Diagnosis of Mycoplasma Mastitis: Validation and Development

Home , Acholeplasma laidlawii, Anaeroplasmatales, Mycoplasma bovis

DIAGNOSIS OF MYCOPLASMA MASTITIS: VALIDATION AND DEVELOPMENT

SUKOLRAT BOONYAYATRA

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY College of Veterinary Medicine

DECEMBER 2010

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of

SUKOLRAT BOONYAYATRA find it satisfactory and recommend that it be accepted.

______Lawrence K. Fox, Ph.D., Chair

______Thomas E. Besser, Ph.D.

______Ashish Sawant, Ph.D.

______John M. Gay, Ph.D.

ACKNOWLEDGMENTS

I would like to thank Dr. Larry Fox, committee chair, for his guidance, support and encouragement of working with mycoplasma mastitis. Additionally, I am very grateful for his acceptance when I transferred from Tennessee. I thank my other committee members, Dr. John

Gay, Dr. Ashish Sawant, and Dr. Tom Besser for their guidance and suggestions on the various aspects and helping me solve many problems with my work.

This thesis dissertation would not have been possible without many people who provided technical assistance in the laboratory. I am very thankful to Dorothy Newkirk for her expert assistance. I also thank to other FDIU staffs for their suggestion and helping me go through all lab work smoothly. I want to thank Dr. Ziv Raviv and Dr. Amy Wetzel for their kindness of teaching me everything about real-time PCR when I was in the Ohio State University.

I am grateful to Dr. Stephen Oliver, other professers and staffs in the University of

Tennessee in Knoxville for providing me a wonderful time in my life when I was in this volunteer state. I want to sincerely thank to the Royal Thai government for the scholarship which allowed me to have this great experience in the United States.

I thank my husband, Veerasak Punyapornwithaya, for helping me solve many problems and being such a good husband and friend. I want to thank my daughter, Minnie, for being my biggest motivation of finishing the research. I am deeply grateful to my parents and sisters for their encouragement and understanding for everything I have done.

Finally, I would like to thank all people who contributed to the successful of all the work in this thesis dissertation.

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DIAGNOSIS OF MYCOPLASMA MASTITIS: VALIDATION AND DEVELOPMENT

Abstract

by Sukolrat Boonyayatra, Ph.D.

Washington State University

December 2010

Chair: Lawrence K. Fox

Microbiological culture of milk samples has been used as a standard diagnosis for

mycoplasma mastitis. It is suggested to perform with fresh samples for optimum diagnosis.

Submission of fresh samples is often difficult given the logistics of collection and shipping of

milk samples from farm to laboratory. Therefore , milk samples are usually chilled or frozen

before culture. A study of the effects of storage methods on the recovery of Mycoplasma species

from milk samples was performed. The results indicated that holding milk at refrigerated

temperatures (5°C) for 5 days and freezing milk samples (-20°C) lowers the number of recovered

Mycoplasma species . Moreover the addition of glycerol prior freezing to achieve 10% and 30%

(v/v) solutions was found to improve the recovery of Mycoplasma species from frozen milk samples.

Even though a Mycoplasma -like-colony is observed by a standard culture method, the diagnosis can be misinterpreted as Acholeplasma species which is indistinguishable by culture.

To validate and suggest techniques used to discriminate between these two genera, a study on the discrimination between Mycoplasma and Acholeplasma using digitonin and nisin disc diffusion assays, and PCR was performed. Findings revealed a high and comparable efficiency of using

nisin and digitonin disc diffusion assays and PCR to distinguish Mycoplasma and Acholeplasma species.

Given the fastidious nature of Mycoplasma species, and the time-consuming nature of the standard culture method, a diagnostic test of mycoplasma mastitis that is more sensitive, less time consuming, and can speciate mycoplasma mastitis pathogens would be valuable. The development of real-time PCR assays to detect 3 major mycoplasma mastitis pathogens: M. bovis, M. californicum and M. bovigenitalium was investigated to provide an alternative diagnostic tool for mycoplasma mastitis. The results given by the novel real-time PCR assays showed a perfect agreement with the gold standard, and the results were obtained within 4-5 hours.

The overall goal of studies reported herein was to investigate improvements in the efficiency of the diagnosis of mycoplasma mastitis in dairy cows. Findings indicate that both phenotypic and genotypic diagnostic techniques can be applied, and in conjunction with current standard techniques, will better identify cows with mycoplasma mastitis.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENT...... iii

ABSTRACT ...... iv

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

CHAPTER 1

1.1. INTRODUCTION ...... 1

1.2. REFERENCES ...... 4

CHAPTER 2 (LITERATURE REVIEW)

2.1. MYCOPLASMA SPECIES ...... 7

2.2. MYCOPLASMA MASTITIS IN DAIRY CATTLE ...... 8

2.3. EFFECT OF STORAGE METHODS ON SURVIVAL OF MYCOPLASMA 10

2.4. DISCRIMINATION BETWEEN MYCOPLASMA AND ACHOLEPLASMA 12

2.5. DIGITONIN ...... 13

2.6. NISIN ...... 14

2.7. NESTED PCR TECHNIQUE ...... 16

2.8. SPECIATION OF MYCOPLASMA SPECIES ...... 17

2.9. REAL-TIME PCR ...... 19

2.10. CONCLUSION AND HYPOTHESIS ...... 23

CHAPTER 3

3.1. INTRODUCTION ...... 44

3.2. MATERIALS AND METHODS ...... 46

3.3. RESULTS ...... 47

3.4. DISCUSSION ...... 48

3.5. ACKNOWLEDGMENT...... 51

3.6. REFERENCES ...... 52

CHAPTER 4

4.1. INTRODUCTION ...... 56

4.2. MATERIALS AND METHODS ...... 58

4.3. RESULTS ...... 62

4.4. DISCUSSION ...... 65

4.5. ACKNOWLEDGMENT...... 68

4.6. REFERENCES ...... 68

CHAPTER 5

5.1. INTRODUCTION ...... 80

5.2. MATERIALS AND METHODS ...... 81

5.3. RESULTS ...... 87

5.4. DISCUSSION ...... 89

5.5. ACKNOWLEDGMENT...... 92

5.6. REFERENCES ...... 93

CHAPTER 6

CONCLUSIONS...... 106

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APPENDIX

A. RAW DATA

A.1: Chapter 3 (Refrigeration experiment) ...... 108

A.2: Chapter3 (Freezing experiment) ...... 118

A.3: Chapter 4 and 5 ...... 131

B. METHODS

B.1: Cloning and filtration techniques for Mycoplasmas ...... 143

B.2: DNA extraction and purification using Purelink  Genomic DNA kit 144

B.3: PCR to amplify 16S-23S rRNA intergenic spacer region of Mycoplasma species

and Acholeplasma species ...... 146

B.4: Restricted fragment length polymorphism (RFLP) ...... 147

B.5: PCR to amplify 16S rRNA genes using universal primers ...... 147

B.6: Gel electrophoresis ...... 148

B.7: Real-time PCR using Quantitect  Probe PCR reaction kit for Applied

Biosystem StepOne Plus real-time PCR machine...... 149

B.8: Digitonin disc diffusion assay ...... 150

B.9: Nisin disc diffusion assay ...... 151

C. COPY RIGHT RELEASES ...... 152

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LIST OF TABLES

CHAPTER 2

1. Taxonomy of the class of Mollicutes ...... 38

CHAPTER 3

1. Mycoplasma counts from fresh and refrigerated milk samples ...... 54

2. Mycoplasma counts from fresh and frozen milk samples with various concentrations of glycerol addition ...... 55

CHAPTER 4

1. Sources of reference strains of Mycoplasma species and Acholeplasma species ...... 72

2. Sequences of primers and targeted regions ...... 73

3. Distribution of isolates of Mycoplasma species and Acholeplasma species as identified by 16S

rRNA partial sequencing...... 74

4. Descriptive statistics of zone of growth inhibition (mm) observed with the digitonin disc

diffusion assay ...... 75

5. Agreement of genera determination by digitonin disc diffusion assay with 16S rRNA partial

sequencing...... 76

CHAPTER 5

1. Mycoplasma species and other bacterial species tested for specificity of the novel real-time

PCR assays ...... 96

2. Sequences of primers and probes used in the study and their targeted regions ...... 98

3. Distribution of isolates of Mycoplasma species and Acholeplasma species as identified by

16S rRNA partial sequencing ...... 99

4. Identification of Mycoplasma species and Acholeplasma species isolates using PCR-RFLP

and real-time PCR ...... 100

LIST OF FIGURES

CHAPTER 2

1. Chemical structure of digitonin ...... 39

2. Structure of nisin A ...... 40

3. General model of the ‘barrel stave’ mechanism of pore formation by peptides ...... 41

4. Models of pore formation by nisin...... 42

5. Step-wise reaction of real-time PCR using a fluorogenic probe...... 43

CHAPTER 4

1. Example plate of Mycoplasma species with a zone of inhibition only presented around

digitonin disc ...... 77

2. Example plate of Acholeplasma species with a zone of inhibition only presented around nisin

discs with both 103.2 µg (N0) and 10.32 µg (N-1) ...... 78

3. Electrophoretic gel of amplicons of some reference strains of Mycoplasma species and

Acholeplasma species ...... 79

CHAPTER 5

1. Electrophoretic gel of amplicons before digestion and after digestion by ASE1 ...... 101

2. Digested amplicons obtained from 6 M. bovigenitalium isolates ...... 102

3. Standard curve of M. bovis real-time PCR assay using pIDTSMART-AMP plasmids cloned

with the target sequence in fusA gene of M. bovis ...... 103

4. Standard curve of M. bovigenitalium real-time PCR assay using pIDTSMART-AMP

plasmids cloned with the target sequence in 16S-23S intergenic spacer region of M.

bovigenitalium ...... 104

5. Standard curve of M. californicum real-time PCR assay using pIDTSMART-AMP plasmids

cloned with the target sequence in rpoB gene of M. californicum ...... 105

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Dedication

This dissertation is dedicated to my father, Surachart Boonyayatra, my mother, Urai

Kwinram and my sisters, Aree and Kanyarpak Boonyayatra, for their support since I came to the

United States. I would also like to dedicate this dissertation to my husband, Veerasak

Punyapornwithaya, and my lovely daughter, Ruangkhao (Minnie) Punyapornwithaya, for their encouragement and support as a family for the past three years in Pullman.

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CHAPTER 1

INTRODUCTION

Mycoplasm a species are Mollicutes known to be able to cause clinical and subclinical mastitis in dairy cattle. Mastitis associated with Mycoplasma has been reported worldwide (Fox

et al., 2005). Numerous species of Mycoplasma have been reported to be associated with mastitis

in dairy cattle. Among all reported cases, Mycoplasma bovis, M. californicum, M. alkalescens,

M. bovigenitalium and M. canadense are the species most commonly associated with bovine

mastitis (Gonzalez and Wilson, 2003). Mycoplasma is considered a contagious pathogen, capable

of transmitting easily from cow-to-cow (Gonzalez and Wilson, 2003) and treatment is usually

unsuccessful (Bushnell, 1984). Therefore, control of mycoplasma mastitis may be best achieved through the identification and segregation or culling of infected cows, which are the infection reservoirs (Fox et al., 2005). Mycoplasma species are difficult to isolate which makes diagnosis of infection challenging. Because specialized media, Modified Hayflicks, and incubation for several days in 10% CO 2 are required (Hogan, 1999), many laboratories may not routinely culture milk for mycoplasma organisms. Additionally, cows with mycoplasma mastitis may shed the pathogen intermittently and shedding of Mycoplasma species into milk of infected cows may be as low as <100 colony forming unit (CFU)/ml, below the threshold of detection by standard culture methods (Biddle et al., 2003). These difficulties increase the importance of proper milk sample handling before culture in an effort to avoid false negative results. Most milk samples are either stored in a refrigerator with the temperature approximately 4 to 7 °C or kept frozen with the temperature approximately -18 to -20 °C before culturing in the laboratory. A study has shown that freezing milk cultures can be detrimental to the recovery of mycoplasma mastitis

pathogens (Biddle et al., 2004), resulting in false negative reporting of Mycoplasma species

positive milk samples. However, the effect of storing milk samples at refrigeration temperature

on survival of Mycoplasma species has never been reported. Moreover, application of a

cryopreservative such as glycerol may be able to improve the recovery of Mycoplasma species

from frozen milk samples. Therefore, effects of storing milk samples at a refrigeration

temperature (5 °C) and effects of adding glycerol to milk samples prior freezing (-20 °C) should

be determined.

Even with cultivable samples, identification of Mycoplasma species can still be confused

with other Mollicutes especially Acholeplasma species. Although Acholeplasma species can

cause mastitis (Pan and Ogata, 1969; Counter, 1978), prevalence of clinical mastitis with this

microbe is very rare and thus its appearance is generally regarded as a contaminant. The

digitonin disc diffusion technique is the standard method to discriminate Mycoplasma from

Acholeplasma when culture is used (Gardella et al., 1983). The principle of this technique is

based on the requirement of sterol for growth by Mycoplasma species. Digitonin can interact with cholesterol to increase membrane permeability and consequently kill Mycoplasma .

Acholeplasma species are less fastidious with respect to their sterol requirement and are more resistant to digitonin. Thus digitonin can be used to distinguish Acholeplasma species from

Mycoplasma species by measuring differences in resistance to the same concentration. In addition to the digitonin disc diffusion assay, utilization of an antimicrobial peptide, nisin can also be used to differentiate between Acholeplasma species from Mycoplasma species when culture techniques are applied. Nisin is an antimicrobial peptide produced by Lactococcus lactis .

Mycoplasma species and Acholeplasma species have different sensitivities to the peptide (Abu-

Amero et al., 1996). Nisin can inhibit growth of Acholeplasma species whereas no growth

inhibition is observed for Mycoplasma species.

Mycoplasma species and Acholeplasma species can be distinguished using genomic

analysis, specifically a duplex-nested polymerase chain reaction (PCR) method for detection has

been reported (Tang et al., 2000). Based on the presence of >1 operon of tRNA genes in the

targeted 16S-23S rRNA intergenic spacer region of A. laidlawii , they were able to discriminate

A. laidlawii from other Mycoplasma species. Even though all these 3 techniques; digitonin disc diffusion assay, nisin disc diffusion assay, and duplex-nested PCR, have been used to distinguish between cultures of these species, no study has been completed to demonstrate a side by side comparison to demonstrate assay differences in sensitivities and specificities of detection. A validation of techniques used to discriminate Mycoplasma species from other Mollicutes is

needed to avoid false positive results for mycoplasma mastitis.

To overcome problems with the standard culture techniques, such as length of culture

time and loss of viable organisms with storage, many PCR-based strategies have been developed

for mycoplasma mastitis diagnosis. Most of these studies have focused only on the identification

of M. bovis in milk samples (Ghadersohi et al., 1997; Hayman and Hirst, 2003; Hotzel et al.,

1999; Hotzel et al., 1996; Pinnow et al., 2001). A nested PCR followed by restricted fragment

length polymorphism (RFLP) (Tang et al., 2000) has been applied to speciate cultures of

Mycoplasma -like-colonies in our laboratory. Genus differentiation of Mycoplasma and

Acholeplasma species can be performed at the nested PCR steps. The nested PCR products are

digested using a restriction enzyme where species unique DNA fragment patterns can be used to

distinguish Mycoplasma species. Real-time PCR technology has recently been exploited in

clinical microbiology. It is a promising tool with an excellent sensitivity and specificity, very fast

and suitable for high throughput of samples with an inherent quantitative ability (Cai et al., 2005;

Espy et al., 2006; Koskinen et al., 2009; Taponen et al., 2009). Development of a real-time PCR technique to detect major mycoplasma mastitis causing pathogens directly from milk samples may be a superior choice for the diagnosis of this disease given its abilities to rapidly determine, quantify, and distinguish Mycoplasma species.

REFERENCES

Abu-Amero, K.K., Halablab, M.A., Miles, R.J., 1996, Nisin resistance distinguishes

Mycoplasma spp. from Acholeplasma spp. and provides a basis for selective growth

media. Appl Environ Microbiol 62, 3107-3111.

Biddle, M.K., Fox, L.K., Hancock, D.D., 2003, Patterns of mycoplasma shedding in the milk of

dairy cows with intramammary mycoplasma infection. J Am Vet Med Assoc 223, 1163-

1166.

Biddle, M.K., Fox, L.K., Hancock, D.D., Gaskins, C.T., Evans, M.A., 2004, Effects of storage

time and thawing methods on the recovery of Mycoplasma species in milk samples from

cows with intramammary infections. J Dairy Sci 87, 933-936.

Bushnell, R.B., 1984, Mycoplasma mastitis. Vet Clin North Am Large Anim Pract 6, 301-312.

Cai, H.Y., Bell-Rogers, P., Parker, L., Prescott, J.F., 2005, Development of a real-time PCR for

detection of Mycoplasma bovis in bovine milk and lung samples. J Vet Diagn Invest 17,

537-545.

Counter, D.E., 1978, A severe outbreak of bovine mastitis asssociated with Mycoplasma

bovigenitalium and Acholeplasma laidlawii . Vet Rec 103, 130-131.

Espy, M.J., Uhl, J.R., Sloan, L.M., Buckwalter, S.P., Jones, M.F., Vetter, E.A., Yao, J.D.,

Wengenack, N.L., Rosenblatt, J.E., Cockerill, F.R., 3rd, Smith, T.F., 2006, Real-time

PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol

Rev 19, 165-256.

Fox, L.K., Kirk, J.H., Britten, A., 2005, Mycoplasma mastitis: a review of transmission and

control. J Vet Med B Infect Dis Vet Public Health 52, 153-160.

Gardella, R.S., DelGiudice, R.A., Tully, J.G., 1983, Section F7 Immunofluorescence, In: Tully,

J.G., Razin, S. (Eds.) Methods in mycoplasmology I. Academic Press, New York, pp.

431-440.

Ghadersohi, A., Coelen, R.J., Hirst, R.G., 1997, Development of a specific DNA probe and PCR

for the detection of Mycoplasma bovis . Vet Microbiol 56, 87-98.

Gonzalez, R.N., Wilson, D.J., 2003, Mycoplasmal mastitis in dairy herds. Vet Clin North Am

Food Anim Pract 19, 199-221.

Hayman, B., Hirst, R., 2003, Development of a semi-nested PCR for the improved detection of

Mycoplasma bovis from bovine milk and mucosal samples. Vet Microbiol 91, 91-100.

Hogan, J.S., Gonzalez, R.N., Harmon, R.J., Nickerson, S.C., Oliver, S.P. Pankey, J.W., Smith,

K.L., 1999, Laboratory handbook on bovine mastitis. National Mastitis Council, Inc,

Wisconsin, pp. 151-188.

Hotzel, H., Heller, M., Sachse, K., 1999, Enhancement of Mycoplasma bovis detection in milk

samples by antigen capture prior to PCR. Mol Cell Probes 13, 175-178.

Hotzel, H., Sachse, K., Pfutzner, H., 1996, Rapid detection of Mycoplasma bovis in milk samples

and nasal swabs using the polymerase chain reaction. J Appl Bacteriol 80, 505-510.

