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A Working Group of the Society of Toxicologic Pathology's Scientific and Regulatory Policy Committee conducted a technical and scientific review of current practices relating to the fixation, trimming, and sectioning of the nonrodent eye to identify key points and species-specific anatomical landmarks to consider when preparing and evaluating eyes of rabbits, dogs, minipigs, and nonhuman primates from ocular and general toxicity studies. The topics addressed in this Points to Consider article include determination of situations when more comprehensive evaluation of the globe and/or associated extraocular tissues should be implemented (expanded ocular sampling), and what constitutes expanded ocular sampling. In addition, this manuscript highlights the practical aspects of fixing, trimming, and sectioning the eye to ensure adequate histopathological evaluation of all major ocular structures, including the cone-dense areas (visual streak/macula/fovea) of the retina for rabbits, dogs, minipigs, and nonhuman primates, which is a current regulatory expectation for ocular toxicity studies.[Box: see text].
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Técnicas Histológicas , Pruebas de Toxicidad , Animales , Perros , Políticas , Conejos , Retina , Porcinos , Porcinos EnanosRESUMEN
Dysfunction of the visual system remains a leading cause of human disability worldwide. Preclinical studies are a key component of efforts to develop drugs and devices to ameliorate visual impairment. Although new opportunities for the delivery of targeted ocular therapeutics have been created, clinical success has been confounded by unique challenges of drug development for the eye. This Special Issue brings together a broad range of articles that augment our current understanding of the visual system and highlight methods for assessing ocular toxicity and some of the current challenges in ocular drug development. Topics addressed include the anatomy, developmental anatomy, and/or immunobiology of the visual system and associated lymphoid tissues; animal models; methods for assessing ocular toxicity; spontaneous background and procedure-related microscopic findings and common artifacts in histologic sections of ocular tissues; and novel ocular drug delivery systems.
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Ojo , Preparaciones Farmacéuticas , Administración Tópica , Animales , Sistemas de Liberación de Medicamentos , Desarrollo de Medicamentos , HumanosRESUMEN
The goal of this study was to develop methods for the evaluation of green fluorescent protein (GFP) and GFP transcript biodistribution in paraformaldehyde-fixed paraffin-embedded (PFPE) eye sections to assess the effectiveness of Adeno-associated virus (AAV) gene delivery in an experimental ocular toxicity study. Female C57BL/6NTac mice were administered AAV2-enhancedGFP vector once via subretinal injection. One group also received anti-inflammatory therapy (meloxicam). Immunohistochemistry (IHC) and RNA in situ hybridization (ISH) for GFP were performed on PFPE serial eye sections and evaluated using semiquantitative methods. On day 43, GFP labeling in both IHC and ISH sections was greatest in the retinal pigment epithelium, compared with other retinal layers in which expression was negative to moderate. Despite the presence of IHC GFP labeling in the photoreceptor layer (PRL) in some animals, only low numbers of transduced cells were detected by ISH in the PRL. Simultaneous analysis of IHC and ISH may be needed for comprehensive assessment of gene transduction and protein biodistribution. This study demonstrates approaches for semiquantitative evaluation of IHC and ISH that allow interpretation and reporting of GFP expression in toxicity studies.
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Dependovirus , Vectores Genéticos , Animales , Dependovirus/genética , Dependovirus/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Retina/metabolismo , Distribución TisularRESUMEN
Identification of test article-related microscopic findings in ocular toxicology studies requires a working knowledge of the artifacts and procedure-related or background findings commonly encountered in such studies. The objective of this article is to provide a mini-atlas of the artifacts and procedure-related or spontaneous background findings commonly observed in ocular tissues from animals in toxicology studies of ocular drug candidates. Artifacts in the eye are often related to collection or fixation procedures and include swelling and vacuolation of lens fibers, separation of the neuroretina from the retinal pigment epithelium (RPE), and vacuolation of the optic nerve. Common in-life procedure-related findings include intravitreal injection needle tracks in the sclera and ciliary body pars plana and foci of RPE hypertrophy and/or hyperpigmentation at subretinal injection sites. Common background findings include corneal mineralization, uveal mononuclear cell infiltrates, and peripheral displacement of photoreceptor nuclei in the retina. A few uncommon spontaneous background findings that may be confused with test article-related findings, such as bilateral optic atrophy in macaques, are also included.
