Lecture #5

CVM 723B Veterinary Virology

Lecture Overview

This lecture is a continuation of Lecture 4, focusing on the advancements in diagnostic tests used by veterinarians to identify illnesses associated with infectious agents. It emphasizes that despite the various rapid diagnostic options available, the costs of these diagnostics can be a significant burden for pet owners. The primary focus is on diagnostic techniques that gauge the host's immunological response to viral infections, specifically through antibody detection.

Lecture Objectives

1. Laboratory Diagnosis of Viral Infections: Understand the importance of quick and accurate viral diagnosis.

2. Sample Collection Practices: Recognize the criteria for selecting and collecting samples for laboratory viral diagnosis.

3. Virus Detection Techniques: Identify various techniques used to detect viruses in specimens and provide examples.

4. Detection of Antibodies: Enumerate methods used to detect antibodies produced by hosts in response to viral infections.

5. Limitations of Diagnostic Methods: Assess factors such as turnaround time, cost, sensitivity & specificity, and correlation with clinical disease as limitations of diagnostic techniques.

Laboratory Techniques to Diagnose a Virus Infection

Virus Detection Techniques

• Virus Isolation: Methods include:

  • Cell culture and observation of cytopathic effect (CPE).

  • Usage of laboratory animals (historical significance).

  • Egg culture as a traditional technique.

• Virus Visualization Techniques:

  • Electron Microscopy (EM)

• Viral Antigen Detection Methods:

  • Enzyme-linked immunosorbent assay (ELISA).

  • In situ Hybridization.

  • Immunohistochemistry / Immunofluorescence.

• Characteristic Gene Sequence Identification:

  • Polymerase chain reaction (PCR).

  • Whole genome sequencing.

  • Metagenomics.

Antibody Detection Techniques

• Common Techniques for Antibody Detection:

  • ELISA.

  • Immunofluorescent Antibody Test (IFAT).

  • Serum Neutralization Testing (SN).

  • Hemagglutination Inhibition Testing.

  • Agar Gel Immunodiffusion (AGID/Ouchterlony).

Important Considerations for Antibody Detection

• The ELISA gives immediate results indicating positive or negative findings.

• Serology tests rely on a principle that seeks a four-fold rise in antibody levels between acute and convalescent serum samples.

• There are significant challenges in collecting a second serum sample, as the initial sample may already contain antibodies due to existing infections or from maternal colostrum.

• These issues regarding antibody detection pose problems primarily in the context of diagnosis rather than in serological surveys aimed at determining infection prevalence in populations.

Enzyme-linked Immunosorbent Assay (ELISA)

• Process:

  1. Serum Sample being tested is analyzed to determine the presence of antibodies.

  2. Virus antigen is attached to the plate, capturing any existing antibodies.

  3. Detecting antibodies are introduced, leading to a color change as an indicator of antibody presence (typically blue).

Serum Virus Neutralization Test to Determine Antibody Titer

• Testing Steps:

  1. Perform serial dilutions of serum (e.g., 1:2, 1:4, 1:8, …, 1:512).

  2. Add each dilution to separate wells containing cultured cells.

  3. Introduce equal amounts (100 plaque-forming units) of the virus to each well.

  4. Evaluate the last dilution that prevents plaque formation to determine the antibody titer.

Immunofluorescence Antibody Test (Titer)

• Requirements:

  • Microscope equipped with fluorescence capability.

• Procedure:

  • Demonstrates the presence of antibodies in the serum through the use of an anti-IgG secondary antibody marked with a fluorescent label.

  • Serial dilutions of the patient's serum can help identify the last well with fluorescence, indicating the titer.

Hemagglutination Inhibition Testing for Antibodies

• Methodology:

  • Certain viruses can agglutinate red blood cells (RBCs).

  • Serial dilutions of the patient's serum are mixed with a known quantity of the virus.

  • The test detects the presence of antibodies by noting the dilution where agglutination is inhibited.

  • Utilization of paired sera (acute and convalescent samples) taken about two weeks apart aids in determining rises in antibody titer.

AGID – Agar Gel Immunodiffusion (Ouchterlony)

• Purpose and Uses:

  • This method is particularly relevant in underdeveloped countries and remains a test of choice for certain diseases.

  • Also useful for screening and in research related to developing new diagnostic tests (like ELISA).

