Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen

Journal of Virological Methods 85 (2000) 65 – 73 www.elsevier.com/locate/jviromet

Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen C.B. Smits a, C. Van Maanen b, R.D. Glas b, A.L.W. De Gee b, T. Dijkstrab b, J.T. Van Oirschot a, F.A.M. Rijsewijk a,* a

Institute for Animal Science and Health (ID-DLO) Department of Mammalian Virology, PO Box 65, 8200 AB Lelystad, The Netherlands b Animal Health Ser6ice, PO Box 9, 7400 AA De6enter, The Netherlands Received 31 December 1997; received in revised form 1 October 1999; accepted 4 October 1999

Abstract Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Bovine herpesvirus 1; Polymerase chain reaction; Semen

1. Introduction Bovine herpesvirus 1 (BHV 1) is an important pathogen economically in cattle causing different * Corresponding author. Tel.: +599-320-238210; fax: + 599-320-238225. E-mail address: [email protected] (F.A.M. Rijsewijk)

syndromes such as infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis (IPV) in cows and infectious pustular balanoposthitis (IPB) in bulls. BHV 1 infection can also cause reproduction problems such as abortion and reduced fertility (Wyler et al., 1989; Miller, 1991). Transmission occurs normally by contact with infected animals but can also take

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place through virus-contaminated semen collected from BHV 1 infected bulls (Kupferschmied et al., 1986; Philpott, 1993). Following infection, the virus establishes a latent state in the ganglia of the nerves that innervate the infected region (Pastoret et al., 1984; Wyler et al., 1989; Van Engelenburg et al., 1995). Under circumstances such as stress or by corticosteroid treatment the latent virus can be reactivated and can be re-excreted at the original site of infection (Pastoret et al., 1979). Therefore, bulls infected genitally may, long after the primary infection, shed BHV 1 in their semen (Dennet et al., 1976) and must be regarded as lifelong carriers and potential shedders. Transmission of BHV 1 is also possible by artificial insemination, whereby samples of a single ejaculate may be inseminated into many females (Drew et al., 1987). The infectivity of BHV 1 may be reduced by cryopreservation treatment, but remains stable after this reduction during storage of semen in liquid nitrogen (Chapman et al., 1979). To prevent transmission of BHV 1, bovine semen should be screened for BHV 1 before it is used for artificial insemination. The-used commonly method to detect BHV 1 in bovine semen is virus isolation (VI) in cell culture, which is an expensive and time consuming method. Virus isolation is difficult because of the natural cytotoxicity of semen and its overshadowing effect on the viral cytopathic effect (CPE) (Weiblen et al., 1992). Different PCR assays have been developed for detection of BHV 1 DNA in cryopreserved bovine semen (extended semen) for artificial insemination (Miller, 1991; Van Engelenburg et al., 1993; Wiedman et al., 1993; Vilcek et al., 1994; Wagter et al., 1996). The advantage of a PCR assay for the detection of BHV 1 in bovine semen has been shown clearly: it is more sensitive and much more rapid than virus isolation. This prompted the need for a PCR assay that could provide results within 8 h and that would be suitable for routine diagnosis of fresh semen that could be used for artificial insemination without any cryopreservation treatment. In the study reported below three different PCR assays were compared for use for routine detection of BHV 1 in fresh bovine semen.

2. Materials and methods

2.1. Viruses and cells A BHV 1 strain was used which was isolated from semen of a bull at a Dutch artificial insemination centre to infect the bulls (Van Oirschot et al., 1993). To propagate BHV 1, Embryonic Bovine Trachea (EBTr) cells were used which have been described previously (Van Engelenburg et al., 1993).

2.2. Experimental design Five seronegative Holstein-Friesian bulls, 11– 14 months of age and housed in an isolation stable, were inoculated with 2× 106 7 50% tissue culture infective doses (TCID50) of the BHV 1 strain. Two millilitres of a virus suspension of 106.7 TCID50/ml has been applied to the preputial cavity of each bull, essentially as described by Van Engelenburg et al. (1995). After the infection, the titre of the virus suspension was re-established and found to be still 106.7 TCID50/ml. All bulls received 0.1 mg dexamethasone (Intervet, Boxmeer, The Netherlands)/kg body weight intramuscularly from 44–48 days post infection (dpi) to reactivate putatively latent virus (Kaashoek et al., 1994).

