Bacteriology: Burkholderia, Bordetella, Fusobacterium, Mycobacteria

Burkholderia Genus

• Gram-negative, aerobic, rod-shaped bacteria
• Found in acidic soil, associated with soil fungi, with over 100 species
• Two species important in veterinary medicine: Burkholderia mallei and Burkholderia pseudomallei, both producing pyogranulomatous disease and designated as category B bioterrorism agents

Glanders Disease (Burkholderia mallei)

• Contagious, acute or chronic, and usually fatal disease
• Characterized by nodule development in upper respiratory tract, lungs, and skin
• Infectious to humans, with 95% fatality rate if left untreated due to septicemia
• Commonly transmitted through contaminated food and water, less commonly through contaminated fomites and aerosols
• Affects horses (Equidae), dogs, cats, camels, goats, and sheep, with felids being particularly susceptible
• Endemic in parts of Asia, Africa, and South America

Acute Glanders Disease

• Incubation period of 3 days to 2 weeks
• Fatal in ~2 weeks, predominantly in donkeys and felids
• Clinical signs include septicemia, high fever, weight loss, thick mucopurulent nasal discharge, respiratory signs, and death

Chronic Glanders Disease

• More common in horses, with donkeys and mules being affected more severely
• Animals may live for years, disseminating the organism
• Three manifesting forms:
  • Nasal Form: nodules in nasal mucosa, degenerating into deep ulcers
  • Pulmonary Form: small tubercle-like nodules in lungs, with consolidation of lung tissue and pneumonia
  • Cutaneous Form ("Farcy"): nodules along lymph vessels, degenerating and ulcerating

Diagnosis and Control of Glanders

• Mallein test: interpalpebral inoculation of mallein, with hypersensitive horses developing purulent conjunctivitis and eyelid swelling within 24 hours
• Treatment with antimicrobials is deemed inappropriate, as it does not reliably produce a cure and infection can remain persistent
• Equine imports from endemic regions are mallein tested, and if positive, euthanized

Burkholderia pseudomallei (Whitmore's Disease)

• Gram-negative, aerobic, rod-shaped bacteria that is facultative intracellular
• Causes pyogranulomatous disease called Melioidosis, affecting a wide host range including humans
• Superficially resembles Glanders, but has a large host range and is a saprophyte

Transmission and Habitat

• Found in soil and water in subtropical and some extratropical regions
• Can be transmitted through direct contact with contaminated soil or surface water, inhalation, ingestion, wound infections, and arthropod bites
• Can also be transmitted transplacental in goats

Melioidosis (Whitmore's Disease)

• Can be systemic or localized, acute, chronic, subclinical, latent, or fulminant
• Manifestation depends on the extent and distribution of the lesions, strain differences, and the host's innate and adaptive immune status
• Treatment is costly, prolonged, and usually unsuccessful, with no commercially available vaccines

Species Diseases

• Horses: mimics Glanders
• Cattle: acute and chronic diseases localizing in lungs, joints, and uterus
• Sheep: arthritis and lymphadenitis
• Goats: loss of condition, respiratory and CNS disturbances, arthritis, and mastitis
• Swine: similar to goats, with abortions and diarrhea
• Dogs: febrile, localized suppurative foci

Epidemiology of Melioidosis

• Clinical disease is usually sporadic
• Endemic in humans in SE Asia and Northern Australia
• Host range is virtually unlimited, with non-mammalian cases documented in birds, tropical fish, and reptiles
• Humans, especially veterinarians, can acquire infection from a wide range of animals

Mechanism of Pathogenesis

• Microorganisms are taken up via phagocytosis and survive inside the cell by being resistant to lysosomal contents and escaping phagosomes and phagolysosomes
• Bacteria gain actin-based motility and undergo 'budding' from affected and unaffected cells
• Infected cells release inflammatory cytokines and undergo apoptosis

Pathology

• Primary pyogranulomatous lesions characterized by single to multiple suppurative or caseous nodules in organs
• Commonly affects lungs, spleen, liver, and associated lymph nodes
• Small abscesses tend to coalesce into large foci

Genus Bordetella

• Gram-negative, small rods that tend to look like coccobacilli
• 16 species, with 3 species causing respiratory diseases in animals
• Found in animal respiratory tracts and has an affinity for ciliated epithelial cells
• Capable of intracellular survival, and healthy animals are carriers for B. bronchiseptica
• Environmental reservoir is amoebae

