Molecular characterization of Brucella species from Zimbabwe

Maphuti Betty Ledwaba; Calvin Gomo; Kgaugelo Edward Lekota; Philippe Le Flèche; Ayesha Hassim; Gilles Vergnaud; Henriette van Heerden

doi:10.1371/journal.pntd.0007311

Abstract

Brucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world.

Author summary

Brucellosis is endemic in Zimbabwe. This article describes the use of various assays such as AMOS, Bruce-ladder, MLVA, and whole genome sequencing to characterize Brucella species isolated from different animals in Zimbabwe. Choice of which assays to use in the laboratory is generally done considering reproducibility, robustness, expertise and affordability in a given setting. As evidenced in this study, most laboratories in Africa lack resources especially finances, equipments and expertise to perform necessary tests for diagnosis and identification of specific pathogens. The study shows that the differentiation of species can be correctly concluded from the analysis with AMOS, Bruce-ladder and MLVA16 assays. Furthermore, MLVA16 can be used as an epidemiological tool and traceback of outbreaks. These PCR assays can therefore add to the control and eradication of brucellosis, since the Brucella species (B. ovis, B. abortus, B. suis and B. canis) existing in Zimbabwe could be identified and characterized.

Citation: Ledwaba MB, Gomo C, Lekota KE, Le Flèche P, Hassim A, Vergnaud G, et al. (2019) Molecular characterization of Brucella species from Zimbabwe. PLoS Negl Trop Dis 13(5): e0007311. https://doi.org/10.1371/journal.pntd.0007311

Editor: Ana LTO Nascimento, Instituto Butantan, BRAZIL

Received: July 29, 2018; Accepted: March 18, 2019; Published: May 20, 2019

Copyright: © 2019 Ledwaba et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported financially by the ITM Belgium and National Research Foundation (NRF South Africa). Work by PLF and GV is supported by the ANR project Microtype-14-ASMA-0002-02. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Brucellosis is a worldwide infectious disease affecting a wide range of domestic and wildlife animals and humans [1]. Brucellosis is caused by species in the genus Brucella, which consists of six classic species, Brucella abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae [2]. Recently, the genus has expanded to include B. ceti and B. pinnipedialis from marine animals [3], B. microti from the common voles (Microtus arvalis) [4] and red foxes [5], B. inopinata isolated from a human breast implant [6], B. papionis from baboons (Papio spp.), B. vulpis from red foxes (Vulpes vulpes) and novel Brucella spp. in amphibians and fish [7, 8, 9, 10].

In African countries, brucellosis is reported to be a serious threat; although, under-reported due to a limited number of studies conducted and the lack of epidemiological evidence [11]. B. abortus and B. melitensis have been reported frequently when livestock have been tested. However, there is limited information on the prevalence of brucellosis in small ruminants as compared to cattle [12]. In Zimbabwe, only B. abortus and B. melitensis have been reported to cause brucellosis in animals [13], with B. abortus biovar (bv.) 1 and to a lesser extent B. abortus bv. 2 reported to be the most prominent cause of bovine brucellosis [13]. However, reports might be biased because investigations/ testing targeted mostly bovine rather than other species. The authors used biotyping and AMOS-PCR to identify Brucella isolates from commercial and communal cattle farms in Zimbabwe and also reported a single B. melitensis bv. 1 isolate from a goat. Brucellosis has been demonstrated by serology to be present in Zimbabwean wildlife including African buffalo, eland, zebra, giraffe and impala [14] as well as in domestic dogs [15]. B. abortus bv. 1 was isolated from waterbuck (Kobus ellipsiprymnus) and eland (Taurotragus oryx) [16]. This complicates the control of the disease since the animals in areas bordering the National parks interact with wildlife and it is almost impossible to vaccinate wildlife. Bovine brucellosis is endemic in the country in most regions with high sero-prevalence of up to 53% reported in commercial herds as compared to 16% from small-scale farmers in Zimbabwe [17, 18].

General classification of Brucella species and biovars is still based on phenotypic characteristics, with minimal standards previously defined [19] well before the development of modern genomics and the discovery of new Brucella species. Biotyping is time-consuming and often difficult to interpret due to limited standardization of the typing reagents [20]. Moreover, the efficacy of biotyping is moderate and since it includes the manipulation of the live agent, it poses a biosafety and public health risks of laboratory infections to the personnel involved [20]. Initial assays based on DNA analysis by PCR amplification were genus-specific and not sufficient to assist brucellosis control programs [21, 22] in the endemic regions of Zimbabwe. Most programs for brucellosis control employ genus-specific serology tests which are confirmed by species-specific culturing since the associated regulatory methods are species dependent [23]. AMOS-PCR is a multiplex PCR assay that differentiates B. abortus bv. 1, 2 and 4, B. melitensis, B. ovis, B. suis bv. 1, B. abortus vaccine strains S19 and RB51 based on the genetic element IS711 [24, 25]. AMOS and Bruce-ladder multiplex PCRs use species- and strain-specific genetic differences to distinguish among Brucella species [26, 27]. The initial Bruce-ladder assay identifies almost all Brucella species including the vaccine strains B. abortus S19, RB51 and B. melitensis Rev1 but will occasionally incorrectly identify some B. canis strains as B. suis [27]. The original Bruce-ladder assay had limited utility for distinguishing the more recently described species such as B. ceti, B. pinnipedialis, B. microti, and B. inopinata; but was later updated [28; 29, 30]. Multi-locus variable number tandem repeats (VNTR) assays (MLVA) is a genetic approach with high discriminatory power in the Brucella genus, clearly identifying species and providing fine-scale resolution among isolates [31, 32]. The most commonly used MLVA scheme consists of 16 VNTR markers, including eight moderately variable minisatellites (panel 1) and eight highly polymorphic microsatellites (panel 2A and 2B) [20, 33] that has the capacity to distinguish Brucella species and their biovars. Accurate discrimination between species and biovars achieved with the high resolution MLVA is necessary to determine the source, origin and geographical spread of infection [34]. Finally, characterisation of the genome of Brucella species with whole genome sequencing (WGS) provides the ultimate genetic resolution and can enable the determination of other features such as virulence factors [35]. The availability of whole genome sequences covering B. melitensis [36], B. suis [37], and B. abortus [38] has contributed to our understanding of the pathogenicity and diagnosis of brucellosis [35, 36]. WGS combined with single nucleotide polymorphism (SNP) analysis provides greater resolution and fine-scale differentiation of Brucella species [39, 40, 41] that cannot be obtained with multiplex PCR assays or MLVA.

Brucella abortus bv.1 is the most frequently isolated species in the cattle industry in Zimbabwe, with B. abortus bv.2 occasionally detected [13]. However, the control program is compulsory for commercial farming but is only optional for communal cattle production systems so may be missing most cases of brucellosis [14]. Various strains have been isolated from samples collected between 1990 and 2009 from various host animals throughout the country at the Central Veterinary Laboratory (CVL) in Zimbabwe, with only some of the isolates previously identified to the species level using biotyping. The aim of this study was to characterize these Brucella strains using AMOS-PCR, Bruce-ladder and MLVA, to evaluate genotyping approaches and develop a toolkit to support a nation-wide eradication program at a sustainable cost. Finally, based on the data obtained with the abovementioned techniques, three isolates were further characterized with WGS [42].

Methods

Ethics statement

All experimental protocols were approved by the Animal Experiments and Ethics Committee of the University of Pretoria (V096-15 AEC Approval) and the Section 20 approval obtained from DAFF (SDAH-Epidem 15012613530_ Section 20) for the use of animals and animal products.

