The Development of a Dual Target Mycoplasma Bovis Taqman Real-Time PCR System for the Rapid Analysis of Bovine Semen THESIS Pres

The Development of a Dual Target Mycoplasma bovis TaqMan real-time

PCR System for the Rapid Analysis of Bovine Semen

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Kristina Marie McDonald, B.A.

Graduate Program in Veterinary Preventive Medicine

The Ohio State University

2012

Master's Examination Committee:

Dr. Gustavo Schuenemann, Advisor

Dr. Ziv Raviv

Dr. Päivi Rajala-Schultz

Copyrighted by

Kristina Marie McDonald

2012

Abstract

Mycoplasma bovis is a major bovine pathogen causing respiratory disease, arthritis, otitis media, mastitis, and genital disorders in cattle worldwide.

M. bovis infections tend to persist in affected herds and are often resistant to antibiotics. Spread by close contact of infected animals or a heavily contaminated environment, M. bovis can colonize mucosal surfaces and disseminate to other organ sites. Rapid identification and isolation of diseased animals is critical to prevent the spread of infection.

Artificial insemination with M. bovis contaminated semen is a common source of infection of the female bovine genital tract. In this study, we report the development and validation of a dual target TaqMan real-time PCR system for the rapid detection of M. bovis in bovine semen. The assays target the housekeeping genes fusA and oppD/F. Both assays exclusively amplified M. bovis when tested against a panel 15 bovine mycoplasma species including 59 field and laboratory strains of M. bovis. Quantification was determined by testing serial dilutions of plasmids containing the target sequences; both the fusA and oppD/F assays demonstrated a detection limit of 1 plasmid copy/uL and efficiencies of 1.975 and 1.977 respectively.

Bovine semen was spiked with serial dilutions of M. bovis in order to compare the detection limits of the real-time PCR assays and culture in clinical samples. Culture demonstrated a detection limit of 310 organisms/µl, while the fusA and oppD/F assays detected 3.1 organisms/µl. Fresh semen ejaculates from 26 commercial bulls were obtained from an artificial insemination center and evaluated for M. bovis by real-time PCR and culture. All samples were negative for the organism by both real-time PCR and culture.

Although real-time PCR systems have been described for the detection of

M. bovis in other bovine fluids and tissues, no PCR assays have been developed to address the specific evaluation of M. bovis in bovine semen. In this study, we present two highly specific and sensitive TaqMan real-time PCRs for the rapid detection and quantification of M. bovis in bovine semen.

iii

This thesis is dedicated to my cherished husband Rick, who supported me unconditionally throughout this journey.

Acknowledgments

I would like to thank my advisor, Dr. Ziv Raviv, for his guidance and support throughout my time in the Veterinary Preventive Medicine Department.

His enthusiasm and dedication to my program ensured that I gained exceptional knowledge, not only about mycoplasmas, but about many molecular techniques as well.

I would also like to recognize the members of my master’s examination committee, Dr. Päivi Rajala-Shultz and Dr. Gustavo Schuenemann, for their support and advice, which made this document possible.

Finally, I would like to thank Select Sires, Inc. for funding my education and research. Throughout my career they have continued to support all of my ambitions and goals.

Vita

1999 - Rutherford B. Hayes High School, Delaware, OH

2007 - B.A. Microbiology, Ohio Wesleyan University

2007 to present - Select Sires, Inc., Plain City, OH

2010 to present - Graduate Student, The Ohio State University

Publications

McDonald, K., Wetzel, A., Raviv, Z. The Development of Dual Target Mycoplasma bovis TaqMan Real-Time PCRs for Rapid Analysis of Bull Semen. Publication Pending

Wetzel, A., Lefevre, K., Raviv, Z. Revised Mycoplasma synoviae vlhA PCRs. Avian Diseases 2010; 54:1292-1297.

Fields of Study

Major Field: Veterinary Preventive Medicine

Studies in Mycoplasma

Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... x

Vita ...... vi

List of Tables ...... ix

List of Figures ...... x

Chapter 1 Introduction ...... 1 1.1 List of References ...... 4

Chapter 2 Literature review ...... 8 2.1 The Class Mollicutes...... 8 2.2 The Genus Mycoplasma ...... 9 2.3 Bovine Mycoplasmas ...... 11 2.4 Mycoplasma bovis ...... 13 2.4.1 The distribution and economic impact of M. bovis ...... 13 2.4.2 Pathogenesis of M. bovis ...... 15 2.4.3 Transmission of M. bovis ...... 16 2.4.4 M. bovis in the bovine genital tract...... 18 2.4.5 M. bovis Detection ...... 20 2.4.6 M. bovis treatment, prevention, and control ...... 23 2.5 Conclusion ...... 26 2.6 List of References ...... 30

vii

Chapter 3 The development of a dual target Mycoplasma bovis TaqMan real-time PCR system for rapid the rapid analysis of bovine semen ...... 36 3.1 Abstract ...... 36 3.2 Introduction ...... 37 3.3 Materials and Methods ...... 38 3.4 Results ...... 46 3.5 Discussion ...... 48 3.6 List of References ...... 69

Chapter 4 Conclusions and further directions ...... 74 4.1 List of References ...... 78

Bibliography ...... 81

viii

List of Tables

Table 2.1 Gross properties of bacterial genomes ...... 28

Table 2.2 Properties distinguishing Mollicutes from other Eubacteria ...... 29

Table 3.1 Mycoplasma isolates utilized in this study ...... 54

Table 3.2 Primer and probe sequences used for PCR systems ...... 58

Table 3.3 Detection limits and reproducibility of the FusA real-time PCR ...... 59

Table 3.4 Detection limits and reproducibility of the OppD/F real-time PCR .. 60

Table 3.5 Summary of M. bovis spiked semen results ...... 61

List of Figures

Figure 3.1 Protocol for the isolation of mycoplasmas from semen ...... 62

Figure 3.2 Alignment of FusA sequences ...... 63

Figure 3.3 Alignment of OppD/F sequences ...... 65

Figure 3.4 FusA standard curve ...... 67

Figure 3.5 OppD/F standard curve ...... 68

Chapter 1

Introduction

The genus Mycoplasma, belonging to the class Mollicutes, contains small prokaryotic organisms which lack a cell wall and are bound by a plasma membrane. Mycoplasmas’ genome is the smallest known among free living and self-replicating organisms and the G/C content of their genome is low (23-40%).

Because of their restricted genomic potential, mycoplasmas have limited functional metabolic pathways, and live as saprophytes or parasites in their hosts. Among the over 200 known species, several mycoplasmas are recognized as important human and animal pathogens [1].

Mycoplasma bovis (Mb) is the most economically significant and pathogenic bovine mycoplasma of intensely farmed cattle in countries free of contagious bovine pleuropneumonia [2-7]. M. bovis colonizes mucosal surfaces producing several diseases including pneumonia, arthritis, otitis media, and meningitis, in calves, and mastitis and genital infections in adult bovines [8, 9]. M. bovis is a contagious mastitis pathogen and the most important causative agent of mycoplasma mastitis [10-12].

M. bovis is a common inhabitant of the bovine genital mucosa producing infections including vaginitis, endometritis, oophoritis, salpingitis, and abortion in cows as well as seminal vesiculitis, epididymitis, orchitis, and decreased semen quality and fertility in bulls [13-16]. Artificial insemination with Mb contaminated semen is a common source of infection of the female genital tract [9, 16, 17].

Shedding of Mb in semen can occur after colonization of the prepuce and distal urethra [18, 19], furthermore the organism can remain active in frozen bovine semen for years [20].

Although the mechanisms of pathogenesis are poorly understood, adherence, antigenic variation, and invasion are imperative for successful Mb colonization. M. bovis infections often follow a chronic course and the majority of host cell damage results from immunopathology [8]. Variable surface lipoproteins

(Vsps) play a crucial role in adherence and immune system evasion. Vsps exhibit highly frequency changes in size and phase variation contributing to the chronic nature of Mb infections [21].

M. bovis responds poorly to antibiotics, and although several bacterins are available in the U.S., there is no field data demonstrating their efficacy [9]. The main strategy in controlling mycoplasma disease is to identify and isolate the infected cattle, thus minimizing exposure to the remaining herd [11].

To date, culture is still the “gold standard” method for Mb infection diagnosis; however, many laboratories do not routinely culture milk, semen, or other body fluids for Mycoplasma sp. due to the fastidious nature of the

2 organism, compound media requirements, and the lengthy culture duration (up to

10 days). Semen in particular presents further culture challenges due to the potential presence of mycoplasma growth inhibitors and the extra supplies, facilities, and effort required to isolate Mb from semen [22-24]. Serology is often employed in the detection of Mb; however, antibody titers only emerge 10-14 days after infection, which delays diagnosis. Moreover serology is often not sensitive enough to identify chronic carriers [25].

To address the limitations of Mb culture and serology, a variety of polymerase chain reaction (PCR) systems have been proposed for the diagnosis of Mb. However, the developed PCR assays have variable sensitivity when performed on clinical samples, as well as cross reactivity with other closely related mycoplasmas (e.g., M. agalactiae) [22, 24, 26-34]. Moreover, none of the developed assays have addressed the specific evaluation of semen.

1.1 List of References

1. Razin, S., D. Yogev, and Y. Naot, Molecular Biology and Pathogenicity of Mycoplasmas. Microbiology and molecular biology reviews : MMBR, 1998. 62(4): p. 1094-156.

2. Brice, N., D. Finlay, D.G. Bryson, J. Henderson, W. McConnell, and H.J. Ball, Isolation of Mycoplasma bovis from cattle in Northern Ireland, 1993 to 1998. The Veterinary record, 2000. 146(22): p. 643-4.

3. Burnens, A.P., P. Bonnemain, U. Bruderer, L. Schalch, L. Audige, D. Le Grand, F. Poumarat, and J. Nicolet, The Seroprevalence of Mycoplasma bovis in lactating cows in Switzerland, particularly in the Republic and Canton of Jura. Schweizer Archiv fur Tierheilkunde, 1999. 141(10): p. 455- 60.

4. Byrne, W.J., R. McCormack, N. Brice, J. Egan, B. Markey, and H.J. Ball, Isolation of Mycoplasma bovis from bovine clinical samples in the Republic of Ireland. The Veterinary record, 2001. 148(11): p. 331-3.

5. Kusiluka, L.J., B. Ojeniyi, and N.F. Friis, Increasing prevalence of Mycoplasma bovis in Danish cattle. Acta veterinaria Scandinavica, 2000. 41(2): p. 139-46.

6. Thomas, A., H. Ball, I. Dizier, A. Trolin, C. Bell, J. Mainil, and A. Linden, Isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium. The Veterinary record, 2002. 151(16): p. 472-6.

7. Nicholas, R., R. Ayling, and L. McAuliffe, Mycoplasma mastitis. The Veterinary record, 2007. 160(11): p. 382; author reply 383.

8. Maunsell, F.P., A.R. Woolums, D. Francoz, R.F. Rosenbusch, D.L. Step, D.J. Wilson, and E.D. Janzen, Mycoplasma bovis infections in cattle. Journal of veterinary internal medicine / American College of Veterinary Internal Medicine, 2011. 25(4): p. 772-83.

9. Nicholas, R.A. and R.D. Ayling, Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci, 2003. 74(2): p. 105-12.

10. Fox, L.K. and J.M. Gay, Contagious mastitis. The Veterinary clinics of North America. Food animal practice, 1993. 9(3): p. 475-87.

11. Fox, L.K., J.H. Kirk, and A. Britten, Mycoplasma mastitis: a review of transmission and control. Journal of veterinary medicine. B, Infectious diseases and veterinary public health, 2005. 52(4): p. 153-60.

