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Abstract Background Brucella is an intracellular pathogen capable of infecting animals and humans. There are six recognized species of Brucella that differ in their host preference. The genomes of the three Brucella species have been recently sequenced. Comparison of the three revealed over 98% sequence similarity at the protein level and enabled computational identification of common and differentiating genes. We validated these computational predictions and examined the expression patterns of the putative unique and differentiating genes, using genomic and reverse transcription PCR. We then screened a set of differentiating genes against classical Brucella biovars and showed the applicability of these regions in the design of diagnostic tests. Results We have identified and tested set of molecular targets that are associated in unique patterns with each of the sequenced Brucella spp. A comprehensive comparison was made among the published genome sequences of B. abortus, B. melitensis and B. suis. The comparison confirmed published differences between the three Brucella genomes, and identified subsets of features that were predicted to be of interest in a functional comparison of B. melitensis and B. suis to B. abortus. Differentiating sequence regions from B. abortus, B. melitensis and B. suis were used to develop PCR primers to test for the existence and in vitro transcription of these genes in these species. Only B. suis is found to have a significant number of unique genes, but combinations of genes and regions that exist in only two out of three genomes and are therefore useful for diagnostics were identified and confirmed. Conclusion Although not all of the differentiating genes identified were transcribed under steady state conditions, a group of genes sufficient to discriminate unambiguously between B. suis, B. melitensis, and B. abortus was identified. We present an overview of these genomic differences and the use of these features to discriminate among a number of Brucella biovars.

Porcine epidemic diarrhea virus (PEDV) causes substantial economic duress due to high mortality rates (80-100%) in neonatal piglets. While endemic in other parts of the world, PEDV was first reported in April 2013 in US. It quickly spread to majority of the states resulting in a loss of about one quarter of swine population. The rapid development of vaccines is a critical component of successful pandemic preparedness plans. The enormous and urgent need for technology to develop rapid response vaccines is the focus of this study. To combine safety and efficacy advantages of inactivated and attenuated vaccines, we disrupted the integrity of the viral genomic RNA, without alternating viral structure to develop the PEDV rapid-response vaccine candidate. Four-week-old piglets were immunized with developed vaccine and challenged with same PEDV virus strain. Vaccinated piglets mounted strong spike-protein specific binding antibody responses and virus neutralization titers. Post challenge viral shedding and intestinal lesions as assessed were undetectable in vaccinated pigs, while they were evident in control pigs, indicating complete protection. No shedding of the vaccine virus prior to challenge or any untoward reactions at the site of injection or in tissues were noted. Hence the developed method showed significant promise in terms of efficacy, safety and rapidness of development; characteristics which were targeted for vaccine development in outbreak or pandemic situations., Gagandeep Sing * (G1), Pankaj Singh (1), Angela Pillatzki (2), Eric Nelson (2), Brett Webb (3), Seven Dillberger-Lawson (2) and Sheela Ramamoorthy (1) (1) Department of Microbiological Sciences, N. Dakota [...]

TTSuVI of family Anelloviridae, has negative sense, single-stranded, circular DNA genomes and is distributed globally in swine population. As TTSuV found in both diseased and healthy domestic pigs, it's role in causing specific swine disease is still unclear. Experimental studies in gnotobiotic pigs indicated that TTSuV1 can be a primary or co-infecting pathogen in clinical syndromes such as the porcine dermatitis and nephropathy syndrome and post-weaning multi-systemic wasting syndrome. However, it is not known whether cross-species transmission of TTV's occur. In this study by using TTSuV-1 - specific ELISA and a panel of TTSuV1-specific PCRs targeting the conserved untranslated regions or more variable open reading frames, the combined serological and molecular epidemiological profile of TTSuV1 infection in five different species (equine, canine, bovine, ovine and elk) was determined using sera samples from the target species. Significant levels of antibody responses to TTSuV1 were detected bovine, ovine and equine samples as compare to canine and elk. TTSuV1 DNA was detected in 46.7% of equines, 70% of canine, 100% of bovine, 40% of ovine and 93.3% of elk by the real-time targeting the UTR region. Detection by PCR's targeting the ORF's was variable. The data from this study provides preliminary evidence that TTSuV1 could infect other species. However, further studies are required to demonstrate the health risk to these animal species from TTSuV-1 infection., Gagandeep Singh * (G) and Sheela Ramamoorthy Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND Support: This project was funded by NIH P30 GM103332 from the National Institute [...]

