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Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams
Veterinary Microbiology 92 (2003) 65–72
Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams L. Manterolaa, A. Tejero-Garce´sa, A. Ficapalb, G. Shopayevaa,d, J.M. Blascoc, C.M. Marinc, I. Lo´pez-Gon˜ia,* a
Departamento de Microbiologı´a, Universidad de Navarra, Pamplona 31008, Spain Laboratori de Sanitat Ramadera, Generalitat de Catalunya, La Seu d’Urgell, Lleida, Spain c Servicio de Investigacio´n Agraria, Diputacio´n General de Arago´n, Zaragoza, Spain d Department of Infectious Diseases, Kazakh National Medical University, Almaty, Kazakhstan b
Received 20 June 2002; received in revised form 29 August 2002; accepted 11 September 2002
Abstract The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams. # 2002 Elsevier Science B.V. All rights reserved. Keywords: PCR; Brucella ovis; Brucellosis; Sheep bacteria; Diagnosis bacteria
1. Introduction Brucella ovis causes genital disease of sheep, characterised by testicular alterations and reduced fertility in rams, and by endometritis and occasional abortions in ewes. This infection represents an important economic problem in many countries where sheep are raised. The control of the disease is mainly based on serological detection and culling of * Corresponding author. Tel.: þ34-948-425600; fax: þ34-948-425649. E-mail address: [email protected] (I. Lo´pez-Gon˜i).
0378-1135/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 3 1 0 - 3
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L. Manterola et al. / Veterinary Microbiology 92 (2003) 65–72
seropositive rams. Several diagnostic techniques have been applied to detect antibodies against B. ovis, of which gel diffusion, complement fixation and ELISA tests are the most effective and widely used (Marin et al., 1989; Blasco, 1990). However, indirect diagnosis based on antibody detection is not totally satisfactory since false-negative serological results are often seen in infected animals (Marin et al., 1989). Bacteriological isolation of B. ovis is the most specific test to establish a firm diagnosis. However, this is difficult because of the lack of a good selective media, a fact that results in overgrowth of contaminants (Blasco, 1990). The frequent existence of semen samples heavily contaminated with other bacteria or containing low numbers of B. ovis could justify the use of PCR as a supplementary direct diagnostic tool. There is increasing information on the diagnostic value of the specific Brucella DNA detection by PCR (Fekete et al., 1992; Leal-Klevezas et al., 1995; Romero et al., 1995a; Rijpens et al., 1996; Gallien et al., 1998; C¸etinkaya et al., 1999; Amin et al., 2001) but a PCR-based assay has never been evaluated for the diagnosis of B. ovis in rams. The purpose of the present study was to compare the sensitivity and specificity of a PCR-based assay with that of bacteriological culture and serological tests for the diagnosis of B. ovis infection in rams.
2. Material and methods 2.1. Samples A total of 192 swabs with semen samples of rams from different sheep flocks were used. Semen was taken from the preputial cavity after electroejaculation. Swabs collected from 35 Brucella-free rams were tested as negative controls. Fifty-six swabs were collected periodically over 8 weeks after inoculation from 14 previously Brucella-free rams inoculated conjunctivally and intrapreputially with a total of 2 109 CFU/ram of the virulent B. ovis PA strain. Finally, serum and swabs with semen samples were collected from 101 rams from flocks naturally infected with B. ovis. The serological and bacteriological status of these 101 animals has been previously reported (Ficapal et al., 1998). Swabs and serum samples were stored at 20 8C until the extraction of bacterial DNA or serological studies were performed. 2.2. Bacteriological and serological tests The methods of preparation of B. ovis PA suspensions and the experimental infection procedure have been described (Blasco et al., 1993). Bacteriological infection of the 14 rams experimentally infected with B. ovis PA was confirmed at necropsy carried out 8 weeks after inoculation, using the samples and the methods described (Blasco, 1990). Each of the 192 semen swabs collected was smeared on at least two plates of modified Thayer–Martin medium (Marin et al., 1996a,b) and the plates were incubated in 10% CO2 for at least 7 days at 37 8C. Suspected colonies were identified and typed according to standard procedures (Alton et al., 1988). The serum samples from the 101 rams from naturally infected flocks were tested for antibodies to B. ovis, by both gel diffusion and complement fixation tests with hot saline B. ovis antigen, as described (Marin et al., 1989; Ficapal et al., 1998).
