Genotyping of Indian antigenic , vaccine , and field Brucella spp . using multilocus sequence typing

Introduction: Brucellosis is one of the most important zoonotic diseases that affects multiple livestock species and causes great economic losses. The highly conserved genomes of Brucella, with > 90% homology among species, makes it important to study the genetic diversity circulating in the country. Methodology: A total of 26 Brucella spp. (4 reference strains and 22 field isolates) and 1 B. melitensis draft genome sequence from India (B. melitensis Bm IND1) were included for sequence typing. The field isolates were identified by biochemical tests and confirmed by both conventional and quantitative polymerase chain reaction (qPCR) targeting bcsp 31Brucella genus-specific marker. Brucella speciation and biotyping was done by Bruce ladder, probe qPCR, and AMOS PCRs, respectively, and genotyping was done by multilocus sequence typing (MLST). Results: The MLST typing of 27 Brucella spp. revealed five distinct sequence types (STs); the B. abortus S99 reference strain and 21 B. abortus field isolates belonged to ST1. On the other hand, the vaccine strain B. abortus S19 was genotyped as ST5. Similarly, B. melitensis 16M reference strain and one B. melitensis field isolate were grouped into ST7. Another B. melitensis field isolate belonged to ST8 (draft genome sequence from India), and only B. suis 1330 reference strain was found to be ST14. Conclusion: The sequences revealed genetic similarity of the Indian strains to the global reference and field strains. The study highlights the usefulness of MLST for typing of field isolates and validation of reference strains used for diagnosis and vaccination against brucellosis.


Introduction
Ever since the discovery of Brucella causative agent more than a century ago, brucellosis remains major problem for animals and mankind [1].Crossborder epizootic [2] and regional outbreak incidences [3] observed in last few decades and the continuous changing trend in its epidemiological distribution across the globe led Brucella to be recognized as a reemerging pathogen [1].It is also credited as a biological warfare agent due to its highly infectious nature and airborne transmission [4].The disease has been successfully eradicated in the cattle population of only a handful of developed countries [5], but it is still highly prevalent and often regionally neglected in many parts of the world, including the Indian subcontinent [6,7].
India has been recognized to be among the regions with the highest prevalence of brucellosis in the world [8], with annual economic losses to the tune of US $58.8 million.B. abortus S99 antigen extract-based diagnostics for surveillance and B. abortus S19 vaccination were recently implemented in the country for control of brucellosis in the bovine population.Knowledge about the prevalent strains in a given geographical area is a component of major importance in surveillance systems in order to define control strategies.However, the strain typing of existing and new isolates within the genus Brucella is a great challenge because members of the genus have highly conserved genomes with more than 90% DNA-DNA hybridization homology [9], have almost identical 16S rRNA [10], and share more than 98% sequence identity [11,12].
Multilocus sequence typing (MLST) has been recently demonstrated as useful tool [13] for genotyping and for phylogenetic and global epidemiology studies of genetically conserved pathogens like Brucella [14,15].Using the MLST technique, nine new sequence types, which were quite different from those observed in other countries, were defined in China [16].Similarly, genetic differences of novel strains of Brucella inopinata were also recently documented [14].To date, genotying of field Brucella isolates of India and comparing these to global isolates had not yet been done.Comparative analysis between the prevalent Brucella strains in India and those found in other regions is still not possible because there is no genotyping information about Brucella isolated in India.Therefore, this study was undertaken to type a limited number of Indian field isolates from domestic ruminants along with reference strains used for vaccine and diagnostics for preliminary study.

Isolation and biochemical characterization of Brucella spp.
The isolations were carried out using samples that included placenta, fetal stomach contents, fetal membrane, fetal heart blood, and vaginal discharge from recently aborted animals, collected by a convenient sampling approach in Karnataka, Assam, Gujarat, and Punjab.All the aborted animals included were serologically screened for brucellosis using the Rose Bengal plate test (RBPT) (Institute of Animal Health & Veterinary Biologicals (IAH &VB), Bengaluru, India) [17].Similarly, samples were screened by protein G based indirect enzyme-linked immunosorbent assay (iELISA) (ICAR-NIVEDI, Bengaluru, India) using smooth lipopolysaccharide antigen from standard strain B. abortus S99.The cutoff values established for diagnosis were decided after thorough screening and validation of the assay [18].Any sample of percent positivity value below 55%, between 55% and 65%, and more than 65% were considered, negative, moderate positive, and strong positive, respectively.Isolations were carried out in Brucella selective broth (Condaa Pronadisa Laboratories, Conda, S.A. Madrid, Spain,) containing antibiotic supplements (nystatin, bacitracin, polymyxin-B, cycloheximide, nalidixic acid) in a laboratory that had a class II biosafety cabinet facility.Sample inoculated tubes were incubated with and without 10% CO2 at 37C for 72 hours.A loopful of broth culture from both sets were streaked onto Brucella selective agar with supplements and incubated with and without 10% CO2 at 37C until the appearance of growth or up to one week.Purified colonies recovered from the processed clinical samples along with four reference strains (B.abortus S99, B. melitensis 16M, B. suis 1330, and vaccine strain B. abortus S19) procured from the National Culture Repository, Indian Veterinary Research Institute, Izatnagar, India, were identified by Gram staining, CO2 requirement, H2S production, catalase, urease, oxidase tests, and inhibition of growth by basic fuchsin and thionin dyes [17].All the isolates were grown to stationary phase at 37°C in tryptic soy broth (TSB) or tryptic soy agar (TSA) (Becton Dickinson, Franklin Lakes, USA) and preserved without extensive laboratory passage.

