Detection of Anaplasma marginale and Anaplasma phagocytophilum in bovine peripheral blood 1 ...

33 Pages·2010·0.55 MB·English

JCM Accepts, published online ahead of print on 12 May 2010
J. Clin. Microbiol. doi:10.1128/JCM.02405-09
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Detection of Anaplasma marginale and Anaplasma phagocytophilum in bovine peripheral blood
2 samples by diplex real-time reverse transcriptase-polymerase chain reaction assay
3 James B. Reinbolda; Johann F. Coetzeeb; Kamesh R. Sirigireddya; Roman R. Gantaa*.
4
5 a Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas
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6 State University; Manhattan, KS 66506, USA www
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7 b Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Kansas State aaa
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8 University; Manhattan, KS 66506, USA ddd

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10 * Correspondent footnote. Current address: Department of Diagnostic Medicine/Pathobiology, ttpttpttp
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11 College of Veterinary Medicine, Kansas State University, 1800 Denison Avenue, Manhattan, KS, jcjcjc
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12 66506, E-mail address: [email protected] sss
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14 Acknowledgements nnn

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15 This study was supported by the following grants: AI070908 from the National Institute of rrr
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16 Allergy and Infectious Diseases, NIH, and USDA CREES 1433 grant (AES Project Number: , , ,
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18 The authors would like to thank Chuanmin Cheng for invaluable technical assistance during the
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19 development of these assays. sss
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20 ABSTRACT
21
22 Insufficient diagnostic sensitivity and specificity coupled with the potential for cross-reactivity
23 among closely-related Anaplasma species has made the accurate determination of infection
24 status problematic. A method for the development of simplex and diplex real-time qRT-PCR
25 assays is described for the detection of 16S rRNA of A. marginale and A. phagocytophilum D
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26 infection in plasma-free, bovine peripheral blood samples. The diplex assay was able to detect as n
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27 few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same d
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28 reaction. The ratio of 16S rRNA to 16S DNA copies for A. marginale was determined to be fr
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29 117.9 (95 % confidence interval, 100.7,135.2):1. Therefore, the detection limit is the minimum h
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30 infective unit of one A. marginale bacterium. The diplex assay detected non-equivalent molar :/
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31 ratios of up to 100-fold. Additionally, the diplex assay and a cELISA were used to screen 237
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32 samples collected from anaplasmosis endemic herds. When evaluating the cELISA by the results m
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33 of the qRT-PCR, the sensitivity and specificity for detecting A. marginale infection was 65.2% g
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34 (95% CI, 55.3,75.1) and 97.3% (95% CI, 94.7,99.9), respectively. A. phagocytophilum infection
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35 was not detected in the samples analyzed. One- and two-way receiver operator characteristic ril
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36 curves were constructed to recommend the optimum negative cut-off value for the cELISA. A 2
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37 percent inhibition of 20 and 15.3% were recommended for the one- and two-way curves, 9

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38 respectively. In conclusion, the diplex real-time qRT-PCR assay is a highly sensitive and specific g
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39 diagnostic tool for the accurate and precise detection of A. marginale and A. phagocytophilum st
40 infections in cattle.
41 INTRODUCTION
42
43 Many species of the genus Anaplasma induce different and distinct forms of
44 anaplasmosis in cattle. The Office International des Epizooties (OIE) Animal Health Code
45 categorizes anaplasmosis as a notifiable disease due to socioeconomic impact and international
46 trade restrictions (22). However, the significance of anaplasmosis is frequently underestimated D
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47 due to seasonal outbreaks and stability in endemic areas (29). Anaplasmosis, caused by n
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48 Anaplasma marginale, is one of the most prevalent tick-transmitted, rickettsial diseases of cattle d
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49 worldwide (18). Vaccination with Anaplasma centrale is a common practice used to reduce fr
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50 disease morbidity in cattle subsequently infected with A. marginale (21). Infection with one h
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51 Anaplasma species does not confer immunity from infection with other Anaplasma species. Co- :/
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52 infection with two or more Anaplasma species occurs in cattle due to ubiquitous disease
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53 susceptibility and animal husbandry practices such as vaccination with live A. centrale bacteria m
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54 in countries such as Israel, Africa, Australia, and in parts of South America(17). g
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55 Although A. marginale causes significant economic and health impacts in infected cattle
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56 worldwide, A. phagocytophilum also causes self-limiting, economically significant disease ril
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57 (within 9 days post-infection) and persistent infection in cattle with this pathogen is also reported 2
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58 (14, 24, 26, 36). Cattle that survive acute infection by A. marginale and A. phagocytophilum 9

