Background: Brucella abortus is a highly pathogenic zoonotic agent, tempting for the development of a rapid diagnostic method to enable adequate treatment and prevent further spread. Enrichment of the bacteria is often used as a first step in diagnostics to increase the bacterial number above the detection limit of the real-time PCR. The enrichment of Brucella spp. takes at least 3 days, which might be avoidable if sensitive PCR methods can be used. Since many matrices contain PCR inhibitors, the limit of detection (LOD) must be determined for each separate matrix. Another aim of this study was the determination of survival of Brucella abortus in the analyzed matrices. Methods: The LOD for the detection of B. abortus in 14 matrices, relevant for human medicine, veterinary medicine and food and feed safety, was determined to evaluate the need of a pre-enrichment step prior to real-time PCR. The survival of B. abortus in the spiked matrices was tested by plate count in a 7-day interval for 132 days. 3 4 Results: The limit of detection for B. abortus in most matrices was in the range of 10 –10 CFU/g for cultivation 4 5 and 10 –10 CFU/g for direct real-time PCR. The survival time of B. abortus was less than 21 days in apple purée and stomach content and 28 days in water while B. abortus remained viable at day 132 in milk, blood, spinach and minced meat. Conclusions: A direct PCR analysis without enrichment of bacteria saves at least 3 days. However, the limit of detection between direct PCR and plate count differs in a 10 fold range. We conclude that this lower sensitivity is acceptable in most cases especially if quick analysis are required. Keywords: Brucella abortus, Limit of detection, Diagnostics, Survival time Background usually transmitted in animals through semen, aborted Brucellosis is a bacterial disease that can affect many dif- embryos and discharge but also by inhalation and oral ferent animal species. The bacteria are 0.6 × 1 μm sized intake. Infected animals rarely show symptoms but dur- Gram-negative coccobacilli that may grow facultative ing pregnancy, fetuses may be aborted, which can also intracellularly. be followed by long-lasting vaginal discharge . Currently, there are 12 species described, most of Some Brucella species such as B. abortus have a high which are highly host specific. Brucella abortus occurs zoonotic potential and are a common source of human in- mainly in cattle while Brucella melitensis occurs in goat fection. Human brucellosis caused by B. abortus is called and Brucella canis in dogs. Brucella infections are Bang’s disease and is characterized by prolonged and re- current undulating fever that can last for many months or even years if not treated. The infection may persist in * Correspondence: email@example.com brain or bone tissue. The mortality rate is low, but effect- National Veterinary Institute, Uppsala, Sweden ive treatment is challenging and there is no prophylaxis. Swedish Forum for Biopreparedness Diagnostics, Umeå, Uppsala and Solna, Sweden The infectious dose is 10–100 bacteria for B. abortus. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kaden et al. BMC Infectious Diseases (2018) 18:259 Page 2 of 6 Depending on the temperature, B. abortus is able to sur- Thermo Fischer Scientific. No animal was euthanized vive up to 114 days in tap water . The most common for the purpose of this study. routes of infection for human brucellosis are inhalation, All the matrices were free from artificial preservatives. or via skin wounds and mucous membranes (e.g. in con- Each sample was spiked with 1 to 10 bacteria per ml. tact with infected animals, in slaughterhouses or in labora- For each matrix a non-spiked sample was used as nega- tory settings) and ingestion of contaminated food, tive control. Spiking was performed directly in case of li- primarily unpasteurized dairy products. The bacterium is quid matrices while 2 g of the solid matter was weighed fastidious, and may persist in the environment for pro- up in 50 ml falcon tubes, mixed with 18 ml physiological longed periods of time. Hence, it is of interest to have ro- NaCl and homogenized by vortexing with 10 glass beads bust methods of analysis for several kinds of matrices that with a diameter of 3 mm, prior to the addition of may harbor the bacterium, e.g. dairy products, feed and bacteria. A volume of 250 μl of the swab liquid and 0.4 g clinical animal samples. placenta were used for analysis, due to access to limited The focus of this study was the laboratory need for ro- sample amounts of