Coxiella burnetii DNA in goat milk after vaccination with Coxevac®

Vaccine 29 (2011) 2653–2656

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Short communication

Coxiella burnetii DNA in goat milk after vaccination with Coxevac® Mirjam H.A. Hermans a,∗ , C. (Ronald) J.J. Huijsmans a , Jeroen J.A. Schellekens a , Paul H.M. Savelkoul b , Peter C. Wever c a

Molecular Diagnostics, Jeroen Bosch Hospital, P.O. Box 90153, 5200 ME ’s-Hertogenbosch, The Netherlands Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands c Department of Medical Microbiology and Infection Control, Jeroen Bosch Hospital, P.O. Box 90153, 5200 ME ’s-Hertogenbosch, The Netherlands b

a r t i c l e

i n f o

Article history: Received 22 November 2010 Received in revised form 31 December 2010 Accepted 30 January 2011 Available online 12 February 2011 Keywords: DNAlactia Coxiella burnetii Goat Milk Vaccination Q fever

a b s t r a c t Q fever is a zoonotic disease caused by Coxiella burnetii, a species of bacteria that is distributed globally. A large Q fever epidemic is currently spreading throughout the Netherlands with more than 3500 human cases notified from 2007 to 2009. Governmental measures to prevent further spread of the disease imposed in December 2009 included vaccination of all dairy goats and sheep and, in parallel, bulk tank milk testing to identify contaminated goat and sheep farms. When bulk tank milk was found to contain C. burnetii DNA, pregnant ruminants were culled. An important, but unsolved issue in this policy was whether vaccine-derived C. burnetii DNA is excreted in milk after vaccination. Using real time PCR and single nucleotide polymorphism (SNP) genotyping techniques, we show here that within hours and up to 9 days after vaccination with Coxevac® , vaccine-derived C. burnetii DNA can be detected in the milk of dairy goats. This is the first report describing DNAlactia of vaccine-derived DNA after vaccination with a completely inactivated vaccine. This finding had implications for the Dutch policy to combat the Q fever epidemic. A 2-week interval was introduced between vaccination and bulk tank milk testing to identify infected farms. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction The largest outbreak of Q fever ever reported started in 2007 in the Netherlands and is currently ongoing. Livestock, which may excrete billions of Coxiella burnetii bacteria in ruminant birth products, poses the greatest risk to public health [1]. By the end of 2009, more than 3500 human cases were notified. The majority of the Q fever patients lived within a 5 km radius of C. burnetii infected goat or sheep farms [2]. However, until November 2009, information regarding the location of these farms was withheld from the public, and no breeding ban on these farms was imposed. It was only in December 2009 that the Ministry of Agriculture, Nature and Food Quality decided to release publication of the locations of infected farms and to preventively slaughter the pregnant goats therein [3]. To identify infected farms, C. burnetii DNA quantities were measured in bulk tank milk samples using PCR techniques [4]. All farms with over 50 dairy goats or sheep were obliged to have their bulk milk tested fortnightly. The bulk tank milk samples were tested by the Animal Health Service Deventer, a leading organization in animal health and animal production that, among others, provides laboratory diagnostic services. If a sample was found positive, the

∗ Corresponding author. Tel.: +31 73 699 2106; fax: +31 73 699 2136. E-mail address: [email protected] (M.H.A. Hermans). 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.01.111

