CVM6202: Microbiology

Rutherford

Anaerobic Bacteria
Important Species
Gram-negative Bacteroides Fusobacterium Veillonella Gram-positive Peptococcus Peptostreptococcus Eubacterium Propionibacterium Clostridium Arachnia Bifidobacterium

Many are commensals of the intestinal tract and are ubiquitous in nature Most are weak pathogens Many genera produce toxins Cause a wide range of diseases Many infections are mixed

Two classes of anaerobes: Aerotolerant- resemble facultative organisms by growing with or without oxygen, Aerotolerant but their metabolism remains fermentative. Obligate- strict anaerobes that cannot tolerate O2, due to lack of superoxide Obligate dismutase. Many lack catalase and peroxidases. 2 O2- + 2H+ -----------------> H2O2 + O2
Superoxide dismutase catalase

H2O + O2 Obligate anaerobes are difficult to recover from samples: poor transport technique overgrowth by other organisms collection of inappropriate samples failure to consider anaerobes
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CVM6202: Microbiology

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Suspect anaerobic involvement when:

tissue necrosis is observed infection is near a mucous membrane infection is secondary to a bite wound the specimen has a foul odor gas is present in tissue blood is present in exudate black discoloration to exudate (Bacteroides) failure of all organisms observed in the Gram stain to grow in vitro previous antibiotic therapy

Collection of samples
Critical for recovery and culture. ? Perform a direct smear for staining, especially if you are not culturing teh sample immediately. ? Samples with normal flora are not suitable for culture. ? Aspirate material from lesion with a syringe, expel the air, then cap the needle tightly. ? Swabs must be protected from drying by placing them in anaerobic broth. ? Tissue samples should be large or sealed in a bag under N2. ? Ship at 4 C.

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Culture of anaerobic organisms If anaerobic involvement is suspected:

perform a direct exam first; this tells you the # and types of bacteria present. inoculate both aerobic and anaerobic media. use pre-reduced media. inoculate both solid and liquid media. use platinum or pure nickel wire loops; nichrome is unacceptable. place in anaerobic environment ASAP. do not open to air for at least 2 days.

Anaerobic Jar- a prepackaged kit contains a catalyst that, when activated by water, will generate hydrogen and carbon dioxide gases to exclude atmospheric oxygen. The Gaspack system can reduce the oxygen concentration to < 0.4% in 100 minutes.

Anaerobic Chamber- A non-mobile unit that is self-enclosed. It is attached to a nitrotgen gas source that will keep atmospheric oxygen out. Glove access permits a full working environment. A small chamber on the side can open to the outside for sample entry. It is then flushed with nitrogen prior to opening to the anaerobic chamber itself.

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CVM6202: Microbiology

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Anaerobic Culture Media

Anaerobic blood agar (ABA) Non-selective Blood agar plus: yeast extract (amino acids, vitamins, minerals) hemin- Fe+3 chelator vitamin K1 L-cystine cysteine-lowers redox potential Thioglycollate broth Non-selective TG broth plus: hemin Vitamin K1 CaCO4 Indicator

ABA with phenylethyl alcohol (APE or PEA) Selective against Gram-negative facultative anaerobes ABA plus: phenylethyl alcohol (2.5 g/l) inhibits G-neg facultative aerobes Kanamycin and vancomycin (KVA) Selects for Gram-negative obligate anaerobes ABA plus: kanamycin (100 mg/l) vancomycin (7.5 mg/l) Bacteroides Bile Esculin (BBE) Selects for pathogenic Bacteroides species Trypticase agar plus: gentamycin (100 g/ml) 20% bile- inhibits most non-pathogenic Bacteroides spp. 0.1% esculin
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CVM6202: Microbiology

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Genus: Clostridium
Learning objectives
To learn the general characteristics of the genus To know by which mechanism important species cause disease Cause disease by two mechanisms: I. Tissue invasion- produce weak toxins Cl. chauvoei Cl. haemolyticum Cl. perfringens Cl. difficile Cl. novyi Cl. spiroforme Cl. septicum Cl. colinum II. Toxin forming, non-invasive Cl. tetani Cl. botulinum Cl. piliforme To understand how toxins contribute to pathogenesis or clinical severity

General characteristics of the genus Large Gram-positive rods Produce irregular, small colonies in 2-3 days Spore-forming Easily decolorized Catalase-negative Motile QuickTime and a Ubiquitous distribution in soil and associated with animals Photo CD Decompressor At least 83 species recognized

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Clostridium spp. showing apparent Gram-negative rods due to over-decolorization.

