonly used method to genotype organisms – it is relatively fast, simple,
and cost effective. However, there are disadvantages to this technique, including poor
reproducibility, and interpretation challenges due to subjectivity of visual comparisons.
Recently, techniques that have been reported that could be more sensitive and
reproducible than RAPD. These include: gene-targeted sequencing [6], and rRNA
Intergenic Spacer Region Sequence [7]. These methods appear to still be easy to run and
financially acceptable within a laboratory, but appear to be advanced in their ability to
be reproducible in other laboratories as well as more sensitive or targeted within the
samples to get further objective and defined data. Genotyping is crucial to
understanding relationships and linkages between strains being researched, so it is
fortunate that techniques in addition to RAPD have been discovered and refined to help
further understand these important characteristics of additional organisms as well as
Mycoplasma gallisepticum.
It was important to identify and compare the unknown strains of inoculum to known
strains of MG to determine their possible similarities or differences, to help further
understand the epidemiology of the organisms collected. Data collected from Fan
Primers was not clear enough to make a viable assessment of the inoculums collected
(Fig. 1), thus running an additional PCR using further primers was recommended. Geary
Primers were used, and showed a clearer association between lanes 7-14 (Fig. 2).
Geary Primers produce a smaller number of bands on an agarose gel than Fan Primers,
and so it makes sense that the banding patterns for all of the inoculums on the Geary
Primer gel could appear similar, but appear slightly different on the Fan Primers gel. Fan
Primers produce slightly more differentiation between the inoculums in each well, but
once looking at the data it is clear that these differences can be associated with the
location and time the samples were collected – all of the inoculums collected in CA in
2013 appear similar, while the inoculums in NC and VA appear to have a slightly
different banding pattern. It could be said, therefore, because more bands are produced
on this gel, that it is showing the slight changes that occur in the strains as they travel
over distance and time spreading from bird to bird, while the Geary Primer is proving
that the inoculums are all still linked to each other at their most basic level, and thus
verifying the ‘single strain’ hypothesis discussed earlier. A possible conclusion,
therefore, would be that inoculums collected in VA, NC and CA can be coupled with
known House Finch strains of MG.
K.C. Venters, J.M. Barahona, D.H. Ley
House Finch Mycoplasma gallisepticum Genotyping by Random Amplified
Polymorphic DNA (RAPD) Analysis
Materials and Methods
House finches with eye lesions were sampled for mycoplasma culture by conjunctival swabs.
Swabs from both eyes were inoculated to Frey’s broth supplemented with 15% swine serum (FMS)
or BD-Copan UTM (mycoplasma transport medium), transported to the laboratory at 4C, and then
incubated at 37C in FMS until growth was detected. Mycoplasma colonies on FMS agar were
identified as MG by immunofluorescence using species-specific fluorescein-conjugated rabbit
antiserum [3].
For RAPD genotyping, DNA was extracted and purified from broth cultures of each inoculum by
Qiagen kit, and then used in two previously described RAPD methods (5,6): the Geary method (5)
used a single primer, and the Fan method used 3 primers [4]. DNA and primers were added to a
‘master mix’ and placed in a thermocycler for PCR. Resulting amplicons were detected by
electrophoresis in 2% agarose gels post-strained with GelRed (a nucleic acid stain) [5], then viewed
and imaged using a BioRad Gel Imaging System. Seven inoculum test samples were run with DNA
base pair size standards (bp ladders), a reference strain (MG R-strain), three MG vaccine strains (F,
ts-11, 6/85), and the index house finch strain (VA1994) (Table 1).
Introduction
An emergence of conjunctivitis in wild house finches (Haemorhous mexicanus) was noted in 1994 beginning in
the mid-Atlantic states then spreading throughout central and western North America over the next several years
[1]. Initially, the etiology was unknown, but through a succession of diagnostic methods including histopathology,
mycoplasma culture, genotyping, and sequencing, it was determined that the cause was Mycoplasma gallisepticum
(MG). Random amplification of polymorphic DNA (RAPD), a PCR-based method of genotyping, showed that MG
isolates from house finches were similar to each other and different from poultry isolates. This suggested that the
ongoing outbreak in house finches was caused by a uniquely identifiable MG strain likely resulting from a single,
point source introduction [2]. RAPD and other genotyping methods can help place MG strains on a phylogenic tree
to help us understand the epidemiology of disease, and relationships among strains and their possible linkages,
sources, or mutations over time and distance [1]. Selected MG isolates from house finches made since 1994 had
been expanded in culture to make stocks for use as inoculums in experimental infections. Seven inoculums made
since 2013 had not been genotyped to check for identity compared to MG vaccine strains or the ‘house finch
strain’. Herein we report the results of RAPD genotyping to check strain identities of seven recent MG isolates from
house finches.
Fig. 1A. Lanes 3-6 are the individual reference and vaccine strains, and appear to have unique banding patterns from all other lanes represented on the gel.
Lanes 7 (known House Finch strain) and 8 (the first unknown strain from VA in 2013) appear similar in this gel, and lanes 11-14 (the last four unknown
inoculums from CA in 2013) appear similar to each other but slightly different in their banding pattern from 7-8. Both lanes 9 (NC 2014) and 10 (CA 2015)
appear slightly different from all other lanes on this gel.
Fig. 1B. Lanes 3-6 represent the reference and known vaccine strains, and so have their own unique banding patterns shown in this gel. Lane 7 is the known
house finch strain, and lanes 8-14 appear very similar to it in their banding patterns.
MG Inoculum Samples That Were Run Using Gel Electrophoresis
Lane Contained MG Inoculum Samples
1 DNA bp Ladder
2 Negative Control
3 MG R-strain
4 MG ts-11 Vaccine
5 MG 6/85 Vaccine
6 VA1994 House Finch index
7 VA2013 (2013.089-15-2P) 9/13/13
8 NC2014 (2014.120-1-2P) 8/14/15
9 CA2015 (2015/022-3-2P) 8/16/15
10 CA2015 (2015.022-3-2P) 8/16/15
11 CA2013a (2013.151-1-2P) 8/24/15
12 CA2013b (2013.080-1-3-P) 9/18/15
13 CA2013c (2013.081-1-3P) 9/19/15
14 CA2013d (2013.103-1-3PA) 9/17/15
15 DNA bp Ladder
Table 1. DNA bp Ladder represents DNA base pair size standards. MG R-strain is
a reference strain. MG F-strain, ts-11 strain, and 6/85 strain are all modified-live
vaccinations currently used in the poultry industry. The rest are the remaining
MG inoculums from the 20 free-ranging House Finch inoculums produced from
selected isolates since 1994. VA2013 2013.089-15 (2P) 9/13/13 identifies where
and when the sample was collected (VA2013), the accession number (2013.089),
the bird number (-15) of origin, the passage number (2P), and the date that the
stock was completed and frozen (9/13/13).
Figure 1A. M16S, S10L M13F Fan Primers.
Figure 1B. Geary 125 Geary Primers.
Lanes 1 and 15 = bp ladders, lane 2 = negative control,
lane 3 = MG R-strain, lane 4 = F-strain, land 5 = ts-11 strain,
lane 6 = 6/85 strain, lane 7= known House Finch strain 7994-1,
lanes 8-14 = unknown cultures.
RAPD Analysis of MG Inoculums,
Reference & Vaccine Strains
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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