Central Veterinary Institute Wageningen, the national reference laboratory for the Dutch government, retested the sample for confirmation. If two subsequent fortnightly bulk tank milk samples tested positive, i.e. above 100 CFU/mL, in both facilities, the farm was considered contaminated and all carrying goats were killed. In December 2009, the Dutch government announced another measure to prevent disease spreading. Before June 1st 2010, all dairy goats and sheep had to be vaccinated with Coxevac® , a vaccine dedicated to protect against Q fever, produced by animal drug company CEVA-Phylaxia, Budapest, Hungary. This vaccine contains formalininactivated C. burnetii (strain RSA 493/Nine Mile phase I). CEVA states that Coxevac® has the advantage of being a so-called “phase I” – as opposed to “phase II” – vaccine which, when used for prevention, not only improves clinical impact, but also considerably reduces or even eliminates excretion of C. burnetii, thereby limiting contamination of the external environment [5]. During the first months of 2010, a number of goat farmers claimed that their tank milk had turned positive in the mandatory tests for C. burnetii DNA as a result of vaccination. According to the Dutch government this was not due to the vaccination since a dead vaccine is used which is not excreted in milk. To obtain evidence whether vaccine-derived DNA was excreted in milk, we investigated the presence of C. burnetii DNA in their milk in the days following inoculation.

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2. Materials and methods 2.1. Vaccination of goats Coxevac® , a phase I C. burnetii vaccine was obtained from CEVA Santé Animale BV, Naaldwijk, The Netherlands. At two different goat farms (designated site A and B) that were declared C. burnetiifree by the Dutch Ministry of Agriculture, Nature and Food Quality, a veterinarian vaccinated six adult milk-producing goats designated A1 (1 year old), A2 (6 years old), A3 (2 years old), B1 (11 years old), B2 (3 years old), and B3 (2 years old) with 2 mL of Coxevac® subcutaneously twice with a 3-weeks interval (day 1 and day 22). At each site, a non-vaccinated adult milk-producing goat designated A4 (4 years old) and B4 (3 years old), residing in the same area as the vaccinated goats, served as control. 2.2. Milk sampling Starting from the day before vaccination (day 0), milk samples were taken aseptically – foremilk was discarded – on a daily basis for 35 days in 50 mL plastic containers and transported at 4 ◦ C to the laboratory at the Jeroen Bosch Hospital in ’s-Hertogenbosch. 2.3. DNA extraction To efficiently extract DNA, proteinase K digestion was performed prior to DNA extraction. Two hundred-and-fifty ␮L of Coxevac® vaccine or individual goat milk samples was digested with 25 ␮L of proteinase K (20 mg/mL; Roche Diagnostics GmbH, Mannheim, Germany) and 225 ␮L digestion solution (produced by adding 1 mL of 1 M Tris–HCl buffer pH 8.0 and 2.5 mL 10% SDS to 44 mL of ultrapure water). The solution was incubated overnight in a thermoshaker at 55 ◦ C and 1400 rpm. DNA was extracted using the NucliSens® EasyMAG extraction system (Biomerieux, Boxtel, The Netherlands) [6]. Five hundred ␮L of digested sample, together with 10 ␮L of Phocine Herpes Virus (PhHV), which served as internal control (to monitor DNA extraction and PCR inhibition) [7], was added to 2 mL NucliSens® Lysis buffer and incubated for 10 min at room temperature. Magnetic silica particles were diluted 1:1 (V/V) with ultrapure water. One hundred ␮L of diluted magnetic silica particles was added to the lysates. The samples were further processed according to the manufacturer’s instructions and eluted in 60 ␮L of elution buffer.