these sample types. Cultivation and bust methods for analysis of samples relevant for human DNA extraction was done directly after spiking the medicine, veterinary medicine and food and feed safety. matrices. The aims were to determine the survival time of Brucella Cultivation was carried out on selective Farrell agar − 1 − 2 in several matrices and to evaluate analytical sensitivity as plates with dilutions of 10 and 10 of the samples limit of detection (LOD) for detection of Brucella in these that were spiked with 1 to 10 bacteria per 1 ml in matrices by selective plate cultivation and real-time PCR addition to the undiluted approach. A volume of 100 μl on direct extractions from the samples. of each matrix and each dilution was spread on Farrell agar. Due to sample consistency and size, placenta and Methods swab samples were incubated from an inoculation Bacterial strain and cultivation streak. Plates were incubated at 37 °C, in 10% CO for T T T B. abortus biovar 1 544 (ATCC 23448 , NCTC 10093 ) 4–5 days to allow for colony formation of B. abortus. were streaked from glycerol stocks (− 80 °C) onto Farrell agar [2, 3] and incubated at 37 °C with 10% CO for Molecular analysis 4 days. Based on plate count experiments, it was DNA was extracted from 200 μl samples using the EZ-1 determined that OD = 1 corresponds to approximately DNA tissue kit and the EZ-1 extraction robot (Qiagen). 5×10 colony forming units (CFU) per ml. The placental samples were first incubated with protein- ase K and G2-buffer (QIAGEN) at 56 °C for 15 min due Spiking of matrices to the high porosity of the material. All other samples Tenfold dilution series with a CFU ranging from 2 × 10 were processed without proteinase K treatment. Prior to to 2 × 10 were prepared in physiological NaCl (0.9%) extraction, 195 μl of each sample were mixed with 5 μl for spiking. The actual bacterial concentration was seal herpesvirus 1 virions (PhHV-1) to a final concentra- determined by plate count on Farrell agar [2, 3], and tion of 10 virions per ml, as an internal process control used as correction factors throughout this study. (IPC) . The matrices for the spiking experiments were: The PCR target sequence for Brucella was the IS711 intergenic spacer gene fragment present in all Brucella 1) Food: low pasteurized milk (3.8–4.5% fat), minced species . The genome of B. abortus has 7 copies of meat, wheat flour, spinach leaves, apple puree, tap IS711 of which one is truncated . As the other com- water, and ground white peppercorns. mon target, the 16S rDNA  only exist in B. abortus in 2) Feed: hay and samples of feed mill scrapings (FMS). 3 copies  the IS711-tageted real time PCR is expected 3) Clinical samples: bovine vaginal e-swab, defibrinated to be more sensitive than the corresponding 16S based sheep blood, bovine placenta, bovine semen, and method. For the IPC, a gB-polymerase gene fragment bovine stomach contents. was used as the target. The real-time PCR was performed as described pre- The samples were chosen based on a clinical, veterin- viously . Amplification was performed in the ABI ary and biosecurity perspective. Bovine semen and 7500 fast thermo cycler (Applied Biosystems) using bovine vaginal e-swab were remaining lab samples from 95°C initial denaturation for 5 min and 45 cycles of standard care of the animal obtained by veterinary 95°C denaturation for 15 s followed by 60°C amplifi- standard procedures from living individuals. Bovine pla- cation for 60 s. centa was taken after birth. Bovine stomach content was At least 3 no template controls (NTC) and 3 positive provided by the Lövsta slaughterhouse (Uppsala, controls (clean B. abortus DNA) were applied in Sweden). Defibrinated sheep blood was ordered from addition to the IPC to evaluate the real-time PCR. The Kaden et al. BMC Infectious Diseases (2018) 18:259 Page 3 of 6 real-time PCR data analysis was done using the ABI recovery rate was determined by plate count and real-time 7500 software version 2.0.6 with a manually selected PCR. threshold of 0.12. The real-time PCR was validated at Brucella abortus is a representative species of the the National Veterinary Institute (SVA) and at the Public genus Brucella due to the high occurrence and the Health Agency of Sweden (FOHM) (data not shown). impact that this organism causes worldwide. Thus, B. T T T Each spiking experiment was performed in triplicates abortus biovar 1 544 (ATCC 23448 , NCTC 10093 ) on three different days for statistical reliability. Addition- was used for the experiments of this study. ally, each