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Artefactos , Enfermedades de la Retina , Animales , Animales de Laboratorio , Retina , Epitelio Pigmentado de la RetinaRESUMEN
Inhibitors of Bruton's tyrosine kinase (BTK) are under development as potential therapies for various autoimmune diseases. In repeat-dose toxicity studies, small-molecule BTK inhibitors (BTKi) have been reported to cause a constellation of histologic effects at the pancreatic endocrine-exocrine interface in male rats; however, similar findings were not reported in other species. Since the BTKi-induced pancreatic effect is morphologically similar to well-documented spontaneous changes (predominantly characterized by insular/peri-insular hemorrhage, pigment deposition, chronic inflammation, and fibrosis) that are known to vary by rat strain, we investigated potential strain-dependent differences in the pancreatic effects of a small-molecule BTKi, LY3337641. Following 13 weeks of LY3337641 treatment, Crl:CD(SD) rats were most sensitive, Crl:WI(Han) rats were of intermediate sensitivity, and Hsd:SD rats were least sensitive. These strain differences appear to be related to differences in rate of weight gain across strains and sexes; however, a definitive mechanism was not determined. This study demonstrated that BTKi-induced pancreatic effects were highly dependent on rat strain and correlated with differences in the incidence and severity of the spontaneous background change. When considered with the lack of pancreas effects in nonrat species, these changes in rats are unlikely predictive of similar changes in humans administered a BTK inhibitor.
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Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Páncreas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/toxicidad , Animales , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Vehicle control Harlan RCCHan™:WIST rats were examined to provide control data for subsequent studies. The rats (180 male and 180 female) were dosed daily via oral gavage with reverse osmosis water for up to 104 weeks. At necropsy, body weights and macroscopic findings were recorded and tissues were collected for histopathology. The mean body weight at terminal sacrifice was 687 g for males and 466 g for females. The overall survival rate was 62% for males and 59% for females. The most common cause of death for males and females found dead or examined following unscheduled euthanasia was pituitary neoplasia with an incidence of 13.9% for males and 18.9% for females. Macroscopic and neoplastic and nonneoplastic microscopic findings are presented by body system.
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Ratas Wistar , Animales , Femenino , Masculino , RatasRESUMEN
Clinical and nonclinical studies have implicated glucagon-like peptide-1 (GLP-1) receptor agonist therapy as a risk factor for acute pancreatitis in patients with type 2 diabetes. Therefore, it is critical to understand the effect that dulaglutide, an approved GLP-1 receptor agonist, has on the exocrine pancreas. Dulaglutide 8.15 mg/kg (approximately 500 times the maximum recommended human dose based on plasma exposure) was administered twice weekly for 12 months to cynomolgus monkeys. Serum amylase and lipase activities were measured and 6 sections of each pancreas were examined microscopically. Ductal epithelial cell proliferation was estimated using Ki67 labeling. Dulaglutide administration did not alter serum amylase or lipase activities measured at the end of treatment compared to control values. An extensive histologic evaluation of the pancreas revealed no changes in the acinar or endocrine portions and no evidence of pancreatitis, necrosis, or pancreatic intraepithelial neoplasia. An increase in goblet cells noted in 4 of the 19 treated monkeys was considered an effect of dulaglutide but was not associated with dilation, blockage, or accumulation of mucin in the pancreatic duct. There was no difference in cell proliferation in ductal epithelium between control and dulaglutide-treated monkeys. These data reveal that chronic dosing of nondiabetic primates with dulaglutide does not induce inflammatory or preneoplastic changes in exocrine pancreas.
