VPM 201: Veterinary Bacteriology and Mycology 17/10/2010. LABORATORY 9-10 : Bacillus, Clostridium and Mycoplasmas

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1 VPM 201: Veterinary Bacteriology and Mycology 17/10/2010 LABORATORY 9-10 : Bacillus, Clostridium and Mycoplasmas A. BACILLUS AND CLOSTRIDIUM The genera Bacillus and Clostridium are gram-positive rods and produce spores. The former grow aerobically while the latter are invariably strict anaerobes. Bacillus anthracis (not provided) forms capsules, and is non-hemolytic. Other Bacillus species, such as B. cereus, are hemolytic and largely found as contaminants in clinical specimens. Aged or oxygen exposed clostridial cells become gram-negative. It is not uncommon to find both grampositive and gram-negative cells in a smear of a pure culture. Smears of C. perfringens usually show short, thick rods however some Clostridia (C. septicum) are filamentous. The presence of spores varies depending on source of sample (direct smear) and species. For example spores are seen infrequently in smears prepared from C. perfringens colonies. In contrast, gram-stained smears of C. perfringens prepared from infected tissues show oval and subterminal spores. C. perfringens forms a double-zone of hemolysis on blood agar. B. MYCOPLASMAS Members of the class Mollicutes (trivial name mycoplasma) are prokaryotic organisms bounded by a single membrane; they lack a peptidoglycan layer and are incapable of synthesizing cell wall precursors. They are the smallest free-living bacteria and coccoid forms average m. They are highly pleomorphic, varying in shape from coccoid to helical or filamentous. Most species (not the hemoplasmas) are facultative anaerobes and grow on serum and yeast extract enriched media (ie. Friis, Hayflicks or Frey s media). The colonies are small ( mm) and have a characteristic "fried-egg" appearance, with the exception of M. ovipneumoniae. Culture plates are typically incubated at 37 C in a humid atmosphere under both: 5 % CO % N 2, and in air with 5-10 % CO 2. Most animal mycoplasmas grow well in air with 5-10 % CO 2. Growth usually takes 4 days, but plates are incubated for one week or longer. A few (M. hyopneumoniae and M. dispar) will usually only be isolated on plate media following growth in liquid media. Haemoplasmas (trivial name) are a recently assigned cluster of organisms within the

2 Mollicutes that are largely derived from genera (Haemobartonella and Eperythrozoon) previously within the order Rickettsiales. These hemotrophic mycoplasmas are characterized by their affinity for host RBC s and subclinical to clinical hemolytic anemia. Mycoplasma (previously Haemobartonella felis) haemofelis is an example of a haemoplasma and causes feline infectious anemia (previously referred to as hemobartonellosis). The fastidious nature of the Mycoplasmas has proven challenging to producers and diagnosticians. PCR and serological testing have become increasingly available and can provide an effective and rapid alternative to culture and identification. PCR-based testing is currently the only confirmatory diagnostic tool for the haemoplasmas. The flowcharts below summarize the testing currently available in the absence and presence of culture. 1. Serological and PCR-based testing in the absence of culture. ** SPA and HI serological assays are typically available for Avian Mycoplasmas 2

3 2. Mycoplasma/Ureaplasma culture and identification flowchart. C. DEMONSTRATIONS 1. Bacillus and Clostridium a. Examine the BA cultures of Bacillus cereus and Clostridium perfringens. Note the colonial morphology. B. cereus C. perfringens b. Examine the Gram-stained smears and note morphology. B. cereus (10-1) C. perfringens (10-2) and C. tetani (8-2) How do C. perfringens, C. tetani differ from Bacillus? c. Examine the tissue section of muscle showing myositis caused by Clostridium septicum. d. Examine some vaccines against clostridial diseases. 3

4 2. Mycoplasma a. Specimen submission for Mycoplasma isolation. b. Isolation methods/culture media. c. Mycoplasma growth: (i) in broth medium (ii) colonies on plates d. Examine the photographs and documents showing colony growth inhibition tests to differentiate Genus and species PCR testing: (i) Digitonin growth inhibition test (ii) Serum growth inhibition test e Commercial PCR tests f. Some Mycoplasma vaccines 3. What am I? A holstein cow presents with raised, scabs covering 20 to 30% of her back. She does not appear febrile, appetite is good and according tho the farmer she is the only animal that has these scabs. After some questioning the farmer indicates old Bessy is bit independent when it comes time to come in for milking and that she prefers to stay outside in all kinds of weather. You know it has been hot, wet and humid of late. You note that the scabs remove relatively easily and reveal a mild-moderate exudative dermatitis underneath. The scabs themselves a re 1-2 centimeters in diameter and with the hair entrapped remind you of a paintbrush when viewed from the side. You decide do an impression smear from the bottom of one scab and gram stain the material. You also submit scabs for standard bacterial as well as fungal culture and identification. The scabs, a BA plate (no growth on MAC or fungal plates) and direct smear are provided. a. What is the bug? Can you diagnose this condition from the direct smear? If so why? b. What is are some of the common names for this condition in cattle, sheep and horses? c. You decide to treat the cow with an antibiotic - which one would you choose? 4

5 4. What am I? Swabs taken from exudative dermatitis on a number of sheep are submitted for standard culture and identification. The clinician that submitted the sample indicated that there was a greenish-discoloration on wool adjacent to the areas of inflammation. BA and MAC plates are provided as well as a gram-stained slide prepared from a BA colony. An oxidase test is also provided and the organism has been subcultured on MHA (Mueller Hinton Agar). Look at the flow-chart provided to help with your identification. a. What is the most likely Genus and species? b. What is the common name of the infection? c. Does intervention require antibiotics or management changes? 5. What am I? A horse presents with muscle rigidity in the head, neck and limbs. The horses stance is unusual and would be described as sawhorse with a rigid extension of neck, back and limbs and an elevated tail head. Examination of the horse and questioning of the owner reveals: a puncture wound on the lower left hind foot and no recent history of vaccination. You are able to aspirate material from the puncture wound and prepare a gram-stained direct smear. a. What is your diagnosis? b. Is there a vaccine? D. QUESTIONS 1. Two sheep in a flock were found dead, and you suspect clostridial enterotoxemia from the history and signs. What samples would you submit to the laboratory? Can you do any quick test for a tentative diagnosis? 2. Are the vaccines against black leg effective? 3. Which antibiotic would you most likely use for treatment of black leg in early stages? 4. What conventional colony growth inhibition test is used to differentiate Mycoplasma from Acholeplasma genera? 5

6 5. a. Name the disease condition(s) caused by these Mycoplasmas: M. gallisepticum M. bovis M. haemofelis M. hyopneumoniae b. Name one test that can be used to identify these pathogens. 6. Name 2 antimycoplasmal drugs used to treat animal mycoplasmosis. 6