A Computationally Designed Serological Assay for Porcine Epidemic Diarrhea Virus.

The periodic emergence of new infectious agents and the genetic and antigenic evolution of existing agents necessitate the improvement of technology for the rapid development of diagnostic assays. The porcine epidemic diarrhea virus (PEDV) emerged in the United States in 2013, causing severe economic damage to the pork industry. The primary goal of this study was to develop methods to reduce the lead time for serological assay development. An approach involving the computational prediction of diagnostic targets, followed by a rapid synthesis of antigens, was adopted to achieve this objective. To avoid cross-reactivity with other closely related swine coronaviruses, the N protein sequences of PEDV were analyzed to identify sequences unique to PEDV. The potential antigenicity of the identified sequence was predicted computationally using the Jameson-Wolf method. A sequence with a high antigenic index was rapidly synthesized using an in vitro transcription and translation system to yield the diagnostic antigen. The computationally designed enzyme-linked immunosorbent assay (ELISA) was validated using 169 field sera, whose statuses were determined by a PEDV-specific immunofluorescence assay. Comparison of the computationally designed ELISA to a conventionally developed ELISA, using bacterially expressed N protein, and to the immunofluorescence assay showed a high degree of agreement among the three tests (mean kappa statistic, 0.842). The sensitivity and specificity, compared to the conventionally developed assay, were 90.62 and 95.18, respectively. Therefore, the described approach is useful in reducing the development time for serological assays in the face of an infectious disease outbreak.

Trans-replicase helper activity of porcine circoviruses promotes the synergistic replication of torque teno virus.

While the primary pathogenic potential of torque teno viruses (TTVs) is yet to be defined, TTVs are often co-detected with other pathogens and are suspected of exacerbating clinical disease in coinfections. Swine TTVs (TTSuVs) enhance clinical signs of porcine circovirus type 2 (PCV2) in a gnotobiotic pig model. However, the mechanisms involved are unknown. In this study, we observed that co-culture of TTSuV1 and PCV1, and specifically supplementing TTSuV1 cultures with the PCV replicase protein in trans consistently resulted in higher levels of replication of TTSuV1 when compared to TTSuV1 cultured alone. Therefore, the hypothesis that the PCV replicase (rep) protein has trans-replicase helper activity for TTSuV1 was examined. Based on EMSA and reporter gene assays, it was determined that the PCV1 rep directly interacted with the TTSuV1 UTR. The TTSuV1 rep trans-complemented a PCV rep null mutant virus, indicating that the TTSuV1 and PCV1 replicase proteins supported the replication of both viruses. In mice, the administration of plasmids encoding the PCV1 rep and a TTSuV1 infectious clone resulted in the production of higher TTSuV1 genome copies in dually exposed mice when compared to singly exposed mice. Higher sero-conversion and lymphoid hyperplasia were also observed in the dually exposed experimental mice. Thus, this study provides evidence for trans-replicase activity of PCVs and TTVs as a novel mechanism of explaining enhanced viral replication in coinfections involving both viruses.

Screening and selection of essential oils for an intranasal spray against bovine respiratory pathogens based on antimicrobial, antiviral, immunomodulatory, and antibiofilm activities.