Koskinen, M.T., Holopainen, J., Pyorala, S., Bredbacka, P., Pitkala, A., Barkema, H.W., Bexiga,

R., Roberson, J., Solverod, L., Piccinini, R., Kelton, D., Lehmusto, H., Niskala, S.,

Salmikivi, L., 2009, Analytical specificity and sensitivity of a real-time polymerase chain

reaction assay for identification of bovine mastitis pathogens. J Dairy Sci 92, 952-959.

Pan, I.J., Ogata, M., 1969, New sero-types of Mycoplasma laidlawii isolated from mastitic milk

and urogenital tracts of cattle. II. Typing of M. laidlawii by biological and serological

methods. Nippon Juigaku Zasshi 31, 313-324.

Pinnow, C.C., Butler, J.A., Sachse, K., Hotzel, H., Timms, L.L., Rosenbusch, R.F., 2001,

Detection of Mycoplasma bovis in preservative-treated field milk samples. J Dairy Sci 84,

1640-1645.

Tang, J., Hu, M., Lee, S., Roblin, R., 2000, A polymerase chain reaction based method for

detecting Mycoplasma/Acholeplasma contaminants in cell culture. J Microbiol Methods

39, 121-126.

Taponen, S., Salmikivi, L., Simojoki, H., Koskinen, M.T., Pyorala, S., 2009, Real-time

polymerase chain reaction-based identification of bacteria in milk samples from bovine

clinical mastitis with no growth in conventional culturing. J Dairy Sci 92, 2610-2617.

BIBLIOGRAPHY OF PUBLISHED AND SUBMITTED MANUSCRIPTS

Boonyayatra, S., Fox, L.K., Besser, T.E., Sawant, A., Gay, J.M., 2010, Effects of storage

methods on the recovery of Mycoplasma species from milk samples. Vet Microbiol 144,

210-213.

CHAPTER 2

LITERATURE REVIEW

2.1. Mycoplasma species

Mycoplasmas are members of the class Mollicutes . Taxonomy of this class is outlined in

Table 1. Mycoplasmas are the smallest self-replicating organisms with the diameter as small as

300 nm. Mycoplasma was first isolated in 1898 as reported by Nocard and Roux (Nocard and

Roux, 1990). The first species to be isolated is M. mycoides subsp. mycoides causing disease in cattle herds at that time. The current number of recognized Mycoplasma species is 102 (Razin et al., 1998). Members of Mollicutes such as Mycoplasma species and Acholeplasma species, are distinguished phenotypically from other bacteria by their minute size (diameters ranging from

0.3 µm to 0.8 µm) and total lack of a cell wall. The genome size range of Mycoplasmas is 580-

1350 kb, about one-sixth that of Escherichia coli , with a low G+C content of 23-40%. They are parasites or commensal organisms of humans and animals. Most human-associated and animal- associated Mycoplasmas adhere to host cells. Only a few species are recognized to be able to internalize into host cells (Razin et al., 1998). Mycoplasma species commonly found to infect cattle include M. alkalescens, M. alvi, M. arginini, M. bovigenitalium, M. bovirhinis, M. bovis,

M. bovoculi, M. californicum, M. canadense, M. dispar, M. mycoides subsp. mycoides , and M. verecundam . Some of these species are highly pathogenic whereas others are either moderately pathogenic or apparently nonpathogenic. Phase variation is a common mechanism among

Mycoplasma species and is thought to be involved in microbial survival, leading to the emergence of varied intra-clonal populations that adapt quickly to new environments.

Mycoplasma bovis and M. mycoides subsp. mycoides are the two bovine mycoplasmas which

have been extensively studied for their pathogenicities (Rosengarten et al., 1994; Behrens et al.,

1994; Pilo et al., 2007). A major characterized virulence factor shared between these two species

and among other Mycoplasma species is the phase or antigenic variation which is mediated by

the characteristics of variable surface proteins, Vsp family proteins. Variability of these proteins

on Mycoplasmas helps them to avoid recognition by the host immune system, which may lead to development of chronic infections.

2.2. Mycoplasma mastitis in dairy cattle

Mycoplasma mastitis has been characterized as a contagious mastitis pathogen. However, the pattern of its outbreak generally associates with farms with low bulk tank milk somatic cell counts with frequent cases of clinical mastitis (Fox et al., 2005). The primary source of new mycoplasma mastitis outbreaks in dairy herds is suspected to be the introduction of replacement animals (Bushnell, 1984; Gonzalez and Wilson, 2003; Jasper, 1981). Therefore, large expanding herds with relatively high rates of importation of animals usually have higher risk for mycoplasma mastitis (Fox et al., 2003; Thomas et al., 1981). As a contagious pathogen,

Mycoplasma appears to be transmitted from cow-to-cow at milking time via fomites or through direct contact during other times (Fox et al., 2005). It is also suspected that Mycoplasma can be internally transferred from an extramammary organ to the mammary gland (Biddle et al., 2004;

Jain et al., 1969). Mycoplasmas can be isolated from mucous membranes of various body sites especially the respiratory and urogenital tracts of healthy animals; therefore, other than transmitting from udder to udder during milking time, the colonization by Mycoplasmas of the respiratory system may also be the reservoir for intramammary infection (Mackie et al., 2000).

Antibiotics targeting cell wall synthesis, such as beta lactam antibiotics, should not be used to

treat mycoplasma infections as this pathogen group lacks a cell wall. Bushnell (1984) suggested

that treatment is not very efficient and can prolong the mycoplasma mastitis outbreak within the

herd despite results of some studies where showed success in using aminoglycoside, macrolide,

and fluoroquinolone antibiotics for mycoplasma mastitis therapy was reported (Ball and

Campbell, 1989; Ayling et al., 2000). Thus, the often recommended strategy to control

mycoplasma mastitis is to identify infected animals and prevent exposure of those infected

animals to naive animals by segregation and/or culling.

In 1961, mycoplasma bovine mastitis was first reported in the United States in

Connecticut (Hale et al., 1962). The causative agent was later identified as M. bovis .

Mycoplasma bovis is the most common Mycoplasma to be associated with mastitis outbreaks in

dairy cows. Additionally, 11 other Mycoplasma species have also been reported to be associated with outbreaks including M. alkalescens, M. arginini, M. bovigenitalium, M. bovirhinis, M. californicum, M. canadense, M. dispar, Mycoplasma species group 7, Mycoplasma F-38,

Acholeplasma laidlawii , and A. axanthum (Gonzalez and Wilson, 2003). Among these species,

M. bovis, M. californicum, M. bovigenitalium, M. alkalescens and M. canadense are 5 major species causing mastitis in dairy herds.

Mycoplasma bovis is the most important mycoplasma mastitis agent. Other than

intramammary infection, it can also cause abortion, infertility, arthritis, keratoconjunctivitis,

otitis media, pneumonia, and subcutaneous abscesses in cattle (Bushnell, 1984; Fish et al., 1985;

Kinde et al., 1993; Rosendal and Martin, 1986; Walz et al., 1997). Mycoplasma californicum and

M. bovigenitalium are also frequently reported as causative agents for mycoplasma mastitis,

especially in California for the past 50 years (Jasper, 1982; Kirk et al., 1997). Mycoplasma

canadense has been also consistently reported and M. alkalescens has been reported many times

in California (Jasper, 1982). Clinical mastitis caused by M. bovigenitalium, M. californicum, M.

canadense and M. alkalescens appear to be less severe, and infected cows can recover sooner compared to cows with M. bovis infection (Jasper, 1982).

Acholeplasma laidlawii has been considered to be a nonpathogenic saprophytic contaminant when it is found in milk (Bushnell, 1984; Jasper, 1979). However, it is possible that this organism can be associated with mastitis outbreak (Pan and Ogata, 1969; Counter, 1978).

Therefore, distinguishing between ‘saprophytic’ and ‘parasitic’ Acholeplasma laidlawii should be performed when it is necessary to determine an outbreak of this microorganism.

2.3. Effect of storage methods on survival of Mycoplasma

For control and prevention purposes, monitoring of bulk tank milk cultures for

Mycoplasma species has been used for a screening test of presence of Mycoplasma in a herd.

Because Mycoplasma species are fastidious species, the sensitivity of mycoplasma mastitis detection from culturing milk directly onto an agar plate may be as low as 60% (Guterbock and

Blackmer, 1984). Milk samples are usually cultured for identification of infected animals.

Although culturing fresh milk samples has been suggested as a means of maximizing the detection of Mycoplasma species (Biddle et al., 2004), the submission of fresh milk samples is often not practical for many dairy operators because of their distance from diagnostic laboratories and the convenience of storing samples for analysis in batches. Thus milk samples waiting for culture are often kept in a refrigerator or frozen for an extended period of time before submission to a laboratory.

The effect of storage of milk samples at refrigerator temperatures on recovery of

Mycoplasma species is not well understood. There are a number of studies that have reported

changes in viability of Mycoplasma species while stored in sterile liquid media. Human

associated Mycoplasma species were reported to survive less than 30 days at 4 °C in culture

broth (Ford, 1962). In contrast, Nagatomo et al. (2001) reported a longer survival period for

Mycoplasma species associated with animals, with some species still viable at 59 day in

pleuropneumonia-like-organism (PPLO) broth stored at refrigerated temperatures. Cheng et al.

(2007) reported the decreasing recovery of M. hyorhinis from biopharmaceutical process with

6 the reduction up to 3 log 10 of titers after 4 weeks of high-dose cultures (~10 CFU/ml) stored at 4

°C. A study on the survival of M. bovis in milk sample stored at 5 °C was recently published

(Vyletelova, 2010). Here it was reported that the total counts of M. bovis in milk samples was reduced approximately 0.5 log 10 after 5 weeks of refrigeration. Thus, it is likely that survival of

Mycoplasma species in milk samples are reduced when refrigerated for an extended period of time.

Storage time and temperature may affect survival of pathogens in milk samples. Even though many studies have shown that freezing has no effect on the recovery of Staphylococcus aureus from milk samples (Bashandy and Heider, 1979; Luedecke et al., 1972; Murdough et al.,

1996; Schukken et al., 1989), several studies have shown a significant decrease in the recovery of various other mastitis pathogens from frozen milk samples including Eschericia coli ,

Streptococcus agalactiae and Streptococcus non-agalactiae species (Bashandy and Heider, 1979;

Luedecke et al., 1972; Storper et al., 1982; Pankey et al., 1987; Schukken et al., 1989).

Moreover, Biddle et al. (2004) observed that freezing suppressed recovery of Mycoplasma species from milk samples. They showed that a 3-4 log 10 reduction in viable counts of

Mycoplasma species with frozen storage for 4 weeks. Moreover, their study also indicated recovery of Mycoplasma from frozen milk samples decreased with increasing episodes of

freezing and thawing. Similar findings were reported recently by Vyletelova (2010). After 5 weeks of freezing at -30 °C and -80 °C, the viable count of M. bovis in milk samples was decreased as much as 0.7 and 0.1 log 10 , respectively. These findings suggest that Mycoplasma species are highly sensitive to freezing and thawing.

Glycerol is an example of a widely used cryoprotective additive in microbiology that might improve the recovery of this pathogen from frozen milk samples. As a highly hydrophilic molecule, glycerol can easily enter the cell through water channels of cell membranes. Inside the cell, glycerol molecules colligate intracellular water, which consequently decreases the freezing- point. This phenomenon can protect cells from damage during cooling process by precluding excessive dehydration, inhibiting osmotic shock and preventing the formation of large ice crystals within the cell (Hubalek, 2003). Even though the cryoprotective effect of glycerol on mastitis pathogens in milk has not been reported, the addition of glycerol to the milk sample as a cryopreservative might enhance the recovery of Mycoplasma species from frozen milk samples at culture and consequently reduce false negative results from culture.

2.4. Discrimination between Mycoplasma and Acholeplasma

Microbiological culture from a milk sample is still the standard for the diagnosis of mycoplasma mastitis (Hogan et al., 1999). Isolation of Mycoplasma is usually performed on modified Hayflick’s or Hayflick medium. Growth may be seen after 3 days of incubation at 37

°C in a moist 10% CO 2 incubator or 5-7 days when the pathogen is incubated in a moist candle jar. Plates are examined for colonies under low power on a stereomicroscope. Incubation should proceed 7-10 days before plates are diagnosed as negative.

Acholeplasma species can also grow and form Mycoplasma -like-colonies on modified

Hayflick’s or Hayflick medium. Therefore, discrimination between Mycoplasma and

Acholeplasma is recommended for mycoplasma mastitis diagnosis by culture. A major difference between Mycoplasma species and Acholeplasma species and other bacterial L-forms is their nutrient requirement for sterol. Mycoplasma species are unable to synthesize sterols or fatty acids

and thus require exogenous cholesterol (or a related sterol) and an appropriate long-chain fatty

acid for growth (McElhaney, 1983). Sterol constitutes from 20% to 40% of the total membrane

lipid in M. mycoides (Rodwell, 1963) and M. hominis (Rottem et al., 1973). Acholeplasma

species are also unable to synthesize sterols but do not require exogenous cholesterol for growth

(Saito et al., 1978). Acholeplasma species are capable of synthesizing straight-chained saturated

fatty acids; however, some members of this genus also require exogenous unsaturated fatty acids

for optimal growth, whereas others do not (McElhaney, 1983). Cholesterol may constitute from

8%-12% of the cell membrane (de Kruyff et al., 1972; Razin, 1975).

2.5. Digitonin

The tests currently used to evaluate sterol needs include an indirect procedure, which is

based on the measurement of growth inhibition by digitonin, and a direct and quantitative

analysis of the growth response of the mycoplasma to cholesterol (Tully, 1983). Digitonin is a

plant glycoalkaloid saponin detergent obtained from Digitalis purpurea (Elias et al., 1978;

Nishikawa et al., 1984). Structure of digitonin is shown in Figure 1. Digitonin is able to form

water-insoluble 1:1 complexes called digitonides with cholesterol and other 3 β-hydroxysteroles

(Severs and Robenek, 1983). When a cell is treated with digitonin, digitonin can form a complex

in the membrane (Akiyama et al., 1980), thus reducing the freely available cholesterol capable of

interacting with other membrane constituents, and apparently causing considerable

rearrangements in the lipid bilayer that lead to increased permeability and cell lysis (Rothblat and

Smith, 1961; Rottem and Razin, 1972). The digitonin sensitivity test was first adapted to a paper

disc growth inhibition test called digitonin disc diffusion assay by Freundt et al. (1973). The test

essentially involves the culture in broth of the test organism followed by transfer of the growth

media onto an agar plate. A paper disc saturated with 1.5% digitonin is placed on the surface of

the agar, followed by incubation of the culture and measurement of zones of inhibited growth

around the paper disc can indicate the sensitivity of the organism to digitonin. Sterol-requiring

members of the Mollicutes like Mycoplasma species give growth inhibition zones of ≥ 5 mm.

Although Acholeplasmas are generally resistant with no zone of inhibition surrounding the digitonin disc, some species can show a zone of growth inhibition up to 3 mm. (Freundt et al.,

1973).

2.6. Nisin

Nisin is a pentacyclic peptide consisting of 34 amino acids produced by Lactococcus lactis subsp. lactis . Within these 34 residues, 13 residues have been post-translationally modified. Two kinds of nisin are naturally produced by lactococci; nisin A and Z, which are different by only a single amino acid residue at position 27; histidine in nisinA and asparagine in nisin Z. Structure of nisin A is shown in Figure 2. Nisin can inhibit growth of various gram positive bacteria (Gross and Morell, 1971), and causing damage to the outer membrane of some gram negative bacteria such as Escherichia coli and Salmonella species. (Stevens et al., 1991).

Nisin can create pores in the membrane which consequently results in an extensive efflux of various compounds from the cytoplasm, such as amino acids, potassium, inorganic phosphate,

pre-accumulated rubidium, glutamate and ATP (Abee et al., 1994; Ruhr and Sahl, 1985) as depicted in Figure 3. The increase in the membrane permeability causes a complete dissipation of both transmembrane potential ( ψ ) and pH gradient ( pH) which are components of the proton motive force (PMF; p) (Abee et al., 1994; Bruno et al., 1992; Bruno and Montville,

1993; Garcera et al., 1993). The PMF drives ATP synthesis and transporting of ions and metabolites (Harold, 1986). Collapse of PMF leads to cell death through a rapid cessation of all biosynthetic processes. The cationic nature of nisin allows it to bind to lipid bilayers through electrostatic interactions with phospholipid head groups; therefore, a large amount of anionic lipids are required for efficient nisin binding (Breukink et al., 2000; Breukink et al., 1997;

Driessen et al., 1995; Giffard et al., 1997; Martin et al., 1996; Moll et al., 1997; Winkowski et al., 1996). The process of pore formation by nisin is shown in Figure 4. As explained by Bauer and Dicks (2005), the process of pore formation is started by binding of the C-terminus of nisin to the anionic lipids via electrostatic interactions. After that, the N-terminus of nisin is inserted into the lipid phase of the membrane. Then, a transient pore is formed by distortion of lipid bilayer through the short-lived transmembrane orientation of the nisin molecules.

Action of nisin on Mollicutes was first revealed in 1986. In a study of a wide range of bacteria and fungi, Henning et al. (1986) found that growth of A. laidlawii was more sensitive to nisin than that of M. mycoides . Moreover, Kordel and Sahl (1986) also showed that 1 mM nisin did not affect the intracellular ATP concentration of M. hominis confirming its resistance to nisin. In 1996, Abu-Amero et al. suggested a possibility of performing a nisin disc diffusion assay to distinguish Mycoplasma from Acholeplasma (Abu-Amero et al., 1996). They studied 11

Mycoplasma species and 5 Acholeplasma species and showed that when filter paper discs containing 20 nmol of nisin were applied on agar plates covered with Acholeplasma species, they

gave clear inhibition zones ranged from 3.5 to 7.0 mm after incubation. In contrast, there were no zones of inhibition detected from any Mycoplasma species. In this study, they also investigated some mode of nisin action against Acholeplasmas . They found that nisin was able to stimulate oxygen uptake by Acholeplasma but not by Mycoplasma species. Their explanation is that the nisin resistance of Mycoplasma species could be due to the nature of their transport systems, particularly for sugars via phosphoenolpyruvate-dependent sugar phosphotransferase transport system (PTS), which is absent in Acholeplasmas . Moreover, they also postulated that the resistance of Mycoplasmas to nisin might be due to the high cholesterol content of their membranes causing a reduction of membrane fluidity. Because the activity of nisin increases as membrane fluidity increases (Ming and Daeschel, 1993), nisin can effectively inhibit growth of

Acholeplasmas but not Mycoplasmas .

2.7. Nested PCR technique

Molecular biology techniques have been used for many bacterial genus and species identification. For Mycoplasma and Acholeplasma species, most of molecular techniques are developed for identification of its contamination into cell culture. Tang et al. (2000) suggested a nested PCR method (2-step PCR) for detecting Mycoplasma species and Acholeplasma species that contaminate cell cultures. They designed two sets of primers to develop a duplex nested

PCR assay. One set is specific for Mycoplasma species and another set is specific only for

Acholeplasma laidlawii . Because A. laidlawii has at least two rRNA operons with the insertion of tRNA genes in the 16S-23S rRNA intergenic spacer region (Nakagawa et al., 1992), the target region of the reaction, a double banded PCR product with the size of 426 bp and 219 bp was consistently detected. In contrast, a single DNA amplicon ranging from 236 to 365 bp was

produced from all commonly Mycoplasma contaminants. This feature provides a practical way to discriminate A. laidlawii from Mycoplasma species. However, the system proposed will identify

Mycoplasma after culture and thus it is not expected that sensitivity of detection due to inadequate DNA recovery will be of concern. Thus a one-step PCR will be performed in this study using a second set of primers.