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CVM6202: Microbiology

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Clostridium chauvoei
Associated with Blackleg in ruminants Infection and pathogenesis
Exposure is via ingestion of spores or through puncture wounds or during teething. Spores germinate and the organism replicates. The organism moves through the lymph to the blood. Organism localizes in muscle and liver. Cl. chauvoei remains dormant in tissue until trauma occurs. Replication and production of toxins. Sudden onset of fever and tissue necrosis with swelling and gas. ? Death can occur in 1-3 days due to toxemia. The infection can self-cure, with the tissue damage repairing itself.

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Tissue samples from cattle suffering from Blackleg.

Toxins alpha toxin: lethal and necrotizing beta toxin: DNase activity gamma toxin: hyaluronidase delta toxin: hemolytic

Vaccination
Vaccines against either the whole bacterium or the alpha toxin are available, but are recommended to be used yearly.

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Clostridium septicum
Associated with malignant edema in sheep, horses and cattle and (rarely) with gastroenteritis in carnivores. Recently associated with necrotic dermatitis in poultry. Infection and Pathogenesis
Malignant edema Intestinal commensal and therefore a common soil contaminant. Enters via cuts or wounds. Induces a rapidly spreading watery swelling; tissue appears gelatinous due to connective tissue degradation. Little or no gas is produced. High fever induction with appearance of intoxication. Toxemia induces a rapid death in 12-48 hours.
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Tissues from animals suffering from Cl. septicum

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Necrotic dermatitis Intestinal commensal and therefore a common contaminant of litter. Enters via cuts or wounds, but occurance increases with stress/crowding. Disease is sporadic. Observe lameness, red/black patches on skin. Prostration. Rapid death in 12-24 hours. Toxins alpha: necrotizing and lethal beta: DNase gamma: hyaluronidase delta: hemolytic
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Clostridium novyi
Associated with gas gangrene and infectious necrotic hepatitis (Black disease) in cattle, sheep and humans, and big head in rams.

Infection and Pathogenesis

Two types of disease: Type A- Gas gangrene and Big Head Invasion of spores or bacterium via wounds, particularly in yearling rams as a result of fighting Replication and toxin production Type A produces alpha, gamma, and epsilon toxins Capillary damage exacerbates swelling Type B- Black Disease The organism grows in liver infarcts caused by liver damage (flukes?). Local liver damage widens Toxins are produced and get into bloodstream Type B produces alpha, beta and zeta toxins Death is due to alpha toxin which is a phospholipase C (lecithinase). The toxin destroys host cell membranes, especially on RBCs, causing hematuria. Death is due to anoxia resulting from a loss of RBCs. NOTE- this disease is very similar in appearance, mechanism of pathogenesis, and etiology as Red water disease in cattle caused by Cl. haemolytica.

There is a Type C Cl. novyi that does not produce toxins and is not pathogenic (?)

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CVM6202: Microbiology

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Clostridium perfringens
Associated with gas gangrene and fatal enterotoxemia in sheep, pigs, horses, dogs, and humans. Commensal of the large intestine and skin and is found in the soil. Exposure is due to ingestion of or exposure to fecal material. Sanitation is most important for control.

Infection and pathogenesis

The organism usually exists in the intestinal tract. Sporulation can occur spontaneously, in alkaline conditions, following antimicrobial therapy, immunosuppression, or dietary alterations. Toxins are released during sporulation and enter bloodstream. The hallmark of Cl. perfringens infection is diarrhea that lasts a few days. There are 5 types of Cl. perfringens that produce up to 15 different exotoxins: Type A: produces a lethal alpha toxin that kills muscle cells and immune cells: associated with gas gangrene and avian necrotic enteritis (Yellow lamb disease). This is probably the most commonly encountered type. Infection can be fatal within 6-12 hours. Type A is also a common cause of necrotizing myositis in horses at wounds or injection sites.
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Cl. perfringens on BA plate showing complete hymolysis.