2.3. Collection of semen samples and sera Semen was collected 1 day before infection and twice a week for more than 15 weeks. A separate sterile artificial vagina was used for each bull at each day of semen collection. From each fresh ejaculate, 10 v/v% was tested in a virus isolation test (VI), 100 ml of each ejaculate was tested by PCR A, PCR B, and PCR C. Of the remaining ejaculate, 2 ml was diluted 1:20 with a Trisbuffered-fructose-glycerol-yolk extender (Foote, 1970) and aliquoted in insemination straws. These straws were stored in liquid nitrogen as a reference panel and were also used for a virus isolation and PCR C on extended semen. Sera were collected from all bulls at 1, 6, 13, 20, 44, 58 and 107 dpi.

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2.4. Virus isolation The method of virus isolation was essentially described by Van Oirschot et al., (1993). The whole 10% of the fresh semen was tested in the virus isolation. For each virus isolation 100 ml semen was used, e.g. when 10 v/v% of the semen contained 500 ml, five fractions of 100 ml were tested. For virus isolation on extended semen, two straws were thawed and pooled and a 100 ml sample was tested.

2.5. PCR A PCR A was a modified version of the procedure described by Van Engelenburg et al. (1993) (Table 1). The isolation of DNA was adapted to give an assay that is more suitable for routine work and for fresh semen instead of extended semen. Briefly, a 100 ml sample of seminal fluid was spiked with an estimated number of 200 molecules of non linearised internal control template p629

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(Fig. 1) and incubated with two volumes of lysis buffer [final concentrations: 0.15 M NaCl, 20 mM Tris (pH 7.5), 10 mM EDTA, 0.5% v/v Tween 20, 1 mg proteinase K (Boehringer Mannheim, Mannheim, Germany), 2.5 mg bacteriophage lambda DNA (Pharmacia LKB Biotechnology, Uppsala, Sweden)/ml] at 60°C for 60 min. The lysate was subjected to a standard phenol extraction. The DNA present in the extracted fraction was directly purified by chromatography on a QIAamp tissue column (QlAgen, Hilden, Germany) according to the manufacturers’ instructions. To elute the DNA from the column, 50 ml preheated (70°C) Tris–HCl (pH 9) was added and the columns were incubated for 10 min at 70°C and centrifuged for 5 min at 6000× g. The PCR products were analysed using an ethidium bromide-stained agarose gel.. A batch of fresh semen samples from a BHV 1-negative bull was used to check for false-positives. The negative semen was obtained from a test sire calf that was fed with anti-BHV 1 colostrum and was BHV

Table 1 Summary of the conditions used for the three different polymerase chain reactionsa PCR A

PCR B

PCR C/PCR CE

Gene target

Glycoprotein C-gene (gC)

gD gene

gD gene

Primers

Pl= 5%-CTG CTG TTC GTA GCC CAC AAC G-3’ P2 = 5%-TGT GAC TTG GTG CCC ATG TCG C-3% 38 Repeated cycles 15 Cycles at 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min 23 cycles at 95°C for 1 min, 60° for 1 min, and 72°C for 1 min plus a 4-s auto segment extension (viral PCR product of 173 bp)

P1 =5%-GTC GAG GTC CGC TAC GCG ACG-3% P2 =5%-CC GCG AGC CGC GCC GAG TTT GCA-3% 35 Repeated cycles At 94°C for 15 s, 67°C for 15 s, and 72°C for 60 s, and completed with a final extension of 5 min at 72°C (viral PCR product of 430 bp)

Pl =5%-CGG CCG CTG TAC TAC ATG GA-3% P2= 3% GAT A.CG TCA GGC GCA GAA CC-3% Same as in PCR B (Viral PCR product of 343 bp)

p629–200 Molecules/sample (PCR product of 278 bp)

No intemal control split sample method (spiking with 30–300 TCID50 BHVI/sample)

400 molecules/sample (PCR product of 920 bp)

A sample of water

Same as in PCR B

Agarose gel electroforese with ethid- Same as in PCR A ium bromide staining

Same as in PCR A

PCR cycling conditions

Internal control

Negative control DNA of BHVI-negative semen Analysis methods a

PCR A, B and C are executed on fresh undiluted semen and PCR CE is executed on extended semen.