2.8. Speciation of Mycoplasma species

The standard methods for definitive speciation of Mycoplasma species are based on reactivity with specific antisera. The first serological test and the most commonly used technique is the growth inhibition (GI) test (Edward and Fitzgerald, 1954). In addition to GI test, metabolism inhibition (MI; Taylor-Robinson, 1983) and immunofluorescence (IF; Bradbury,

1998; Gardella et al., 1983) are the other two techniques frequently used for identification of

Mycoplasma and Acholeplasma species. These methods are based on either the ability of specific antisera to inhibit growth or metabolism or the demonstration of reactivity with specific antisera using either a fluorescence or chromogen-based detection system, and require the isolation of

Mycoplasma -like-colonies prior to conducting the test. The serological test adds an additional 24 to 48 hours to complete the identification of Mycoplasma species after 7 to 10 days of growing

Mycoplasma -like-colonies. Given the contagious nature of the disease, this long period of diagnosis can increase the likelihood that Mycoplasma infected animals will contact and transmit the pathogen to naïve animals. A less complicated and reduced time dependent method to identify cows with mycoplasma mastitis is needed.

Other techniques that do not require the isolation of viable Mycoplasma species, such as enzyme linked immunosorbant assays (ELISA) (Byrne et al., 2000; Ghadersohi et al., 2005;

Nunez et al., 2008) can be used in place of culture for the diagnosis of mycoplasma mastitis.

However, only ELISA tests detecting M. bovis have been reported. Moreover, except for one

study by Byrne et al. (2000) that performed ELISA on milk samples, the ELISA tests demand the

collection of blood samples, which is more invasive than collection of milk samples. It should be

emphasized that ELISA may not be specific to mastitis because it detects antibodies presented in

any pathogen-contacted animals including animals having infection of organs other than

mammary glands. Application of polymerase chain reaction (PCR) based techniques

(Ghadersohi et al., 1997; Hotzel et al., 1999; Hotzel et al., 1996; Pinnow et al., 2001) is another

approach to detect Mycoplasma species directly from milk samples without requirement for isolation. Ghadersohi et al. (1997) successfully developed a specific probe for M. bovis with a very low sensitivity of detection, 10 – 20 CFU/ml in milk when it is used in a dot blot hybridization assay after performing PCR. Hotzel et al. (1999) developed a monoclonal antibodies specific for M. bovis and used it to capture antigen prior to PCR to reduce the problem of the presence of PCR inhibitors in DNA extracted from milk. Pinnow and co-workers (2001) were able to detect M. bovis from 2-year preservative-treated frozen milk samples using nested-

PCR with a very low detection limit at 5.1 CFU/ml of milk. However, even though PCR based

techniques are very promising with a very low detection limit (5 – 500 CFU/ml), they are not

widely used by standard laboratories for primary Mycoplasma species identification. Not all mycoplasma IMI are caused by M. bovis. The development of a culture-independent method to detect all major mycoplasma mastitis agents directly from milk samples, either preserved or frozen, would improve mycoplasma mastitis diagnosis.

To overcome problems of long time spent for definitive identification of Mycoplasma species, many PCR-based strategies have been developed for detection and speciation of this

genus. Hirose et al. (2001) successfully developed a PCR method based on the amplification of

16S rRNA gene to detect M. alkalescens, M. bovigenitalium, M. bovirhinis , and M. bovis in milk.

However, with highly conserved regions of 16S rRNA sequences, cross-amplification of primers with a species other than the target species can easily occur (Kobayashi et al., 1998). In 1999, a nested PCR amplifying 16S-23S rRNA intergenic spacer region was developed (Baird et al.,

1999) for identification of Mycoplasma species after the enrichment cultures of milk samples.

Differential gradient gel electrophoresis (DGGE) of 16S rRNA PCR products was also reported to successfully identify 13 bovine Mycoplasmas (McAuliffe et al., 2005). However, there are some drawbacks with this technique including a long period of electrophoresis (18 hours), the difficulties in differentiation of M. bovis from M.canadense , and the apparent inability of this method to identify Mycoplasma species directly from milk samples. In our laboratory, a nested

PCR followed by RFLP (Tang et al., 2000) has been applied to speciate cultures of Mycoplasma - like-colonies. This method employs PCR to differentiate Mycoplasma species from

Acholeplasma species. For speciation the nested PCR product has to be digested using a

restriction enzyme giving a species-specific pattern of DNA fragments after performing gel

electrophoresis. This technique requires at least 24 hours to complete the speciation, and it has

not been successfully used directly with milk samples. Therefore, development of a new

technique that can complete the diagnosis including speciation of Mycoplasma species within

hours of milk sample collection would be an improvement over the current procedures.

2.9. Real-time PCR

Real-time PCR technology has recently been exploited in clinical microbiology. It is a

promising tool with an excellent sensitivity and specificity, very fast and suitable for high

throughput of samples with an inherent quantitative ability (Espy et al., 2006). The real-time

PCR system is capable of detecting PCR products as they accumulate and will accurately quantify nucleic acid. When the real-time PCR was first developed (Higuchi et al., 1992;

Higuchi et al., 1993), the system consisted of an adapted thermal cycler to irradiate the samples with ultraviolet light, the intercalator ethidium bromide in each amplification reaction, with detection of the resulting fluorescence with a computer-controlled cooled charge-coupled device

(CCD) camera. Amplification during the PCR reaction produces increasing amounts of double- stranded DNA of PCR products. Double-stranded DNA bound by ethidium bromide results in increasing fluorescent signals.

Two kinds of chemistries have been used in real-time PCR: double-stranded DNA binding dyes and fluorogenic probes. The intercalator ethidium bromide was first used in real- time PCR by Higuchi et al. (1992). SYBR Green is another double-stranded DNA binding dye that has been developed and has become a more commonly used dye for real-time PCR

(Morrison et al., 1998). Even though use of a DNA binding dye for real-time detection of PCR allows detection of double-stranded PCR products produced during PCR, the fluorescent signals come from both specific and non-specific products. Thus, any mis-priming events will generate false positive signals when a double-stranded DNA binding dye is used for real-time detection.

Moreover, because multiple molecules of dye can bind to a single molecule of PCR product, the amount of fluorescent signal is dependent on the mass of double-stranded DNA produced in the reaction. Therefore, if the amplification efficiencies are the same, amplification of a longer product will generate more signal than a shorter one giving lower sensitivity of the reaction for the shorter PCR product. In contrast to using dye, using fluorogenic probes in the 5’ nuclease assay allows the real-time PCR reaction to detect only specific amplification products. The

property of 5’ nuclease activity of Taq DNA polymerase enables the target-specific product to be detected when the target probe is cleaved during PCR (Holland et al., 1991). A probe is a short piece of sequence, usually 20 to 30 nucleotides, complementary to a region of target sequence, which is able to anneal to its target sequence at a higher annealing temperature before the annealing of primers. This allows the probe to be cleaved during amplification by the 5’ nuclease activity of Taq DNA polymerase when the enzyme extends from an upstream primer into the region of the probe. The first developed probe was labeled with 32 P on its 5’ end and blocked at its 3’ end so it could not act as a primer (Holland et al., 1991). In 1993, Lee et al. developed a fluorogenic probe to reduce the time spent for post-PCR processing for analysis of probe degradation. A fluorogenic probe has both a reporter fluorescent dye and a quencher dye attached. Three parameters affect the performance of the 5’ nuclease assay: the quenching of the intact probe, its hybridization efficiency and the efficiency of cleavage by the polymerase. While the probe is intact, the proximity of the quencher inhibits the fluorescence emission from the reporter dye by Förster resonance energy transfer (FRET) through space. Thus, during amplification, increasing cleavage of probe from increasing production of target PCR products, results in increased fluorescence from the reporter dye that is not quenched (Figure 5). The advantage of fluorogenic probes over DNA binding dyes is that specific hybridization between probe and target is required to generate a fluorescent signal. Non-specific amplification due to mis-priming or primer-dimer artifact does not generate a signal. Moreover, probe and target can be labeled with different, distinguishable reporter dyes allowing the detection of multiple targets in a single PCR reaction (multiplex real-time PCR).

Real-time PCR has recently been applied to use for the diagnosis of mastitis causing pathogens (Cai et al., 2005; Gillespie and Oliver, 2005; Koskinen et al., 2009; Taponen et al.,

2009). Gillespie and Oliver (2005) had successfully employed a multiplex real-time PCR assay

for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae and Strep. uberis

directly from milk samples. With an overnight enrichment of milk samples before performing

DNA extraction, their designed real-time PCR assay showed an excellent detection limit of less

than 1 CFU/ml with an extremely high sensitivity and specificity of detection of these 3 major

mastitis pathogens. Another real-time PCR assay detecting bovine mastitis pathogens was

recently reported (Koskinen et al., 2009). The study revealed the accuracy of a commercial PCR

reaction kit, the PathoProof Mastitis PCR Assay (Finzymes Oy) which is reported to detect 11

major pathogen species or species groups responsible for bovine mastitis with 100% sensitivity

and 100% specificity within 4 hours (Koskinen et al., 2009). Additionally, another study

reported the ability of this real-time PCR assay (the PathoProof Mastitis PCR Assay) to detect

mastitis pathogens in milk samples from bovine clinical mastitis with no growth in conventional

culturing (Taponen et al., 2009). Using real-time PCR technique, results of this study

demonstrated successful detection of mastitis pathogens in 43% of samples that did not have

cultivable bacteria. These data revealed a potential ability to reduce false negative results of the

conventional culture method for identification of mastitis pathogens from milk samples.

However, this real-time PCR reaction kit cannot detect Mycoplasma species.

In the past 5 years, a number of real-time PCR assays have been developed to detect

mastitis caused by infection of M. bovis . Cai et al. (2005) reported the development of a real-

time PCR targeting 16S rRNA gene for detection of M. bovis in milk and lung samples. As

mentioned previously, DNA sequence in 16S rRNA region is very similar between M. bovis and

M. agalactiae ; therefore, their designed real-time PCR assay amplified the targeted region of both species. However, in their study, they were able to specifically differentiate M. bovis from

M. agalactiae based on their different melting peaks of the fluorescence resonance energy

transfer (FRET) probes. Based on their applied DNA extraction method and real-time PCR

assay, a detection limit was reported at 550 CFU/ml of M. bovis from a milk sample. In 2009,

Sachse et al. described a novel real-time PCR assay with the capability to detect M. bovis directly from milk, nasal swabs and conjunctival swabs. Their designated probe and primers targeted 3’- terminal region of the oppD gene with an excellent detection limit of 1 copy number of DNA from culture and 100 CFU/ml of M. bovis in milk (Sachse et al., 2009). Another study utilizing real-time PCR to detect M. bovis directly from milk and tissue samples was published this year

(Rossetti et al., 2010). They described a successful new specific real-time PCR assay targeting the uvrC gene of M. bovis directly from milk and tissue lysate. When milk lysate of the samples were tested by real-time PCR and by culture method in parallel, the test sensitivity of their designed real-time PCR was equivalent to the test sensitivity of the culture method with the detection limit of 2.0 x 10 3 ± 2.8 x 10 2 CFU/ml (Rossetti et al., 2010). Although the assay described by Cai et al. (2005) and Rossetti et al. (2010) appears to have applicability to mycoplasma mastitis diagnosis, sensitivities of detection suggest both assays would miss perhaps as many as 20% of the infected cows (Biddle et al., 2003). Of the three most recently described real-time PCR techniques, none detect mycoplasma organisms other than M. bovis , which may be the cause of a mycoplasma mastitis outbreak.

2.10. Conclusion and Hypothesis

The ability to detect Mycoplasma species in milk samples of cows with mastitis may not be sufficiently sensitive given the difficulties associated with culture and shedding rates in cows.

Additionally, standard culture is used as a presumptive technique and identification is made at

the level of a mollicute, a mycoplasma like organism. Improvements to the standard culture procedures that would result in a more sensitive Mycoplasma identification system should be investigated.

A previous study demonstrated that freezing samples reduced the recovery of viable

Mycoplasma species from milk samples (Biddle et al., 2004). The authors of this study recommended the culture of fresh milk samples for optimum diagnosis of mycoplasma mastitis.

Submission of fresh samples for mycoplasma culture is often difficult given the logistics of collection and shipping of milk samples from farm to laboratory. An evidence of a detrimental effect of storing a milk sample at the refrigerated temperature (5 °C) prior culturing was recently reported (Vyletelova, 2010). The study showed a decline of M. bovis counts from fresh to 1, 2, 3,

4 and 5 weeks of storing milk in a refrigerator. Therefore, a first study was conducted to test the hypotheses that storing milk samples at the refrigerated temperature (5 °C) for duration of 1 to 5 days would reduce the mycoplasma counts recovered from standard culture of milk samples. An ancillary study was run in parallel to determine if the addition of glycerol, a common cryopreservative, to milk samples prior to freezing would improve the recovery of Mycoplasma from frozen milk samples.

The study to determine longevity of Mycoplasma in refrigerated and frozen milk was designed to test a procedure to improve the sensitivity of standard culture. To improve the specificity, a method to discriminate between Mycoplasma species and Acholeplasma species would be valuable. The digitonin disc diffusion assay has been used as a standard method to distinguish between these two genera. Two other techniques, nisin disc diffusion assay and PCR, have also been used as procedures to distinguish between Mycoplasma species and

Acholeplasma species. It was hypothesized that the digitonin, nisin and PCR tests have the

equivalent efficiency to discriminate between the two genera, and allow proper classification of

Mycoplasma species that cause mastitis.

Given the characteristic of fastidious growing conditions needed by Mycoplasma , and

the time-consuming nature of the standard culture method, a diagnostic test of mycoplasma

mastitis that is more sensitive, less time consuming, and can speciate mycoplasma mastitis

pathogens would be valuable. The development of real-time PCR assays to detect 3 major

mycoplasma mastitis pathogens: M. bovis, M. californicum and M. bovigenitalium was proposed to prevail over the limitations of the standard culture procedures. It was hypothesized that a real- time PCR assays can detect the three major mycoplasma mastitis agents with a high degree of agreement (>95% agreement) with the identification by 16S rRNA partial sequencing. This study was designed to be a first step in the development of a rapid speciation of mycoplasma mastitis agents directly from a milk sample.

In conclusion, the overall goal of the studies proposed was to improve the efficiency of the diagnosis of mycoplasma mastitis in dairy cows. Accurate diagnosis is required to most effectively control and prevent contagious mycoplasma mastitis by dairy managers and consulting veterinarians.

REFERENCES

Abee, T., Rombouts, F.M., Hugenholtz, J., Guihard, G., Letellier, L., 1994, Mode of Action of

Nisin Z against Listeria monocytogenes Scott A Grown at High and Low Temperatures.

Appl Environ Microbiol 60, 1962-1968.

Abu-Amero, K.K., Halablab, M.A., Miles, R.J., 1996, Nisin resistance distinguishes

Mycoplasma spp. from Acholeplasma spp. and provides a basis for selective growth

media. Appl Environ Microbiol 62, 3107-3111.

Akiyama, T., Takagi, S., Sankawa, U., Inari, S., Saito, H., 1980, Saponin-cholesterol interaction

in the multibilayers of egg yolk lecithin as studied by deuterium nuclear magnetic

resonance: digitonin and its analogues. Biochemistry 19, 1904-1911.

Ayling, R.D., Baker, S.E., Peek, M.L., Simon, A.J., Nicholas, R.A.J., 2000

Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline,

spectinomycin and tilmicosin against recent field isolates of Mycoplasma bovis . Vet

Record 146, 745–747.

Baird, S.C., Carman, J., Dinsmore, R.P., Walker, R.L., Collins, J.K., 1999, Detection and

identification of Mycoplasma from bovine mastitis infections using a nested polymerase

chain reaction. J Vet Diagn Invest 11, 432-435.

Ball, H.J., Campbell, J.N., 1989, Antibiotic treatment of experimental Mycoplasma californicum

mastitis. Vet Rec 125, 377-378.

Bashandy, E.Y., Heider, L.E., 1979, The effect of freezing of milk samples on the cultural

results. Zentralbl Veterinarmed B 26, 1-6.

Bauer, R., Dicks, L.M., 2005, Mode of action of lipid II-targeting lantibiotics. Int J Food

Microbiol 101, 201-216.

Behrens, A., Heller, M., Kirchhoff, H., Yogev, D., Rosengarten, R., 1994, A family of phase-

and size- variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis .

Infect Immun 62, 5075-5084.

Biddle, M.K., Fox, L.K., Hancock, D.D., 2003, Patterns of mycoplasma shedding in the milk of

dairy cows with intramammary mycoplasma infection. J Am Vet Med Assoc 223, 1163-

1166.

Biddle, M.K., Fox, L.K., Hancock, D.D., Gaskins, C.T., Evans, M.A., 2004, Effects of storage

time and thawing methods on the recovery of Mycoplasma species in milk samples from

cows with intramammary infections. J Dairy Sci 87, 933-936.

Bradbury, J.M., 1998, Identification of mycoplasmas by immunofluorescence. Methods Mol Biol

104, 119-125.

Breukink, E., Ganz, P., de Kruijff, B., Seelig, J., 2000, Binding of Nisin Z to bilayer vesicles as

determined with isothermal titration calorimetry. Biochemistry 39, 10247-10254.

Breukink, E., van Kraaij, C., Demel, R.A., Siezen, R.J., Kuipers, O.P., de Kruijff, B., 1997, The

C-terminal region of nisin is responsible for the initial interaction of nisin with the target

membrane. Biochemistry 36, 6968-6976.

Bruno, M.E., Kaiser, A., Montville, T.J., 1992, Depletion of proton motive force by nisin in

Listeria monocytogenes cells. Appl Environ Microbiol 58, 2255-2259.

Bruno, M.E., Montville, T.J., 1993, Common Mechanistic Action of Bacteriocins from Lactic

Acid Bacteria. Appl Environ Microbiol 59, 3003-3010.

Bushnell, R.B., 1984, Mycoplasma mastitis. Vet Clin North Am Large Anim Pract 6, 301-312.

Byrne, W.J., Ball, H.J., Brice, N., McCormack, R., Baker, S.E., Ayling, R.D., Nicholas, R.A.,

2000, Application of an indirect ELISA to milk samples to identify cows with

Mycoplasma bovis mastitis. Vet Rec 146, 368-369.

Cai, H.Y., Bell-Rogers, P., Parker, L., Prescott, J.F., 2005, Development of a real-time PCR for

detection of Mycoplasma bovis in bovine milk and lung samples. J Vet Diagn Invest 17,

537-545.

Cheng, H.S., Shen, C.W., Wang, S.R., 2007, Effect of storage conditions on detection of

mycoplasma in biopharmaceutical products. In Vitro Cell Dev Biol Anim 43, 113-119.

Counter, D.E., 1978, A severe outbreak of bovine mastitis asssociated with Mycoplasma

bovigenitalium and Acholeplasma laidlawii . Vet Rec 103, 130-131. de Kruyff, B., Demel, R.A., van Deenen, L.L., 1972, The effect of cholesterol and epicholesterol

incorporation on the permeability and on the phase transition of intact Acholeplasma

laidlawii cell membranes and derived liposomes. Biochim Biophys Acta 255, 331-347.

