Gram Positive Rods

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Gram Positive Rods
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Listeria

• Small Gram positive rods or coccobacilli (lt2µm)
• Tolerate wide temperature and pH range
• Small haemolytic colonies on blood agar (ß
  haemolysis)
• Facultative anaerobes
• Catalase positive, oxidase negative
• Tumbling motility at 25 degrees
• Aesculin hydrolysed
• Do not grow on MacConkey agar
• Environmental
• Outbreaks related to silage

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Listeria spp

• L. monocytogenes meningoencephalitis
• septicaemia
• abortion
• pyogenic infection
• L. ivanovii abortion, systemic infection
• L. innocua non-pathogenic
• L. seeligeri
• L. welshimeri
• L. grayi

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Epidemiology

• Common commensal (tonsils, intestine) and
  environmental organisms
• grow at temperatures 4 - 45C, pH 6.0 - 9.6
• Incidence relates to management/husbandry,
  (silage feeding), seasonal
• predisposed by trauma, immunocompromise, hormonal
  alterations

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Diagnosis

• Specimens
• Visceral form liver, kidney or spleen
• Neural form spinal fluid, brain stem
• Abortion placenta, foetal abomasal contents
• Blood smear is not very informative
• Isolation grow aerobically on both blood and
  MacConkey agars at 37C for 24-48h
• Colonies appear transparent after 24 hours and
  greyish in 48 hours. Pathogenic strains show ß
  haemolysis
• L. Monocytogenes is CAMP ve with Staph aureus
• L. Ivanovii is CAMP ve with Rhodococcus equi

CAMP test
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Listeriosis - Ruminants

• Meningoencephalitis
• Most common form circling disease (small
  ruminants)
• animal circles in one direction only
• unilateral facial paralysis, difficulty in
  swallowing
• fever, blindness, headpressing
• paralysis, death in 2 - 3 days

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Listeriosis - Ruminants

• In pregnant animals, may localize in placentomes
• cross-over to amniotic fluid, multiplies
• ingested by fetus, causes fetal death, abortion
• In milking cows, mammary gland can be involved
• subclinical mastitis, contamination of milk
• may survive low temp pasteurization inside MØ
• lengthy survival in nature
• growth at low temperatures
• Entry also by nasal mucosa, conjunctivae
• Direct access to nervous system via dental plates
  of trigeminal ganglia

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Pathogenesis
tooth loss cutting naïve/neonatal
pregnancy animal ? ? ?
oral inoculation epithelial invasion
? ? trigeminal nerve
bacteraemia ? ? ? brain stem
neonatal placentitis ?
septicaemia ? meningoencephalitis
abortion circling disease
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Silage and sheep
Listeria encephalitis (bacteria in brain)
A Circling disease in sheep B Cranial nerve
paralysis
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Pathogenic mechanisms

• Facultative intracellular parasites surviving in
  macrophages and epithelial cells
• Cell uptake induced by bacterial protein
  internalin (inlA)
• Inside the cell they escape the phagolysosome,
  multiply in the cytoplasm and via actin based
  motility spread laterally to adjacent cells
• They escape epithelium and are taken up by PMN
  and macrophages
• These cells are killed and the organism may
  spread systemically

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Pathogenic mechanisms

• Bacteria polymerize actin, form tails
• hollow mesh forms on surface, left behind as
  bacterium moves through cytoplasm, invade
  adjacent cells
• actin depolymerized as organism moves (turnover)

• Major virulence factor mediating intracellular
  survival is a cholesterol binding cytolysin
  called listeriolysin (LLO)
• Shares 40-50 aa similarity to other thiol
  activated toxins
• Mediates escape from phagocytic vesicle

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LLO

• Cholesterol-directed pore-forming cytolysin (gt22
  members)
• Bind to cholesterol-containing membranes
• Insertion
• Oligomerization (20-80 mers)
• Pore (20-30 nM) formation