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Gangrenous tissue caused by Cl. perfringens. QuickTime and a

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Type B: produces alpha, beta, and epsilon toxins; causative agent of lamb dysentery but is not found in the US. Type C: produces alpha and beta toxins; hemorrhagic enterotoxemia (bloody scours) in piglets, calves, and lambs that ranges from subclinical to fatal. Babies dont nurse, and there is increased incidence of piglet crushing. Vaccinating sows can reduce incidence. Type D: produces alpha and epsilon toxins; associated with overeating disease (pulpy kidney disease) in sheep, an enterotoxemia The epsilon toxin is converted by gastric juices, gets into the blood, and damages kidneys. The brain can also be affected. Diagnose by neural signs, no fever, no vomiting, and history. Post-mortum, kidneys are swollen and soft. Vaccinate against epsilon toxin, but treatment is difficult once neural signs are noted.

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Sheep showing leaning and head-thrust behavior associated with Cl. perfringens toxemia.

Type E: produces alpha and iota toxins; a very uncommon cause of an enterotoxemia in calves and lambs. Toxins alpha toxin is lethal and is produced by all types of Cl. perfringens beta toxin is produced by types B, C, and E and is responsible for intestinal mucosa loss leading to diarrhea epsilon toxin produced by types B and D, it is converted to a lethal toxin in the gut kappa and lambda toxins break down collagenase
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CVM6202: Microbiology

Rutherford

Clostridium botulinum
Causative agent of food poisoning in a wide variety of hosts. Member of toxin forming, non-invasive group of Clostridia. Forms highly resistant spores that are released during vegetative growth. There are at least 7 types of C. botulinum. Humans, horses, and cattle are particularly susceptible. Cats have no reported natural cases of disease.

Infection and Pathogenesis

C. botulinum makes at least 7 neurotoxins (A-G) which are used to type strains. Most animal infections are caused by types C and D. The toxins can be detected in feces, blood, and food source. Expsoure is usually via ingestion of preformed toxins, but vegetative growth of bacteria in gut can occur. Ingestion of toxins Toxins move to blood Invasion of peripheral nerves (hours to days) Blocks release of acetylcholine resulting in flaccid paralysis, usually of ocular and respiratory muscles first Animals remain aware and can feel pain Limberneck in birds quadraplegia in mammals blurred vision swallowing difficulty mortality depends on toxin dose; recovery length depends on dose but can be as long as 1 month. Recovery is usually complete.

Diagnosis/Vaccination/Treatment
Diagnosis is based on finding the toxin in foodsource, urine, feces, or vomitus. An electromyography can show abnormal nerve transduction. Toxoid vaccines are of questionable efficacy, but administration of neutralizing antibodies can help clear unbound toxins. Supportive care involves feeding, preventing secondary infections, and expression of body wastes. Penicillin is effective for killing Cl. botulinum.
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CVM6202: Microbiology

Rutherford

Clostridium tetani
Causative agent of lockjaw in mammals, especially horses and humans. Member of the toxin-forming, non-invasive group. Ubiquitous distribution of spores in soil and feces. Ten types based on flagella, but the toxin is the same in all types.

Infection and Pathogenesis

Spores enter deep tissues with low oxygen tension. Spores germinate and toxins are produced. In 3-21 days, tetanospasmin toxin blocks neurotransmitter (glycine and gammaaminobutyric acid) release. Toxins spread along: * Peripheral nerves: ascending tetanus (from wound to trunk) * Hematogenous through lymph: descending tetanus (lockjaw) Vascillating spasms occur; respiratory distress; victim remains conscious. ? Respiratory arrest, and death occur if toxin dose is high enough. Toxins tetanospasmin- a neurotoxin that causes clinical disease; highly lethal hemolysin- tissue necrosis nonspasmogenic toxin- function?

Cl. tetani showing spores at one end of cell.

Diagnosis/Vaccination/Treatment
Diagnosis based on clinical signs and organism in smears from lesion. Antitoxins can neutralize unbound toxins, but bound toxins must be cleared naturally. Supportive care includes removal of stimulation, giving food and fluids, irrigating site of infection, and antibiotics to kill vegetative organisms. Muscle relaxants can help minimize discomfort. Can also vaccinate with toxoid (?). But recovery can take lots of time and $. Yearly vaccination is recommended for horses.
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Clostridium piliforme
Causative agent of Tyzzers Disease in mammals, especially laboratory rodents. Gram-negative; used to be in the genus Bacillus. Obligate intracellular parasite. Commensal of the intestinal tract of rodents. Dogs and cats susceptible, but most cases are associated with rodent contact.