of a number of strain specific discriminatory SNPs in the C. burnetii genome. SNP profiles were determined from DNA isolated from 4 milk samples obtained 1–3 days after Coxevac® vaccination. In addition, SNP profiles were generated from several dilutions (between 100 and 2000 genome equivalents per mL) of Coxevac® DNA, and DNA isolated from 12 samples from naturally infected Dutch livestock (1 cow colostrum, 6 goat bulk milk, 1 cow bulk milk, 1 goat placenta and 3 individual goat milk). Three SNP assays were used: SNP 7087 – reference sequence AE01682.2 –, nucleotides T/C; SNP 7726, nucleotides C/G and SNP 7974, nucleotides A/G. SNP amplification assays were used according to the manufacturer’s instructions. Twenty-five ␮L of PCR contained 20 mmol/L Tris–HCl, pH 8.4, 50 mmol/L KCl, 5 mmol/L MgCl2 (prepared from 10× PCR buffer (Invitrogen BV) and 50 mmol/L MgCl2 solution (Applied Biosystems, Foster City, CA, USA)), 0.75 units of TaKaRa Ex Taq HS polymerase (TaKaRa Bio, Otsu, Japan), 0.01% bovine serum albumine (molecular biology grade; Westburg BV Benelux, Leusden, The Netherlands), 200 ␮mol/L of each dNTP (Invitrogen BV), 0.5 ␮L of Rox reference dye (Invitrogen BV), 1.25 ␮L of SNP primerprobe pool (Applied Biosystems) containing two primers and two MGB TaqMan probes (5 VIC for allele 1, 5 FAM for allele 2, a 3 black hole quencher), and 11.25 ␮L of eluted target DNA. The ABI Prism sequence detection system 7500 Fast (Applied Biosystems) was used for amplification and detection (10 95 ◦ C, 45 cycles of 3 95 ◦ C and 30 60 ◦ C and ∞ at 25 ◦ C in Fast 7500 mode). Data analysis was performed using SDS software version 1.3.1. Linear amplification plots were analysed with a baseline from cycle 6 to 15 for both reporters. 3. Results 3.1. C. burnetii DNA content of Coxevac® vaccine Analyses of the C. burnetii DNA content of the Coxevac® vaccine by real time PCR revealed a Ct value of 8.3 ± 0.1 (mean ± SD; n = 4), which corresponds to 3.6 × 109 genome equivalents per mL [7]. 3.2. C. burnetii DNA detection in goat milk after vaccination

The real time PCR to detect C. burnetii was carried out in duplicate with 10 ␮L of DNA eluate for each PCR as described [8] with 40 cycles of amplification. The quantity of C. burnetii DNA in Coxevac® vaccine and goat milk samples was estimated based on the cycle threshold (Ct) values obtained and those of a quantified Nine Mile strain stock [8]. If during the 40 cycles no fluorescent signal above the threshold was observed, the Ct value was considered 40 for calculation purposes. Mean Ct values from duplicate analyses of individual milk samples were subtracted from 40 (40 minus mean Ct value) and the outcome was used for graphical depiction of DNA excretion over time. PCRs with PhHV Ct values ≤29.70 were considered not inhibited. In all runs we included DNA isolation controls (NC; a mock isolation submitted to PCR) and no-template controls (NTC; a PCR with PCR ingredients and water instead of sample) to monitor the presence of contaminants in isolation and/or PCR reagents.

Initial milk samples from all eight goats tested negative for C. burnetii DNA on the day before vaccination. In the six vaccinated goats, C. burnetii DNA became detectable in milk samples from a few hours after the first vaccination until day 10 (Fig. 1A). Of the 18 milk samples that tested positive, eight samples tested positive in one of the duplicate PCRs. In these eight samples, the Ct value was 35.2 ± 0.9 (mean ± SD; n = 8). In the 10 samples that tested positive in duplicate, the Ct value was 34.5 ± 0.9 (mean ± SD; n = 20). The lowest Ct value measured was 33.2 corresponding to approximately 100 Nine Mile RSA 493 genome equivalents per mL [8]. After the booster vaccination on day 22, C. burnetii DNA was detected in milk samples from two out of six vaccinated goats (A3 and B1) from day 24 until day 26 (Fig. 1B). The two milk samples from goat A3 tested positive in one of the duplicate PCRs. The one milk sample from goat B1 tested positive in both duplicate PCRs. No C. burnetii DNA was detected in any of the samples obtained from day 12 until day 21, and from day 33 until day 35 (data not shown). Milk samples from the two non-vaccinated goats remained PCR-negative throughout the whole experiment. Two milk samples (goat A2 on day 31 and 34) could not be evaluated due to inhibition of the PCRs. All NCs and NTCs tested throughout the experiment remained negative.