Bacterial strains and bacteriology

Sixteen Brucella strains (Table 1) were isolated at CVL from samples of domestic animals collected and isolated isolated between 1990 and 2009 in Zimbabwe and used to evaluate the feasibility and the need of large scale surveillance in the country. At the time of the study (2011–2013) there was no surveillance going on, the study isolates were obtained from farms/clients samples submitted to CVL for routine screening. They were characterized as Brucella by bacteriological methods (urease, catalase, oxidase, H₂S, indole and sensitivity to dyes (thionin and basic fuchsin)) as indicated by previously [43]. Due to financial constraints, it was not possible for the laboratory to buy PCR reagents at that time; thus, only 7 of the 16 cultures were further classified to species level with the available reagents (S1 Table) according to standard bacteriological methods (excluding the phage lysis test) [43].

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Table 1. List of field strains isolated from samples collected and isolated from 1990–2009 in Zimbabwe and the reference strains used in the study as well as their spp. identity, hosts and place of origin.

https://doi.org/10.1371/journal.pntd.0007311.t001

DNAs from 17 reference strains obtained from National and OIE/FAO Animal Brucellosis Reference Laboratory in France were included as controls for PCR assays (Table 1). Genotyping information of Brucella strains from previous studies [20, 31, 32, 44] that were used in MLVA in this study can be accessed from MLVA database [45].

DNA preparation

DNA was extracted from each strain grown on Brucella selective media and blood agar using Qiagen DNA mini kit (Qiagen) at CVL in Zimbabwe and quantified with BioTek Take3 Micro-Volume Plate used in BioTek Microplate reader using the Gen5 pre-programmed quantification protocol at the University of Pretoria, South Africa. The study controls were amplified with Genomiphi DNA Amplification Kit (GE Healthcare Life Sciences AEC-Amersham) to increase their quantity.

AMOS-PCR and Bruce-ladder

AMOS-PCR was done as described previously [25, 26]. The PCR mixture contained 1X MyTaq mix (Bioline), a combination of five primer sets specific for B. abortus, B. melitensis, B. ovis, B. suis (0.2 μM) and IS711 (1 μM), respectively, and 10 ng DNA per 25 μl reaction. The PCR conditions consisted of an initial denaturation at 95°C for three minutes followed by 35 cycles of 95°C for one minute, 55.5°C for two minutes and 72°C for two minutes.

Bruce-ladder PCR was also done as described previously [27]. PCR reactions (25 μl) composed of 1X MyTaq mix (Bioline), 0.4μM of each primer of the eight primer pairs and 10ng template DNA. PCR conditions consisted of initial denaturation at 95°C for three minutes, followed by 25 cycles at 95°C for 30 sec, 64°C for 45 sec and 72°C for three minutes and a final extension of 72°C for five minutes on an ABI 2720 Thermal Cycler (Applied Biosystems).

To confirm the identity of strains identified as B. suis and B. canis with Bruce-ladder, the previously described Suis-ladder multiplex PCR assay [46] was used.

PCR products were separated by gel electrophoresis on a 1.5% agarose gel subsequently stained with ethidium bromide and photographed under UV light.

MLVA

MLVA16 was performed as previously described [20, 31]. The 16 locus set was divided in three groups namely panel 1 (bruce06, bruce08, bruce11, bruce12, bruce42, bruce43, bruce45 and bruce55), panel 2A (bruce18, bruce19, bruce21) and panel 2B (bruce04, bruce07, bruce09, bruce16 and bruce30). PCR was performed in 15 μl reactions containing 3–15 ng of DNA template, 1X PCR buffer (Promega), 200 μM of each deoxynucleotide triphosphate, 0.5 μM of each flanking primer [20,31] and 1U GoTaq Hotstart polymerase (Promega). The PCR conditions included an initial denaturation step of 96°C for five minutes, followed by 30 cycles of 96°C for 30 seconds, 60°C for 30 seconds, extension at 72°C for one minute, followed by a final extension step of 72°C for 5 minutes. The PCR reaction products (5 μl) were separated on agarose gels in 1x TAE buffer using electrophoresis until the bromophenol blue has run for 20 cm on the agarose gel. The 16M B. melitensis reference strain was included as a control since each VNTR locus size is known. Brucella reference strains that have already been characterized using the MLVA16 markers panel 1, panel 2A and panel 2B were included to ensure accurate evaluation of field strain genotypes. For Panel 1 VNTRs, 2% agarose gel was used with GeneRuler 100 bp plus DNA ladder (Thermo Scientific). For panel 2 VNTRs, 3% standard agarose gel and low molecular weight DNA ladder 766–25 bp (New England Biolabs) were used. The ethidium bromide stained gels were visualized by UV light. Genotype was scored by visual analysis of the gel images or BioNumerics software version 6.6 (Applied-Maths).

Data analysis

Band size estimates were converted to repeat units following the published allele numbering system version 3.6 [45] (S1 Table). MLVA data were analysed as a character data set within BioNumerics software (version 6.6) (Applied Maths). Clustering analysis was performed using the categorical coefficient and UPGMA (unweighted pair group method using arithmetic averages). A different weight was given to the markers depending on their panel: Panel 1 markers were assigned an individual weight of 2 (total weight for panel 1: 16), panel 2A markers a weight of 1 (total weight for panel 2A: 3), and markers of panel 2B a weight of 0.2 (total weight for panel 2B: 1) [20]. The MLVA16 results were compared with MLVA16 published data of Brucella reference and other strains [20, 31, 44] (S1 Table). Minimum spanning tree (MST) analysis was performed using MLVA8 (panel 1) in BioNumerics as well.

Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses

Zimbabwean B. suis strains ZW043 (GenBank accession CP009094.1 and CP009095.1) and ZW046 (GenBank accession CP009096.1 and CP009097.1) and B. abortus strain ZW053 (GenBank CP009098.1 and CP009099.1) [47] were selected for WGS since these strains were isolated from cattle in different regions of Zimbabwe and represented different MLVA genotype subclades. In addition, 23 B. abortus and 17 B. suis complete genomes were retrieved from GenBank and used for comparison and phylogenetic analyses (S2 Table). Sequenced reads from B. abortus and B. suis strains were aligned to B. abortus str. 9–941 (Accession no: NC_006932.1, NC_006933.1) and B. suis 1330 (Accession no: NC_017251, NC_017250) respectively, using Burrows-Wheeler Aligner (BWA) [48]. SAMtools [49] was used to sort and index the aligned reads of Brucella genomes. Sequence reads of the complete and draft Brucella genomes were simulated using SAMtools [49]. Picard-tools (http://picard.sourceforge.net/) were used to mark duplicate reads and to build binary index of the samples. Repeated regions of the Brucella sequenced reads were excluded from this analysis. For variant detection, Unified Genotyper method in GATK [50] was used to call for SNPs. Variant filtration and selection of SNPs was achieved using GATK. SNPs positioning sets were deducted from the aligned genomes using molecular evolutionary genetics analysis (MEGA) tool version 6 [51]. Only SNP positions that could be called in all genome sequences were used (core genome analysis) for phylogenetic analysis. A phylogenetic tree was constructed using (MEGA) tool version 6 [51] from the coreSNPs of the Brucella genomes. The trees were generated using maximum likelihood method with 500 bootstrap replicates.

Results

Bacteriology

All Brucella spp. strains from Zimbabwe were non-motile, gram-negative coccobacilli, positive for modified Ziehl-Neelsen stain, negative for indole production, and oxidase and catalase production positive. Only a few of the strains (S1 Table) were further characterized using growth characteristics and biochemical profiles (phage lysis was not determined).

AMOS-PCR and Bruce-ladder

All strains except ZW100 and ZW377 were successfully genotyped using AMOS-PCR (Fig 1A). ZW002 and ZW005 were identified as B. ovis, ZW011, ZW040, ZW043, ZW045-048, ZW201 as B. suis and ZW053, ZW248, ZW283 and ZW323 as B. abortus (Fig 1A and Table 2).

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Fig 1.

