Session #510
Susan L. Clubb, DVM
Loxahatchee, Florida
Bruce L. Homer, DVM, PhD
Department of Pathology, College of Veterinary Medicine, University of Florida
Gainesville, Florida
John Pisani, PhD
Micrim Laboratories, Inc.
Miami, Florida
Candi Head
Loxahatchee, Florida
Bordetella sp. have long been recognized as significant pathogens of the upper respiratoy system in many mammalian species as well as poultry. Gerlach reports that psittacines and finches are also susceptible to infection with Bordetella but gives no description of the disease in these species.5
Bordetella avium was isolated from cockatiel chicks exhibiting nestling mortality, rhinitis, sinusitis and temporomandibular rigidity ("lock jaw" syndrome). The clinical syndrome was reproduced by inoculation of the cockatiel chicks. Bordetella bronchiseptica and B. avium was isolated from a group of ostrich chicks exhibiting stunting, tracheitis, impaction and mortality.
An Outbreak Of Bordetella Avium In Cockatiel Chicks
A small commercial psittacine aviary experienced intermittent nestling mortality in cockatiels for several years. Mortality occurred primarily in the summer months. Chicks were usually between 2 and 4 weeks of age. Morbid chicks, and chicks for necropsy were first presented in June 1992. The chicks were emaciated, dehydrated and most had intestinal stasis prior to death. Some chicks had serous nasal discharge but lockjaw was not observed. The crops and intestines were dilated with fluid contents (the owner diluted the formula with an electrolyte solution). Chicks were fed a formula based on commercial monkey diet.
The clinical syndrome was suggestive of immunosuppression and multiple secondary infections. Aerobic cultures of the crops and cloacas revealed scant to 3+ yeast, 3+ mixed gram positive, and gram negative bacterial growth typically with 50-100% E coli. Some chicks also had Klebsiella and Pseudomonas. Wet mount examination of feces revealed protozoa morphologically similar to Hexamita. Fecal floatation was negative. Crop and cloacal gram stains revealed moderate gram-positive rods, scant to heavy yeast, and 0 to 10 % gram-negative rods. Electron microscopic examination of intestinal contents was negative for viruses. Chlamydial and viral isolation attempts were negative chick embryo inoculation. Cloacal swabs and liver and spleen suspensions were negative on ELISA (Kodak-Surecell) for chlamydia. Histopathology revealed koilin dysplasia and ventricular megabacteriosis (upper respiratory tract was not submitted). Borderella was not isolated but could easily have been overlooked in the heavy growth off E coli and Klebsiella.
Affected chicks were treated individually with enrofloxacin, ketoconazole, ciprofloxacin, nystatin, amikacin nitrofurazone, doxycycline, and gentian violet. None of these treatment regimens satisfactorily resolved the problem and mortality continued.
Aviary husbandry practices were evaluated. A soft food mix containing shredded carrots, endive and whole wheat bread was fed to the breeder cockatiels each morning. Enough was prepared for three days and stored under refrigeration. The soft food was fed on top of the seed mix each morning. Cultures of the soft food from the refrigerator revealed the contamination with yeast and E coli.
Feeding soft food was discontinued and food hygiene was improved. Treatment of the flock for Hexamita was attempted with metronidazole administered both as a feed mix and by individual parenteral therapy for known shedding breeders. Birds that were rechecked after treatment were usually still shedding. The entire flock was treated with nitrofurazone in the water for 7 days, after which time they were still positive for protozoa but mortality was reduced. The breeder elected to discontinue breeding during the hot summer months. Mortality in the following year was moderate.
In November, 1993, the aviculturist again experienced significant chick mortality. Several chicks in the group developed "lock jaw" Clinically, the chicks were debilitated, dehydrated, were sneezing and had a serous nasal discharge. They begged to be fed, but the mouth was tightly locked and could not be opened enough to introduce the syringe tip for feeding. The suborbital chamber of the infraorbital sinus appeared swollen, displacing the lower lid dorsally over the cornea so the eye appeared half closed. The eyes were glassy. Affected chicks were irritable and hyperexcitable, in apparent pain, especially during attempts to pry the mouth open. If the mouth could be opened enough to feed the chick, the chance of aspiration was great. Affected chicks usually died within 24 hours.