Introduction: The rise in antibiotic resistant pathogens associated with bovine respiratory disease (BRD) poses a serious challenge, particularly to the beef feedlot industry, as they currently depend on antibiotics to prevent BRD to mitigate the financial burden (approx. $1 billion annual loss) inflicted by BRD-associated high mortality and morbidity in feedlot cattle. Thus, there is an impetus need for the development of antimicrobial alternative strategies against BRD. This study aimed to screen and select candidate essential oils (EOs) for the development of an intranasal EO spray that can inhibit BRD pathogens and promote microbiota-mediated respiratory health. Methods: The effects of selected EOs (ajowan, cinnamon leaf, citronella, grapefruit, fennel, and thyme) on a bovine nasopharyngeal microbiota culture were evaluated using 16S rRNA gene sequencing. The microbiota culture was enriched by incubating nasopharyngeal swabs obtained from finishing beef heifers in brain heart infusion broth with and without EOs (0.025%, v/v). These EOs were then also evaluated for their immunomodulatory effects on bovine turbinate (BT) cells by analyzing the concentrations of 15 cytokines and chemokines in cell culture after 24 h incubation. The crystal violet assay was done to assess the antibiofilm activity of EOs against Escherichia coli UMN026 strain. Finally, 15 EOs were screened for their antiviral activity against the bovine viral diarrhea virus 1 (BVDV-1) using BT cells and a fluorescence-based method. Results: Ajowan, fennel, and thyme resulted in a moderate reduction of overall nasopharyngeal microbiota growth with significant alterations of both alpha and beta diversity, and the relative abundance of predominant bacterial families (e.g., increasing Enterobacteriaceae and decreasing Moraxellaceae) compared to the control (p < 0.05). Co-incubation of BT cells with selected EOs resulted in minimal alterations in cytokine and chemokine levels (p > 0.05). Ajowan, thyme, fennel, and cinnamon leaf exhibited antibiofilm activity at concentrations of 0.025 and 0.05%. Reduction of BVDV-1 replication in BT cells was observed with thyme (strong), and ajowan and citronella (moderate) at 0.0125% concentration. Discussion: Accordingly, ajowan, thyme, fennel, cinnamon leaf, and citronella EOs were selected for further development as an intranasal EO spray to prevent and control of BRD pathogens in feedlot cattle.

Chimeric porcine circoviruses (PCV) containing amino acid epitope tags in the C terminus of the capsid gene are infectious and elicit both anti-epitope tag antibodies and anti-PCV type 2 neutralizing antibodies in pigs.

A chimeric porcine circovirus (PCV1-2) with the capsid gene of pathogenic PCV2 cloned into the genomic backbone of nonpathogenic PCV1 is attenuated in pigs but elicits protective immunity against PCV2. In this study, short epitope tags were inserted into the C terminus of the capsid protein of the chimeric PCV1-2 vaccine virus, resulting in a tractable marker virus that is infectious both in vitro and in vivo. Pigs experimentally infected with the epitope-tagged PCV1-2 vaccine viruses produced tag-specific antibodies, as well as anti-PCV2 neutralizing antibodies, indicating that the epitope-tagged viruses could potentially serve as a positive-marker modified live-attenuated vaccine.

Multiplex method for simultaneous serological detection of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the "gold standard" enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P < 0.001) for PRRSV and 0.80 (P < 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.

Restricted enzooticity of hepatitis E virus genotypes 1 to 4 in the United States.

Hepatitis E is recognized as a zoonosis, and swine are known reservoirs, but how broadly enzootic its causative agent, hepatitis E virus (HEV), is remains controversial. To determine the prevalence of HEV infection in animals, a serological assay with capability to detect anti-HEV-antibody across a wide variety of animal species was devised. Recombinant antigens comprising truncated capsid proteins generated from HEV-subgenomic constructs that represent all four viral genotypes were used to capture anti-HEV in the test sample and as an analyte reporter. To facilitate development and validation of the assay, serum samples were assembled from blood donors (n = 372), acute hepatitis E patients (n = 94), five laboratory animals (rhesus monkey, pig, New Zealand rabbit, Wistar rat, and BALB/c mouse) immunized with HEV antigens, and four pigs experimentally infected with HEV. The assay was then applied to 4,936 sera collected from 35 genera of animals that were wild, feral, domesticated, or otherwise held captive in the United States. Test positivity was determined in 457 samples (9.3%). These originated from: bison (3/65, 4.6%), cattle (174/1,156, 15%), dogs (2/212, 0.9%), Norway rats (2/318, 0.6%), farmed swine (267/648, 41.2%), and feral swine (9/306, 2.9%). Only the porcine samples yielded the highest reactivities. HEV RNA was amplified from one farmed pig and two feral pigs and characterized by nucleotide sequencing to belong to genotype 3. HEV infected farmed swine primarily, and the role of other animals as reservoirs of its zoonotic spread appears to be limited.

