Besnoitia besnoiti, an apicomplexan protozoan parasite, is the causative agent of bovine besnoitiosis. This infection may
dramatically affect body condition, and, in males, lead to irreversible infertility. While identification of clinical cases and their
histopathological confirmation is relatively simple to carry out, finding subclinical forms of infection is more difficult, thus a more
sensitive test for the identification of the etiological agent may be an appropriate diagnostic tool. We have developed the ITS1
rDNA-sequence-based conventional and real-time PCR which are highly sensitive and specific for the detection of B. besnoiti
infection in cattle. A recombinant internal positive control was introduced to assess possible sample-related inhibitory effects
during the amplification reaction and, in order to prevent false-positive results, a pre-PCR treatment of potentially contaminating
dU-containing PCR product with uracyl-DNA-glycosylase (UDG) was followed.
# 2007 Elsevier B.V. All rights reserved

Cortes, Helder C.E.

Gottstein, Bruno

Hemphill, Andrew

Leitão, Alexandre

Müller, Norbert

Reis, Yara

Veterinary Parasitology

Repositório Científico da Universidade de Évora

 1 Application of conventional and real-time fluorescent ITS1 rDNA PCR for 1 detection of Besnoitia besnoiti infections in bovine skin biopsies 2  3  4 HELDER C. E. CORTES1*, YARA REIS2, BRUNO GOTTSTEIN3, ANDREW 5 HEMPHILL3, ALEXANDRE LEITÃO2 AND NORBERT MÜLLER3* 6  7 1* Laboratório de Parasitologia, Núcleo da Mitra, ICAM, Universidade de Évora, 8 Apartado 94, 7000-554 Évora, Portugal  9  10 2 Instituto de Investigação Científica Tropical, CIISA, Faculdade de Medicina 11 Veterinária, Av. da Universidade Técnica, 1300-447 Lisboa, Portugal. 12  13 3 Institute of Parasitology, Länggass-Str. 122, P.O. Box 8466, CH-3001 Berne, 14 Switzerland. 15  16 *Corresponding authors: Phone: 00351 266760860, Fax: 00351 266 760912 17 Electronic mail address: hcec@uevora.pt (HCEC); Phone: 0041 31 631 2474; 18 Fax. 0041 31 631 2477; email: nmueller@ipa.unibe.ch (NM) 19  20 RUNNING TITLE: PCR and real-time PCR for Besnoitia besnoiti 21  22  23  24  2 We have developed ITS1 rDNA-sequence-based conventional and real-time 25 PCR (with an internal control) for sensitive specific and quantitative 26 detection of Besnoitia besnoiti infection in cattle. The assay, with 27 sensitivity equivalent to one B. besnoiti, also provides a tool to explore 28 parasite-host interaction and therapeutical aspects of B. besnoiti 29 infections in experimental and natural infection. 30  31  3 Besnoitia besnoiti is a cyst-forming coccidian parasite of cattle, mainly in 32 the sub-Saharan Africa, with high veterinary relevance (4,14). In Europe, it has 33 been recently reported in France (P. J Bourdeau, et al., Abstr. IX European 34 Multicolloquium of Parasitology, pp 459-460, 2004), Spain (11,12) and Portugal 35 (5,6). The first clinical manifestations of the disease, consisting mainly of 36 respiratory disorders, are seldom recognised as B. besnoiti infection. The 37 subsequent chronic stage includes the formation of dermal lesions, dramatic 38 thickening, hardening and wrinkling of the skin, hyperkeratosis and alopecia and 39 leads to caquexia (1,3,14) and irreversible infertility in males (6). 40 Serological diagnosis of B. besnoiti infection using indirect 41 immunofluorescence, ELISA and western blot has been described (7,16,17). 