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2.3. Extraction of DNA Protocol 1: Semen swabs were twirled in 500 ml of sterile distilled water and the samples were incubated at 100 8C for 10 min. After cooling on ice for 2 min, 1 ml of sample was processed for PCR. Protocol 2: Alternatively, swabs were twirled in 200 ml of NET buffer (50 mM NaCl, 125 mM EDTA, 50 mM Tris–HCl pH 7.6) and the sample was centrifuged (13,000 g for 2 min). After incubation of the supernatant at 80 8C for 10 min, the mixture was cooled on ice and digested with sodium dodecyl sulphate (SDS, final concentration 8%) and proteinase K (final concentration 650 mg/ml) at 50 8C for 1 h. DNA was extracted by using the NucleoSpin (Macherey-Nagel, Germany) system according to the manufacturer’s directions. The DNA was stored at 20 8C until further use. An amount of 1 ml of this DNA solution was added to the PCR cocktail reaction. In all experiments, one sample of sterile water was included as negative control. To determine the specificity of the PCR, DNA was extracted also from Ochrobactrum intermedium and Ochrobactrum anthropi (the closest known relatives of the genus Brucella) and from several bacterial species known to be etiologic agents of epididymitis in rams (Blasco, 1990): Actinobacillus seminis, Chamydophila abortus (formerly Chlamydia abortus), Corynebacterium ovis, Histophilus ovis, Mannheimia haemolytica (formerly Pasteurella haemolytica, Staphylococcus aureus, and Streptococcus spp. (Table 1). All these bacteria (with the exception of C. abortus and H. ovis) were grown on tryptic soy or brain hearth infusion agar plates at 37 8C for 2–3 days. Genomic DNA was extracted from pure cultures as described previously (Romero et al., 1995b). The DNA from C. abortus was extracted from a commercial live vaccine (Unisolve, Intervet, Spain), and DNA from H. ovis was kindly provided by Dr. G. Aduriz (Neiker, Bizkaia, Spain). DNA obtained from B. ovis PA, B. abortus 2308 and B. melitensis 16M were used as positive PCR controls. Table 1 PCR results with DNAs from Brucella strains, bacteria phylogenetically related to Brucella and bacteria known to be etiologic agents of epididymitis in rams Bacterium
Strain
Sourcea
PCR
Brucella ovis Brucella abortus Brucella melitensis Ochrobactrum intermedium Ochrobactrum anthropi Chamydophila abortus Actinobacillus seminis Corynebacterium ovis Histophilus ovis Mannheimia haemolytica Staphylococcus aureus Streptococcus spp.
PA 2308 16M 3301 3331
INRA NADC CVL LMG LMG Unisolve Field isolateb Field isolate Field isolate Field isolate Field isolate Field isolate
þ þ þ
a INRA: Institut Nationale de la Recherche Agronomique, Tour, France; NADC: National Animal Disease Center, Ames, IA, USA; CVL: Central Veterinary Laboratory, Weybridge, England; LMG: Culture Collection of the Laboratory for Microbiology Ghent, Belgium. b Isolated from semen samples from rams.
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2.4. Amplification and detection of Brucella ovis DNA by PCR The oligonucleotide primers used were ISP1 (50 -GGTTGTTAAAGGAGAACAGC-30 ) and ISP2 (50 -GACGATAGCGTTTCAACTTG-30 ), designed from the nucleotide sequence of the Brucella IS6501 (Ouahrani-Bettache et al., 1996). These primers were used because of the possibility of enhanced sensitivity because of its presence in B. ovis in multiple copies (Ouahrani-Bettache et al., 1993). PCR was performed in a 25 ml volume containing final concentration of 1 reaction buffer (Perkin-Elmer), 200 mM each dNTP, 1 mM each primer, 1 mM MgCl2, 0.5 unit of DNA Taq polymerase (Perkin-Elmer) and 1 ml of extracted DNA. To reduce the effects of PCR inhibitors, bovine serum albumin (Promega Corporation, Madison, WI, USA) was added (80 mg/ml) to the reaction mixture. The amplification was performed in a GeneAmp PCR System 2400 (Perkin-Elmer). Cycling conditions consisted of an initial denaturation step at 95 8C for 5 min, followed by 30 cycles of 95 8C for 35 s, 62 8C for 45 s, 72 8C for 45 s and a final extension at 72 8C for 6 min. The positive control contained 80 ng of B. ovis DNA as template, and the negative control consisted of sterile water instead of DNA template. After amplification, 7 ml of the reaction mixture was electrophoresed in a 0.8% agarose gel, stained with ethidium bromide, and photographed on an UV transilluminator. A clear-cut band of 700 bp was regarded as a positive result. Each sample was tested at least in duplicate. 2.5. Procedures to avoid cross-contamination during PCR Four separate rooms were used for: (a) sample preparation and extraction of DNA; (b) buffers and reagents preparation (Taq DNA polymerase excluded); (c) addition of the Taq DNA polymerase to the amplification reaction mixture; (d) amplification and product analysis. Other procedures, e.g. recombinant plasmid preparation, were also performed in different rooms. Laminar flow hoods with UV light were used for sample preparation, extraction of DNA and reaction mixture preparation. Aerosol-resistant pipette tips were used to ensure no cross-contamination occurred. Enzymes, primers, dNTPs, buffers, water and other reagents were aliquoted to minimize handling. If false-positive reactions took place, all current aliquots were discarded and new fresh aliquots used. Negative and positive controls were run daily with PCR assays, to monitor for reagent and laboratory contamination. 2.6. Statistics The proportion of observed test agreement was calculated with the Win Episcope 2.0 program with a 99.5% confidence level.