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Receptor del Péptido 1 Similar al Glucagón/agonistas , Péptidos Similares al Glucagón/análogos & derivados , Hipoglucemiantes/toxicidad , Fragmentos Fc de Inmunoglobulinas/toxicidad , Páncreas Exocrino/efectos de los fármacos , Proteínas Recombinantes de Fusión/toxicidad , Animales , Péptidos Similares al Glucagón/toxicidad , Macaca fascicularis , Masculino , Páncreas Exocrino/patologíaRESUMEN
In spring of 2005, the authors implemented and evaluated a process at the Iowa State University College of Veterinary Medicine in which third-year students evaluated fourth-year students' performances on an advanced case-analysis assignment. This assignment, called the case correlation assignment, required a thorough integration and explanation of all ante- and post-mortem data for a specific hospital patient. Using a 21-point rubric, the necropsy course instructor and third-year students rated these assignments. Fourth-year students' performances on this assignment were used as an indicator of the success of the pathology curriculum. The authors evaluated the assessment process for feasibility, reliability, and validity. Many-facet Rasch analysis was used to determine item, case, and rater agreement. The assessment process produced good agreement among items and cases (VM4 student competence). Furthermore, most third-year students were able to reliably rate the case correlation assignments with no special training. The evaluation process was cost effective and occurred in the context of regular course assignments, thereby making it feasible. A case can be made that the overall process provides a valid measure of the pathology program's success in preparing students in the area of veterinary pathology.
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Educación en Veterinaria/métodos , Evaluación Educacional/métodos , Evaluación Educacional/normas , Patología/educación , Grupo Paritario , Aprendizaje Basado en Problemas/métodos , Adulto , Autopsia/veterinaria , Competencia Clínica/normas , Curriculum , Femenino , Humanos , Iowa , Masculino , Evaluación de Programas y Proyectos de Salud , Distribución Aleatoria , Facultades de Medicina Veterinaria , Estudiantes , Encuestas y Cuestionarios , Adulto JovenRESUMEN
OBJECTIVE: To report the prevalence of bovine viral diarrhea virus (BVDV) in calves and calf groups (ie, calves from the same farm) in beef breeding herds and evaluate the ability of biosecurity risk assessment questionnaires to identify calf groups with positive results for BVDV. DESIGN: Nonrandom survey. ANIMALS: 12,030 calves born in spring from 102 operations. PROCEDURES: Cow-calf producers that voluntarily enrolled in a screening project submitted ear notch specimens from calves and answered a 29-question survey instrument. Ear notch specimens were tested for BVDV with an antigen-capture ELISA (ACE), and ear notch specimens with positive ACE results for BVDV were immediately retested by performing immunohistochemistry (IHC). Follow-up testing, 3 to 4 weeks after initial positive ACE results, was done by use of a second IHC test and virus isolation on a subsequently submitted ear notch specimen from the same calves to identify those that were persistently infected (PI). RESULTS: 102 producers submitted ear notch specimens for BVDV screening. Initially, 24 of 12,030 calves had positive ACE results for BVDV. A second ear notch specimen was submitted for 20 of these 24 calves. Of 20 retested calves, 12 had positive ICH results for BVDV, confirming PI status. The 12 PI calves came from 4 calf groups (3 singletons and 1 calf group with 9 PI calves). CONCLUSIONS AND CLINICAL RELEVANCE: Prevalence of BVDV in calf groups was low, and questions designed to identify high-risk biosecurity behaviors had little value in identifying calf groups with positive results for BVDV.