Histopathology revealed chronic active inflammation in the nasal cavity and infraorbital sinuses with inflammation extending into area skeletal muscles. There was hyperplasia of the mucous membranes of the infraorbital sinus, associated with loss of cilia. Inflammation extended into the proximal trachea with multifocal to focally extensive heterophihc tracheitis. In some birds, infection extended into the lungs with bacterial bronchitis. Bordetella avium was isolated from the choana and cloaca of 6 of 10 birds cultured.
Subsequent bacterial cultures from the choana of 16 symptomatic chicks in the nursery revealed 14 which were positive for Bordetella. Treatment was attempted with enrofloxacin, amikacin, piperacillin, and doxycycline alone and in combination with each other and always with an antifungal agent. All chicks were euthanized due to poor therapeutic response and fear of developing a carrier state. Another 20 chicks were cultured: 10 were asymptomatic cockatiel chicks as they were pulled from the nest (3 were positive), and 10 were symptomatic cockatiel chicks from the nursery (7 were positive). Two Wagler's conures (Aratinga wagleri), a jenday conure: (Aratinga jandaya), a moluccan cockatoo (Cacatua moluccensis), and a nobles macaw (Ara nobilis) began displaying similar clinical signs and cultured positive for Bordetella. While most of the larger birds showed clinical improvement following antibiotic therapy, they often had chronic nasal discharge and cultured positive for Bordetella following therapy. Again all positive cockatiel chicks were euthanized, but asymptomatic chicks soon infected others in the nursery.
Screening of the breeder flock was elected in order to detect carriers. Twenty breeders were initially cultured and 2 were found positive. Both were males and Bordetella was not found in their mates. These pairs also produced chicks that succumbed in the 1992 outbreak. One of these male birds was frequently used as a foster parent and chicks had cultured positive when when removed from his nest. Approximately 24 pairs were cultured with 5 birds found positive. Positive birds were eliminated from the breeding flock. Nest boxes were replaced and the aviary sanitized. At the time of this writing (June, 1994) the aviculturist is again experiencing mortality in the nest and the nursery and further control methods are being investigated.
An Outbreak of Bordetellosls In an Ostrich "Grow-Out" Facility
A group of approximately 40 ostrich chicks, 10 to 12 weeks old, were stable and growing well in a South Florida grow-out facility after release from quarantine. Another group of chicks approximately the same age and belonging to the same owner, were held in a similar grow-out facility in another state. These chicks were dying and were brought to this facility for treatment. The ill chicks were extremely thin and under size. Most were impacted with gravel and sand, and mortality was high. Heavy growths of mixed gram-negative flora including Pseudomonas, Proteus, Klebstella and E coli were isolated from the choana and cloaca. Direct fecal examinations revealed 3 different flagellated protozoa resembling Giardia and Trichomonas.
Shortly after the sick chicks arrived, two stable chicks at the facility began to lose weight and exhibit depression. WBC's were elevated. One chick injured its leg and responded well to topical treatment and bandaging, but was found dead after the wound healed. The second chick died approximately 10 days after first exhibiting clinical signs.
A gross post-mortem exam of the first chick revealed pseudomembranous tracheitis. The liver was enlarged and friable with multiple yellow foci. Lungs and other organs appeared grossly normal. Histopathology revealed severe peracute necrotizing tracheitis, advancing septicemia, and hepatic necrosis. Bordetella bronchiseptica was isolated from the trachea but was not found in the lung or liver. Bordetella was isolated from the trachea of the second chick prior to its death. Gross and microscopic lesions were similar to those present in the first chick.
The proximal tracheas of the surviving, stunted, and chronically ill chicks that were initially brought to the South Florida facility were cultured. Bordetella bronchtseptica was isolated in 6 of 11 chicks cultured. Most also had other gram negative bacteria such as E coli and Klebsiella. These chicks had previously been treated with enrofloxacin and ceftiofur. In an attempt to eliminate the Bordetellosis, the birds were treated with amikacin intratracheally and oxytetracycline in the drinking water. Bordetella was not isolated on post treatment cultures.
Another approximately 4 month old became acutely ill showing depression, coughing, slinging the head and mucous from the mouth. Bordetella avium was isolated from the proximal trachea. This chick was also treated with oral oxytetracycline and intratracheal amikacin and responded well to therapy.