Comparative immunopathogenesis and biology of recently discovered porcine circoviruses.

Porcine circoviruses are important pathogens of production swine. Porcine circovirus type 1 (PCV1) is non-pathogenic, and discovered as a contaminant of a porcine kidney cell line, PK-15. The discovery of pathogenic variant, PCV2, occurred in the late 90s in association with post-weaning multi-systemic wasting disease syndrome (PMWS), which is characterized by wasting, respiratory signs and lymphadenopathy in weanling pigs. A new PCV type, designated as PCV3, was discovered in 2016, in pigs manifesting porcine dermatitis and nephropathy syndrome (PDNS), respiratory distress and reproductive failure. Pathological manifestations of PCV3 Infections include systemic inflammation, vasculitis and myocarditis. A fourth PCV type, PCV4, was identified in 2020 in pigs with PDNS, respiratory and enteric signs. All the pathogenic PCV types are detected in both healthy and morbid pigs. They cause chronic, systemic infections with various clinical manifestations. Dysregulation of the immune system homeostasis is a pivotal trigger for pathogenesis in porcine circoviral infections. While the study of PCV3 immunobiology is still in its infancy lessons learned from PCV2 and other circular replication-associated protein (Rep)-encoding single stranded (ss) (CRESS) DNA viruses can inform the field of exploration for PCV3. Viral interactions with the innate immune system, interference with dendritic cell function coupled with the direct loss of lymphocytes compromises both innate and adaptive immunity in PCV2 infections. Dysregulated immune responses leading to the establishment of a pro-inflammatory state, immune complex associated hypersensitivity, and the necrosis of lymphocytes and immune cells are key features of PCV3 immunopathogenesis. A critical overview of the comparative immunopathology of PCV2 and PCV3/4, and directions for future research in the field are presented in this review.

Harnessing the Genetic Plasticity of Porcine Circovirus Type 2 to Target Suicidal Replication.

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.

Torque teno viruses in health and disease.

Torque teno viruses (TTVs) are small, ubiquitous, viruses with a highly diverse, single-stranded, negative sense DNA genome and wide host range. They are detected at high rates in both healthy and diseased individuals and are considered a significant part of the mammalian virome. Similar to human TTVs, swine TTVs (TTSuVs) are epidemiologically linked to several coinfections including porcine circovirus types 2 and 3 and the porcine reproductive and respiratory disease syndrome virus. Experimental infection of gnotobiotic pigs with TTSuVs resulted in lesions in multiple organs and exacerbation of coinfections, making TTSuVs the only members of the Anelloviridae family with experimental evidence for pathogenicity. However, due to the lack of reliable cell culture and animal models, mechanistic studies on viral immunity and pathogenesis are limited. The objective of this review is to summarize the current status of knowledge regarding the biology, detection, pathogenesis and public health significance of TTSuVs, while identifying gaps in knowledge which limit the field.

Targeted Alteration of Antibody-Based Immunodominance Enhances the Heterosubtypic Immunity of an Experimental PCV2 Vaccine.

Despite the availability of commercial vaccines which can effectively prevent clinical signs, porcine circovirus type 2 (PCV2) continues to remain an economically important swine virus, as strain drift, followed by displacement of new subtypes, occurs periodically. We had previously determined that the early antibody responses to the PCV2 capsid protein in infected pigs map to immunodominant but non-protective, linear B cell epitopes. In this study, two of the previously identified immunodominant epitopes were mutated in the backbone of a PCV2b infectious clone, to rationally restructure the immunogenic capsid protein. The rescued virus was used to immunize 3-week-old weanling piglets, followed by challenge with a virulent heterologous PCV2d strain. As expected, immunodominant antibody responses to the targeted epitopes were abrogated in vaccinated pigs, while a broadening of the virus neutralization responses was detected. Vaccinated pigs were completely protected against challenge viral replication, had reduced microscopic lesions in lymphoid organs and gained significantly more body weight when compared to unvaccinated pigs. Thus, the experimental PCV2 vaccine developed was highly effective against challenge, and, if adopted commercially, can potentially slow down or eliminate new strain creation.

