Development and application of direct and indirect diagnostic techniques in the new paradigm of porcine circovirus diagnostics
Date
2024-08
Authors
Kroeger, Molly Elizabeth
Major Professor
Advisor
Piñeyro, Pablo P
Miller, Cathy
Roth, James
Tuggle, Christopher
Diel, Diego
Committee Member
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Abstract
Porcine circovirus 2 (PCV2) is the etiological agent of porcine circovirus associated disease (PCVAD), resulting in devasting health outcomes and tremendous economic losses to the global swine industry since the 1990s. While great strides have been made in PCV research and vaccination strategies over the past couple of decades, the emergence of new PCV2 variants and coinfections with porcine reproductive and respiratory syndrome virus (PRRSV) has called vaccine efficacy into question. Over the past several years, the description of two new, novel porcine circoviruses, porcine circovirus 3 (PCV3) and porcine circovirus 4 (PCV4), has created a new landscape of circovirus dynamics and increased diagnostic complexities within swine herds. The aim of this dissertation research is to explore the development and application of direct and indirect diagnostic techniques in the new paradigm of porcine circovirus diagnostics.
First identified in the United States of America (USA) in 2016, PCV3 has been reported globally and is most commonly associated with reproductive failure, multisystemic inflammation, and subclinical infection. While the presence of PCV3 has been reported retrospectively in several countries, the prevalence of PCV3 in the USA before its first description has not been reported. Therefore, the PCV3 and PCV3/PCV2 coinfection farm prevalence, PCV3 evolution and selection pressure forces, and Capsid (Cap) morphological changes were evaluated in USA grower-finisher farms from 2000, 2006, and 2012. Our study revealed that PCV3 was endemic in the USA swine industry before the first description. Additionally, the prevalence of PCV3 and PCV3/PCV2 coinfection declined from 2000 to 2012. While multiple PCV3 whole genome and Cap subtypes were identified from obtained sequences, Cap nucleotide identity remained relatively high ranging from 97.8-100%. Selection pressure analysis and Cap molecular modeling support that amino acids 24 and 27 underwent diversifying selection, which may have contributed to a change in the Cap secondary structure from alpha helix to coil at these residues. Furthermore, other amino acid mutations were identified in a predicted T-cell epitope and areas of uncharacterized immunological relevance. Thus, this study broadened current knowledge of the prevalence and molecular evolution of PCV3 in the USA swine herd from 2000-2012.
Information regarding the PCV3 humoral response following experimental infection is limited. Additionally, the antibody dynamics following maternal antibody transfer have not been characterized. Therefore, the objective was to characterize the dynamic of PCV3 Cap and Rep IgG antibodies following maternal antibody transfer and experimental infection. Additionally, the cross-reactivity of convalescent serum from PCV2 and PCV3 experimentally infected pigs was evaluated. Maternally derived Cap and Rep IgG antibodies were detectable in piglet serum seven-nine weeks and five weeks post-farrowing, respectively. Following experimental PCV3 inoculation, Cap and Rep IgG were detected at two weeks post-inoculation and four weeks post- inoculation, respectively. Furthermore, convalescent serum from either PCV2 or PCV3 methods displayed no cross-reactivity by IFA and ELISA. The increased understanding of the dynamics of maternally derived antibodies and development of the humoral response following infection may aid in the establishment of husbandry practices and the potential application of prophylactic strategies to control PCV3 clinical disease.
Questions regarding PCV2 vaccine efficacy have arisen in recently due to heterologous PCV2 infection and coinfections with virulent, contemporary PRRSV isolates. Therefore, the objective was to assess clinical, pathological, and immunological parameters in PCV2 homologous and heterologous vaccinated pigs following PCV2d/PRRSV 1-7-4 co-challenge. PCV2 viral load in tissues, viremia, lesions associated with lymphoid depletion, and amount of PCV2 antigen detected in tissues was significantly lower in the PCV2d vaccinates, correlating to subclinical infection 28 days post-challenge. In contrast, approximately 20-40% of the PCV2a vaccinates had moderate to severe lymphoid depletion with moderate to severe antigen detection and levels of viremia, corresponding to clinical disease. Prior to the co-challenge on day 28 (28 days post-vaccination), PCV2d vaccinates had significantly higher neutralizing antibody titers against the PCV1-2d challenge chimeric virus, which may partially explain the better clinical protection in the PCV2d vaccinates. Therefore, homologous vaccination may provide greater protection in virulent coinfection scenarios in the field, potentially due to the earlier, enhanced development of homologous neutralizing antibodies.
Increased use of PCR methodologies has spurred interest in applying indirect, noninvasive strategies to understand PCV2 viral circulation. While processing fluids (PF) have increased in use for PCV2 diagnostics over the past several years, the applicability of this sample matrix to drive sow herd vaccination strategies needs to be better understood. Therefore, the objectives were to analyze differences in sow circulating antibody levels and the amount of PCV2 detected in PFs pre and post-whole sow herd vaccination and determine the correlation between sow humoral status and viral load in PFs. While the levels of sow antibodies significantly increased post-whole sow herd vaccination, the average PCV2 PF Ct value remained statistically similar. Higher levels of sow antibodies were not correlated to lower amounts of PCV2 detected in PFs. Low R squared values revealed that sow antibody levels poorly explain the amount of PCV2 detected in PFs. Thus, the amount of PCV2 detected in PFs may not be useful to gauge the sow herd immune status and drive management decisions associated with sow vaccination strategies.
Since PCV4 was first described in 2019, the virus has been identified in several countries in Southeast Asia and Europe, but PCV4 has not been described in the USA. Most studies have been limited to detecting PCV4 by PCR, resulting in an unclear association between the presence of PCV4 and clinical disease. In this study, the presence of PCV4 was confirmed by direct and indirect diagnostic methods in several tissues for the first time in the USA. PCV4 was detected in 8.6% of samples with an average Ct value of 33 in clinical samples submitted to the Iowa State University Veterinary Diagnostic Laboratory from June-September 2023. Direct detection of PCV4 by RNAscope confirmed viral replication in lymph nodes and the small intestine. Characterization of immune cell populations revealed viral replication in lymph node germinal centers overlapped with in areas of B lymphocytes and macrophages. In contrast, viral replication in the lamina propria of the small intestine overlapped in areas of histiocytic and T lymphocyte infiltration. While PCV4 detection was most commonly observed in nursery to finishing aged pigs displaying respiratory and enteric disease, coinfection with PCV2, PCV3, and other endemic pathogens was frequently observed. Thus, future studies are necessary to determine the role of PCV4 as a primary pathogen and the outcome of disease pathogenesis in PCV4 and endemic pathogen coinfection scenarios.
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Veterinary Diagnostic and Production Animal Medicine
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