12. Gonzalez, R.N. and Wilson D.J., Mycoplasma Mastitis in Dairy Herds. . Veterinary Clinics of North America: Food Animal Practice, 2003. 19: p. 199-221.

13. Eaglesome, M.D., M.M. Garcia, and R.B. Steward, Microbial Agents Associated with Bovine Genital Tract Infections and Semen. Part II. Haemophilus somnus, Mycoplasma spp and Ureaplasma spp, Chlamydia; Pathogens and Semen Contaminants; Treatment of Bull Semen with Antimicrobial Agents. Veterinary Bulletin, 1992. 62(9): p. 887-910.

14. Eaglesome, M.D. and M.M. Garcia, The Effect of Mycoplasma bovis on Fertilization Processes In Vitro with Bull Spermatozoa and Zona-free Hamster Oocytes. Veterinary microbiology, 1990. 21(4): p. 329-37.

15. Irons, P.C., C. J. V. Trichard, and A. P. Shutte, Bovine Genital Mycoplasmosis, in Infectious Diseases of Livestock. 2004, Oxford University Press: Oxford. p. 2076-2082.

16. Kumar, A., A.K. Verma, and A. Rahal, Mycoplasma bovis, A Multi Disease Producing Pathogen: An Overview. Asian Journal of Animal and Veterinary Advances, 2011. 6(6): p. 537-546.

17. Givens, M.D. and M.S. Marley, Pathogens that cause infertility of bulls or transmission via semen. Theriogenology, 2008. 70(3): p. 504-7.

18. LaFaunce, N.A. and K. McEntee, Experimental Mycoplasma bovis seminal vesiculitis in the bull. The Cornell veterinarian, 1982. 72(2): p. 150-67.

19. Petit, T., J. Spergser, J. Aurich, and R. Rosengarten, Examination of semen from bulls at five Austrian artificial insemination centres for chlamydiae and mollicutes. Veterinary Record, 2008. 162(24): p. 792-793.

20. Pfutzner, H. and K. Sachse, Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Rev Sci Tech Oie, 1996. 15(4): p. 1477-1494.

21. Beier, T., H. Hotzel, I. Lysnyansky, C. Grajetzki, M. Heller, B. Rabeling, D. Yogev, and K. Sachse, Intraspecies polymorphism of vsp genes and expression profiles of variable surface protein antigens (Vsps) in field

isolates of Mycoplasma bovis. Veterinary microbiology, 1998. 63(2-4): p. 189-203.

22. Hotzel H., B.D., K. Sachse, A. Pflitsch, and H. Pfutzner, Detection of Mycoplasma bovis Using in vitro Deoxyribonucleic acid Amplification. Rev. sci. tech. Off. int. Epiz., 1993. 12(2): p. 581-591.

23. Nicholas, R.A., Bovine mycoplasmosis: silent and deadly. The Veterinary record, 2011. 168(17): p. 459-62.

24. Sachse, K., H. Pfützner, H. Hotzel, B. Demuth, M. Heller, and E. Berthold, Comparison of various diagnostic methods for the detection of Mycoplasma bovis. Revue Scientifique Et Technique (International Office Of Epizootics), 1993. 12(2): p. 571-580.

25. Hotzel, H., J. Frey, J. Bashiruddin, and K. Sachse, Detection and differentiation of ruminant mycoplasmas. Methods in molecular biology, 2003. 216: p. 231-45.

26. Ayling, R.D., R.A. Nicholas, and K.E. Johansson, Application of the polymerase chain reaction for the routine identification of Mycoplasma bovis. The Veterinary record, 1997. 141(12): p. 307-8.

27. Bashiruddin, J.B., J. Frey, M.H. Konigsson, K.E. Johansson, H. Hotzel, R. Diller, P. de Santis, A. Botelho, R.D. Ayling, R.A. Nicholas, F. Thiaucourt, and K. Sachse, Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial. Veterinary journal, 2005. 169(2): p. 268-75.

28. Boonyayatra, S., L.K. Fox, T.E. Besser, A. Sawant, J.M. Gay, and Z. Raviv, A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis. Journal of dairy science, 2012. 95(1): p. 196-205.

29. Chavez Gonzalez, Y.R., C. Ros Bascunana, G. Bolske, J.G. Mattsson, C. Fernandez Molina, and K.E. Johansson, In vitro amplification of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR. Veterinary microbiology, 1995. 47(1-2): p. 183-90.

30. Gabinaitiene, A., J. Siugzdaite, H. Zilinskas, R. Siugzda, and S. Petkevicius, Mycoplasma bovis and bacterial pathogens in the bovine respiratory tract. Veterinarni Medicina, 2011. 56(1): p. 28-34.

31. Hotzel, H., K. Sachse, and H. Pfutzner, Rapid detection of Mycoplasma bovis in milk samples and nasal swabs using the polymerase chain reaction. J Appl Bacteriol, 1996. 80(5): p. 505-10.

32. Sachse, K., H.S. Salam, R. Diller, E. Schubert, B. Hoffmann, and H. Hotzel, Use of a novel real-time PCR technique to monitor and quantitate Mycoplasma bovis infection in cattle herds with mastitis and respiratory disease. Veterinary journal, 2010. 186(3): p. 299-303.

33. Subramaniam, S., D. Bergonier, F. Poumarat, S. Capaul, Y. Schlatter, J. Nicolet, and J. Frey, Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR. Molecular and cellular probes, 1998. 12(3): p. 161-9.

34. Thomas, A., I. Dizier, A. Linden, J. Mainil, J. Frey, and E.M. Vilei, Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis. Veterinary journal, 2004. 168(1): p. 100-2.

35. Daxboeck, F., G. Khanakah, C. Bauer, M. Stadler, H. Hofmann, and G. Stanek, Detection of Mycoplasma pneumoniae in serum specimens from patients with mycoplasma pneumonia by PCR. International journal of medical microbiology : IJMM, 2005. 295(4): p. 279-85.

36. Espy, M.J., J.R. Uhl, L.M. Sloan, S.P. Buckwalter, M.F. Jones, E.A. Vetter, J.D. Yao, N.L. Wengenack, J.E. Rosenblatt, F.R. Cockerill, 3rd, and T.F. Smith, Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clinical microbiology reviews, 2006. 19(1): p. 165-256.

Chapter 2

Literature Review

2.1 The Class Mollicutes

Mollicutes represent a class of organisms phenotypically distinguished from other prokaryotes by their small size and total lack of a cell wall. Named from the Latin ‘mollis,’ meaning ‘soft,’ and ‘cutis,’ meaning ‘skin,’ they house the smallest genome known among free living and self-replicating organisms [1].

Phylogenic data determined by 16S rRNA sequencing, classifies

Mollicutes as a member of the phylum Firmicutes and divides them into 5 families: Mycoplasmataceae, Entomoplasmataceae, Spiroplasmataceae,

Acholeplasmataceae, and Anaeroplasmataceae. Although mollicutes are considered minimalist organisms, they evolved late from their gram-positive ancestors, approximately 600 million years ago, by degenerative evolution. While maintaining the essential genes for life, the class lost a significant amount of their ancestral genome. As a consequence of genome reduction, mollicutes have limited functional metabolic pathways. The gross properties of the Mycoplasma genitalium and E. coli genomes are contrasted in Table 2.1 [1].

Mollicutes have assumed a parasitic role, and rely heavily on their host for nutrients. Mycoplasma genitalium and Mycoplasma pneumoniae contain no genes for amino acid biosynthesis and minimal genes for cofactor biosynthesis.

The majority of mollicutes cannot synthesize fatty acids and lack the ability to regulate membrane fluidity, which is overcome by incorporating exogenous cholesterol into their membrane [1].

Most mollicutes live commensally with their hosts which include humans, animals, insects, and plants. Pathogenic mycoplasmas often follow a chronic disease course and are resistant to rifampin as well as antibiotics that affect the cell wall. The permanent lack of cell wall accounts for the “fried egg” appearance of their colony morphology on solid growth media. General mollicutes properties that discriminate them from other bacteria are summarized in Table 2.2 [1, 2].

2.2 The Genus Mycoplasma

Microorganisms from the genus Mycoplasma exist mainly as commensals and are generally host specific. Pathogenic mycoplasma tend to colonize on the respiratory, ocular, or genital mucosa causing persistent, chronic infections [2].

Rarely causing fulminant type disease, mycoplasma infections progress slowly with high morbidity and low mortality [3]. Dissemination can occur following mucosal colonization, particularly in hosts with weakened immune systems [2].

The basis of molecular pathogenicity for mycoplasmas is poorly understood. Mycoplasmas do not release potent toxins. Metabolic by-products,

9 such as hydrogen peroxide and superoxide radicals, can cause oxidative injury, but it is suggested that host cell damage mainly occurs due to host immune and inflammatory responses (immunopathology). Close contact with cells, as well as intracellular invasion, may also affect host cell integrity and function [1, 4]. While most mycoplasmas remain extracellular, species such as M. pneumonia, M. genitalium, M. penetrans, and M. gallisepticum can penetrate into non-phagocytic host cells evading host defenses as well as antibiotic therapy [1, 4].

Successful colonization of mycoplasmas requires adherence to the mucosal epithelium. M. pneumonia, the causative agent of primary atypical pneumonia, uses an attachment organelle to adhere to the respiratory tract. The membrane bound polarized structure allows localization of adhesins and subsequent colonization on the epithelium. M. gallisepticum, a poultry pathogen causing chronic respiratory disease, in addition to a putative attachment organelle (similar to the M. pneumonia) possesses adhesion-like component encoded by a multigene family, which is subject to spontaneous variation. The modulation of the two systems, the attachment organelle and the vlhA (variable lipoprotein hemagglutinin A) gene family are suggested to be the crucial element for transition from a localized infection into an invasive systemic infection [3].

Antigenic variation plays a key role in mycoplasmas’ evasion of the host humoral immune response. Multiple pathogenic mycoplasma species undergo high-frequency, reversible mutations that affect both the size and expression of cell surface lipoproteins. Surface antigenic variation is also reported to stem from

10 the loss or gain of entire genes or gene sets. M. hyorhinis, a swine pathogen, demonstrates multiple levels of gene plasticity in its vlp gene family, which encodes for major coat proteins and surface antigens. Strain variation occurs in such that the organism can contain 3, 6, or 7 vlp genes. M. hyorhinis also exhibits Vlp size variation in a given population, as well as selective variant expression in the presence of host serum [3].

Mycoplasmas also play an important role as tissue culture contaminants.

Cell cultures are frequently an artificial habitat for mycoplasmas. It has been reported that 10 to 87% of mammalian cell cultures are infected by mycoplasmas

[1]. Their small size and absence of a cell wall allows mycoplasma to pass through biological filters and subsequently compete for nutrients with the host cells. Depending on the cell culture type, competition with mycoplasmas can lead to changes in metabolism, proliferation, and gene expression, and consequently false data interpretation [5].

2.3 Bovine Mycoplasmas

Presently, 30 species of mycoplasma have been isolated from cattle.

Although many species are commensals, bovine mycoplasmosis is considered a major emerging infectious disease affecting animal production units worldwide.

Mycoplasmas produce a range of clinical conditions including respiratory disease, mastitis, arthritis, otitis, and reproductive disorders [6-9].

Bovine respiratory disease (BRD) is a major health problem affecting housed cattle worldwide. In addition to poor growth rates, increased morbidity and mortality has led to vast economic losses for the cattle industry [6, 10].

Contagious Bovine Pleuropneumonia (CBPP), the only bacterial disease classified by the Office International des Epizooties (OIE) as a list A disease, is caused by M. mycoides ssp mycoides small colony variant. Contagious Bovine

Pleuropneumonia is considered the most significant mycoplasma disease, however, apart from Sub-Saharan Africa, it has been eradicated [11]. Over the last decade, substantial evidence has indicated that infections with mycoplasma are major primary and contributory causes to BVD infection worldwide [6].