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59 progress to become subclinical carriers of infection. The carrier animals can serve as reservoirs g
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60 of infection for naïve cattle despite vaccination practices with live A. centrale bacteria and st
61 treatment in countries where domestic ruminants are vaccinated (3, 4, 17, 18). In the absence of
62 effective treatment strategies, vaccine availability, and problematic vector control, anaplasmosis
63 control strategies are primarily focused on determination of infection status and prevention of
64 transmission (27).
65 Disease prevention strategies are centered on reliable diagnostic tests for accurately and
66 precisely identifying infected cattle. The subinoculation of whole blood into splenectomized
67 cattle has served as the gold standard for determination of A. marginale infection status.
68 Currently, one of the most common diagnostic techniques used in commercial lab settings today,
69 cELISA, relies upon the identification of bovine anti-major surface protein 5 (MSP-5) antibodies
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70 that recognize the MSP5 protein epitope of A. marginale (16). Due to the establishment of a o
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71 carrier states in infected animals, the cELISA is regarded as a reliable screening test for lo
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72 identifying A. marginale-infected cattle. However, cross-reactivity among Anaplasma species d

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73 has been reported when using cELISA to classify cattle infected with A. marginale and A.
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74 phagocytophilum (9, 35). Additionally, the lag time between infection and anti-MSP5 antibody t
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75 response may allow for the misclassification of cattle subacutely infected with anaplasmosis (5, jc
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76 27). a
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77 The selection of an appropriate target for the accurate and precise determination of .o
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78 infection status is critical for the development of a robust diagnostic method. Due to their role in
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79 the translation of genetic code, ribosomes, as well as ribosomal RNA, are present in high copy
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80 numbers when compared to a single copy of DNA. The extensive conservation of the primary il 9
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81 and secondary structures of rRNA implies an ancient origin of these macromolecules (13). The 0
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82 16S rRNA gene segment is a common structure of bacterial rRNA genes, including that of the b
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83 genus Anaplasma (30). The 16S rRNA gene sequence has been shown to be identical among u
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84 isolates of A. marginale and A. phagocytophilum, but differs considerably among closely related
85 Anaplasma species (7, 10). However, sequence analysis has shown there are highly conserved
86 and specific regions of the 16S rRNA gene segment of the family Anaplasmataceae (33).
87 In this study, methods are described for the development of simplex real-time qRT-PCR
88 assays for the detection of 16S rRNA gene sequences of A. marginale and A. phagocytophilum
89 and a diplex assay for the simultaneous detection 16S rRNA gene sequences of these two
90 organisms in plasma-free bovine peripheral blood samples. The diplex real-time qRT-PCR assay
91 and a cELISA were also used to screen field samples from cattle originating from anaplasmosis
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92 endemic herds. This analysis aided in evaluating the differences in the diplex assay and the o
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93 cELISA as anaplasmosis diagnostic tools. lo
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94 d

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95 MATERIALS AND METHODS m
96 h
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97 This study was approved by the Kansas State University (KSU) Institutional Animal Care and :/
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98 Use Committee (protocol #2517) and Institutional Biosafety Committee (protocol #524).
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99 Design of primers and probes for the RT-PCR assays m
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100 The 16S rRNA gene sequences for A marginale (M60313) and A. phagocytophilum g
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101 (M73220) were downloaded from the GenBank nucleotide sequence database and aligned with
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102 other related species of the genera Anaplasma and Ehrlichia by use of the University of ril
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103 Wisconsin Genetic Computer Group program, Pileup and Pretty (33). Anaplasma and Ehrlichia 2
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104 genera-specific regions were identified for the design of PCR primers. Species-specific regions 9

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105 were used to design TaqMan probes in support of real-time pathogen detection. TaqMan probes g
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106 were designed with specific fluorescent reporter dyes and quencher molecules to facilitate the t
107 diplex assay (Table 1).
108
109 A. marginale and A. phagocytophilum positive control plasmids
110 A whole blood sample collected from a cow infected with the Florida isolate of A.
111 marginale was previously stored at -80°C in a 16.7% glycerol solution. Genomic DNA was
112 isolated from 300 µL of this sample by use of a Puregene DNA purification kit (Gentra Systems;
113 Minneapolis, MN) as described by the manufacturer’s recommendations. The isolated DNA
114 pellet was re-hydrated with 100 µL of the kit-supplied DNA hydration solution and stored at -
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115 80°C. o
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116 Genomic DNA of A. marginale was used as the template to amplify a 0.48-kb 16S rRNA lo
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117 gene segment. The PCR was performed with 200 ng of genomic DNA by use of the d