Molecular characterization of cultures
Cultures were grown on TSA at 37°C for 48 hours, inactivated for 2 hours at 80°C, and DNA was extracted with the QIAamp DNA mini kit (Qiagen, Dusseldorf, Germany).For Brucella genus-specific detection, sequences were amplified by genus-specific primers [19].Species were identified as B. abortus, B. melitensis, and B. suis by multiplex Bruce ladder polymerase chain reaction (PCR) [20]; biovar typing was done by modified abortus-melitensis-ovis-suis (AMOS) PCR (primers by Eurofins Genomics Pvt.Ltd, Bengaluru, India) [21].Similarly, the isolates were subjected to both genus and speciation real-time quantitative polymerase chain reaction (qPCR) assays that are routinely carried out in the laboratory [22,23].

MLST typing and sequence analysis
Genotyping was done for 22 Brucella field isolates and four reference strains by amplifying nine distinct genetic loci (seven housekeeping and one each outer membrane protein and intergenic fragment) using primers (Table 1) [24].The PCR-amplified products were purified by a QIAquick PCR purification kit (Qiagen) and sequenced at M/S Eurofins Genomics India, (Bangalore, India) using an ABI 3730 sequencer.The sequences were edited using Chromas Lite 2.01 software (http://www.technelysium.com.au/chromas_lite.html).MLST sequences (accession numbers AM694191 through AM695630) of the strains were downloaded from the GenBank database.Distinct alleles at each of the loci were given a numerical designation according to the sequence of defined alleles.The unique allelic pattern over all loci was identified as a sequence type (ST).The sequences of the nine loci were concatenated to produce a sequence of 4,396 bp for each genotype.Genotype relatedness analysis along with global isolates obtained in the database was carried out using MEGA 6.06 software (http://www.megasoftware.net/mega.php).

Results
All the aborted animals' serum samples were positive by RBPT and protein G based iELISA.From these samples, 22 Brucella isolates were recovered; 21 were identified as B. abortus and 1 as B. melitensis.These isolates were from different geographical regions of the country: four B. abortus isolates from Punjab and ten from Gujarat, two from Assam and five from Karnataka, and one isolate of B. melitensis from Karnataka (Tables 2 and 3) .In the PCR, all the isolates showed 223 bp amplification specific for Brucella genus (Figure 1) and species-specific amplification products by multiplex Bruce ladder PCR (Figure 2).In AMOS PCR, a 498 bp amplicon was observed in all the B. abortus isolates, confirming their biovars to be either of I, II, or IV biovar; 731 bp in B. melitensis strains and a 285 bp product in B. suis biovar I (Figure 3).MLST typing revealed five distinct STs; the B. abortus S99 reference strain (antigenic strain) and 21 B. abortus field isolates belonged to ST1, whereas B. abortus S19 (vaccine strain) was found to be ST5.Similarly, the B. melitensis 16M reference strain and one B. melitensis field isolate (B.melitensis ADMAS-G1) were clustered into ST7.Recently, a draft genome sequence of B. melitensis Bm IND1 field isolate was reported to be ST8, and B. suis 1330 reference strain was recognized as ST14 (Table 4).The sequence divergence by neighbor-joining dendrogram showing the genetic relatedness among the five STs of the 27 Indian isolates (including four reference strains) and nine STs of the 24 global isolates is represented in Figures 4 and 5