Mycoplasma-associated pneumonia can be caused by M. bovis, M. dispar, and

Ureaplasma spp. Multiple other species of mycoplasma have been isolated from the respiratory tract but have not been shown to cause disease [8].

Mycoplasmas are a principle agent of intramammary infections and mastitis outbreaks globally. Mycoplasmas colonize the epithelium of the mammary gland eliciting a massive inflammatory response resulting in decreased milk production. As the disease progresses, mycoplasmas can invade the tissue and disseminate systemically to other body sites. M. bovis is the most common cause of mycoplasma mastitis infections, however 11 other species have been associated with mastitis including M. alkalescens, M. bovigenitalium,

M. californicum, and M. canadense as common isolates [12, 13].

Many mycoplasma species, as well as the mollicutes Ureaplasma diversum and Acholeplasma laidlawii, are common inhabitants of the bovine reproductive tract in diseased and presumably healthy animals. Established bovine genital mycoplasma pathogens include M. bovis, M. bovigenitalium, M. canadense and U. diversum. Diseases caused by genital mycoplasmosis include vaginitis, endometritis, oophoritis, salpingitis, and abortion in cows as well as seminal vesiculitis, epidiymitis, orchitis, and decreased semen quality and fertility in bulls [7, 14-16]. Mycoplasma-associated genital infections have resulted in considerable losses in production, and careful attention should be given to prevent the potential transmission of mycoplasmosis to susceptible animals [2,

7].

2.4 Mycoplasma bovis

2.4.1 Distribution and Economic Impact of M. bovis

Mycoplasma bovis (Mb) is the most important pathogenic and economically significant bovine mycoplasma in countries free of CBPP [9, 17-21].

M. bovis infections predominantly affect intensively farmed cattle causing a variety of diseases including pneumonia, arthritis, otitis media, and meningitis, in calves, and mastitis and genital infections in adult bovines [22, 23].

The US was the first to isolate Mb in 1961 from a severe mastitis case in cattle. The microorganism was originally identified as M. agalactiae var bovis due to similarities in clinical signs, as well as antigenicity and biochemical properties,

13 of the sheep pathogen M. agalactiae. In 1976, technological advancements led to its species ranking based on 16S rRNA sequencing. M. bovis has continued to spread worldwide via animal movements to countries including Israel (1964),

Spain (1967), Australia (1970), France (1974), mainland Britain (1975),

Czechoslovakia (1975), Germany (1977), Denmark (1981), Switzerland (1983),

Morocco (1988), South Korea (1989), Brazil (1989), Northern Ireland (1993),

Republic of Ireland (1994), Chile (200), South Africa (2005), and the Czech

Republic (2007). Presently, there are very few countries, apart from Norway and

New Zealand, which are free of Mb [16, 23-25].

M. bovis is a major contributor to calf pneumonia and is often the predominate isolate from affected calves [26]. The association of Mb with viral infections leading to bovine respiratory disease (BRD) has also been reported

[27]. Of the estimated 2 million cattle affected annually by respiratory disease in

Europe, it is believed that Mb is responsible for 25-33% of the infections. This translates to total losses of up to 190 million euros. In the U.S, the loss of weight gain and carcass value due to Mb infections is estimated to be $32 million per year [6, 25].

M. bovis is the principle causative agent of mycoplasma mastitis and is considered a contagious mastitis pathogen [12, 13, 28]. Clinical signs, often latent, include drastic drops in milk production and signs of systemic illness [22].

In the United States, losses due to Mb bovine mastitis are estimated to be $108

14 million per year. Once Mb has been introduced into the herd, infection can rates of up to 70% have been reported [16, 25, 29].

2.4.2 Pathogenesis of M. bovis

The ability of Mb to colonize mucosal surfaces, invade tissues, and persist at sites of disease despite an aggressive immune response, is exemplified by its unique interaction with its host. Although the mechanisms of pathogenicity are poorly understood, molecules involved in adherence, antigenic variation, and invasion are important for successful colonization [28, 30]. The lack of a cell wall permits exposed Mb membrane proteins to interface with host cells facilitating adherence to mucosal surfaces [22]. The majority of host cell damage results from the host immune and inflammatory responses (immunopathology).

Secondary injury may occur from bacterial metabolic by-products and intimate contact with host cells [3]. M. bovis can also form biofilms providing increased resistance to desiccation and heat stress [22].

Major antigenic targets of the host immune system include variable surface lipoproteins (Vsps), as well as unrelated proteins pMB67 and P48 [10].

The vsp gene family plays a critical role as mediators for adherence to epithelial cells, maintaining parasitism, and evading the immune system. Vsps exhibit highly dynamic and spontaneous changes in size and phase variations in their expression level by high-frequency rearrangement of genes [28]. The frequency of surface antigen variation ranges from 103 to 105 per cell per generation [4],

15 and extensive strain variation in their coding sequences has been demonstrated

[22]. In vitro experiments have shown that the addition of Vsp specific antibodies to Mb cultures results in the selection of phase-variant mycoplasmas with OFF expression of that Vsp. Subsequent removal of the antibodies results in reversion to the original phenotype [31]. Vsps play an important role in inflammation by inducing the secretion of proinflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin 1, and interleukin 6 [28].

Alveolar macrophages are important for early clearance of mycoplasmas; however, inappropriate activation of alveolar macrophages by Mb and the subsequent excessive production of TNF-α initiates a detrimental inflammatory response. Large amounts of inflammatory mediators released by excessive neutrophil recruitment can also occur. Overstimulation of the immune system is directly correlated with the severity of Mb disease [22].

In 2011, the genome sequence of Mb type strain PG45 was published.

The complete sequence can facilitate the prediction of phenotypic and genotypic instability in populations. These characteristics may be important in assigning phenotypes to pathogenic field isolates and subsequently developing effective control measures [32].

2.4.3 Transmission of M. bovis

Generally, Mb is introduced into M. bovis-free herds by clinically healthy calves or young cattle shedding mycoplasma. M. bovis is not ubiquitous but

16 widely spread within an infected bovine population, and once established becomes very difficult to eradicate. M. bovis can colonize mucosal surfaces and persist without causing clinical disease.

Cattle infected with Mb via the respiratory tract act as reservoirs, shedding

Mb for months or even years [22, 25]. Calves can become infected in their respiratory tract not only horizontally through aerosol droplets and nose to nose contact, but also from infected milk and vertically through the uterus [16].

Fomite-mediated transmission can also occur as Mb has been shown to survive for long periods in bedding, cooling ponds, and dirt lots [22, 33]. Following established infection, dissemination of Mb from the upper respiratory tract to the lower respiratory tract, middle ear, and meninges can occur. Joints are a frequent site of secondary colonization following hematologic dissemination of infection

[26].

M. bovis is characterized as a contagious mastitis pathogen and infected herds are generally marked by medium to low rates of clinical mastitis with progressively increasing somatic cell counts [12]. There are multiple routes of transmission making it difficult to identify the source of infection. M. bovis introduction into a herd can occur from replacement animals, putting large herds with more cow turnover at greater risk [12]. Asymptomatic carriers can spread the disease at milking times by mechanical transfer via fomites such as milking machines, teat cups, and milker’s hands. Additionally, mycoplasma mastitis outbreaks have been reported in previously clean herds without introduction of

17 animals or a history of previous intramammary treatment [28]. It has been suggested that internal transfer of Mb from extramammary organ sites to the mammary gland can occur. M. bovis was isolated from the blood and other organ systems of cows experimentally inoculated with mycoplasma in a single mammary quarter following the establishment of infection. Furthermore, multiple mammary quarters became infected, suggesting haematogenous spread and/or lymphatic transmission [12].

Infection of the bovine genital tract of both males and females can occur through a heavily contaminated environment or by direct contact of cattle shedding Mb [16]. Artificial insemination with semen infected with Mb is also a common source of infection of the female genital tract [16, 23, 28]. M. bovis shedding in semen occurs following infection of the prepuce, urethra, epididymis and testes of seemingly healthy bulls [34]. Mycoplasmas have also been shown to survive in cryopreserved semen for several years [15, 16, 33].

2.4.4 M. bovis in the Bovine Genital Tract

Although most Mb infections are non-reproductive in nature, the male and female bovine genital tract have been found to harbor the organism in both normal and diseased cattle [35]. Due to the systemic nature of Mb, as well as the intermittent shedding pattern of the organism, transmission via the genital tract remains a concern for the cattle industry.

The female bovine genital tract can become colonized by Mb via an ascending infection [33]. Uterine infusion of Mb into heifers has resulted in salpingitis, endometritis, and peritonitis. The intrauterine inoculation of Mb in pregnant cows produced placentitis, fetal death, and abortion. Additionally, heifers inseminated with Mb contaminated semen demonstrated chronic salpingitis and other breading problems [35]. Vertical transmission of Mb to newborn calves and fetuses from infected genital organs and amnion has been reported [14]. Furthermore, Mb has been recovered from the uterus of slaughtered cows and from aborted fetuses, during and after Mb-mastitis [33].

Mb infection of the male genital tract ascends through the prepuce following transmission via a contaminated environment or by the licking of shedder animals. Infections following colonization of Mb in the prepuce and distal urethra include seminal vesiculitis and orchitis and result in the shedding of Mb in semen [33, 36].

Mb has been associated with reduced fertility in bulls. In vitro studies of the effect of Mb contaminated semen on fertilization have shown reduced sperm penetration and embryo development rates [15, 37]. Furthermore, scanning electron microscopy images show Mb adhesion to the surface of sperm cells following washing and swim-up isolation, as well as the surface of zona pellucida-intact embryos [37].

2.4.5 M. bovis Detection

Control of mycoplasma disease is largely dependent on early identification of the pathogen [38]. Laboratory diagnosis of Mb infections is essential due to the lack of characteristic clinical and pathological signs [23]. To date, culture is still the “gold standard” method for Mb infection diagnosis; however, many laboratories do not routinely culture milk, semen, or other body fluids for

Mycoplasma sp. due to the fastidious nature of the organism, compound media requirements, and the lengthy culture duration (up to 10 days). False-negative results are often produced due to bacterial contamination, intermittent shedding, and antibiotic use in the affected herds [6, 23, 28].

Mycoplasma culture requires complex media, specialized equipment, and technical skill. The sensitivity of culture in clinical mastitis is low and can be affected by intermittent shedding, uneven distribution of Mb in diseased tissue, suboptimal sample handling or culture conditions, overgrowth by saprophytic mycoplasmas and contaminants, and the presence of mycoplasma inhibitors

[22]. Mycoplasma colonies are suggested by their “fried egg” morphology on solid media but walled bacteria in L-Phase should be considered. Following isolation, speciation by immunofluorescence or PCR is necessary since variety of pathogenic and nonpathogenic mollicutes can inhabit the infection site [26].

Culture procedures for the isolation of Mb from bovine fluids and tissues can be found in “Mycoplasmosis in Animals: Laboratory Diagnosis” [8].

Mb-specific antibodies can be detected within two weeks of infection by various methods including enzyme-linked immunosorbent assay (ELISA), immunobinding (IBA), and immunohistochemistry [16, 22]. Serology is generally used for screening purposes only. In natural infections, individual animal titers are poorly correlated with infection for several reasons: Not all diseased animals develop high titers, high titers can persist for months, and high-titers in calves can result from maternal antibody transfer [22]. The sensitivity of ELISA is often insufficient to identify chronically infected carriers [39].

