Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.

Genetic diversity and prevalence of Atypical Porcine Pestivirus in the Midwest of US swine herds during 2016-2018.

Atypical porcine pestivirus (APPV), a highly divergent pestivirus, has a wide geographical distribution around the world. APPV is known to cause type A-II congenital tremors in newborn piglets. The main objective of this study is to access APPV prevalence in the US swine herds utilizing a newly developed quantitative real-time RT-PCR assay. Retrospective analysis of 1,785 samples revealed a 19.0% prevalence in Midwest swine herds over a period of three years (2016-2018). Among all clinical and field samples that were APPV positive, 82 samples (24.19%) were also positive for one or more swine viral pathogens. Two APPV US strains identified in this study demonstrated significant sequence diversity (~12% in full genome) compared to the first reported APPV strain from the United States in 2014. Of the two strains identified in this study, USA/023005/2016 is closer to two strains identified in Germany, and USA/047310/2017 shares more similarities with two US strains including Minnesota-1 and ISDVDL2014016573. Partial NS5B sequences (9127-9836 nt of the polyprotein gene) obtained from 54 APPV-positive samples revealed considerable sequence diversity, ranging from 85.8% to 100% nucleotide identity, within the US strains in samples from different geographic regions. Analysis of all US samples indicates high prevalence of APPV in Minnesota (37.35%), followed by Illinois (32.86%), Iowa (30.60%) and Kansas (21.89%). APPV was detected in 15.48% of samples assayed from 2017, slightly higher than that in 2016 (13.08%), but much lower than 2018 (28.77%). Among the various sample types tested, oral fluid samples had the highest prevalence and lowest average Ct value suggesting their suitability as a reliable diagnostic specimen for APPV detection. Overall, sequence variation among APPV strains and prevalence of the pathogen within the United States provides a basis for understanding the genetic diversity and molecular epidemiology of APPV in the US swine herds.

Evaluation by polymerase chain reaction assay of persistent shedding of pathogenic leptospires in the urine of dogs with leptospirosis.

BACKGROUND: Persistent leptospiruria in naturally infected dogs occurs despite appropriate antibiotic treatment. HYPOTHESIS/OBJECTIVES: To determine the frequency of persistent leptospiruria in naturally infected dogs and the association of persistent leptospiruria with different antibiotic treatments. ANIMALS: Thirty-two dogs of varying age and breed diagnosed with leptospirosis via urine polymerase chain reaction assay (PCR). METHODS: A prospective observational study of dogs diagnosed with leptospirosis was undertaken to determine the frequency of persistent leptospiruria as determined by PCR. Clinical presentation of leptospirosis, antibiotic treatment, serum creatinine concentration, and outcome were recorded. RESULTS: Fifteen of 32 dogs had a negative urine PCR on the first submission in the study, 5 of 15 received only an aminopenicillin. The remaining 17 dogs had a negative urine PCR on the second (n = 6 dogs), third (n = 5), fourth (n = 5), and eighth (n = 1) submissions. Acute kidney injury was reported in 32/32 dogs. Two of 32 dogs developed chronic kidney disease. CONCLUSIONS AND CLINICAL IMPORTANCE: Persistent leptospiruria is common despite treatment with antibiotics frequently recommended for treatment. Follow-up urine PCR to confirm clearance of the organism is recommended in all dogs. In dogs with persistent leptospiruria, chronic kidney disease can develop after acute kidney injury.

Development of a quantitative real time RT-PCR assay for sensitive and rapid detection of emerging Atypical Porcine Pestivirus associated with congenital tremor in pigs.

Atypical Porcine Pestivirus (APPV) is reported as the etiologic agent for type AII congenital tremors in newborn piglets. Initial PCR-based diagnostic tests to detect APPV were designed based on the limited sequence information and are not capable of detecting the majority of APPV strains. A sensitive and reliable PCR-based diagnostic test is critical for accurate detection of APPV. In this study, a quantitative reverse transcription PCR (RT-qPCR) assay was developed for reliable detection of all currently known APPV strains. The assay design also included swine 18S rRNA gene as an internal control to monitor RNA extraction efficiency. Two APPV gene fragments, one each from NS5b and NS3, were cloned and used to determine the dynamic range of detection, linearity and analytical sensitivity/limit of detection (LOD). Both individual and multiplex assays (duplex and triplex) had correlation coefficients of >0.99 and PCR amplification efficiencies of >90 %. Comparison of detection limit and analytical sensitivity between individual, and multiplex assays indicated no inhibition of PCR sensitivity upon multiplexing. The detection limit for APPV target, based on analytical sensitivity, is 7.75 copies (NS5b) and 5.2 copies (NS3) per reaction. Assay specificity was verified by testing nucleic acids of other closely related pestiviruses and clinical samples that are positive for other common swine pathogens. Assay sensitivity was also assessed on synthesized gene fragments of the most divergent China strains. Testing 339 known APPV-positive and 202 negative clinical samples demonstrated a good diagnostic sensitivity and specificity. Data from six independent runs, including 5 replicates of three clinical samples with three Ct ranges, were utilized to assess inter-assay repeatability and intra-assay reproducibility. This analysis demonstrated intra-assay/inter-assay coefficients of variation of 0.71 % and 0.01 %, respectively, with a PCR efficiency of 92.71 % for the triplex assay. Testing of 1785 clinical samples revealed ∼19 % prevalence of APPV in the US swine herds and oral fluids demonstrates to be a reliable specimen for viral detection. This multiplex RT-qPCR assay offers a rapid and reliable detection of APPV in swine herds and serves as useful tool in APPV surveillance and epidemiological investigations.

Development of a differential multiplex real-time PCR assay for porcine circovirus type 2 (PCV2) genotypes PCV2a, PCV2b and PCV2d.

A multiplex quantitative real-time polymerase chain reaction (mqPCR) assay was developed and validated for detection and differentiation of porcine circovirus type 2 (PCV2) genotypes, PCV2a, PCV2b and PCV2d. Single nucleotide polymorphism in primers or probes was deployed for different genotype detections, while conserved sequence in the 3' end of a primer and in the middle of a probe was used for the targeted genotype. In silico analysis of 2601 PCV2 ORF2 sequences showed that the predicted strain coverage of the assay was 93.4 % (409/438) for PCV2a, 95.1 % (1161/1221) for PCV2b and 93.6 % (882/942) for PCV2d strains. The PCR amplification efficiencies were 94.5 %, 100.2 %, and 99.2 % for PCV2a, PCV2b and PCV2d, respectively, with correlation coefficients >0.995 for all genotypes. The limits of detection (LOD) were 1.58 × 10-4 TCID50/mL for PCV2a, 5.62 × 10-4 TCID50/mL for PCV2b, and 3.16 × 10-3 TCID50/mL for PCV2d. Sanger sequencing of 74 randomly selected PCV2 positive clinical samples confirmed the genotypes of strains identified by the mqPCR. Validation with clinical samples co-positive for target and non-target pathogens demonstrated that the mqPCR assay specifically detected targeted viruses without cross reacting to each other or to other common porcine viruses.

Development of a real-time PCR assay for detection of African swine fever virus with an endogenous internal control.

Real-time PCR assays are highly sensitive, specific and rapid techniques for the identification of ASF virus (ASFV) (Section 3.8, OIE Terrestrial Manual, 2019). Although an ASFV p72 gene-based real-time PCR assay (a.k.a. the Zsak assay) (Journal of Clinical Microbiology, 2005, 43, 112) has been widely used for ASFV detection, several more ASFV whole genome sequences have become available in the 15 years since the design of the Zsak assay. In this study, we developed a new ASFV p72 gene-based real-time PCR after analysis of all currently available sequences of the p72 gene and multiplexed the new assay with a modified Zsak assay aiming to have a broader coverage of ASFV strain/isolates. To reduce false-negative detections, porcine house-keeping gene, beta actin (ACTB), was applied as an internal control. Eight ACTB sequences from the GenBank and 61 partial ACTB sequences generated in this study, and 1,012 p72 sequences from the GenBank and 23 p72 sequences generated at FADDL, were used for ACTB and ASFV primer and probe designs, respectively, to ensure broader host and ASFV coverage. Multiplexing ACTB in the reaction did not affect ASFV amplification. The multiplex assay was evaluated for strain/isolate coverage, sensitivity and specificity. The in silico analysis showed high ASFV strain/isolate coverage: 98.4% (978/994) of all p72 sequences currently available. The limit of detection (LOD) was 6 plasmid copies or 0.1-1 TCID50 /ml of ASFV isolates per reaction. Only targeted ASFV isolates and the viruses in the positive clinical samples were detected, indicating that the assay is highly specific (100% specificity). The test results of 26 ASFV isolates with different country origins showed that this newly developed multiplex assay performed better than the Zsak assay that has been widely accepted and used worldwide, indicating that it may be used as an alternative assay for ASFV detection.

Development of a nested PCR assay for detection of Streptococcus equi subspecies equi in clinical equine specimens and comparison with a qPCR assay.

Streptococcus equi subsp. equi is a Gram positive bacterial pathogen commonly associated with strangles in horses, a respiratory disease characterized by abscessation of submandibular and retropharyngeal lymph nodes which can lead to obstruction of the airway. Several real-time PCR (qPCR) assays have been developed for detection of S. equi from horses with many targeting conserved regions of the S. equi cell wall-associated M-protein (SeM), a major virulence factor and immunogen of S. equi. Our objective was to develop a nested PCR (nPCR) targeting SeM and an 18S rRNA internal control gene for detection of S. equi from horses with potential improvement in detection sensitivity compared to a qPCR. Primers and probes from the Kansas State Veterinary Diagnostic Laboratory (KSVDL) S. equi clinical testing assay were utilized for all qPCR testing. Primers flanking the SeM qPCR target region were selected for an initial end-point PCR step of the nested assay; PCR product from the end-point reaction then served as template for the qPCR reaction step of the nested assay. Sample nucleic acid was also tested directly with qPCR to allow for assay comparison. Nucleic acid from clinical specimens (n = 188) submitted to KSVDL were tested in parallel with each assay. The nPCR and qPCR assays identified 22.9% (43/188) and 13.3% (25/188) of samples positive for S. equi, respectively. None of the samples positive by qPCR were negative by nPCR. The PCR products from all positive samples were submitted for DNA sequencing. Each of the 25 samples positive by both assays had a high nucleotide identity match (>96%) to the SeM gene. Among the samples positive by nPCR but negative by qPCR, 17 of 18 were sequence confirmed for SeM at greater than 96% nucleotide identity. Based on the nPCR Ct (37.8) of the one sequence un-confirmed case, it is likely that the S. equi bacterial load in this sample was below the necessary concentration for successful sequencing. Limit of detection (LOD) for the nPCR was established at a Ct of 37, and based both on the LOD of the qPCR assay (Ct of 37), as determined by standard curve data, and on the highest nPCR Cts (~37) of clinical samples able to result in SeM sequence-confirmation. As demonstrated by sequencing confirmation, the nPCR assay targeting the SeM gene is highly specific to S. equi. The increased sensitivity of the nPCR, compared to the qPCR, may reduce the number of false negative sample results in clinical testing and provide a superior detection method during low bacterial shedding periods.

Genetic diversity and prevalence of porcine circovirus type 3 (PCV3) and type 2 (PCV2) in the Midwest of the USA during 2016-2018.

In recent years, reports indicated that PCV3 may be involved in porcine dermatitis and nephropathy syndrome (PDNS)-like disease similar to that linked to PCV2. A total of 2,125 porcine samples from 910 cases were collected during 2016-2018 and tested for presence of PCV3 and PCV2 by real-time PCR assays. Results showed high prevalence of PCV3 and PCV2: 28.4% samples from 41.2% cases were PCV3 positive and 16.4% samples from 16.7% cases were PCV2 positive. The overall coinfection rate was 5.4% and 8.4% at the sample and case level, respectively. Temporal analysis indicated that PCV3 positive case rate increased from 31.6% in 2016, 40.9% in 2017, to 55.6% in 2018. Although its prevalence was lower, PCV2-positive case rate in 2018 (28.8%) doubled that in 2017 (14.4%). The coinfection case rate also increased from 3.4% in 2016, 8.0% in 2017 to 16.1% in 2018. The high positive rate of PCV3 (56.9%) and PCV2 (33.8%) in oral fluids, PCV3 in foetuses (57.1%) and PCV2 in tonsils (54.8%) implied viral transmission route and tissue tropism. In phylogenetic analysis, two small PCV3 clusters (1 and 2) were separated but others were clustered with low bootstrapping values indicating overall low genetic diversity. Genotypes, PCV2a-h, were confirmed by analysing 2,944 strains, with a new genotype proposed as PCV2i. In this study, 61 PCV3 unique whole genomes were sequenced; 12 belonged to a separate cluster that were characterized by five consistent amino acid changes in the capsid protein (24V, 27K, 56D, 98R and 168K) and may be associated with potential differences in immunogenicity. Among the 43 unique PCV2 whole genomes sequenced, 31 belonged to PCV2d, 7 to PCV2a and 5 to PCV2b. Thus, our study demonstrates that PCV2d is the predominant genotype and PCV3 is widely circulating in the Midwest of the USA.

Development of a multiplex real-time RT-PCR assay for simultaneous detection and differentiation of influenza A, B, C, and D viruses.

Influenza is a common and contagious respiratory disease caused by influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV). A multiplex real-time RT-PCR assay was developed for simultaneous detection of IAV, IBV, ICV, and IDV. The assay was designed to target unique sequences in the matrix gene of IBV and ICV, the RNA polymerase subunit PB1 of IDV, and combined with USDA and CDC IAV assays, both target the matrix gene. The host 18S rRNA gene was included as an internal control. In silico analyses indicated high strain coverages: 97.9% for IBV, 99.5% for ICV, and 100% for IDV. Transcribed RNA, viral isolates and clinical samples were used for validation. The assay specifically detected target viruses without cross-reactivity, nor detection of other common pathogens. The limit of detection was approximately 30 copies for each viral RNA template, which was equivalent to a threshold cycle value of ~37.

A multiplex real-time PCR assay for the detection and differentiation of five bovine pinkeye pathogens.

Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is one of the most common eye diseases in cattle. Several pathogens have been associated with IBK cases, however, Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and bovine herpesvirus type 1 (BHV-1) are most frequently observed. A multiplex real-time PCR assay using two reactions was developed for the detection and differentiation of these five pathogens. Detection sensitivities of the multiplex assays were compared to singleplex reactions testing for the same targets. Correlation coefficients (R2) of >0.99, and PCR efficiencies between 92 and 106% were demonstrated in all singleplex and multiplex real-time PCR reactions. The limits of detection (LOD) of multiplex assays for Moraxella bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi and BHV-1 were 19, 23, 25, 24 and 26 copies per reaction, respectively. No cross amplification was observed for specificity testing of 179 IBK positive clinical samples and 55 non-target clinical samples. Percentage of clinical samples positive for Mycoplasma bovoculi, Moraxella bovoculi, Moraxella bovis, BHV-1 and Mycoplasma bovis were 88.8% (159/179), 75.9% (136/179), 60.3% (108/179), 11.7% (21/179) and 10.0% (18/179), respectively. Moraxella bovis, Moraxella bovoculi and Mycoplasma bovoculi were more prevalent than Mycoplasma bovis and BHV-1 in IBK samples collected from animals in this study population. Our data indicates that the multiplex real-time PCR panel assay is highly sensitive and highly specific for the detection and differentiation of the five major pathogens associated with bovine pinkeye.

A multiplex real-time PCR assay for the detection and differentiation of the newly emerged porcine circovirus type 3 and continuously evolving type 2 strains in the United States.

A multiplex quantitative real-time polymerase chain reaction (mqPCR) assay was developed and validated for the detection and differentiation of porcine circovirus type 3 (PCV3) and type 2 (PCV2) strains. The assay coverage was 97.9% (184/188) for PCV3 and 99.1% (1889/1907) for PCV2 sequences that were available from the current GenBank database. The PCR amplification efficiencies were 98-99% for plasmids, and 92-96% for diagnostic samples, with correlation coefficients all greater than 0.99. The limit of detection (LOD) determined as plasmid copies per reaction was 17 for PCV3 and 14 for PCV2. The assay specifically detected the targeted viruses without cross reacting to each other or to other common porcine viruses. Among 336 swine clinical samples collected in 2018, 101 (30.1%) were PCV3 positive, 56 (16.7%) were PCV2 positive and 18 (5.4%) were co-positives. Sixty selected PCV3 positives were confirmed by Sanger sequencing, and 53 of the 56 PCV2 positive samples were tested positive by another validated PCR assay.

