Wood Lamp Technique in Veterinary Microbiology

Introduction

Principle

Technique / Procedure

1. Warm-up: Allow the lamp to warm up for at least 5 minutes before use - this stabilizes the UV emission and prevents false negatives due to insufficient UV output.
2. Dark room: Examination must be performed in a completely darkened room to properly visualize fluorescence. The room should be dark enough for the examiner's eyes to adapt.
3. Distance: Hold the lamp 4-5 cm from the animal's skin/hair coat for optimal fluorescence detection.
4. Examination: Systematically scan affected hair-bearing areas - scalp (in small animals), skin lesions, claws, and suspicious areas. In cats, the entire coat should be examined.
5. Observation: Look for apple-green (yellow-green) fluorescence along the hair shaft, particularly at the base near the follicle, where actively growing infected hair is located.
6. Sample collection: Fluorescing hairs should be epilated (plucked) with forceps and submitted for fungal culture (DTM/Sabouraud's dextrose agar) and microscopic KOH examination to confirm the diagnosis.

Organisms and Fluorescence Patterns

Applications in Veterinary Medicine

• Primary screening: For dermatophytosis (ringworm) in cats, dogs, horses, cattle, and other species susceptible to M. canis
• Selecting hairs for culture: Fluorescing hairs are specifically targeted for fungal culture and microscopy (KOH prep), improving diagnostic yield
• Monitoring therapy: Can be used to assess treatment response - reduction or disappearance of fluorescent hairs indicates successful therapy
• Shelter medicine: Useful cost-effective screening tool for animal shelters to identify infected cats before integration with populations

Limitations

1. Species-specific: Only screens for M. canis - does not detect Trichophyton spp. or M. gypseum, which do not produce fluorescent metabolites
2. Strain variation: Not all M. canis strains fluoresce; reported sensitivity is around 66-71%, meaning a negative result does not rule out infection
3. False positives: Scale, topical medications, certain soaps, petrolatum, and some bacteria (e.g., Pseudomonas) can produce fluorescence - true M. canis fluorescence is bright apple-green along the hair shaft specifically, not the skin surface
4. Examiner dependence: Requires proper technique (adequate warm-up, dark room) and experience to differentiate true from false fluorescence
5. Not confirmatory: A positive Wood lamp result must always be confirmed by fungal culture (gold standard)

Interpretation Summary

• Positive (apple-green fluorescence on hair shaft) = presumptive M. canis infection; collect fluorescing hairs for culture
• Negative fluorescence = does NOT rule out dermatophytosis; proceed with culture and microscopy if clinical suspicion remains

Conclusion

B.V.Sc. & A.H. Second Professional Examination 2025

Veterinary Microbiology Paper II - Complete Answers

Q.1 Fill in the Blanks (20 × 0.5 = 10)

Correct answer: B cells are absent from the paracortex of lymph nodes (the paracortex is the T-cell-dependent zone).

Q.2 MCQ Answers (Write the correct option number)

Note: Peyer's patches are also secondary lymphoid organs. But among the options, Spleen (3) is the most clearly secondary lymphoid organ. (If Peyer's patches is the intended answer as option 1, that is also secondary - both 1 and 3 are secondary. The answer is likely 3 - Spleen as primary lymphoid organs are bone marrow and thymus only.)

Q.3 Short Answer Questions (2-3 lines each) - Attempt any 10

C3 → (spontaneous hydrolysis) → C3(H₂O)
C3(H₂O) + Factor B → C3(H₂O)B
C3(H₂O)B + Factor D → C3(H₂O)Bb (Initial C3 convertase) + Ba
↓ cleaves C3
C3b (deposits on surface)
C3b + Factor B → C3bB
C3bB + Factor D → C3bBb (Amplification C3 convertase) + Ba
Properdin (Factor P) stabilizes C3bBb
C3bBb cleaves more C3 → more C3b (amplification loop)
C3bBbC3b = C5 CONVERTASE (Alternative pathway)
↓ cleaves C5
C5a + C5b → MAC formation

1. Infectious bovine rhinotracheitis (IBR) - caused by Bovine herpesvirus 1 (BHV-1)
2. Marek's disease in poultry - caused by Marek's disease virus (MDV/Gallid alphaherpesvirus 2)
3. Equine herpesvirus infection (rhinopneumonitis/abortion) - caused by EHV-1 and EHV-4
4. Canine herpesvirus infection (fading puppy syndrome) - caused by Canine herpesvirus 1 (CHV-1)
5. Duck enteritis (Duck plague) - caused by Anatid alphaherpesvirus 1
6. Pseudorabies (Aujeszky's disease) in swine - caused by Suid herpesvirus 1