The NC229 multi-station research consortium on emerging viral diseases of swine: Solving stakeholder problems through innovative science and research.

The NC229 research consortium was created in 1999 in response to the emergence of porcine reproductive and respiratory syndrome virus (PRRSV), a viral agent responsible for devastating economic losses to the swine industry. The project follows the traditional "consortium" approach for Multistate Agricultural Research driven through the US State Agricultural Experiment Stations (SAES), wherein stakeholder-driven needs to combat swine infectious diseases are identified and scientific solutions pursued by combining funds from federal, state, commodity groups, and the animal health industry. The NC229 consortium was the main driving force in successfully competing for a USDA multi-station Coordinated Agricultural Project (PRRS CAP-I) in 2004-2008, immediately followed by a renewal for 2010-2014 (PRRS CAP-II)-, resulting in an overall record achievement of almost $10 million dollars. The CAP funding was not only useful for quality research, extension, and education in PRRS and related diseases, but also instrumental in enabling the group to leverage swine industry funding of more than $34 million dollars, distributed between creative research and extension on PRRS during the last 20 years. The North American/International PRRS Symposium, now recognized by the community as a highly effective platform for the exchange of basic research findings and fundamental translational technology, is directly derived from the NC229 consortium. Other significant offshoots from NC229 include the PHGC (PRRS Host Genomic Consortium), a platform for discoveries on the role of host genetics during PRRSV infection, since 2007. Since 2009, the NC229 consortium has expanded its collective research interests beyond PRRSV to include nine other emerging viral diseases of swine. In the current project (2019-2024), African Swine Fever Virus (ASFV) retains a central focus, with the goal of harnessing the group's expertise in promoting preparedness for the global control of ASFV.

Efficacy of a Commercial PCV2a Vaccine with a Two-Dose Regimen Against PCV2d.

Porcine circovirus type 2, the causative agent of porcine circovirus associated diseases (PCVAD), consists of three major genotypes PCV2a, 2b and 2d. Current commercial vaccines contain the first-identified PCV2a's capsid protein or whole virions. Outbreaks of PCVAD, caused by the recently identified PCV2d in vaccinated herds have raised concerns regarding the efficacy of current PCV2a vaccines against PCV2d. Thus, the primary objective of this study was to assess the efficacy of a two-dose regimen for the recently reformulated Fostera PCV MetaStim vaccine, to determine if reformulation with the squalene oil adjuvant and two-dose regimen improves the threshold of protection enough to eliminate viremia in a vaccination and challenge model. Two groups of seven pigs each were vaccinated with the commercial vaccine or PBS, and challenged with the PCV2d virus. Strong pre-challenge virus neutralizing responses were detected against all three genotypes. Post-challenge viremia was not completely eliminated as expected but a 2 log10 mean reduction in viral load was achieved in vaccinated pigs. Vaccinated pigs had a mean score of 0 for pathological evaluation, while unvaccinated pigs had a score of 6.6. In conclusion, the reformulated Fostera PCV MetaStim PCV2a-based vaccine provided significant heterologous protection and was effective against PCV2d.

Delivery of a thermo-enzymatically treated influenza vaccine using pulmonary surfactant in pigs.