Multiplex PCR assays (A) AMOS-PCR and (B) Bruce-ladder differentiation of Zimbabwean and reference Brucella strains. Lane 2–10: B. suis bv. 1 (1330, BCCN R12), ZW011, ZW040, ZW043, ZW045, ZW046, ZW047, ZW048, ZW201; lane 11–13: B. canis (RM 6/66, BCCN R17), ZW100, ZW377; lane 14–15: B. melitensis bv. 1 (16M, BCCN R1), B. melitensis rev 1; lane 16–23: B. abortus bv. 1 (544, BCCN R4), ZW053, ZW086, ZW248, ZW283, ZW323, B. abortus RB51, B. abortus S19; lane 24 negative (water) control; lane 1 Fermentas 100 bp marker plus.

https://doi.org/10.1371/journal.pntd.0007311.g001

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Table 2. Summary table indicating the results obtained with AMOS, Bruce-ladder and MLVA testing the isolates from Zimbabwe.

https://doi.org/10.1371/journal.pntd.0007311.t002

Bruce-ladder gave identical results (Fig 1B and Table 2) as AMOS-PCR and in addition could identify strains ZW100 and ZW377 as B. canis. Using the complementary Suis-ladder multiplex PCR [46], both strains were confirmed to be B. canis and strains ZW011, ZW40, ZW043, ZW045-048, ZW201 were confirmed as B. suis bv. 1 (S1 Fig).

MLVA

Due to lack of sufficient DNA, strains ZW002 and ZW005 (both B. ovis), and ZW248 and ZW283 (both B. abortus) could not be genotyped using MLVA16. MLVA data derived from the seven reference strains were as expected from previously published data, with the exception of reference strains 16M, 63/290 and RM 6/66. The 16M strain used in the present study differs from the B. melitensis reference 16M strain at locus Bruce07, which is not unexpected due to high variability at this locus [25]. The reference strain RM 6/66 we used differed from B. canis reference RM 6/66 strain at Bruce07, Bruce09 and Bruce16 loci whereas 63/290 differs at loci Bruce09 and Bruce16. The difference between control strains used in this study and the reference strains may be due to amplification of the reference DNA using Genomiphi (GE Healthcare Life Sciences AEC-Amersham) due to low quantities of DNA from these strains available in our study.

The Zimbabwean strains consisted of eight MLVA16 genotypes and clustered into three groups when analyzed together with MLVA data from [45]. All eight B. suis bv. 1 strains (ZW011, 040, 043, 045, 046, 047, 048 and 201) belong to MLVA8 genotype 6 like the vast majority of B. suis bv. 1 strains in the MLVA bank and are most closely related to B. suis bv. 1 reference strain 1330 in the B. suis bv. 1, 3, 4 / B. canis MLVA cluster (Fig 2). ZW100 and ZW377 (both isolated from dogs in Harare) formed a sub-cluster with B. canis REF RM 6/66 in the B. suis bv. 1, 3, 4 / B. canis cluster (Fig 2). B. abortus strain ZW323 (MLVA8 genotype 28) was identical at all 16 VNTR loci to B. abortus bv. 1 strain (LNIV-416Ba1-07) from Portugal [44] while B. abortus bv. 1 ZW053 strain also belonged to MLVA8 genotype 28. The clustering obtained with the Minimum Spanning Tree (MST) analysis is similar to the UPGMA clustering (S2 Fig).

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Fig 2. UPGMA algorithm cluster analysis of 54 Brucella strains (published data and Zimbabwean (ZW numbers)) using MLVA16.

The dendrogram is based on 47 genotypes obtained from 54 strains. The color code reflects the grouping of Brucella species using minimal spanning tree of MLVA8 (S1 Fig) with white color-coded Zimbabwean Brucella strains, brown B. canis and B. suis bv. 3 and 4, red B. suis bv. 1, 2 and 5, yellow B. ovis, dark blue B. melitensis, green B. abortus and pink B. neotomae. The last three columns indicate MLVA8 (panel 1), MLVA11 (panel 1 & 2A) and MLVA16 (panel 1; 2A & 2B) genotype identification.

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Whole genome sequence and WGS-SNP analysis

WGS-SNP phylogenetic analysis of 19 B. suis and 24 B. abortus genomes was defined by 7104 and 4549 core SNPs respectively. Phylogenetic analysis of the Brucella genomes showed that B. abortus ZW053 clustered in B. abortus bv. 1 and 2 clade alongside B. abortus bv.2 86/8/59, while B. suis ZW043 and ZW046 strains are grouped within the B. suis bv. 1 clade (Fig 3). Comparative SNP analysis between ZW053 and B. abortus bv. 2 str. 86/8/59 resulted in 35 SNPs as compared to 90 SNPs obtained when comparing the strain with B. abortus bv.1 str 9–941.

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Fig 3.

Phylogenetic trees of (A) Brucella suis and (B) B. abortus using SNPs from whole genomes. Dendrograms were generated using maximum likelihood with 500 bootstrap replicates, using 7104 and 4549 SNPs of B. suis and B. abortus genomes respectively.

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Discussion

Fast and accurate diagnosis of brucellosis is important for control programs [23] and since the choice of the assay to use depends on the affordability and availability of expertise in a given country, it is always a trade-off between the two requirements. Eradication and control program based on compulsory calf vaccination with B. abortus strain S19 was introduced in Zimbabwe in the early 1980s, but only to commercial farms and was voluntary to communal ones [17, 18]. However, infections caused by B. abortus and B. melitensis have been reported from both the communal and the commercial areas of Zimbabwe [13, 14, 15]. PCR-based assays can be used as a supplement or even a replacement to biotyping for the identification of Brucella species and/or biovars [23, 25], as genotyping is often essential for accurate epidemiological inference. Biotyping is time consuming, labour intensive and requires good expertise specific for this pathogen. In addition, it involves handling of live cultures that poses risks of laboratory exposure and infection [52]. The purpose of the study was to explore the practical suitability of PCR assays (MLVA, AMOS-PCR and Bruce-ladder) for laboratories that do not have biotyping capabilities as was the case with CVL, Zimbabwe at the time of the identification of these Brucella strains isolated from cattle, pigs, dogs and sheep. B. abortus and B. melitensis are the most prominent species in Africa and were previously reported in Zimbabwe from livestock and wildlife [13, 14]. The occurrence of these species in wildlife complicates the control of bovine brucellosis since it is almost impossible to vaccinate wildlife. Furthermore, interaction between wild life and animals in areas bordering the National parks could result in possible transmission of the disease.

In the present report, eight strains including five isolated from bovine and two from pigs were identified as B. suis bv. 1. The strains were identified as B. suis using AMOS-PCR [24, 25] and Bruce-ladder [26]. Suis-ladder [43] and MLVA identified these isolates to be B. suis bv. 1. Four strains isolated from cattle were identified as B. abortus bv.1 with AMOS-PCR and Bruce-ladder in this study. The identification could be confirmed by MLVA in two cases only due to limited DNA availability. In a previous study [13], B. abortus bv. 1 was shown to be the main cause of bovine brucellosis in Zimbabwe; however, in this study B. suis bv. 1 was most frequently isolated strain even from cattle. The isolation of B. suis bv.1 from both pigs and cattle might be the result of either mixed farming or the interaction of animal species in the grazing areas and drinking points. MLVA, Bruce-ladder and Suis-ladder assays identified two strains ZW100 and ZW377 as B. canis. This is the first report of B. canis in Zimbabwe. Due to low quantity of DNA, two B. ovis strains (ZW002 and ZW005) were only identified with AMOS and Bruce-ladder PCR but not with MLVA. Brucella ovis has been indicated by OIE reports as present in Zimbabwe [19].

Two B. suis bv. 1 strains isolated from cattle were selected for draft whole genome sequencing since B. suis had not been reported from pigs in Zimbabwe in literature but was detected in samples from both cattle and pigs in this study. WGS indicated that the two strains are separated from B. suis bv. 1 reference strain 1330 [37] by only five SNPs. A third strain, identified as B. abortus bv.1 was shown by WGS-SNP analysis to be closest to a strain independently recovered from Zimbabwe.