3. Results and discussion To determine the specificity of the ISP1 and ISP2 primers, DNA from the Brucella controls, the seven other pathogens that may cause epididymitis and from both the Ochrobactrum spp. were tested by PCR. Only DNA from Brucella species produced
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Table 2 Detection of B. ovis by culture and PCR in semen swabs taken from 14 experimentally infected ramsa at different intervals after infection Animal
Weeks after inoculation 4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total a
5
6
8
Culture
PCR
Culture
PCR
Culture
PCR
Culture
PCR
þ þ þ
þ þ
þ þ þ þ
þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
3
2
4
2
7
8
9
7 9
The Brucella-free rams were inoculated conjunctivally and intrapreputially with a total of 2 10 CFU/ ram of the virulent B. ovis PA strain. All were successfully infected as demonstrated by the isolation of B. ovis after slaughter (8 weeks after infection) from the epididymides, seminal vesicles and ampullae in all cases.
positive PCR amplicons (Table 1). In addition, the negative control swabs with semen samples from the 35 Brucella-free rams were always negative by PCR, independently of the protocol used for the extraction of DNA (see Section 2.3). These results confirm the high specificity reported for the IS6501 primers (Ouahrani-Bettache et al., 1996), which was 100% compared to the bacteriological culture in these experiments. The comparison of the sensitivity of the classical semen culture with that of the PCR in the 14 rams experimentally infected with B. ovis PA, is shown in Table 2. Culture (a total of 23 samples were positive) was slightly more sensitive than the PCR (19 samples positive), particularly during the first 5 weeks after experimental infection. B. ovis was isolated from four rams at week 5 post-infection, but only two of these animals were positive by PCR. In contrast, at 6 weeks post-infection B. ovis DNA was detected by PCR in eight samples, but only seven of these were culture-positive. In week 8, nine samples were culture-positive, but only seven of these were PCR-positive. Although not every culture-positive sample was PCR-positive, the PCR was able to detect all 10 rams that were positive by culture on at least one sampling date. Necropsy examinations confirmed that all 14 rams experimentally infected by B. ovis PA had bacteriological evidence of infection. The main target organs of infection for all animals were the epididimies, seminal vesicles and ampullae. Despite the fact that all these organs were the source of various semen components, only 10 out of the 14 rams excreted B. ovis in the semen as determined by culture or PCR. This is consistent with previous
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L. Manterola et al. / Veterinary Microbiology 92 (2003) 65–72
Table 3 Comparison of PCR, bacteriological culture and serological results in 101 rams belonging to naturally B. ovis infected flocks No. of rams with Culture þvea, serology þveb
Culture ve, serology þve
Culture ve, serology ve
PCR-positive PCR-negative
22 4
5 21
3 46
Total
26
26
49
a b
Rams in which B. ovis was isolated by culture from the corresponding semen swab. Rams showing positive results in gel diffusion and/or complement fixation tests.