42 However, detection of the parasite is exclusively based on visual observation of 43 cysts on the sub-conjuntiva (15) and on histopathology (2,10). The latter,  based 44 on the morphological characteristics of the cyst wall (9), is specific and 45 conclusive but only applicable when the number of cysts is high. Here, we 46 describe a specific and sensitive conventional and a real-time ITS (internal 47 transcribed spacer) 1 rDNA PCR test which allows detection of the parasite in  8 48 mm diameter bovine skin biopsies through the amplification of parasite specific 49 DNA sequences.  50 Samples of DNA were extracted from skin using the DNAeasy tissue kit 51 system (Qiagen, Basel, Switzerland) with an additional step of three freezing-52 thawing cycles prior to addition of ethanol in methodical step 4. Conventional 53 PCR was performed in a 25 µl mixture containing 2.5 µl 10xGene Amp™ PCR 54 buffer (Applied Biosystems, Basle, Switzerland), 0.2 mM each dATP, dGTP and 55 dCTP, 0.4 mM dUTP (Invitrogen, Dübendorf, Switzerland), 0.25 µM each B. 56  4 besnoitia-specific forward ITS1F (5'-TGACATTTAATAACAATCAACCCTT-3') 57 and reverse ITS1R1 (5'-GGTTTGTATTAACCAATCCGTGA-3') primers, 1.25 58 units of AmpliTaq™ DNA polymerase (Applied Biosystems) and 0.5 units of 59 heat-labile uracyl DNA glycosylase (UDG) (Roche Diagnostics, Basle, 60 Switzerland). To remove eventual dUTP containing carry-over contaminations 61 from previous diagnostic reactions, UDG and dUTP (instead of dTTP) was 62 included in the reaction mixture according to a method elaborated by Longo et 63 al. (13). For UDG-mediated decontamination prior to PCR, the reaction mixture 64 was initially incubated for 10 min at 20 °C. This incubation was followed by a 2 65 min incubation step at 95 °C to inactivate UDG and denature the DNA. 66 Subsequently, amplification was done in 45 cycles of denaturation at 94 °C for 67 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min; this was 68 followed by a final 15 min extension at 72°C and a 4 ºC hold at the completion of 69 the profile. As observed by agarose gel electrophoresis, the amplification 70 product of the conventional PCR had the expected size of 231 base pairs (bp) 71 (see Fig. 1). 72 To control for false-negative results, a recombinant PCR inhibition control 73 (13) was done with plasmid Bluescript KS plus (pBS+) (Stratagene) DNA using 74 chimeric primers containing the B. besnoiti-forward primer sequence plus a 75 sequence representing nt positions 986-1004 on the plasmid (chimeric forward 76 primer BbICF: 5‘-77 TGACATTTAATAACAATCAACCCTTGAATCGGCCAACGCGCG-3)’ and the 78 Besnoitia reverse primer sequence plus the reverse sequence from nt positions 79 1275-1293 on the pKS (chimeric reverse primer BbICR: 5‘-80 GGTTTGTATTAACCAATCCGTGATATAGTCCTGTCGGGTTTC-3’). These 81  5 chimeric primers produced a 355 bp pBS+ amplification product with the 82 Besnoitia-specific primer sequences incorporated at the ends. This amplification 83 product was then cloned into the pGEM™-Teasy vector (Promega) according to 84 the instructions of the manufacturer. About 10 molecules from the resulting 85 recombinant plasmid (subsequently referred to as inhibition control) were added 86 as a control to a duplicate from each sample reaction to monitor possible 87 inhibitory effects within the PCR (Fig. 1). 