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118 AmpliTaqPCR reagent kit (Applied Biosystems; Foster City, CA). Thermal cycles were defined
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119 by an initial denaturation cycle for 3 minutes at 94°C, 45 cycles of 94°C for 30 seconds, 52°C for t
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120 30 seconds, and 72°C for 3 minutes. The PCR product was resolved on a 1% agarose gel in 1X jc
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121 Tris-acetate EDTA buffer (40mmol/L Tris-acetate, 1mmol/L EDTA, pH 8.0) containing 0.1 a
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122 µg/mL of ethidium bromide and visualized under UV light (32). The amplicon was ligated into .o
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123 chemically competent Escherichia coli by use of a PCR product cloning kit (TOPO TA Cloning-
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124 TOP10, pCR 2.1-TOPO version U, Invitrogen Corp.; Carlsbad, CA). The E. coli strain was
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125 streaked on LB media plates containing ampicillin (100µg/mL) and 20 µL of kanamycin (50 il 9
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126 µg/mL) applied to the surface of the plates. Transformants containing the A. marginale 0
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127 recombinant plasmid were isolated and propagated at 37°C in an LB media solution containing b
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128 kanamycin (50 µg/mL). u
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129 An A. phagocytophilum positive control plasmid was used in the development of this
130 qRT-PCR assay as previously described (33). Similarly, transformants of the A.
131 phagocytophilum plasmid were re-established by growth in the LB media solution. A boiling
132 preparation method (32) was used to extract plasmid DNA from the transformants. Plasmid DNA
133 was linearized with a restriction enzyme digest by use of SpeI and BamHI for A. marginale and
134 A. phagocytophilum, respectively (32). The sites for these restriction enzymes are located at the
135 3' ends of the insert in the multiple-cloning site region of the plasmids, but are absent within the
136 cloned inserts. This allowed the plasmids to linearize downstream to the inserts for use in the
137 synthesis of in vitro transcripts with the T7 polymerase. The A. marginale plasmid insert was
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138 verified by sequencing with a thermo-sequencing reaction kit (USB Corp., Cleveland, OH) o
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139 according to the manufacturer’s recommendations. A comparison was made between reported lo
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140 nucleotide sequences in the GenBank database and the nucleotide sequence results for the d

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141 plasmid insert of A. marginale and as previously reported for the plasmid insert of A.
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142 phagocytophilum (33). t
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143 m
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144 In vitro transcripts s
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145 In vitro transcripts were prepared for use in the quantitative RT-PCR (qRT-PCR) assay .
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146 development according to the following procedures. Linearized inserts of A. marginale and A. /
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147 phagocytophilum were purified with a phenol-chloroform extraction technique (31). Two A
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148 micrograms of each of the purified linear insert was used to generate recombinant in vitro il
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149 transcripts with the aid of a T7 polymerase kit as recommended by the manufacturer 0
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150 (MEGAscript kit, Ambion, Inc.; Austin, TX). The recombinant in vitro transcripts were purified b
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151 free of plasmid DNA by treating with DNAse I and by use of an RNA purification kit u
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152 (MEGAclear kit, Ambion, Inc.; Austin, TX).
153
154 Real-Time qRT-PCR
155 TaqMan-based real-time amplification (15, 20) assays were performed by use of a Smart
156 Cycler II system (Cepheid; Sunnyvale, CA). Simplex and diplex real time quantitative qRT-PCR
157 assays were developed by use of previously designed forward and reverse primers (33) and
158 TaqMan probes designed as a part of this study (Table 1). A commercially available RT-PCR
159 assay kit (SuperScript III Reverse Transcriptase, Invitrogen Corporation; Carlsbad, CA) was
160 used for development of the simplex and diplex assays. A standard curve of RNA concentrations
161 were made from ten-fold serial dilutions of known quantities of the in vitro transcripts (ranging
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162 from 1 billion to one molecule) and were analyzed in triplicate to optimize these assays for o
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163 sensitivity in each simplex reaction and species specificity for diplex pathogen detection. The lo
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164 temperature cycles used for the qRT-PCR assay were: an initial cDNA generation cycle at 48°C d