findings (Blasco, 1990) confirming that the shedding of B. ovis in semen can be intermittent or even absent in some infected animals. This, rather than a low sensitivity of the Thayer– Martin’s modified medium for semen culture, explains the existence of infected rams with negative semen culture (i.e. rams’ number 2, 4, 9 and 10, in Table 2). Thus, assuming the sensitivity of the bacteriological results obtained after necropsy was 100%, the sensitivity of the detection of B. ovis-infected rams over the examination period, by either semen culture or PCR on semen swabs, was 71.4% in both cases. The sensitivity of the PCR was also evaluated (Table 3) with 101 semen swabs from field rams belonging to flocks naturally infected with B. ovis, of which the serological and semen culture status was known. A total of 52 rams showed serological evidence of B. ovis infection, but only 26 (50%) were positive on semen culture. The existence of infected seropositive animals giving negative semen culture is a widely reported finding because of the intermittent shedding of B. ovis in semen or its localisation in organs that are not a source of semen components (Blasco, 1990). A total of 22 (84.6%) out of these 26 rams positive on both serology and culture were also positive in the PCR test. This somewhat higher sensitivity of the culture is consistent with the results obtained in the previous experiment with semen samples taken from the 14 experimentally infected rams (Table 2). By contrast, five PCR-positive samples were obtained in the 26 seropositive but culturenegative rams. These positive PCR tests in culture-negative but seropositive rams could be due to the culture failure because of overgrowth of contaminations or the existence of dead or nonviable B. ovis cells. Three semen samples out of the 49 taken from rams negative on both serology and culture were positive in the PCR-based test. The meticulous attention to prevent cross-contamination of the PCR assay and the exhaustive controls included during the extraction of bacterial DNA and PCR protocols rules out the existence of nonspecific PCR reactions or laboratory contaminations. Excretion of B. ovis in semen of seronegative rams has been reported (Bulgin, 1990). It is therefore likely that the same happened in these three animals. However, only a future study testing the B. ovis excretion in flocks performed over time will confirm this hypothesis. When the two protocols of extraction of DNA were compared (see Section 2.3), 29 out of the 49 PCR-positive results obtained in this study (Tables 2 and 3) were positive only when the DNA was extracted by protocol 2. This demonstrated that this protocol was able to
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eliminate the PCR inhibitors that could account for negative results when the DNA was extracted by protocol 1. Considering the serological results as the gold standard, the relative sensitivities for the diagnosis of B. ovis infection were 50% for semen culture and 51.9% for PCR. Altogether, the comparison of the semen culture and PCR results with the 192 semen samples tested, showed an observed proportion of agreement of 0.91 (level of confidence of 99.5) between both tests. Therefore, our results show that the sensitivity of the PCR-based assay with respect to the culture was equivalent. Although we used a protocol of extraction of DNA that reduced PCR inhibitors present in the DNA, the persistence of such inhibitors in some samples could reduce, at least in part, the sensitivity of the PCR. To increase the sensitivity of the detection of Brucella by PCR, Da Costa et al. (1996) have described a nested PCR assay using genomic DNA. However, in our experience the use of nested PCR in bacteriological diagnosis increases the risk of DNA contamination and results in frequent false-positive results. In conclusion, the PCR test, described here, with sensitivity similar to that of semen culture, could be used as a complementary test, and will improve our capacity to diagnose B. ovis infections in rams when semen samples were heavily contaminated with other bacteria or were containing low numbers of B. ovis.
Acknowledgements Fellowship support to L. Manterola from Ministerio de Ciencia y Tecnologı´a (Spain) and to G. Shopayeva from NATO are gratefully acknowledged. This work was partially supported by Ministerio de Ciencia y Tecnologı´a from Spain (AGL2000-0305-C02), and by Gobierno de Navarra (Spain).
References Alton, G.G., Jones, L.M., Angus, R.D., Verger, J.M., 1988. Techniques for the brucellosis laboratory. Institut National de la Recherche Agronomique, Paris. Amin, A.S., Hamdy, M.E.R., Ibrahim, A.K., 2001. Detection of Brucella melitensis in semen using the polymerase chain reaction assay. Vet. Microbiol. 83, 37–44. Blasco, J.M., 1990. Brucella ovis. In: Nielsen, K., Duncan, J.R., (Eds.), Animal Brucellosis. CRC Press, Boca Raton, FL, pp. 351–382. Blasco, J.M., Marin, C.M., Jime´ nez De Bagu¨ es, M.P., Barberan, M., 1993. Efficacy of Brucella suis strain 2 vaccine against Brucella ovis in rams. Vaccine 11, 1291–1294. Bulgin, M.S., 1990. Brucella ovis excretion in semen of seronegative, clinically normal breeding rams. J. Am. Vet. Med. Assoc. 96, 313–315. ¨ ngo¨ r, H., Muz, A., Ertas, H.B., Kalender, H., Erdogan, H.M., 1999. Detection of Brucella C¸etinkaya, B., O species DNA in the stomach content of aborted sheep fetuses by PCR. Vet. Rec. 144, 239–240. Da Costa, M., Guillou, J.P., Garin-Bastuji, B., Thie´ baud, M., Dubray, G., 1996. Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification. J. Appl. Bacteriol. 81, 267–275. Fekete, A., Bantle, J.A., Halling, S.M., 1992. Detection of Brucella by polymerase chain reaction in bovine fetal and maternal tissues. J. Vet. Diagn. Invest. 4, 79–83. Ficapal, A., Jordana, J., Blasco, J.M., Moriyo´ n, I., 1998. Diagnosis and epidemiology of Brucella ovis infection in rams. Small Rum. Res. 29, 13–19.