Q.4 Long Short Answers (8-10 lines each) - Attempt any 6

• Four polypeptide chains: two identical heavy (H) chains (~50 kDa each) and two identical light (L) chains (~25 kDa each), held together by disulfide bonds and non-covalent interactions.
• Variable (V) regions: The N-terminal portions of both H and L chains form the variable region, which contains the antigen-binding site (Fab). Each V region has three complementarity-determining regions (CDRs) that directly contact antigen, and four framework regions.
• Constant (C) regions: The C-terminal portions determine the antibody class (isotype) and mediate effector functions.
• Fab fragment: (Fragment antigen binding) - consists of one VH + CH1 of heavy chain + entire light chain. Two Fab arms provide bivalency.
• Fc fragment: (Fragment crystallizable) - consists of CH2 + CH3 domains of both heavy chains. Mediates complement activation, binding to Fc receptors on phagocytes, placental transfer (IgG).
• Hinge region: A flexible region between CH1 and CH2 that allows movement of the two Fab arms. Rich in cysteine and proline residues; site of inter-heavy-chain disulfide bonds.
• Light chains: Either kappa (κ) or lambda (λ) type; each light chain has one VL and one CL domain.
• Antibodies exist in five isotypes (IgG, IgA, IgM, IgE, IgD) determined by the type of heavy chain (γ, α, μ, ε, δ respectively).

1. Sensitization phase: On first exposure, antigen is processed by APCs (dendritic cells, macrophages) and presented via MHC class II to CD4+ T helper cells (Th1 subtype), which become sensitized memory T cells.
2. Effector phase (re-exposure): Memory Th1 cells recognize antigen-MHC II complexes and release cytokines: IFN-γ (activates macrophages), TNF-α, IL-2 (T cell proliferation), and MCP-1 (recruits monocytes).
3. Activated macrophages release lysosomal enzymes, reactive oxygen species, and more cytokines, causing tissue damage.
4. In some cases (e.g., persistent antigen), a granuloma forms (e.g., in tuberculosis).

1. Serial passage in non-natural host or cell culture: Repeated passaging in heterologous cells/animals selects for mutations adapting the virus to the new host while losing virulence for the natural host. Example: Pasteur attenuated rabies virus by serial intracerebral passage in rabbits; CDV Onderstepoort strain by serial cell culture passages.
2. Physical methods: Cultivation at suboptimal/elevated temperatures selects for cold-adapted or temperature-sensitive mutants. Example: Cold-adapted influenza vaccines.
3. Chemical mutagenesis: Treatment with mutagens (e.g., nitrous acid, 5-fluorouracil) introduces mutations that reduce virulence.
4. Genetic engineering (recombinant DNA technology): Targeted deletion of virulence genes (gene-deleted vaccines). Example: BHV-1 gE-deleted vaccine (DIVA vaccine), MDV deletion mutants.
5. Cultivation on non-natural substrates: Growing bacteria on abnormal media. Example: BCG (Bacillus Calmette-Guérin) was attenuated by serial passage on bile-potato-glycerin medium over 13 years (230 passages).
6. UV irradiation: Causes mutations in nucleic acid, reducing replicative ability.
7. Desiccation: Used for some bacterial vaccines (e.g., anthrax spore vaccine).

• Order: Mononegavirales
• Family: Rhabdoviridae
• Key genera in veterinary medicine:
  • Lyssavirus - Rabies virus (RABV), Australian bat lyssavirus, Lagos bat virus
  • Vesiculovirus - Vesicular stomatitis virus (VSV) - Indiana and New Jersey serotypes
  • Ephemerovirus - Bovine ephemeral fever virus (BEFV)
  • Novirhabdovirus - Infectious hematopoietic necrosis virus (IHNV) in fish
  • Tibrovirus, Curiovirus - other genera
• Morphology: Bullet-shaped (cylindrical with flat end and rounded end), 180 × 75 nm, enveloped, with helical nucleocapsid containing negative-sense ssRNA.
• Genome: Single-stranded, negative-sense RNA (~12 kb), encoding 5 proteins: N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein - surface spikes, responsible for neutralizing antibody induction), L (large protein/RNA-dependent RNA polymerase).