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Epidemiology of Human Listeriosis
enteric Listeria in animals ? contamination of
carcase, milk or food crops ? Ingestion (pate,
soft cheese, coleslaw) ? colonisation of tonsils
and intestine ? ? immunocompetent immunocompr
omised (neonate, elderly, pregnancy)
? ? asymptomatic
septicaemia meningitis
abortion
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Consequences of human L. monocytogenes infection
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Associated Foods

• Milk products raw pasteurised
• Cheeses
• Meat and poultry products Raw, cooked ready to
  eat meat and poultry (sporadic and epidemic)
• Seafood fresh, frozen and processed seafood

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Consequences of human L. monocytogenes infection

• Entry via GI tract, 20 h incubation period
• usually asymptomatic/mild, influenza-like
  symptoms in adult humans, transient
  gastroenteritis
• more serious infection immunocompromised
• CNS infections (encephalitis, meningitis), fatal
  bacteremia
• puerperal sepsis
• crosses placenta ? in utero fetal infection
• stillbirths, preterm labor
• infant born with systemic infection

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Cutaneous listeriosis (in vets)

• 17 cases, all with lesions on fore or upper arms,
  or hands
• 16 farmers, veterinarians
• 1 butcher
• Most developed lesions 1-4 days after attending
  congenitally infected bovines

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Erysipelothrix rhusiopathiae

• Gram positive rods (1-2.5µm)
• Commensal widespread in animals, infects man
• Grows 4-37C
• Catalase negative, oxidase negative
• Facultative anaerobic, non-motile
• Opaque, pin-point, non-haemolytic colonies
• infection disease mainly in pigs sheep,
  turkeys, others
• Smooth (S) and rough (R) forms associated with
  different diseases
• Acute septicaemia in pigs, turkeys, sub-acute
  skin lesions in pigs S
• Chronic arthritis in sheep, endocarditis in pigs
  - R

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Erysipelothrix rhusiopathiae
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Pathogenesis
commensal tonsils, RES, bone marrow, many organs
mainly R or R/S depression of host
defences multiplication of virulent strains
(reversion to S?) entry via tonsils or
cuts/abrasions invades neutrophils ? ?
? septicaemia urticarial form arthritis/endoca
rditis fever malaise
persistent anorexia fever
erosive DIC DIC chronic
inflammation haemorrhage lymphadenitis epidermal
lesions fatal ("Diamonds")
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Swine erysipelas (diamond skin disease)
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Actinomycete Group

• Gram positive
• Catalase positive
• Many are acid fast (mycolic acid in cw)
• Slow growing - survive in macrophages
• Pleomorphic (coccobacilli to filamentous). Very
  small (lt1µm)
• Many saprophytic some opportunist
• Pathogenic genera
• Actinomyces spp.
• Nocardia spp.
• Arcanobacterium spp.
• Dermatophilus spp.
• Corynebacterium spp.
• Eubacterium spp.
• Rhodococcus spp.

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Actinomycete diseases

• Pyogenic
• Granulomatous
• Include abscesses, pyelonephritis,
  lymphadenitis, osteomyelitis
• Chronic inflammation focal lesions
• Some highly-host adapted
• CMI protective A/bodies mainly non-protective
• In vitro - sensitive to some antibacterials
• In vivo poor response - intracellular location

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Actinomyces

• Actinomyces bovis, Actinomyces viscosus,
  Actinomyces suis
• (Most) non-acid fast branching rods
  
• non-motile
• microaerophilic or anaerobic
• Colonies are non-haemolytic, small and white
• Produce pyogenic, granulomatous reactions -
  sulphur granules

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Actinomyces bovis

• Normal flora - anaerobic
• Thick yellow pus sulphur granules
• (occ. confused with wooden tongue)
• Causes actinomycosis or Lumpy jaw in cattle
  (other tissues). Invasion through wound/rough
  feed/damaged mucosa Bone infection
  osteomyelitis animal stops eating, loses weight
• Soft tissue infection
• Mastitis