A previous study [24] compared the AMOS, Bruce-ladder and MLVA8 assays for typing of Brucella species and found only Bruce-ladder correctly identified all tested Brucella strains as MLVA8 does not resolve the very closely related B. canis and B. suis bv. 4. Both MLVA11 and MLVA16 resolve the two species however and also allows comparison to a worldwide Brucella MLVA dataset [53]. As shown in previous studies [39, 40, 41], WGS-SNP analysis provides better resolution than MLVA16, and much stronger phylogenetic support although there are still fewer strains from more limited geographic areas available for comparisons as compared to the MLVA database. Importantly the number of public whole genome sequences, particularly sequence reads archives, is rapidly growing with already more than 1000 datasets available.

The status of B. suis as a single species has been questioned in light of a broader host specificity [54]. Isolation of B. suis bv. 1 from bovines in Zimbabwe was first reported in 2014 [47]. The present study further emphasizes the occurrence of B. suis bv. 1 in cattle and pigs. There are several reports of isolation of B. suis bv. 1 from cattle [55, 56] in which the infection appears to be noncontagious with limited induced pathology and no induction of abortions [19, 54]. The presence of B. suis bv. 1 in pigs and bovines in Zimbabwe could be due to the predominance of smallholdings with mixed populations of livestock [57]. Therefore, the use of multiplex PCR assays that will distinguish the four species (B. ovis, B. abortus, B. suis and B. canis) present in Zimbabwe as confirmatory test will strengthen the control programs since most serology assays are based on smooth lipopolysaccharides (LPS) which cannot detect B. ovis and B. canis as they are rough strains.

WGS analysis showed that the ZW053 strain from a bovine in Zimbabwe [47] has large insertions and deletions as described in other B. abortus genomes [38, 58]. In spite of the variations observed in the genome sequences (S2 Table), whole-genome sequencing of the three strains and their comparison to reference genomes indicate that the isolates were B. suis (ZW043 and ZW046) and B. abortus (ZW053) respectively, thus corresponding with the data obtained with the Bruce-ladder, AMOS, Suis-ladder and MLVA PCR assays. Isolates from sub-Saharan countries and those from Europe have been shown to respectively cluster together, although heterogeneity within these species especially B. abortus do exist [12, 59]. This was also the case with ZW053 as it grouped with a Portuguese strain, and we hypothesize that this might be the result of socio-economic, migration or colonization links among Zimbabwe, Mozambique and Portugal or more generally European countries. Clustering of B. abortus bv. 2 strain 86/8/59 within biovar 1 and 2 clade and alongside ZW053 (Fig 3) and other B. abortus in WGS-SNP analysis was also shown in a previous study [41] that indicated that it might either be due to the paraphyletic nature of the biovar 1, 2 and 4 clade and the biovar classification not consistently reflecting genetic relationship in this species and/or that the biochemical biotyping to biovar level is unreliable. The present results further indicate the usefulness of MLVA and WGS-SNP in support of disease control. However, to perform the abovementioned assays requires a purified DNA template which may prove difficult to obtain due to the difficulty of culturing Brucella. Furthermore, brucellosis is endemic in sub-Saharan countries including Zimbabwe thus, the use of affordable high-throughput assays is necessary. More importantly, tests that can detect all the species that exit in a specific country should be considered.

Since most laboratories in Africa lack resources and expertise to do biotyping of Brucella to the species level, PCR assays like Bruce-ladder, AMOS and MLVA can contribute to the identification and can furthermore be used as an epidemiological tool and traceback of outbreaks. However, the choice of assays should be made considering reproducibility, robustness, expertise and affordability in a given setting and in most cases this choice will be a compromise. Brucellosis control programs in most countries are based on serological tests which includes Rose Bengal test (RBT), milk ring test, (MRT), complement fixation test (CFT), enzyme-linked immunosorbent assay (ELISA), the fluorescence polarisation assay (FPA) etc. [60]. These tests have varying sensitivity and specificity and they are prone to cross-reactions with other bacteria that have the smooth lipopolysaccharide used as the antigen in these assays [60]. Therefore, to complement these limitations, molecular assays can be used since most of them are robust, less expensive and can differentiate between Brucella spp. at genus, species and biovar levels [25, 26, 27, 28, 33, 44]. The development of standardised, safe and efficient DNA extraction procedures sufficient to produce a few micrograms of DNA of a good quality allowing long term conservation will be essential for this purpose.

Bruce-ladder and AMOS assays are species-specific simple and robust multiplex PCRs. Even though the initial AMOS PCR assay was more limiting as it has the capability of detecting only B. abortus bv 1, 2 and 4, B. melitensis bv. 1 and B. suis but not B. canis; it was subsequently enhanced and currently can detect B. abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3 as well [61]. Furthermore, its subsequent use alongside Bruce-ladder is also an advantage. Moreover, a previous study [13] in which AMOS PCR assay was used, also indicated the presence of brucellosis in Zimbabwe with infections mainly caused by B. abortus bv. 1 (84.6%) and B. abortus bv. 2 (15.4%). The MLVA16 assay provides a clustering of strains that is in accordance with all currently recognized Brucella species and biovars [11, 32, 43].

Considering affordability and reproducibility; Bruce-ladder can be used as it allows identification of all known Brucella species including the vaccine strains simultaneously in one run. This study has confirmed that species differentiation can be correctly deduced from both MLVA16 and Bruce-ladder analysis. These PCR assays can therefore add to the control and eradication of brucellosis, since B. ovis, B. abortus, B. suis and B. canis could be identified. The latter two species are reported for the first time in Zimbabwe. Additionally, more strains, whole genome sequences, and epidemiological data from Zimbabwe are needed to accurately draw conclusions on the clustering and circulation of strains.

Supporting information

S1 Table. Reference strains and Zimbabwean Brucella spp. isolates identified by Bruce-ladder and repeat copy number of the indicated loci.

https://doi.org/10.1371/journal.pntd.0007311.s001

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S2 Table. Brucella abortus and B. suis genome sequences retrieved from GenBank, used in the study for comparison of whole genome single nucleotide polymorphisms (WGS-SNPS) phylogenetic analysis.

https://doi.org/10.1371/journal.pntd.0007311.s002

(PDF)

S1 Fig. Suis-ladder multiplex PCR assay of Brucella DNA from Zimbabwe and reference strains.

https://doi.org/10.1371/journal.pntd.0007311.s003

(TIF)

S2 Fig. Minimum spanning tree analysis of published data and Zimbabwean Brucella isolates using the MLVA8 data (Panel 1 genotypes).

https://doi.org/10.1371/journal.pntd.0007311.s004

(TIF)

Acknowledgments

Special appreciation to bacteriology staff from CVL Harare, particularly Musari Shuvai for helping in bacteriology typing of strains.

