Lecture 13 Study Guide

• Biological Traits:     * Metabolism: Aerobic organisms.     * Physical State: Non-spore-forming and non-motile.     * Morphology: Classified as Gram-positive (G+) rods.     * Acid-Fast Properties: They are Acid-fast bacilli (AFB) due to the presence of mycolic acids and high lipid content in the cell wall.         * Functional Benefits of Cell Wall: These components protect the bacteria against phagocytosis, extreme pH levels, and various environmental stresses.         * Staining Restrictions: They cannot be stained using the standard Gram stain because of the waxy cell wall.

• Staining Procedures and Identification:     * Ziehl-Neelsen (ZN) / Acid-Fast Staining: This is the primary method used for visualization.     * Preliminary Diagnosis: If AFB are observed on a stain, it constitutes a preliminary diagnosis of mycobacterial infection.     * Mechanism: Lipids in the cell wall bind to carbol fuchsin (a red dye). They resist decolorization even when treated with alcohol, which is the definition of "acid-fast."     * Counter-Staining: Methylene blue is used as a counter-stain.     * Visual Outcome: On the slide, the background appears blue while the Mycobacterium organisms appear red.

• Growth and Diagnosis Timeline:     * Growth Rate: These organisms grow very slowly, with generation times up to $5\text{ hours}$, leading to delayed diagnosis.     * Species-Specific Growth Times:         * M. tuberculosis and M. bovis: Can take up to $4\text{ weeks}$ to grow.         * M. avium: May require more than $4\text{ months}$.         * Non-Tuberculous Mycobacteria (NTM): Typically grow within $1-3\text{ weeks}$.     * Laboratory Protocol: Practitioners must explicitly inform the laboratory if a Mycobacterial infection is suspected, as these organisms require specialized, complex media to grow.

• Host Dynamics and Pathogenesis:     * Host Range: Wide range of hosts are susceptible.     * Intracellular Nature: They are facultative intracellular pathogens of macrophages. Mycolic acids and lipids are critical here as they prevent the fusion of the phagosome and the lysosome.     * Nature of Infection: Infections are typically chronic and progressive, capable of persisting for years without showing overt clinical disease, which often results in delayed diagnosis.         * Example: In Johne's Disease, calves may be infected but will not show clinical signs until they are at least $2\text{ years}$ old.

• Species Classification:     * Obligate Pathogens: Includes M. bovis, M. tuberculosis, and M. paratuberculosis.         * Characteristics: Grow slowly; survive in the environment for extended periods; transmitted primarily by diseased animals.     * Saprophytes: Known as NTMs or Atypical Mycobacteria.         * Characteristics: Grow faster (days to one week); survive long-term in the environment; usually cause opportunistic infections.

• Immunopathology:     * Granulomatous Infections: Mycobacteria cause granulomatous inflammation. The host immune response forms granulomas, which are specialized structures composed primarily of macrophages, to wall off the bacteria.

Bovine Tuberculosis ($M. bovis$)

• Etiological Agent: Mycobacterium bovis.

• Target Population: Primarily cattle, though it has a wide host range. Notably, chickens are resistant to M. bovis.

• Encounter and Environmental Survival:     * Source: Infected animal populations.     * Transmission Routes: Respiratory droplets, sputum, milk, feces, urine, and various bodily discharges.     * Persistence: The organism can survive in the environment for $4\text{ days}$ during the summer and up to $28\text{ days}$ during the winter.     * Replication: Importantly, M. bovis does not replicate in the environment; it only survives there. Transmission is significantly aided by crowded living conditions.

• Pathogenesis: Entry, Multiplication, and Spread:     * Route of Entry: Inhalation or ingestion.     * Cellular Uptake: Bacteria are taken up by macrophages where they multiply intracellularly.     * Local Spread: They travel from the site of entry to the local lymph nodes (LN).     * Systemic Spread: If the bacteria enter the lymphatics or the general circulation, they can disseminate widely throughout the body.

• Outcomes of Infection:     * Latency: Occurs when adequate cell-mediated immunity is able to control the infection without eliminating it.     * Systemic Granulomas (Tuberculosis): Occurs when bacterial multiplication is unchecked.

• Pathology of the Granuloma:     * Formation: Engulfed bacilli trigger the arrival of T cells to induce killing. T lymphocytes produce cytokines that increase the recruitment of more macrophages.     * Structure: The accumulation of these cells forms the granuloma.         * Outer Layer: Composed of T cells, macrophages, giant cells, and fibroblasts.         * Inner Core: Characterized by caseous necrosis.     * Pathognomonic Lesions: Granulomas themselves are considered pathognomonic for these infections.