• Results: A positive result is indicated by a precipitin band showing the presence of antigen-antibody interaction.

Selecting the Appropriate Diagnostic Test

Before selecting a diagnostic test, one should consider:

• The disease stage.

• Knowledge of the sensitivity and specificity of the test.

• Suspected notifiable diseases.

• When in doubt, consult the diagnostic laboratory for guidance.

Limitations of Diagnostic Techniques

• Despite being cost-effective, the ELISA's reliability may vary.

• Virus isolation methods are usually more expensive but beneficial for developing vaccines.

• PCR methods are widely available and efficient, but require advanced laboratory setups.

• Sequencing and metagenomics also necessitate sophisticated laboratories.

• Understanding that antibody detection primarily indicates past exposure, rather than the timing of infections, is crucial for disease correlation.

Considerations in Diagnostic Test Selection

The determination of which diagnostic test to utilize should be based on factors such as:

• Speed of results.

• Sensitivity and specificity.

• Simplicity and accessibility of testing options.

• Consultation with diagnostic laboratories for localized trends or outbreaks can assist in decision-making.

Interpretation of Laboratory Findings

• Diagnostic laboratories provide critical information to clinicians, but the term "diagnostic laboratory" can be misleading as these labs do not offer definitive diagnoses.

• Clinicians must consider specimen collection sites, virus identification, known pathogenicity, and clinical presentations.

• Sensitivity and specificity of tests are vital considerations in the diagnosis process.

• For serological tests, evaluating the age of the animal with respect to colostrum antibodies and vaccination status is also essential.

• The implications of reporting findings—such as confirming a rabies diagnosis—should be thoughtfully considered.

Comparison of Diagnostic Methods

Advantages and Disadvantages

1. Virus Isolation:

   • Advantages: Sensitive, allows for further study, generally accessible.

   • Disadvantages: Identification can be time-intensive, requires specific conditions.

2. Electron Microscopy:

   • Advantages: Rapid, identifies non-cultivable viruses and mixed infections.

   • Disadvantages: High costs, limitations in sensitivity.

3. Serological Tests:

   • Advantages: Rapid results from various methods, including ELISA.

   • Disadvantages: Not applicable for all viruses, challenging results interpretation.

4. Nucleic Acid Tests (PCR):

   • Advantages: Fast and generally specific.

   • Disadvantages: Not suitable for all virus types, result interpretation can be complicated.

Types of Veterinary Diagnostic Laboratories (VDLs) in the US

1. State VDLs: Affiliated with Departments of Agriculture/Veterinary Schools, like the Louisiana Animal Disease Diagnostic Lab (LADDL).

2. National Veterinary Reference Laboratories (USDA): Includes NVSL and specialized facilities like the National Bio and Agro-Defense Facility (NBAF).

3. Commercial Laboratories: Such as Antech and IDEXX.

4. Practitioner Labs (In-house): Facilities run directly by veterinary practitioners.

Roles and Importance of VDLs

• VDLs play a fundamental role in ensuring accuracy and precision in test results through regular audits and proficiency testing.

• Their mission includes diagnostics, research, outreach, and educational support for stakeholders in the veterinary field.

Importance of Laboratory Diagnosis of Infectious Diseases

• The emergence of infectious diseases necessitates recognition of new agents and timely diagnosis to guide treatment.

• Zoonotic diseases pose public health risks and require vigilant cross-species monitoring.

• Effective control strategies depend heavily on accurate and rapid diagnoses of disease outbreaks in herds and populations, impacting economic stability in various sectors connected to animal health, including livestock and companion animal markets.

Diagnostic Medicine Across Multiple Species

• Species Covered: Companion animals, cattle, poultry, equine, sheep, goats, swine, exotic pets, fish, among others.

• Client Diversity: Includes veterinary teaching hospitals, private clinics, state and federal officials, and individual animal owners or farmers.

National Animal Health Laboratory Network (NAHLN)

• Established in 2002 to look after animal disease diagnostics and surveillance.

• Collaboration spans various stakeholders including the USDA and veterinary diagnostic laboratories across states.

• Responsibilities include early detection of diseases and response strategies for livestock.

Current Standardized Methods Provided by National Veterinary Services Laboratories

• Standard operating procedures (SOPs) are in place for various significant animal diseases such as ASF, BSE, and CWD.

• Resources include standardized reagents, drill exercises, and electronic result messaging for enhanced laboratory efficiency.