Monoclonal antibodies have been utilized in the development of multiple methods of Mb diagnosis and speciation. However, due to genetic similarities, monoclonal antibodies often cross react with other mycoplasma species including M. agalactiae and M. gallisepticum [16].

Although serology is rapid, and often the most inexpensive method of detection, it has limitations as an Mb diagnostic tool. The sensitivity of ELISA is 2 or more orders of magnitude lower than polymerase chain reaction (PCR), rendering it unable to identify inapparent shedders. Diagnosis is delayed since antibody titers emerge only after 10-14 days and specificity is limited due to cross-reactivity with other mycoplasmas [39].

The introduction of PCR has provided a rapid and more sensitive tool for the diagnosis of Mb infection. Multiple PCR assays have been developed for the detection of Mb from milk and lung tissue [24, 38-49]. Early PCRs targeting the

16S rRNA gene showed high cross reactivity with M. agalactiae [40, 43]; and are

21 generally not suitable for differentiation of closely related organisms [47]. To address this problem, PCRs targeting housekeeping genes such as replication repair gene uvrC [47, 49] and the oppD/F gene involved in peptide transport, have been developed [38, 41]. Sensitivity of PCR is also dependent on mycoplasma genomic DNA extraction from clinical specimens. Proteins, enzymes, and other milk and tissue components can interfere with the PCR reaction [28].

In recent years, real-time PCR technology has revolutionized the way clinical microbiology laboratories diagnose infectious diseases. The combination of excellent sensitivity and specificity, ease of performance, speed, and inherent quantitative nature, has made real-time PCR an appealing alternative to end- point PCR methods [50, 51]. Specificity can be increased by the utilization of fluorescent sequence specific probes (e.g., hydrolysis probes) that recognize nucleotides between primers. Real-time PCR does not require additional sample handling post-amplification, which reduces the risk of ‘carry-over’ contamination and results in faster analysis [38]. The use of multiplexing in real-time PCR allows for the simultaneous detection of multiple species. A recent publication in the Journal of Dairy Science describes the development and validation of a real- time PCR assay that detects the 3 most common mycoplasma mastitis agents:

M. bovis, M. bovigenitalium, and M. californicum. The assay can serve as a screening tool or a confirmation test for mycoplasma mastitis [41].

Since multiple pathogenic and nonpathogenic mycoplasmas can cohabitate a single site, detection methods to identify mixed mycoplasma infection have been developed. PCR amplification of the 16s rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) has successfully differentiated 13 bovine mycoplasma species [29]. However, it is laborious and complex to perform and interpret [6]. A recent study introduced DNA microarray technology as a platform to differentiate mycoplasma species. The assay, which targets the 16S-23S intergenic spacer region, was shown to identify cultured strains of 37 mycoplasma species [52].

2.4.6 M. bovis Treatment, Prevention, and Control

Treatment of Mb infection is contingent on the associated disease. The efficacy of antimicrobials is largely dependent on how early treatment is initiated

[22]. Mycoplasmas are inherently resistant to antibiotics that target the cell wall, i.e. penicillins and cephalosporins, as well as rifampin and sulfonamides since mycoplasmas varied in the structure of the RNA polymerase β subunit and do not synthesize folic acid, respectively [1, 22]. Over the past decade, studies have shown Mb strains becoming increasingly resistant to common antibiotics used to treat bovine respiratory disease (BRD), including fluoroquinolones [6], oxytetracyclines, tilmicosin, and spectinomycin [23]. In the United States, tulathromycin and florfenicol are approved for the treatment of Mb associated

BRD [22]. Short-term use of anti-inflammatory drugs combined with antibiotics

23 has shown to be beneficial to some degree in treating BRD [26]. Overall, the response of Mb to antibiotic treatment is poor, and it is unlikely that the pathogen is ever completely eliminated [29].

There is very little data on the antibiotic treatment of naturally occurring

Mb associated diseases other than BRD. Treatment with fluoroquinolones, tetracyclines, and macrolides is recommended for cattle with Mb associated arthritis, but poor response to antibiotics is seen without early aggressive treatment [22]. Uterine Mb infections have been treated with infusion of the vagina or uterus with antibiotics as well as vaginal douche with tetracyclines, however, insufficient data exists to support the efficacy of this treatment. To date, no effective treatment for the elimination of Mb from the bull reproductive tract has been developed [14].

In general, vaccines developed against Mb have remained ineffectual.

Currently there are several Mb bacterins licensed for marketing in the U.S.; however, there is no data demonstrating their efficacy in the field [23]. There are no Mb vaccines commercially available in Europe [22]. The lack of a reliable and reproducible animal model remains one of the biggest obstacles in vaccine development [6]. Various vaccines have appeared promising in experimentally infected cattle, but remain ineffective or resulted in increased disease severity in the field [22]. A 2004 study in the UK presented an Mb bacterin able to prevent respiratory disease in calves 3 weeks after vaccination, however when used in a field trial, the vaccinated group displayed an increased rate of severity of

24 respiratory disease [29]. Increased severity of clinical mastitis has also been reported in cows vaccinated with an Mb bacterin compared to non-vaccinated cow after intramammary inoculation of Mb [22]. This phenomenon may be due to

Mb strain selection. Other factors to consider in vaccine development include differences in virulence of Mb isolates [6], the ability of Mb to rapidly change its surface antigens [28], and the importance of local immunity [22].

In the absence of effective chemotherapy and vaccination, the main strategy in controlling mycoplasma disease is to identify and isolate the infected animal thus minimizing the exposure to healthy cattle [12]. M. bovis can spread swiftly and silently among herds, making rapid diagnosis crucial [6]. Proper hygiene measures for handling sick cattle and the disinfection of mechanical surfaces reduce fomite-mediated disease transmission [12].

Prevention of mycoplasma disease is largely dependent on the implementation of biosecurity practices targeted to the individual operation.

Important procedures include maintaining a closed herd or the screening and quarantine of purchased animals [22]. To prevent calf pneumonia, it is recommended that calves be separated from adults as early as possible.

Additional measures including the reduction of environmental stress, adequate housing with good air circulation, and pasteurization of feeding milk [22, 23].

Routine screening of bulk tank milk in dairy herds, as well as the employment of strict milking time hygiene practices, are effective control strategies against Mb mastitis [12].

The application of stringent hygienic and sanitary procedures is important in the prevention of genital mycoplasma infections [14]. During artificial insemination (AI), sanitary sleeves should be placed over the insemination pipette to prevent infection from being transmitted between animals.

Mycoplasmas are frequently isolated from the semen and prepuce of apparently healthy bulls at artificial insemination centers [34-37, 53-55]. Hygienic practices during collection and processing, as well as regular bedding replacement and decreased stock density, are often implemented to control the spread of disease

[11]. As a protective measure, antibiotics including lincomycin, spectinomycin, and tylosin are added to semen prior to processing [14]. However, it is suggested that the antibiotic combination is only effective under certain conditions, i.e. extender, incubation time, frozen semen [56-58]. In order to reduce the risk of transmitting Mb to herds, Certified Semen Services (CSS), the U.S. Artificial

Iinsemination industry’s quality assurance program, restricts the use of non- frozen semen for artificial insemination [57]. New Zealand, a mycoplasma-free country, has implemented biosecurity measures to ensure no semen collected from a bull that has ever tested positive for Mb enters the country [59].

2.5 Conclusions

Mycoplasma bovis is a primary cattle pathogen and the etiological agent of several serious and economically costly diseases of cattle. The prevalence of mycoplasmas is most likely underestimated as a disease agent since it is

26 frequently not investigated due to the fastidious nature of the organism [6, 23]. M. bovis associated diseases are often chronic, debilitating, and respond poorly to chemotherapy [26]. In the absence of an effective vaccine, mycoplasma control depends significantly on biosecurity measures. Strict hygiene practices and the rapid identification and isolation of infected animals are crucial in preventing the spread of disease [12]. Real-time PCR as a rapid tool for the detection Mb in both early and chronic infections is an appealing alternative to traditional culture and serology. However, difficulties in cross-reactivity and inhibitory actions of milk and tissue components remain obstacles [47]. Continued efforts to increase the sensitivity and specificity of real-time PCR methods are needed.

Property M. genitalium E. coli No. of base pairs 580,070 4,639,221 G + C content (mol%) 32 50 No. of putative coding sequences (ORFs) 479 4,288 No. of ORFs tentatively identified 468 2,659 No. of ORFs with no functional prediction or data base match 11 1,629 aAdapted from reference 1

Table 2.1 Gross Properties of Bacterial Genomesa

Property Mollicutes Other eubacteria Cell wall Absent Present Cholesterol present in most Plasma membrane species Cholesterol absent Genome size 580-2,220 kb 1,050 - > 10,000 kb G + C content 23-40 mol% 25 -75 mol% No. of rRNA operons 1 or 2b 1 -10 5SrRNA length 104-113 ntc >114 nt 30 (M. capricolum), 84 (B. subtilis), No. of tRNA genes 33 (M. pneumoniae) 86 (E. coli) Tryptophan codon in Mycoplasma, Ureaplasma, Spiroplasma, UGA codon usage Mesoplasma Stop codon RNA polymerase Rifampin resistant Rifampin sensitive a Adapted from reference 1 b Three rRNA operons in Mesoplasma lactucae c nt, nucleotides

Table 2.2 Properties Distinguishing Mollicutes from other Eucbacteriaa

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Chapter 3

The Development of Dual Target Mycoplasma bovis TaqMan real-time PCRs

for Rapid Analysis of Bovine Semen

3.1 Abstract

Mycoplasma bovis is a major bovine pathogen causing respiratory disease, arthritis, otitis media, mastitis, and genital disorders in cattle worldwide.

36 field and laboratory strains of M. bovis. Quantification was determined by testing serial dilutions of plasmids containing the target sequences; both the fusA and oppD/F assays demonstrated a detection limit of 1 plasmid copy/µl and efficiencies of 1.975 and 1.977, respectively.

Although real-time PCR systems have been described for the detection of

3.2 Introduction

The genus Mycoplasma, belonging to the class Mollicutes, contains small prokaryotic organisms which lack a cell wall and are bound by a plasma membrane. Mycoplasmas’ genome is the smallest known among free living and self-replicating organisms, the G/C content of their genome is low (23-40%), and

37 they have limited functional metabolic pathways. Among the over 200 known species, most mycoplasmas live in their hosts as commensals, however several are known as important human and animal pathogens [1].

Mycoplasma bovis (Mb) is the most important pathogenic bovine mycoplasma in countries free of contagious bovine pleuropneumonia [2-7], and is a worldwide pathogen of intensively farmed cattle. The US was the first to isolate

Mb in 1961 from a mastitic cow [8] and the UK reported it in the respiratory tract of a calf in 1975 [9]. Common manifestations of Mb infections include pneumonia, arthritis, otitis media, and meningitis, in calves, and mastitis and genital infections in adult bovines [10, 11]. Mycoplasma bovis is the most important causative agent of mycoplasma mastitis and is considered a contagious mastitis pathogen [12-14].

Genital Mb infections can lead to vaginitis, endometritis, oophoritis, salpingitis, and abortion in cows as well as seminal vesiculitis, epididymitis, orchitis, and decreased semen quality and fertility in bulls [8, 15-17]. Naturally occurring infection in the male genital tract can be caused by systemic dissemination via other infected tissues, contact with other animals, or a heavily contaminated environment. A common route for female genital tract mycoplasma infection is artificial insemination with infected semen [8, 11, 18]. Shedding of Mb in the semen may occur after colonization of the prepuce, urethra, epididymis, and testes [19-21], and the organism can remain active in frozen bovine semen for years [22]. Concern of Mb infection due to bovine semen is starting to spread

38 across the world. Recent bovine semen importation restrictions have been implemented in New Zealand requiring that “donors have never recorded a positive test for Mycoplasma bovis” [23].

