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Balantioides coli is a zoonotic protozoan parasite whose main reservoir is pigs. Recent studies have shown that B. coli variant A but not B has zoonotic potential. While B. coli infection has been reported in different animals and countries, the prevalence of the zoonotic variant is limited due to a lack of molecular information. Therefore, this study investigated the prevalence of B. coli in domestic pigs in Korea and assessed its zoonotic potential. A total of 188 pig fecal samples were collected from slaughterhouses in Korea. B. coli was identified by microscopy and molecular methods. B. coli was identified in 79 (42.9%) and 174 (94.6%) samples by microscopy and polymerase chain reaction (PCR), respectively. This study also developed a PCR-restriction fragment length polymorphism (PCR-RFLP) method to differentiate B. coli variant A from B without sequence analysis. Using this method, 62 (33.7%) and 160 (87.0%) samples were positive for variants A and B, respectively, and 48 (26.1%) samples were co-infected with both variants. Sequence and phylogenetic analyses showed a high genetic diversity of B. coli in pigs in Korea. To our knowledge, this is the first study to develop a method to differentiate B. coli variants A and B without sequence analysis and to assess the molecular epidemiology of B. coli in pigs. Continuous monitoring of zoonotic B. coli in pigs should be performed as pigs are the main source of human balantidiasis.

Simple Summary Balantioides coli is a protozoan parasite that can infect humans, and its main reservoir is pigs. Recent studies suggested that one of B. coli variants, named variant A, has zoonotic potential. Previous studies have reported B. coli infection in pigs in different countries; however, the prevalence of the zoonotic variant is limited due to a lack of molecular information. In this study, we developed a molecular technique-based method that could differentiate B. coli variant A from B without sequence analysis. Using the method, 174/188 (94.6%) pig fecal samples collected in domestic pigs in Korea were positive for B. coli, and of the samples, 62 (33.7%) were the zoonotic variant. To our knowledge, this is the first study to develop a method to differentiate B. coli variants A and B without sequence analysis and to assess the molecular epidemiology of B. coli in pigs. Abstract Balantioides coli is a zoonotic protozoan parasite whose main reservoir is pigs. Recent studies have shown that B. coli variant A but not B has zoonotic potential. While B. coli infection has been reported in different animals and countries, the prevalence of the zoonotic variant is limited due to a lack of molecular information. Therefore, this study investigated the prevalence of B. coli in domestic pigs in Korea and assessed its zoonotic potential. A total of 188 pig fecal samples were collected from slaughterhouses in Korea. B. coli was identified by microscopy and molecular methods. B. coli was identified in 79 (42.9%) and 174 (94.6%) samples by microscopy and polymerase chain reaction (PCR), respectively. This study also developed a PCR-restriction fragment length polymorphism (PCR-RFLP) method to differentiate B. coli variant A from B without sequence analysis. Using this method, 62 (33.7%) and 160 (87.0%) samples were positive for variants A and B, respectively, and 48 (26.1%) samples were co-infected with both variants. Sequence and phylogenetic analyses showed a high genetic diversity of B. coli in pigs in Korea. To our knowledge, this is the first study to develop a method to differentiate B. coli variants A and B without sequence analysis and to assess the molecular epidemiology of B. coli in pigs. Continuous monitoring of zoonotic B. coli in pigs should be performed as pigs are the main source of human balantidiasis.

Salmonella enterica serotype Choleraesuis causes swine paratyphoid, with clinical findings of enterocolitis and septicemia. However, the clinicopathological features of S. Choleraesuis infections in pigs have not been reported in Korea. We describe the pathological findings of two weaned pigs with S. Choleraesuis infections, presenting with diarrhea, cough, and sudden death. Pathological examination indicated severe necrotic colitis in pig 1 and septicemic lesions in pig 2. Multidrug-resistant S. Choleraesuis was isolated from the pigs’ lungs and intestinal contents. Further research is required for the surveillance of S. Choleraesuis infections in pigs and the virulence estimation in the S. Choleraesuis isolates.