Bordetella spp. Virulence Factors

• Adhesins, biofilms, LPS, outer membrane proteins, and multiple iron acquisition systems are involved in Bordetella virulence
• Tracheal cytotoxin damages ciliated epithelial cells, interfering with DNA synthesis
• DNT (dermonecrotizing toxin) causes dermal necrosis following injection into the skin
• Exotoxins alter the actin cytoskeleton of affected cells, inhibiting differentiation of osteoblasts in bone tissues, leading to turbinate atrophy
• Adenylyl cyclase toxin has hemolytic and pore-forming activity, increasing intracellular CAMP levels and disrupting iron and fluid regulation

Transmission of Bordetella

• B. bronchiseptica can cause respiratory disease in nearly all animals, primarily through airborne transmission via aerosols
• Environmental contamination and direct transmission through contaminated water and litter are also significant factors
• B. avium is directly transmitted in turkeys via contaminated water and litter

Pathogenesis of Bordetella

• Bacterial attachment to ciliated epithelial cells leads to expression of adhesins with toxic or pro-inflammatory properties, production of ciliostasis, and initiation of inflammation
• Adherence to immune cells and release of adenyl cyclase toxin and effector proteins interferes with phagocytosis, leading to proliferation and active inflammation
• Mucus and fluid accumulation in the upper respiratory tract results in loss of control in iron and fluid regulation, leading to secondary pneumonia

Consequences of Bordetella-induced Changes

• Depression of respiratory clearance mechanisms facilitates complications
• In pigs, B. bronchiseptica causes nasal irritation, making turbinates susceptible to P. multocida toxin, leading to progressive atrophic rhinitis or non-progressive, mild, reversible turbinate hypoplasia

Pathology of Bordetella Infection

• Clinical disease caused by Bordetella is characterized by pathogen attachment, functional impairment, and destruction of ciliated respiratory epithelium
• The disease process begins with a catarrhal phase, followed by a suppurative phase, which may result in pneumonia or air sacculitis
• Compromised host clearance mechanisms in the upper respiratory tract may result in pneumonia or air sacculitis, enhanced by secondary bacterial pathogens

Bordetella Infections

• Swine:
  • Atrophic rhinitis: progressive form is a combined infection with P. multocida and B. bronchiseptica, while non-progressive form is caused by B. bronchiseptica alone
  • Transient and self-limiting, affecting piglets 3-4 weeks old
  • Pneumonia: usually a secondary infection, but primary pneumonia can occur in neonatal piglets (3-4 days old), causing coughing, dyspnea, high morbidity, and high mortality at times

Canine Infectious Tracheobronchitis (Kennel Cough)

• Caused by B. bronchiseptica
• Clinical disease most common in young dogs or mature dogs with a lowered immune system
• Often associated with viral infections, including canine influenza virus (CIV), canine parainfluenza virus (CPV), canine adenovirus 1 or 2 (CAV-1/2), and canine herpesvirus (CHV)
• Disease outbreaks occur rapidly in kennels and animal hospitals, with an incubation period of approximately 1 week
• Clinical symptoms:
  • Acute coughing
  • Gagging and retching (severe cases)
  • Recovery within a few weeks without treatment, but bacteria can persist and cause a relapse infection
• Pneumonia: usually secondary and associated with other pathogens

Feline Upper Respiratory Disease

• B. bronchiseptica is becoming increasingly important in terms of feline respiratory infections
• Clinical signs:
  • Mild tracheobronchitis
  • Conjunctivitis
  • Nasal discharge
  • Sneezing
  • Coughing
• Signs resolve after 10 days, but cats can carry the organism asymptomatically for up to 19 weeks following recovery

Guinea Pigs

• Extremely susceptible to B. bronchiseptica infections
• Acute development of disease associated with high mortality
• Clinical signs:
  • Depression
  • Ruffled fur
  • Labored breathing
  • Sneezing
  • Anorexia
  • Mucopurulent or catarrhal exudate in the nares (may be present)
• Without quick treatment, usually progresses to fatal pneumonia
• Necropsies show consolidation of cranioventral lung tissue, fibrinous suppurative pleuritis, and exudate in the airways
• Guinea pigs that survive infection will become chronic, asymptomatic carriers