To date, culture is still the “gold standard” method for Mb infection diagnosis; however, many laboratories do not routinely culture milk, semen, or other body specimens for Mycoplasma sp. due to the fastidious nature of the organism, compound media requirements, and the lengthy culture duration (up to

To address the limitations of Mb culture, a variety of polymerase chain reaction (PCR) systems have been proposed for the diagnosis of Mb. However, the developed PCR assays have variable sensitivity when performed on clinical samples, as well as cross reactivity with other closely related mycoplasmas (e.g.,

M. agalactiae) [9, 24, 26-34]. Moreover, none of the developed assays have addressed the specific evaluation of semen.

The objective of this study was to develop and validate a rapid assay for the detection of Mb in bovine semen. Herein we describe a dual target, highly specific and sensitive Mb TaqMan real-time PCR system and its applicability for evaluation of Mb in bull semen.

3.3 Materials and Methods

Mycoplasma Isolates. Pure cultures representing of 15 bovine mycoplasma species, including 59 strains of M.bovis isolated from milk and bovine tissues were included in this study (Table 3.1).

Mycoplasma Culture Methods. Isolates were propagated in either SP-4 broth, SP-4 broth containing arginine, or Hayflick’s broth with horse serum aerobically at 37°C [37]. Stock cultures were stored with 10% (v/v) glycerol at

-80°C.

Mycoplasma was cultured from semen using Hayflick’s media containing porcine serum according to the method published in Mycoplasmosis in Animals:

Laboratory Diagnostics [37] (Figure 3.1). Briefly, four 10-fold serial dilutions prepared in broth and plated (20 µl) on days 0, 2, and 4; 20 µl of undiluted semen was also plated on day 0. All media was incubated aerobically at 37°C in a sealed chamber. Plates were examined for colonies under magnification every 2 days and were discarded as negative after 10 days.

Mycoplasma Genomic DNA Extraction. Mycoplasma genomic DNA extraction from broth media (200 µl) was performed using the QIAamp DNA Mini

Kit (QIAGEN, Valencia, CA) following the manufacturer’s recommendations.

Mycoplasma genomic DNA was extracted from semen using the QIAamp

DNA Mini Kit (QIAGEN, Valencia, CA) according to the following protocol: semen in the volume of 200 µl was combined with 200 µl Tris buffer containing 2%

Triton X. Samples were incubated for 1 hour at 110°C with 10 sec vortexing every 10 min. After incubation, 200 µl ATL buffer and 40 µl Proteinase K were added to each sample and incubated for 1 hour at 56°C with 10 sec vortexing every 20 min. After incubation, 400 µl AL buffer was added followed by a 10 min incubation at 70°C. The samples were centrifuged for 2 min at 14,000 rpm, the supernatant was removed, and mixed with 400 µl absolute ethanol. The mixture was bound to a QIAmp mini spin column by centrifugation for 1 min at 8,000 rpm.

The column was washed with 500 µl AW1 buffer by centrifuging for 1 min at

8,000 rpm followed by a wash with 500 µl AW2 buffer by centrifugation for a total of 4 min at 14,000 rpm. Elution buffer (35 µl) was added to the column and the samples were incubated for 5 min at room temperature before eluting by centrifugation for 2 min at 8,000 rpm. DNA was stored at -20°C until further use.

Mycoplasma bovis TaqMan Real-Time PCRs. Two separate TaqMan real-time PCR assays were designed with the Primer3 software

(http://frodo.wi.mit.edu/primer3/input.htm) to specifically target 2 sites on the Mb genome, the fusA and oppD/F genes. The assay designs were based on the 41

GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) sequences and the fusA and oppD/F end-point PCR product sequences of 45 & 28 Mb strains respectively (Figure 3.2 & 3.3). The reactions’ oligonucleotide sequences were evaluated for species specificity with the NCBI BLAST tool utilizing both the

Megablast and the Blastn algorithms on the Nucleotide Collection database, and analyzed for interactions using the Operon Oligo Analysis tool

(http://www.operon.com/technical/toolkit.aspx). The sequences of the oligonucleotides that comprise each reaction are presented in Table 3.2.

The assays were conducted as 2 separate simplex reactions. Each real- time PCR reaction was prepared in a volume of 25 µl containing 12.5 µl of

LightCycler 480 Probes Master mix 2X concentration (Roche, Indianapolis, IN), primers to a final concentration of 0.4 µM, probe to a final concentration of 0.2

µM, 5.0 µl of water, and 5.0 µl of DNA template solution. The reaction was performed in the LightCycler 480 (Roche, Indianapolis, IN) using the following thermocycle program: pre-incubation at 95°C for 10 min; 40 cycles of amplification at 95°C for 10 sec, 60°C for 50 sec, and 72°C for 1 sec with optic

ON; cooling for 10 sec at 40°C. Any reaction that had a recorded crossing point

(Cp) value was considered positive and any reaction that had no recorded Cp value was considered negative. Only samples positive for both fusA and oppD/F

TaqMan real-time PCRs were considered positive for Mb.

Mycoplasma End-Point PCRs. Primers for both the fusA and oppD/F genes were designed with the Primer3 software 42

(http://frodo.wi.mit.edu/primer3/input.htm) to sequence broad spanning regions of the Mb TaqMan real-time PCRs’ target sites. The assay designs were based on

GenBank sequence data (http://www.ncbi.nlm.nih.gov/Genbank/index.html), and the primer sequences are presented in Table 3.2. The reaction mix of both assays was prepared using the Roche FastStart High Fidelity PCR System

(Roche, Indianapolis, IN) in a total volume of 50 µl containing 1X reaction buffer

(1.8 mM MgCl2), 200 µM of each deoxynucloside triphosphate, 0.4 µM forward and reverse primers, and 2.5 U High Fidelity Enzyme Blend (Roche, Indianapolis,

IN). Both reactions were performed in a MJ Mini Thermocycler (BioRad

Laboratories, Hercules, CA) using the following program: hot start at 95°C for 3 min; 40 cycles of amplification at 94°C for 30 sec, 55°C (fusA) or 56° (oppD/F) for

30 sec, and 72°C for 1 min; final extension of 72°C for 5 min.

Speciation primers were also designed for this study to sequence a portion of the 16S rRNA gene of Mycoplasma isolates. The primer sequences presented in Table 3.2 were designed with the Primer3 software

(http://frodo.wi.mit.edu/primer3/input.htm) and were based on GenBank sequence data (http://www.ncbi.nlm.nih.gov/Genbank/index.html). The 50 µl conventional PCR reaction mix was prepared using the Roche FastStart High

Fidelity PCR System (Roche, Indianapolis, IN) containing 1X reaction buffer (1.8 mM MgCl2), 200 µM of each deoxynucloside triphosphate, 0.4 µM forward and reverse primers, and 2.5 U High Fidelity Enzyme Blend (Roche, Indianapolis, IN).

The reaction was performed in a MJ Mini Thermocycler (BioRad Laboratories,

Hercules, CA) using the following program: hot start at 95°C for 3 min; 40 cycles of amplification at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min; final extension at 72°C for 5 min.

PCR products were separated by electrophoresis on a 1% agarose gel containing 0.5X Tris-borate-ethylenediaminetetraacetic acid buffer and 0.5 mg/ml ethidium bromide. Amplicons were visualized by ultraviolet trans-illumination.

Sequencing and Analysis of End-Point PCR Products. PCR products were purified using the QIAquick®PCR Purification Kit (Qiagen, Valencia, CA) according to manufacturer’s recommendation. Both forward and reverse strands of purified PCR products were submitted for Sanger sequencing (GENEWIZ,

Inc., NJ).

Sequence data was complemented, edited, and consensuses were constructed using the SeqMan program (Lasergene; DNASTAR, Inc.; WI).

Sequences were aligned with the MegAlign program (Lasergene; DNASTAR, Inc.

WI), using the Clustal V method with a gap penalty of 10 (Figure 3.2 & 3.3).

Quantitative Analysis and Detection Limit of the TaqMan Real-Time

PCRs. The fusA and oppD/F real-time PCR target gene sequences were cloned into pIDTSMART-KAN plasmids (Integrated DNA Technologies, Coralville, IA).

The cloned plasmids were reconstituted in TE buffer, and the DNA concentration

(ng/µl) was measured by spectrophotometry with the NanoDrop 1000 device

(Thermo Scientific, Wilmington, DE). The plasmid copies/µl was calculated using

44 the following formula: (X g/µl DNA / [plasmid length in basepairs x 660]) x 6.022 x

1023 = plasmids/µl

(http://www.qiagen.com/resources/info/guidelines_rtpcr/quantifying.aspx). The concentration was adjusted to 109 plasmids/µl in TE buffer and 10-fold serial dilutions were prepared in TE buffer down to 100.

To determine the quantitation and detection limit of each TaqMan real- time PCR assay, the 10-fold serial dilutions of the reaction specific plasmid were ran in triplicate. A standard curve was generated for each reaction by plotting the mean Cp values vs. log10 of the standard DNA control copy numbers of three independent runs. The efficiency (E) of each reaction was calculated by the following equation: E=10(-1/slope).

Bull Semen Spiked with M.bovis Experiment. Semen was spiked with

M. bovis to compare the recovery limits of the TaqMan real-time PCRs and culture. Serial dilutions of a fresh Mb culture with a concentration of 3.436 x 106 cells/µl (calculated by quantitative real-time PCR) were prepared in Hayflick’s broth. A single ejaculate known to be Mb free was collected with an artificial vagina at Select Sires, Inc. (Plain City, Ohio) and transported to the lab at 5°C.

For each treatment, 1ml of semen was combined with 100 µl of 10 fold serial dilution of the Mb culture, further diluting the Mb cells by 10 times. The final concentrations of the 5 spiked treatments were 3.1 x 104, 3.1 x 103, 3.1 x 102, 3.1 x 101, 3.1 x 100 cells/µl semen. Mycoplasma genomic DNA was extracted from the spiked semen samples and real-time PCR analysis was performed using the 45 methods described in this paper. Simultaneous culture was performed using the published culture method for isolating mycoplasmas from semen. [37].

Analysis of Bovine Semen Field Samples Experiment. Fresh bull semen ejaculates from 26 commercial bulls were collected using an artificial vagina and transported to the lab at 5°C. Genomic Mycoplasma DNA was extracted from 200 µl of semen and tested for Mb using the developed TaqMan real-time PCR assays. Simultaneous culture was performed using the published semen culture method [37]. Colonies from plates positive for mycoplasma were subcultured into Hayflick’s broth and allowed to incubate for 48 hours at 37°C before genomic DNA was extracted for organism’s species determination.

Mycoplasma speciation was performed by amplification and subsequent sequencing of a portion of the 16S rRNA gene (described above). Species identification was determined by the NCBI BLAST tool, using the Megablast algorithm (http://blast.ncbi.nlm.nih.gov).

3.4 Results

Assay Specificity. To assess the species specificity and cross-reactivity of the developed assays, DNA extracts from 15 different bovine mycoplasma species, including 59 Mb field and laboratory isolates, were tested with both the fusA and oppD/F TaqMan real-time PCR assays. All Mb isolates produced

46 amplification signals with recorded Cp values <40. No real-time PCR signals were detected for the other bovine mycoplasma species, including M. agalactiae.