Clinical Case Examples

Case 1: Pseudocowpox

• Findings: Pathognomonic ring-shaped lesion, confirmed via electron microscopy.

Case 2: Eastern Equine Encephalitis (EEE)

• Symptoms: Depression, blindness, ataxia.

• Findings: Virus isolation, ELISA positivity, histopathology confirmation.

Case 3: Canine Influenza

• Symptoms: Elevated temperature, coughing, nasal discharge.

• Findings: Virus isolation, ELISA positive, PCR confirmed similarity to equine isolates.

Lessons from the COVID-19 Pandemic

• Highlighting the intersection of animal and human health, emphasizing the concepts of One Health.

• Need for effective communication, knowledge acquisition, and informed decision-making based on primary research rather than popular media.

Key Concepts

1. Importance of Quick and Accurate Diagnosis: Essential for controlling diseases effectively, identifying zoonotic threats, and managing herd health.

2. Specimen Selection: Critical for gathering useful information for managing diseases.

3. Antigen Detection: Useful for identifying viral presence in tissues and fluids.

4. Antibody Detection Techniques: ELISA and titers provide valuable insights regarding immunity.

5. Limitations in Diagnostics: Complexity in laboratories, costs, and the importance of appropriate specimen selection and interpretation.

Conclusion

Emphasizing the importance of vaccination in disease prevention. A call to rely on credible resources for continuous knowledge enhancement regarding infectious disease prevention and treatment.

Lecture Overview

This lecture is a comprehensive continuation of Lecture 4, centered on advancements in diagnostic methodologies used by veterinarians to identify infectious agents. It highlights that despite the rapid evolution of diagnostic technologies, the economic burden on pet owners remains a crucial factor in clinical decision-making. The primary focus shifts toward gauging the host's secondary immunological response to viral infections, primarily through the detection of specific antibodies (humoral immunity).

Lecture Objectives

1. Laboratory Diagnosis of Viral Infections: Understand the clinical urgency and pathophysiological relevance of rapid viral identification.

2. Sample Collection Practices: Master the criteria for timing (acute vs. convalescent phases) and site selection (swabs, blood, tissue) to maximize diagnostic yield.

3. Virus Detection Techniques: Differentiate between direct detection (virion visualization, antigen detection) and indirect detection (host response).

4. Detection of Antibodies: Define and compare various serological assays used to quantify the host's immune response.

5. Limitations of Diagnostic Methods: Evaluate the trade-offs between turnaround time, cost-efficiency, $sensitivity$, $specificity$, and the predictive value of a positive result.

Laboratory Techniques to Diagnose a Virus Infection

Virus Detection Techniques

• Virus Isolation (The 'Gold Standard'):

  • Cell Culture: Monitoring for Cytopathic Effect (CPE) such as syncytia formation, inclusion bodies, or cell lysis.

  • Laboratory Animals: Historically used for viruses that do not grow in vitro, though largely replaced by alternative methods due to ethical concerns.

  • Embryonated Egg Culture: A classic technique specifically used for influenza and poxviruses.

• Virus Visualization Techniques:

  • Electron Microscopy (EM): Directly visualizes viral morphology ($e.g.$, helical vs. icosahedral); essential for non-cultivable viruses ($e.g.$, Norovirus).

• Viral Antigen Detection Methods:

  • ELISA: Rapid detection of viral proteins.

  • In situ Hybridization (ISH): Detects viral nucleic acids within specific tissue sections to correlate infection with pathology.

  • Immunohistochemistry (IHC) / Immunofluorescence (IF): Uses labeled antibodies to visualize viral antigens in tissue biopsies or smears.

• Characteristic Gene Sequence Identification:

  • Polymerase Chain Reaction (PCR): Amplifies specific DNA segments; Reverse Transcriptase PCR (RT-PCR) is used for RNA viruses.

  • Whole Genome Sequencing (WGS) & Metagenomics: Investigates the entire genetic makeup of a sample to identify novel or emerging pathogens without prior target knowledge.

Antibody Detection Techniques (Serology)

• Common Assays:

  • ELISA: Often the first-line screening tool.

  • Immunofluorescent Antibody Test (IFAT): Visualizes antibody binding on virus-infected cells.

  • Serum Neutralization Testing (SN): Determines the ability of antibodies to block viral entry into cells.