References

1. Godfroid J. Brucellosis in wildlife. Rev Sci Tech OIE. 2002;21: 277–86.
- View Article
- Google Scholar
2. Osterman B, Moriyón I. International Committee on Systematics of Prokaryotes. Subcommittee on the taxonomy of Brucella. Report of the meeting, 17 September 2003, Pamplona, Spain. Int J Syst Evol Micro. 2006;56: 1173–5.
- View Article
- Google Scholar
3. Foster G, Osterman BS, Godfroid J, Jacques I, Cloeckaert A. Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. Int J Syst Evol Micro. 2007;57: 2688–93.
- View Article
- Google Scholar
4. Scholz HC, Hubalek Z, Nesvadbova J, Tomaso H, Vergnaud G, Le Flèche P, et al. Isolation of Brucella microti from soil. Emerg Infect Dis. 2008;14: 1316–7. pmid:18680668
- View Article
- PubMed/NCBI
- Google Scholar
5. Scholz HC, Hubalek Z, Sedlácek I, Vergnaud G, Tomaso H, Al Dahouk S, et al. Brucella microti spp. nov. isolated from the common vole Microtus arvalis. Int J Syst Evol Micro. 2008;58: 375–82.
- View Article
- Google Scholar
6. Scholz HC, Nöckler K, Gollner C, Bahn P, Vergnaud G, Tomaso H, et al. Brucella inopinata spp. nov., isolated from a breast implant infection. Int J Syst Evol Micro. 2010;60: 801–8.
- View Article
- Google Scholar
7. Whatmore AM, Davison N, Cloeckaert A, Al Dahouk S, Zygmunt MS, Brew SD, et al. Brucella papionis spp. nov., isolated from baboons (Papio spp.). Int J Syst Evol Microbiol. 2014;64: 4120–28 pmid:25242540
- View Article
- PubMed/NCBI
- Google Scholar
8. Scholz HC, Revilla-Fernández S, Al Dahouk S, Hammerl JA, Zygmunt MS, Cloeckaert A, et al. Brucella vulpis spp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes). Int J Syst Evol Microbiol. 2016;66(5): 2090–98 pmid:26928956
- View Article
- PubMed/NCBI
- Google Scholar
9. Al Dahouk S, Köhler S, Occhialini A, Jiménez de Bagüés MP, Hammer JA, Eisenberg T, et al. Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts. Scientific Reports 2017; 7: 44420. pmid:28300153
- View Article
- PubMed/NCBI
- Google Scholar
10. Eisenberg T., Riße K., Schauerte N., Geiger C., Blom J.& Scholz H.C. Isolation of a novel ‘atypical’ Brucella strain from a bluespotted ribbontail ray (Taeniura lymma). Antonie van Leeuwenhoek 2017;110: 221–234. pmid:27785661
- View Article
- PubMed/NCBI
- Google Scholar
11. McDermott JJ, Arimi SM. Brucellosis in sub- Saharan Africa: epidemiology, control and impact. Vet. Microbiol. 2002;90: 111–134. pmid:12414138
- View Article
- PubMed/NCBI
- Google Scholar
12. Ducrotoy M, Bertu WJ, Matope G, Cadmus S, Conde-Álvarez R, Gusi AM, et al. Brucellosis in Sub-Saharan Africa: Current challenges for management, diagnosis and control. Acta Trop. 2017;165:179–93 pmid:26551794
- View Article
- PubMed/NCBI
- Google Scholar
13. Matope G., Bhebhe E., Muma J.B. Skjerve E, Djonne B. Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR. BMC Res Notes. 2009; 2: 261–7. pmid:20028545
- View Article
- PubMed/NCBI
- Google Scholar
14. Madsen M, Anderson EC. Serological survey of Zimbabwe wildlife for brucellosis. J Zoo Wildlife Med. 1995;26: 240–5.
- View Article
- Google Scholar
15. Chinyoka S., Dhliwayo S., Marabini L., Dutlow K., Matope G. & Pfukenyi D.M. Serological survey of Brucella canis in dogs in urban Harare and selected rural communities in Zimbabwe. J S Afr Vet. Assoc. 2014;85(1)
- View Article
- Google Scholar
16. Condy JB, Vickers DB. Brucellosis in Rhodesian wildlife. J S Afr Vet Assoc. 1972;43: 175–79. pmid:4217834
- View Article
- PubMed/NCBI
- Google Scholar
17. Madsen M. The current state of brucellosis in Zimbabwe. Zimbabwe Vet. J 1989;20: 133–149.
- View Article
- Google Scholar
18. Mohan K, Makaya PV, Muvavarirwa P, Matope G, Mahembe E, Pawandiwa A. Brucellosis surveillance and control in Zimbabwe: bacteriological and serological investigation in dairy herds. Onderstepoort J Vet Res 1996;63: 47–51. pmid:8848302
- View Article
- PubMed/NCBI
- Google Scholar
19. Corbel MJ. Brucellosis: an overview. Emerg Infect Dis.1997;3: 213–21. pmid:9204307
- View Article
- PubMed/NCBI
- Google Scholar
20. Whatmore AM. Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infect Genet Evol. 2009;9: 1168–84. pmid:19628055
- View Article
- PubMed/NCBI
- Google Scholar
21. Cortez A, Scarcelli E, Soares RM, Heinemann MB, Sakamoto SM, Genovez ME, et al. Detection of Brucella DNA from aborted bovine foetuses by polymerase chain reaction. Australian Vet J 2001;79:500–1.
- View Article
- Google Scholar
22. Keid LB, Soares RM, Vasconcellos SA, Chiebao DP. A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches. Theriogenol 2007;68:1260–70.
- View Article
- Google Scholar
23. Al Dahouk S, Le Flèche P, Nöckler K, Jacques I, Grayon M, Scholz HC, et al. Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Meth. 2007;69: 137–45.
- View Article
- Google Scholar
24. Weiner M, Iwaniak W, Szulowski K. Comparison of PCR-based AMOS, Bruce-ladder and MLVA assays for typing of Brucella species. Bull Vet I Pulawy. 2011;55: 625–30.
- View Article
- Google Scholar
25. Bricker BJ, Halling SM. Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin Microbiol. 1995;33: 1640–42. pmid:7650203
- View Article
- PubMed/NCBI
- Google Scholar
26. Bricker BJ, Halling SM. Differentiation of Brucella abortus bv 1, 2 and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv 1 by PCR. J Clin Microbiol. 1994;32: 2660–6. pmid:7852552
- View Article
- PubMed/NCBI
- Google Scholar
27. García-Yoldi D, Marín CM, de Miguel MJ, Muñoz PM, Vizmanos JL, López-Goñi I, et al. Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin Chem. 2006;52: 779–81. pmid:16595839
- View Article
- PubMed/NCBI
- Google Scholar
28. López-Goñi I, García D, Marín CM, de Miguel MJ, Muñoz PM, Balsco JM, et al. Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J Clin Microbiol. 2008;46: 3484–7. pmid:18716225
- View Article
- PubMed/NCBI
- Google Scholar
29. Mayer-Scholl A, Draeger A, Göllner C, Scholz CH, Nöckler K. Advancement of a multiplex PCR for the differentiation of all currently described Brucella species. J Microbiol Meth. 2010;80: 112–4.
- View Article
- Google Scholar
30. Ferreira AC, Chambel L, Tenreiro T, Cardoso R, Flor L, Dias IT, et al. MLVA16 typing of Portuguese human and animal Brucella melitensis and Brucella abortus isolates. PLoS ONE 2012;7(8): e42514. pmid:22905141
- View Article
- PubMed/NCBI
- Google Scholar
31. Le Flèche P, Jacques I, Grayon M, Al Dahouk S, Bouchoni P, Denoeud F, et al. Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol. 2006;6: 9. pmid:16469109
- View Article
- PubMed/NCBI
- Google Scholar
32. Vergnaud G, Hauck Y, Christiany D, Daoud B, Pourcel C, Jacques I. Genotypic expansion within the population structure of classical Brucella species revealed by MLVA16 typing of 1404 Brucella isolates from different animal and geographic origins, 1974–2006. Front. Microbiol. 2018;9: 1545. pmid:30050522
- View Article
- PubMed/NCBI
- Google Scholar
33. Whatmore AM, Shankster SJ, Perrett LL, Murphy TJ, Brew SD, Thirlwall RE, et al. Identification and characterization of variable number tandem-repeat markers for typing of Brucella spp. J Clin Microbiol. 2006;44: 1982–93. pmid:16757588