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Anticuerpos Antivirales/sangre , Diarrea Mucosa Bovina Viral/epidemiología , Virus de la Diarrea Viral Bovina/inmunología , Encuestas Epidemiológicas , Medición de Riesgo , Animales , Antígenos Virales/inmunología , Diarrea Mucosa Bovina Viral/diagnóstico , Bovinos , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Inmunohistoquímica/veterinaria , Masculino , Tamizaje Masivo/veterinaria , Sensibilidad y Especificidad , Estudios SeroepidemiológicosRESUMEN
Instructors of the senior necropsy rotation at Iowa State University experienced difficulty ensuring similarity of case exposure and workload during the year. This was problematic during times of low caseload, as, without necropsy cases, there was no uniform method for training or assessing students. A new assignment, the Case Correlation Assignment (CCA), was created in order to improve the educational rigor and consistency of the rotation, increase utilization of necropsy cases as teaching material, and provide students more opportunities to correlate clinical pathology data with lesions. The CCA provides an opportunity for senior students to present and explain the antemortem and postmortem findings from an ISU-VTH patient in case report format. This illustrated report is submitted via WebCT. Students receive feedback on WebCT through a scoring rubric and written comments from the instructor. Since 2002, approximately 420 students have completed this assignment. The average score on the assignment over the four-year period is 94.7%. Despite complaints about the hard work required, students generally report that writing the CCA is a valuable learning experience. The CCA allows for greater utilization of necropsy cases and the incorporation of clinical pathology into necropsy cases. Currently, the CCA is used in a peer-assessment assignment in the junior pathology course and has been incorporated into case-based teaching courses. The CCA has been revised and expanded over the past four years in response to student feedback and to the discovery of new ways to utilize the completed assignments as teaching material.
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Competencia Clínica , Curriculum , Educación en Veterinaria/métodos , Patología Clínica/educación , Humanos , Internet , Iowa , Facultades de Medicina VeterinariaRESUMEN
A potent anti-vascular endothelial growth factor (VEGF) biologic and a compatible delivery system were co-evaluated for protection against wet age-related macular degeneration (AMD) over a 6month period following a single intravitreal (IVT) injection. The anti-VEGF molecule is dimeric, containing two different anti-VEGF domain antibodies (dAb) attached to a human IgG1 Fc region: a dual dAb. The delivery system is based on microparticles of PolyActive™ hydrogel co-polymer. The molecule was evaluated both in vitro for potency against VEGF and in ocular VEGF-driven efficacy models in vivo. The dual dAb is highly potent, showing a lower IC50 than aflibercept in VEGF receptor binding assays (RBAs) and retaining activity upon release from microparticles over 12months in vitro. Microparticles released functional dual dAb in rabbit and primate eyes over 6months at sufficient levels to protect Cynomolgus against laser-induced grade IV choroidal neovascularisation (CNV). This demonstrates proof of concept for delivery of an anti-VEGF molecule within a sustained-release system, showing protection in a pre-clinical primate model of wet AMD over 6months. Polymer breakdown and movement of microparticles in the eye may limit development of particle-based approaches for sustained release after IVT injection.
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Anticuerpos/farmacología , Neovascularización Coroidal/prevención & control , Fragmentos Fc de Inmunoglobulinas/farmacología , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos/inmunología , Neovascularización Coroidal/inmunología , Preparaciones de Acción Retardada , Portadores de Fármacos , Liberación de Fármacos , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Inyecciones Intravítreas , Rayos Láser , Macaca fascicularis , Microesferas , Tamaño de la Partícula , Poliésteres/química , Polietilenglicoles/química , Conejos , Degeneración Macular Húmeda/prevención & controlRESUMEN
OBJECTIVE: To determine the association between the existence of a calf persistently infected (PI) with bovine viral diarrhea virus (BVDV) and pen morbidity. ANIMALS: 5,041 calves in 50 pens at a feedlot in Iowa. PROCEDURE: In a longitudinal study, ear notches were collected from cattle and tested for BVDV antigen. Characteristics of each pen (owner, sex, disease rate, number of groups, and source) were recorded. The association between the existence of a BVDV-PI calf and morbidity in each pen was examined. RESULTS: Commingling was associated with an increase in respiratory tract disease (odds ratio [OR], 3; 95% confidence interval [CI], 2.5 to 3.6). Ten BVDV-PI calves (10/5,041 [0.2%]) were identified in 8 of 50 pens. A BVDV-PI calf was associated with reduced pen-level respiratory tract disease (OR, 0.7; 95% CI, 0.5 to 0.9). Disease prevalence (mean +/- SD morbidity, 7.9 +/- 3.1%) was lowest in pens containing single-source cattle and a BVDV-PI calf (4 pens containing 302 cattle), compared with single-source cattle with no BVDV-PI calf (mean morbidity, 11.89 +/- 9.7%; 31 pens containing 3,093 cattle), commingled cattle with no BVDV-PI calf (mean morbidity, 29.3 +/- 16.22%; 11 pens containing 1,127 cattle), and commingled cattle with a BVDV-PI calf (mean morbidity, 28.6 +/- 10.1%; 4 pens containing 519 cattle). CONCLUSIONS AND CLINICAL RELEVANCE: Commingling was the greatest risk factor associated with morbidity in each pen. A BVDV-PI calf in a pen was not associated with increased disease prevalence in commingled groups.