The broad-spectrum lytic capability of Salmonella bacteriophages against various Salmonella species was evaluated to determine their potential as an alternative for antibiotics, and the safety and preventive effects of the bacteriophages were assessed on mice and pigs. Four bacteriophage cocktails were prepared using 13 bacteriophages, and the lytic capability of the four bacteriophage cocktails was tested using Salmonella reference strains and field isolates. Bacteriophage cocktail C (SEP-1, SGP-1, STP-1, SS3eP-1, STP-2, SChP-1, SAP-1, SAP-2; ≥109 pfu/ml) showed the best lytic activity against the Salmonella reference strains (100% of 34) and field isolates (92.5% of 107). Fifty mice were then orally inoculated with bacteriophage cocktail C to determine the distribution of bacteriophages in various organs, blood and feces. The effects of bacteriophages on Salmonella infection in weaned pigs (n=15) were also evaluated through an experimental challenge with Salmonella Typhimurium after treatment with bacteriophage cocktail C. All mice exhibited distribution of the bacteriophages in all organs, blood and feces until 15 days post infection (dpi). After 35 dpi, bacteriophages were not detected in any of these specimens. As demonstrated in a pig challenge study, treatment with bacteriophage cocktail C reduced the level of Salmonella shedding in feces. The metagenomic analyses of these pig feces also revealed that bacteriophage treatment decreased the number of species of the Enterobacteriaceae family without significant disturbance to the normal fecal flora. This study showed that bacteriophages effectively controlled Salmonella in a pig challenge model and could be a good alternative for antibiotics to control Salmonella infection.

The alternative splicing pattern of transcription units can be influenced by the genotype of a neighboring locus, which is termed splicing quantitative trait locus (sQTL). Here we report a comprehensive catalog of sQTLs discovered from the public RNA-seq and matched genotype datasets of three European ancestries. Each pair of RNA-seq and genotype dataset was analyzed with IVAS, a locally developed R/Bioconductor package for sQTL discovery. A meta-analysis was applied to the three result sets to reach a consensus of 2525 sQTLs (FDR

Slow speed of the Next-Generation sequencing data analysis, compared to the latest high throughput sequencers such as HiSeq X system, using the current industry standard genome analysis pipeline, has been the major factor of data backlog which limits the real-time use of genomic data for precision medicine. This study demonstrates the DRAGEN Bio-IT Processor as a potential candidate to remove the “Big Data Bottleneck”. DRAGENTM accomplished the variant calling, for ~40× coverage WGS data in as low as ~30 minutes using a single command, achieving the over 50-fold data analysis speed while maintaining the similar or better variant calling accuracy than the standard GATK Best Practices workflow. This systematic comparison provides the faster and efficient NGS data analysis alternative to NGS-based healthcare industries and research institutes to meet the requirement for precision medicine based healthcare.

To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy.

Between 2011 and 2012, a total of 896 pig fecal samples were collected from nine provinces in Korea, and 50 salmonella enterica susp. enterica serovar Typhimurium (S. Typhimurium) was isolated. The characteristics of the 50 strains were analyzed, and 4 strains were identified as Salmonella enterica subsp. enterica serovar 4,[5],12:i:-. Salmonella 4,[5],12:i:- could not be distinguished from S. Typhimurium through phage typing, antimicrobial resistance testing or multiple-locus variable-number tandem repeat analysis (MLVA). However, among the four Salmonella 4,[5],12:i:- strains, one (KVCC-BA1400078) was identified as a Salmonella 4,[5],12:i:- clone isolated from humans in the United States, and another (KVCC-BA1400080) was identified as DT193, which has been primarily isolated from humans and animals in European countries. The presence of Salmonella 4,[5],12:i:- in Korea poses a significant threat of horizontal transfer between pigs and humans.

(Received: July 23, 2014; Revised: September 16, 2014; Accepted: October 13, 2014)Abstract : In Korea, brucellosis has been reported periodically in cattle and rarely in dogs; however, it has not previouslybeen screened in domestic animals such as elk, pigs and goats. To investigate the serological prevalence, serum sampleswere taken from the aforementioned animals annually during 2007-2013 and screened by the rose-bengal test (RBT)or modified RBT, after which positive sera were evaluated by the standard tube agglutination test (STAT). Finally,RBT and STAT-positive sera were confirmed by competitive-ELISA. Brucella abortus biovar 1 was isolated from threeelk that were shown to be positive serologically in 2008. There was no evidence of brucellosis in pigs. Based onserological monitoring and investigation of etiological agents, there is no evidence of outbreak of brucellosis in elk,pigs or goats of Korea since 2008. However, the possibility for brucellosis from cattle to affect these other livestockexists; therefore, extensive and continuous serological monitoring is required to maintain their brucellosis-free status.Keywords : brucellosis, competitive-ELISA, rose-Bengal test, serological test, standard tube agglutination test

Genome-wide association (GWA) studies have found many important genetic variants that affect various traits. Since these studies are useful to investigate untyped but causal variants using linkage disequilibrium (LD), it would be useful to explore the haplotypes of single-nucleotide polymorphisms (SNPs) within the same LD block of significant associations based on high-density variants from population references. Here, we tried to make a haplotype catalog affecting body mass index (BMI) through an integrative analysis of previously published whole-genome next-generation sequencing (NGS) data of 7 representative Korean individuals and previously known Korean GWA signals. We selected 435 SNPs that were significantly associated with BMI from the GWA analysis and searched 53 LD ranges nearby those SNPs. With the NGS data, the haplotypes were phased within the LDs. A total of 44 possible haplotype blocks for Korean BMI were cataloged. Although the current result constitutes little data, this study provides new insights that may help to identify important haplotypes for traits and low variants nearby significant SNPs. Furthermore, we can build a more comprehensive catalog as a larger dataset becomes available.

The plasmid-mediated colistin resistance gene mcr-1 was first reported in China in 2013, and has since been identified in both humans and livestock from many other countries (1-3,5).….

Streptococcus species are emerging potential pathogens in marine mammals. We report the isolation and identification of Streptococcus halichoeri and Streptococcus phocae in a Steller sea lion (Eumetopias jubatus) in South Korea.

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis . The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6 pg/μl by DNA dilution, or 3 × 10 3 colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis . Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis , and the new assay will provide a valuable tool for the diagnosis of B. canis infection.

A confirmatory serological test, the standard tube agglutination test (STAT) is evaluated for the diag-nostic efficiency in brucellosis Korea. A total of 345 bovine samples were collected from regional vet-erinary branch under national brucellosis monitoring program from January 2010 to June 2012 in Korea. These samples were diagnosed as suspected serum and brucellosis positive by the Rose Bengal test (RBT) and the STAT, respectively. The STAT was compared and evaluated with three serological test such as the indirect-enzyme linked immunosorbent assay (I-ELISA), competitive-enzyme linked im-munosorbent assay (C-ELISA) and fluorescence polarisation assay (FPA) prescribed for international trade by OIE. Among the 345 bovine serum samples, 302 (87.5%) were diagnosed as positive in the STAT, while 215 (62.3%), 223 (64.6%) and 194 (56.2%) serum samples were diagnosed as positive for brucellosis in the I-ELISA, C-ELISA and FPA, respectively. The STAT showed quite high positive results as compared with three prescribed tests of OIE. FPA, I-ELISA and C-ELISA have shown 60.6%, 64.9% and 67.2% correlation, respectively as compared to the STAT. However correlations of three pre-scribed tests ranged high 84.1∼97.7%. Especially, correlation between I-ELISA and C-ELISA is quite high, 97.7%. These results suggest that the STAT has shown many false-positive reactions. Therefore, additional serological test, such as ELISAs and FPA, would be necessary to adopt as a confirmatory test in the national surveillance program of bovine brucellosis in Korea.

From the end of July 2012, several cases of abortion have been happened at the Korean indigenous cattle farm with 124 heads in Chungbuk province, Korea. Serological tests such as Rose-bengal test (RBT) and standard tube agglutination test (STAT) have been performed according to the standard official protocols of bovine brucellosis and 41 cattle turned out to be brucellosis-positive simultaneously. To find out the main factors of brucellosis outbreaks and spreads, additional serological, etiological and molecular investigation were applied. Totally, 11 B. abortus were isolated from 10 cattle's specimens including lymph-nodes and/or testis, and drinking water in cowhouse. In genotyping by multi-locus VNTR assay (MLVA) using 17 loci markers, the present B. abortus isolates were shown all the same pattern, D1 genotype, which has been reported in Gyeonggi and Gangwon province, Korea. These results suggest that the input of brucellosis might come from neighboring farms directly or indirectly, even if by unknown factor and expansion within farm would accelerate by materials related with aborting cows.

Altogether 7720 Enterococcus faecalis and 3939 E. faecium isolated from food animals and animal carcasses during 2003-2014 in Korea were investigated to determine if linezolid-resistant (LR) enterococci (≥8μg/ml) are present. Overall, 12 E. faecalis and 27 E. faecium recovered from chickens (n=32), pigs (n=6), and cattle (n=1) were resistant to linezolid and were further characterized using molecular methods Most LR isolates were also resistant to chloramphenicol (97.44%) and florfenicol (92.31%). Molecular analysis showed no mutations in the 23S ribosomal RNA and in the ribosomal protein L3. The optrA gene was found in 89.74% of the LR enterococci, including 12 E. faecalis and 23 E. faecium isolates. Among them, 30 optrA-positive isolates co-carried phenicol exporter gene fexA. Seven LR E. faecium isolates had Asn130Lys mutations in the ribosomal protein L4, of which six also carried optrA gene. None of the isolates carried the mutliresistance gene cfr. Transfer of optrA gene was observed in 16 of the 35 optrA-positive isolates by conjugation. Pulsed-field gel electrophoresis demonstrated that the vast majority of Enterococcus strains carrying optrA gene were genetically heterogeneous. Multi-locus sequence typing revealed eight novel Sequence types among E. faecalis and E. faecium strains. To our knowledge, this is the first report of optrA gene in isolates from cattle and animal carcasses. This is also the first report of optrA gene in Korea. Active surveillance of optrA in enterococci is urgently warranted.

Closely related species share large genomic segments called syntenic regions, where the genomic elements such as genes are arranged co-linearly among the species. While synteny is an important criteria in establishing orthologous regions between species, non-syntenic regions may display species-specific features. As the first step in cataloging human- or primate- specific genomic elements, we surveyed human genomic regions that are not syntenic with any other non-primate mammalian genomes sequenced so far. Based on the data compiled in Ensembl databases, we were able to identify 10 such regions located in eight different human chromosomes. Interestingly, most of these highly human- or primate- specific loci are concentrated in subtelomeric or pericentromeric regions. It has been reported that subtelomeric regions in human chromosomes are highly plastic and filled with recently shuffled genomic elements. Pericentromeric regions also show a great deal of segmental duplications. Such genomic rearrangements may have caused these large human- or primate- specific genome segments.

Genome-wide association (GWA) studies may benefit from the inclusion of imputed SNPs into their dataset. Due to its predictive nature, the imputation process is typically not perfect. Thus, it would be desirable to de-velop a scheme for filtering out the imputed SNPs by maximizing the concordance with the observed geno-types. We report such a scheme, which is based on the combination of several parameters that are calculated by PLINK, a popular GWA analysis software program. We imputed the genotypes of 8,842 Korean individuals, based on approximately 2 million SNP genotypes of the CHB＋JPT panel in the International HapMap Project Phase II data, complementing the 352k SNPs in the original Affymetrix 5.0 dataset. A total of 333,418 SNPs were found in both datasets, with a median con-cordance rate of 98.7%. The concordance rates were calculated at different ranges of parameters, such as the number of proxy SNPs (NPRX), the fraction of success-fully imputed individuals (IMPUTED), and the information content (INFO). The poor concordance that was ob-served at the lower values of the parameters allowed us to develop an optimal combination of the cutoffs (IMPUTED≥0.9 and INFO≥0.9). A total of 1,026,596 SNPs passed the cutoff, of which 94,364 were found in both datasets and had 99.4% median concordance. This study illustrates a conservative scheme for filtering imputed SNPs that would be useful in GWA studies.Keywords: genome-wide association, HapMap, PLINK, SNP imputation