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165 for 30 minutes, 3 minutes at 94°C followed by 45 cycles of 94°C for 15 seconds, 50°C for 30
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166 seconds, and 60°C for 60 seconds. The RT-PCR product formation was monitored in real-time t
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167 by measuring the emitted fluorescence associated with exponential growth of the PCR product jc
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168 during the log-linear phase. A reaction was qualified as positive for the presence of a template a
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169 when seven fluorescent units for the emission channel of the fluorescent probe were detected. .o
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170 The PCR cycle at which fluorescence occurs, which was template concentration-dependent in the
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171 reaction, was regarded as the cycle threshold (Ct) value. Linear regression was used to correlate
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172 the reported Ct value to the number of 16S rRNA template molecules in the 25 µL reaction. il 9
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173 0
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174 Evaluation of blood samples from anaplasmosis endemic herds y

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175 Field sample collection was arranged during phone consultation with producers seeking u
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176 recommendations for the control of anaplasmosis. The collection of samples from anaplasmosis
177 endemic herds resulted in the screening of 237 cattle from northeastern and southeastern Kansas
178 during September and October of 2008. These samples were collected from adult cows and bulls
179 of Bos taurus influence. Samples were collected in no-additive evacuated tubes and evacuated
180 tubes containing EDTA during 2008. Typically, the samples were collected on-site, transported
181 on ice, and processed within 48 hours. However, some samples were shipped overnight on ice
182 packs and processed within 48 hours of receipt.
183
184 Anaplasmosis cELISA
185 Serum was collected from no-additive evacuated tubes and analyzed by cELISA. A
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186 commercially available cELISA (Anaplasma Antibody Test Kit, cELISA, VMRD Inc.; Pullman, w
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187 WA) was used in accordance with the method described by the OIE and recommended by the a
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188 manufacturer (22, 40). The optical density (OD) of each well was measured by use of an ELISA d

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189 plate reader at a wavelength of 620 nm. The percent inhibition (% I) of each sample was m

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190 calculated by use of the following equation: t
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191 %I = 100 – [(sample OD X 100) /mean OD of negative control sample] jc
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192 A binary reporting format was used to report cELISA results. Reports with a %I of < 30% were s
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193 recorded as negative (0), whereas reports that were > 30% were recorded as positive (1) (22, 35, o
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194 40). o
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195 RNA extraction ril
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196 Samples collected in evacuated tubes containing EDTA were centrifuged at 2,750 X g at ,

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197 4º C for five minutes. Plasma was removed by a single use pipette while ensuring the buffy coat 1
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198 and red blood cell fraction were not disturbed. The plasma-free sample was vortexed to mix the y

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199 cell fractions. Extraction of RNA from the homogenous, plasma-free sample occurred according e
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200 to the manufacturer’s recommendations of a commercially available product (TRI Reagent,
201 Sigma-Aldrich; Saint Louis, MO). The RNA pellet was resolubilized with 50 µL of nuclease free
202 water. Samples from a known A. marginale carrier and a naïve cow were extracted and analyzed
203 simultaneously for monitoring qRT-PCR assay performance and quality of the RNA extraction
204 technique. Samples were stored in a -80°C freezer until analysis by qRT-PCR. Extracted samples
205 were not subjected to a DNAse treatment; however, contamination was assessed by comparing
206 qRT-PCR and qPCR assay results by replacing reverse-transcriptase (SuperScript III Reverse
207 Transcriptase, Invitrogen Corporation; Carlsbad, CA) with Taq polymerase (Platinum Taq DNA
208 Polymerase, Invitrogen Corporation; Carlsbad, CA) in the optimized A. marginale simplex
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209 assay. o
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210 Ratio of 16S rRNA to 16S DNA of A. marginale d
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211 The number of 16S rRNA molecules present in Anaplasma species is unknown. This d

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212 provides uncertainty for determining the minimum detection sensitivity of the assay in a 250 µL m

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213 plasma-free sample of bovine peripheral blood. A comparison was made by extracting RNA and t
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214 DNA from a 200 µL sample of ten plasma-free blood samples preserved in 50% glycerol as jc
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215 described. The extraction of RNA proceeded as described. The extraction of DNA was s
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216 performed by use of a Wizard SV Genomic DNA Purification System (Promega Corporation; o
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217 Madison, WI) according to the manufacturer’s instructions and a protocol modification for blood o
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218 products (23). The extracted DNA and RNA were each re-hydrated in 500 µL of nuclease-free p
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219 water. 9
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220 Stability of RNA in plasma-free blood samples stored in 50% glycerol 9

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221 The stability of RNA from whole blood samples stored in glycerol is unknown. Ten y

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222 samples were processed on the day of sampling with the RNA extraction method described e
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223 above and the optimized A. marginale simplex assay. Aliquots of these samples were stored in
224 50% glycerol at -80°C (as described) for an extended time period. Samples were thawed and
225 underwent a similar extraction and analysis as the fresh samples. A percent difference change in