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Crianza de Animales Domésticos/métodos , Diarrea Mucosa Bovina Viral/transmisión , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/virología , Virus de la Diarrea Viral Bovina/patogenicidad , Vivienda para Animales , Enfermedades Respiratorias/veterinaria , Animales , Diarrea Mucosa Bovina Viral/epidemiología , Bovinos , Enfermedades de los Bovinos/transmisión , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Oído Externo/virología , Inmunohistoquímica/veterinaria , Iowa/epidemiología , Modelos Logísticos , Estudios Longitudinales , Morbilidad , Enfermedades Respiratorias/prevención & controlRESUMEN
Glucagon-like peptide-1 (GLP-1) receptor agonists, used for the treatment of type 2 diabetes, have caused hyperplasia/neoplasia of thyroid C cells in rodent carcinogenicity studies. Studies in monkeys have not identified an effect of GLP-1 receptor agonists on thyroid C cells; however, group sizes were small. Dulaglutide is a once-weekly, long-acting human GLP-1 receptor agonist recently approved in the United States and the European Union. The objective of this study was to determine whether dulaglutide altered C-cell mass in monkeys. Male cynomolgus monkeys (20 per group) were sc injected with dulaglutide 8.15 mg/kg (â¼500-fold maximum human plasma exposure) or a vehicle control twice weekly for 52 weeks. Basal and calcium gluconate-stimulated serum calcitonin concentrations were obtained at 3, 6, 9, and 12 months. Thyroid glands were weighed, fixed, and sectioned at 500-µm intervals. C-cell volumes were measured using an automated image analysis. C-cell proliferation was estimated using Ki67/calcitonin colabeling and cell counting. Administration of dulaglutide 8.15 mg/kg twice weekly for 52 weeks did not increase serum calcitonin in monkeys or affect thyroid weight, histology, C-cell proliferation, or absolute/relative C-cell volume. This study represents a comprehensive evaluation of the monkey thyroid C cells after dosing with a GLP-1 receptor agonist, with a large group size, and measurement of multiple relevant parameters. The lack of effect of dulaglutide on C cells is consistent with other studies in monkeys using GLP-1 receptor agonists and suggests that nonhuman primates are less sensitive than rodents to the induction of proliferative changes in thyroid C cells by GLP-1 receptor agonists.
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Calcitonina/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Péptidos Similares al Glucagón/análogos & derivados , Hipoglucemiantes/farmacología , Fragmentos Fc de Inmunoglobulinas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Glándula Tiroides/efectos de los fármacos , Animales , Calcitonina/sangre , Gluconato de Calcio/farmacología , Receptor del Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón/farmacología , Macaca fascicularis , Masculino , Tamaño de los Órganos/efectos de los fármacos , Receptores de Glucagón/agonistas , Glándula Tiroides/metabolismo , Glándula Tiroides/patologíaRESUMEN
The tumorigenic potential of dulaglutide was evaluated in rats and transgenic mice. Rats were injected sc twice weekly for 93 weeks with dulaglutide 0, 0.05, 0.5, 1.5, or 5 mg/kg corresponding to 0, 0.5, 7, 20, and 58 times, respectively, the maximum recommended human dose based on plasma area under the curve. Transgenic mice were dosed sc twice weekly with dulaglutide 0, 0.3, 1, or 3 mg/kg for 26 weeks. Dulaglutide effects were limited to the thyroid C-cells. In rats, diffuse C-cell hyperplasia and adenomas were statistically increased at 0.5 mg/kg or greater (P ≤ .01 at 5 mg/kg), and C-cell carcinomas were numerically increased at 5 mg/kg. Focal C-cell hyperplasia was higher compared with controls in females given 0.5, 1.5, and 5 mg/kg. In transgenic mice, no dulaglutide-related C-cell hyperplasia or neoplasia was observed at any dose; however, minimal cytoplasmic hypertrophy of C cells was observed in all dulaglutide groups. Systemic exposures decreased over time in mice, possibly due to an antidrug antibody response. In a 52-week study designed to quantitate C-cell mass and plasma calcitonin responses, rats received twice-weekly sc injections of dulaglutide 0 or 5 mg/kg. Dulaglutide increased focal C-cell hyperplasia; however, quantitative increases in C-cell mass did not occur. Consistent with the lack of morphometric changes in C-cell mass, dulaglutide did not affect the incidence of diffuse C-cell hyperplasia or basal or calcium-stimulated plasma calcitonin, suggesting that diffuse increases in C-cell mass did not occur during the initial 52 weeks of the rat carcinogenicity study.