Multiple-locus variable number of tandem repeats analysis (MLVA) is useful for epidemiological investigations. In this study, MLVA-18 was used to assess the genetic and epidemiological relationships of Brucella canis isolates among a domestic dog and dogs from two breeding farms. MLVA-18 assay showed variation in copy numbers in 6 of the 18 loci. Interestingly, the YJ02-1 strain isolated from the pet dog from Yangju-si showed only one tandem repeat change in Bruce 09 locus as compared with that of NYJ-1 strain isolated from a dog from the Namyangju-si farm. The two strains were shown to have originated from the same source or a closely related strain.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as one of the most common serotypes related to human salmonellosis. In this study, the 22 isolates of S. 4,[5],12:i:- from food animals were identified by a specific multiplex polymerase chain reaction between 2009 and 2012. The isolation rate of S. 4,[5],12:i:- accounted for 1.7% (22/1271) of Salmonella spp. isolates from food animal origins: more specifically, 7.6% (18/235) from pigs and 0.6% (4/686) from chickens. The predominant S. 4,[5],12:i:- isolates in Korea belonged to phage type DT193 (12/22) with ampicillin-streptomycin-sulfonamide-tetracycline (ASSuT) resistance pattern (9/22). The XbaI-pulsed-field gel electrophoresis (PFGE) analysis revealed 11 different pulsotypes, and the major X-1 pattern was shared by 8 isolates. The isolates belonging to pattern X-1 were further subdivided into three BlnI-PFGE patterns and four variable-number tandem-repeat analysis (MLVA) allele combinations. The combining of MLVA and PFGE data could be valuable in characterizing highly clonal strains and discriminating their epidemiological relationship.

To diagnose brucellosis effectively, many genus-and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific singlenucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/μl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy.

A rapid and accurate diagnosis of brucellosis is required to reduce and prevent the spread of disease among animals and the risk of transfer to humans. In this study, a Brucella abortus-specific (Ba) LAMP assay was developed, that had six primers designed from the BruAb2_0168 region of chromosome I. The specificity of this LAMP assay was confirmed with Brucella reference strains, B. abortus vaccine strains, B. abortus isolates and phylogenetically or serologically related strains. The detection limit of target DNA was up to 20 fg/μl within 60 min. The sensitivity of the new LAMP assay was equal to or slightly higher than other PCR based assays. Moreover, this Ba-LAMP assay could specifically amplify all B. abortus biovars compared to previous PCR assays. To our knowledge, this is the first report of specific detection of B. abortus using a LAMP assay. The Ba-LAMP assay can offer a rapid, sensitive and accurate diagnosis of bovine brucellosis in the field.

The current brucellosis serodiagnostic assays are chiefly based on detecting anti-LPS (lipopolysaccharide) antibodies. However, cross-reaction with some gram-negative bacteria can occasionally induce due to similar O-polysaccharide (OPS) structure. Therefore, the aim of the present study was to identify new candidate antigens from Brucella abortus RB51, a mutant strain lacking the LPS portion, which might be valuable in brucellosis diagnosis. To detect potential antigens, immobilized pH gradients (IPG) strips with three ranges (pH 3–5.6, 4–7 and 6–11) were applied. After separating the insoluble proteins of B. abortus RB51 using two-dimensional electrophoresis (2-DE), their immunogenicity was evaluated by western blotting using four types of antisera – B. abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7-positive, and B. abortus-negative bovine sera. Among the several immunogenic spots, the spots showing specific reactivity with only the B. abortus-positive antisera, were considered as candidate antigens. Overall, eleven immuno-reactive proteins were identified, as follows: Cu/Zn superoxide dismutase, histidinol dehydrogenase, chaperonin DnaK, chaperonin GroES, beta-ketoadipyl CoA thiolase, two-component response regulator, the cell-division protein FtsZ, aldehyde dehydrogenase, 50s ribosomal protein L10 and invasion protein B. These selected highly immunogenic protein spots might be useful as alternative antigens for brucellosis and helpful in reducing the cross-reactivity.