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Gallien, P., Dorn, C., Alban, G., Staak, C., Protz, D., 1998. Detection of Brucella species in organs of naturally infected cattle by polymerase chain reaction. Vet. Rec. 142, 512–514. Leal-Klevezas, D.S., Martı´nez-Va´ zquez, I.O., Lo´ pez-Merino, A., Martı´nez-Soriano, J.P., 1995. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J. Clin. Microbiol. 33, 3087–3090. Marin, C.M., Jime´ nez De Bagu¨ es, M.P., Blasco, J.M., Gamazo, C., Moriyo´ n, I., Dı´az, R., 1989. Comparison of three serological tests for Brucella ovis infection of rams using different antigenic extracts. Vet. Rec. 125, 504–508. Marin, C.M., Alabart, J.L., Blasco, J.M., 1996a. Effect of antibiotics contained in two Brucella selective media on the growth of Brucella abortus, Brucella melitensis and Brucella ovis. J. Clin. Microbiol. 34, 426–428. Marin, C.M., Jime´ nez De Bagu¨ es, M.P., Barberan, M., Blasco, J.M., 1996b. Comparison of two selective media for the isolation of Brucella melitensis from naturally infected sheep and goats. Vet. Rec. 138, 409–411. Ouahrani-Bettache, S., Michaux, S., Widada, J.S., Bourg, G., Tournebize, R., Ramuz, M., Liautard, J.P., 1993. Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies. J. Gen. Microbiol. 139, 3265–3273. Ouahrani-Bettache, S., Soubrier, M.P., Liautard, J.P., 1996. IS6501-anchored PCR for the detection and identification of Brucella species and strains. J. Appl. Bacteriol. 81, 154–160. Rijpens, N.P., Jannes, G., Asbroeck, M.V., Rossau, R., Herman, L.M.F., 1996. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Appl. Environ. Microbiol. 62, 1683–1688. Romero, C., Pardo, M., Grillo´ , M.J., Dı´az, R., Blasco, J.M., Lo´ pez-Gon˜ i, I., 1995a. Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle. J. Clin. Microbiol. 33, 3198–3200. Romero, C., Gamazo, C., Pardo, M., Lo´ pez-Gon˜ i, I., 1995b. Specific detection of Brucella DNA by PCR. J. Clin. Microbiol. 33, 615–617.
Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams L. Manterolaa, A. Tejero-Garce´sa, A. Ficapalb, G. Shopayevaa,d, J.M. Blascoc, C.M. Marinc, I. Lo´pez-Gon˜ia,* a
Departamento de Microbiologı´a, Universidad de Navarra, Pamplona 31008, Spain Laboratori de Sanitat Ramadera, Generalitat de Catalunya, La Seu d’Urgell, Lleida, Spain c Servicio de Investigacio´n Agraria, Diputacio´n General de Arago´n, Zaragoza, Spain d Department of Infectious Diseases, Kazakh National Medical University, Almaty, Kazakhstan b
Received 20 June 2002; received in revised form 29 August 2002; accepted 11 September 2002
Abstract The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams. # 2002 Elsevier Science B.V. All rights reserved. Keywords: PCR; Brucella ovis; Brucellosis; Sheep bacteria; Diagnosis bacteria
1. Introduction Brucella ovis causes genital disease of sheep, characterised by testicular alterations and reduced fertility in rams, and by endometritis and occasional abortions in ewes. This infection represents an important economic problem in many countries where sheep are raised. The control of the disease is mainly based on serological detection and culling of * Corresponding author. Tel.: þ34-948-425600; fax: þ34-948-425649. E-mail address: [email protected] (I. Lo´pez-Gon˜i).
0378-1135/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 3 1 0 - 3
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seropositive rams. Several diagnostic techniques have been applied to detect antibodies against B. ovis, of which gel diffusion, complement fixation and ELISA tests are the most effective and widely used (Marin et al., 1989; Blasco, 1990). However, indirect diagnosis based on antibody detection is not totally satisfactory since false-negative serological results are often seen in infected animals (Marin et al., 1989). Bacteriological isolation of B. ovis is the most specific test to establish a firm diagnosis. However, this is difficult because of the lack of a good selective media, a fact that results in overgrowth of contaminants (Blasco, 1990). The frequent existence of semen samples heavily contaminated with other bacteria or containing low numbers of B. ovis could justify the use of PCR as a supplementary direct diagnostic tool. There is increasing information on the diagnostic value of the specific Brucella DNA detection by PCR (Fekete et al., 1992; Leal-Klevezas et al., 1995; Romero et al., 1995a; Rijpens et al., 1996; Gallien et al., 1998; C¸etinkaya et al., 1999; Amin et al., 2001) but a PCR-based assay has never been evaluated for the diagnosis of B. ovis in rams. The purpose of the present study was to compare the sensitivity and specificity of a PCR-based assay with that of bacteriological culture and serological tests for the diagnosis of B. ovis infection in rams.