Infection and Pathogenesis

Infection is linked to immunosuppression. Bacteria or spores are ingested. Bacteria grow in intestine until stress or some event permit entry into liver. Clinical appearance is characterized by: Rodents- diarrhea, necrotic intestinal epithelium, and infiltration of inflammatory cells. Dogs/cats- show rapid onset of lethargy anorexia, and abdominal pain. Hepatomegaly and focal lesions on liver are apparent at necropsy. Liver enzymes (AAT, SDH) are often elevated. Death can occur within 24 hours of clinical signs.

Diagnosis/Vaccination/Treatment
Diagnosis is usually based on necropsy results. Culture of the organism is difficult and time-consuming. Treatment is as yet not successful, in part due to rapid progression of the disease. Vaccination is not practiced, although formalin-inactivated vaccines are successful in mice.

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CVM6202: Microbiology

Rutherford

Genus: Bacteroides
Learning objectives
To learn the general characteristics of the genus. To understand the mechanism of pathogenesis for the important species. Other species Important species B. nodosus B. melaninogenicus B. fragilis B. pyogenes B. suis To understand the synergism with Fusobacterium.

General characteristics of the genus

Numerous species exist, but few are pathogenic Gram-positive rods Non-spore forming. Many species produce black pigment on KVA plates. Many fluoresce under long-wave UV. Commensals of the GI tract. Require complex media (vitamin K1, hemin) for growth. Extremely intolerant of O2. Can be speciated by gas chromatography by determining the fatty acids formed when grown on glucose or peptone. Usually, indirect immunofluorescence assays are used. Associated with necrotic infections or abscesses.

Bacteroides fragilis
The most commonly isolated anaerobe from human infections. Found in >80% of human peritonitis, and is very often found in soft tissue abscesses. Reported neonatal infections in sheep. Constitutes 1-2% of normal flora in humans, and is an intestinal commensal in many mammals. Secretes an enterotoxin that has metalloprotease activity that damages tight junctions, leading to diarrhea or blood leakage.
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CVM6202: Microbiology

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Bacteroides or Dichelobacter nodosus

Causative agent of virulant foot rot in ruminants. Pili are required for virulence. There are at least 14 serotypes. Data suggests a susceptibility link to the MHC.

Infection and Pathogenesis

Enters wound in hoof from soil. Is aided by coinfection with Fusobacterium necrophorum and/or Actinomyces pyogenes, wet weather, or rocky terrain. Produces a protease which dissolves the keratin of the hoof. Also produces a factor which enhances growth of F. necrophorum. The horn will rapidly separate from the foot; very painful. A foul smell is produced. Many animals recover spontaneously.
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Normal (left) sheep hooves. The middle shows a hoof during virulent foot rot infection. Note the horn separation and matted tissue and and soil. On right is the same hoof after cleaning to raise the oxygen tension in the lesion.

Diagnosis/Vaccination/Treatment
Diagnosis is usually based on clinical signs, culture of the organism, or PCR. Treatment involves clearing dead tissue, footbaths, and killing the organism. Vaccines are available for many serotypes. Administer prior to pasture.
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Genus: Fusobacterium
Causative agent of necrotic infections in mammals and occaisionally poulty.

General characteristics of the genus

F. necrophorum Gram-negative long rods Commensal of the alimentary canal and therefore shed in feces. Requires complex media for growth. Colonies are fuzzy and show complete hemolysis. Speciated by gas chromatography of fermentation products or IFA assay.
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Infection and Pathogenesis

Causes a number of necrotic lesions: Interdigital necrobacillosis in sheep and cattle. Non-invasive; requires sut or lesion to intiate infection. Mixed infection with A. pyogenes, B. nodosus, or B. melaninogenicus. Swelling between digits, resulting in lameness, but disease is not as severe as B. nodosus. Produces a foul smell.

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Footrot due to Fusobacterium in a sheep (left) and cow (arrow, right). Note the absence of purulence and keratin damage as with B. nodosus.

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Oral necrobacillosis in pigs and cattle. Abscess forms in mouth or on tongue, often following penetration of a foreign object Animals lose appetite QuickTime and a Foul breath
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Oral necrobacillosis on tongue due to Fusobacterium.

Calf diphtheria Initiated as oral necrobacillosis Material is aspirated into lungs Fatal supperative pneumonia

Liver necrobacillosis in cattle, usually neonates Organism invades at navel Septicemia for 5-7 days Localizes in liver and spleen causing focal necrosis Anorexia and fever observed.
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Bullnose in pigs Necrotic rhinitis initiates from ringing or clipping needle teeth Strong cellular response results in severe facial swelling Foul-smelling nasal discharge

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