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Actinomyces bovis

• Infection endogenous
• Organism lives in the mouth normally
• Pathology the result of tissue trauma, lesions
  or prolonged irritation
• Treat lesions small, circumscribed surgery,
  abscesses drained, packed with gauze and iodine
  penicillin

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Sulphur granules
Gram stain, A. bovis
48 hour culture, A. bovis
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Lumpy Jaw
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Actinomyces viscosus

• Mainly dogs (cats, pigs, goats, cattle, horses).
• Fimbriae - adherence to teeth plaque
• Similar lesions to Nocardia (Nocardia rarely
  produces granules)
• Localised, pyogranulomatous lesions
• Two main conditions - Thoracic lesions (pleural,
  pericardial fluid, lung lesions) and
  osteomyelitis
• Treatment prolonged
• Actinomyces suis mastitis in pigs (sows),
  trauma initiates disease

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Nocardia

• Strictly aerobic
• Gram-positive, pleomorphic (filaments, rods,
  cocci branching)
• Widely distributed soil, water, air, sewage
• 0.5 1.2 µm in size
• Acid fast
• Non-motile
• Non-spore-forming

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Nocardia

• 12 species pathogenic to birds, goats, cats,
  dogs, fish, horses, cattle and humans
• Nocardia asteroides most frequent nocardial
  pathogen subcutaneous infections in dogs
• N. brasiliensis pneumonia in horses
• Colonies are vivid white occasionally pigmented

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Nocardia
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Nocardia

• Organism inhaled, in wound or ingested
• Direct or haematogenous spread
• Prevents phagocytosis
• Chronic invasive pyogenic infections (no sulphur
  granules)
• 3 clinical forms cutaneous, respiratory
  (pyothorax) and systemic (pyrexia, cough,
  neurological, resembles distemper)
• Dogs 3x more common in males, Cats mainly
  thoracic infection
• Treatment difficult and prolonged not
  penicillin

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Dog, Nocardia
Fluid from chest
4 day culture from fluid
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Dermatophilus congolensis

• Facultatively anaerobic
• Gram positive, non acid fast
• filamentous, branching filaments mature they
• fragment and release motile flagellate spores
• Septation of filaments
• zoospores (motile) Beaded, Tram-track the
  infectious form
• CO2 chemotactic to zoospores zoospores
  germinate form filament form new zoospores
  repeating the cycle
• Pathogenesis
• Commensal/spore entry injury/ wet damage
• Colonisation keratinase production. Aid spread
  and growth
• Strong host response, neutrophil and lymphocyte
  exudation.
• EPIDERMIS ONLY

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Dermatophilus congolensis

• Dermatitis cattle, dog, cat, man
• Sheep lumpy wool, strawberry foot
• Horse mud fever, greasy heel, rain scald
• Treatment Pen/Strep. Tetracyclines
• No effective vaccine

Dermatophilus in a horse
Dermatophilus lesions, SHEEP
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Corynebacterium

• Small pleomorphic, Gram-positive rods
• Chinese letters
• Facultatively anaerobic, non-motile
• Catalase positive, oxidase negative
• Pyogenic
• Common commensals
• Colonies are white, small, dry, non-haemolytic
  (except C.pseudotuberculosis).
• Genus originally created for the important human
  pathogen C. diphtheriae, the cause of diphtheria
  in man

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CAMP Test
CAMP-Inhibition Test
1. Staphylococcus aureus 2. C.pseudotuberculosis 3
. C.renale 4. Rhodococcus equi
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Corynebacterial species of veterinary interest

• C. renale group
• C. renale
• C. pilosum
• C. cystitidis
• All cause cystitis and pyelonephritis in cattle
• Diphtheria group
• C. diphtheriae
• C. ulcerans
• C. pseudotuberculosis
• Various diseases of cats, cattle,
• horses, small ruminants and humans

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Corynebacterium renale group