Poultry and Birds

• Two species cause diseases in birds and are associated with stunted growth and tracheal collapse:
  • B. avium (‘Turkey coryza’): affects young turkeys, leading to tracheobronchitis, sinusitis, and air sacculitis
  • B. hinzii: commensal in poultry respiratory tract, causing clinical signs such as nasal exudate, conjunctivitis, sneezing, and dyspnea
• Morbidity can be high, mortality is generally low except for cases of secondary infections
• Recovery can begin after approximately 2 weeks, but illness can persist for 6 weeks

Gram-Negative Anaerobes

• Most anaerobes are part of normal microbiota in the mouth, GI tract, upper respiratory tract, and female urogenital tract in various species
• Genera of anaerobic bacteria associated with animal diseases include:
  • Bacteriodes
  • Dichelobacter
  • Treponema
  • Porphyromonas
  • Prevotella
  • Fusobacterium

General Pathogenesis of Anaerobic Bacteria

• Microenvironments:
  • Process of establishing infection is dependent on the site involved
  • Bacteria need to generate a local anaerobic microenvironment or be able to tolerate oxygen exposure
  • Microenvironments may be created by bacterial damage to the host or because of synergistic interactions with facultative anaerobic bacteria that can utilize oxygen
• Protective enzymes:
  • Many anaerobes can grow at low oxygen tension by using enzymes that protect them against oxygen, such as superoxide dismutase (SOD), catalase, and nicotinamide adenine dinucleotides (NAD) oxidase
• Bacterial products = damage:
  • Tissue destruction may occur from host immune responses and inflammation that are triggered by bacterial products
  • Local release of a wide variety of inflammatory cytokines
  • Fermentation products create characteristic putrid odor of anaerobic infections and have inflammatory cytotoxic effects, including:
    • Lactic acid
    • Butyric acids
    • Ammonia

Genus Fusobacterium

• Gram-negative, obligate anaerobic, non-motile, spindle-shaped (fusiform) or long filamentous bacilli
• 17 species, all producing large amounts of butyric acid as a fermentation product
• Species of relevance to veterinary medicine and public health:
  • Fusobacterium necrophorum: major animal pathogen
  • Fusobacterium canifelinum, nucleatum, and russi: members of normal oral microbiota in dogs and cats, frequently isolated from bite wounds in humans
  • Fusobacterium equinum: normal part of oral microbiota in horses, isolated from oral-associated and respiratory diseases of horses
• Fusobacterium necrophorum:
  • 2 subspecies: necrophorum and funduliforme, varying in cellular morphology, colonial characteristics, growth patterns in broth, extracellular enzymes, virulence factors, and general virulence and infrequency of occurrence in infections
  • Natural habitat: mouth, gastrointestinal tract, urogenital tract, and soil, especially well-manured soil
• Fusobacterium necrophorum types of diseases and pathologic changes:
  • Major bovine pathogen, causing necrobacillosis and associated with numerous necrotic disease conditions
  • Hepatic necrobacillosis (liver abscesses): occurs secondarily to damage of the rumen wall epithelium, resulting in rumen acidosis and epithelial damage, which is required for F. necrophorum to colonize

Genus Mycobacterium

• 188 species, mostly saprophytic, obtaining food by absorbing dissolved organic material
• Non-flagellated, rod-shaped, and aerobic, with varying lengths
• Acid-fast, resisting decolorizing after staining, and cytochemically Gram-positive
• Characterized by a mycolic acid layer or mycomembrane, conferring high resistance to chemicals and antibiotics

Pathogenic Species: Host-Adapted Species

• Mycobacterium tuberculosis (humans): host-adapted, rarely infects other animals, and has limited environmental survival capability
• Mycobacterium bovis (cattle, humans): zoonotic, transmitted bidirectionally via aerosols and ingestion of unpasteurized milk
• Mycobacterium pinnipedii (seals): host-adapted strain
• Mycobacterium caprae (goats): host-adapted strain
• Mycobacterium mungi (banded mongoose): host-adapted strain
• Mycobacterium microti (voles, humans): host-adapted strain
• Mycobacterium canettii (unknown): host range unknown