Driessen, A.J., van den Hooven, H.W., Kuiper, W., van de Kamp, M., Sahl, H.G., Konings,

R.N., Konings, W.N., 1995, Mechanistic studies of lantibiotic-induced permeabilization

of phospholipid vesicles. Biochemistry 34, 1606-1614.

Edward, D.G., Fitzgerald, W.A., 1954, Inhibition of growth of pleuropneumonia-like organisms

by antibody. J Pathol Bacteriol 68, 23-30.

Elias, P.M., Goerke, J., Friend, D.S., Brown, B.E., 1978, Freeze-fracture identification of sterol-

digitonin complexes in cell and liposome membranes. J Cell Biol 78, 577-596.

Espy, M.J., Uhl, J.R., Sloan, L.M., Buckwalter, S.P., Jones, M.F., Vetter, E.A., Yao, J.D.,

Wengenack, N.L., Rosenblatt, J.E., Cockerill, F.R., 3rd, Smith, T.F., 2006, Real-time

PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol

Rev 19, 165-256.

Fish, N.A., Rosendal, S., Miller, R.B., 1985, The Distribution of Mycoplasmas and Ureaplasmas

in the Genital Tract of Normal Artificial Insemination Bulls. Can Vet J 26, 13-15.

Ford, D.K., 1962, Culture of Human Genital "T-Strain" Pleuropneumonia-Like Organisms. J

Bacteriol 84, 1028-1034.

Fox, L.K., Hancock, D.D., Mickelson, A., Britten, A., 2003, Bulk tank milk analysis: factors

associated with appearance of Mycoplasma sp. in milk. J Vet Med B Infect Dis Vet

Public Health 50, 235-240.

Fox, L.K., Kirk, J.H., Britten, A., 2005, Mycoplasma mastitis: a review of transmission and

control. J Vet Med B Infect Dis Vet Public Health 52, 153-160.

Freundt, E.A., Andrews, B.E., Erno, H., Kunze, M., Black, F.T., 1973, The sensitivity of

mycoplasmatales to sodium-polyanethol-sulfonate and digitonin. Zentralbl Bakteriol Orig

A 225, 104-112.

Garcera, M.J., Elferink, M.G., Driessen, A.J., Konings, W.N., 1993, In vitro pore-forming

activity of the lantibiotic nisin. Role of protonmotive force and lipid composition. Eur J

Biochem 212, 417-422.

Gardella, R.S., DelGiudice, R.A., Tully, J.G., 1983, Section F7 Immunofluorescence, In: Tully,

J.G., Razin, S. (Eds.) Methods in mycoplasmology I. Academic Press, New York, pp.

431-440.

Ghadersohi, A., Coelen, R.J., Hirst, R.G., 1997, Development of a specific DNA probe and PCR

for the detection of Mycoplasma bovis . Vet Microbiol 56, 87-98.

Ghadersohi, A., Fayazi, Z., Hirst, R.G., 2005, Development of a monoclonal blocking ELISA for

the detection of antibody to Mycoplasma bovis in dairy cattle and comparison to

detection by PCR. Vet Immunol Immunopathol 104, 183-193.

Giffard, C.J., Dodd, H.M., Horn, N., Ladha, S., Mackie, A.R., Parr, A., Gasson, M.J., Sanders,

D., 1997, Structure-function relations of variant and fragment nisins studied with model

membrane systems. Biochemistry 36, 3802-3810.

Gillespie, B.E., Oliver, S.P., 2005, Simultaneous detection of mastitis pathogens, Staphylococcus

aureus, Streptococcus uberis , and Streptococcus agalactiae by multiplex real-time

polymerase chain reaction. J Dairy Sci 88, 3510-3518.

Gonzalez, R.N., Wilson, D.J., 2003, Mycoplasmal mastitis in dairy herds. Vet Clin North Am

Food Anim Pract 19, 199-221.

Gross, E., Morell, J.L., 1971, The structure of nisin. J Am Chem Soc 93, 4634-4635.

Guterbock, W.M., Blackmer, P.E., 1984, Veterinary interpretation of bulk tank milk.

Vet Clin North Am Food Anim Pract 6, 257–268.

Hale, H.H., Helmboldt, C.F., Plastridge, W.N., Stula, E.F., 1962, Bovine mastitis caused by a

Mycoplasma species. Cornell Vet 52, 582-591.

Harold, F.M., 1986, Transcellular ion currents in tip-growing organisms: where are they taking

us? Prog Clin Biol Res 210, 359-366.

Henning, S., Metz, R., Hammes, W. P., 1986, Studies on the mode of action of nisin. Int J Food

Microbiol 3, 121-134.

Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992, Simultaneous amplification and

detection of specific DNA sequences. Biotechnology (N Y) 10, 413-417.

Higuchi, R., Fockler, C., Dollinger, G., Watson, R., 1993, Kinetic PCR analysis: real-time

monitoring of DNA amplification reactions. Biotechnology (N Y) 11, 1026-1030.

Hirose, K, Kawasaki, H, Kotani, K, Tanaka, A., Abiko, K., Ogawa, H., 2001,

Detection of Mycoplasma in mastitic milk by PCR analysis and culture

method. J Vet Med Sci 63, 691-693.

Hogan, J.S., Gonzalez, R.N., Harmon, R.J., Nickerson, S.C., Oliver, S.P. Pankey, J.W., Smith,

K.L., 1999, Laboratory handbook on bovine mastitis. National Mastitis Council, Inc,

Wisconsin, pp. 151-188.

Holland, P.M., Abramson, R.D., Watson, R., Gelfand, D.H., 1991, Detection of specific

polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of

Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88, 7276-7280.

Hotzel, H., Heller, M., Sachse, K., 1999, Enhancement of Mycoplasma bovis detection in milk

samples by antigen capture prior to PCR. Mol Cell Probes 13, 175-178.

Hotzel, H., Sachse, K., Pfutzner, H., 1996, Rapid detection of Mycoplasma bovis in milk samples

and nasal swabs using the polymerase chain reaction. J Appl Bacteriol 80, 505-510.

Hubalek, Z., 2003, Protectants used in the cryopreservation of microorganisms. Cryobiology 46,

205-229.

Jain, N.C., Jasper, D.E., Dellinger, J.D., 1969, Serologic response of cows to Mycoplasma under

experimental and field conditions. Am J Vet Res 30, 733-742.

Jasper, D.E., 1979, Bovine mycoplasmal mastitis. J Am Vet Med Assoc 175, 1072-1074.

Jasper, D.E., 1981, Bovine mycoplasmal mastitis. Adv Vet Sci Comp Med 25, 121-157.

Jasper, D.E., 1982, The role of Mycoplasma in bovine mastitis. J Am Vet Med Assoc 181, 158-

162.

Kinde, H., Daft, B.M., Walker, R.L., Charlton, B.R., Petty, R., 1993, Mycoplasma bovis

associated with decubital abscesses in Holstein calves. J Vet Diagn Invest 5, 194-197.

Kirk, J.H., Glenn, K., Ruiz, L., Smith, K., 1997, Epidemiologic analysis of Mycoplasma spp

isolated from bulk-tank milk samples obrained from dairy herds that were members of

milk cooperative. J Am Vet Med Assoc 211, 1036-1038.

Kobayashi, H., Hirose, K., Worarach, A., Paugtes, P., Ito, N., Morozumi, T., Yamamoto, K.,

1998, In vitro amplification of the 16S rRNA genes from Mycoplasma bovirhinis,

Mycoplasma alkalescens and Mycoplasma bovigenitalium by PCR. J Vet Med Sci 60,

1299-1303.

Kordel, M., Sahl, H.G., 1986, Susceptibility of bacterial, eukaryotic and artificial membranes to

the disruptive action of the cationic peptides Pep 5 and nisin. FEMS Microbiol Lett 34,

139-144.

Koskinen, M.T., Holopainen, J., Pyorala, S., Bredbacka, P., Pitkala, A., Barkema, H.W., Bexiga,

R., Roberson, J., Solverod, L., Piccinini, R., Kelton, D., Lehmusto, H., Niskala, S.,

Salmikivi, L., 2009, Analytical specificity and sensitivity of a real-time polymerase chain

reaction assay for identification of bovine mastitis pathogens. J Dairy Sci 92, 952-959.

Lee, L.G., Connell, C.R., Bloch, W., 1993, Allelic discrimination by nick-translation pcr with

fluorogenic probes. Nucleic Acids Res 21, 3761-3766.

Luedecke, L.O., Forster, T.L., Williams, K., Hillers, J.K., 1972, Effect of freezing and storage at

-20 C on survival of mastitis pathogens. J Dairy Sci 55, 417-418.

Mackie, D.P., Finlay, D., Brice, N., Ball, H.J., 2000, Mixed mycoplasma mastitis outbreak in a

dairy herd. Vet Rec 147, 335-336.

Martin, I., Ruysschaert, J.M., Sanders, D., Giffard, C.J., 1996, Interaction of the lantibiotic nisin

with membranes revealed by fluorescence quenching of an introduced tryptophan. Eur J

Biochem 239, 156-164.

McAuliffe, L., Ellis, R.J., Lawes, J.R., Ayling, R.D., Nicholas, R.A., 2005, 16S rDNA PCR and

denaturing gradient gel electrophoresis; a single generic test for detecting and

differentiating Mycoplasma species. J Med Microbiol 54, 731-739.

McAuliffe, O., Ross, R.P., Hill, C., 2001, Lantibiotics: structure, biosynthesis and mode of

action. FEMS Microbiol Rev 25, 285-308.

McElhaney, R.N., 1983, Section D4 Manipulation of membrane lipid composition, In: Tully,

J.G., Razin, S. (Eds.) Methods in mycoplasmology I. Academic Press, New York, pp.

235-239.

Ming, X.T., Daeschel, M.A., 1993, Nisin resistance of foodborne bacteria and the specific

resistance responses of Listeria monocytogenes scott-a. J. Food Prot. 56, 944-948.

Moll, G.N., Clark, J., Chan, W.C., Bycroft, B.W., Roberts, G.C., Konings, W.N., Driessen, A.J.,

1997, Role of transmembrane pH gradient and membrane binding in nisin pore

formation. J Bacteriol 179, 135-140.

Morrison, T.B., Weis, J.J., Wittwer, C.T., 1998, Quantification of low-copy transcripts by

continuous SYBR Green I monitoring during amplification. Biotechniques 24, 954-958,

960, 962.

Murdough, P.A., Deitz, K.E., Pankey, J.W., 1996, Effects of freezing on the viability of nine

pathogens from quarters with subclinical mastitis. J Dairy Sci 79, 334-336.

Nagatomo, H., Takegahara, Y., Sonoda, T., Yamaguchi, A., Uemura, R., Hagiwara, S., Sueyoshi,

M., 2001, Comparative studies of the persistence of animal mycoplasmas under different

environmental conditions. Vet Microbiol 82, 223-232.

Nakagawa, T., Uemori, T., Asada, K., Kato, I., Harasawa, R., 1992, Acholeplasma laidlawii has

tRNA genes in the 16S-23S rRNA intergenic spacer of the rRNA operon. J Bacteriol 174,

8163-8165.

Nishikawa, M., Nojima, S., Akiyama, T., Sankawa, U., Inoue, K., 1984, Interaction of digitonin

and its analogs with membrane cholesterol. J Biochem 96, 1231-1239.

Nocard, Roux, 1990, The microbe of pleuropneumonia. 1896. Rev Infect Dis 12, 354-358.

Nunez, C., Salinas, E.M., Maya, J.J.M., Hernandez, L., 2008, Detection of subclinical bovine

mastitis caused by mycoplasmosis by indirect ELISA test and isolation. Veterinaria

Mexico 39, 161-171.

Paila, Y.D., Pucadyil, T.J., Chattopadhyay, A., 2005, The cholesterol-complexing agent digitonin

modulates ligand binding of the bovine hippocampal serotonin 1A receptor. Mol Membr

Biol 22, 241-249.

Pan, I.J., Ogata, M., 1969, New sero-types of Mycoplasma laidlawii isolated from mastitic milk

and urogenital tracts of cattle. II. Typing of M. laidlawii by biological and serological

methods. Nippon Juigaku Zasshi 31, 313-324.

Pankey, J.W., Wadsworth, J.K., Metha, H., Murdough, P.A., 1987, Effects of storage no viability

of mastitis pathogens. J Dairy Sci 70(Suppl.1), 132 (Abstr.).

Pilo, P., Frey, J., Vilei, E.M., 2007, Molecular mechanisms of pathogenicity of

Mycoplasma mycoides subsp. mycoides SC. Vet J 174, 513-521.

Pinnow, C.C., Butler, J.A., Sachse, K., Hotzel, H., Timms, L.L., Rosenbusch, R.F., 2001,

Detection of Mycoplasma bovis in preservative-treated field milk samples. J Dairy Sci 84,

1640-1645.

Razin, S., 1975, Cholesterol incorporation into bacterial membranes. J Bacteriol 124, 570-572.

Razin, S., Yogev, D., Naot, Y., 1998, Molecular biology and pathogenicity of mycoplasmas.

Microbiol Mol Biol Rev 62, 1094-1156.

Rodwell, A.W., 1963, The Steroid Growth-Requirement of Mycoplasma mycoides . J Gen

Microbiol 32, 91-101.

Rosendal, S., Martin, S.W., 1986, The association between serological evidence of mycoplasma

infection and respiratory disease in feedlot calves. Can J Vet Res 50, 179-183.

Rosengarten, R., Behrens, A., Stetefeld, A., Heller, M., Ahrens, M.,Sachse, K., Yogev, D.,

Kirchhoff, H., 1994, Antigen heterogeniety among isolates of Mycoplasma bovis is

generated by high frequency variation of diverse membrane surface proteins. Infect

Immun 62, 5066-5074.

Rossetti, B.C., Frey, J., Pilo, P., 2010, Direct detection of Mycoplasma bovis in milk and tissue

samples by real-time PCR. Mol Cell Probes in press .

Rothblat, G.H., Smith, P.F., 1961, Nonsaponifiable lipids of representative pleuropneumonia-like

organisms. J Bacteriol 82, 479-491.

Rottem, S., Cirillo, V.P., de Kruyff, B., Shinitzky, M., Razin, S., 1973, Cholesterol in

mycoplasma membranes. Correlation of enzymic and transport activities with physical

state of lipids in membranes of Mycoplasma mycoides var. capri adapted to grow with

low cholesterol concentrations. Biochim Biophys Acta 323, 509-519.

Rottem, S., Razin, S., 1972, Isolation of mycoplasma membranes by digitonin. J Bacteriol 110,

699-705.

Ruhr, E., Sahl, H.G., 1985, Mode of action of the peptide antibiotic nisin and influence on the

membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles.

Antimicrob Agents Chemother 27, 841-845.

Sachse, K., Salam, H.S.H., Diller, R., Schubert, E., Hoffmann, B., Hotzel, H., (in press) 2009,

Use of a novel real-time PCR technique to monitor and quantitate Mycoplasma bo vis

infection in cattle herds with mastitis and respiratory disease. The Veterinary Journal.

Saito, Y., Silvius, J.R., McElhaney, R.N., 1978, Membrane lipid biosynthesis in Acholeplasma

laidlawii b: elongation of medium- and long-chain exogenous fatty acids in growing

cells. J Bacteriol 133, 66-74.

Schukken, Y.H., Grommers, F.J., Smit, J.A., Vandegeer, D., Brand, A., 1989, Effect of freezing

on bacteriologic culturing of mastitis milk samples. J Dairy Sci 72, 1900-1906.

Severs, N.J., Robenek, H., 1983, Detection of microdomains in biomembranes. An appraisal of

recent developments in freeze-fracture cytochemistry. Biochim Biophys Acta 737, 373-

408.

Stevens, K.A., Sheldon, B.W., Klapes, N.A., Klaenhammer, T.R., 1991, Nisin treatment for

inactivation of Salmonella species and other gram-negative bacteria. Appl Environ

Microbiol 57, 3613-3615.

Storper, M., Ziv, G., Saran, A. 1983. Effect of storing milk samples at -18 °C on the viability of

certain udder pathogens. Refuah Vet 39, 1-2.

Tang, J., Hu, M., Lee, S., Roblin, R., 2000, A polymerase chain reaction based method for

detecting Mycoplasma/Acholeplasma contaminants in cell culture. J Microbiol Methods

39, 121-126.

Taponen, S., Salmikivi, L., Simojoki, H., Koskinen, M.T., Pyorala, S., 2009, Real-time

polymerase chain reaction-based identification of bacteria in milk samples from bovine

clinical mastitis with no growth in conventional culturing. J Dairy Sci 92, 2610-2617.

Taylor-Robinson, D., 1983, Section F4 Metabolism Inhibition Tests, In: Tully, J.G., Razin, S.

(Eds.) Methods in mycoplasmology I. Academic Press, New York, pp. 411-418.

Thomas, C.B., Willeberg, P., Jasper, D.E., 1981, Case-control study of bovine mycoplasmal

mastitis in California. Am J Vet Res 42, 511-515.

Tully, J.G., 1983, Section E5 Tests for digitonin sensitivity and sterol requirement, In:

Tully, J.G., Razin, S. (Eds.) Methods in mycoplasmology I. Academic Press,

New York, pp. 355-362.

Vyletelova, M., 2010, The survival of Mycoplasma bovis at different temperatures,

Czech J Food Sci 28, 74-78.

Walz, P.H., Mullaney, T.P., Render, J.A., Walker, R.D., Mosser, T., Baker, J.C., 1997, Otitis

media in preweaned Holstein dairy calves in Michigan due to Mycoplasma bovis . J Vet

Diagn Invest 9, 250-254.

Winkowski, K., Ludescher, R.D., Montville, T.J., 1996, Physiochemical characterization of the

nisin-membrane interaction with liposomes derived from Listeria monocytogenes . Appl

Environ Microbiol 62, 323-327.

TABLES

TABLE 1. Taxonomy of the class of Mollicutes (adapted from Razin et al., 1998)

Classification Number of Genome Cholesterol Distinctive Habitat

recognized size requirement properties

species (kb)

Order I: Mycoplasmatales Yes Humans and

Family I: Mycoplasmataceae animals

Genus I: Mycoplasma 102 580-1350 Optimum growth

at 37 °C

Genus II: Ureaplasma 6 760-1170 Urea hydrolysis

Order II: Entomoplasmatales Insects and

Family I: Entomoplasmataceae plants

Genus I: Entomoplasma 5 790-1140 Yes Optimum growth

at 30 °C

Genus II: Mesoplasma 12 870-1100 No 0.04% Tween 80

required in serum-

free medium

Family II: Spiroplasmataceae Helical motile Insects and

Genus I: Spiroplasma 33 780-2220 Yes filaments plants

Order III: Acholeplasmatales Animals,

Family I: Acholeplasmataceae Optimum growth some plants,

Genus: Acholeplasma 13 1500-1650 No at 30 – 37 °C insects

Order IV: Anaeroplasmatales Oxygen-sensitive Bovine/Ovin

Family: Anaeroplasmataceae anaerobes e rumen

Genus I: Anaeroplasma 4 1500-1600 Yes

Genus II: Asteroleplasma 1 1500 No

Undefined taxonomic status Not defined 640-1185 Not known Uncultured in vitro Insects,

Phytoplasma plants

FIGURES

FIGURE 1. Chemical structure of digitonin (Paila et al., 2005, permission granted by Journal of Molecular Membrane Biology, Taylor & Francis Ltd, Philadelphia, PA, USA)

FIGURE 2. Structure of nisin A (McAuliffe et al., 2001, permission granted by FEMS

Microbiology Review, John Wiley and Sons, Inc, Hoboken, NJ, USA).