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Fig. 1. Construction of control templates for PCR A and PCR C. (A) Construction of the control template for PCR A. A 314-bp Eco47III/Sac I site of the BHV 1 gC gene was subcloned in pUC18 (p615). To obtain clone p629, a 105-bp EagI fragment derived from the BHV 1 gE region was inserted into the EagI site of clone p615. (B) Construction of the control template for PCR C. A dimer of the two primers with an EcoRV site in between was inserted in a bluescript plasmid. A 873-bp PstI/SaI fragment (fimX gene of B. bronchiseptica) derived from clone pIVB3-426 (Savelkoul, 1992) was inserted into the EcoRV site. The PCR primers are indicated by arrowheads.

1-seronegative for 1 year. As a waiting bull this animal remained BHV 1-seronegative for at least another 2 years (Dr J. Bosch, personal communication). Between every three samples one sample of this negative bull was interspersed and all samples were tested simultaneously.

2.6. PCR B PCR B was essentially as described by Wagter et al. (1996). Briefly, the procedure is as follows: One hundred microlitres of semen was treated with proteinase-K reagent followed by centrifugation to precipitate the spermatozoa. After phenol, chloroform, isoamyl-alcohol extraction and centrifugation the supernatant fraction was mixed with water-saturated ether to isolate the DNA. The DNA was precipitated and resuspended in 50 ml ultrapure water. The split sample method was used for PCR in which a duplicate of each semen sample was spiked with an aliquot of a virus suspension containing 30-300 TCID50 BHV 1 and tested in parallel with a control sample. A water sample was tested in parallel with every test series to check for carry-over contamination

2.7. PCR C/PCR CE PCR C is a modified version of the PCR described by Wagter et al. (1996). The split sample

procedure was replaced by the use of an internal control template. The data showed for PCR C on fresh semen were the result of the following procedure: After isolation of DNA for PCR B, the residual DNA fractions dissolved in 50 ml aqua dest were directly stored at − 80°C. After thawing these samples, 25 ml was spiked with an estimated amount of 400 molecules of the internal control template and tested with a gD primer set: forward: 5%-CGGCCGCTGTACTACATGGA-3%, reversed: 5%GATACGTCAGGCGCAGAACC-3%. The predicted size of the amplified viral product and of the internal control are, respectively 343 and 920 base pairs (Fig. 1). For PCR C on extended semen (PCR CE) a slightly modified procedure was used. Briefly, a 100 m1 aliquot of semen was incubated in 200 m1 proteinase K (1.5 mg/ml proteinase K, 0.75% sodium-N-laurylsarcosine, 0.15 M NaCl) for 1 h at 56°C, followed by centrifugation (12 000× g, 1 min) to pellet the spermatozoa. The supernatant was used in a multiple phenol-chloroform-isoamylalcohol extraction procedure. DNA was isolated by precipitation with iso-propanol and 3 M NaAc and dissolved in 50 ml water. An 25 ml aliquot was added to the PCR-mixture with a final test volume of 100 ml. The PCR was performed with the gD primer set as described for PCR C.

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2.8. Serological test Sera were tested for the presence of anti BHV 1 gB antibodies by a blocking ELISA (Kramps et al., 1994).

3. Results

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samples only one of the aliquots was positive. Thirteen of the 16 ‘full positive’ samples were collected during the acute phase of the infection. With extended semen virus was isolated from only 13 samples and 12 of these 13 samples were collected during the acute phase of the infection and one during the dexamethasone treatment.