• C. renale gt C. cystitidis gt C. pilosum
• Opportunist highly adapted
• Causes cystitis, pyelonephritis, balanoposthitis
• Predisposing factors - pregnancy, parturition,
  post mating
• 90 bulls C. cystitidis - prepuce

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Cow with C. renale infection
Urine, C. renale
C. renale on milk agar
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Corynebacterium renale group

• Virulence factors
• Pili - adherence
• Renalin - cell lysis
• Urease
• Caseinase

• Pathogenesis
• Adhere to urogenital mucosa
• Stress
• Proliferation
• Ascending infection
• Inflammation
• Cystitis/pyelonephritis

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The diphtheria group

• Highly-related (based on DNA hybridisation
  studies)
• C. diphtheriae diphtheria (humans)
• C. pseudotuberculosis various pyogenic
  infections
• C. ulcerans nasal congestion (cats), mastitis
  (cattle)
• Susceptible to infection with ß-corynephages
• C. pseudotuberculosis C. ulcerans zoonotic
  pathogens
• C. diphtheriae normally only a human pathogen

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Corynebacterium diphtheriae

• Occasionally isolated from infected wounds in
  horses (potential environmental reservoir?)
• Ironically, horses were the original heroes in
  fight against diphtheria

An illustration from the Nov. 17, 1894, issue of
Scientific American, showing doctors drawing
blood from a horse to produce antitoxin for
diphtheria
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Corynebacterium ulcerans

• Significant increase in human infections caused
  by C. ulcerans
• Same organism isolated from several domestic cats
  with bilateral nasal discharge (2002-2003)
• Strains isolated from domestic cats were found to
  exhibit the predominant types observed among
  human clinical isolates, suggesting that C.
  ulcerans isolated from cats could be a potential
  reservoir for human infection

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Corynebacterium pseudotuberculosis

• C. pseudotuberculosis (pseudes-tuberculosis)
• Thought to have spread from Europe with expanding
  colonial powers
• 2 biotypes identified
• ovis non-nitrate reducing, infect sheep/goats
  (CLA)
• equi nitrate reducing, infects predominantly
  horses

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Caseous lymphadenitis (CLA)

• Widespread globally
• Fibrous, encapsulated lesions at various sites
• Devastating visceral form reported in USA
• Significant financial losses to producers/animal
  welfare
• deaths, carcass condemnations at slaughter
• decreased wool, milk production
• decreased reproductive performance

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Pathogenesis

• C. pseudotuberculosis gains entry to host through
  wounds (shearing, fighting, etc.)
• Normally transfer of pus (direct contact or
  flies)
• Pus contains millions of
• bacterial cells
• Aerosol route of disease
• transfer also suggested
• From point of entry,
• drains to local drainage
• lymph node

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Pathogenesis

• Cells are phagocytosed as part of innate immune
  response survive and replicate within
  macrophages
• Focus of infection established within lymph node
• Multiple microscopic foci coalesce to form
  pyogranuloma
• Lesion encased within fibrous capsule.
  Infiltration of immune cells forms caseous
  abscess
• Classic onion ring appearance

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Pathogenesis

• Trafficking of infected phagocytes to other
  tissues allows dissemination of infection
• Cutaneous CLA Superficial lymph nodes,
  occasionally skin
• Visceral CLA (25) lungs, kidneys, brain,
  internal lymph nodes
• Mediastinal abscesses place pressure on trachea ?
  restricted rumination ? chronic wasting

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Pathogenesis

• Virulence factors
• Phospholipase D (PLD)
• Sphingomyelinase, antichemotactic, lethal for
  neutrophils, complement depletion, increased
  vascular permeability
• Mycolic acid (cell wall lipid)
• Toxicity, survival within macrophages
• Serine protease (Cp40)
• Undefined function, possibly survival within
  macrophages
• Siderophore
• Acquisition of iron from host

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Pathogenesis

• Aerosol transmission?
• Disease spread in absence of external abscesses
• C. Pseudotuberculosis cultured from trachea of
  infected sheep
• Potential for coughing to aerosolise pus