Complete Genome Sequence of an Influenza C Virus Strain Identified from a Sick Calf in the United States.

Influenza C virus (ICV) has been identified for the first time from bovine respiratory disease complex (BRDC) samples in the United States. Here, we report the complete genome sequence of the strain C/bovine/Montana/12/2016, identified from a nasal swab sample collected from a sick calf with clinical signs of respiratory disease in Montana.

Influenza C Virus in Cattle with Respiratory Disease, United States, 2016-2018.

We identified influenza C virus (ICV) in samples from US cattle with bovine respiratory disease through real-time PCR testing and sequencing. Bovine ICV isolates had high nucleotide identities (≈98%) with each other and were closely related to human ICV strains (≈95%). Further research is needed to determine bovine ICV's zoonotic potential.

Molecular detection and characterization of transient bovine viral diarrhea virus (BVDV) infections in cattle commingled with ten BVDV persistently infected cattle.

Fifty-three cattle of unknown serologic status that were not persistently infected (PI) with bovine viral diarrhea virus (BVDV) were commingled with 10 cattle that were PI with different strains of BVDV, and were monitored for an extended commingle period using a reverse-transcription real-time PCR (RT-rtPCR) BVDV assay on various sample types. Transient infections with BVDV were also assessed by virus isolation, virus neutralization (VN) assays, and direct buffy coat 5'-UTR sequencing. Infections were demonstrated in all cattle by RT-rtPCR; however, the detection rate was dependent on the type of sample. Buffy coat samples demonstrated a significantly greater number of positive results ( p ≤ 0.05) than either serum or nasal swab samples. Presence of elevated BVDV VN titers at the onset inversely correlated with the number of test days positive that an individual would be identified by RT-rtPCR from buffy coat samples, and directly correlated with the average Ct values accumulated over all RT-rtPCR test days from buffy coat samples. Both single and mixed genotype/subgenotype/strain infections were detected in individual cattle by direct sample 5'-UTR sequencing. A BVDV-2a strain from a PI animal was found to be the predominant strain infecting 64% of all non-PI cattle; BVDV-1b strains originating from 3 PI cattle were never detected in non-PI cattle. Although direct sample 5'-UTR sequencing was capable of demonstrating mixed BVDV infections, identifying all strains suspected was not always efficient or possible.

Genetic diversity in envelope genes of contemporary U.S. porcine reproductive and respiratory syndrome virus strains influences viral antigenicity.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases in swine caused by porcine reproductive and respiratory syndrome virus (PRRSV). Genome sequences of sixty-six PRRSV strains were obtained using metagenomic sequencing of serum samples collected in the U.S. in 2014 to explore contemporary genetic diversity. Phylogenetic analysis of the genes encoding the envelope proteins identified four to eight distinct lineages with >87% intraclade identity. To explore the effect of the observed genetic diversity on antigenicity, the genome regions encoding either GP2a-GP3-GP4 or GP5-M in strain SD95-21 were replaced with alleles from each of eight distinct PRRSV strains using reverse genetics. The GP2a-GP3-GP4 region from only four of the eight strains yielded viable recombinant virus. When viable, both GP2a-GP3-GP4 and GP5-M variably affected antigenicity. A strain-dependent significant loss in cross reactivity was variably observed by indirect immunofluorescence assays using antisera from pigs vaccinated with commercial modified-live vaccines following replacement of GP2a-GP3-GP4 or GP5-M. Significantly reduced neutralization titers were similarly measured using antisera from naturally PRRSV-exposed pigs. These results illustrate the need to consider genomic regions besides GP5 for PRRSV epidemiology and vaccination.

Evaluation of conjunctival bacterial flora in a herd of goats in the Midwestern United States.