The aim of this study was to develop in situ hybridization for detection of Mycoplasma hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from pigs with polyserositis. M. hyorhinis was isolated from the spleen (2 pigs) and pericardium (1 pig). M. hyorhinis DNA was detected 16 out of 20 pigs with polyserositis. In situ hybridization produced a distinct positive signal for the M. hyorhinis p37 gene in inflammatory cells in the polyserositis. In situ hybridization developed in the present study present diagnostic tools capable of detection of M. hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from the naturally infected pigs.

To investigate the epidemiologic relatedness of Brucella abortus isolates from Chinese water deer (Hydropotes inermis) and goral (Naemorhedus goral raddeanus) in 2010–2011, 22l isolates from livestock (including domestic elk, Cervus canadensis) were analyzed using the multilocus variable-number tandem repeats analysis. In the clustering analysis, Korean B. abortus isolates were divided into 40 genotypes by 18 markers, and 2 B. abortus isolates from wildlife were clustered with those of domestic cattle. Based on the minimum spanning tree, B. abortus isolates from wildlife were closely related to or had originated from livestock. Control measures are necessary to be able to block the transmission of Brucella between domestic and wild animals, and continuous monitoring of wildlife will be necessary to eradicate brucellosis in South Korea.

Brucellosis causes a great economic loss to the livestock industries through abortion, infertility, birth of weak and dead offspring, increased calving interval and reduction of milk yield and it is endemic in Bangladesh. The present study was performed to know the seroprevalence of brucellosis for 1000 ruminants (135 Buffaloes, 465 cattle, 230 goats and 170 sheep) in five different districts of Bangladesh by four conventional serological tests such as: Rose Bengal Plate Test (RBT), tube agglutination test (TAT), competitive enzyme-linked immunosorbent assay (C-ELISA), and Fluorescent polarization assay (FPA). Sheep has the highest prevalence (8.24%) of brucellosis. The seroprevalence of brucellosis was significantly higher in animals with previous abortion record in case of buffaloes, cattle, goats and sheep than that with no abortion record. C-ELISA can be the most suitable choice for extensive use in many kinds of livestocks and accurate estimation of Brucella antibodies in ruminants in Bangladesh.

Background Accurate classification into genotypes is critical in understanding evolution of divergent viruses. Here we report a new approach, MuLDAS, which classifies a query sequence based on the statistical genotype models learned from the known sequences. Thus, MuLDAS utilizes full spectra of well characterized sequences as references, typically of an order of hundreds, in order to estimate the significance of each genotype assignment. Results MuLDAS starts by aligning the query sequence to the reference multiple sequence alignment and calculating the subsequent distance matrix among the sequences. They are then mapped to a principal coordinate space by multidimensional scaling, and the coordinates of the reference sequences are used as features in developing linear discriminant models that partition the space by genotype. The genotype of the query is then given as the maximum a posteriori estimate. MuLDAS tests the model confidence by leave-one-out cross-validation and also provides some heuristics for the detection of 'outlier' sequences that fall far outside or in-between genotype clusters. We have tested our method by classifying HIV-1 and HCV nucleotide sequences downloaded from NCBI GenBank, achieving the overall concordance rates of 99.3% and 96.6%, respectively, with the benchmark test dataset retrieved from the respective databases of Los Alamos National Laboratory. Conclusions The highly accurate genotype assignment coupled with several measures for evaluating the results makes MuLDAS useful in analyzing the sequences of rapidly evolving viruses such as HIV-1 and HCV. A web-based genotype prediction server is available at http://www.muldas.org/MuLDAS/.

In situ hybridization and immunohistochemistry with different types of antibody (monoclonal vs. polyclonal, natural vs. synthetic) was compared to detect porcine circovirus 2 (PCV2) in formalin-fixed, paraffin-embedded tissues from pigs with experimentally and naturally occurring postweaning multisystemic wasting syndrome. PCV2 DNA and antigen was detected in tissues from both experimentally and naturally infected pigs by in situ hybridization and immunohistochemistry, respectively. Statistical evaluation revealed that more PCV2 positive signals were significantly detected in both experimentally and naturally infected pigs by in situ hybridization compared with immunohistochemistry (P