Quantification Analysis and Detection Limit of the TaqMan Real-Time

PCRs. Quantification and detection limits of both assays were determined by generating standard curves (Figure 3.4 & 3.5) using Cp values obtained from 3 independent runs (Table 3.3 & 3.4) of 6 serial dilutions of plasmids containing the fusA or oppD/F gene target sequences. Mean Cp values were plotted against the log10 of the standard DNA control copy number. Mean Cp values that deviated from the linear phase of the plotted curve were excluded from the standard curve. Regression formulas, linearity (R2), and efficiency (E=10(-1/slope)) were calculated from the standard curves: fusA: y = -3.3821x + 43.329, R2 =

0.998, E=1.975 (Figure 3.4); oppD/F: y = -3.3762x + 41.707, R2 = 0.9994,

E=1.977 (Figure 3.5). The fusA and oppD/F reactions lost linearity below 102 and

104 template copies, respectively. Both the fusA and oppD/F assays demonstrated a detection limit of 1 plasmid copy/µl (Table 3.3 & 3.4).

Assay reproducibility was evaluated by calculating the standard error of the mean (SEM) for each plasmid dilution. The fusA assay SEM varied from 0.06 to 0.50 for the whole range of plasmid copies (Table 3.3). The oppD/F assay

SEM varied between 0.02 and 0.18 for the range of 107 to 102 plasmid copies, and between 1.46 and 2.07 for 101 and 100 plasmid copies, respectively (Table

3.4). Overall, both assays showed low variance between samples indicating reliable reproduction of results. 47

Bull Semen Spiked with M. bovis Experiment. To access the correlation of the developed TaqMan real-time PCR assays and culture, bovine semen was spiked with serially diluted Mb culture and analyzed. Both the fusA and oppD/F assays were able to detect Mb in all spiked treatments. For most treatments, Mb detection by real-time PCR differed by 1-2 orders of magnitude from the actual genome copy number. However, in the most diluted treatment,

3.1 Mb genomes/µl of semen, the fusA reaction detected 3 genomic copies/µl and the oppD/F reaction detected 6 genomic copies/µl. Mycoplasma bovis was detected by culture in only the 3 most concentrated samples with a detection limit of 310 cells/µl. Real-time PCR and culture results are summarized in Table 3.5.

Analysis of Bovine Semen Field Samples Experiment. Fresh bovine semen samples (n=26) were analyzed for Mb by the developed TaqMan real- time PCR assays as well as culture. All field semen samples were negative for

Mb by real-time PCR and culture. Field semen cultures positive for mycoplasma were speciated by 16S rRNA gene partial sequencing and identified as M. bovigenitalium or M. zarardi2.

3.5 Discussion

Mycoplasma bovis costs the U.S cattle industry an estimated $140 million per year [8], yet is often underestimated as a pathogen due to difficulties in diagnosis [38]. An important route of Mb infection is through the reproductive tract by natural and artificial insemination [8, 11, 14]. Culture is still considered 48 the method of choice for isolating mycoplasma from semen; however, the fastidious nature of this group of microorganism presents an obstacle in rapid identification. Furthermore, semen presents additional culture challenges due to the potential presence of mycoplasma growth inhibitors and the extra supplies, facilities, and effort required to isolated Mb from semen [24-26]. Moreover, culture cannot discriminate directly between bovine reproductive tract pathogenic and nonpathogenic mycoplasmas; M. bovirhinis, M. alkalescens, and M arginini,

M. verecundum, M. alvi, and Acholeplasma laidlawii are among several non- disease causing mycoplasmas that inhabit the bovine reproductive tract [17, 37].

Serology is often employed in the detection of Mb; however, antibody titers only emerge 10-14 days after Mb introduction, which delays diagnosis.

Moreover, serology is often not sensitive enough to identify chronic carriers [39].

To address the limitation of Mb culture and serology, PCR systems have been proposed for the detection of Mb in bovine fluids and tissues [9, 24, 27-29, 31-34,

39-42], but no PCR assays have been developed to address the specific evaluation of Mb in bovine semen.

In this study we describe the development and validation of a dual target

TaqMan real-time PCR method and its applicability for the rapid evaluation of Mb in bovine semen. Both the fusA and oppD/F assays were demonstrated to be highly specific when tested with genomic DNA from multiple bovine mycoplasma species. The developed assays exclusively amplified genomic DNA extracted from 59 Mb strains isolated from milk and various bovine tissues acquired from

49 laboratories across the U.S. It is important to note the lack of cross-reactivity of both developed assays with M. agalactiae; previously developed PCR systems have had mixed success in differentiating the 2 species [27, 29], which have a

16S rRNA similarity of 99.8% [11]. The assays developed here target the housekeeping genes fusA and oppD/F. FusA encodes for elongation factor G, an ATPase participating in elongation and ribosome recycling [43]. OppD/F, an oligopeptide permease, is a component of the ABC-transporter system which aids in nutrition and peptide transport [44].

The developed real-time PCRs demonstrated a detection limit of 3.1 organisms per µl and were completed in 5 hours, while culture was able to detect

3.1 x 102 organisms per µl and took 10 days. The process of culturing mycoplasma from semen is confounded by the presence of unknown mycoplasma growth inhibitors, and therefore requires multiple serial dilutions and enrichment; for each semen sample, 4 broths and 13 plates are required to complete culture isolation. This phenomenon was demonstrated in the experiment in which semen was spiked with Mb and recovered by culture. For example, only 2 out of 13 plates were positive for Mb colonies during culture of semen spiked with 3.1 x 102 organisms/µl. The 2 positive plates were from dilution 10-2 plated on days 2 & 4 post broth inoculation. In addition, growth was only seen in diluted and enriched samples for semen spiked with 3.1 x 103 mycoplasmas/µl, in which only 4 plates were positive for Mb colonies, dilutions

10-2 and 10-3 plated on days 2 & 4 post broth inoculation (Data not shown).

A major component of this study was the development of specific protocols for the efficient recovery and detection of Mb in bull semen material.

Extracting mycoplasma genomic DNA from semen material presents major challenges. Efficient recovery of the mycoplasma DNA from a DNA rich material

(spermatocytes DNA) was one challenge. Furthermore, the adhesion properties of Mb can interfere with the extraction of genomic mycoplasma DNA in naturally infected samples. Scanning electron microscope images show that Mb can adhere to the disulfide bond-rich plasma membrane of spermatozoa following swim up isolation and repeated washing [45, 46]. To address this concern, buffers containing different reducing agents (e.g. dithiothreitol, Triton-X), as well as various periods of incubation at varying temperatures, were tested to obtain optimal Mb genomic recovery from semen samples. In addition, mycoplasma genomic DNA extraction from semen was optimized by comparing different extraction kits, buffers, and incubation times to obtain optimal Mb recovery.

Moreover, real-time PCR, in comparison with end-point PCR, offers a more rapid and quantitative alternative with high-throughput capability and hence selected for this application. The employment of sequence specific TaqMan probes can further increase the specificity and sensitivity [32]. In addition, by amplifying 2 sites on the Mb genome, the reliability as a diagnostic assay is increased, further adding to the robustness of the system. Initially, the developed real-time PCRs were intended to be utilized as a multiplex reaction; however, during validation and optimization, it was observed that at low template

51 concentrations the use of simplex reactions proved more sensitive than the duplex reaction.

M. bovis was not detected by real-time PCR or culture from any field semen samples. Other countries have also reported low recovery rates of Mb from semen including Austria [20], Canada [47], Germany [48], and Northern

Ireland [49]. However, Mb remains an important pathogen of the bovine genital tract causing multiple reproductive diseases in both males and females including seminal vesiculitis, abortion, and infertility [37]. Semen used for artificial insemination (AI) that is contaminated with Mb can serve as a vector for transmission to female cattle [15, 21, 47]. M. bovis colonization of mucosal surfaces is often followed by dissemination throughout the host. Reports show that internal transfer of mycoplasma from an external site to the mammary gland is possible [13]. Vertical transmission from cow to fetus/calf leading to respiratory infections via the genital organs has also been reported [15]. Mycoplasma infected semen is seen as a risk factor for the introduction of Mb to previously uninfected herds [50], making rapid and reliable Mb screening and diagnosis in semen a pressing need for the AI industry.

In summary, we have developed a dual target TaqMan real-time PCR system for the rapid detection and quantification of M. bovis in bovine semen.

Both the fusA and oppD/F assays are highly specific, sensitive, and reproducible.

These assays can be completed in 5 hours, in contrast to culture, which requires up to 10 days. In the future, the fusA and oppD/F assays can be used as

52 diagnostic and research tools for the detection and quantification of Mb in bovine semen samples.

Mollicute Species Strain Designation Strain Information Source M. bovigenitalium 19852 Bovine genital tract ATCC M. bovigenitalium 14173 Bovine genital tract ATCC M. californicum 33461 Bovine mastitic milk ATCC M. californicum Cs687 N/A Washington State University M. californicum ST6 N/A University of Flordia M. alkalescens N/A N/A Washington State University M. agalactiae 35890 Caprine milk ATCC M. agalactiae N/A Clinical isolate University of Flordia M. canadense 29418 Bovine genital tract ATCC M. canis SCI-IA N/A Iowa M. canis N/A N/A University of Flordia 54 M. dispar 27140 Calf lung ATCC M. dispar SDO N/A Iowa M. dispar N/A Clinical isolate University of Flordia M. bovirhinis PG-43 N/A University of Flordia M. bovirhinis 352i N/A Washington State University Acholeplasma laidlawii B, 14192 Sewage ATCC A. laidlawii PG8 N/A University of Flordia Ureaplasma diversum 49783 Male bovine urogenital tract ATCC U. diversum 132vu Clinical isolate Washington State University M. arginini 23243 Sheep lung ATCC M. arginini G-230 N/A University of Flordia M. leachii (formerly serogroup 7) 27369 Calf joint ATCC Continued

Table 3.1 Mycoplasma isolates utilized in this study

Table 3.1 Continued

Mollicute Species Strain Designation Strain Information Source M. verecundum 27862 Bovine eye ATCC M. bovoculi M165-69 N/A University of Flordia M. bovoculi FS8-7 N/A Iowa M. bovoculi C52 N/A Washington State University M. bovis 27368 Bovine milk ATCC M. bovis 25025 Bovine mastitic milk ATCC M. bovis 25523 Bovine mastitis isolate ATCC M. bovis 2383-10 N/A Ohio Department of Agriculture M. bovis 26335-09 N/A Ohio Department of Agriculture

55 M. bovis 27634-09 N/A Ohio Department of Agriculture

M. bovis 27861-09 N/A Ohio Department of Agriculture M. bovis 27876-09 N/A Ohio Department of Agriculture M. bovis 29049-09 N/A Ohio Department of Agriculture M. bovis 29932-09 N/A Ohio Department of Agriculture M. bovis 30172-09 N/A Ohio Department of Agriculture M. bovis 3020-10 N/A Ohio Department of Agriculture M. bovis 31128-09 N/A Ohio Department of Agriculture M. bovis 4336-10 N/A Ohio Department of Agriculture M. bovis 5608-10 N/A Ohio Department of Agriculture M. bovis 595-10 N/A Ohio Department of Agriculture M. bovis 7688-10 N/A Ohio Department of Agriculture M. bovis 7805-10 N/A Ohio Department of Agriculture M. bovis 79-10 N/A Ohio Department of Agriculture Continued

Table 3.1 Continued

Mollicute Species Strain Designation Strain Information Source M. bovis 8986-10 N/A Ohio Department of Agriculture M. bovis 9110-10 N/A Ohio Department of Agriculture M. bovis #137 188.47 Calf ear Washington State University M. bovis #134 188.41 Bovine lung Washington State University M. bovis #142 188.57 Calf lung Washington State University M. bovis #133 188.39 Bovine lung Washington State University M. bovis #129 188.27 Bovine ear Washington State University M. bovis #140 188.53 Calf lung Washington State University M. bovis 56 #143 188.59 Calf ear Washington State University