2. Material and methods 2.1. Samples A total of 192 swabs with semen samples of rams from different sheep flocks were used. Semen was taken from the preputial cavity after electroejaculation. Swabs collected from 35 Brucella-free rams were tested as negative controls. Fifty-six swabs were collected periodically over 8 weeks after inoculation from 14 previously Brucella-free rams inoculated conjunctivally and intrapreputially with a total of 2 109 CFU/ram of the virulent B. ovis PA strain. Finally, serum and swabs with semen samples were collected from 101 rams from flocks naturally infected with B. ovis. The serological and bacteriological status of these 101 animals has been previously reported (Ficapal et al., 1998). Swabs and serum samples were stored at 20 8C until the extraction of bacterial DNA or serological studies were performed. 2.2. Bacteriological and serological tests The methods of preparation of B. ovis PA suspensions and the experimental infection procedure have been described (Blasco et al., 1993). Bacteriological infection of the 14 rams experimentally infected with B. ovis PA was confirmed at necropsy carried out 8 weeks after inoculation, using the samples and the methods described (Blasco, 1990). Each of the 192 semen swabs collected was smeared on at least two plates of modified Thayer–Martin medium (Marin et al., 1996a,b) and the plates were incubated in 10% CO2 for at least 7 days at 37 8C. Suspected colonies were identified and typed according to standard procedures (Alton et al., 1988). The serum samples from the 101 rams from naturally infected flocks were tested for antibodies to B. ovis, by both gel diffusion and complement fixation tests with hot saline B. ovis antigen, as described (Marin et al., 1989; Ficapal et al., 1998).
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67
2.3. Extraction of DNA Protocol 1: Semen swabs were twirled in 500 ml of sterile distilled water and the samples were incubated at 100 8C for 10 min. After cooling on ice for 2 min, 1 ml of sample was processed for PCR. Protocol 2: Alternatively, swabs were twirled in 200 ml of NET buffer (50 mM NaCl, 125 mM EDTA, 50 mM Tris–HCl pH 7.6) and the sample was centrifuged (13,000 g for 2 min). After incubation of the supernatant at 80 8C for 10 min, the mixture was cooled on ice and digested with sodium dodecyl sulphate (SDS, final concentration 8%) and proteinase K (final concentration 650 mg/ml) at 50 8C for 1 h. DNA was extracted by using the NucleoSpin (Macherey-Nagel, Germany) system according to the manufacturer’s directions. The DNA was stored at 20 8C until further use. An amount of 1 ml of this DNA solution was added to the PCR cocktail reaction. In all experiments, one sample of sterile water was included as negative control. To determine the specificity of the PCR, DNA was extracted also from Ochrobactrum intermedium and Ochrobactrum anthropi (the closest known relatives of the genus Brucella) and from several bacterial species known to be etiologic agents of epididymitis in rams (Blasco, 1990): Actinobacillus seminis, Chamydophila abortus (formerly Chlamydia abortus), Corynebacterium ovis, Histophilus ovis, Mannheimia haemolytica (formerly Pasteurella haemolytica, Staphylococcus aureus, and Streptococcus spp. (Table 1). All these bacteria (with the exception of C. abortus and H. ovis) were grown on tryptic soy or brain hearth infusion agar plates at 37 8C for 2–3 days. Genomic DNA was extracted from pure cultures as described previously (Romero et al., 1995b). The DNA from C. abortus was extracted from a commercial live vaccine (Unisolve, Intervet, Spain), and DNA from H. ovis was kindly provided by Dr. G. Aduriz (Neiker, Bizkaia, Spain). DNA obtained from B. ovis PA, B. abortus 2308 and B. melitensis 16M were used as positive PCR controls. Table 1 PCR results with DNAs from Brucella strains, bacteria phylogenetically related to Brucella and bacteria known to be etiologic agents of epididymitis in rams Bacterium
Strain
Sourcea
PCR
Brucella ovis Brucella abortus Brucella melitensis Ochrobactrum intermedium Ochrobactrum anthropi Chamydophila abortus Actinobacillus seminis Corynebacterium ovis Histophilus ovis Mannheimia haemolytica Staphylococcus aureus Streptococcus spp.
PA 2308 16M 3301 3331
INRA NADC CVL LMG LMG Unisolve Field isolateb Field isolate Field isolate Field isolate Field isolate Field isolate
þ þ þ
a INRA: Institut Nationale de la Recherche Agronomique, Tour, France; NADC: National Animal Disease Center, Ames, IA, USA; CVL: Central Veterinary Laboratory, Weybridge, England; LMG: Culture Collection of the Laboratory for Microbiology Ghent, Belgium. b Isolated from semen samples from rams.