3.2. PCR on fresh semen

3.1. Virus isolation On the 149 semen samples examined in this study only 22 were positive by virus isolation on fresh semen (Table 2). Sixteen of these samples were positive in all the 100 ml aliquots used in the isolation procedure, while of the remaining six

During the acute phase of the infection (between 2 and 13 dpi), all three PCRs on fresh semen scored the same semen samples as positive, with the exception of one sample which was found negative in PCR B (Table 2). Also after the dexamethasone treatment at 48 and 55 dpi all

Table 2 Results of the six assays to detect BHV1 in the 149 bovine semen samplesa

a The assays were: virus isolation (VIF), PCR A, PCR B and PCR C on fresh undiluted semen, and virus isolation (VIE) and PCR CE on extended semen. The results of the VlFs are given as the ratio of the number of positive samples on the total number of tested samples. The last column gives the total number of samples that have been tested positive per assay per bull. All serum samples tested positive for anti-BHV1-gB antibodies from 13 dpi onwards. The day the dexamethasone treatment started is indicated with a shaded area; +, tested positive; −, tested negative; N.S., no semen collected.

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Table 3 Results of the PCR assays A, B and C performed on undiluted fresh bovine semen and PCR CE and VIE on extended semen (straw) compared to virus isolation on 10% of the undiluted fresh ejaculate (VIF)a Assay

VIF (+)

VIF (−)

Total

PCRA(+)

22

23

45

PCRA(−)

0

104

104

PCRB(+)

18

21

39

PCRB(−)

4

106

110

PCR C(+)

22

44

66

PCRC(−)

0

83

83

PCR CE(+)

15

6

21

PCR CE(−)

7

121

128

VIE(+)

13

0

13

VIE(−)

9

127

136

k

Cl

0.57

(0.43–0.72)

0.49

(0.34–0.65)

0.36

(0.23–0.48)

0.65

(0.49–0.81)

0.71

(0.56–0.86)

a In each assay, 149 semen samples were tested. The k-values represent the agreement beyond chance between two assays and Cl represents the 95% confidence interval.

semen samples were found positive in all three PCRs with the exception of one sample which was found negative in PCR B. In the period between the acute phase and the dexamethasone treatment semen samples were found positive in PCR C alone, in PCR C in combination with PRC A, or in all three PCRs. In contrast, in the period after 58 dpi, most of the positive semen samples were only found by one PCR at a time. In total 45 semen samples were found positive in PCR A, 39 in PCR B and 66 in PCR C (Table 3).

3.3. PCR C on extended semen During the acute phase, virus excretion was found in semen samples of all five bulls. After dexamethasone treatment at 48 dpi, the semen samples of two bulls were found positive it this PCR assay and at 58 dpi, the semen samples of two other bulls were found positive. All other semen samples remained negative after 48 dpi. In all, 21 semen samples were found positive in this PCR assay.

3.4. Comparison of the assays Thirteen samples were found positive by all six assays (4 PCRs and two virus isolations (Table 2). Virus was isolated from an additional nine samples that were positive for PCR A and PCR C. Another 15 samples were found positive by PCR A, PCR B and PCR C and another seven samples were found positive by PCR A and PCR C. One sample was positive by only PCR C on both fresh and extended semen. In all, 45 samples were found positively at least two assays. One sample was positive only by PCR A, six samples were positive only by PCR B and 21 samples were positive only by PCR C. To distinguish between true and false positives, a sample was considered as a true positive when it was positive by at least two assays. Based on this approach, PCR C on fresh semen has a sensitivity of 100% because samples 45/45 were found positive. PCR A has a sensitivity of 98% (44/45), PCR B, 73% (33/45) VIF of 49% (22/45), VIE of 29% (13/45) and PCR CE has a sensitivity of 47% (21/45). However, if the VI on fresh semen is taken as a standard, the sensitivity of PCR A and PCR C is 100% and of PCR B is 82%.

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3.5. Serological analysis Antibodies against gB of BHV 1 were first detected in all inoculated bulls at 13 dpi and remained detectable until the end of the study (data not shown).