  • Hemagglutination Inhibition (HI) Testing: Measures antibodies that prevent viruses from clumping red blood cells.

  • Agar Gel Immunodiffusion (AGID/Ouchterlony): A precipitation-based assay common for EIA (Equine Infectious Anemia) testing.

Important Considerations for Antibody Detection

• The $4$-Fold Rise Rule: A definitive diagnosis often requires comparing antibody levels in an acute sample (taken at the onset of illness) and a convalescent sample (taken $2-4$ weeks later). A $4$-fold increase in titer ($e.g.$, from $1:4$ to $1:16$) indicates an active or very recent infection.

• Confounding Factors:

  • Maternal Antibodies: Colostrum-derived antibodies can persist for weeks, leading to false positives in young animals.

  • Previous Vaccination: Distinguishing between vaccine-induced antibodies and natural infection antibodies often requires specific DIVA (Differentiating Infected from Vaccinated Animals) vaccines and tests.

Enzyme-linked Immunosorbent Assay (ELISA)

• Mechanism:

  1. Antigen Coating: Known viral antigens are adsorbed onto a plastic plate.

  2. Primary Binding: Patient serum is added; if antibodies are present, they bind to the antigens.

  3. Secondary Binding: An enzyme-conjugated anti-species antibody ($e.g.$, anti-canine IgG) is added.

  4. Substrate Reaction: A colorless substrate is added; the enzyme converts it into a colored product (usually blue or yellow), indicating a positive result.

Serum Virus Neutralization (SVN) and Titers

• Protocol for Titration:

  1. Create a serial dilution of the patient's serum ($1:2, 1:4, 1:8, 1:16, 1:32, \dots, 1:n$).

  2. Mix each dilution with a constant, known concentration of the virus ($e.g., 100$ TCID50).

  3. Inoculate cell cultures with the mixtures.

  4. Titer Definition: The titer is the reciprocal of the highest dilution of serum that completely inhibits the viral effect (prevents plaque formation or CPE).

Selecting the Appropriate Diagnostic Test

• Sensitivity: The probability that a test correctly identifies an infected animal ($\text{Sensitivity} = \frac{TP}{TP + FN}$).

• Specificity: The probability that a test correctly identifies a non-infected animal ($\text{Specificity} = \frac{TN}{TN + FP}$).

• Clinical Context:

  • Early stage: Focus on direct virus detection (PCR, Antigen ELISA).

  • Late stage/Recovery: Focus on antibody detection (Serology).

  • Notifiable Diseases: Suspected cases of Rabies, Foot and Mouth Disease, or HPAI must be reported to state/federal authorities immediately.

Comparison of Diagnostic Methods

Veterinary Diagnostic Laboratories (VDLs) in the US

1. State VDLs: Usually affiliated with universities ($e.g.$, LADDL at LSU). They handle the bulk of clinical diagnostics for local vets.

2. National Reference Labs (USDA/APHIS):

   • NVSL (Ames, Iowa): The premier reference lab for animal health in the US.

   • NBAF (Manhattan, KS): Designed for high-consequence foreign animal diseases (BSL-4).

3. Commercial Labs: Highly automated labs ($e.g.$, IDEXX, Antech) specializing in volume and speed.

National Animal Health Laboratory Network (NAHLN)

• A coordinated network of federal, state, and university laboratories.

• Focus: Early detection of high-consequence 'Foreign Animal Diseases' (FADs).

• Standardization: Uses validated SOPs (Standard Operating Procedures) to ensure that a test result in Oregon is identical in meaning to a test result in Louisiana.

Clinical Case Studies

• Pseudocowpox: Visualized via EM; recognized by the pathognomonic 'cocoon' or 'fingerprint' appearance of the virion.

• Eastern Equine Encephalitis (EEE): A zoonotic Alphavirus. Diagnosis combines clinical signs (neurological) with IgM ELISA (indicating recent exposure) and post-mortem histopathology.

• Canine Influenza (H3N8/H3N2): Diagnosis via RT-PCR on nasal swabs. Understanding the shift from equine to canine species highlights the danger of host-switching.

Conclusion

Effective diagnostic medicine requires a synergy between clinical observation and laboratory precision. Decisions should be based on peer-reviewed research and consultation with laboratory specialists to ensure appropriate sample selection and result interpretation, ultimately leading to better patient outcomes and public health safety.