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Ruminant Mycoplasmas PCR 231 16 Detection and Differentiation of Ruminant Mycoplasmas Helmut Hotzel, Joachim Frey, John Bashiruddin, and Konrad Sachse 1. Introduction 1.1. Importance of the Agents More than 20 different species of mollicutes, most of them belonging to the genus Mycoplasma, have been identified from ruminant hosts to date. While a considerable part of this group is conceived to be of minor epidemiological relevance, it contains some important pathogenic agents that have specific host ranges. Due to several peculiar properties of mycoplasmas, which include the absence of a cell wall and the capability of surface antigen variation, diseases caused by mycoplasmas are difficult to control in the conventional fashion by chemotherapy or immunoprophylaxis. Another general feature of mycoplasma infections is their protracted and occasionally chronic course. Five mycoplasmas of economic and welfare importance are mentioned here and serve as examples for the variety of clinical and diagnostic circumstances that can be resolved using polymerase chain reaction (PCR). Mycoplasma (M.) bovis, one of the etiological agents of bovine mycoplasmosis occurring most frequently in Europe and North America, was associated with mastitis in cows, arthritis and pneumonia in calves and young cattle, as well as genital disorders in bulls and cows (1). While mastitis outbreaks mainly occur in the larger dairy herds, pneumonia and arthritis in calves represent typical mycoplasma diseases in small farms. M. agalactiae causes contagious agalactia, a severe infectious disease of sheep and goats expressed clinically as mastitis, arthritis, or keratoconjunctivi- From: Methods in Molecular Biology, vol. 216: PCR Detection of Microbial Pathogens: Methods and Protocols Edited by: K. Sachse and J. Frey © Humana Press Inc., Totowa, NJ 231
232 Hotzel et al. tis (2). Particularly the countries of the Mediterranean and the Middle East are affected, but the agent seems to be much more widespread. M. mycoides subsp. mycoides small colony type (MmmSC) is the causative agent of contagious bovine pleuropneumonia (CBPP), a contagious disease of cattle causing severe losses in livestock production. The disease is especially widespread in Africa, where mortality rates of 30–80% were reported in affected herds (3). Due to its high sanitary, economic, and socio-economic impact, CBPP is a disease of List A of the Office International des Epizooties and, consequently, belongs to the animal diseases which must be eradicated. A phylogenetically closely related pathogen, known as Mycoplasma mycoides subsp. mycoides large colony type (MmmLC) or as Mycoplasma mycoides subsp. capri has been isolated mainly from goats, more rarely from sheep and cattle, and was reported to be responsible for cases of mastitis, keratoconjunctivitis, arthritis, pulmonary disease, and septicemia (4). M. conjunctivae was demonstrated to be the etiological agent of infectious keratoconjunctivitis in domestic sheep and goats, as well as in European alpine ibex and chamois (5). The disease is characterized by inflammation of the conjunctiva and cornea. 1.2. Diagnostic Methods There are several specific reasons why a change from conventional detection methods to DNA-based techniques is bound to bring about significant improvements in the diagnosis of mycoplasma-associated diseases. First of all, the slow growth of mycoplasmas represents a great obstacle. Identification by culture usually takes 5–10 d, which may be late for effective control measures in infected herds to be taken. In contrast, a PCR assay delivering results in 1 d can make all the difference, for instance, in animal trade. In herd diagnosis, antigen enzyme-linked immunosorbent assay (ELISA) may be an economical and relatively fast alternative, but specificity is often limited due to cross-reactions with related mycoplasmas, and its sensitivity is inferior to that of PCR by two or more orders of magnitude, thus rendering it unsuitable for the identification of clinically inapparent shedders. Similarly, antibody detection ELISAs have their limitations in mycoplasma diagnosis, since antibody titers emerge only 10–14 d after infection at the earliest, and sensitivity is often insufficient to identify chronically infected carriers. Meanwhile, PCR-based detection assays for all important ruminant mycoplasmas have been published. Being the most extensively studied genomic region of many mycoplasma species, 16S ribosomal (r) RNA genes emerged as a favorite target of amplification assays (6). As a result of evolutionary processes, they harbor both conserved and variable sequences, thus allowing the
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TM Methods in Molecular Biology VOL
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4 Sachse 2. Selection of Target Seq
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Table 1 Target Sequences Used in PC
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8 Sachse Fig. 1. Structural organiz
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10 Sachse proteins (66-68), invasio
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12 Sachse Since there appears to be
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14 Sachse In the context of identif
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16 Sachse to partially compensate t
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18 Sachse nostic potential of PCR.
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20 Sachse product has to be confirm
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22 Sachse 7. van Camp, G., Fierens,
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24 Sachse 33. Rappelli, P., Are, R.