88 The real-time PCR in the LightCycler Instrument was performed with 1 89 µl of 1:10 diluted DNA sample (in absence and presence of inhibition control)  90 using the LightCycler DNA Master Hybridization Probes Kit (Roche 91 Diagnostics) in a standard reaction containing 0.25µM of each primer and 92 supplemented with 3 mM MgCl2. After heat-activation of the Taq-polymerase 93 and simultaneous denaturation of DNA for 15 min at 95oC, amplification was 94 done in 50 cycles (including denaturation: 95oC, 15 s; annealing: 56oC, 15 s; 95 extension: 72oC, 30 s; ramp rates in all cycle steps were 20oC/s) with 1 µl of 96 1:10 diluted DNA samples. Fluorescence was measured after an increase of the 97 temperature to 82ºC at the end of each annealing phase in the “single“ mode. 98 Fluorescence signals from the amplification products were quantitatively 99 assessed by applying the standard software (version 3.5.3) according to the 100 instructions for the LightCycler Instrument.  101 In order to determine the sensitivity of the conventional and the real-time 102 ITS1 rDNA PCR, amplification reactions on DNA equivalent to 10’000, 1’000, 103 100, 10, 1 and 0.1 in vitro propagated parasites (8) were performed. The 104 sensitivity of the amplification reactions was extremely high in that it consistently 105 allowed detection of 1 B. besnoiti cell by both conventional (not shown) and real-106  6 time PCR (Fig. 2). The high specificity of the PCRs was demonstrated in that 107 exclusively B. besnoiti DNA was amplified from a panel of apicomplexan 108 parasite DNAs (B. besnoiti, Neospora caninum, Toxoplasma gondii, Sarcocystis 109 neurona, S. cruzi, S. tenella, S. muris, S. spellei, S. miescheriana, S. zamari, S. 110 singapurencei, S. gigantea, S. moulei, S. capracanis, S. arieticanis, S. peeri) as 111 well as from bovine genomic DNA (not shown).  112 Both, the conventional and the real-time ITS1 rDNA PCR were tested on 113 43 skin biopsies from B. besnoiti-infected and non-infected cattle from the South 114 of Portugal and selected after histopathological analysis (6) and indirect 115 immunofluorescence antibody test (IFAT) (16), defining three groups: (i) non-116 infected animals as confirmed by negative IFAT and histopathology (21 117 animals), (ii) infected animals positive in IFAT and negative in histopathology (10 118 animals), and (iii) infected animals positive in both tests (12 animals). The latter 119 group contained one animal that exhibited macroscopic skin lesions. Only 3 120 samples (N0 23, 35, and 37) were inhibitory i.e. negative in diagnostic PCR and 121 inhibitory in parallel inhibition control DNA reaction (Table 1). The analytical 122 features of inhibitory samples as well as non-inhibitory B. besnoiti-positive and 123 negative samples are exemplified in Fig 1. In contrast, none of the samples 124 inhibited the inhibition control reaction when tested by real-time PCR (Table 1). 125 The 12 samples that contained histologically detectable cysts (animals N0 26, 126 29, 30, 31, 33, 34, 36, 39, 40, 41, 42, and 43) were positive in both diagnostic 127 PCR techniques. Significantly, 3/5 samples that were non-inhibitory by PCR, 128 and negative by histopathology (animals N0 1, 16, 28, 32, and 38, see Table 1) 129 were positive by real time PCR (animals Nº 16, 28 and 32), emphasising the 130 great sensitivity of the PCR test. Interestingly all 3 animals had previously been 131  7 exposed to the parasite (titer >1:256 in IFAT, as previously described (16)). The 132 conventional PCR was somewhat less sensitive and only identified 2 of these 133 samples (animals N0 16 and 28, see Table 1) to be positive. Conversely, the 20 134 samples (N0 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 21, 22, and 135 25) that were non-inhibitory in PCR, and scored negative in IFAT-based 136 serology (see Table 1) were also negative in the two PCR tests. 