FIGURE 3. General model of the ‘barrel stave’ mechanism of pore formation by peptides

(McAuliffe et al., 2001, permission granted by FEMS Microbiology Review, John Wiley and

Sons, Inc, Hoboken, NJ, USA)

FIGURE 4. Models of pore formation by nisin. (A) Barrel-stave pore. (B, C) General models for pore formation. Step1: binding of nisin via its C-terminal. Step2: insertion of nisin into the membrane. The depth of insertion depends on the percentage of anionic lipids and nisin concentration. Step3: wedge/magainin-like pore. B and C represent pore formation initiated by translocation of the C-terminus and N-terminus, respectively. Step 4: translocation of the peptide to the inside of the membrane. (Bauer and Dicks, 2005, permission granted by Internationa

Journal of Food Microbiology, Elsevier Limited, Oxford, United Kingdom)

FIGURE 5. Step-wise reaction of real-time PCR using a fluorogenic probe (the Taqman assay,

Applied Biosystems, Foster City, CA, USA)

CHAPTER 3

Effects of storage methods on the recovery of Mycoplasma species from milk samples

INTRODUCTION

Mycoplasm a species are Mollicute s known to be able to cause clinical and subclinical mastitis in dairy cattle. Mastitis associated with Mycoplasma has been reported worldwide (Fox et al., 2005). Numerous species of Mycoplasma have been reported to be associated with mastitis in dairy farms. Among all reported cases, Mycoplasma bovis, M. californicum, M. alkalescens,

M. bovigenitalium and M. canadense are the most commonly found to cause bovine mastitis

(Gonzalez and Wilson, 2003). Mycoplasma species are classified as a contagious pathogen, capable of transmitting easily from cow-to-cow (Gonzalez and Wilson, 2003) and treatment is usually unsuccessful (Bushnell, 1984). Therefore, control of mycoplasma mastitis may be best

achieved through the identification and segregation or culling of infected cows, which are the

infection reservoirs (Fox et al., 2005). Mycoplasma species are difficult to isolate which makes

diagnosis of infection challenging. Because specialized media, modified Hayflicks, and

incubation for several days in 10% CO 2 are required (Hogan et al., 1999), many laboratories may

not routinely culture milk for mycoplasma organisms. Additionally, cows with mycoplasma

mastitis may shed the pathogen intermittently and shedding of Mycoplasma species into milk of

infected cows may be as low as <100 colony forming unit (CFU)/ml, below the threshold of

detection by standard culture methods (Biddle et al., 2003). These difficulties increase the

importance of milk sample handling before culture in an effort to avoid false negative results.

Although culturing fresh milk samples has been suggested as a means of maximizing the

detection of Mycoplasma species (Biddle et al., 2004), the submission of fresh milk samples is

often not practical for many dairy operators because of their distance from diagnostic

laboratories and the convenience of storing samples for analysis in batches. Thus milk samples

waiting for culture are often kept in a refrigerator or frozen for an extended period of time before

submission to a laboratory. Storage time and temperature may affect survival of pathogens in

milk samples. Several studies have shown a significant decrease in the recovery of various

mastitis pathogens from frozen milk samples (Luedecke et al., 1971; Bashandy et al., 1979;

Storper et al., 1982; Pankey et al., 1987; Schukken et al., 1989). Moreover, Biddle and

colleagues (2004) observed that freezing suppressed recovery of Mycoplasma species from milk samples. Glycerol is an example of a widely used cryoprotective additive in microbiology that might improve the recovery of this pathogen from frozen milk samples. Being a highly hydrophilic molecule, glycerol can easily enter the cell through water channels of cell membranes. Inside the cell, glycerol molecules colligate intracellular water which consequently decreases the freezing-point. This phenomenon can protect cells from damage during cooling process by precluding excessive dehydration, inhibiting osmotic shock and preventing the formation of large ice crystals within the cell (Hubalek, 2003). However, the cryoprotective effect of glycerol on mastitis pathogens in milk has not been reported.

The effect of refrigerating a milk sample on recovery of Mycoplasma species by standard culture has not been assessed. Also, the addition of glycerol to the milk sample as a cryopreservative might enhance the recovery of Mycoplasma species from frozen milk samples at culture. The objectives of this study were: 1) to determine the effects of refrigerating milk samples for various periods of time on Mycoplasm a species viability assessed by culture; and 2) to determine the effects of glycerol addition at different concentrations on the viability of several species of Mycoplasma as determined by culture of milk samples.

MATERIALS AND METHODS

Mycoplasma strains

Two strains of each of 5 species: M. bovis, M. californicum, M. bovigenitallium, M. canadense and M. alkalescens , were selected, original sources being the American Type Culture

Collection (ATCC, Manassas, VA, USA), and the diagnostic laboratories at the University of

California (UCD, Davis, CA, USA) and Cornell University (CU, Ithaca, NY, USA). Strains were: M. bovis ATCC25523, M. bovis ATCC27368, M. californicum ATCC33461, M. californicum UCD8, M. bovigenitalium ATCC19852, M. bovigenitalium ATCC14173, M. canadense ATCC29418, M. canadense CU21113, M. alkalescens CU21146 and M. alkalescens

CU22261. Two hundred microliters (µl) of each strain were cultured with 10 ml of plueropneumonia-like organism (PPLO or Mycoplasma) broth and incubated at 37 °C, 10% CO 2 for 4 days.

Inoculation of milk samples

The PPLO cultures were centrifuged at 5000 x g for 30 minutes to sediment the bacteria.

The mycoplasma cell pellets were resuspended with phosphate buffer saline solution (PBS) and adjusted for their concentration by measuring the optical density at wavelength of 540 nm

(OD 540 ) using a spectophotometer. All Mycoplasma -PBS solutions were adjusted to have OD 540

= 0.2, which was expected to contain approximately 10 8 CFU/ml of Mycoplasma . Ten-fold serial dilutions were then made. One milliliter of each of 3 dilutions of resuspended culture was inoculated in 9 ml of bulk tank milk that was free of Mycoplasma to create 3 different starting concentrations of Mycoplasma in milk: high, medium and low concentration. The expected concentrations of Mycoplasma in inoculated milk samples for high, medium and low were 10 6,

10 4 and 10 2 CFU/ml, respectively. For each of the three concentrations, milk was spilt into 4 different tubes to determine the effect of refrigeration, Experiment I. The first tube, a fresh sample, was the control for the experiment. The 3 refrigeration treatments included refrigeration

(5° C) for 1 day, for 3 days, and for 5 days. l. In Experiment II, four treatments of glycerol addition (v/v) to the inoculated milk included 0% glycerol, 10% glycerol, 30% glycerol and 50% glycerol, which were then frozen at -20 °C for 1 week. After refrigeration and after thawing, 100

µl of each milk sample was removed and cultured on Modified Hayflicks agar plates (Hogan et

al., 1999) and incubated for 7-10 days at 37 °C, 10% CO 2 before enumeration of colonies. The

experiment was replicated with the identical methods.

Statistical analysis

Mycoplasma count data were log 10 transformed before analysis. Mycoplasma counts for

different species and different storage methods were compared using analysis of variance

(ANOVA). Mixed model analysis was performed using Proc Mixed by SAS version 9.1 (SAS

Institute, Cary, NC). Storage methods and species were analyzed as fixed effects whereas

starting concentrations, strains, and replication were analyzed as random effects. The criteria for

statistical significant was P <0.05.

RESULTS

A total of 540 inoculated milk samples were assayed; 240 samples for refrigeration study

and 300 samples for glycerol addition study. The effects of refrigeration are shown in Table 1.

Refrigerated milk mycoplasma counts were lower than those from fresh milk samples after 5

days of refrigeration (P<0.05). The effects of freezing milk samples with various concentrations

of glycerol addition are shown in Table 2. All frozen milk samples had lower number of

mycoplasma counts compared to those of fresh milk. Frozen milk with 10% and 30% glycerol

had greater mycoplasma counts compared to those of frozen milk without glycerol (P<0.05). No

significant interactions of species and storage methods were detected in this study (P>0.05).

DISCUSSION

The trend indicated a reduction in the recovery of Mycoplasma species from milk over time with refrigerated storage and refrigeration beyond 3 days had a significant negative effect on the recovered number of Mycoplasma species from milk. The reduction in the amount of

Mycoplasma species can increase the probability of having false negative results from in vitro culture (Biddle et al., 2004). Several studies reported the change in viability of Mycoplasma species based on their survival in sterile liquid media. Human mycoplasmas were reported to survive less than 30 days at 4 °C in cultured broth (Ford, 1962). In contrast, Nagatomo et al.

(2001) reported a longer survival period for animal mycoplasmas under similar refrigerated conditions with some species still viable at 59 day in PPLO broth. A study on the viability of M. bovis in milk sample stored at 5 °C was recently published (Vyletelova, 2010). They reported that the total count of M. bovis in milk samples was reduced approximately 0.5 log 10 after 5 weeks of refrigeration. Mycoplasma species in milk not collected aseptically, such as bulk tank milk samples, may suffer from refrigerated storage as a result of the overgrowth of other bacteria which may change the physicochemical properties of the milk media. Celestino and colleagues

(1996) revealed that the proportion of psychrotrophic bacteria in the total plate count increased from 47% in fresh milk to 80% after storage at 4 °C for two days. Moreover, they reported an increase in enzyme activities, notably lipolysis and proteolysis, occurred after two days in

refrigerated milk. These changes subsequently increased free fatty acids and lowered pH in milk.

Because Mycoplasma species are sensitive to changing pH and overgrowth of other bacteria

(Boughton, 1979; Nicholas and Baker, 1998), refrigerating milk samples for long periods of time

may increase the risk of false-negative tests for mycoplasma mastitis. It had been suggested that

when a milk sample has to be stored for more than 2 days before culturing for suspected

Mycoplasma species, the milk sample should be frozen or kept in liquid nitrogen (Hogan et al.,

1999). However, most frozen milk samples usually are kept in the freezing part of the

conventional refrigerator with the temperature around -20 °C, which is warmer than the preferred

temperature at -30 °C or lower (Hogan et al., 1999). The data presented in the current study

indicates that storage at -20 °C reduces mycoplasma counts, which may increase the chance of

false negative results. False negative results could increase from to 20% to 45% when milk

samples were frozen for 1 week to 4 weeks, respectively (Biddle et al., 2004). The addition of

glycerol to milk as a cryopreservative was considered to improve the recovery of Mycoplasma

species from frozen milk. We conjectured that glycerol would rapidly penetrate into mycoplasma

cells and effectively alleviate cell damage caused by the formation of intracellular and

extracellular ice crystalization (Raccach et al., 1975). Our results showed that adding glycerol to

milk prior freezing at the concentration from 10% to 30% can improve the number of recovered

Mycoplasma species from frozen milk. Moreover, adding glycerol at higher concentrations, such

as 50%, may be detrimental to the recovery of Mycoplasma species. Although freezing milk with any addition of glycerol led to a decrease in recoverable Mycoplasma species , the decrease was not as great as without glycerol addition. Thus, this study reveals a possibility of adding glycerol to milk to preserve Mycoplasma species before isolation in laboratories when frozen storage of samples is the best option.

The aim of mastitis diagnosis is to isolate the causative pathogen from a milk sample.

The mean reduction of the number of recovered Mycoplasma species reported in this study was approximately 0.3 log 10 when fresh milk samples were compared to 5-day refrigerated milk samples and approximately 1.0 log 10 when fresh milk samples were compared to frozen milk samples without glycerol addition. Reductions of this magnitude may not significantly increase the risk of a false negative result. Sensitivity of detection will be in large part a function of the number of pathogens in the milk sample and the volume of sample cultured. When samples have relatively large number of mycoplasma pathogens in milk such as 10 x 10 6 CFU/ml in some infected mammary quarters, a 1.0 log 10 reduction in the number of recovered pathogens due to storage will not affect sensitivity of detecting an infected cow. However, it may affect the diagnosis when an infected cow sheds a very small amount the organism. As indicated by Biddle and co-workers (2003), infected cows can shed as less than 10,000 CFU/ml of Mycoplasma species for more than 20% (it will be greater than 10%, 2 of 10 cows virtually never shed this organism) of the time. It is very critical to find the infected cows to prevent the transmission of this contagious pathogen. Therefore it would be advisable to culture fresh milk samples to maximize the detection of Mycoplasma species in milk because sample handling by refrigeration

or freezing can reduce the number of the pathogens.

In conclusion, evidence from this study indicates that milk samples should be stored

under refrigerated temperature for less than 5 days prior to submission for culture for

Mycoplasma species. In addition to refrigeration, findings confirm that freezing milk samples

reduces the number of Mycoplasma species recovered by culture. Moreover, the addition of

glycerol at 10% to 30% (v/v) can be used as a cryopreservative to enhance Mycoplasma species

recovery from frozen milk. For dairy operators and attending veterinarians, this study suggests

that milk samples submitted for mycoplasma diagnosis should be sent as fresh as possible because the number of viable Mycoplasma species decreases with time which may result in failure to detect the pathogen. Transit time of 3 days from farm to laboratory, if samples are held at refrigerated temperatures, should not significantly decrease recovery of Mycoplasma species from milk. If milk samples need to be held longer before culturing, freezing milk after adding

10% to 30% of glycerol (v/v) as a cryoprotective additive is suggested. This application can reduce damage from freezing and thawing methods, and improve the recovery of Mycoplasma species from frozen milk.

ACKNOWLEDGMENTS

We are grateful to Dorothy Newkirk, and Veerasak Punyapornwithaya for their excellent assistance. This study was published as a short communication in Veterinary Microbiology July

29, 2010, volume 144, page 210-213. Permission to reproduce the article was granted by Elsevier on October 12, 2010.

REFERENCES

Bashandy, E. Y., Heider, L. E., 1979. The effect of freezing milk samples on the cultural results.

Zentralbl. Veterinaermed. Reihe B. 26, 1-6.

Biddle, M. K., Fox, L. K., Hancock, D. D., 2003. Patterns of mycoplasma shedding in the milk

of dairy cows with intramammary mycoplasma infection. J. Am. Vet. Med. Assoc. 223,

1163-1166.

Biddle, M. K., Fox, L. K., Hancock, D. D., Gaskins, C. T., Evans, M. A., 2004. Effects of

storage time and thawing methods on the recovery of Mycoplasma species in milk

samples from cows with intramammary infections. J. Dairy Sci. 87, 933-936.

Boughton, E., 1979. Mycoplasma bovis mastitis. Vet. Bull. 49, 377-387.

Bushnell, R. B., 1984. Mycoplasma mastitis. Vet. Clin. N. Am. Food Anim. Pract. 6, 301-312.

Celestino, E. L., Lyer, M., Roginski, H., 1996. The effects of refrigerated storage on the quality

of raw milk. Aust. J. Dairy Technol. 51, 59-63.

Ford, D. K., 1962. Culture of human genital “T-strain” pleuropneumonia-like organisms. J.

Bacteriol. 84, 1028-1034.

Fox, L. K., Kirk, J. H., Britten, A., 2005. Mycoplasma mastitis: a review of transmission and

control. J. Vet. Med. B. 52, 153-160.

Gonzalez, R. N., Wilson, D. J., 2003. Mycoplasma mastitis in dairy herds. Vet. Clin. North Am.

Food Anim. Pract. 19, 199-221.

Hogan, J. S., Gonzalez, R. N., Harmon, R. J., Nickerson, S. C., Oliver, S. P., Pankey, J. W.,

Smith, K. L., 1999. Laboratory handbook on bovine mastitis. National Mastitis Council,

Wisconsin, pp. 151-153.

Hubalek, Z. 2003. Protectants used in the cryopreservation of microorganisms. Cryobiology. 46,

205-229.

Luedecke, L. O., Forster, T. L., Williams, K., Hillers, J. K., 1971. Effect of freezing and storage

at -20 °C on survival of mastitis pathogens. J. Dairy Sci. 55, 417-418.

Nagatomo, H., Takegahara, Y., Sonoda, T., Yamagushi, A., Uemura, R., Hagiwara, S.,

Sueyoshi, M., 2001. Comparative studies of the persistence of animal mycoplasmas

under different environmental conditions. Vet. Microbiol. 82, 223-232.

Nicholas, R., Baker, S., 1998. Recovery of Mycoplasmas from animals. In: Miles, R., Nicholas,

R. (Eds.), Mycoplasma Protocols, Humana Press, Totowa, New Jersey, pp. 37-43.

Pankey, J. W., Wadsworth, J. K., K. Metha, H., Murdough, P. A., 1987. Effects of storage on

viability of mastitis pathogens. J. Dairy Sci. 70(Suppl.1), 132. (Abstr.)

Raccach, M., Rottem, S., Razin, S., 1975. Survival of frozen Mycoplasmas. Appl. Microbiol. 30,

167-171.

SAS/STAT 9.1 User’s Guide, 2008. SAS Institute Inc., North Carolina

Schukken, Y. H., J. Smit, A. H., Grommers, F. J., Vandegeer, D., Brand, A., 1989. Effect of

freezing on bacteriologic culturing on mastitis milk samples. J. Dairy Sci. 72, 1900-1906.

Storper, M., Ziv, G., Saran, A. 1983. Effect of storing milk samples at -18 °C on the viability of

certain udder pathogens. Refuah Vet. 39, 1-2.

TABLES

TABLE 1. Mycoplasma countsa from fresh and refrigerated milk samples

Storage conditions Mycoplasma counts Significant differences b

Least squares mean ± SE

(Log 10 CFU/ml)

Fresh 4.43 ± 0.28 A

Refrigeration (5°C) for 1day 4.45 ± 0.27 A

Refrigeration (5°C) for 3days 4.29 ± 0.28 AB

Refrigeration (5°C) for 5days 4.10 ± 0.29 B

bDifferent letters indicate statistically significant differences in least square means of

mycoplasma counts between treatments, P<0.05.

TABLE 2. Mycoplasma counts a from fresh and frozen milk samples with various concentrations of glycerol addition b

Storage conditions Mycoplasma counts Significant differences c

Least squares mean ± SE

(Log 10 CFU/ml)

Fresh 4.56 ± 0.22 A

0% glycerol in frozen milk 3.71 ± 0.27 C

10% glycerol in frozen milk 4.16 ± 0.23 B

30% glycerol in frozen milk 4.06 ± 0.22 B

50% glycerol in frozen milk 3.45 ± 0.26 C

a Mycoplasma counts are a compilation of the mean counts of all isolates for the respective treatment. Isolates were two strains of each of 5 species: M. bovis, M. californicum, M. bovigenitallium, M. canadense and M. alkalescens . b Glycerol addition was made to achieve final concentrations of 10%, 30% and 50%, volume to volume.

cDifferent letters indicate statistically significant differences in least square means of mycoplasma counts between treatments, P<0.05.