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26 Sachse 60. Furrer, B., Candrian,
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28 Sachse 89. Bloch, W. (1991) A bi
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30 Sachse
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32 Rådström et al. Fig. 1. Illust
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34 Rådström et al. immunoglobulin
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36 Rådström et al. and the captur
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Table 2 Comparison of the Performan
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40 Rådström et al. explain these
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42 Rådström et al. 5.1. Proteins
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44 Rådström et al. neous. Consequ
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46 Rådström et al. 11. Powell, H.
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48 Rådström et al. 39. Katcher, H
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50 Rådström et al. 73. Nagai, M.,
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52 Hoorfar and Cook 2. FOOD-PCR: A
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54 Hoorfar and Cook Fig. 2. Practic
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56 Hoorfar and Cook Fig. 4. The str
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Table 58 3 Hoorfar and Cook Test Co
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60 Hoorfar and Cook The choosing an
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62 Hoorfar and Cook 10. Demanding L
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64 Hoorfar and Cook 15. Anon. (1995
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66 Lübeck and Hoorfar ods for the
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68 Lübeck and Hoorfar amplificatio
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70 Lübeck and Hoorfar It may be ne
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72 Lübeck and Hoorfar specificity
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74 Lübeck and Hoorfar The latter i
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76 Lübeck and Hoorfar A simple che
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78 Lübeck and Hoorfar 13. Hanai, K
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80 Lübeck and Hoorfar 43. Mitchell
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82 Lübeck and Hoorfar 70. Lawrence
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84 Lübeck and Hoorfar 98. Lantz, P
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88 Frey Table 1 Presence of the Var
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90 Frey 13. Lysis buffer: 100 mM Tr
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92 Frey as template for PCR. In add
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94 Frey strains, including all sero
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96 Frey
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98 Ewalt and Bricker (named for the
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100 Ewalt and Bricker 4. Ethidium b
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102 Ewalt and Bricker Primer Cockta
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104 Ewalt and Bricker Fig. 2. Typic
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106 Ewalt and Bricker with betaine
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108 Ewalt and Bricker unused ethidi
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110 Englen et al. which campylobact
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112 Englen et al. 18. Dehydrated cu
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114 Englen et al. 3.1.3. Fecal Samp
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116 Englen et al. respectively), an
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118 Englen et al. Fig. 1. Identific
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120 Englen et al. 3. Goossens, H.,
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122 Englen et al.
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124 Sachse and Hotzel Table 1 Compa
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126 Sachse and Hotzel Table 2 Prime
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128 Sachse and Hotzel 7. Phenol: sa
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130 Sachse and Hotzel 4. Vortex mix
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132 Sachse and Hotzel Fig. 2. Speci
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134 Sachse and Hotzel In the latter
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136 Sachse and Hotzel 17. Longbotto
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138 Popoff spastic paralysis. The g
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Table 3 Primers for Toxin Gene Dete
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142 Popoff 18. Agarose gel loading
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144 Popoff Taq reaction buffer 1X,
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146 Popoff almost all C. perfringen
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148 Popoff 2. Add to each PCR tube
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150 Popoff 7. Hielm, S., Hyyttia, E
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152 Popoff 35. Wieckowski, E., Bill
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154 Berri et al. 2. Materials 1. C.
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156 Berri et al. 3.3.2. Vaginal Swa
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158 Berri et al. Fig. 1. Trans-PCR.
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160 Berri et al. Fig. 3. Sensitivit
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162 Berri et al.
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164 Gallien The first description o
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166 Gallien
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168 Gallien 2. Materials 2.1. Bacte
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170 Gallien 10. Ethidium bromide: 1
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Table 2 Components (in µL) of 25-
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Table 4 Components (in µL) of 25-
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176 Gallien 3.3. Specific Isolation
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Table 6 PCR Conditions for Subtypin
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Page 191 and 192: 194 Lester and LeFebvre titers (4).
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Page 221 and 222: 224 Khan Simultaneous detection of
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282 Malorny and Helmuth Table 2 Vol
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284 Malorny and Helmuth Table 4 PCR
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286 Malorny and Helmuth 3. Wallace,
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288 Malorny and Helmuth
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290 Wastling and Mattsson is an imp
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292 Wastling and Mattsson 2. Remove
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294 Wastling and Mattsson Fig. 1. B
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296 Wastling and Mattsson time PCR
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298 Wastling and Mattsson
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300 Pozio and La Rosa Table 1 Princ
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302 Pozio and La Rosa regions; spot
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304 Pozio and La Rosa 3. Proteinase
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306 Pozio and La Rosa Table 3 PCR-R
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308 Pozio and La Rosa 2. Pepsin sho
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310 Pozio and La Rosa
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312 Knutsson and Rådström The ref
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314 Knutsson and Rådström Fig. 1.
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316 Knutsson and Rådström 2.4. El
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318 Knutsson and Rådström the swa
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320 Knutsson and Rådström Fig. 3.
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322 Knutsson and Rådström 4. Alek
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324 Knutsson and Rådström 34. Hee
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PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

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