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2.4. Amplification and detection of Brucella ovis DNA by PCR The oligonucleotide primers used were ISP1 (50 -GGTTGTTAAAGGAGAACAGC-30 ) and ISP2 (50 -GACGATAGCGTTTCAACTTG-30 ), designed from the nucleotide sequence of the Brucella IS6501 (Ouahrani-Bettache et al., 1996). These primers were used because of the possibility of enhanced sensitivity because of its presence in B. ovis in multiple copies (Ouahrani-Bettache et al., 1993). PCR was performed in a 25 ml volume containing final concentration of 1 reaction buffer (Perkin-Elmer), 200 mM each dNTP, 1 mM each primer, 1 mM MgCl2, 0.5 unit of DNA Taq polymerase (Perkin-Elmer) and 1 ml of extracted DNA. To reduce the effects of PCR inhibitors, bovine serum albumin (Promega Corporation, Madison, WI, USA) was added (80 mg/ml) to the reaction mixture. The amplification was performed in a GeneAmp PCR System 2400 (Perkin-Elmer). Cycling conditions consisted of an initial denaturation step at 95 8C for 5 min, followed by 30 cycles of 95 8C for 35 s, 62 8C for 45 s, 72 8C for 45 s and a final extension at 72 8C for 6 min. The positive control contained 80 ng of B. ovis DNA as template, and the negative control consisted of sterile water instead of DNA template. After amplification, 7 ml of the reaction mixture was electrophoresed in a 0.8% agarose gel, stained with ethidium bromide, and photographed on an UV transilluminator. A clear-cut band of 700 bp was regarded as a positive result. Each sample was tested at least in duplicate. 2.5. Procedures to avoid cross-contamination during PCR Four separate rooms were used for: (a) sample preparation and extraction of DNA; (b) buffers and reagents preparation (Taq DNA polymerase excluded); (c) addition of the Taq DNA polymerase to the amplification reaction mixture; (d) amplification and product analysis. Other procedures, e.g. recombinant plasmid preparation, were also performed in different rooms. Laminar flow hoods with UV light were used for sample preparation, extraction of DNA and reaction mixture preparation. Aerosol-resistant pipette tips were used to ensure no cross-contamination occurred. Enzymes, primers, dNTPs, buffers, water and other reagents were aliquoted to minimize handling. If false-positive reactions took place, all current aliquots were discarded and new fresh aliquots used. Negative and positive controls were run daily with PCR assays, to monitor for reagent and laboratory contamination. 2.6. Statistics The proportion of observed test agreement was calculated with the Win Episcope 2.0 program with a 99.5% confidence level.
3. Results and discussion To determine the specificity of the ISP1 and ISP2 primers, DNA from the Brucella controls, the seven other pathogens that may cause epididymitis and from both the Ochrobactrum spp. were tested by PCR. Only DNA from Brucella species produced
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Table 2 Detection of B. ovis by culture and PCR in semen swabs taken from 14 experimentally infected ramsa at different intervals after infection Animal
Weeks after inoculation 4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total a
5
6
8
Culture
PCR
Culture
PCR
Culture
PCR
Culture
PCR
þ þ þ
þ þ
þ þ þ þ
þ þ
þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ
3
2
4
2
7
8
9
7 9
The Brucella-free rams were inoculated conjunctivally and intrapreputially with a total of 2 10 CFU/ ram of the virulent B. ovis PA strain. All were successfully infected as demonstrated by the isolation of B. ovis after slaughter (8 weeks after infection) from the epididymides, seminal vesicles and ampullae in all cases.
positive PCR amplicons (Table 1). In addition, the negative control swabs with semen samples from the 35 Brucella-free rams were always negative by PCR, independently of the protocol used for the extraction of DNA (see Section 2.3). These results confirm the high specificity reported for the IS6501 primers (Ouahrani-Bettache et al., 1996), which was 100% compared to the bacteriological culture in these experiments. The comparison of the sensitivity of the classical semen culture with that of the PCR in the 14 rams experimentally infected with B. ovis PA, is shown in Table 2. Culture (a total of 23 samples were positive) was slightly more sensitive than the PCR (19 samples positive), particularly during the first 5 weeks after experimental infection. B. ovis was isolated from four rams at week 5 post-infection, but only two of these animals were positive by PCR. In contrast, at 6 weeks post-infection B. ovis DNA was detected by PCR in eight samples, but only seven of these were culture-positive. In week 8, nine samples were culture-positive, but only seven of these were PCR-positive. Although not every culture-positive sample was PCR-positive, the PCR was able to detect all 10 rams that were positive by culture on at least one sampling date. Necropsy examinations confirmed that all 14 rams experimentally infected by B. ovis PA had bacteriological evidence of infection. The main target organs of infection for all animals were the epididimies, seminal vesicles and ampullae. Despite the fact that all these organs were the source of various semen components, only 10 out of the 14 rams excreted B. ovis in the semen as determined by culture or PCR. This is consistent with previous
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Table 3 Comparison of PCR, bacteriological culture and serological results in 101 rams belonging to naturally B. ovis infected flocks No. of rams with Culture þvea, serology þveb
Culture ve, serology þve
Culture ve, serology ve
PCR-positive PCR-negative
22 4
5 21
3 46
Total
26
26
49
a b
Rams in which B. ovis was isolated by culture from the corresponding semen swab. Rams showing positive results in gel diffusion and/or complement fixation tests.