• Genome: Linear double-stranded DNA (26-45 kb) with inverted terminal repeats (ITRs) and covalently attached terminal protein (TP) at each 5' end.
• Replication site: Nucleus (forms Cowdry type A intranuclear inclusion bodies).
• Strategy: Uses a protein-primed strand displacement mechanism:
  1. The terminal protein (TP) acts as a primer - its serine residue is covalently linked to dCMP to initiate synthesis.
  2. Adenovirus DNA polymerase (AdPol) performs strand displacement synthesis, starting from one or both ends of the linear genome.
  3. Early genes (E1A, E1B, E2, E3, E4) are transcribed first by host RNA Pol II; E2A and E2B encode DNA-binding protein (DBP) and AdPol respectively.
  4. Late genes encode structural proteins (hexon, penton, fiber).
  5. New linear dsDNA is produced without an RNA primer requirement.
• Notable: Host cell DNA polymerase is NOT used; adenoviruses encode their own DNA polymerase.

• Genome: Circular, single-stranded DNA (negative sense), ~2-4 kb - smallest known animal DNA viruses.
• Members: Torque teno virus (TTV), Torque teno mini virus (TTMV), Torque teno micro virus (TTMiV); Porcine circovirus 1 (PCV1) and PCV2 were formerly in this family.
• Replication site: Nucleus.
• Strategy: Uses a rolling circle replication (RCR) mechanism:
  1. Host cell DNA polymerase converts the incoming ssDNA to double-stranded replicative form (RF) using a complementary strand.
  2. A virus-encoded Rep protein (replication initiator protein) nicks the positive strand of the dsDNA RF at the origin of replication.
  3. Rolling circle amplification proceeds - the 3'-OH of the nick serves as primer, and host DNA polymerase extends it around the circular template, displacing the old strand.
  4. Multiple copies of ssDNA genome are generated, which are then circularized and packaged into capsids.
• Key difference from Adenoviridae: Anelloviridae rely almost entirely on host cell DNA polymerase (G2/S phase of cell cycle), while Adenoviridae encode their own polymerase.

Q.5 Long Essay Questions (Attempt any 2)

• Order: Piccovirales
• Family: Parvoviridae
• Subfamily Parvovirinae (infects vertebrates):
  • Genus Protoparvovirus: Canine parvovirus 2 (CPV-2), Feline panleukopenia virus (FPV), Mink enteritis virus
  • Genus Amdoparvovirus: Aleutian mink disease parvovirus (AMDV)
  • Genus Aveparvovirus: Chicken parvovirus, Turkey parvovirus
  • Genus Bocaparvovirus: Bovine parvovirus (BPV), Canine minute virus (MVC/CPV-1)
  • Genus Tetraparvovirus: Human parvovirus B19 (Primate erythroparvovirus 1)
  • Genus Dependoparvovirus: Adeno-associated viruses (AAV) - require helper virus (adenovirus)
• Subfamily Densovirinae (infects invertebrates/insects)

• Size: 18-26 nm - smallest DNA animal viruses
• Symmetry: Icosahedral (T=1), non-enveloped
• Capsid: Consists of VP1 (minor), VP2 (major), and VP3 (in some); no envelope
• Genome: Single-stranded DNA (negative or positive sense), ~5 kb, linear, with hairpin loop structures (palindromic sequences) at both 3' and 5' ends - serve as self-primers for replication
• Replication: Requires dividing cells (S-phase); occurs in nucleus
• Stability: Highly resistant to heat, acids, lipid solvents, disinfectants due to non-enveloped nature

• Route of entry: Oronasal exposure to feces-contaminated material; CPV-2 is shed in feces in extremely large quantities (up to 10^9 virions/g feces).
• CPV-2 first replicates in lymphoid tissue of oropharynx (tonsils, mesenteric lymph nodes) → enters bloodstream → viremia (Day 1-5 post-infection).
• During viremia, virus targets rapidly dividing cells:
  • Intestinal crypt epithelium (in duodenum and jejunum): destruction of crypt cells → inability to replace villous epithelium → villous blunting → malabsorption, protein-losing enteropathy, severe hemorrhagic diarrhea
  • Bone marrow: destruction of rapidly dividing hematopoietic precursors → leukopenia (lymphopenia + neutropenia) → immune suppression, susceptibility to secondary bacterial infections, septicemia
  • Thymus (in young pups): thymic atrophy
  • Myocardium (CPV-2 myocarditis form - now rare due to maternal antibody protection): seen in pups <8 weeks; cardiac myocyte necrosis → acute heart failure or sudden death
• Clinical signs: severe hemorrhagic gastroenteritis, projectile vomiting, profuse bloody diarrhea, dehydration, leukopenia, fever → death in untreated cases within 48-72 hours.