137 In conclusion, the present study has demonstrated the practicability and 138 advantages of PCR-based diagnosis of B. besnoiti infections in bovine skin 139 samples, providing possible PCR-inhibitory effects of the samples are excluded. 140 The assays, particularly the real-time PCR are a useful improvement on current 141 procedures because they allow detection of B. besnoiti even in those skin 142 samples that were collected from sero-positive but subclinically infected animals. 143 As a quantitative assay, the real-time ITS1 rDNA PCR will be useful for 144 epidemiological, clinical and pharmacological studies, as well as for 145 investigations elucidating the consequences of immunological and (immuno-146 )pathological effects on growth of the parasite in both natural and experimental 147 hosts. 148 We are deeply thankful to Michael Parkhouse for critical reviewing and 149 providing valuable suggestions about the manuscript. This work was supported 150 by CIISA, ICAM, and the ”Hans-Sigrist”-Foundation, and was part of the EU 151 research collaboration COST Action 854. 152  8 REFERENCES 153  1.  Basson, P. A., R. M. McCully, and R. D. Bigalke. 1970. Observations 154 on the pathogenesis of bovine and antelope strains of Besnoitia besnoiti 155 (Marotel, 1912) infection in cattle and rabbits. Onderstepoort J.Vet.Res. 156 37:105-126. 157  2.  Besnoit, C. and V. Robin. 1912. Sarcosporidioses cutanée chez une 158 vache. Rec.Vet. 37:649. 159  3.  Bigalke, R. D. 1960. Preliminary observation on the mechanical 160 transmission of cyst organisms of Besnoitia besnoiti  (Marotel, 1912) from 161 a chronically infected bull to rabbits by Glossina brevipalpis Newstead, 162 1910. J.S.Afr.Vet.Assoc. 31:37-44. 163  4.  Bigalke, R. D., J. W. van Niekerk, P. A. Basson, and R. M. McCully. 164 1967. Studies on the relationship between Besnoitia of blue wildebeest 165 and impala, and Besnoitia besnoiti of cattle. Onderstepoort J.Vet.Res. 166 34:7-28. 167  5.  Cortes, H., M. L. Ferreira, J. F. Silva, R. Vidal, P. Serra, and V. Caeiro. 168 2003. Contribuição para o estudo da besnoitiose bovina em Portugal. 169 Revista Portuguesa de Ciências Veterinárias 98, nº 545:43-46. 170  6.  Cortes, H., A. Leitão, R. Vidal, M. J. Vila-Vicosa, M. L. Ferreira, V. 171 Caeiro, and C. A. Hjerpe. 2005. Besnoitiosis in bulls in Portugal. Vet 172 Rec. 157:262-264. 173  7.  Cortes, H., S. Nunes, Y. Reis, D. Staubli, D. Vidal, H. Sager, A. Leitão, 174 and B. Gottstein. 2006. Improved immunodiagnosis of Besnoitia 175  9 besnoiti-infection in cattle by the use of ELISA and Western blot. Vet 176 Parasitol. in press. 177  8.  Cortes, H. C. E., Y. Reis, H. Waap, R. Vidal, H. Soares, I. Marques, I. 178 Pereira da Fonseca, I. Fazendeiro, M. L. Ferreira, V. Caeiro, V. Shkap, 179 A. Hemphill, and A. Leitão. 2006. Isolation of Besnoitia besnoiti from 180 infected cattle in Portugal. Vet Parasitol. In press. 181  9.  Dubey, J. P., V. Shkap, E. Pipano, L. Fish, and D. L. Fritz. 2003. 182 Ultrastructure of Besnoitia besnoiti tissue cysts and bradyzoites. 183 J.Eukaryot.Microbiol. 50:240-244. 184  10.  Franco, E. E. and I. Borges. 1915. Nota sobre a sarcosporidiose bovina. 185 Revista de Medicina Veterinária Ano XIV:255-268. 186  11.  Irigoien, M., E. Del Cacho, M. Gallego, F. Lopez-Bernad, J. Quilez, 187 and C. Sanchez-Acedo. 2000. Immunohistochemical study of the cyst of 188 Besnoitia besnoiti. Vet.Parasitol. 91:1-6. 189  12.  Juste, R. A., L. A. Cuervo, J. C. Marco, and L. M. Oregui. 1990. La 190 besnoitiosis bovina: desconocida en España? Medicina Veterinária 191 7:613-618. 192  13.  Longo, M. C., M. S. Berninger, and J. L. Hartley. 