CHAPTER 4

Discrimination between Mycoplasma and Acholeplasma using digitonin disc diffusion assay,

nisin disc diffusion assay, and conventional PCR

INTRODUCTION

Mycoplasma species have been reported to cause severe mastitis in dairy cattle

worldwide (Fox et al., 2005). Mycoplasma species are categorized as contagious pathogens

which can be readily transmitted from cow-to-cow. Treatment of mycoplasma mastitis is not

very effective and can lead to a chronic and/or subclinical infection (Bushnell, 1984). Therefore,

segregation and culling of infected animals has been suggested to prevent exposure of infected

animals to naïve animals in an infected herd. Precise detection of infected animals at an early

stage is a critical component of a mycoplasma mastitis control strategy.

Direct culture of a milk sample on blood agar plates has been the standard initial step in

the determination of most etiologic agents causing mastitis (Hogan et al., 1999). Mycoplasma

species are fastidious pathogens and have special growth requirements. Isolation of these

organisms requires specific medium; modified Hayflick’s (Hogan et al., 1999). Then inoculated

media is incubated at 37 °C in a moist 10% CO 2 environment as Mycoplasma require low

oxygen tension. Acholeplasma laidlawii is a common non pathogenic saprophytic contaminant in

the dairy environment and is occasionally found in both tank-milk and cow-milk samples

(Bushnell, 1984; Jasper, 1982). Acholeplasma laidlawii is normally considered a non-pathogenic agent (Jasper, 1981) although some studies reported the isolation of this species from mastitic milk (Pan and Ogata, 1969; Counter, 1978). Acholeplasma species can not be distinguished from

Mycoplasma species on modified Hayflick’s medium. Therefore, additional discriminatory tests

are needed to distinguish between Mycoplasma and Acholeplasma. A major difference between

Mycoplasma species and Acholeplasma species is their sterol requirement. Mycoplasma species

are unable to synthesize sterols or fatty acids and thus require exogenous sterol from media

(McElhaney, 1983). Acholeplasma species do not require exogenous cholesterol for growth

(Saito et al., 1978). The digitonin disc diffusion assay can be used to distinguish between

Mycoplasma and Acholeplasma based on the different sterol nutrient requirements. Digitonin can

form a complex with sterol, interrupting the uptake of exogenous sterol by Mycoplasma but not

Acholeplasma. Thus growth of Mycoplasma but not Acholeplasma is inhibited by digitonin.

An antimicrobial peptide nisin can also be used to differentiate between Acholeplasma

species and Mycoplasma species. Nisin is produced by Lactococcus lactis and can inhibit growth of most gram-positive and some gram-negative bacteria. Abu-Amero et al. (1996) reported that nisin readily inhibited growth of Acholeplasma species whereas Mycoplasma species were

substantially less affected by nisin. Therefore, they proposed that a nisin disc diffusion assay on

agar plates could be used to distinguish between these two genera.

Mycoplasma species and Acholeplasma species can also be distinguished based on their genotypic characteristics. A duplex-nested PCR method for detection has been reported (Tang et al., 2000). This PCR assay was designed to target 16S-23S rRNA intergenic spacer region (ITS).

Because more than one operon of tRNA genes in the targeted ITS region are usually presented in the genome of Acholeplasmas but not in the genome of Mycoplasmas , more than one size of amplicon are usually detected with Acholeplasmas whereas only one size of amplicon is

presented with Mycoplasmas . As a result Tang et al., (2000) were able to discriminate

Acholeplasma species from other Mycoplasma species using a PCR assay.

Discrimination of Acholeplasma species from Mycoplasma species has been practiced using the digitonin and/or the nisin disc diffusion assay, and by PCR methods, for several years.

These are tests that can be commonly employed by veterinary diagnostic laboratories. Yet what is not known is how comparable these tests of mollicute genera and if discrimination between genera by these tests can be made using isolates from mastitic milk representing several different species. A comparison of techniques used to discriminate Mycoplasma species from other

Mollicutes is needed to improve the accuracy of diagnosis of mycoplasma mastitis. The objective of this study was to determine the agreement for genus identification of Mycoplasma species and

Acholeplasma species obtained from the digitonin and nisin disc diffusion assays, and PCR; to the 16S rRNA partial sequence analysis as a gold standard.

MATERIALS AND METHODS

Organisms.

A total of 268 isolates of unknown species from bovine sources, primarily milk samples from cows with mastitis, and presumptively identified as Mycoplasma from isolation on modified Hayflick’s agar (BBL , Franklin Lakes, NJ, USA), were included in the study.

Additionally, 12 reference strains were chosen (Table 1). Filtration-cloning technique was applied to all isolates as described by Tully (1983). In brief, all isolates were subcultured and filtered through 0.45 µm three times to obtain a dominant clone from stock cultures. A single colony of each isolate was punched from the agar plate and grown in PPLO broth (Hardy

Diagnostics, Santa Maria, CA, USA), in a 10 % CO 2, environment for 4 days. Glycerol (30% v/v) was added to cultured broth before storage at -85 °C until use.

Digitonin disc diffusion assay.

The digitonin disc diffusion assays were performed as described by Tully (1983).

Digitonin stock solution of 1.5% (w/v) in 95% ethanol was made by adding 75 mg of digitonin

(Sigma-Aldrich, St. Louis, MO, USA) to 5 ml of 95% ethanol and storing at 4 °C. Digitonin

discs were made by adding 25 µl of the stock solution to 6 mm blank paper disc (BD, Sparks,

MD, USA). All digitonin discs were dried overnight at room temperature and stored at 4 °C until

use.

Both unknown and reference mollicutes were cultured in PPLO broth and incubated at

10% CO 2 for 4 days. Then, 200 µl of each culture was spread on modified Hayflick’s agar plate.

Digitonin discs were pressed gently to the surface of the agar after the inoculum had dried. Plates were incubated for 4 – 7 days at 37 °C in a 10 % CO 2 incubator. After incubation, all plates were examined under the stereomicroscope and the zones of inhibition were measured from the edge of the disc to the edge of clear zone of no growth (mm). Positive digitonin tests were considered when the clear zone was >5 mm, negative digitonin tests had zones <3 mm, and all else was considered an ambiguous result.

Nisin disc diffusion assay.

Nisin solution at the concentration of 5.16 mg/ml (ImmuCell Corp., Portland, ME, USA) was used as a stock solution. A ten fold dilution was made from the stock solution. Twenty microliters from the stock solution (N0: 103.2 µg) and the dilution (N-1: 10.32 µg) was added to a separate 6 mm blank paper disc (BD, Sparks, MD, USA). All nisin discs were dried overnight at room temperature and stored at 4 °C until use. Test organisms were cultured and plated as described for the digitonin disc assay. Nisin discs, both N0 and N-1, were applied to the agar

plates and incubated for 4 – 7 days at 37 °C in a 10 % CO 2 incubator. After incubation, all plates were examined under the stereomicroscope and measured for the zone of inhibition (mm).

According to the results reported by Abu-Amero et al. (1996), a positive nisin test, was indicated by the presence of a visible growth inhibition zone around the nisin disc. A negative nisin test was indicated by the lack of any inhibition zone.

Genomic DNA extraction for PCR.

Mycoplasma isolates were re-cultured in PPLO broth and incubated at 37 °C in a 10%

CO 2 incubator for 4 days. After incubation, the DNA extraction was performed on all cultures by following instructions for extraction of gram-negative bacterial genomic DNA (Invitrogen,

Carlsbad, CA, USA). Briefly, all cultured isolates were centrifuged at 5000 x g for 25 minutes to sediment mycoplasma cells. The mycoplasma cell pellets were re-suspended with a digestion buffer. Cell lysate was prepared by adding 20 µl of Proteinase K to the re-suspension and incubated at 55 °C for 45 minutes. After incubation, 20 µl of RNase A was added to each cell lysate and incubated at room temperature for 2 minutes. Then, 200 µl of lysis-binding buffer and

200 µl of 96 – 100 % ethanol were added to the cell lysate. The cell lysate was then transferred into a DNA binding column. The cell lysate loaded DNA binding column was centrifuged at

10,000 x g for 1 minute. The DNA binding column was washed twice; first with 500 µl of washing buffer 1 and centrifuged at 10,000 x g for 1 minute, and then with 500 µl of washing buffer 2 and centrifuged at maximum speed for 3 minutes. After washing, the genomic DNA was eluted out by adding 75 µl of elution buffer, and then the tube was centrifuged at maximum speed for 1 minute. All genomic DNA extracts were frozen at -80 °C until used for PCR.

Discrimination between Acholeplasma species and Mycoplasma species using conventional

PCR.

A one-step PCR was performed as adapted from Tang et al. (2000). PCR was performed

in a total volume of 50 µL containing 1x PCR buffer (20 mM Tris-HCl, 2 mM MgCl2, and 50

mM KCl; pH 8.4 ), 50 µM of each of deoxynucleoside triphosphates (dNTPs), 20 pmol of each

primer, and 1 U Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). A set of primers

including F2, R2 and R34 was used to amplify the 16S-23S rRNA intergenic spacer regions of

Mycoplasma and Acholeplasma . Sequences and targets of these primers are shown in Table 2.

Five microliters of DNA extract was used as a template by adding into a 45 µL of reaction

mixture. The thermal cycling protocol was programmed: initial denaturation at 94 °C for 30 s, followed by 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 2 minutes,

and extension at 72 °C for 2 minutes. A final extension was performed at 72 °C for 5 minutes.

The final PCR products were electrophoresed on a 2% agarose gel and DNA bands were

visualized by UV fluorescence. The presence of a single band of PCR product indicated the

presence of Mycoplasma species. The presence of > 1 band of PCR product indicated the

presence of Acholeplasma species.

16S rRNA partial sequencing.

Identifications of all 281 isolates were confirmed using 16S rRNA partial sequencing as a gold standard. A region of approximately 1500 base pairs of the 16S rRNA gene was amplified using a universal primer pair; pH and pA as listed in Table 2 (Stakenborg et al., 2005). The PCR reaction mixture in the total volume of 50 µL included 3 U of Taq DNA polymerase (Invitrogen,

Carlsbad, CA, USA), 1x PCR buffer (20 mM Tris-HCl, 1.5 mM MgCl2, and 50 mM KCl; pH

PCR products were purified according directions (Roche, Indianapolis, IN, USA) and submitted for sequencing using primer pA (Macrogen Corp., Rockville, MD, USA). Nucleotide sequences were compared with GenBank database using the Basic Local Alignment Search Tool (BLAST;

Altschul et al., 1990). A nucleotide sequence was identified as a species giving the highest hit of at least 98% identity from BLAST search.

Statistical analysis.

All isolate assays were replicated. If results of each replicate did not agree, a third assay was performed. The definitive identification of an isolate was then given when results of 2 out of

3 replicates agreed. Genera classification by digitonin disc and nisin disc diffusion assay, and

PCR, were compared with results from 16S rRNA partial sequencing and presented as percentage agreement. For all techniques exact kappa analysis with 95% confident interval (CI) was used as a statistical test of agreement (Stokes et al., 2000) with the gold standard, 16S rRNA partial sequencing technique using PROC FREQ in SAS 9.2 (SAS Institute, Cary, NC, USA).

RESULTS

Mycoplasma isolates identified by 16S rRNA partial sequencing.

Two hundred and eighty Mycoplasma isolates from various sources were included in the study, 234 isolates (83.57%) were originally isolated from milk samples and 46 isolates

(16.43%) were originally isolated from other organs. In this collection, 278 isolates (99.29%)

were identified as Mycoplasma species based on the results of 16S rRNA partial sequencing.

Two isolates (0.71%) of A. axanthum were identified; one was a reference isolate (ATCC25176) and the other was from an ear swab solution of a dairy cow in Pennsylvania State. The distribution of Mycoplasma and Acholeplasm a isolates was listed in Table 3.

Discrimination between Mycoplasma and Acholeplasma using digitonin disc diffusion assay.

Of 280 isolates, 275 isolates showed zones of growth inhibition (+ and +/-) around the discs, ranging in size from 3 to 20 mm, mean ± SE of 11.07 ± 0.12 mm. An example of positive

results (Mycoplasma species) is shown in Figure 1, and an example of negative results

(Acholeplasma species) is shown in Figure 2. Descriptive statistics of zones of growth inhibition were indicated in Table 4. Zones of inhibition observed from M. bovigenitalium and M. bovis

were the largest with the means ± SE of 14.67 ± 0.70 and 11.85 ± 0.08, respectively. Five

isolates were categorized as negative which included 2 isolates of A. axanthum , 2 isolates of M.

yeatsii and 1 isolate of M. cottewii (Table 4).

All of the M. bovis isolates tested (n=231) had zones of growth inhibition of > 5 mm

threshold. Thus at this threshold M .bovis had 100% agreement with the 16S rRNA partial

sequencing technique. Equivalent agreements with M. bovigenitalium (6/6) and M. agalactiae

(4/4) isolates are also observed. Percentages of agreement for M. calfornicum and M. alkalescens

were only 34.78% (8/23) and 50% (2/4), using the 5 mm threshold (Table 4). The overall

Mycoplasma species agreement of digitonin disc diffusion assay to 16S rRNA partial sequencing

technique was 90.36% (253/280) using the ≥ 5 mm threshold, a significant agreement between

these two tests (P=0.0111) with the kappa of 0.1131 (95% CI: -0.0309, 0.2571).

In contrast, when the presence of zones of inhibition ≥ 3 mm was considered to be the threshold for a positive test, accuracy of digitonin disc diffusion assay presented as agreement with 16S rRNA partial sequencing technique was 100% for all Mycoplasma species except M. yeatsii (0%) and M. cottewii (0%) as shown in Table 5. Using the 3 mm threshold the overall agreement of digitonin disc diffusion assay to 16S rRNA partial sequencing technique was

98.93% (277/280) giving a significant agreement between these two tests (P<0.0001) with the kappa of 0.5670 (95% CI: 0.1277, 1.0000) as shown in Table 5.

Discrimination between Mycoplasma and Acholeplasma using nisin disc diffusion assay.

No zones of nisin inhibited growth were seen by any Mycoplasma tested. Both isolates of

A. axanthum had zones of growth inhibition with the nisin disc diffusion assay for both N0 and

N-1 discs. An example of negative results ( Mycoplasma species) was shown in Figure 1, and an

example of positive results ( Acholeplasma species) was shown in Figure 2. Ranges of zones of

inhibition were 3 – 8 mm and 1 – 6 mm for N0 and N-1, respectively. Mean ± SE of zones of inhibition for nisin disc diffusion assay were 5 ± 1.08 mm and 2.5 ± 1.19 mm for N0 and N-1.

Medians of zones of inhibition of nisin disc diffusion assay were 4.5 mm and 1.5 mm for N0 and

N-1. All Mycoplasma and Acholeplasma isolates were correctly identified with the percentage

agreement with 16S rRNA partial sequencing of 100%.

Discrimination between Mycoplasma and Acholeplasma using PCR technique.

The amplicons of all Mycoplasma and Acholeplasma isolates correctly identified each

genus; Mycoplasma always yielded a single amplicon and Acholeplasma yielded 2 amplicons as

demonstrated in Figure 3. The PCR test resulted in a 100% agreement with 16S rRNA partial

sequencing.

DISCUSSION

The thrust of the study was to contrast accuracy of Mycoplasma species and

Acholeplasma species identification using the digitonin, nisin disc diffusion, and PCR assays as compared to the gold standard 16S rRNA partial sequencing technique. All isolates tested by nisin test and PCR tests were in perfect agreement (100%) with the gold standard. Results obtained from the digitonin disc diffusion assay, using a threshold either >5 mm or >3 mm for the presence of zone of growth inhibition, showed a significant agreement with the gold standard. Moreover, the discordant results observed from only 9 isolates out of 234 bovine milk isolates (3.85 %). Therefore, the results obtained from the digitonin disc diffusion assay were still reasonably to be used to distinguish between the two genera originally isolated from mastitis cases. These findings indicate that all 3 techniques performed in this study can be used to distinguish Mycoplasma species and Acholeplasma species isolated from bovine origin.

Two hundred and thirty four isolates tested in this study (83.57%) were from milk samples, cows with clinical mastitis. Of these mastitis isolates, 91.88% (215/234), 5.13%

(12/234), and 0.85% (2/234) were M. bovis, M. californicum, and M. bovigenitalium , and only

2.14% (5/234) were identified to be other species. This finding is consistent with other studies done in California that M. bovis is the most prevalent Mycoplasma species, followed by M. californicum and M. bovigenitalium, isolated from samples of clinical mastitis cases (Jasper,

1980) and bulk-tank milk samples (Kirk et al., 1997).

Digitonin disc diffusion assay has been used as a standard method to discriminate

Acholeplasma species from Mycoplasm a species in many laboratories capable of culturing

Mollicutes as suggested by Subcommittee on the Taxonomy of Mollicutes (1995). In the current

study, all isolates identified to be M. bovis , M. bovigenitalium and M. agalactiae were accurately distinguished to be Mycoplasma based on the presence of zone of growth inhibition using either

>5 mm or >3 mm as a threshold. Using the zone of growth inhibition threshold of >5 mm as

suggested by Tully (1983), 65.22% (15/23) of M. californicum isolates and 50% (2/4) of M.

alkalescens isolates were misclassified. When the presence of zone of growth inhibition of >3 mm was considered to be the threshold, all mycoplasma isolates originally isolated from milk samples were correctly categorized to be Mycoplasma species. There were only 3 isolates originally isolated from bovine lung tissues; 2 M. yeatsii isolates, and 1 M. cottewii isolate that were slightly sensitive to digitonin (1 mm of zone of growth inhibition) and thus would be misclassified regardless of the threshold of zone of inhibition used. Our results for these 3 isolates differs from another report where the sensitivity to digitonin was greater as measured by zone of growth inhibition of 6 to 11 mm for M. yeatsii and M. cottewii (DaMassa et al., 1994).

It appears that zones of growth inhibition by the digitonin disc diffusion assay vary by species. Larger zones of growth inhibition (mean ± SE) were observed for M. bovigenitalium

(14.67 ± 0.70 mm) and M. bovis (11.85 ± 0.08 mm) compared to M. californicum (5.33 ± 0.19 mm) , M. arginini (3.88 ± 0.30 mm) and M. alkalescens (3.88 ± 0.30 mm). Thurmond et al.

(1989) reported larger zones for M. arginini (12 mm) and M. californicum (9.74 mm) whereas small zones were observed for M. canadense (8.35 mm) and M. alkalescens (7.87 mm). In the

current study the digitonin disc diffusion assay correctly identified Acholeplasma isolates.

However, there were only 2 isolates of A. axanthum tested. There are some studies which

demonstrated some variation of zones of inhibition of Acholeplasma species isolated from

bovine origin. Freundt et al. (1973) and Jurmanová (1975) reported the zones of inhibition of

Acholeplasma species to digitonin ranged from 0.5 to 1.5 mm. Thurmond et al. (1996) reported

zones of inhibition among 20 isolates of A. laidlawii ranged from 1-4 mm. More isolates of

Acholeplasma species included in the study would be necessary to investigate variation of zone of inhibition by digitonin.