findings (Blasco, 1990) confirming that the shedding of B. ovis in semen can be intermittent or even absent in some infected animals. This, rather than a low sensitivity of the Thayer– Martin’s modified medium for semen culture, explains the existence of infected rams with negative semen culture (i.e. rams’ number 2, 4, 9 and 10, in Table 2). Thus, assuming the sensitivity of the bacteriological results obtained after necropsy was 100%, the sensitivity of the detection of B. ovis-infected rams over the examination period, by either semen culture or PCR on semen swabs, was 71.4% in both cases. The sensitivity of the PCR was also evaluated (Table 3) with 101 semen swabs from field rams belonging to flocks naturally infected with B. ovis, of which the serological and semen culture status was known. A total of 52 rams showed serological evidence of B. ovis infection, but only 26 (50%) were positive on semen culture. The existence of infected seropositive animals giving negative semen culture is a widely reported finding because of the intermittent shedding of B. ovis in semen or its localisation in organs that are not a source of semen components (Blasco, 1990). A total of 22 (84.6%) out of these 26 rams positive on both serology and culture were also positive in the PCR test. This somewhat higher sensitivity of the culture is consistent with the results obtained in the previous experiment with semen samples taken from the 14 experimentally infected rams (Table 2). By contrast, five PCR-positive samples were obtained in the 26 seropositive but culturenegative rams. These positive PCR tests in culture-negative but seropositive rams could be due to the culture failure because of overgrowth of contaminations or the existence of dead or nonviable B. ovis cells. Three semen samples out of the 49 taken from rams negative on both serology and culture were positive in the PCR-based test. The meticulous attention to prevent cross-contamination of the PCR assay and the exhaustive controls included during the extraction of bacterial DNA and PCR protocols rules out the existence of nonspecific PCR reactions or laboratory contaminations. Excretion of B. ovis in semen of seronegative rams has been reported (Bulgin, 1990). It is therefore likely that the same happened in these three animals. However, only a future study testing the B. ovis excretion in flocks performed over time will confirm this hypothesis. When the two protocols of extraction of DNA were compared (see Section 2.3), 29 out of the 49 PCR-positive results obtained in this study (Tables 2 and 3) were positive only when the DNA was extracted by protocol 2. This demonstrated that this protocol was able to
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eliminate the PCR inhibitors that could account for negative results when the DNA was extracted by protocol 1. Considering the serological results as the gold standard, the relative sensitivities for the diagnosis of B. ovis infection were 50% for semen culture and 51.9% for PCR. Altogether, the comparison of the semen culture and PCR results with the 192 semen samples tested, showed an observed proportion of agreement of 0.91 (level of confidence of 99.5) between both tests. Therefore, our results show that the sensitivity of the PCR-based assay with respect to the culture was equivalent. Although we used a protocol of extraction of DNA that reduced PCR inhibitors present in the DNA, the persistence of such inhibitors in some samples could reduce, at least in part, the sensitivity of the PCR. To increase the sensitivity of the detection of Brucella by PCR, Da Costa et al. (1996) have described a nested PCR assay using genomic DNA. However, in our experience the use of nested PCR in bacteriological diagnosis increases the risk of DNA contamination and results in frequent false-positive results. In conclusion, the PCR test, described here, with sensitivity similar to that of semen culture, could be used as a complementary test, and will improve our capacity to diagnose B. ovis infections in rams when semen samples were heavily contaminated with other bacteria or were containing low numbers of B. ovis.
Acknowledgements Fellowship support to L. Manterola from Ministerio de Ciencia y Tecnologı´a (Spain) and to G. Shopayeva from NATO are gratefully acknowledged. This work was partially supported by Ministerio de Ciencia y Tecnologı´a from Spain (AGL2000-0305-C02), and by Gobierno de Navarra (Spain).
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