2.5. Single nucleotide polymorphism (SNP) genotyping

3.3. Genotyping of C. burnetii derived DNA

The SNP genotyping assay for C. burnetii was recently developed in our laboratory [9]. The method is based on the detection

To discriminate vaccine-derived (Nine Mile strain) C. burnetii DNA from environmental-derived C. burnetii DNA, a recently devel-

2.4. Real time PCR

40 - Ct value C. burnetii real time PCR

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dilutions and the in silico predicted profile from the Nine Mile sequence AE01682.2. These profiles clearly differed from the profiles obtained from 11 milk and 1 tissue samples of naturally infected Dutch livestock [9]. Fig. 2 shows a representative allelic discrimination/scatter plot (of SNP 7087). The signals generated with DNA from goat milk after vaccination clustered with the Coxevac® vaccine derived signals, while the signals derived from other Dutch livestock samples visibly form a distinct entity. 4. Discussion

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Days Fig. 1. Shedding of C. burnetii DNA in goat milk after Coxevac® vaccination. At goat farms A and B, six adult milk-producing goats, designated A1, A2, A3, B1, B2 and B3, were subcutaneously inoculated by a veterinarian with 2 mL of Coxevac® at day 1 and day 22 as indicated by the two arrows. Two adult milk-producing control goats, designated A4 and B4, were not inoculated. The real time PCR to detect C. burnetii DNA in milk of the individual goats was carried out as described in Section 2 with 40 cycles of amplification. The X-axis plots time in days of the experiment. The Yaxis plots excretion of C. burnetii DNA depicted as 40 minus the mean Ct value of individual milk samples tested in a duplicate PCR analysis. (A) Results after the first inoculation; (B) results after the booster vaccination.

oped SNP genotyping assay was used. Because DNA quantities after vaccination in milk were low, only three SNPs located on the multicopy gene IS1111 could be used for strain differentiation and only four milk samples obtained 1–3 days after vaccination could be genotyped. The allelic profile of DNA isolated from the milk samples of the goats after vaccination was SNP 7087 nucleotides T and C; SNP 7726 nucleotide C; and SNP 7974 nucleotide A. This profile was identical to the SNP profile of the Coxevac® vaccine

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VIC fluoresence (T) Fig. 2. Allelic discrimination/scatter plot of real time PCR analyses of single nucleotide polymorphism 7087 of the C. burnetii multicopy gene IS1111. Grey squares represent the signals obtained from DNA derived from milk/tissue samples of naturally infected Dutch livestock. The black squares represent signals obtained from dilutions of Coxevac® vaccine DNA (Nine Mile strain). The four white triangles represent the signals obtained from 4 different milk samples obtained 1–3 days after Coxevac® vaccination.