M. bovis #132 188.37 Bovine lung Washington State University Bovine regropharyngeal lymph M. bovis #145 188.63 node Washington State University M. bovis #136 188.45 Bovine lung Washington State University M. bovis #98 179.27 CU 21528 N/A Washington State University M. bovis #125 188.19 Bovine lung Washington State University M. bovis #112 179.55 UCD 3 N/A Washington State University M. bovis #128 188.25 Bovine lung Washington State University M. bovis #123 188.11 Bovine lung Washington State University M. bovis #127 188.23 Bovine lung Washington State University M. bovis #131 188.35 Bovine lung Washington State University M. bovis #121 188.7 Bovine lung Washington State University M. bovis #57 175.43 Bovine mastitic milk Washington State University M. bovis #111 179.53 UCD 2 N/A Washington State University Continued

Table 3.1 Continued

Mollicute Species Strain Designation Strain Information Source M. bovis #106 179.43 CU 22257 N/A Washington State University M. bovis #118 188.1 Bovine ear Washington State University M. bovis #107 179.45 CU 22258 N/A Washington State University M. bovis #105 179.41 CU 22256 N/A Washington State University M. bovis #103 179.37 CU 22254 N/A Washington State University M. bovis #101 179.33 CU 22251 N/A Washington State University M. bovis #110 179.51 UCD 1 N/A Washington State University M. bovis #100 179.31 CU 22144 N/A Washington State University M. bovis #124 188.13 Bovine ear Washington State University

M. bovis #99 179.29 CU 22117 N/A Washington State University 57

M. bovis #61 175.53 Female bovine genital tract Washington State University

M. bovis #102 179.35 CU 22253 N/A Washington State University M. bovis N/A Bovine milk isolate Washington State University M. bovis N/A Bovine milk isolate Washington State University M. bovis N/A Bovine milk isolate Washington State University M. bovis N/A Bovine milk isolate Washington State University M. bovis N/A Bovine milk isolate Washington State University M. bovis N/A Bovine milk bulk tank isolate Devrie

Oligonucleotide Sequence

Fus Forward Primer 5' TAA TGC ACG CAA ACT CTC GTA GT 3'

Fus Reverse Primer 5' TGT CAC CAG TTG TTG TGC CTT 3'

Fus Probe 5' 56-FAM ACC AAC AGC AGC AAC AAT ATC ACC TGC-3BHQ 3'

OppD Forward Primer 5' ATG AGC GCT TAT CTC GGC TA 3'

OppD Reverse Primer 5' GTG CAA AAG GGT CAC CGA TA 3'

OppD Probe 5' 5Cy5 AGT TTG CCA TAT CTG GTG GGG TTC CT-3BHQ 3'

Fus Broad Spanning Primer Forward 5' GAT GAT TGA CGC CGT TGT TGA T 3'

5 Fus Broad Spanning Primer Reverse 5' CCA GCG ATA ATT GTT TGA CCT GT 3'

Opp Broad Spanning Primer Forward 5' CCA GCT CAC CCT TAT ACA TGA 3'

Opp Broad Spanning Primer Reverse 5' CAC CAA TTA GAC CGA CTA TTT CAC 3'

16S rRNA Universal Primer Forward 5' ACT CCT ACG GGA GGC AGC AG 3'

16S rRNA Universal Primer Reverse 5' CTC ACG ACA CGA GCT GAC GA 3'

Table 3.2 Primer and probe sequences used for PCR systems

Log 10 Plasmid Copies per Average reaction Cp1 Cp2 Cp3 CpA SEMB 0 38.82 39.11 37.6 38.51 0.46 1 40 40 38.49 39.50 0.50 2 36.66 36.49 36.7 36.62 0.06 3 33.02 33.23 32.92 33.06 0.09 4 28.85 29.95 30.08 29.63 0.39 5 26.69 27.02 27.1 26.94 0.13 6 23.06 22.8 22.46 22.77 0.17 7 19.13 20 19.82 19.65 0.27 A Mean crossing point (Cp) value from three independent runs utilized to construct the standard curve B Standard error of the mean Cp values

Table 3.3 Detection limits and reproducibility of the FusA real-time PCR

Log 10Plasmid Copies per Average reaction Cp1 Cp2 Cp3 CpA SEMB 0 33.77 37.91 35.84 2.07 1 40 37.08 38.54 1.46 2 33.24 32.98 32.85 33.02 0.11 3 30.67 31.26 30.8 30.91 0.18 4 28.48 28.44 28.32 28.41 0.05 5 24.78 24.55 24.61 24.65 0.07 6 21.31 21.43 21.45 21.40 0.04 7 18.121 17.63 18.07 17.94 0.16 8 14.76 14.84 14.78 14.79 0.02 9 11.28 11.44 11.43 11.38 0.05 A Mean crossing point (Cp) value from three independent runs utilized to construct the standard curve B Standard error of the mean Cp values

Table 3.4 Detection limits and reproducibility of the OppD/F real-time PCR

Detected fus oppD oppD M.bovis by fus Cp genomic Cp genomic cells/uL Culture Value copies/uL Value copies/uL 3.1 x 104 + 32.1 4.2 x 102 29.07 1.1 x 103 3.1 x 103 + 34.59 76 32.96 77.9 3.1 x 102 + 37.09 13 34.55 26.3 3.1 x 101 - 37.98 7.6 37.31 4 3.1 x 100 - 39.31 3 36.56 6

Table 3.5 Summary of M. bovis spiked semen results

Plate Day 0 Plate Day Fresh Semen Ejaculate 10-1 3mL 0, 2, & 4 Plate Day broth 10-2 3mL 0, 2, & 4 Plate Day broth 10-3 3mL 0, 2, &4 broth 10-4 3mL Plate Day 20 uL broth 0, 2, & 4

Figure 3.1 Protocol for the isolation of mycoplasmas from semen adopted from

reference 37

Continued

Figure 3.2 Alignment of fusA sequences, full sequences will be submitted to GenBank at a later date.

Figure 3.2 Continued

Continued

Figure 3.3 Alignment of oppD/F sequences, full sequences will be submitted to GenBank at a later date.

Figure 3.3 Continued

fusA Standard Curve 40

35 y = -3.3821x + 43.329 R² = 0.9981

30 E= 1.975487

CpValue 20

10 1 2 3 4 5 6 7 8 9 10 Log 10 Plasmid DNA Copy #

Figure 3.4 FusA standard curve

oppD/F Standard Curve 40

35 y = -3.3762x + 41.707 R² = 0.9994

30 E= 1.977839 25

CpValue 20

10 2 3 4 5 6 7 8 9 10 Log 10 Plasmid DNA Copy #

Figure 3.5 OppD/F standard curve

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13. Fox, L.K., J.H. Kirk, and A. Britten, Mycoplasma mastitis: a review of transmission and control. Journal of veterinary medicine. B, Infectious diseases and veterinary public health, 2005. 52(4): p. 153-60.

14. Gonzalez, R.N., Wilson D.J., Mycoplasma Mastitis in Dairy Herds. . Veterinary Clinics of North America: Food Animal Practice, 2003. 19: p. 199-221.

15. Eaglesome, M.D., M.M. Garcia, and R.B. Steward, Microbial Agents Associated with Bovine Genital Tract Infections and Semen. Part II. Haemophilus somnus, Mycoplasma spp and Ureaplasma spp, Chlamydia; Pathogens and Semen Contaminants; Treatment of Bull Semen with Antimicrobial Agents. Veterinary Bulletin, 1992. 62(9): p. 887-910.

16. Eaglesome, M.D. and M.M. Garcia, The Effect of Mycoplasma bovis on Fertilization Processes In Vitro with Bull Spermatozoa and Zona-free Hamster Oocytes. Veterinary microbiology, 1990. 21(4): p. 329-37.

17. Irons, P.C., C. J. V. Trichard, and A. P. Shutte, Bovine Genital Mycoplasmosis, in Infectious Diseases of Livestock. 2004, Oxford University Press: Oxford. p. 2076-2082.

18. Givens, M.D. and M.S. Marley, Pathogens that cause infertility of bulls or transmission via semen. Theriogenology, 2008. 70(3): p. 504-7.

19. LaFaunce, N.A. and K. McEntee, Experimental Mycoplasma bovis seminal vesiculitis in the bull. The Cornell veterinarian, 1982. 72(2): p. 150-67.

20. Petit, T., J. Spergser, J. Aurich, and R. Rosengarten, Examination of semen from bulls at five Austrian artificial insemination centres for chlamydiae and mollicutes. Veterinary Record, 2008. 162(24): p. 792-793.

21. Fish, N.A., S. Rosendal, and R.B. Miller, The distribution of mycoplasmas and ureaplasmas in the genital tract of normal artificial insemination bulls. The Canadian veterinary journal. La revue veterinaire canadienne, 1985. 26(1): p. 13-5.

22. Pfutzner, H. and K. Sachse, Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Rev Sci Tech Oie, 1996. 15(4): p. 1477-1494.

23. Import Health Standard For Bovine Semen, M.o.A.a.F.B.N. Zealand, Editor 2011, Manager Animal Imports and Exports: Wellington, New Zealand.

24. Hotzel H., B.D., K. Sachse, A. Pflitsch, and H. Pfutzner, Detection of Mycoplasma bovis Using in vitro Deoxyribonucleic acid Amplification. Rev. sci. tech. Off. int. Epiz., 1993. 12(2): p. 581-591.

25. Nicholas, R.A., Bovine mycoplasmosis: silent and deadly. The Veterinary record, 2011. 168(17): p. 459-62.

26. Sachse, K., H. Pfützner, H. Hotzel, B. Demuth, M. Heller, and E. Berthold, Comparison of various diagnostic methods for the detection of Mycoplasma bovis. Revue Scientifique Et Technique (International Office Of Epizootics), 1993. 12(2): p. 571-580.

31. Hotzel, H., K. Sachse, and H. Pfutzner, Rapid detection of Mycoplasma bovis in milk samples and nasal swabs using the polymerase chain reaction. J Appl Bacteriol, 1996. 80(5): p. 505-10.

37. Ruhnke, H.L., Mycoplasmas Associated with Bovine Genital Tract Infections, in Mycoplasmosis in Animals: Laboratory Diagnosis. 1994, Iowa State University Press: Iowa.

38. Clothier, K.A., D.M. Jordan, C.J. Thompson, J.M. Kinyon, T.S. Frana, and E.L. Strait, Mycoplasma Bovis Real-Time Polymerase Chain Reaction Assay Validation and Diagnostic Performance. Journal of Veterinary Diagnostic Investigation, 2010. 22(6): p. 956-960.

39. Hotzel, H., J. Frey, J. Bashiruddin, and K. Sachse, Detection and differentiation of ruminant mycoplasmas. Methods in molecular biology, 2003. 216: p. 231-45.

40. Cai, H.Y., P. Bell-Rogers, L. Parker, and J.F. Prescott, Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 2005. 17(6): p. 537-45.

41. Hotzel, H., M. Heller, and K. Sachse, Enhancement of Mycoplasma bovis detection in milk samples by antigen capture prior to PCR. Molecular and cellular probes, 1999. 13(3): p. 175-8.

42. Rossetti, B.C., J. Frey, and P. Pilo, Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR. Molecular and cellular probes, 2010. 24(5): p. 321-3.

43. Chen, Y., R.K. Koripella, S. Sanyal, and M. Selmer, Staphylococcus aureus elongation factor G--structure and analysis of a target for fusidic acid. The FEBS journal, 2010. 277(18): p. 3789-803.

44. Hiles, I.D., M.P. Gallagher, D.J. Jamieson, and C.F. Higgins, Molecular characterization of the oligopeptide permease of Salmonella typhimurium. Journal of Molecular Biology, 1987. 195(1): p. 125-142.