- View Article
- PubMed/NCBI
- Google Scholar
34. Whatmore AM, Koylass MS, Muchowski J, Edwards-Smallbone J, Gopaul KK, Perrett LL. Extended multilocus sequence analysis to describe the global population structure of the genus Brucella: Phylogeography and relationship to biovars. Front Microbiol. 2016; 7: 2049. pmid:28066370
- View Article
- PubMed/NCBI
- Google Scholar
35. Wattam AR, Foster JT, Mane SP, Beckstrom-Sternberg SM, Beckstrom-Sternberg JM, Dickerman AW, et al. Comparative phylogenomics and evolution of the Brucellae reveal a path to virulence. J Bacteriol. 2014;196: 920–30. pmid:24336939
- View Article
- PubMed/NCBI
- Google Scholar
36. DelVecchio VG, Kapatral V, Redkar RJ, Patra G, Mujer C, Los T, et al. The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci USA 2002;99: 443–8. pmid:11756688
- View Article
- PubMed/NCBI
- Google Scholar
37. Paulsen IT, Seshadri R, Nelson KE, Eisen JA, Heidelberg JF, Read TD, et al. The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Pro Natl Acad Sci USA 2002;99: 13148–53.
- View Article
- Google Scholar
38. Halling SM, Peterson-Burch BD, Bricker BJ, Zuerner RL, Qing Z, Li LL. Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis. J Bacteriol 2005;187: 2715–26. pmid:15805518
- View Article
- PubMed/NCBI
- Google Scholar
39. Sankarasubramanian J, Vishnu US, Sridhar J, Gunasekaran P, Rajendhran J. A genome-wide SNP-based phylogenetic analysis distinguishes different biovars of Brucella suis. Infect Genet Evol 2016;41: 213–17. pmid:27085292
- View Article
- PubMed/NCBI
- Google Scholar
40. Foster JT, Price LB, Beckstrom-Sternberg SM, Pearson T, Brown WD, Kiesling DM, et al. Genotyping of Brucella species using clade specific SNPs. BMC Microbiol. 2012;12: 110. pmid:22712667
- View Article
- PubMed/NCBI
- Google Scholar
41. Foster JT, Beckstrom-Sternberg SM, Pearson T, Beckstrom-Sternberg JS, Chain PSG, Roberto FF, et al. Whole genome-based phylogeny and divergence of the genus Brucella. J Bacteriol 2009; 191:2864–70. pmid:19201792
- View Article
- PubMed/NCBI
- Google Scholar
42. Garofolo G, Di Giannatale E, Platone I, Zilli K, Sacchini L, Abass A, et al. Origins and global context of Brucella abortus in Italy. BMC Microbiol 2017;17: 28. pmid:28152976
- View Article
- PubMed/NCBI
- Google Scholar
43. Alton G, Jones LM, Angus RD, Verger JM. Techniques for the brucellosis laboratory, Institut National de la Recherche Agronomique, Paris (France) pp. 81–134.
44. Maquart M, Le Flèche P, Foster G, Tryland M, Ramisse F, Djonne B, et al. MLVA-16 typing of 295 marine mammal Brucella isolates from different animal and geographic origins identifies 7 major groups within Brucella ceti and Brucella pinnipedialis. BMC Microbiol. 2009;9: 145. pmid:19619320
- View Article
- PubMed/NCBI
- Google Scholar
45. MLVA database (http://microbesgenotyping.i2bc.paris-saclay.fr/databases/public).
46. López-Goñi I, García-Yoldi D, Marín CM, de Miguel MJ, Barquero-Calvo E, Guzman-Verri C, et al. New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis. Vet Microbiol. 2011;154: 152–5. pmid:21782356
- View Article
- PubMed/NCBI
- Google Scholar
47. Allele numbering system version 3.6 (http://mlva.u-psud.fr/brucella/spip.php?article93).
48. Ledwaba MB, Mafofo J, van Heerden H. Genome sequences of Brucella abortus and Brucella suis strains isolated from bovine in Zimbabwe. Genome Announc. 2014;2: e01063–14.
- View Article
- Google Scholar
49. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25: 1754–60. pmid:19451168
- View Article
- PubMed/NCBI
- Google Scholar
50. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009;25: 2078–79. pmid:19505943
- View Article
- PubMed/NCBI
- Google Scholar
51. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. "The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data." Genome Res. 2010;20(9): 1297–1303. pmid:20644199
- View Article
- PubMed/NCBI
- Google Scholar
52. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30: 2725–2729. pmid:24132122
- View Article
- PubMed/NCBI
- Google Scholar
53. Wojno JM, Moodley C, Pienaar J, Beylis N, Jacobsz L, Nicol MP, et al. Human brucellosis in South Africa: Public health and diagnostics pitfalls. S. Afr. Med. J. 2016; 106: 883–5. pmid:27601111
- View Article
- PubMed/NCBI
- Google Scholar
54. Gandara B, Merino AL, Rogel A, Martinez-Romero E. Limited genetic diversity of Brucella spp. J Clin Microbiol. 2001;39: 235–40. pmid:11136777
- View Article
- PubMed/NCBI
- Google Scholar
55. Ewalt DR, Payeur JB, Rhyan JC, Geer PL. Brucella suis biovar 1 in naturally infected cattle: a bacteriological, serological, and histological study. J Vet Diagn Invest 1997;9: 417–20. pmid:9376434
- View Article
- PubMed/NCBI
- Google Scholar
56. Tae H, Shallom S, Settlage R, Hawkins GN, Adams LG, Garner HR. Complete genome sequence of Brucella suis VB122, isolated from bovine milk. J Bacteriol 2012;194: 910. pmid:22275106
- View Article
- PubMed/NCBI
- Google Scholar
57. Gandiwa E. Local knowledge and perceptions of animal population abundances by communities adjacent to the northern Gonarezhou National Park, Zimbabwe. Trop Conserv Sci 2012;5: 255–69.
- View Article
- Google Scholar
58. Suárez-Esquivel M, Ruiz-Villalobos N, Castillo-Zeledón A, Jiménez-Rojas C, Roop RM II, Comerci DJ, et al. Brucella abortus Strain 2308 Wisconsin Genome: Importance of the Definition of Reference Strains Front. Microbiol. 2016;7: 1557
- View Article
- Google Scholar
59. Bertu WJ, Ducrotoy MJ, Munoz PM, Mick V, Zúniga-Ripa A, Bryssinckx W, et al. Phenotypic and genotypic characterization of Brucella strains isolated from autochthonous livestock reveals the dominance of B. abortus biovar 3a in Nigeria. Vet. Microbiol. 2015;180: 103–108. pmid:26315770
- View Article
- PubMed/NCBI
- Google Scholar
60. Christopher S, Umapathy BL, Ravikumar KL. Brucellosis: review on the recent trends in pathogenicity and laboratory diagnosis. J Lab Physicians 2010;2(2): 55–60. pmid:21346896
- View Article
- PubMed/NCBI
- Google Scholar
61. Ocampo-Sosa AA, Agüero-Balbίn J, Garcίa-Lobo JM. Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3. Vet. Microbiol. 2005;110: 41–51. pmid:16029934
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Godfroid J. Brucellosis in wildlife. Rev Sci Tech OIE. 2002;21: 277–86.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Osterman B, Moriyón I. International Committee on Systematics of Prokaryotes. Subcommittee on the taxonomy of Brucella. Report of the meeting, 17 September 2003, Pamplona, Spain. Int J Syst Evol Micro. 2006;56: 1173–5.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Foster G, Osterman BS, Godfroid J, Jacques I, Cloeckaert A. Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. Int J Syst Evol Micro. 2007;57: 2688–93.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. Scholz HC, Hubalek Z, Nesvadbova J, Tomaso H, Vergnaud G, Le Flèche P, et al. Isolation of Brucella microti from soil. Emerg Infect Dis. 2008;14: 1316–7. pmid:18680668
View Article
PubMed/NCBI
Google Scholar

[11] View Article

[12] PubMed/NCBI

[13] Google Scholar

[ref5] 5. Scholz HC, Hubalek Z, Sedlácek I, Vergnaud G, Tomaso H, Al Dahouk S, et al. Brucella microti spp. nov. isolated from the common vole Microtus arvalis. Int J Syst Evol Micro. 2008;58: 375–82.
View Article
Google Scholar

[15] View Article

[16] Google Scholar

[ref6] 6. Scholz HC, Nöckler K, Gollner C, Bahn P, Vergnaud G, Tomaso H, et al. Brucella inopinata spp. nov., isolated from a breast implant infection. Int J Syst Evol Micro. 2010;60: 801–8.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref7] 7. Whatmore AM, Davison N, Cloeckaert A, Al Dahouk S, Zygmunt MS, Brew SD, et al. Brucella papionis spp. nov., isolated from baboons (Papio spp.). Int J Syst Evol Microbiol. 2014;64: 4120–28 pmid:25242540
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref8] 8. Scholz HC, Revilla-Fernández S, Al Dahouk S, Hammerl JA, Zygmunt MS, Cloeckaert A, et al. Brucella vulpis spp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes). Int J Syst Evol Microbiol. 2016;66(5): 2090–98 pmid:26928956
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref9] 9. Al Dahouk S, Köhler S, Occhialini A, Jiménez de Bagüés MP, Hammer JA, Eisenberg T, et al. Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts. Scientific Reports 2017; 7: 44420. pmid:28300153
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref10] 10. Eisenberg T., Riße K., Schauerte N., Geiger C., Blom J.& Scholz H.C. Isolation of a novel ‘atypical’ Brucella strain from a bluespotted ribbontail ray (Taeniura lymma). Antonie van Leeuwenhoek 2017;110: 221–234. pmid:27785661
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref11] 11. McDermott JJ, Arimi SM. Brucellosis in sub- Saharan Africa: epidemiology, control and impact. Vet. Microbiol. 2002;90: 111–134. pmid:12414138
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref12] 12. Ducrotoy M, Bertu WJ, Matope G, Cadmus S, Conde-Álvarez R, Gusi AM, et al. Brucellosis in Sub-Saharan Africa: Current challenges for management, diagnosis and control. Acta Trop. 2017;165:179–93 pmid:26551794
View Article
PubMed/NCBI
Google Scholar

[41] View Article

[42] PubMed/NCBI

[43] Google Scholar

[ref13] 13. Matope G., Bhebhe E., Muma J.B. Skjerve E, Djonne B. Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR. BMC Res Notes. 2009; 2: 261–7. pmid:20028545
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref14] 14. Madsen M, Anderson EC. Serological survey of Zimbabwe wildlife for brucellosis. J Zoo Wildlife Med. 1995;26: 240–5.
View Article
Google Scholar

[49] View Article

[50] Google Scholar

[ref15] 15. Chinyoka S., Dhliwayo S., Marabini L., Dutlow K., Matope G. & Pfukenyi D.M. Serological survey of Brucella canis in dogs in urban Harare and selected rural communities in Zimbabwe. J S Afr Vet. Assoc. 2014;85(1)
View Article
Google Scholar

[52] View Article

[53] Google Scholar

[ref16] 16. Condy JB, Vickers DB. Brucellosis in Rhodesian wildlife. J S Afr Vet Assoc. 1972;43: 175–79. pmid:4217834
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref17] 17. Madsen M. The current state of brucellosis in Zimbabwe. Zimbabwe Vet. J 1989;20: 133–149.
View Article
Google Scholar

[59] View Article

[60] Google Scholar

[ref18] 18. Mohan K, Makaya PV, Muvavarirwa P, Matope G, Mahembe E, Pawandiwa A. Brucellosis surveillance and control in Zimbabwe: bacteriological and serological investigation in dairy herds. Onderstepoort J Vet Res 1996;63: 47–51. pmid:8848302
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref19] 19. Corbel MJ. Brucellosis: an overview. Emerg Infect Dis.1997;3: 213–21. pmid:9204307
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref20] 20. Whatmore AM. Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infect Genet Evol. 2009;9: 1168–84. pmid:19628055
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref21] 21. Cortez A, Scarcelli E, Soares RM, Heinemann MB, Sakamoto SM, Genovez ME, et al. Detection of Brucella DNA from aborted bovine foetuses by polymerase chain reaction. Australian Vet J 2001;79:500–1.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref22] 22. Keid LB, Soares RM, Vasconcellos SA, Chiebao DP. A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches. Theriogenol 2007;68:1260–70.
View Article
Google Scholar

[77] View Article

[78] Google Scholar

[ref23] 23. Al Dahouk S, Le Flèche P, Nöckler K, Jacques I, Grayon M, Scholz HC, et al. Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Meth. 2007;69: 137–45.
View Article
Google Scholar

[80] View Article

[81] Google Scholar

[ref24] 24. Weiner M, Iwaniak W, Szulowski K. Comparison of PCR-based AMOS, Bruce-ladder and MLVA assays for typing of Brucella species. Bull Vet I Pulawy. 2011;55: 625–30.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref25] 25. Bricker BJ, Halling SM. Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin Microbiol. 1995;33: 1640–42. pmid:7650203
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref26] 26. Bricker BJ, Halling SM. Differentiation of Brucella abortus bv 1, 2 and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv 1 by PCR. J Clin Microbiol. 1994;32: 2660–6. pmid:7852552
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref27] 27. García-Yoldi D, Marín CM, de Miguel MJ, Muñoz PM, Vizmanos JL, López-Goñi I, et al. Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin Chem. 2006;52: 779–81. pmid:16595839
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref28] 28. López-Goñi I, García D, Marín CM, de Miguel MJ, Muñoz PM, Balsco JM, et al. Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J Clin Microbiol. 2008;46: 3484–7. pmid:18716225
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref29] 29. Mayer-Scholl A, Draeger A, Göllner C, Scholz CH, Nöckler K. Advancement of a multiplex PCR for the differentiation of all currently described Brucella species. J Microbiol Meth. 2010;80: 112–4.
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref30] 30. Ferreira AC, Chambel L, Tenreiro T, Cardoso R, Flor L, Dias IT, et al. MLVA16 typing of Portuguese human and animal Brucella melitensis and Brucella abortus isolates. PLoS ONE 2012;7(8): e42514. pmid:22905141
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref31] 31. Le Flèche P, Jacques I, Grayon M, Al Dahouk S, Bouchoni P, Denoeud F, et al. Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol. 2006;6: 9. pmid:16469109
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref32] 32. Vergnaud G, Hauck Y, Christiany D, Daoud B, Pourcel C, Jacques I. Genotypic expansion within the population structure of classical Brucella species revealed by MLVA16 typing of 1404 Brucella isolates from different animal and geographic origins, 1974–2006. Front. Microbiol. 2018;9: 1545. pmid:30050522
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref33] 33. Whatmore AM, Shankster SJ, Perrett LL, Murphy TJ, Brew SD, Thirlwall RE, et al. Identification and characterization of variable number tandem-repeat markers for typing of Brucella spp. J Clin Microbiol. 2006;44: 1982–93. pmid:16757588
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref34] 34. Whatmore AM, Koylass MS, Muchowski J, Edwards-Smallbone J, Gopaul KK, Perrett LL. Extended multilocus sequence analysis to describe the global population structure of the genus Brucella: Phylogeography and relationship to biovars. Front Microbiol. 2016; 7: 2049. pmid:28066370
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref35] 35. Wattam AR, Foster JT, Mane SP, Beckstrom-Sternberg SM, Beckstrom-Sternberg JM, Dickerman AW, et al. Comparative phylogenomics and evolution of the Brucellae reveal a path to virulence. J Bacteriol. 2014;196: 920–30. pmid:24336939
View Article
PubMed/NCBI
Google Scholar

