Bacteria causing respiratory diseases in chicken

Bacterial pathogens play an important role in causing respiratory disease in domestic poultry species. In many cases, the bacterial component of a respiratory disease colonizes the respiratory system only after a primary viral or environmental insult. Colonization of the air sacs of a chicken by Escherichia coli following an infectious bronchitis virus infection is an example of secondary bacterial invasion. In other cases, the bacterial component of the respiratory disease is the primary initiating cause of the disease. Examples of primary bacterial respiratory disease are infectious coryza in chickens and fowl cholera in chickens and turkeys.

Bacteria DiseasePasteurella multocida Fowl cholera

Haemophilus paragallinarum Infectious coryzaBordetella avium BordetellosisEscherichia coli Colibacillosis

Ornithobacterium rhinotracheale Ornithobacterium rhinotracheale

Pasteurella multocida:CharacteristicsPasteurella multocida is spherical, ovoid or rod-shaped cells 0.3-1.0µm in diameterand 1.0-2.0µm in length. Cells are Gram negative, and occur singly, or in pairs or short chains. Bipolar staining may be seen and capsules may be present. It’snon-motile, and facultatively anaerobic.

Principles of IdentificationColonies on blood agar are identified by colonial morphology, Gram stain, oxidase test and catalase production.

IdentificationMicroscopic AppearanceGram stain Pasteurella species are spherical, ovoid or rod-shaped Gram negative rods or coccobacilli which occur singly or in pairs or short chains. Bipolar staining is common, capsules may be present.

Primary Isolation MediaBlood agar incubated in 5-10% CO2 at 35-37°C for 16–48hr.

Colonial Appearance Colonies are grey and viscous but rough irregular colonies occur frequently.

Biochemical testsOxidase test: Pasteurella multocida is oxidase positive.” have oxidase enzyme that oxidizes the reagent to deep purple color

Catalase test: Pasteurella multocida is catalase positive

Haemophilus paragallinarum:CharacteristicsHaemophilus species are Gram negative spherical, oval or rod-shaped cells less than 1µm in width, variable in length, with marked pleomorphism, and sometimes forming filaments. The optimum growth temperature is 35–37°C. They are facultatively anaerobic and non-motile.

Principles of IdentificationColonies on blood or chocolate agar may be presumptively identified by colonial morphology, Gram stain, haemolysis and requirement for X and V factors and CO2. Identification is confirmed by commercial biochemical testsIdentification Microscopic AppearanceGram stain Haemophilus species are small coccobacilli or longer rod-shaped Gram negative cells, variable in length with marked pleomorphism and sometimes forming filaments.

Primary Isolation MediaChocolate agar incubated in 5-10% CO2 at 35-37°C for 24-48hr.Colonial AppearanceHaemophilus species are small, round, convex colonies, which may be iridescent and develop after 24hr incubation on chocolate agar.

Biochemical testsCatalase test Haemophilus paragallinarum is catalase negative.

Oxidase test Haemophilus paragallinarum is oxidase negative.

Urease test: Red color as a positive result since H. paragallinarum has urease enzyme and able to split urea to ammonia.

Bordetella aviumCharacteristicsBordetella avium is gram negative coccobacilli 0.2-0.5 x 0.5-2.0µm.Microscopically they appear arranged singly or in pairs and rarely in chains 4. They often exhibit bipolar staining. Cells may be motile or non-motilPrinciples of IdentificationColonies isolated on Bordetella selective agar are identified preliminarily by colonial appearance, Gram stain and slide agglutination with polyvalent antiserum. Biochemical and other additional tests are used to distinguish between species of the genus Bordetella and to differentiate Bordetella from similar organisms. e. They are strictly aerobic.Microscopic AppearanceGram stain Gram negative, thin coccobacilli occurring singly or in pairs, rarely in chains. Some strains may be capsulated.

Primary Isolation MediaCharcoal selective agar, incubated aerobically with high humidity and good circulation of air, for 7 days at 35°C-37°C is used for primary isolation..Colonial AppearanceColonies of B. avium on charcoal blood agar with cefalexin are smooth, convex, pearly and glistening, greyish-white and butyrous and appear in 3 days on subculture,longer on primary isolation.

Biochemical testsOxidase test B. avium is oxidase positive “have oxidase enzyme that oxidizes the reagent to deep purple color”

Catalase test B. avium is catalase positive “have catalase enzyme that splits H2O2”

Citrate test B. avium is citrate positive “utilizese citrate (source of C) resulting in alkaline product”

Escherichia coli: Is a Gram negative rod. Most strains are motile and possess the flagellar antigen H7.They are facultatively anaerobic. On sorbitol MacConkey agar (SMAC) or SMAC containing cefixime and tellurite (CT-SMAC), the colonies are colourless and 2- 3mm in diameter.

Principles of identification Investigation of faecal specimens for enteric pathogens recommends that all diarrhoeal stools are screened for the presence of E. coli. Presumptive isolates from primary culture are identified by colonial appearance on CTSMAC, serology (agglutination with specific antisera) and biochemical tests.

Identification: Microscopic appearance Gram stain Gram negative rods.

Primary isolation media Cefixime tellurite-sorbitol MacConkey (CT-SMAC) agar incubated in air at 35-37°C for 16-24hr. CT-SMAC agar is used since classical sorbitol non-fermenting are relatively resistant to potassium tellurite compared with other E. coli. There is a risk of isolation failure on SMAC agar lacking cefixime and tellurite.Colonial appearance On CT-SMAC agar, typical colonies are smooth, colorless or slightly grayish in appearance which may appear with an orange-colored halo and are 2- 3mm in diameter. Some rare variant strains of VTEC O157 ferment sorbitol and may grow poorly on CT-SMAC/SMAC.

Biochemical tests Oxidase test: E. coli is oxidase negative. Screening with oxidase test may be helpful and should be done on a media containing non-fermentable carbohydrates.

Catalase test: Effervescence of gases as a positive result for presence of E. coli that contains catalase enzyme which split H2O2.

Agglutination test Use VTEC O157 antiserum (latex or other commercial reagent). It is important to perform the appropriate control for auto agglutination.

Ornithobacterium rhinotracheale:

is a gram negative rod shaped bacterium causing respiratory disease in turkeys, chickens and other avian species. It is the only bacterium currently classified as within the Ornithobacterium genus. The bacterium is non-haemolytic and can tolerate a range of aerobic/anaerobic conditions and colonise a variety of agars. Colonies are smooth and non-pigmented.

Principles of identification  is most commonly isolated and cultured from the trachea and lungs, but is possible from other organs. Blood agar is usually used for culture but overgrowth of other bacteria is a common complication. PCR, immuno-histochemical staining and immunofluorescence are also possible.

Identification: Microscopic appearance: Gram stain Gram negative rod bacteria

Primary isolation:Optimal growth of ORT strains occurs in air enriched with 7.5-10% C02 (micro aerophilic condition) for at least 48h at 37 C.The organism grows well (but slowly) on 5-10% sheep blood agar or chocolate agar. At 24hr,pinpoint colonies (<1 mm) are observed.By 48hr.

Colonial appearance colonies are 1-2mm in diameter, circular, grey to grey-white, convex with an entire edge and non haemolytic. The colonies sometimes have a reddish glow and always give off a distinct odour, similar to that of butyric acid.

Biochemical tests

Oxidase ORT is oxidase positive. “Have oxidase enzyme that oxidizes the reagent to deep purple color”

Urease testORT is urease positive. “Secretes urease enzyme that split Urea into ammonia”

Sugar (CHO) fermentation test ORT ferment glucose to produce acid without gas