Swine influenza A virus (IAV-S) infections are a major cause of economic losses for the swine industry. The vast genetic and antigenic diversity often results in mismatch between the vaccine and field strains, necessitating frequent updates of vaccines. Inactivated IAV-S vaccines are of questionable efficacy. Intra-nasally administered live vaccines are more effective but are associated with safety concerns. The objective of this study was to develop a first-generation vaccine which combines the safety and efficacy advantages of inactivated and attenuated vaccines respectively. The approach targeted fragmentation of viral nucleic acids while preserving structure. Hence, cultures of influenza A/CA/04/09 H1N1 were exposed to 44 °C for 10 min. to reversibly denature the capsid, followed by RNase treatment to digest the genomic RNA and then refolded at lower temperatures. As targeted, treated virions retained an intact structure and were not detected in the first passage in infected cells. To improve intra-nasal delivery of the vaccine antigen, the vaccine antigen was delivered in porcine lung surfactant. Both the treated vaccine alone or vaccine in combination with the surfactant elicited strong anti-HA and virus neutralizing antibodies, protection against viral shedding and lung lesions in 3-week-old piglets. There were no significant differences between the groups. Vaccine viral replication was not detected in the vaccinated pigs. The described approach can advance current immunization practices against swine influenza viruses due to the relative simplicity, high efficacy and safety and ease of adaptation to newly emerging field strains.

An amphiphilic invertible polymer as a delivery vehicle for a M2e-HA2-HA1 peptide vaccine against an Influenza A virus in pigs.

Influenza A viruses (IAVs) are a group of genetically diverse and economically important zoonotic pathogens. Despite decades of research, effective and broadly protective vaccines are yet to be developed. Recent breakthroughs in epitope-based immunization for influenza viruses identify certain conserved regions of the HA2 and M2e proteins as capable of inducing broad protection against multiple influenza strains. The M2e and HA2 peptides have been evaluated in mice but not as a combination in pigs, which play an important role in the transmission and evolution of IAV. Peptides are inherently weak immunogens; and effective delivery of peptide antigens is challenging. To enhance the delivery and immunogenicity of peptide-based vaccines, the conserved M2e and HA2 and a strain-specific HA1 epitope of Influenza A (H1N1) pdm09 were expressed as a chain in a bacterial expression system and entrapped in a novel amphiphilic invertible polymer made from polyethyelene glycol (PEG, molecular weight 600 g/mol) and polytetrahydrofuran (PTHF, molecular weight 650 g/mol), PEG600PTHF650. Piglets vaccinated with polymeric peptide vaccine mounted significantly stronger antibody responses against the peptide construct when compared to piglets immunized with the multi-epitope peptide alone. When vaccinated pigs were challenged with Influenza A (H1N1) pdm09, viral shedding in nasal secretions and lung lesion scores were significantly reduced when compared to the unvaccinated controls and pigs vaccinated with the peptide alone at six days post-challenge. Thus, the combination of the PEG600PTHF650 polymer and trimeric peptide construct enhanced delivery of the peptide antigen, acted as an adjuvant in stimulating strong antibody responses, reduced the effects of viral infection in vaccinated pigs.

A Minimally Replicative Vaccine Protects Vaccinated Piglets Against Challenge With the Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV), is an economically important enteric coronavirus, with over a 90% mortality rate in neonatal piglets. The virus emerged in the US in 2013, resulting in severe production losses. Effective vaccine development against PEDV is a challenge. Inactivated vaccines are of questionable efficacy. Attenuated vaccines, while more effective, require a relatively long lead development time, are associated with safety concerns and are also unable to prevent new field outbreaks. To combine the safety and efficacy advantages of inactivated and attenuated PEDV vaccines, respectively, in this study, we tested the hypothesis that subjecting PEDV virions to heat treatment at 44°C for 10 min to reversibly unfold structural proteins, followed by exposure to RNAse to fragment the genome, would result in a vaccine preparation with intact viral structure/antigenicity but highly diminished replicative abilities. We expected the vaccine to be both safe and effective in a piglet challenge model. Following the heat and RNAse treatment, PEDV virions had an intact electron microscopic ultrastructure and were amplified only in the 3rd passage in Vero cells, indicating that diminished replication was achieved in vitro. Strong PEDV spike-protein specific and virus neutralizing antibody responses were elicited in vaccinated piglets. Upon challenge, all vaccinated pigs were protected against fecal viral shedding and intestinal pathology, while the unvaccinated controls were not. The vaccine virus was not detected in the fecal matter of vaccinated pigs prior to challenge; nor did they develop intestinal lesions. Thus, the described approach has significant promise in improving current approaches for PEDV immunization.