sample was analyzed in duplicates in the real- B. abortus could be cultured from all different matri- time PCR. Furthermore, the experiments were per- ces. The recovery by cultivation one hour after spiking formed in 10-fold dilutions and thus the results of real- was highest in water and milk with more than 96 and time PCR and cultivation should represent the concen- 93% recovery respectively (Fig. 1). Milk is a natural trations of the dilution row in a logical 10-fold sequence. reservoir of many Brucella species  and B. abortus The determination of the LOD with real-time PCR persisted throughout the study for 132 days with a num- and culturing was performed according to the recom- ber of colony forming units (CFU) of 4 × 10 per ml, mendations of the Minimum Information for Publication which is almost the same CFU as at the beginning of the of Quantitative Real-Time PCR Experiments (MIQE) experiment. While the statistical valid LOD obtained guidelines  and the US food and drug administration with cultivation in milk (825 CFU/ml) was lower than (FDA) , respectively. This means for the evaluation the LOD obtained using real-time PCR on extractions of the results of this study, that the LOD was deter- made directly from the samples (8667 CFU/ml) (Fig. 2), the mined by the result in which 95% of the real-time PCR LLD of both methods was approximately 100 CFU/ml. samples gave a positive quantification cycle (cq) signal In tap water, which is the least complex of the studied and in which a number of 25 to 250 CFU was counted matrices, the recovery rate was almost 100% (Fig. 1) and on the agar plates. Furthermore, the recovery rate was the LOD was comparable with the LOD in milk (Fig. 2). determined in % where 100% is equal to the resulting The survival time of B. abortus in tap water was 28 days. concentration of bacteria spiked to the samples. The A rapid decrease of the CFU in water was observed in lowest limit of a possible single detection (LLD) was cal- the beginning of the experiment (Fig. 3). culated according eq. 1 for cultivation experiments and The recovery rate in stomach content from the abo- determined as single occurrence of one positive cq value masum with a naturally low pH was 85% but only 50% in qPCR independent on reproducibility and confidence in the clinical samples: blood, semen, and vaginal swab. level. The LLD is also applicable to estimate the minimal An even lower recovery rate with 10% was observed in required sample weight for a positive detection. spinach, hay, FMS, white pepper, wheat flour, and minced meat (Fig. 1). Load spiked load of bacteria on min… load min LLD ¼ the plate with the lowest number of detected bacteria; n grown bacteria load ¼ LOD if load > 25 min min Discussion While the general expected survival time of B. abortus Equation 1: Calculation of the lowest possible limit of in milk according to former reports is no longer than detection 87 days [13, 14] the bacteria survived 132 days in our study. The differences could be caused by different types Survival analyses of milk or different storage conditions within the Samples with a final concentration of 10 bacteria per g experiments. of matrix as described above were stored at 4°C. A The LLD obtained from cultured and uncultured milk weekly sampling with selective cultivation was samples was approximately 100 CFU/ml, which is in performed for 132 days. The samples were spread agreement with the results published by MER Hamdy − 1 − 2 undiluted and in a dilution of 1:10 and 1:10 , and AS Amin . However, a higher fat content of milk incubated on selective Farrell agar as described above, should correspond to a higher content of casein micells, and the number of CFU was determined according which are binding DNA. This phenomenon might FDA’s recommendations . explain the factor 10 between the culture-based and PCR-based LOD in milk samples. Results B. abortus survived in tap water for 28 days in our For the evaluation of a method for the detection of B. study. Falenski et al. determined a survival time of B. abortus and determination of the survival of the bacterium abortus in mineral water of 63 days . The tap water in food, feed and clinical samples, several matrices were used in our studies was treated with sodium hypochlor- spiked with various concentrations of bacteria. The ide, which probably caused the shorter survival time. Kaden et al. BMC Infectious Diseases (2018) 18:259 Page 4 of 6 Fig. 1 Recovery obtained by cultivation. Legend: Recovery of Brucella abortus from spiked samples; 100% is equal