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Péptidos Similares al Glucagón/análogos & derivados , Hipoglucemiantes/toxicidad , Fragmentos Fc de Inmunoglobulinas/toxicidad , Proteínas Recombinantes de Fusión/toxicidad , Glándula Tiroides/efectos de los fármacos , Neoplasias de la Tiroides/inducido químicamente , Animales , Calcitonina/sangre , Calcitonina/efectos de los fármacos , Pruebas de Carcinogenicidad , Carcinoma Neuroendocrino , Femenino , Receptor del Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón/toxicidad , Hiperplasia , Masculino , Ratones , Ratones Transgénicos , Tamaño de los Órganos , Proteínas Proto-Oncogénicas p21(ras)/genética , Ratas , Receptores de Glucagón/agonistas , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Neoplasias de la Tiroides/patologíaRESUMEN
A field-based case-control study was conducted to assess the strength of association of porcine circovirus type 2 (PCV2) and some major swine viruses with postweaning multisystemic wasting syndrome (PMWS). Cases were defined as individual pigs with a clinical history of progressive weight loss and histopathological lesions characteristic of PMWS. Controls were pigs without clinical signs and histopathological lesions typical of PMWS. A total of 31 cases and 56 controls was identified from diagnostic submissions. Serum and various tissues were collected from all animals and assayed for PCV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus, porcine enterovirus types 1-3, swine influenza virus, porcine respiratory coronavirus, transmissible gastroenteritis virus, porcine endogenous retrovirus, porcine lymphotropic herpesvirus type 1, and bovine viral diarrhea virus. The proportion of case and control pigs positive for each virus was determined and statistically compared for determining the strength of the association that each virus had with PMWS individually or in combinations. Porcine circovirus type 2 had the strongest association (OR = 9.3, P = 0.006) with PMWS among the viruses tested for. Risk for PWMS was much higher (OR = 31.2, P = 0.0009) if the animal was concurrently infected with PCV2 and PRRSV, suggesting that development of PMWS may be enhanced by cofactor(s). Because PCV2 was also found in 62.5% of the controls, PCV2 from 5 cases and 4 controls were selected and genetically compared. No significant genetic difference was observed between PCV2 from PMWS and control pigs.
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Circovirus/patogenicidad , ADN Viral/análisis , Enfermedades de los Porcinos/virología , Síndrome Debilitante/veterinaria , Síndrome Debilitante/virología , Animales , Animales Recién Nacidos , Estudios de Casos y Controles , Infecciones por Circoviridae , Circovirus/genética , Cartilla de ADN , Oportunidad Relativa , Reacción en Cadena de la Polimerasa/veterinaria , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Factores de Riesgo , Porcinos , DesteteRESUMEN
Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations.
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Anticuerpos Antivirales/sangre , Proteínas de la Cápside , Cápside/inmunología , Circovirus/inmunología , Glicoproteínas/inmunología , Animales , Circovirus/clasificación , Ensayo de Inmunoadsorción Enzimática , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , PorcinosRESUMEN
Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa.