Detection of mycoplasmas in goat milk by flow cytometry.

Abstract

The detection of mycoplasma in milk can be performed by either culture techniques or polymerase chain reaction (PCR) based methods. Although PCR can reduce the average diagnostic time to 5 h in comparison with the several days for the isolation of the agent, there is still a need to develop methods, which could give earlier results. For this purpose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk samples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large-colony type, known to cause contagious agalactia in goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat milk samples, from which mycoplasmas have been isolated in broth medium were also analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it was not possible to distinguish between the different mycoplasma species. In our conditions, the detection limit of the technique was of the order of 10(3)-10(4) cells ml(-1). Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC together with SYBR Green I was able to distinguish between mycoplasma cells and debris present in milk samples and gave results in 20-30 min. This is an important first step in developing a robust, routine flow cytometric method for the detection of mycoplasmas in milk samples.

Full text

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Detection of Mycoplasmas in Goat Milk
by Flow Cytometry
Patricia Assunc ¸a ˜o,1*Hazel M. Davey,2Ruben S. Rosales,1Nuno T. Antunes,1
Christian de la Fe,1Ana S. Ramirez,1Carlos M. Ruiz de Galarreta,3Jose B. Poveda1
? Abstract
The detection of mycoplasma in milk can be performed by either culture techniques or
polymerase chain reaction (PCR) based methods. Although PCR can reduce the average
diagnostic time to 5 h in comparison with the several days for the isolation of the agent,
there is still a need to develop methods, which could give earlier results. For this pur-
pose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk sam-
ples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae,
Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma
mycoides subsp. mycoides large-colony type, known to cause contagious agalactia in
goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat
milk samples, from which mycoplasmas have been isolated in broth medium were also
analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it
was not possible to distinguish between the different mycoplasma species. In our condi-
tions, the detection limit of the technique was of the order of 103–104cells ml21.
Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC to-
gether with SYBR Green I was able to distinguish between mycoplasma cells and debris
present in milk samples and gave results in 20–30 min. This is an important first step in
developing a robust, routine flow cytometric method for the detection of mycoplasmas
in milk samples.
' 2007 International Society for Analytical Cytology
? Key terms
mycoplasma; flow cytometry; SYBR Green I; contagious agalactiae; milk
CONTAGIOUS agalactia of small ruminants is a disease that is notifiable to the World
Organization for Animal Health (OIE, old B list) and is responsible for causing severe
economic losses in goat- and sheep-farms. It has a worldwide distribution, being
endemic in the Mediterranean area and in certain African and Asian regions (1–3).
The main causative agent of the disease in sheep and goats is Mycoplasma agalactiae,
however, contagious agalactia can also be caused by Mycoplasma putrefaciens, Myco-
plasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large-
colony type (1). Contagious agalactia causes a variety of clinical syndromes like mas-
titis, arthritis, kerato-conjunctivitis and, occasionally, abortion (1,3). Taken together,
this disease has been estimated to cause annual losses in excess of $30 million in Eu-
ropean countries around the Mediterranean (4) and consequently methods for con-
firming suspected infections are required.
Early detection of infection, or the potential for infection, gives the opportunity
to control further spread of the disease with vaccines or antibiotics (1).
The detection of mycoplasmas in goat milk can be performed by culture techni-
ques or by polymerase chain reaction (PCR) based methods (1,5,6). Culture techni-
ques are the most common but have as a major disadvantage the long time required
to obtain results (several days), making them time consuming and labour demand-
ing. In addition, problems can occur due to growth of contaminants that out-com-
pete the more slowly-growing mycoplasmas. PCR based methods offer a substantial
1Unidad de Epidemiolog? ıa y Medicina
Preventiva, Facultad de Veterinaria,
Universidad de Las Palmas de Gran
Canaria, Arucas, Spain
2Institute of Biological Sciences,
University of Wales, Aberystwyth,
Ceredigion, SY23 3DD Wales, UK
3Departamento de Bioqu? ımica, Biolog? ıa
Molecular y Fisiolog? ıa, Centro de
Ciencias de La Salud, Universidad de
Las Palmas de Gran Canaria, Las
Palmas, Spain
Received 6 June 2007; Accepted 31
August 2007
This article contains supplementary
material available via the Internet at
http://www.interscience.wiley.com/
jpages/1552-4922/suppmat.