1. Antigen detection (ELISA): Fecal ELISA using monoclonal antibodies against VP2 - rapid, in-clinic test; detects CPV antigen in feces (Days 3-10 post-infection); most practical
2. Electron microscopy: Visualization of parvovirus particles in feces (20-22 nm icosahedral particles)
3. Hemagglutination (HA) and Hemagglutination Inhibition (HI): CPV agglutinates pig or rhesus monkey RBCs; used for diagnosis and serology
4. PCR/Real-time PCR: Most sensitive; detects viral DNA in feces, blood; can differentiate CPV-2 variants (2a, 2b, 2c)
5. Virus isolation: CRFK or A72 cells; CPE visible; confirmed by FAT or IEM
6. Histopathology: Intranuclear inclusion bodies in crypt cells; villous blunting; lymphoid depletion

• Live modified virus (LMV) vaccines: Most effective; based on attenuated CPV-2 (or related high-titer FPV strains that protect against CPV); induce strong and long-lasting immunity. Standard schedule: 6-8 weeks, 10-12 weeks, 14-16 weeks, then annually or every 3 years.
• Killed/inactivated vaccines: Available but less immunogenic; require adjuvant and booster.
• Maternal antibody interference: Main challenge in puppies <16 weeks; high-titer vaccines (CPV-2c based) help overcome maternal antibody interference.
• Key point: CPV-2 is a core vaccine antigen per WSAVA guidelines.

• Order: Mononegavirales
• Family: Paramyxoviridae
• Subfamily Orthoparamyxovirinae:
  • Genus Avulavirus: Newcastle Disease Virus (NDV = Avian paramyxovirus 1, APMV-1)
  • Genus Respirovirus: Bovine parainfluenza virus 3 (BPIV-3), Human PIV 1 and 3
  • Genus Morbillivirus: Rinderpest virus (RPV), Canine distemper virus (CDV), Peste des petits ruminants virus (PPRV), Measles virus
  • Genus Rubulavirus: Mumps virus, Canine parainfluenza virus 5
  • Genus Henipavirus: Nipah virus (NiV), Hendra virus (HeV) - uses Ephrin-B2/B3 as receptor
  • Genus Metapneumovirus (subfamily Pneumoviridae): Avian metapneumovirus (aMPV), Human MPV
  • Genus Pneumovirus (subfamily Pneumoviridae): Bovine RSV, Human RSV

• Size: 150-500 nm (pleomorphic), enveloped
• Symmetry: Helical nucleocapsid
• Envelope: Lipid bilayer derived from host cell membrane with two major surface glycoproteins:
  • HN (Hemagglutinin-Neuraminidase): Mediates attachment to sialic acid receptors; neuraminidase activity releases progeny virions; also has hemagglutination activity
  • F (Fusion protein): Mediates fusion of viral envelope with cell membrane; responsible for syncytium formation; synthesized as inactive F0 → cleaved by host proteases into F1+F2 (disulfide linked) = activated
• M (Matrix protein): Lines the inner surface of the envelope; maintains virion integrity
• Nucleocapsid: RNA + N (nucleoprotein) + P (phosphoprotein) + L (large protein/RdRp)
• Genome: Single-stranded, negative-sense, non-segmented RNA (~15 kb); 3'-N-P-M-F-HN-L-5' gene order

• Agent: Newcastle Disease Virus (NDV), officially named Avian orthoavulavirus 1, genus Avulavirus, family Paramyxoviridae
• Host: Primarily domestic poultry (chickens); affects all birds (~250 species); zoonotic potential (mild conjunctivitis in humans)
• Classification of NDV strains by pathotype: Lentogenic (mild) → Mesogenic (moderate) → Velogenic (highly pathogenic) - based on intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI)

1. Attachment via HN protein to sialic acid residues on host cell surface
2. F protein-mediated membrane fusion; nucleocapsid released into cytoplasm
3. Primary transcription: L protein (RdRp) transcribes the negative-sense genome into individual mRNAs (5'-capped, 3'-polyadenylated) in sequential gradient - genes near 3' end transcribed more than genes near 5' end (transcription gradient)
4. Translation of viral proteins; accumulation of N protein triggers switch from transcription to replication
5. Replication: Full-length positive-sense antigenomic RNA (+RNA) is synthesized as template → then copied back to full-length negative-sense genomic RNA
6. New genomes are encapsidated by N protein; M protein drives assembly at plasma membrane
7. Budding: New virions bud from plasma membrane; neuraminidase activity of HN cleaves sialic acid to prevent self-aggregation and release progeny