• Clinical Presentation:     * Asymptomatic State: Many animals have no clinical signs; signs depend heavily on which organs are involved (any organ can develop small "tubercles").     * Respiratory Signs: Moist cough, dyspnea, and weight loss.     * Necropsy Findings: Enlarged bronchial, mediastinal, or mesenteric lymph nodes.     * Mastitis: This is a rare clinical manifestation.

• Diagnostic Methods:     * Caudal Fold Test (CFT): Also known as the Intradermal Skin Test.         * Usage: Primary screening test.         * Mechanism: Measures delayed-type hypersensitivity to tuberculin (injected intradermally).         * Observation: Swelling is assessed at $48-72\text{ hours}$ post-injection.         * Sensitivity: The test becomes positive $3-6\text{ weeks}$ post-infection (PI).         * False Negatives (FN): Can occur if it is too early/late in the disease, in recently calved cows, in old animals, or in cases of advanced progressive disease.     * Comparative Cervical Test (CCT): Used to confirm positive CFT results and rule out cross-reactions with M. avium paratuberculosis.         * Positive Result: Reaction occurs to M. bovis but not to M. avium.     * Interferon Gamma Assay: Used in conjunction with skin testing.     * Necropsy and Histopathology: Visualizing AFB on microscopy and identifying granulomatous lesions using ZN stains.     * Culture: The definitive diagnostic method. Requires BSL-3 facilities and specialized media.     * PCR: Rapid and sensitive method for definitive diagnosis.

• Treatment and Control:     * Treatment: There is NO treatment. Infected animals are sent to slaughter.     * Control Strategies: Test newly introduced animals (isolate and retest after $60\text{ days}$).     * Eradication Program: The National Tuberculosis Eradication Program uses testing and slaughter policies to mostly eliminate the disease.     * Vaccination: No vaccine exists for Bovine Tuberculosis.

Bovine Paratuberculosis (Johne’s Disease)

• Etiological Agent: Mycobacterium avium subsp. paratuberculosis (MAP).

• Target Population: Ruminant species.

• Encounter and Prevalence:     * Contamination: Infected animals contaminate the environment via feces.     * Replication: The organism replicates ONLY inside the host, but it can survive in soil or water for more than $1\text{ year}$.     * US Statistics: Approximately $70\%$ of US dairy herds are currently infected.

• Pathogenesis: Entry and Multiplication:     * Transmission: Fecal-oral infection. Exposure usually occurs shortly after birth via feces, contaminated feed/water, the udder, or milk.     * Target Site: Macrophages ingest the organism within the Peyer’s patches of the ileum.     * Mechanism: Bacteria multiply intracellularly, kill the macrophages, and spread to adjacent cells.     * Consequence: The inflammatory response causes a thickened intestinal wall and a complete loss of absorptive function. Primary infection is localized in the ileum and ileal lymph nodes.

• Clinical Signs:     * Incubation: Infection occurs in the perinatal period, but signs do not develop until after the animal is $2\text{ years}$ old.     * Silent Spreaders: Animals shedding the bacteria are rarely clinically sick.     * Disease Manifestations: Chronic, contagious, and debilitating enteritis. Symptoms include decreased milk production, chronic diarrhea, and weight loss despite maintaining a normal appetite.

• Diagnostics:     * Microscopy: Detection of AFB in a rectal pinch smear or scrape.     * DNA PCR: Approved and sensitive method.     * Serology: Tests like ELISA, INF, and AGID exist but are generally not sensitive.     * Johnin Test: An intradermal skin test used for screening.     * Culture: Definitive diagnosis using feces or ileal lymph nodes.         * Timeline: Takes $3-4\text{ months}$.         * Shedding Issues: Bacteria are shed intermittently, leading to false negatives.         * Protocol: $10-15\text{ g}$ fecal samples are taken at $6\text{-month}$ intervals.

• Control and Management:     * Treatment: None; infected animals must be eliminated.     * Herd Management: Maintain a closed herd or only introduce animals from herds that test negative.     * Hygiene: Ensure calves, lambs, and kids are born in clean environments; avoid fecal contamination of feed and water.     * Culling: Identify all infected animals and isolate or cull them.     * Vaccination: A killed vaccine is available.

Non-Tuberculous Mycobacterium (NTM) / Atypical Mycobacteria

• Ecology: These are saprophytes.

• Clinical Presentation:     * Nature of Infection: Occasional granulomatous infections that can be localized or systemic.     * Common Manifestations: Skin infections (often via wound contamination), pneumonia, and gastrointestinal infections.     * Specific Example: M. smegmatis is known to cause panniculitis in cats.

• Diagnosis:     * Histology: ZN staining reveals pyogranulomatous inflammation.     * Molecular: PCR testing.     * Culture: Sensitivity (C/S) testing is necessary to determine appropriate antibiotic treatment.

• Treatment:     * Medical: Treated with antibiotics based on sensitivity results.     * Surgical: May require surgical intervention in some cases.