[125] View Article

[126] PubMed/NCBI

[127] Google Scholar

[ref36] 36. DelVecchio VG, Kapatral V, Redkar RJ, Patra G, Mujer C, Los T, et al. The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci USA 2002;99: 443–8. pmid:11756688
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref37] 37. Paulsen IT, Seshadri R, Nelson KE, Eisen JA, Heidelberg JF, Read TD, et al. The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Pro Natl Acad Sci USA 2002;99: 13148–53.
View Article
Google Scholar

[133] View Article

[134] Google Scholar

[ref38] 38. Halling SM, Peterson-Burch BD, Bricker BJ, Zuerner RL, Qing Z, Li LL. Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis. J Bacteriol 2005;187: 2715–26. pmid:15805518
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref39] 39. Sankarasubramanian J, Vishnu US, Sridhar J, Gunasekaran P, Rajendhran J. A genome-wide SNP-based phylogenetic analysis distinguishes different biovars of Brucella suis. Infect Genet Evol 2016;41: 213–17. pmid:27085292
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref40] 40. Foster JT, Price LB, Beckstrom-Sternberg SM, Pearson T, Brown WD, Kiesling DM, et al. Genotyping of Brucella species using clade specific SNPs. BMC Microbiol. 2012;12: 110. pmid:22712667
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref41] 41. Foster JT, Beckstrom-Sternberg SM, Pearson T, Beckstrom-Sternberg JS, Chain PSG, Roberto FF, et al. Whole genome-based phylogeny and divergence of the genus Brucella. J Bacteriol 2009; 191:2864–70. pmid:19201792
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref42] 42. Garofolo G, Di Giannatale E, Platone I, Zilli K, Sacchini L, Abass A, et al. Origins and global context of Brucella abortus in Italy. BMC Microbiol 2017;17: 28. pmid:28152976
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref43] 43. Alton G, Jones LM, Angus RD, Verger JM. Techniques for the brucellosis laboratory, Institut National de la Recherche Agronomique, Paris (France) pp. 81–134.

[ref44] 44. Maquart M, Le Flèche P, Foster G, Tryland M, Ramisse F, Djonne B, et al. MLVA-16 typing of 295 marine mammal Brucella isolates from different animal and geographic origins identifies 7 major groups within Brucella ceti and Brucella pinnipedialis. BMC Microbiol. 2009;9: 145. pmid:19619320
View Article
PubMed/NCBI
Google Scholar

[157] View Article

[158] PubMed/NCBI

[159] Google Scholar

[ref45] 45. MLVA database (http://microbesgenotyping.i2bc.paris-saclay.fr/databases/public).

[ref46] 46. López-Goñi I, García-Yoldi D, Marín CM, de Miguel MJ, Barquero-Calvo E, Guzman-Verri C, et al. New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis. Vet Microbiol. 2011;154: 152–5. pmid:21782356
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref47] 47. Allele numbering system version 3.6 (http://mlva.u-psud.fr/brucella/spip.php?article93).

[ref48] 48. Ledwaba MB, Mafofo J, van Heerden H. Genome sequences of Brucella abortus and Brucella suis strains isolated from bovine in Zimbabwe. Genome Announc. 2014;2: e01063–14.
View Article
Google Scholar

[167] View Article

[168] Google Scholar

[ref49] 49. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25: 1754–60. pmid:19451168
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref50] 50. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009;25: 2078–79. pmid:19505943
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

[ref51] 51. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. "The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data." Genome Res. 2010;20(9): 1297–1303. pmid:20644199
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

[ref52] 52. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30: 2725–2729. pmid:24132122
View Article
PubMed/NCBI
Google Scholar

[182] View Article

[183] PubMed/NCBI

[184] Google Scholar

[ref53] 53. Wojno JM, Moodley C, Pienaar J, Beylis N, Jacobsz L, Nicol MP, et al. Human brucellosis in South Africa: Public health and diagnostics pitfalls. S. Afr. Med. J. 2016; 106: 883–5. pmid:27601111
View Article
PubMed/NCBI
Google Scholar

[186] View Article

[187] PubMed/NCBI

[188] Google Scholar

[ref54] 54. Gandara B, Merino AL, Rogel A, Martinez-Romero E. Limited genetic diversity of Brucella spp. J Clin Microbiol. 2001;39: 235–40. pmid:11136777
View Article
PubMed/NCBI
Google Scholar

[190] View Article

[191] PubMed/NCBI

[192] Google Scholar

[ref55] 55. Ewalt DR, Payeur JB, Rhyan JC, Geer PL. Brucella suis biovar 1 in naturally infected cattle: a bacteriological, serological, and histological study. J Vet Diagn Invest 1997;9: 417–20. pmid:9376434
View Article
PubMed/NCBI
Google Scholar

[194] View Article

[195] PubMed/NCBI

[196] Google Scholar

[ref56] 56. Tae H, Shallom S, Settlage R, Hawkins GN, Adams LG, Garner HR. Complete genome sequence of Brucella suis VB122, isolated from bovine milk. J Bacteriol 2012;194: 910. pmid:22275106
View Article
PubMed/NCBI
Google Scholar

[198] View Article

[199] PubMed/NCBI

[200] Google Scholar

[ref57] 57. Gandiwa E. Local knowledge and perceptions of animal population abundances by communities adjacent to the northern Gonarezhou National Park, Zimbabwe. Trop Conserv Sci 2012;5: 255–69.
View Article
Google Scholar

[202] View Article

[203] Google Scholar

[ref58] 58. Suárez-Esquivel M, Ruiz-Villalobos N, Castillo-Zeledón A, Jiménez-Rojas C, Roop RM II, Comerci DJ, et al. Brucella abortus Strain 2308 Wisconsin Genome: Importance of the Definition of Reference Strains Front. Microbiol. 2016;7: 1557
View Article
Google Scholar

[205] View Article

[206] Google Scholar

[ref59] 59. Bertu WJ, Ducrotoy MJ, Munoz PM, Mick V, Zúniga-Ripa A, Bryssinckx W, et al. Phenotypic and genotypic characterization of Brucella strains isolated from autochthonous livestock reveals the dominance of B. abortus biovar 3a in Nigeria. Vet. Microbiol. 2015;180: 103–108. pmid:26315770
View Article
PubMed/NCBI
Google Scholar

[208] View Article

[209] PubMed/NCBI

[210] Google Scholar

[ref60] 60. Christopher S, Umapathy BL, Ravikumar KL. Brucellosis: review on the recent trends in pathogenicity and laboratory diagnosis. J Lab Physicians 2010;2(2): 55–60. pmid:21346896
View Article
PubMed/NCBI
Google Scholar

[212] View Article

[213] PubMed/NCBI

[214] Google Scholar

[ref61] 61. Ocampo-Sosa AA, Agüero-Balbίn J, Garcίa-Lobo JM. Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3. Vet. Microbiol. 2005;110: 41–51. pmid:16029934
View Article
PubMed/NCBI
Google Scholar

[216] View Article

[217] PubMed/NCBI

[218] Google Scholar

Figures

Abstract

Author summary

Introduction

Methods

Ethics statement

Bacterial strains and bacteriology

DNA preparation

AMOS-PCR and Bruce-ladder

MLVA

Data analysis

Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses

Results

Bacteriology

AMOS-PCR and Bruce-ladder

MLVA

Whole genome sequence and WGS-SNP analysis

Discussion

Supporting information

S1 Table. Reference strains and Zimbabwean Brucella spp. isolates identified by Bruce-ladder and repeat copy number of the indicated loci.

S2 Table. Brucella abortus and B. suis genome sequences retrieved from GenBank, used in the study for comparison of whole genome single nucleotide polymorphisms (WGS-SNPS) phylogenetic analysis.

S1 Fig. Suis-ladder multiplex PCR assay of Brucella DNA from Zimbabwe and reference strains.

S2 Fig. Minimum spanning tree analysis of published data and Zimbabwean Brucella isolates using the MLVA8 data (Panel 1 genotypes).

Acknowledgments

References