Grant sponsor: Canarias Government;
Grant number: IDT-LP-04/016.
*Correspondence to: Patr? ıcia Assunc ¸~ ao,
Unidad de Epidemiolog? ıa y Medicina
Preventiva, Facultad de Veterinaria,
Universidad de Las Palmas de Gran
Canaria, Trasmonta~ na s/n 35416,
Arucas, Las Palmas, Spain
Email: passuncao@becarios.ulpgc.es
Published online 30 October 2007 in Wiley
InterScience (www.interscience.
wiley.com)
DOI: 10.1002/cyto.a.20476
© 2007 International Society for
Analytical Cytology
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advantage in that they reduce the average diagnostic time to
about 5 h (6). Positive results from PCR assays enable a full
investigation to take place, however negative results should
not be considered definitive.
Flow cytometry (FC) is a sensitive technique, which
avoids the need for culturing and can be both qualitative
and quantitative (7). FC has been used to detect, enumerate
and differentiate several bacteria with a combination of fluo-
rescent stains, antibodies, or oligonucleotide probes (7–9).
The detection, enumeration, and determination of viability of
mycoplasmas in laboratory culture have been demonstrated
(10,11), however, we are not aware of any efforts to detect,
enumerate, or differentiate mycoplasmas in milk samples by
FC.
The aim of the present study was thus to develop a
method based on FC in order to detect total mycoplasmas in
goat milk samples.
MATERIALS AND METHODS
Strains and Culture Conditions
The reference strains of M. mycoides subsp. mycoides
large-colony type (LC), M. agalactiae (Ma), M. putrefaciens
(Mp), and M. capricolum subsp. capricolum (Mcc) were ob-
tained from the National Collection of Type Cultures (NCTC,
United Kingdom). Furthermore, a field strain of Staphylococ-
cus aureus isolated from a clinical case (goat milk) submitted
to the Department of Epidemiology and Preventive Medicine
of the Las Palmas de Gran Canaria University (Spain) was also
used to show that mycoplasmas could be differentiated from
larger bacteria associated with similar infections. Mycoplasmas
were propagated in PH broth medium under aerobic con-
ditions (12) and S. aureus was propagated in Blood Agar
Medium (Oxoid), both at 378C for 24 h.
Milk Samples
Mycoplasmas [108–103Colony Forming Units (CFU)
ml21] and S. aureus (107CFU ml21) were inoculated sepa-
rately or together into goat milk (collected from healthy ani-
mals from Las Palmas Veterinary Faculty experimental farm,
Spain), or an infant milk formula (Nestle ´, Switzerland), which
was reconstituted in sterile water following the instructions of
the manufacturer. The infant milk formula was used in order
to optimise the method. In addition, three goat milk samples
that had been remitted to our lab for routine diagnostics,
from which Mycoplama spp. had been detected using culture-
based techniques, were also analyzed by FC. The number of
CFU was determined as described elsewhere (13).
Fluorescence Labeling and Flow Cytometric Analysis
Milk samples (100 ll) were diluted to 1 ml with sterile-
filtered saline solution (0.85% NaCl) and stained (15 min at
room temperature in the dark) with the cell-permeant DNA-
fluorochrome Sybr green-I (SYBR, Amresco Inc., Ohio) used
at a final concentration 1:5,000 (vol/vol) of the commercial
stock solution. Neither the molecular weight nor the chemical
formula are provided by the manufacturer, however it is
reported to be [2-[N-(3-dimethylaminopropyl)-N-propyl-
amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methyl-
idene]-1-phenyl-quinolinium] (14). Before FC analysis, to
avoid coincidence, the samples were further diluted between
4 and 4,000 times in a 0.85% NaCl solution, in order to main-
tain the flow-rate below 2,000 events s21.
Sample analysis was performed in a Coulter Epics XL-
MCL flow cytometer (Coulter, Miami, FL) equipped with an
air-cooled 488 nm argon-ion laser (15 mW output). Each cell
was characterised by three optical parameters: Side-Angle-
Scatter (SSC), Forward angle scatter (FSC) and green fluores-
cence for SYBR (525 ? 20 nm, FL1 detector) and data were
acquired on a four-decade logarithmic scale. Green fluores-
cence from SYBR was collected combining a 550 dichroic long
filter and a 525 band pass filter.