The grow-out facility was located next to a commercial boarding kennel and used unfiltered and untreated well water. Culture of chicks at the facility in another state approximately 1 month later revealed B. avium.
In both of these outbreaks, Bordetella was isolated on McConkey's agar grown aerobically at approximately 37C. Small convex .1 to .5 mm in diameter non-pigmentnd colonies were evident at 24 hours. On the second day, colonies were 1 to 1.5 mm in diameter and were umbonate, opaque and grayish. If picked up on a swab, the swab appeared bluish. Oxidase and catalase tests are positive. Initial identification of B. avium was made by the Florida Department of Agriculture, Kissimmee Diagnostic Laboratory utilizing the API NFT kit. Later confirmations of B. avium and B. bronchiseptica were made by Micrim Laboratories, Inc., Miami, FL also using APl NFT tests (now Bio-merieux 20 biochemical strip).
Fulfilling Koch's Postulates
Two cockatiel chicks approximately 2 1/2 weeks of age were obtained from a breeding collection with no history of the "lock jaw" syndrome. Choanal and cloacal cultures revealed only normal gram-positive microflora and no presence of Bordetella. An Isolate of Bordetella avium obtained from cockatiels in the outbreak from the psittacine aviary was grown on McConkey's agar and found to be a pure culture. Approximately 5 mature umbonate colonies were removed from the plate and suspended in 1 ml of sterile normal saline solution. Two drops were instilled into each nostril of the 2 cockatiel chicks. The chicks were held in isolation, and hand feeding was continued by a person having no other contact with birds.
The chicks started to sneeze 1 dpi (days post inoculation) At 2 dpi, the nares became erythemic and sneezing continued. At 4 dpi, the chicks began to refuse food and had a mucous discharge from the mouth. At 5 dpi, one chick developed swelling in the area of the suborbital chamber of the infraorbital sinus. The lower lid rose over the cornea and the eyes were glassy. The mouth cound not be opened more than 1/8 inch. The chick was 4 weeks old, weighed 89 grams, was thin, and had polyuria. When it sneezed, mucous ran out of the nostril. The chick was euthanized. Gross post-mortem examination revealed a thin chick with no abnormal findings other than "lock jaw". A choanal gram stain revealed 10% gram negatives, no yeast, and good gram-positive flora. A cloacal gram stain revealed the same except scant yeast. The WBC count was 11,6000. Bordetella avium was re-isolated from the choana and cloaca.
A histologic examination of multiple tissues revealed lesions predominating in the upper respiratory tract. These included subacute to hyperplastic multifocal intraorbital sinusitis and perisinusitis, with extensions of inflammation into skeletal muscle ventral and medial to the eye; subacute multifocal cellulitis and edema of the face, subacute diffuse rhinitis; mild subacute diffuse rhinitis; and mild subacute multifocal tracheitis fo the upper trachea. There was multifocal loss of cilia and squamous metaplasia in the mucosa of the nasal cavity and infraorbital sinus. Inflammation also extended into the lacrimal gland. The lung had a mild lymphocytic interstitial pneumonia. There was mild to moderate splenic lymphoid depletion. Other viscera were unremarkable.