4. Discussion Three PCR assays have been adjusted and evaluated for rapid routine screening of fresh bovine semen to be used for artificial insemination. The three PCR assays were compared with a standard virus isolation method on both fresh semen and on extended semen, which can be considered as the ‘gold standard’. VI is at present still used routinely on extended semen (Office International des Epizooties, 1996) All three PCR assays are based on the purification of BHV 1 DNA from a semen fraction free from spermatozoa DNA. Van Engelenburg et al. (1993) have shown that, in BHV 1 contaminatedsemen, most of the BHV 1 DNA can be found in the seminal fluid. PCR A uses only the seminal fluid fraction of semen to detect BHV 1 DNA. PCR B and C, however, first treat the whole semen with proteinase K to liberate BHV 1 that may be present in non sperm cells, or is adhering to spermatozoa (Wagter et al., 1996). Another difference between the PCR assays is the method used to check for false negative results; in PCR A and PCR C, 200 or 400 molecules of an internal control DNA template are added to each PCR reaction, respectively. Due to competition be-

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tween the internal control template and the viral template, PCR A and PCR C have a detection limits of 50–100 and : 200 molecules, respectively. In PCR B, 30–300 TCID50 of BHV 1 virus is spiked into one half of each split sample. Assuming that the ratio of viral genomes to infectious particles in semen ranges from 30–100 (Pastoret et al., 1979), the detection limit of PCR B is between 900–30 000 molecules. The semen samples, obtained after an experimental intrapreputial infection of five bulls and collected over a period of more than 15 weeks, form a suitable panel to test these three PCR assays. In the middle of the acute phase of the infection, : 6 dpi, very large quantities of BHV 1 are shed into the semen (Van Engelenburg et al., 1995), whereas at the end of the acute phase and shortly after reactivation, only small quantities of BHV 1 are shed. This is reflected by the 22 positive samples in the VIF, where 13 out of the 16 ‘full positive’ samples (in all 100 ml aliquots) were collected during the acute phase of the infection. The results show that virus isolation on extended semen is less sensitive than the virus isolation on fresh semen, especially during periods when less virus is shed. This may be due to the dilution of the semen. All three PCR assays are clearly more sensitive than the virus isolation (Table 3). Therefore, kvalues of PCR A, B and C as compared with virus isolation on fresh semen (VIF) are B0.6, which is considered a poor agreement. However, one should also take in consideration that shedding of non viable virus will not be detected in the virus isolation. Kappa-values of PCR B and C as com-

Table 4 Results of the PCR assays B and C, performed on undiluted fresh bovine semen, compared to PCR A. In each PCR assay 149 semen samples were testeda PCR assay

A (+)

B(+)

33

6

39

B(−) C(+)

12 44

98 22

110 66

C(−)

1

82

83

a

A(−)

Total

k

Cl

0.70

(0.54–0.86)

0.68

(0.52–0.83)

The k-values represent the agreement beyond chance between two assays and Cl represents the 95% confidence interval.

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pared with PCR A on fresh semen samples are 0.70 and 0.68, respectively (Table 4), which is a good agreement. Are the samples that were positive only by one PCR true or false positives? Due to the use of the internal control template, 200 molecules for PCR A and 400 molecules for PCR C, the amount of BHV 1 genomes present in the tested sample can be estimated. Some of the samples that scored positive in PCR A contained less than 200 molecules, a quantity that might not have been detected by the less sensitive PCR B. This does not necessarily mean that the six samples that are only positive by PCR B are false positive. Due to the proteinase K treatment used in PCR B, more virus particles may have ended up in the seminal fluid. PCR B may be less sensitive but when a large amount of virus is liberated in this way, such samples may still be positive by PCR B. On the other hand spiking with large quantities of BHV 1 may have caused contamination of negative samples. Because the detection limits of PCR A and C are comparable, the higher positive score of PCR C could be due to the proteinase K treatment, although other explanations cannot be excluded. In conclusion, in this study three PCR assays for the detection of BHV 1 DNA in fresh bovine semen have been compared. All three assays are clearly more sensitive than virus isolation and can be completed within 8 h, allowing the artificial insemination station, in case of a positive reaction, to take measures before semen has been distributed. The results also show that virus isolation and PCR are more sensitive on fresh semen than on extended semen. All three PCR assays are a suitable tool for the screening of bovine semen for the presence of BHV 1 DNA for the artificial insemination of fresh semen.

Acknowledgements The authors thank Betty Verstraten for the technical assistance and the animal technicians for taking care of the bulls. In this work the authors cooperated with the Animal Health Service ZuidOost Genetics (Harfsen, The Netherlands). This

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