to the initial concentration of bacteria spiked to the samples The recovery rate from stomach content was 85%. occurring CFU in infected individuals, sufficient for Most Brucella infections occur orally and the most rele- diagnostics. vant source of human infections is unpasteurized milk. Hay and FMS may contain naturally occurring nano- Thus, B. abortus has probably evolutionally adapted to particles that commonly have charged edges or surfaces the low pH in the gastro-intestinal tract. Due to the high . Those particles are able to bind or affect the nega- recovery rate of the bacterium from stomach content, tively charged bacteria and nucleic acids . The high this source of infection should be recognized in health- LOD in hay and FMS obtained by real-time PCR analysis care, even if the risk is limited to individuals with acute supports this theory. The presence of bi- or multiva- intake of Brucella spp. as it may occur in Brucella-en- lent cations or humic substances enhances the demic countries. described effect due to the increased number of pos- The clinical samples: blood, semen, and vaginal swab sible binding sites. are known to contain substances that affect the recovery The highest standard deviation within the culture rate of bacteria. This also applies to placenta, which, in experiments was observed in blood and apple puree. addition, has a surface texture that increases the total The same batch of both matrices was used for all inde- surface. Thus, the LOD for placental samples was pendent experiments. Thus, the observed differences 8250 CFU/g which is high but, in terms of actually might be caused by inhomogeneity of the matrices and Fig. 2 LOD and LLD [cfu/g]. Legend: Limit of detection (LOD) and lowest possible limit of detection (LLD) of Brucella abortus in several matrices obtained by cultivation and real-time PCR Kaden et al. BMC Infectious Diseases (2018) 18:259 Page 5 of 6 Fig. 3 Survival of Brucella abortus. Legend: Survival of B. abortus in different matrices during the first 70 days of the study. B. abortus remained viable during days 71–132 in milk, blood, spinach and minced meat therefore corresponds with the expected variance of re- recommend using real-time PCR-LLD directly. In case sults in real samples. The composition of blood varies of a negative signal, we recommend the treatment of day by day even within the same individual which leads samples and cultivation of bacteria as described in the to the conclusion that it is challenging to create stan- methods section. dardized blood samples. Without this standardization Furthermore, we present the survival times of B. and with the presented standard deviation of our experi- abortus in several matrices, which was less than ments it should be discussed if blood containing media 21 days in apple puree and stomach content and or blood culture, which is a common method not only 28 days in water, while B. abortus survived for more in Brucella diagnostics, could be replaced by more stable than 132 days in milk, blood, spinach and minced standardized methods in the future. meat. Some spices and herbs of the present study are known Abbreviations to have an antimicrobial activity. Spinach contains the ATCC: American Type Culture Collection; CFU: Colony forming units; FDA: US antimicrobial peptides So-D1–7 that might contribute to food and drug administration; FMS: Feed mill scrapings; FOHM: Public Health Agency of Sweden; IS711: Intergenic spacer 711; LLD: Lowest possible limit the low recovery rate . The inhibitory effects of of detection; LOD: Limit of detection; MIQE: Minimum Information for white pepper on bacterial growth observed in this study Publication of qPCR Experiments; MSB: Swedish Civil Contingencies Agency; was also described by E Ceylan and DY Fung . NaCl: Sodium chloride; NCTC: National Collection of Type Cultures England; NTC: No template control; OD : Optical density at wavelength 600 nm; Wheat is known to contain inhibitory peptides, thionins PhHV-1: Phocine (seal) herpesvirus 1; qPCR: Quantitative polymerase chain . However, the specific effect of these inhibitory sub- reaction; SVA: Swedish National Veterinary Institute stances was not tested against Brucella. The results of Acknowledgements this study indicate an inhibitory effect of these matrices We thank Viveca Båverud, Hans Lindmark and Rickard Knutsson for all the on growth and survival of B. abortus but further investi- support during and after the project. This work was supported by the gations are necessary to confirm this observation. Swedish Civil Contingencies Agency (MSB). Funding Conclusions This work was supported by grants (Anslag 2:4 Krisberedskap) distributed by The evaluated method is time saving due to the direct the Swedish Civil Contingencies Agency (MSB). application of the real-time PCR without an enrichment Availability of data and materials of bacteria while detection of B. abortus by cultivation T T The bacterial strain B. abortus biovar 1 544 is available as ATCC 23448 and was in some cases more sensitive. The limit of detection NCTC 10093 from the corresponding strain collections. 