45. Bielanski, A., J. Devenish, and B. Phipps-Todd, Effect of Mycoplasma bovis and Mycoplasma bovigenitalium in semen on fertilization and association with in vitro produced morula and blastocyst stage embryos. Theriogenology, 2000. 53(6): p. 1213-23.

46. Weyrich, A., Preparation of genomic DNA from mammalian sperm. Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.], 2012. Chapter 2: p. Unit 2 13 1-3.

47. Garcia, M.M., R.B. Truscott, J. McLaren, R.B. Stewart, B. Kingscote, and J. Burchak, Absence of Mycoplasma bovis in unprocessed frozen bull semen from Canadian artificial insemination centres. The Veterinary record, 1986. 119(1): p. 11-2.

48. Kirchoff, H.a.B., A., Investigations on the Occurrence of Mycoplasma bovis and other Mycoplasma Species in Cattle in the Northern part of Germany. Journal of Veterinary Medicine Infectious Diseaes and Veterinary Public Health B, 1986. 33: p. 68-72.

49. Ball, H.J., E.F. Logan, and W. Orr, Isolation of mycoplasmas from bovine semen in Northern Ireland. The Veterinary record, 1987. 121(14): p. 322- 4.

50. Flores-Gutierrez, G.H., F. Infante, J.A. Salinas-Melendez, C.B. Thomas, P.C. Estrada-Bellmann, and F. Briones-Encinia, Development of an immunobinding assay with monoclonal antibodies to diagnose Mycoplasma bovis in semen. Veterinary research communications, 2004. 28(8): p. 681-6. 73

Chapter 4

Conclusions and Future Directions

Mycoplasmas are the smallest and simplest known free-living and self- replicating forms of life. Arising by degenerative evolution, mycoplasmas carry a minimal genome, surviving as saprophytes or parasites [1]. Mycoplasmas are distributed worldwide and inhabit an extensive array of hosts including humans, mammals, reptiles, fish, arthropods, and plants [2]. In order to maintain their parasitic lifestyle, mycoplasmas have developed sophisticated mechanisms for the colonization of their host and the evasion of the host immune system; furthermore, a substantial amount of their genome is devoted to adhesion and antigenic variation [1].

Mycoplasma bovis (Mb) is a primary cattle pathogen and the etiological agent of several serious and economically costly diseases of cattle. The prevalence of Mb is most likely underestimated as a disease agent since it is frequently not investigated due to the fastidious nature of the organism [3, 4]. M. bovis can colonize mucosal surfaces, invade, and disseminate to multiple tissue sites. M. bovis associated diseases are often chronic, debilitating , and respond poorly to antibiotics [5]. In general, attempts to vaccinate against Mb have been 74 unrewarding [6]. Mycoplasma control depends significantly on biosecurity measures including strict hygiene practices as well as the rapid identification and isolation of infected animals [7].

M. bovis is an important pathogen of the bovine genital tract causing multiple reproductive diseases in both males and females including seminal vesiculitis, abortion, and infertility [8]. Semen used for artificial insemination (AI) that is contaminated with Mb can serve as a vector for transmission to female cattle [9-11]. M. bovis colonization of mucosal surfaces is often followed by dissemination throughout the host. Reports show that internal transfer of mycoplasma from an external site to the mammary gland is possible [7]. Vertical transmission from cow to fetus/calf leading to respiratory infections via the genital organs has also been reported [9]. Mycoplasma infected semen is seen as a risk factor for the introduction of Mb to previously uninfected herds [12], making rapid and reliable Mb screening and diagnosis in semen a pressing need for the AI industry.

Culture is still considered the method of choice for isolating mycoplasma from semen; however, the fastidious nature of this group of microorganism presents an obstacle in rapid identification. Furthermore, semen presents additional culture challenges due to the potential presence of mycoplasma growth inhibitors and the extra supplies, facilities, and effort required to isolate

Mb from semen [3, 13, 14]. Moreover, culture cannot discriminate directly between bovine reproductive tract pathogenic and nonpathogenic mycoplasmas.

To address the limitation of Mb culture, PCR systems have been proposed for the detection of Mb in bovine fluids and tissues [13, 15-26], but no PCR assays have been developed to address the specific evaluation of Mb in bovine semen.

This thesis focused on the development and validation of a rapid assay for the detection of Mb in bovine semen. The assay, a dual target TaqMan real-time

PCR system targeting the housekeeping genes fusA and oppD/F, was shown to be highly specific when tested with the genomic DNA of 15 bovine mycoplasmas and 59 strains of Mb. The developed real-time PCRs demonstrated a detection limit of 3.1 organisms per µl of semen and were completed in 5 hours, while culture was able to detect 3.1 x 102 organisms per µl of semen and took 10 days.

Mycoplasma genomic DNA extraction from semen was a major challenge in the development of this real-time PCR system. Considerable effort was put forth to ensure efficient recovery of mycoplasma DNA from the DNA-rich spermatocytes and to avoid the interference of Mb adhesion to sperm cells in the extraction process.

Further research into the optimization of the fusA and oppD/F assays as a real-time PCR duplex should be considered. Initially, the developed real-time

PCRs were intended to be utilized as a multiplex reaction; however, during validation and optimization, it was observed that at low template concentrations the use of simplex reactions proved more sensitive than the duplex reaction.

PCR drift or PCR selection could be possible explanations for this phenomenon.

In conclusion, molecular detection of Mb as well as other pathogenic mycoplasmas is an important priority for the AI industry. The dual target Mb

TaqMan real-time PCR system developed in this study is the first developed specifically for semen evaluation, and can be used as a diagnostic and research tool for the rapid detection and quantification of M. bovis in bovine semen samples.

4.1 List of References

1. Razin, S., D. Yogev, and Y. Naot, Molecular Biology and Pathogenicity of Mycoplasmas. Microbiology and molecular biology reviews : MMBR, 1998. 62(4): p. 1094-156.

2. Gonzalez, R.N., Wilson D.J., Mycoplasma Mastitis in Dairy Herds. . Veterinary Clinics of North America: Food Animal Practice, 2003. 19: p. 199-221.

3. Nicholas, R.A., Bovine mycoplasmosis: silent and deadly. The Veterinary record, 2011. 168(17): p. 459-62.

4. Nicholas, R.A. and R.D. Ayling, Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci, 2003. 74(2): p. 105-12.

5. Maunsell, F.P. and G.A. Donovan, Mycoplasma bovis Infections in young calves. The Veterinary clinics of North America. Food animal practice, 2009. 25(1): p. 139-77, vii.

6. Maunsell, F.P., A.R. Woolums, D. Francoz, R.F. Rosenbusch, D.L. Step, D.J. Wilson, and E.D. Janzen, Mycoplasma bovis infections in cattle. Journal of veterinary internal medicine / American College of Veterinary Internal Medicine, 2011. 25(4): p. 772-83.

7. Fox, L.K., J.H. Kirk, and A. Britten, Mycoplasma mastitis: a review of transmission and control. Journal of veterinary medicine. B, Infectious diseases and veterinary public health, 2005. 52(4): p. 153-60.

8. Ruhnke, H.L., Mycoplasmas Associated with Bovine Genital Tract Infections, in Mycoplasmosis in Animals: Laboratory Diagnosis. 1994, Iowa State University Press: Iowa.

9. Eaglesome, M.D., M.M. Garcia, and R.B. Steward, Microbial Agents Associated with Bovine Genital Tract Infections and Semen. Part II. Haemophilus somnus, Mycoplasma spp and Ureaplasma spp, Chlamydia; Pathogens and Semen Contaminants; Treatment of Bull Semen with Antimicrobial Agents. Veterinary Bulletin, 1992. 62(9): p. 887-910.

10. Fish, N.A., S. Rosendal, and R.B. Miller, The distribution of mycoplasmas and ureaplasmas in the genital tract of normal artificial insemination bulls. The Canadian veterinary journal. La revue veterinaire canadienne, 1985. 26(1): p. 13-5.

11. Garcia, M.M., R.B. Truscott, J. McLaren, R.B. Stewart, B. Kingscote, and J. Burchak, Absence of Mycoplasma bovis in unprocessed frozen bull semen from Canadian artificial insemination centres. The Veterinary record, 1986. 119(1): p. 11-2.

12. Flores-Gutierrez, G.H., F. Infante, J.A. Salinas-Melendez, C.B. Thomas, P.C. Estrada-Bellmann, and F. Briones-Encinia, Development of an immunobinding assay with monoclonal antibodies to diagnose Mycoplasma bovis in semen. Veterinary research communications, 2004. 28(8): p. 681-6.

13. Hotzel H., B.D., K. Sachse, A. Pflitsch, and H. Pfutzner, Detection of Mycoplasma bovis Using in vitro Deoxyribonucleic acid Amplification. Rev. sci. tech. Off. int. Epiz., 1993. 12(2): p. 581-591.

14. Sachse, K., H. Pfützner, H. Hotzel, B. Demuth, M. Heller, and E. Berthold, Comparison of various diagnostic methods for the detection of Mycoplasma bovis. Revue Scientifique Et Technique (International Office Of Epizootics), 1993. 12(2): p. 571-580.

15. Ayling, R.D., R.A. Nicholas, and K.E. Johansson, Application of the polymerase chain reaction for the routine identification of Mycoplasma bovis. The Veterinary record, 1997. 141(12): p. 307-8.

16. Bashiruddin, J.B., J. Frey, M.H. Konigsson, K.E. Johansson, H. Hotzel, R. Diller, P. de Santis, A. Botelho, R.D. Ayling, R.A. Nicholas, F. Thiaucourt, and K. Sachse, Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial. Veterinary journal, 2005. 169(2): p. 268-75.

17. Boonyayatra, S., L.K. Fox, T.E. Besser, A. Sawant, J.M. Gay, and Z. Raviv, A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis. Journal of dairy science, 2012. 95(1): p. 196-205.

18. Cai, H.Y., P. Bell-Rogers, L. Parker, and J.F. Prescott, Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 2005. 17(6): p. 537-45.

19. Chavez Gonzalez, Y.R., C. Ros Bascunana, G. Bolske, J.G. Mattsson, C. Fernandez Molina, and K.E. Johansson, In vitro amplification of the 16S

rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR. Veterinary microbiology, 1995. 47(1-2): p. 183-90.

20. Hotzel, H., J. Frey, J. Bashiruddin, and K. Sachse, Detection and differentiation of ruminant mycoplasmas. Methods in molecular biology, 2003. 216: p. 231-45.

21. Hotzel, H., M. Heller, and K. Sachse, Enhancement of Mycoplasma bovis detection in milk samples by antigen capture prior to PCR. Molecular and cellular probes, 1999. 13(3): p. 175-8.

22. Hotzel, H., K. Sachse, and H. Pfutzner, Rapid detection of Mycoplasma bovis in milk samples and nasal swabs using the polymerase chain reaction. J Appl Bacteriol, 1996. 80(5): p. 505-10.

23. Rossetti, B.C., J. Frey, and P. Pilo, Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR. Molecular and cellular probes, 2010. 24(5): p. 321-3.

24. Sachse, K., H.S. Salam, R. Diller, E. Schubert, B. Hoffmann, and H. Hotzel, Use of a novel real-time PCR technique to monitor and quantitate Mycoplasma bovis infection in cattle herds with mastitis and respiratory disease. Veterinary journal, 2010. 186(3): p. 299-303.

25. Subramaniam, S., D. Bergonier, F. Poumarat, S. Capaul, Y. Schlatter, J. Nicolet, and J. Frey, Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR. Molecular and cellular probes, 1998. 12(3): p. 161-9.

26. Thomas, A., I. Dizier, A. Linden, J. Mainil, J. Frey, and E.M. Vilei, Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis. Veterinary journal, 2004. 168(1): p. 100-2.

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