Brief Review of Bordetellosis in Turkeys
Bordetellosis, known as turkey coryza, is a highly contagious upper respiratory tract disease of young turkeys. The disease is caused by Bordetella avium, alone or in combination with environmental stresses and other respiratory pathogens. Colonization of ciliated epithelium by B. avium results in protracted inflammation and distortion of the respiratory mucosa. Bordetellosis in turkeys is characterized by abrupt onset of sneezing, clear oculonasal discharge, mouth breathing, submandibular edema, altered voice, tracheal collapse, stunted growth, and mortality. The disease was previously known as Alcaligenes rhinotracheitis, adenovirus-associated respiratory disease, acute respiratory disease syndrome and turkey bronchiseptica-like and alcaligenes faecalis prior to the name Bordetella avium being proposed and accepted in 1984. (1,3)
Bordetella avium is a gram-negative, non-fermentive, motile, strictly aerobic bacillus which grows readily on McConkey's agar. B. avium has a strict trophism for ciliated epithelium and produces toxins including dermonecrotic (heat labile) toxin, heat-stabile toxin, and tracheal cytotoxin. (1,3)
Most commonly used disinfectants appeart to kill B. avium when used according to manufactures' recommendations. Survival of the organism for at least 6 months in undisturbed litter has been reported. Low temperatures, low humidities, and neutral pH prolong survival. (1,2)
Most strains are sensitive in-vitro to a large number of antibiotics; however, treatment of affected turkeys results in either no effect or a transient reduction in bacterial numbers. (2,4,6)
Major virulence factors of B. avium involve adhesion to cilia of respiratory epithelium, loval mucosal injury attributed to toxins, and systemic effects. a number of systemic effects have been attributed to B. avium infection, including elevation of serum corticosterone, enhanced leukocyte migration, reduced body temperature, and reduced monoamines in brain and lymphoid tissue. Defective immune function has often been reported but not substantiated. (1)
The incubation period in turkey poults is 7-10 days in natural and 4-6 days in experimental infections. Clinical signs include abrupt onset of sneezing in a high percentage of 2-6 week old poults. Clear nasal exudate can be expressed by using gentle pressure between the nostrils. The nares and feathers of the head become coated with exudate. Some birds develop submandibular edema. Mouth breathing, dyspnea, and altered vocalization occur in the second week as the nasal cavity and upper trachea become partially occluded with mucoid exudate. Tracheal softening can be palpated through the skin. Affected birds huddle and become inactive and anorexic. Morbidity is usually high while mortality is low unless birds are concurrently infected with E coli. Distinctive microscopic lesions of diagnostic value include cilia-associated bacterial colonies, cytoplasmic inclusions, cystic mucosal glands, and generalized loss of ciliated epithelium. Most turkeys develop a humeral immune response to infection with B. avium which is detectable within 2 weeks after experimental inoculation. Antibody is also produced by submucosal plasma cells. Due to damaged ciliated mucosa, some convalescent birds are slow to clear B. avium from their respiratory tissues, which may result in re-infection after immunity wanes or transmission to susceptible birds. B. avium is isolated predominantly from the upper trachea. B. avium is highly contagious by direct contact and through contamination of water, feed, and litter. Modified live vaccines and bacterins are available commercially, but efficacy has been questionable as turkeys less than 3 weeks of age are unable to mount an adequate local immune response. While the severity of tracheal lesions is reduced, infection is not prevented. Maternal antibody may confer temporary immunity to newly hatched poults. (1)
Summary
Following the initial outbreak of Bordetellosis in cockatiels, infected and diseased birds were presented from 5 other aviaries in South Florida and one in Texas, and positive cultures were submitted from an aviary in Minnesota. Verbal reports of the "lock jaw" syndrome are widespread. Concurrent superinfection with other common gram-negative bacteria and yeast may overgrow the tiny colonies so Bordetella can be easily overlooked. The potential for immunosuppression and resultant superinfection with other pathogens, including protozoa, should be considered. It would appear that Bordetellosis may be a significant problem for psittacine aviculture and possibly for the ostrich industry. Control measures warrant further study.
References
1. Arp LH, Skeeles JK. Bordetellosis (Turkey Coryza). In:
Diseases of Poultry, 9th ed, 1991
2. Cimiotti WG, Glunder, Hinz KH.
Survival of the bacterial turkey coryza agent.
Vet Rec 1982;110:304-306
3. Kersters K, Hinz KH, Hertle A, et al.
Bordetella avium sp. nov.isolated from the respiratory tracts
of turkeys and other birds.
Int J Syst Bacteriology 1984;34:56-70
4. Ficken MD. Antibiotic aerosolization for treatment of alcaligenes rhinotracheitis. Avian Dis 1983;27:545- 548.
5. Gerlach H. Bacteria. In: Ritchie BW, Harrison GJ, Harrison LR, eds. Avian Medicine: Principles and Application. Lake Worth, FL: Wingers Publishing Co., 1994;959.
6. Skeeles JK, Swafford WS, Wages DP, et al. Studies on the use of a long-acting oxytetracycline in turkeys; efficacy against experimental infections with alcaligenes faecalis and pasturella multocida. Avian Dis 1983;27:1126-1130.