To our knowledge, this is the first publication describing excretion of DNA from a completely inactivated bacterial vaccine in milk after inoculation. The DNA was vaccine-derived and detectable until 9 days after vaccination in quantities up to approximately 100 C. burnetii genome equivalents per mL, a more than 10 million fold dilution of the Coxevac® vaccine that contained 3.6 × 109 genome equivalents per mL. The limit of detection of the real time PCR assay which we used in this study has been published to be 4 genome equivalents per reaction mixture (Ct value 33.5 ± 1.2, mean ± SD; n = 20) [8]. The quantities of C. burnetii DNA that we measured after vaccination were around detection level (Ct values in between 33.2 and 36.4), which probably explains the zigzag pattern in Fig. 1. Still, we believe that our observations are valid, especially since genotyping confirmed that excreted DNA was vaccine-derived. Furthermore, the two goats that received no vaccination remained negative throughout the experiment, as did all the NCs and NTCs. In addition, diagnosis of human acute Q fever on serum samples by real time PCR is often based on Ct values that are also around detection level. Nevertheless, these real time PCR test results appear very reliable when considering seroconversion of C. burnetii antibodies in follow-up serum samples of these patients [8]. DNAlactia of C. burnetii DNA after the boost was shorter in duration, quantitatively lower and detectable in fewer animals compared to DNAlactia following the first vaccination. This can be well explained by the emergence of antibodies directed against C. burnetii from 14 days onward after Coxevac® vaccination [10]. To our knowledge there is only one study describing vaccinederived DNAlactia after Brucella abortus vaccination in water buffaloes [11]. The B. abortus rough mutant strain RB51, a live vaccine strain, could be cultured from milk samples during the first week post-vaccination. In addition, B. abortus DNA could be detected 1–4 weeks post-vaccination in adult water buffaloes. The viability of the B. abortus strain used for vaccination may well account for the relatively long shedding of DNA in milk compared to our observation with a non-viable vaccine. The experiment described here encompassed a small number of goats. We would like to emphasize that a larger study is required to evaluate the effect of mass vaccination on bulk tank milk C. burnetii DNA content. During mass vaccination, high-pressure inoculation pistols are used. This is in contrast to our experiment, where the vaccine was manually inoculated subcutaneously. The mode of administration may influence the uptake of the vaccine in bodily fluids, and hence the proportion of vaccine-derived DNA excreted in milk. In conclusion, this is the first report on excretion of DNA from a completely inactivated bacterial vaccine in milk after inoculation. Following vaccination of adult milk-producing goats with Coxevac® , vaccine-derived DNA could be detected in milk samples until 9 days after vaccination in quantities up to approximately 100 C. burnetii genome equivalents per mL. This observation had implications for the policy of the Dutch Ministry of Agriculture, Nature and Food Quality to combat the spread of the ongoing Q fever epidemic in the Netherlands. The strategy of simultaneous vaccination of dairy goats and bulk tank milk testing to identify contaminated

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farms was reconsidered and a 2-week interval between vaccination and bulk tank milk testing was imposed. Acknowledgements We cordially thank the goat farmers who organized the experiment and provided the milk samples as well as the veterinarians who performed the vaccinations. References [1] Enserink M. Infectious diseases Questions abound in Q-fever explosion in the Netherlands. Science 2010;327(5963):266–7. [2] http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19520 (consulted on November 22nd, 2010): van der Hoek W, Dijkstra F, Schimmer B, Schneeberger PM, Vellema P, Wijkmans C, et al. Q fever in the Netherlands: an update on the epidemiology and control measures. Euro Surveill 2010;15:pii=19520. [3] http://www.minlnv.nl/portal/page? pageid=116,1640363& dad=portal& schema=PORTAL&p news item id=24954 (consulted on November 22nd, 2010). All pregnant animals on infected farms to be culled. Press release 21-12-2009.

[4] Kim SG, Kim EH, Lafferty CJ, Dubovi E. Coxiella burnetii in bulk tank milk samples, United States. Emerg Infect Dis 2005;11(4):619–21. [5] http://www.ceva.com/en/Products/Cattle/Vaccines (consulted on November 22nd, 2010). [6] Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa NJ. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28(3):495–503. [7] van Doornum GJ, Guldemeester J, Osterhaus AD, Niesters HG. Diagnosing herpesvirus infections by real-time amplification and rapid culture. J Clin Microbiol 2003;41(2):576–80. [8] Schneeberger PM, Hermans MH, van Hannen EJ, Schellekens JJ, Leenders AC, Wever PC. Real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever. Clin Vaccine Immunol 2010;17(2):286–90. [9] Huijsmans CJ, Schellekens JA, Wever PC, Toman R, Savelkoul PH, Janse I, et al. Single-Nucleotide-Polymorphism Genotyping of Coxiella burnetii during a Q fever Outbreak in The Netherlands. Appl Environm Microbiol 2011; In Press. [10] Arricau-Bouvery N, Souriau A, Bodier C, Dufour P, Rousset E, Rodolakis A. Effect of vaccination with phase I and phase II Coxiella burnetii vaccines in pregnant goats. Vaccine 2005;23(35):4392–402. [11] Longo M, Mallardo K, Montagnaro S, De Martino L, Gallo S, Fusco G, et al. Shedding of Brucella abortus rough mutant strain RB51 in milk of water buffalo (Bubalus bubalis). Prev Vet Med 2009;90(1–2):113–8.