Characterization of shedding patterns of Porcine circovirus types 2a and 2b in experimentally inoculated mature boars.

Porcine circovirus-2 (PCV-2) is an economically important swine pathogen and causes PCV-associated disease (PCVAD) in pigs worldwide. Currently, 2 genotypes of PCV-2, PCV-2a and -2b, are circulating in U.S. swine herds. The objectives of the current study were to evaluate the amount of PCV-2 DNA present in semen over time, compare and correlate incidence and amount of PCV-2 present in semen samples to that present in serum samples and blood swabs, and determine if there are differences in shedding patterns between PCV-2a and -2b. Fifteen 7-month-old PCV-2-naïve Landrace boars (Sus scrofa) were randomly allocated to 3 treatment groups. The boars in group 1 (n = 3) served as negative controls, and those in groups 2 (n = 6) and 3 (n = 6) were intranasally and intramuscularly inoculated with PCV-2a and -2b, respectively. Semen, serum, and blood swab samples were collected up to 90 days postinoculation (DPI), and necropsies were performed on DPI 23, 48, and 90. Larger quantities of both PCV-2a and -2b DNA were detected earlier in serum and blood swab samples than in raw semen of experimentally inoculated boars. The incidence and duration of presence of PCV-2 DNA in semen varied among boars; however, intermittent shedding was not observed. In all sex glands, PCV-2 DNA was detected by polymerase chain reaction; however, PCV-2 antigen was not detected by immunohistochemistry, and PCV-2 had no effect on sperm morphology. Differences in shedding patterns between PCV-2a and -2b were not observed under the study conditions.

Comparison of three enzyme-linked immunosorbent assays to detect Porcine circovirus-2 (PCV-2)-specific antibodies after vaccination or inoculation of pigs with distinct PCV-1 or PCV-2 isolates.

Porcine circovirus-2 (PCV-2) serology is frequently used to determine PCV-2 status and optimal timing of PCV-2 vaccination in the field. The objectives of the current study are to determine the diagnostic accuracy of 3 currently available commercial anti-immunoglobulin G (IgG) PCV-2 enzyme-linked immunosorbent assays (ELISAs) and to compare the ability of the 3 assays to detect and differentiate between anti-PCV-2a and anti-PCV-2b antibodies, as well as anti-PCV-2 and anti-PCV-1 antibodies. Fifty-five 3-week-old, conventional pigs were randomly allocated to 7 groups: 1) negative controls (n = 7), 2) PCV-2a (n = 8; inoculated with PCV-2 ISU-40895), 3) PCV-2b (n = 8; inoculated with PCV-2 NC-16845), 4) PCV-1 (n = 8), 5) vaccine A (n = 8; Ingelvac CircoFLEX), 6) vaccine B (n = 8; Circumvent PCV2), and 7) vaccine C (n = 8; Suvaxyn PCV2 One Dose). Blood samples were collected weekly, and all sera were tested by 3 different anti-PCV-2 IgG ELISAs. The results indicated that all ELISAs had area under the receiver operating curve values greater than 0.94, detected both anti-PCV-2a and -2b antibodies with no differentiation, and did not detect anti-PCV-1 antibodies in infected animals. One of the ELISAs was able to distinguish pigs vaccinated with vaccine B from pigs inoculated with either PCV-2a or PCV-2b.

Potential for the cross-species transmission of swine torque teno viruses.