3 4 was in a range of 10 –10 CFU/g for cultivation and The raw data generated and analyzed during the current study are not 4 5 publicly available due to restrictions in publishing data relevant for biosafety 10 –10 CFU/g for direct real-time PCR with exception to avoid misuse. Data are available from the corresponding author on of the higher LOD of FMS. The lowest possible limit of reasonable request. detection LLD of with real-time PCR was the most sen- sitive method and the only method to detect B. abortus Authors’ contributions RK wrote the manuscript and performed the spiking and survival in concentrations near the infection dose. Since LLD is experiments. SF planned and performed the lab experiments and evaluated based on a single positive signal the method is appropri- the results. TJ performed the spiking experiments and calculated and ate for a first screening. As a result of this study we evaluated the results. MLi did the molecular analyses and performed the Kaden et al. BMC Infectious Diseases (2018) 18:259 Page 6 of 6 spiking experiments. TW provided and adjusted molecular methods and 13. Falenski A, Mayer-Scholl A, Filter M, Göllner C, Appel B, Nöckler K. Survival of provided the Brucella strain. MLa planned and performed the lab Brucella spp. in mineral water, milk and yogurt. Int J Food Microbiol. 2011; experiments and wrote parts of the manuscript. All authors have read and 145(1):326–30. approved the manuscript. 14. Mitscherlich PE, Marth EH. Microbial survival in the environment - Bacteria and Rickettsiae important in human and animal health. Berlin-Heidelberg- New York-Tokyo: Springer Publishing House; 1984. Ethics approval 15. Hamdy MER, Amin AS. Detection of Brucella species in the milk of infected All the samples were taken for this study according to EU directive 2010/63/ cattle, sheep, goats and camels by PCR. Vet J. 2002;163(3):299–305. EU on the protection of animals used for scientific purposes. Bovine semen 16. Kaden R, Menger-Krug E, Emmerich K, Petrick K, Mühling M, Krolla- and bovine vaginal e-swab were remaining lab samples from standard care Sidenstein P. The dynamic cultivation system: a new method for the of the animal obtained by veterinary standard procedures from living ani- detection of temporal shifts in microbial community structure in clay. Appl mals. Bovine placenta was taken after a common birth. This requires no eth- Clay Sci. 2012;65–66:53–6. ical approval. Stomach content was provided by the Lövsta slaughterhouse 17. Takada-Hoshino Y, Matsumoto N. An improved DNA extraction method in Uppsala, Sweden from a cow which was euthanized for food production using skim milk from soils that strongly adsorb DNA. Microbes Environ. only. Defibrinated sheep blood was obtained from Thermo Fischer Scientific. 2004;19(1):13–9. No animal was euthanized for the purpose of this study. 18. Segura A, Moreno M, Molina A, García-Olmedo F. Novel defensin subfamily from spinach Spinacia oleracea. FEBS Lett. 1998;435(2):159–62. Competing interests 19. Ceylan E, Fung DY. Antimicrobial activity of species 1. J Rapid Methods The authors declare that they have no competing interests Autom Microbiol. 2004;12(1):1–55. 20. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev. 1999;12(4):564–82. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 2 National Veterinary Institute, Uppsala, Sweden. Swedish Forum for Biopreparedness Diagnostics, Umeå, Uppsala and Solna, Sweden. Swedish Joint Laboratory for Food Safety and Biopreparedness, Uppsala, Sweden. Department of Medical Sciences, Uppsala University, Uppsala, Sweden. 5 6 National Food Agency, Uppsala, Sweden. Public Health Agency of Sweden, Solna, Sweden. Received: 21 November 2017 Accepted: 7 May 2018 References 1. Corbel MJ. Brucellosis in humans and animals. Geneva: World Health Organization Publications; 2006. 2. Farrell ID. 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Published: Jun 5, 2018
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