OBJECTIVE: To identify conjunctival bacterial flora in healthy adult and newborn kid goats in the Midwestern United States and to compare vaginal and ocular surface flora in dam-kid pairs. ANIMALS STUDIED: Thirty mixed-breed (crosses between Boer, Kiko, and Syfan Spanish) goats, 20 adult does and 10 newborn kids. PROCEDURES: One eye of 15 adult goats (n = 15 eyes) and 5 kids (n = 5 eyes) was randomly selected. A subset of 5 adults (n = 10 eyes) and 5 kids (n = 10 eyes) underwent bilateral sampling. Each recently kidded dam's vaginal canal (n = 10) was also sampled. Two swabs were collected from each sample site for aerobic bacterial culture and Mycoplasma and Chlamydia spp. PCR. RESULTS: Of the animals with positive cultures, Staphylococcus and Streptococcus were the most common bacterial genera from the conjunctival sac of adult (16/17; 94%) and kid (5/5; 100%) goats; three adults and 5 kids had no growth of bacteria on aerobic culture. Moraxella bovoculi was the most common single bacteria in adults, in 9 eyes (36%) of 8 animals (40%). Staphylococcus equorum was identified in all 5 kids with positive cultures. Mycoplasma sp. DNA was detected in 7 animals. Chlamydia sp. DNA was not detected in any sample. Four of 10 dam-kid pairs had identical bacteria isolated from the dam's vaginal sample and the kid's conjunctival sample. CONCLUSIONS: Staphylococcus and Streptococcus were the most common conjunctival bacterial genera in this goatherd. Moraxella bovoculi was the most common single bacteria isolated from adults, and Staphylococcus equorum was the most common bacteria in kids. Mycoplasma sp. occurred infrequently at the ocular surface of adult and kid goats. A convincing association between dam-kid vaginal-conjunctival samples was not identified.

Tissue localization, shedding, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus following inoculation of 4-week-old feeder pigs.

We determined tissue localization, shedding patterns, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus (PEDV) following inoculation of 4-week-old feeder pigs. Thirty-three pigs were randomly assigned to 1 of 3 groups for the 42-day study: inoculated (group A; n = 23), contact transmission (group B; n = 5), and aerosol transmission (group C; n = 5). Contact transmission occurred rapidly to group B pigs whereas productive aerosol transmission failed to occur to group C pigs. Emesis was the first clinical sign noted at 3 days postinoculation (dpi) followed by mild to moderate diarrhea lasting 5 more days. Real-time PCR detected PEDV in fecal and nasal swabs, oral fluids, serum, and gastrointestinal and lymphoid tissues. Shedding occurred primarily during the first 2 weeks postinoculation, peaking at 5-6 dpi; however, some pigs had PEDV nucleic acid detected in swabs collected at 21 and 28 dpi. Antibody titers were measurable between 14 and 42 dpi. Although feces and intestines collected at 42 dpi were PEDV negative by PCR and immunohistochemistry, respectively, small intestines from 70% of group A pigs were PCR positive. Although disease was relatively mild and transient in this age group, the results demonstrate that 4-week-old pigs are productively infected and can sustain virus replication for several weeks. Long-term shedding of PEDV in subclinically affected pigs should be considered an important source for PEDV transmission.

A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.

Discovery of a novel putative atypical porcine pestivirus in pigs in the USA.

Pestiviruses are some of the most significant pathogens affecting ruminants and swine. Here, we assembled a 11 276 bp contig encoding a predicted 3635 aa polyprotein from porcine serum with 68 % pairwise identity to that of a recently partially characterized Rhinolophus affinis pestivirus (RaPV) and approximately 25-28 % pairwise identity to those of other pestiviruses. The virus was provisionally named atypical porcine pestivirus (APPV). Metagenomic sequencing of 182 serum samples identified four additional APPV-positive samples. Positive samples originated from five states and ELISAs using recombinant APPV Erns found cross-reactive antibodies in 94 % of a collection of porcine serum samples, suggesting widespread distribution of APPV in the US swine herd. The molecular and serological results suggest that APPV is a novel, highly divergent porcine pestivirus widely distributed in US pigs.