Optical alignment was based on an optimised signal from
10 lm fluorescent beads (Flow-check, Beckman-Coulter Inc.,
Fullerton, CA). For absolute counts we used the Coulter Fix
Volume System analysis and the discriminator was set on
green fluorescence (FL1). The number of cells counted was
then converted to cells ml21.
Data Analysis
Data were analyzed with the SYSTEM II software
(Coulter, Miami, FL) and the WinMDI software version 2.8
(Joseph Trotter, The Scripps Research Institute La Jolla, CA).
SPSS (Statistical Package for Social Science) version 12.0 was
used for the statistic analysis; data were analyzed by least
squares linear regression.
RESULTS
In this study, we investigated the potential of FC in com-
bination with a fluorescent dye (SYBR) to rapidly detect and
enumerate mycoplasmas in goat milk samples.
In these experiments, we inoculated the different species
of mycoplasmas in the infant milk and in goat milk samples,
before staining them with SYBR and performing flow cyto-
metric analysis. Samples of each of the pure cultures of myco-
plasmas were also analyzed by FC. Results show that myco-
plasma cells stained with the nucleic acid-specific SYBR dye
were easily distinguished from the debris that was present in
the culture medium or the milk samples. Infant milk and
mycoplasma cultures demonstrated less background noise
than goat milk samples when analyzed by FC, however, even
the higher background present in the goat milk did not inter-
fere with the detection of mycoplasmas. Analysis of clinical
samples known to contain mycoplasmas gave similar results
(Fig. 1).
To assess the sensitivity of the FC method we performed
additional experiments using tenfold dilutions of the different
mycoplasma cultures in milk samples. Data derived from flow
cytometric analysis using dual parameter contour plots of FL1
vs. SCC demonstrated that the detection limit for mycoplasma
cells stained with SYBR was in the order of 103–104cells ml21.
A good correlation (r25 0.96) was obtained between FC and
plate count method.
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Furthermore, in order to validate the FC method, a field
strain of S. aureus was inoculated into milk samples to deter-
mine whether the method was capable of distinguishing myco-
plasmas from other bacteria. As shown by the data in Figure 2,
there is clear evidence that under these conditions mycoplas-
mas could be easily distinguished from S. aureus by the optical
parameters FSC together with SSC and green fluorescence
(SYBR) together with FSC or SSC. Using the flow cytometric
method described results were obtained in \30 min, since,
after a 20 min incubation with SYBR, the flow cytometric
analysis takes approximately 1 min.
DISCUSSION
Techniques based on flow cytometric principles are routi-
nely applied in the dairy industry for measuring the total bac-
terial count within a few minutes (7,8). However, so far, there
aren’t any reports of flow cytometric methods for the detec-
tion of mycoplasmas in milk samples. This may be due to the
fact that mycoplasmas are the smallest self-replicating micro-
organisms known (15), and this may have discouraged
researchers from analysing these microorganisms using flow
cytometric techniques. The small size and correspondingly
lower concentrations of cellular constituents of mycoplasmas
results in smaller optical signals that are more difficult to
resolve from the background noise than those obtained from
mammalian cells or even from larger microorganisms.
In this study, we demonstrate by flow cytometric analysis,
that the four species of mycoplasmas (LC, Ma, Mcc, and Mp),
known to cause contagious agalactiae in goats, can be easily
resolved against the background debris present in both infant
formula and goat milk without any previous treatment of the
samples. Furthermore, we show that the method is capable of
distinguishing between mycoplasmas and S. aureus. In previ-
ous studies related to the detection of bacteria, researchers had
to perform a pre-treatment of the milk samples before being
analyzed by FC (7,8,16–20). Gunasekera et al. (7) reported
that before Escherichia coli and S. aureus could be detected in
milk samples, a prior treatment with proteinase K or savinase
for UHT milk samples and of savinase plus Triton X-100 for
raw milk samples was necessary. Initially, we investigated these
protocols, but we found that these treatments also caused loss
of the mycoplasma cells, thus preventing their detection by
FC. Nonetheless, as shown above we have found that by using
SYBR mycoplasmas can be readily detected without recourse
to time-consuming pre-treatments and thus the FC-based
assay is not only quicker than culture-based methods but also
more rapid than PCR-based techniques.