1990. Use of uracil 193 DNA glycosylase to control carry-over contamination in polymerase chain 194 reactions 195 1. Gene 93:125-128. 196  10  14.  Pols, J. W. 1960. Studies on Bovine besnoitiosis with special reference 197 to the aetiology. Onderstepoort J.Vet.Res. 28:265-356. 198  15.  Sannusi, A. 1991. A simple field diagnostic smear test for bovine 199 besnoitiosis. Vet.Parasitol. 39:185-188. 200  16.  Shkap, V., A. Reske, E. Pipano, L. Fish, and T. Baszler. 2002. 201 Immunological relationship between Neospora caninum and Besnoitia 202 besnoiti. Vet.Parasitol. 106:35-43. 203  17.  Shkap, V., H. Ungar-Waron, E. Pipano, and C. Greenblatt. 1984. 204 Enzyme linked immunosorbent assay for detection of antibodies against 205 Besnoitia besnoiti in cattle. Trop.Anim Health Prod. 16:233-238. 206  207  11  208 FIGURE LEGENDS 209  210 FIG. 1. Agarose gel-electrophoretic analysis (1% gels) of amplification products 211 from conventional Besnoitia besnoiti ITS1 rDNA PCR on skin biopsies (samples 212 21 to 30) from infected and non-infected cattle in absence (A) and presence (B) 213 of inhibition control DNA. Positive (P) and negative (N) PCR-controls are 214 included. On the left, the sizes of the amplification products are indicated in 215 base pairs (bp). Note that PCR-inhibition can be observed in sample 23. 216  217 FIG 2. Sensitvity of the real-time Besnoitia besnoiti ITS1 rDNA PCR. Results as 218 fluorescence signals, representing amplification reactions for 10’000, 1’000, 100, 219 10, 1 parasite(s) and a negative control (0 parasites) are presented. Dilutions of 220 DNA equivalent to < 1 cell (e.g. 0.1 cells) did not consistently result in a 221 detectable amplification reaction (not shown). 222  223  12 TABLE 1. Characterisitcs of animals included in this study 224  225 Animal no. Histopathology/ IFATa Convent. PCR  Real-time PCR  Clin. manifest (titer) Inhib.b Resultc  Inhib.b Resultc 1 - 1:1024 - -  - - 2 - <1:128 - -  - - 3 - <1:128 - -  - - 4 - <1:128 - -  - - 5 - <1:128 - -  - - 6 - <1:128 - -  - - 7 - <1:128 - -  - - 8 - <1:128 - -  - - 9 - <1:128 - -  - - 10 - <1:128 - -  - - 11 - <1:128 - -  - - 12 - <1:128 - -  - - 13 - <1:128 - -  - - 14 - <1:128 - -  - - 15 - <1:128 - -  - - 16 - 1:1024 - +  - + 17 - <1:128 - -  - - 18 - 1:1024 - -  - - 19 - <1:128 - -  - - 20 - <1:128 - -  - - 21 - <1:128 - -  - - 22 - <1:128 - -  - - 23 - <1:128 + ?  - - 24 - 1:1024 - -  - - 25 - <1:128 - -  - - 26 Cysts 1:1024 - +  - + 27 - 1:1024 - -  - - 28 - 1:1024 - +  - + 29 Cysts 1:1024 - +  - + 30 Cysts 1:1024 - +  - + 31 Cysts 1:1024 - +  - + 32 - 1:512 - -  - + 33 Cysts 1:512 - +  - + 34 Cysts 1:512 - +  - + 35 - 1:1024 + ?  - - 36 Cysts 1:1024 - +  - + 37 - 1:1024 + ?  - - 38 - 1:1024 - -  - - 39 Cysts 1:512 - +  - + 40 Cysts 1:1024 - +  - + 41 Cysts 1:1024 - +  - + 42 Cysts 1:1024 - +  - + 43 Cysts/disease 1:1024 - +  - + aIn the IFAT, sera with a titer >1:256 were scored positive 226 bPCRs with (+) or  without (-) inhibition of amplification reaction 227 cPositive (+) or negative (-) PCR results or questionable (?) result due to PCR inhibition 228  229  230 Figure 1ABFigure 2

Application of conventional and real-time fluorescent ITS1 rDNA PCR for detection of Besnoitia besnoiti infections in bovine skin biopsies

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Application of conventional and real-time fluorescent ITS1 rDNA PCR for detection of Besnoitia besnoiti infections in bovine skin biopsies

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Repositório Científico da Universidade de Évora