Discussion
Brucellosis, a persistent bacterial disease of livestock and humans, causes huge economic losses and significant morbidity [25].Accurate identification of the prevailing strains of Brucella in a given geographical area is central to epidemiological surveillance and public health decisions.Since 1947, there were studies pertaining to isolation of Brucella spp. in many regions of the country.Isolation of both B. abortus and B. melitensis from horses [26] and B. abortus bv.I and III [27], bv IV [28], and bv.I [29] from cattle has been reported.Similarly, B. melitensis from cattle [30], bv.I of B. melitensis from sheep and goats [31], B. suis bv.II from a cow [32], and B. suis bv.I and II to V from aborted sows have been reported [33,34].All these studies attempted to identify Brucella species and biovars by conventional methods such as analysis of an array of phenotypic characteristics and serological and phage typing.These conventional methods, though time consuming, biohazardous to laboratory personnel, and difficult to differentiate between vaccine and wild strains, have been used worldwide for isolation and typing over the years.With the advent of molecular tools, the conventional typing methods have slowly been replaced by highly discriminatory sequence-based typing.In India, apart from recently reported draft genome sequences of few Brucella isolates [35,36], there is no documentation of genetic diversity studies of Brucella isolates.
In the study, 13, 7, and 1 B. abortus isolates recovered from cattle, buffalo, and pigs, respectively, and 1 B. melitensis from goats were identified and confirmed by biochemical methods and PCR.It has been reported that B. abortus is the most prevailing species in bovines in the country [37].It is noteworthy that isolation of B. abortus from pigs clearly indicates the possibility of transmission among the livestock species in a mixed farming system.Though Brucella isolates were recovered from distant geographical regions within the country, most of them had similar sequence types.The current study highlights the genetic similarity among Indian isolates and with other global isolates.It has been observed that ST1 of B. abortus was predominant among the limited number of sequenced strains.In further studies, sequencing large numbers of field isolates will provide the most prevalent sequence types in the country.
Among reference strains, B. abortus S99, which is being used for diagnostic antigen preparations in various organizations in the country, belonged to ST1 and was phylogenetically related to the global designate From a public health perspective, data on prevailing strains will be useful in the investigation of pyrexia of unknown origin studies in humans [38], disease outbreaks in domestic ruminants, and to appreciate the appropriateness of antigen/vaccination strains used in the region.This information is also useful to differentiate between naturally occurring outbreaks and bioterrorism.Moreover, the MLST method can also provides a framework for typing any new or emerging Brucella strains. .

Conclusions
The present study is the preliminary work carried as part of the Department of Biotechnology (DBT) -Network project on brucellosis.This small-scale validation of the sequence typing method is significant because in the future, large numbers of isolates can be sequenced and genotyped in a similar order.Also, this study underlines the genetic relatedness of the B. abortus S19 vaccine and B. abortus S99 antigenic strains.
Brucellosis, which is gratefully acknowledged.We are thankful to all the research assistants, junior research fellows, senior research fellows, and Mr. H.M. Manukumar, office assistant, who are involved in the DBT Network Project on Brucellosis.

Table 1 .
Oligonucleotide sequences used for the amplification and sequencing of nine genetic loci.

Table 2 .
Brucella isolates and their geographical region of origin.

Table 3 .
Phenotypic and biochemical profile of Indian field Brucella spp.along with four reference strains.
. Genotypic relatedness analysis classified the STs into different groups.Cluster C1 is divided into 2 clades.Clade 1 comprises B. neotomae and B. inopinata in two close subclades, indicating the close genetic resemblance between these species.Similarly, clade 2 of cluster C1 is divided into many sub-clades comprising different Brucella spp.-B.abortus, B. melitensis, B. ovis, B. suis B. microti, B. ceti, B. pinnipedialis, and B. canis -under different clades and subclades.This cluster is the largest group, comprising eight different Brucella spp.holding five different STs of Indian isolates together with eight different STs of global isolates.

Table 4 .
Allele profile and sequence type of the Brucella species reference stain (B.abortus 9-941).Similarly, B. abortus S19, used for calfhood vaccine preparation is in the ST5 group, has also been found to be genetically related to the other global vaccine strains such as B. abortus S19, 2308, and 13334.The other two reference strains, B. melitensis 16M and B. suis, belonged to ST7 and ST14, respectively.B. melitensis 16M is being used for preparation of plain agglutination antigen for diagnosis of B. melitensis infection in small ruminants and humans in the country.B. suis, on the other hand, is of limited diagnostic and vaccine importance.The phenotypic and genetic typing of reference strains used for vaccine and diagnosis is important, as the Indian Government's Department of Animal Husbandry, Dairying, and Fisheries (DADF) has initiated a nationwide brucellosis control program.In the program, B. abortus S19 calfhood vaccination for female cattle and buffalo calves and surveillance by various diagnostic tests using antigen extracts from B. abortus S99 are recommended.Hence, evaluation of vaccine and diagnostic reference strains is justified.The other two reference strains, B. melitensis 16M and B. suis, belonged to ST7 and ST14, respectively.B. melitensis 16M is being used for preparation of plain agglutination antigen for diagnosis of B. melitensis infection in small ruminants and humans in the country, whereas B. suis is of limited diagnostic and vaccine importance.