S. aureus is the most frequently isolated pathogen known
to be involved in mammary infections of goats (21–25). Con-
sequently, to be a robust method, any protocol for identifying
mycoplasma infection of milk must be capable of distinguish-
ing between Mycoplasma spp. and S. aureus. As shown by the
results in infant milk, the differentiation between mycoplas-
mas and S. aureus is possible due to the differences in size
(FSC), complexity (SSC), and fluorescence due to SYBR stain-
ing (FL1). In the future it may be possible to improve the
methods described here by the use of specific probes such as
fluorescently labelled antibodies or oligonucleotides to effec-
tive diagnosis at the generic or species level. However, suitable
probes that are both reliable and brightly fluorescent are not
yet available commercially for this purpose. In any case, it is
likely that an initial screen of the type described here would be
a useful screening step prior to the use of more costly and
time consuming specific assays.
In the analyses reported here, data were collected for 1
min and this corresponds to 20 ll of sample. This approach
resulted in a detection limit of the order of 103–104cells ml21.
The detection limit could be improved by analyzing a larger
sample volume however it is unlikely that concentrations
below 100 bacteria ml21could be detected since FC is not
adapted to the detection of rare events (26). Nevertheless, Cai
et al. (27) found that when using real-time PCR to detect
Mycoplasma bovis in clinical manifestations of bovine mastitis,
the M. bovis culture-positive milk samples were estimated to
contain between 5 3 104and 7.7 3 108cells ml21. These
values are in the range of detection of mycoplasmas in milk
samples by the FC method thus indicating that the sensitivity
of the method is appropriate for diagnostic purposes.
Figure 1. Dual parameter dot plot of green fluorescence (FL1) versus side angle scatter (SSC) of goat milk samples of three clinical cases
from which Mycoplasma spp. was detected by cultured based techniques. SSC and FL1 data were acquired in a four-decade logarithmic
scale. Mycoplasma spp. (M); Debris (D). FC Mycoplasma spp. cell counts: 1.1 3 107counts ml21(A); 8.1 3 107counts ml21(B); 9.6 3 104
counts ml21(C).
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1036 Mycoplasma Detection in Milk by FC

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Furthermore, it is expected that a slight underestimation
of total counts will be obtained by the flow cytometric analysis
method since mycoplasma clumps will be enumerated as one
unit in FC analysis. This phenomenon also occurs with the
traditional plate count method that is used for this purpose
and with which we compared our flow cytometric results.
It is important to note that the flow cytometric method
presented here does not distinguish between Mycoplasma spe-
cies. This is not considered problematic from a veterinary
viewpoint as knowledge of the species of mycoplasma involved
in the infection process is not important for the establishment
of adequate treatment and control measures.
Figure 2. Differentiation of cultures of mycoplasmas (a mix of the four species at a concentration of 107CFU ml21) from S. aureus (107CFU
ml21) in infant milk by different optical parameters (FSC, SSC, and FL1). FSC, SSC, and FL1 data were acquired in a four-decade logarithmic
scale. Infant milk alone (A), mycoplasmas (B), S. aureus (C), mycoplasmas and S. aureus (D).
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In conclusion, this study represents to our knowledge,
the first step in developing a routine FC based method for the
detection of total mycoplasmas in milk samples. We have
demonstrated the ability of FC to discriminate mycoplasmas
from other bacteria and from debris in milk. The main advan-
tage of the flow cytometric approach, when compared with
culture-based or PCR-based methods, is the increased speed
with which results are available. The flow cytometric approach
allows results to be obtained in 20–30 min. Since the micro-
biological content of raw milk affects quality, shelf life and
safety of milk and dairy products (28), our development of a
method for the detection of bacteria in milk that does not
require time-consuming pre-treatments may have wider
applicability in the dairy industry and for monitoring the gen-
eral health status of livestock. Furthermore, the need for
methods of screening herds for mycoplasma infection is likely
to increase as a consequence of a perceived bioterrorism
threat. Contagious bovine pleuropneumonia, a highly infec-
tious disease of cattle and buffalo which is caused by M.
mycoides subsp. mycoides small-colony type has been identi-
fied as a potential agroterrorism threat (29) and it is likely
that the flow cytometric method would be appropriate for
detection of this pathogen.
ACKNOWLEDGMENT
We thank Esther Dı ´az for her technical assistance.
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1038Mycoplasma Detection in Milk by FC