Torque teno viruses [TTVs] are negative sense, single-stranded, DNA viruses, which are distributed globally in several mammalian hosts such as humans, apes, sheep and swine in a species-specific manner. While the pathogenic potential of TTVs is under debate, recent experimental studies in gnotobiotic pigs indicate that swine TTVs, TTSuV1 in particular, can act as a primary or co-infecting pathogen. Hence, determining whether TTSuV1 can infect other mammals would eventually further our understanding of viral pathogenesis, especially in coinfections. In this study, we tested sera from horses, cattle, sheep, dogs and elk for the presence of TTSuV1 DNA using a panel of TTSuV1-specific primers, and assessed the extent of sero-conversion to TTSuV1 in the selected species. We found that TTSuV1 DNA was detected in 46.7% of equines, 70% of canine, 100% of bovine, 40% of ovine and 93.3% of elk samples. However, significant TTSuV1 specific antibody responses were detected only in the bovine, ovine and equine samples but not the canine or elk samples, indicating that these animals could support the replication of TTSuV1. This combined serological and molecular epidemiological profile of TTSuV1 infection in five different species indicates the host range of species-specific TTVs could be wider than initially believed. Further studies are required to understand the health risks to these animal species from TTSuV-1 infection.

A convenient colorimetric assay for the quantification of porcine epidemic diarrhea virus and neutralizing antibodies.

Neonatal enteritis caused by the porcine epidemic diarrhea virus (PEDV) is an important cause of high mortality and economic losses to the swine industry. Virus neutralization (V/N) assays are commonly requested in diagnostic laboratories for the assessment of protective antibodies. However, the visual assessment of viral cytopathic effects by operators to determine antibody titers or for viral quantification is a tedious, subjective and time-consuming process, especially when high volume testing is involved. To improve the ease of testing, a colorimetric virus neutralization and TCID50 assays were developed and validated in this study using (3-(4,5-dimethylthiazol-2-yl) Tr-2,5-diphenyltetrazolium- bromide) (MTT), a colorimetric agent which measures cell viability. The respective conventional assays were used as the gold standards. An OD cut off value of ≤0.53, selected by receiver operating characteristics analysis, could distinguish between wells with and without CPE accurately. Performance and reproducibility parameters of the colorimetric assays were comparable to the conventional assays. The described methods can reduce testing time in diagnostic laboratories, while significantly improving current protocols.

Prevention of lethal experimental infection of C57BL/6 mice by vaccination with Brucella abortus strain RB51 expressing Neospora caninum antigens.

Bovine abortions caused by the intracellular protozoal parasite Neospora caninum are a major concern to cattle industries worldwide. A strong Th1 immune response is required for protection against N. caninum. Brucella abortus strain RB51 is currently used as a live, attenuated vaccine against bovine brucellosis. Strain RB51 can also be used as an expression vector for heterologous protein expression. In this study, putative protective antigens of N. caninum MIC1, MIC3, GRA2, GRA6 and SRS2, were expressed individually in B. abortus strain RB51. The ability of each of the recombinant RB51 strains to induce N. caninum-specific immunity was assessed in C57BL/6 mice. Mice were immunised by two i.p. inoculations, 4 weeks apart. Five weeks after the second immunisation, spleen cells from the vaccinated mice secreted high levels of IFN-gamma and IL-10 upon in vitro stimulation with N. caninum whole cell lysate antigens. N. caninum-specific antibodies of both IgG1 and IgG2a subtypes were detected in the serum of the vaccinated mice. Mice in the vaccinated and control groups were challenged with 2 x 10(7)N. caninum tachyzoites i.p. and observed for 28 days after vaccination. All unvaccinated control mice died within 7 days. Mice in the MIC1 and GRA6 vaccine groups were completely protected while the mice in the SRS2, GRA2 and MIC3 vaccinated groups were partially protected and experienced 10-50% mortality. The non-recombinant RB51 vector control group experienced an average protection of 69%. These results suggest that expression of protective antigens of N. caninum in B. abortus strain RB51 is a novel approach towards the development of a multivalent vaccine against brucellosis and neosporosis.