Pathogenic Species: Environmentally Adapted Species

• Mycobacterium avium complex: found in surface water, includes distinct species, and causes TB-like infections in birds
• Mycobacterium intracellulare: opportunistic pathogen
• Mycobacterium avium: includes four subspecies, causing infections in birds, swine, humans, and ruminants
• Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium marinum, and Mycobacterium ulcerans: all opportunistic pathogens

Pathogenesis of Mycobacterial Infections

• Transmission: inhalation of aerosols, ingestion of unpasteurized milk, and inoculation through damaged skin
• Pathogenesis: entry via mucosa or skin breach, phagocytosis by macrophages, and avoidance of macrophage activation and bactericidal response

Progression to Clinical Disease

• Clinical disease depends on the number of bacteria, infecting dose, virulence, and development of anti-mycobacterial cell-mediated immunity
• Disease onset: ~3 weeks after infection, mediated by T-helper 1 cells, IL-12, and Interferon Gamma
• Granulomas or tubercles form, containing infected macrophages, epithelioid cells, lymphocytes, and fibroblasts
• Caseous necrosis develops, dependent on disease outcome, and tubercles may enlarge and coalesce, eventually effacing large areas of tissue

General Pathogenesis of TB

• Infected animals mount both humoral and cell-mediated immune responses of varying efficacy
• Interferon gamma is key to host protection
• Granulomas are central to mycobacterial control in the body

M.bovis Pathogenesis

• Transmitted via direct aerosol and shedding into milk
• Initial uptake via alveolar macrophages and epithelial cells
• Binding of mycobacterial cell wall and pattern recognition receptors on macrophages
• Persists in macrophage phagosome and inhibits maturation of phagolysosome
• Proliferates in phagosomal vacuole

M.avium Pathogenesis

• Transmitted via fecal-oral route
• Initial uptake via M cells of intestinal Peyer's Patches
• Binding of mycobacterial cell wall and pattern recognition receptors on macrophages
• Persists in macrophage phagosome and inhibits maturation of phagolysosome
• Proliferates in phagosomal vacuole

Infection Outcomes

• Spontaneous healing
• Containment of the infection (remission)
• Reactivation and dissemination (progression)

Predilection Sites

• M.bovis: lung and lymph nodes of the head and thorax
• M.avium paratuberculosis: small intestine and mesenteric lymph nodes

Clinical Signs

• Weight loss is a common sign of advanced disease
• Respiratory symptoms (M.bovis)
• Intestinal dysfunction symptoms (M.avium paratuberculosis)

Tuberculous Infections in Animals

• Birds: naturally susceptible to primarily M.avium avium
• Ruminants:
  • Cattle: M.bovis, commonly progressive with hematogenous dissemination to the liver and kidneys
  • Sheep and goats: susceptible to M.bovis, M.avium paratuberculosis, and M.avium hominissuis
  • Deer: susceptible to M.bovis
• Dogs and cats:
  • Dogs: M.bovis, lesions resemble foreign body reaction than tubercles
  • Cats: M.avium species, intestinal and abdominal localization
• Horses: rarely infected, usually with M.avium hominissuis
• Swine: infection usually via the alimentary route, M.bovis causes progressive disease with classical lesions
• Wild animals: serve as reservoir for M.bovis

Nontuberculous Infections

• Prevalent in the environment, cause opportunistic TB-like infections in animals and humans
• Infections can cause false positive test results due to cross immune reactions
• Mostly saprophytic species
• Opportunistic, granulomatous infections
• Johne's disease (Paratuberculosis) caused by M.avium paratuberculosis

Johne's Disease (Paratuberculosis)

• Chronic, irreversible wasting disease of ruminants
• Caused by M.avium paratuberculosis
• Clinically affected and asymptomatic shedders
• Main route of infection is fecal-oral
• Signs: reduced milk production, breeding problems, increased incidents of mastitis, early culling, and overall economic losses
• Lesions: characteristic gross corrugated appearance of intestinal mucosa
• No cost-effective antimicrobial treatments
• Prevention and elimination of infected animals is best for overall herd health

Feline Leprosy Syndrome

• Chronic, nodular, and ulcerative mycobacterial infection of the skin
• Several causative species, including M.lepraemurium
• Common locations of lesions suggest transmission by rodent bites or arthropods
• Disease is more common in older cats and progresses slowly
• Lesions: nodules occur in cutis or subcutis, ulceration, and lymph node involvement