Abu-Amero et al. (1996) reported that the nisin disc diffusion assay could be used to discriminate between Acholeplasma from Mycoplasma . They tested the sensitivity of nisin to 5

Acholeplasma species and 11 Mycoplasma species. All 5 Acholeplasma species had zones of inhibition whereas all 11 Mycplasma species did not show any zone of inhibition. Findings herein were similar in that growth of all Acholeplasma and no Mycoplasma were inhibited by the nisin discs at two concentration levels. Again, with only 2 Acholeplasma species tested, the ability to evaluate the variation in Acholeplasma responses to an inhibitory substance, nisin, is considerably diminished. Although the two levels of nisin applied to the filter discs, 103.2 µg

(N0) versus 10.32 µg (N-1) yielded equivalent results, the small population of Acholeplasma species tested makes it difficult to draw conclusions on the optimum level of nisin to use.

The PCR also had 100% agreement with the gold standard. The lack of Acholeplasma species isolates to test lessens the ability to truly determine the accuracy of the PCR technique described by Tang et al. (2000). Yet the results reported herein are consistent with previous findings that identified 2 PCR products by Acholeplasma species and one PCR product by all

Mycoplasma species as described (Tang et al., 2000).

In conclusion, this study suggests a high and comparable efficiency of using nisin and digitonin disc diffusion assays and PCR to distinguish Mycoplasma and Acholeplasma species.

Thus the study confirms those of others that all the assays can distinguish between Acholeplasma

species and Mycoplasma species, although the lack of Acholeplasma species tested in the current

study limits confidence in accuracy of discrimination. Choosing a technique to use to discriminate between Mycoplasma and Acholeplasma species depends upon the instrumentation requirements, labor, and costs and toxicity of chemicals. Our results indicate that the nisin and digitonin disc diffusion assays, and PCR assay, can be used to confirm that a mollicute presumptively identified as a Mycoplasma species is not Acholeplasma .

ACKNOWLEDGMENTS

We would like to thank ImmuCell Corp. (Portland, ME, USA), especially Dr. Joseph

Crabb, for his kindly supply of nisin. We are grateful to Dorothy Newkirk, and Veerasak

Punyapornwithaya for their excellent assistance.

REFERENCES

Abu-Amero, K.K., Halablab, M.A., Miles, R.J., 1996, Nisin resistance distinguishes

Mycoplasma spp. from Acholeplasma spp. and provides a basis for selective growth

media. Appl Environ Microbiol 62, 3107-3111.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990, Basic local alignment

search tool. J Mol Biol 215, 403-410.

Brown, M.B., Gionet, P., Senior, D.F., 1990, Identification of Mycoplasma felis and Mycoplasma

gateae by an immunobinding assay. J Clin Microbiol 28, 1870-1873.

Bushnell, R.B., 1984, Mycoplasma mastitis. Vet Clin North Am Large Anim Pract 6, 301-312.

Counter, D.E., 1978, A severe outbreak of bovine mastitis asssociated with Mycoplasma

bovigenitalium and Acholeplasma laidlawii . Vet Rec 103, 130-131.

DaMassa, A.J., Tully, J.G., Rose, D.L., Pitcher, D., Leach, R.H., Cottew, G.S., 1994,

Mycoplasma auris sp. nov. , Mycoplasma cottewii sp. nov. , and Mycoplasma yeatsii sp.

nov. , new sterol-requiring Mollicutes from the external ear canals of goats. Int J Syst

Bacteriol 44, 479-484.

Delves-Broughton, J., 1990, Nisin and its uses as a food preservative. Food Technol 44, 100-117.

Fox, L.K., Kirk, J.H., Britten, A., 2005, Mycoplasma mastitis: a review of transmission and

control. J Vet Med B Infect Dis Vet Public Health 52, 153-160.

Freundt, E.A., Andrews, B.E., Erno, H., Kunze, M., Black, F.T., 1973, The sensitivity of

mycoplasmatales to sodium-polyanethol-sulfonate and digitonin. Zentralbl Bakteriol Orig

A 225, 104-112.

Gonzalez, R.N., Wilson, D.J., 2003, Mycoplasmal mastitis in dairy herds. Vet Clin North Am

Food Anim Pract 19, 199-221.

Hogan, J.S., Gonzalez, R.N., Harmon, R.J., Nickerson, S.C., Oliver, S.P. Pankey, J.W., Smith,

K.L., 1999, Laboratory handbook on bovine mastitis. National Mastitis Council, Inc,

Wisconsin, pp. 151-188.

International Committee on Systematic Bacteriology Subcommittee on the Taxonomy

of Mollicutes , 1995, Minimum Standards for Description of New Species of the Class

Mollicutes (Division Tenericutes). Int J Syst Bacteriol 45, 605-612.

Jasper, D.E., 1980, Prevalence of mycoplasma mastitis in the Western states. Calif.

Vet 4, 24-26.

Jasper, D.E., 1981, Bovine mycoplasmal mastitis. Adv Vet Sci Comp Med 25, 121-157.

Jasper, D.E., 1982, The role of Mycoplasma in bovine mastitis. J Am Vet Med Assoc 181, 158-

162.

Jurmanová, K., 1975, A contribution to tentative identification of bovine mycoplasmas. Zbl Vet

Med B 22, 529-534.

Kirk, J.H., Glenn, K., Ruiz, L., Smith, E., 1997, Epidemiologic analysis of Mycoplasma spp

isolated from bulk-tank milk samples obtained from dairy herds that were members of a

milk cooperative. J Am Vet Med Assoc 211, 1036-1038.

Liehmann, L., Degasperi, B., Spergser, J., Niebauer, G.W., 2006, Mycoplasma felis arthritis in

two cats. J Small Anim Pract 47, 476-479.

McElhaney, R.N., 1983, Section D4 Manipulation of membrane lipid composition, In: Tully,

J.G., Razin, S. (Eds.) Methods in mycoplasmology I. Academic Press, New York, pp.

235-239.

Pan, I.J., Ogata, M., 1969, New sero-types of Mycoplasma laidlawii isolated from mastitic milk

and urogenital tracts of cattle. II. Typing of M. laidlawii by biological and serological

methods. Nippon Juigaku Zasshi 31, 313-324.

Pfützner, H., Baumgärtner, B., Wehnert, C., 1986, The control of mycoplasma infections in

cattle. Monatshefte für Veterinärmedizin 41, 577-579.

Saito, Y., Silvius, J.R., McElhaney, R.N., 1978, Membrane lipid biosynthesis in Acholeplasma

laidlawii b: elongation of medium- and long-chain exogenous fatty acids in growing

cells. J Bacteriol 133, 66-74.

Stakenborg, T., Vicca, J., Butaye, P., Maes, D., De Baere, T., Verhelst, R., Peeters, J., de Kruif,

A., Haesebrouck, F., Vaneechoutte, M., 2005, Evaluation of amplified rDNA restriction

analysis (ARDRA) for the identification of Mycoplasma species . BMC Infect Dis 5, 46.

Stokes, M.E., Charles, S. D., Gary, G.K., 2000, Chapter 5: the s x r table, In: Stokes, M.E.,

Charles, S. D., Gary, G.K. (Eds.) Categorical data analysis using the SAS ® System,

Second Edition. SAS Institute Inc., North Carolina, pp. 112-116.

Taylor, T.M., Davidson, P.M., Zhong, Q., 2007, Extraction of nisin from a 2.5% commercial

nisin product using methanol and ethanol solutions. J Food Prot 70, 1272-1276.

Tang, J., Hu, M., Lee, S., Roblin, R., 2000, A polymerase chain reaction based method for

detecting Mycoplasma/Acholeplasma contaminants in cell culture. J Microbiol Methods

39, 121-126.

Thurmond, M.C., Holmberg, C.A., Luiz, D.M., 1989, Evaluation of a digitonin disk assay to

discriminate between acholeplasma and mycoplasma isolates from bovine milk. Cornell

Vet 79, 71-81.

Tully, J.G., 1983, Section E5 Tests for digitonin sensitivity and sterol requirement, In:

Tully, J.G., Razin, S. (Eds.) Methods in mycoplasmology I. Academic Press,

New York, pp. 355-362.

TABLES

TABLE 1. Sources of reference strains of Mycoplasma species and Acholeplasma species

Organisms Origin of isolates Source

M. bovis Bovine mastitic milk ATCC a 25025

M. bovis Bovine mastitic milk ATCC 25523

M. bovis Bovine mastitic milk Cornell University 22253

M. californicum Bovine mastitic milk ATCC 33461

M. bovigenitalium Bovine genital tract ATCC 14173

M. alkalescens Bovine nasal cavity ATCC 29103

M. alkalescens Bovine mastitic milk Cornell University 21146

M. agalactiae Mastitic goat milk ATCC 35890

M. arginini Lung of sheep with pneumonia ATCC 23243

M. bovirhinis Bovine respiratory tract ATCC 27748

M. leachii sp. nov Arthritic joint of a calf IOM b Mollicutes Collection; N29 Leach

strains

A. axanthum Murine leukemia tissue culture ATCC 25176

a American Type Culture Collection, Manassas, VA, USA.

b International Organization of Mycoplasmology, West Lafayette, IN, USA.

TABLE 2. Sequences of primers and targeted regions

Primers Sequences Targets

F2 5’-GTG(C/G)GG(A/C)TGGATCACCTCCT-3’ 16S-23 S rRNA intergenic spacer

R2 5’-GCATCCACCA(A/T)A(A/T)AC(C/T)CTT-3’ region of Mycoplasma species

R34 5’-CCACTGTGTGCCCTTTGTTCCT-3’ 16S-23 S rRNA intergenic spacer

region of Acholeplasma species

pA 5’-AGAGTTTGATCCTGGCTCAG-3’ 16S rRNA gene

pH 5’-AAGGAGGTGATCCAGCCGCA-3’

TABLE 3. Distribution of isolates of Mycoplasma species and Acholeplasma species as identified by 16S rRNA partial sequencing

Species identification from Total number of isolates

16S rRNA partial sequencing (number of reference strains)

M. bovis 231 (2)

M. californicum 23 (1)

M. bovigenitalium 6 (1)

A. axanthum 2 (1)

M. alkalescens 4 (2)

M. arginini 4 (1)

M. yeatsii 2

M. cottewii 1

M. bovirhinis 1 (1)

M. felis 1

M. leachii (Bovine serogr.7) 1 (1)

M. agalactiae 4 (1)

Total 280

TABLE 4. Descriptive statistics of zone of growth inhibition (mm) observed with the digitonin disc diffusion assay

Species Min Max Median Mean ± SE

M. bovis 6 20 12 11.85 ± 0.08

M. californicum 3 8 5 5.33 ± 0.19

M. bovigenitalium 11 19 14.5 14.67 ± 0.70

M. alkalescens 3 14 7.5 7.75 ± 0.91

M. agalactiae 6 11 9 9.13 ± 0.52

M. arginini 3 5 4 3.88 ± 0.30

M. bovirhinis 3 4 3.5 3.5 ± 0.50

M. felis 4 5 4.5 4.5 ± 0.50

M. leachii 4 4 4 4 ± 0.00

M. yeatsii 0 1 1 0.75 ± 0.25

M. cottewii 1 1 1 1 ± 0.00

A. axanthum 0 0 0 0

TABLE 5. Agreement of genera determination by digitonin disc diffusion assay with 16S rRNA

partial sequencing

Species Agreement with 16S rRNA partial sequencing technique (%)

Zone of growth inhibition >5 Zone of growth inhibition >3

mm a mm a

M. bovis 100% (231/231) 100% (231/231)

M. californicum 34.78% (8/23) 100% (23/23)

M. bovigenitalium 100% (6/6) 100% (6/6)

M. alkalescens 50% (2/4) 100% (4/4)

M. agalactiae 100%(4/4) 100%(4/4)

M. arginini 0%(0/4) 100%(4/4)

M. bovirhinis 0%(0/1) 100%(1/1)

M. felis 0%(0/1) 100%(1/1)

M. leachii 0%(0/1) 100%(1/1)

M. yeatsii 0%(0/2) 0%(0/2)

M. cottewii 0%(0/1) 0%(0/1)

A. axanthum 100%(0/2) 100%(0/2)

Total 90.36% (253/280) 98.93% (277/280)

aZones of growth inhibition thresholds tested at >5 mm and >3 mm to signify Mycoplasma species.

FIGURES

N0 disc N-1 disc

digitonin disc

FIGURE 1. Example plate of Mycoplasma species with a zone of inhibition only presented

around digitonin disc

N0 disc N-1 disc

digitonin disc

FIGURE 2. Example plate of Acholeplasma species with a zone of inhibition only presented

around nisin discs with both 103.2 µg (N0) and 10.32 µg (N-1)

1 2 3 4 5 6 7 8 9 10 11

FIGURE 3. Electrophoretic gel of amplicons of some reference strains of Mycoplasma species

and Acholeplasma species. Lane 1 and 11: 100 bp molecular ladder, lane 2: M. bovis ATCC

25025, lane 3: M. bovis ATCC 25523, lane 4: M. californicum ATCC 33461, lane 5: M.

bovigenitalium ATCC 14173, lane 6: M. alkalescens ATCC 29103, lane 7: M. bovirhinis ATCC

27748, lane 8: M. agalactiae ATCC 35890, lane 9: M. arginini ATCC 23243, lane 10: A. axanthum ATCC 25176

CHAPTER 5

Identification of Mycoplasma species using novel real-time PCR assays and PCR-RFLP

technique

INTRODUCTION

Mycoplasma species were first recognized as a cause of contagious mastitis in dairy cattle in the United States in 1961 (Hale et al, 1962). Since then, mycoplasma mastitis has been reported throughout the country and many regions of the world (Fox et al, 2005). M. bovis is the most common species to be associated with bovine mastitis, with perhaps as many as 49% to

60% of all mycoplasma mastitis isolates have been speciated as M. bovis (Jasper, 1979; Kirk et

al., 1997). Although less common, M. californicum and M. bovigenitalium have been frequently

reported to be associated with mastitis in dairy cows in many regions through out the world

(Jasper, 1982; Mackie et al., 1986; Jurmanová et al., 1983; Infante-Martínez et al., 1999; Kirk et

al., 1997; Hirose et al., 2001) and are perhaps the next two most prevalent species causing

mycoplasma mastitis.

The slow growth and fastidious nutritional requirements of Mycoplasma species in vitro

are an impediment to identification of this genus in the laboratory. Applications of highly

specific and sensitive polymerase chain reaction (PCR) techniques have been used to overcome

this problem. Nested PCR followed by restricted fragment length polymorphism (RFLP) has

been used to identify isolates of suspected Mycoplasma species (Tang et al., 2000). Even though

this technique seems to be promising for identification of Mycoplasma species directly from

biological samples, it requires 2 steps of PCR and a long period of incubation for enzyme

digestion, requiring approximately 18 to 30 hours to complete the diagnosis.

Application of real-time PCR techniques can overcome the difficulties associated with

the culture of Mycoplasma species in vitro and the length of time need to speciate by

conventional PCR. Real-time PCR methods have been used for more than a decade (Heid et al.,

1996) and facilitate the screening of large numbers of samples within a few hours with an

excellent sensitivity and specificity. Fluorescence detection of specific PCR products and

accumulation of this signal for each PCR cycle in a real-time basis allow quantitation of template

DNA (Leutenegger, 2001). Utilizing fluorgenic probes (TaqMan ) make the real-time PCR assay to be highly specific to detect the pathogen because only a specific target sequence in amplicons is recognized.

This study was proposed to develop 3 novel real-time PCR assays detecting 3 most common mycoplasma mastitis agents including M. bovis , M. californicum and M.

bovigenitalium . To validate these newly developed real-time PCR assays, the conventional PCR

described by Tang et al. (2000) was also performed with the same samples and results were

compared using 16S rRNA gene partial sequencing as a gold standard.

MATERIALS AND METHODS

Organisms.

A total of 268 isolates of unknown species from bovine sources, primarily milk samples

from cows with mastitis, and presumptively identified as Mycoplasma from isolation on

modified Hayflick’s agar, were included in the study. Additionally, 12 reference strains were

chosen (Table 1). Filtration-cloning technique was applied to all isolates as described by Tully

(1983). In brief, all isolates were subcultured and filtered through 0.45 µm three times to obtain a

dominant clone from stock cultures. A single colony of each isolate was punched from the agar

plate and grown in pleuropneumonia-like-organism (PPLO) broth (BBL , Franklin Lakes, NJ,

USA), in a 10 % CO 2 environment for 4 days. Glycerol (30% v/v) was added to cultured broth

before storage at -85 °C until use.

Primers and probes design for M. bovis, M. californicum and M. bovigenitalium .

Primer and probe design was performed using the Qiagen online tool

(http://www1.qiagen.com/products/pcr/quantitect/customassays.aspx ). Three different house

keeping genes; fusA, rpoB , and 16S-23S rRNA intergenic spacer region (ITS) were selected for

M. bovis , M. californicum and M. bovigenitalium , respectively. The fusA gene encodes for elongation factor G. The rpoB encodes for RNA polymerase beta subunit. The ITS encodes for a short region between the 16S and 23S subunits of ribosomal RNA. Primers and probes were tested for interactions using the online Oligo Analysis tool ( http://www.operon.com/technical/

toolkit.aspx ). The oligonucleotide sequences were tested for their specificity by searching against

GenBank database using the Basic Local Alignment Search Tool (BLAST; Altschul et al., 1990).

All primers and probes were found to be specific for the target genes of the target species with no

cross-matching with other bovine Mycoplasma species . The sequences of primers and probes were shown in Table 2.

Quantitative testing of the real-time PCR assays.

Real-time PCR results were given in terms of the cycle threshold (C T) which is defined as

the fractional cycle number at which the fluorescence passes the fixed threshold. To establish the

quantitative ability of each real-time PCR assay, a plot of the initial amount of DNA for a set of

standards versus C T or a standard curve was generated. Target sequences in fusA , rpoB , and ITS

genes were cloned into pIDTSMART-AMP plasmids (Integrated DNA technologies, Coralville,

IA, USA) and used to create standard curves for M.bovis , M. californicum and M. bovigenitalium real-time PCR assays, respectively. A standard curve with 6 dilutions containing 6 copy numbers of targeted DNA: 10 5, 10 4, 10 3, 10 2, 10 and 1 copy; was generated for each species as shown in

Figures 3-5. Quantitation of the amount of target in unknown samples was accomplished by

measuring C T and using the standard curve to determine starting amount of DNA.

Genomic DNA extraction for PCR.

Mycoplasma isolates were re-cultured in PPLO broth and incubated at 37 °C in a 10%

CO 2 incubator for 4 days. After incubation, the DNA extraction was performed on all cultures by

following instructions for extraction of gram-negative bacterial genomic DNA (Invitrogen,

Carlsbad, CA, USA). Briefly, all cultured isolates were centrifuged at 5000 x g for 25 minutes to

sediment mycoplasma cells. The mycoplasma cell pellets were re-suspended with a digestion

buffer. Cell lysate was prepared by adding 20 µl of Proteinase K to the re-suspension and

incubated at 55 °C for 45 minutes. After incubation, 20 µl of RNase A was added to each cell

lysate and incubated at room temperature for 2 minutes. Then, 200 µl of lysis-binding buffer and

200 µl of 96 – 100 % ethanol were added to the cell lysate. The cell lysate was then transferred

into a DNA binding column. The cell lysate loaded DNA binding column was centrifuged at

10,000 x g for 1 minute. The DNA binding column was washed twice; first with 500 µl of

washing buffer 1 and centrifuged at 10,000 x g for 1 minute, and then with 500 µl of washing

buffer 2 and centrifuged at maximum speed for 3 minutes. After washing, the genomic DNA was

eluted out by adding 75 µl of elution buffer, and then the tube was centrifuged at maximum

speed for 1 minute. All genomic DNA extracts were frozen at -80 °C until used for PCR.