The porcine epidemic diarrhea virus (PEDV) was first detected in the U.S in 2013 and caused a loss of roughly one quarter of the swine population and several millions of dollars in 1 year. Reliable methods to culture and quantify the virus are essential for vaccine and diagnostic test development. To circumvent the subjective and time consuming nature of current methods, we have developed a colorimetric assay that is inexpensive, convenient, and adaptable to a high-throughput format for the quantification of PEDV virus. The conventional TCID50 and plaque assays were performed by standard methods. After visual assessment of the end point by an operator, a tetrazolium dye called MTT was added to detect cell viability. Optical density (OD) readings were obtained by spectrophotometry. Receiver Operator Characteristics analysis was performed to determine the sensitivity (Se), specificity (Sp) and OD cutoff values for the developed assay. The Sp and Se of the MTT50 assay in comparison to the conventional TCID50 assay were 95% and 81% respectively at a software specified cutoff value of 0.53. The agreement between the tests as determined by a kappa statistic value was 0.83 with a 95% confidence interval of 0.780 to 0.874. Currently, the colorimetric method is being adapted to titrate virus neutralizing antibody responses. The methods developed in this study will significantly reduce the difficulty associated with the manual examination of a large volume of PEDV test., Jenna Karsky * (U), Pankaj Singh, and Sheela Ramamoorthy Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND Support: This study was supported by the Agricultural Products Utilization Committee [...]

To the Editor: Brucellosis is a serious, globally distributed zoonotic disease. Humans are susceptible to infection by Brucella suis, B. abortus, B. melitensis, and B. canis and can have lifelong [...]

Anelloviridae is a recently described family of small DNA viruses that infect a variety of animals. Torque teno virues (TTVs) were the first anelloviruses discovered, and they remain the most [...]

Purpose of the research: Swine Torque Teno sus viruses (TTSuV or TTV) resemble human TTVs and belong to family Anelloviridae, genus Iotatorquevirus. There are two main swine TTV groups--TTV1 and TTV2. Swine TTV1 is non-enveloped, circular, negative sense single stranded DNA virus of 2.8 kb. The TTV1 genome has three open reading frames--ORF1, 2 and 3 [1]. Although, widely distributed in swine populations, currently there is no data to confirm the replication of DNA and growth of viable swine TTV1 virions in animal cell cultures. Moreover, TTV contamination of mammalian cell cultures used for biomedical experimentation may result in unintended experimental consequences. Methodology: In the current study, DNA was obtained from swine cell lineages--PK15N (NVSL), PK15 (ATCC), ST, 3D4/31, and cell culture media components including trypsin, Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (10%)(complete DMEM) and Hank's balanced salt solution (HBSS). Extracted DNA were examined for the presence of TTV1 DNA using polymerase chain reaction (PCR) with TTV1 specific primers. For all the samples that were positive for TTV1 DNA, the presence of TTV1- specific mRNA was examined by reverse transcription PCR targeting the TTV1 ORF3 which is a spliced gene. Further investigation to assess the presence of TTV1 ORF1 capsid protein expression in DNA positive cell cultures via immunofluorescence assay is underway. Results and conclusions: TTV1 DNA was observed in cell lineages--ST, 3D4/31, as well as complete DMEM and trypsin. However, no viral DNA was found in PK15N, PK15 (ATCC) cells as well as HBSS although the same media was used to culture all cell lines, indicating that the DNA was detected in cells. Further research is being carried out to confirm the mRNA presence and protein expression of TTV1. The presence of the viral DNA highlight that there is a possibility of TTV1contamination in cell cultures and at the same time the evidence may be indicating the actual replication of TTV1 in vitro., References: [1] Niel C, Diniz-Mendes L, Devalle S. (2005) J Gen Virol., 86(5) pp 1343-1347. Anuradha Vegi [1] *, Sheela Ramamoorthy [1] [1] Department of Veterinary & Microbiological Sciences, North [...]

Porcine circovirus strain 2 (PCV2) is the cause of porcine circovirus associated diseases (PCVAD) in pigs, a disease that has caused great economic losses in the pig industry in the [...]

Contemporary vaccines against Porcine Reproductive and Respiratory Disease Syndrome viruses (PRRSV) and Swine Influenza viruses (SIV) are composed of either live attenuated viruses that risk frequent recombination between viral and [...]