• ICPI (Intracerebral Pathogenicity Index): Standard OIE test; allantoic fluid inoculated IC in 1-day-old chicks; scored 0-2 over 8 days. ICPI ≥0.7 = notifiable; ≥1.2 = velogenic
• IVPI (Intravenous Pathogenicity Index): 6-week-old birds; scored 0-3
• Mean Death Time (MDT) in embryonated eggs: Lentogenic >90 hrs; Mesogenic 60-90 hrs; Velogenic <60 hrs
• Molecular pathotyping: RT-PCR + sequencing of F protein cleavage site: Velogenic/mesogenic = polybasic cleavage site (R-R-Q-K-R↓F or similar); Lentogenic = monobasic (G-R-Q-G-R↓L)

1. Virus isolation: Inoculate oropharyngeal/cloacal swabs or organ homogenates into 9-11 day embryonated eggs (allantoic cavity); incubate 4-7 days; harvest allantoic fluid and perform Haemagglutination (HA) test using chicken RBCs - positive = haemagglutinating agent present
2. HI test (Haemagglutination Inhibition): Identify NDV-specific HA using known NDV antiserum; gold standard serological test for identification and serology
3. RT-PCR / Real-time RT-PCR: Most sensitive and rapid; detects NDV RNA from swabs, organs, directly; can detect and differentiate strains; can assess pathotype via F cleavage site sequencing
4. Fluorescent Antibody Test (FAT): Direct/indirect FAT on frozen sections of trachea, lung, brain
5. ELISA: For serosurveillance and vaccine monitoring; detects anti-NDV antibodies in serum
6. Electron microscopy: Pleomorphic enveloped particles with surface spikes

         Peptide-binding groove
         (holds 8-10 aa peptide)
         ___________________
α1 domain| α2 domain|
    ↕ non-covalent     
α3 domain (Ig-like) ← binds CD8 co-receptor
    ↕ non-covalent
β2-microglobulin (β2m) - light chain, not MHC-encoded
    ↕
Cell membrane (transmembrane anchor via α3)

• Expressed on ALL nucleated cells
• Heavy α-chain (MHC-encoded, chromosome 6 in mammals) + β2-microglobulin (chromosome 15)
• α1 + α2 form the peptide-binding groove (closed ends - accepts short 8-10 aa peptides)

         Peptide-binding groove
         (holds 13-25 aa peptide)
         ___________________
α1 domain|  β1 domain |
    ↕ non-covalent
α2 domain (Ig-like) + β2 domain (Ig-like) ← β2 binds CD4 co-receptor
    ↕
Cell membrane (both α and β chains are transmembrane)

• Expressed only on professional APCs (dendritic cells, macrophages, B cells)
• Heterodimer of α-chain + β-chain (both MHC-encoded)
• α1 + β1 form the peptide-binding groove (open ends - accommodates longer peptides, 13-25 aa)

Intracellular protein (viral, tumor)
        ↓
Ubiquitination → degraded by PROTEASOME (26S)
        ↓
Peptide fragments (8-10 aa)
        ↓
Transported into ER by TAP (Transporter associated with Antigen Processing)
        ↓
Peptide loaded onto MHC Class I (α-chain + β2m) in ER → with help of tapasin, calreticulin
        ↓
Stable peptide-MHC I complex → transported via Golgi
        ↓
Expressed on CELL SURFACE
        ↓
Recognized by CD8+ Cytotoxic T Lymphocytes (CTL)
        ↓
CTL kills infected/abnormal cell (perforin/granzyme, Fas-FasL pathway)

Extracellular antigen (bacteria, virus, parasites)
        ↓
ENDOCYTOSIS / PHAGOCYTOSIS by APC (DC, macrophage, B cell)
        ↓
Endosome → Phagolysosome (acidic pH, proteolytic enzymes → peptide fragments 13-25 aa)
        ↓
Meanwhile: MHC Class II synthesized in ER → associated with Invariant chain (Ii/CD74)
           Ii chain blocks peptide-binding groove (CLIP = Class II-associated Ii peptide)
        ↓
MHC II + Ii chain transported in vesicle
        ↓
Vesicle fuses with endolysosome (MIIC compartment)
        ↓
HLA-DM (chaperone) catalyzes removal of CLIP → replaced by processed antigen peptide
        ↓
Stable peptide-MHC II complex → transported to cell surface
        ↓
Recognized by CD4+ T Helper Cells (Th1, Th2, Tfh)
        ↓
Th cells secrete cytokines → activate macrophages, help B cells make antibody