For other gram positive bacteria, genomic DNA was extracted according to the protocol

described by Pitcher et al. (1989). Briefly, DNA was extracted from a cell pellet after overnight

culture. Cell pellet was reconstituted in Tris-EDTA (TE) buffer. Five microliter of lysostaphin

and 100 µl of lysozyme was added to the reconstituted cell pellet, and then incubated at 37 °C for

2 hours. After incubation, 500 µl of lysis buffer and 250 µl of ammonium acetate was added, and then set on ice for 10 minutes. An equal volume of chloroform in isoamyl alcohol was added to the tube, and then centrifuged at 13,000 x g for 15 minutes. The upper aqueous phase containing

DNA was collected and transferred into a new tube. An equal volume of chloroform in isoamyl alcohol was added, and then centrifuged again at 13,000 x g for 15 minutes. The upper aqueous phase was collected and transferred into a new tube. Isopropanol was added at the volume of

0.56x of the volume of the upper aqueous phase liquid collected, and then set at -20 °C for 2

hours. The tube was centrifuged at 13,000 x g for 5 minutes, and supernatant was then removed.

Five repeats of DNA washing were performed by adding 1 ml of absolute ethanol and

centrifuged at 13,000 x g for 5 minutes. The DNA pellet was air-dried. After completely dried,

50 µl of sterile distilled water was added to dissolve the DNA and frozen at -85 °C until use.

Real-time PCR.

The PCR reaction was initiated by combining 5 µL of the extracted DNA with 12.5 µL of

2x Master Mix, 10 pmol of each primer and 5 pmol of probe according to the Qiagen Quantitect

Probe PCR kit’s manual (Qiagen, Valencia, CA, USA). The amplification and detection was

performed on Step One Plus ® (Applied Biosystems, Foster City, CA, USA) real-time PCR

instrument. The thermal cycling protocols which were identical for all 3 assays included 15

minutes of pre-denaturation at 95 °C and 45 cycles of 15 seconds of denaturation at 94 °C and 60

seconds of annealing at 60 °C. Each reaction was classified as positive or negative for a target based on obtaining a C T below or above 37, respectively (Bustin, 2004).

PCR-RFLP.

A one-step PCR followed by RFLP was performed as adapted from Tang et al. (2000).

PCR was performed in a total volume of 50 µL containing 1x PCR buffer (20 mM Tris-HCl, 2 mM MgCl2, and 50 mM KCl; pH 8.4 ), 50 µM of each of deoxynucleoside triphosphates

(dNTPs), 20 pmol of each primer, and 1 U Taq DNA polymerase (Invitrogen, Carlsbad, CA,

USA). A set of primers including F2, R2 and R34 was used to amplify the 16S and 23S rRNA intergenic spacer regions of Mycoplasma and Acholeplasma . Sequences and targets of these primers are shown in Table 2. Five microliters of DNA extract was used as a template by adding into a 45 µL of reaction mixture. The thermal cycling protocol was programmed: initial denaturation at 94 °C for 30 seconds, followed by 35 cycles of denaturation at 94 °C for 30 seconds, primer annealing at 55 °C for 2 minutes, and extension at 72 °C for 2 minutes. A final extension was performed at 72 °C for 5 minutes. The final PCR products were electrophoresed on a 2% agarose gel and DNA bands were visualized by UV fluorescence. After performing

PCR, all PCR products were digested by a restriction enzyme, ASE1. The digested PCR products were electrophoresed on a 2% agarose gel and patterns of DNA fragments were visualized by

UV fluorescence. Identification of M. bovis , M. californicum , M. bovigenitalium and other species was given to each isolate by comparing the patterns of DNA fragments to the patterns observed with the reference strains of M. bovis , M. californicum , and M. bovigenitalium .

16S rRNA partial sequencing.

Identifications of all 280 isolates were confirmed using 16S rRNA partial sequencing as a gold standard. A region of approximately 1500 base pairs of the 16S rRNA gene was amplified using a universal primer pair; pH and pA as listed in Table 2 (Stakenborg et al., 2005). The PCR reaction mixture in the total volume of 50 µL included 3 U of Taq DNA polymerase (Invitrogen,

Carlsbad, CA, USA), 1x PCR buffer (20 mM Tris-HCl, 1.5 mM MgCl 2, and 50 mM KCl; pH

8.4), 50 µM of each of dNTPs, 10 pmol of each primer and 1 µL of the genomic DNA as template. The thermal cycling protocol involved initial denaturation at 95 °C for 3 minutes followed by 35 cycles of denaturation at 94 °C for 15 seconds, primer annealing at 57 °C for 15 seconds and extension at 72 °C for 2 minutes. A final extension was performed at 72 °C for 5 minutes. The PCR products were purified according directions (Roche, Indianapolis, IN, USA) and submitted for sequencing using primer pA (Macrogen Corp, Rockville, MD, USA).

Nucleotide sequences were compared with GenBank database using the Basic Local Alignment

Search Tool (BLAST; Altschul et al., 1990). A nucleotide sequence was identified as a species giving the highest hit of at least 98% identity from BLAST search.

Statistical analysis.

All isolate assays were replicated. If results of each replicate did not agree, a third replicate was performed. Therefore, a definitive identification by real-time PCR was given to a sample when a consistent presence of C T <37 for an assay was observed in two different replicates. For PCR-RFLP, a definitive identification of an isolate was given when the similar

DNA fragment pattern was observed. For both techniques exact kappa analysis with 95% confident interval (CI) was used as a statistical test of agreement (Stokes et al., 2000) with the

gold standard, 16S rRNA partial sequencing technique using PROC FREQ in SAS 9.2 (SAS

Institute, Cary, NC, USA).

RESULTS

Mycoplasma isolates identified by 16S rRNA partial sequencing.

Two hundred and eighty Mycoplasma isolates from various sources were included in the

study, 234 isolates (83.57%) were originally isolated from milk samples and 46 isolates

(16.43%) were from other organs. In this collection, 278 isolates (99.29%) were identified as

Mycoplasma species based on the results of 16S rRNA partial sequencing. Two isolates (0.71%) of A. axanthum were identified; one was a reference isolate (ATCC25176) and the other was from an ear swab solution of a dairy cow in Pennsylvania State. The distribution of Mycoplasma and Acholeplasm a isolates was listed in Table 3.

Identification of M. bovis , M. californicum , and M. bovigenitalium using PCR-RFLP.

The patterns of amplicons before and after digestion by ASE1 of M. bovis , M.

californicum and M. bovigenitalium were illustrated in Figure 1. The sizes of digested amplicon

for M. bovis were 140, 125 and 115 bp and for M. bovigenitalium were 265 and 115 bp.

Amplicon of M. californicum (370 bp) was not cut by ASE1. All M.bovis isolates (231/231) and

all M. californicum isolates (23/23) were correctly identified compared with the identification by

16S rRNA partial sequencing as the gold standard; perfect agreement (100%) for both species as

shown in Table 1. Four of 6 M. bovigenitalium isolates were identified correctly, 66% agreement

with the gold standard (Table 1). Amplicons of two M. bovigenitalium isolates were not digested

by ASE1 resulting in an electrophoretic pattern similar to those of M. californicum isolates as

shown in Figure 2, lanes 2 and 4. These 2 isolates that the gold standard identified as M. bovigenitalium were misidentified by PCR-RFLP and thus would be considered as M. bovigenitalium false negative results. Since these isolates were identified as M. californicum by

PCR-RFLP, they would be considered as false positive M. californicum results. Based on the miss-interpretation of those two M. bovigenitalium isolates, agreement analyses for identification of M. bovigenitalium (kappa = 0.7965, 95% CI: 0.5214, 1.0000, P <0.0001) and M. californicum

(kappa = 0.9544, 95% CI: 0.8916, 1.0000, P <0.0001) using PCR-RFLP were statistically significant.

Test specificity and detection limit of novel real-time PCR assays

Genomic DNA of 15 eubacterial species and 13 representative Mollicutes were tested to define the specificity of detection (Table 4). All M. bovis , M. californicum and M. bovigenitalium strains produced the expected amplification signal, and a log-linear dependence of CT value on mycoplasma copy number with the specific real-time PCR assay was observed

(Figures 3-5). No specific products from other Mycoplasma species and eubacterial species were detected by real-time PCR, and cross-reactivity was not observed with related ruminant

Mycoplasmas , such as M. agalactiae , M. bovirhinis , M. arginini , M. alkalescens , M. ovipneumonia and M. leachii .

To determine detection limits of the novel real-time PCR assays, the pIDTSMART-AMP plasmids cloned with target sequences of each real-time PCR assay were used as template DNA.

The detection limit of all 3 assays with the plasmids was as low as 1-10 copies of targeted DNA

(Figures 3-5). A C T of 37 is the detection limit of 1 copy of M. californicum, and approximately

10 copies of M. bovis and M. bovigenitalium.

Identification of M. bovis , M. californicum , and M. bovigenitalium using novel real-time

PCR assays.

All M.bovis (231/231), M. californicum (23/23), and M. bovigenitalium (6/6) isolates were correctly identified compared to the identification by 16S rRNA partial sequencing being a gold standard, giving a perfect agreement (100%) for all 3 species as shown in Table 1.

However, there were 4 isolates which were identified by the gold standard as M. bovis. These isolates by real-time PCR had CT values below the threshold (C T <37) with M. californicum assay. To investigate the possibility of cross amplification, targeted region for M. californicum assay was amplified for those 4 isolates using primers used with real-time PCR assay for M. californicum . Amplicons obtained from those 4 isolates were sequenced and searched for similarity against GenBank data base. All 4 amplicons were 100% matched with the target sequence of M. californicum suggesting that truly both M. bovis and M. californicum were presented and that 16S rRNA sequence analysis did not detect M. californicum . Assuming that the gold standard was infallible, and these 4 samples contained M. bovis only, then the exact kappa analysis of M. californicum real-time PCR assay indicated a significant agreement with the gold standard (kappa = 0.9122, 95% CI: 0.8271, 0.9973, P<0.0001).

DISCUSSIONS

The goal of the study was to develop real-time PCR assays to detect the 3 most common mycoplasma mastitis agents: M. bovis , M. californicum , and M. bovigenitalium . All 3 assays correctly detected the specific target regions selected without cross amplification with other

Mycoplasma species or other bacteria. Several real-time PCR assays have been reported to detect

M. bovis in many kinds of biological samples. Cai et al. (2005) successfully developed a real-

time PCR assay using fluorescence resonance energy transfer (FRET) probes to detect M. bovis

in milk and lung samples. Their system targeted a region in 16S rRNA gene which gave positive

results with both M. bovis and M. agalactiae . To distinguish between these species they

measured the melting temperature of probes. The detection limits they reported were 550

CFU/ml of milk (Cai et al., 2005). Sachse et al. (2009) reported a novel real-time PCR assay

detecting M. bovis from nasal and conjunctival swabs and from milk samples. This real-time

PCR assay targeted the 3’-terminal region of the oppD gene encoding for an oligopeptide permease and member of the ABC-transporter family. Sachse et al. (2009) reported the detection limit of their assay to be 100 CFU/ml of milk. Recently, Rossetti et al. (2010) developed a new real-time PCR assay to detect M. bovis from milk lysate. Their assay targeted a region in uvrC gene encoding for a part of exinuclease. The detection limit they reported was 2,000 CFU/ml of milk (Rossetti et al., 2010). Our newly developed real-time PCR assay targeted fusA gene

encoding for elongation factor G of M. bovis . The real-time PCR assays detecting M. bovis

targeting oppD (Sachse et al., 2009), uvrC (Rossetti et al., 2010) and fusA have the advantages

that they appear to be free of cross-amplification with DNA template of M. agalactiae .

The current study is a first reporting successful development of real-time PCR assays to detect M. californicum and M. bovigenitalium . Both M. californicum and M. bovigenitalium have been reported to be associated with mastitis in the United States and other countries (Jasper,

1981; Jurmanova et al., 1983; Mackie et al., 1986; Osman et al., 2008). Even though the severity of the disease caused by both species seems to be less compared with the disease caused by M. bovis , they are also characterized by their ability to transmit from cow-to-cow (Jasper, 1981).

Therefore, these newly developed real-time PCR assays enhance the opportunity to detect not only M. bovis , but other common mycoplasma mastitis agents. A number of techniques have

been developed to detect Mycoplasma species, primarily for detection of cell culture

contamination (Hay et al., 1989; Harasawa et al., 1993). We have applied a PCR-RFLP

technique (Tang et al., 2000) to speciate Mycoplasma species in our laboratory. This technique

was developed to detect cell culture contamination and has never been tested with Mycoplasma

strains found in cattle. Excellent agreement of PCR-RFLP identificiation of M. bovis and M.

californicum isolated from milk with 16S rRNA partial sequencing was found. The PCR-RFLP

failed to correctly identify 2 M. bovigenitalium isolates; one was originally from a vaginal swab and another one was originally from mastitic milk.

In this study a cut-off value of C T<37 was used to discriminate between a positive detection as opposed to no detection of an isolate using the real-time PCR assay. This cut-off value gave a detection limit for the M. californicum assay of 1 copy of target DNA (Figure 5) whereas the detection limits for M. bovis and M. bovigenitalium assays were approximately 10 copies of target DNA (Figures 3 and 4). The choice of a cut-off value of C T<37 is consistent with that used by Bustin (2004) who suggested the use of C T<37 value to guard against a false positive result due to a technical errors as noted by weak signals present in negative controls

(Bustin, 2004). Using this cut-off value of C T<37, four M. bovis isolates that were thought to be of a single species after filter cloning were identified as positive with both M. bovis and M. californicum . Those isolates were later confirmed to have both M. bovis and M. californicum.

Thus, the specificity using this cut-off value was still excellent although 4 false positive results

based on comparisons to the gold standard; 16S rRNA partial sequencing, were observed.

Because 16S rRNA partial sequencing assay is a qualitative PCR-based technique, only one

dominant species presented in a sample can be detected. Real-time PCR assays can detect and

quantify very low amounts of DNA template in a sample. Therefore real-time PCR assays can

detect more than one species even though those species may be presented in a very low amount in a sample. For this reason, the novel real-time PCR assays may be more superior compared to other conventional nucleic acid detection techniques used to identify mixed infection samples.

These novel real-time PCR assays were developed to overcome the difficulties of the diagnosis of mycoplasma mastitis using the in vitro culture technique. The developed real-time

PCR assays have the potential to be more rapid compared with the standard culture technique if the assays could be applied directly to milk samples. Currently speciation using the developed real-time PCR is made from isolates previously cultured and presumptively identified. Yet the use of primers and probes that are specific and that have excellent agreement with the gold standard tests for the two most prevalent mycoplasma mastitis species indicates that the procedure has potential to be developed into a routine mastitis diagnostic technique. Further development of the real-time PCR assays, combining all three in a single multiplex real test that can be used directly with milk samples should be attempted.

ACKNOWLEDGMENTS

We are grateful to Dorothy Newkirk, Daniel Righter and Veerasak Punyapornwithaya for their excellent assistance. We would like to thank Dr. Ziv Raviv and Dr. Amy Wetzel from the

Ohio State University for their cooperation. This study was partially granted by 2009 Graduate

Student and House Officer Research Awards, College of Veterinary Medicine, Washington State

University.

REFERENCES

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990, Basic local alignment

search tool. J Mol Biol 215, 403-410.

Bustin, S.A., 2004, Quantification of nucleic acids by PCR, In: Bustin, S.A. (Ed.) An A-Z of

quantitative PCR. IUL Press, California, pp. 3-29.

Cai, H.Y., Bell-Rogers, P., Parker, L., Prescott, J.F., 2005, Development of a real-time PCR for

detection of Mycoplasma bovis in bovine milk and lung samples. J Vet Diagn Invest 17,

537-545.

Fox, L.K., Kirk, J.H., Britten, A., 2005, Mycoplasma mastitis: a review of transmission and

control. J Vet Med B Infect Dis Vet Public Health 52, 153-160.

Hale, H.H., Helmboldt, C.F., Plastridge, W.N., Stula, E.F., 1962, Bovine mastitis caused by a

Mycoplasma species. Cornell Vet 52, 582-591.

Harasawa, R., Mizusawa, H., Nozawa, K., Nakagawa, T., Asada, K., Kato, I., 1993, Detection

and tentative identification of dominant mycoplasma species in cell cultures by restriction

analysis of the 16S-23S rRNA intergenic spacer regions. Res Microbiol 144, 489-493.

Hay, R.J., Macy, M.L., Chen, T.R., 1989, Mycoplasma infection of cultured cells. Nature 339,

487-488.

Heid, C.A., Stevens, J., Livak, K.J., Williams, P.M., 1996, Real-time quantitative PCR. Genome

Res 6, 986-994.

Hirose, K, Kawasaki, H, Kotani, K, Tanaka, A., Abiko, K., Ogawa, H., 2001,

Detection of Mycoplasma in mastitic milk by PCR analysis and culture

method. J Vet Med Sci 63, 691-693.

Infante, M. F., Aguado S. J., Jasper, D.E., 1999, Mastitis outbreak due to Mycoplasma

californicum and Mycoplasma canadense in a commercial dairy herd in the

state of Jalisco, México. Rev Lat-Amer Microbiol 41, 117-120.

Jasper, D.E., 1979, Bovine mycoplasmal mastitis. J Am Vet Med Assoc 175, 1072-1074.

Jasper, D.E., 1981, Bovine mycoplasmal mastitis. Adv Vet Sci Comp Med 25, 121-157.

Jasper, D.E., 1982, The role of Mycoplasma in bovine mastitis. J Am Vet Med Assoc 181, 158-

162.

Jurmanová, K., Hájková, M., Védova, J., 1983, Further evidence of the involvement of

Mycoplasma californicum in bovine mastitis in Europe. Vet Rec 112, 608.

Kirk, J.H., Glenn, K., Ruiz, L., Smith, K., 1997, Epidemiologic analysis of Mycoplasma spp

isolated from bulk-tank milk samples obrained from dairy herds that were members of

milk cooperative. J Am Vet Med Assoc 211, 1036-1038.

Leutenegger, C.M., 2001, The real-time TaqMan PCR and applications in veterinary medicine.

Vet Sci Tomorrow 1, 1-15.

Mackie, D.P., Ball, H.J., Logan, E.F., 1986, Mycoplasma californicum mastitis in the dry dairy

cow. Vet Rec 119, 350-351.

Osman, K.M., Abd El-Razik, K.A., Barbar, E.E., ELShafey, D.Y.H., Arafa, A.A., 2008,

Molecular typing of Mycoplasma species recovered from bovine mastitis. Global

Veterinaria 2, 360-368.

Pitcher, D.G., Saunders, N.A., Owen, R.J., 1989, Rapid extraction of bacterial genomic DNA

with guanidine thiocyanate. Lett Appl Microbiol 8, 151-156.

Rossetti, B.C., Frey, J., Pilo, P., (in press) 2010, Direct detection of Mycoplasma bovis in milk

and tissue samples by real-time PCR. Mol Cell Probes.