History and Clinical Findings

Within a flock of 4-day-old broiler breeder pullet chicks on a farm in northeast Georgia, birds in 3 houses had an increased mortality rate. A representative sample of live and dead birds was submitted for diagnostic evaluation. Two of the affected houses had bird capacities of 10,800 birds, and the third had a capacity of 8,000 birds. At 7 days of age, flock mortality was reported to be between 2.24% and 3.8%. The chicks were observed to have neurologic signs of tremors, circling, and torticollis; evidence of ocular opacity and swelling; and lameness. At 14 days of age, flock mortality was reported to be between 5.14% and 6.1%.

At the hatchery, the pullets were vaccinated for Marek disease and had received a DNA immunostimulant and a full dose of gentamicin in ovo. At 1 day of age, pullets were spray vaccinated against coccidial infection and infectious bursal disease, vaccinated against Marek disease by SC injection, and administered ceftiofur SC.

Three to 10 pullets from each affected house that were dead or had to be euthanized because they were moribund were submitted to the Georgia Poultry Laboratory Network and the Poultry Diagnostic and Research Center at the University of Georgia for evaluation; various tissue specimens underwent histologic examination and bacteriologic culture. While laboratory results were pending, antimicrobial treatment of the remaining birds in the flock was initiated via the water supply. The pullets received tetracycline for 6 days.

Gross lesions consisted of retained yolks, femoral head necrosis, pericarditis, airsacculitis, peritonitis, cranial cellulitis, and panophthalmitis with and without hypopyon. Corneas appeared intact and without gross lesions. One pullet had an opaque white to yellow area in the cerebrum that was circular and slightly irregular (Figure 1). It was slightly raised and appeared edematous, compared with the other side of the cerebrum. In addition, the cerebellum appeared somewhat swollen.

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Photographs of the heads (skin partially removed) of two 4-day-old commercial broiler breeder pullets from a flock that had an increase in mortality rate. Affected birds in the flock had tremors, circling, torticollis, ocular swelling, and lameness. A—In 1 bird, gross signs of panophthalmitis, including severe hypopyon, are present. B—In the other bird, there is evidence of ocular cellulitis (caret), and pale-yellow focal discoloration of the cerebrum is visible through the cranium (asterisk).

Citation: Journal of the American Veterinary Medical Association 260, 1; 10.2460/javma.19.05.0253

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Histopathological and Ancillary Findings

The observed cerebellar lesions grossly corresponded histologically to perivascular edema with multifocal lymphocytic and heterophilic infiltration of the parenchyma; perivascular cuffs were observed along with necrosis, vasculitis, and numerous bacterial colonies composed of bacilli. Brown and Hopps staining revealed that those bacilli were gram negative. Findings in the cerebellum included a multifocal necrotizing lymphocytic meningitis that extended into the molecular, Purkinje, and granular cell layers. Multifocal regions of cerebellar fibrinoheterophilic exudate and intralesional bacterial colonies were present (Figure 2) in the meninges and adjacent parenchyma of the folia.

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Photomicrographs of sections of the cerebellum (A) and eyes (B through F) from commercial broiler breeder pullets that died or were moribund and euthanized by cervical dislocation as a result of tremors, circling, torticollis, ocular swelling, and lameness. A—Findings in the cerebellum included severe multifocal necrotizing and lymphocytic meningitis. H&E stain; bar = 2 mm. B—Ocular findings included severe panophthalmitis. H&E stain; bar = 2 mm. C—Lens degeneration is apparent. H&E stain; bar = 500 μm. D—At higher magnification, notice the presence of morgagnian globules in the lens (asterisk). H&E stain; bar = 5 μm. E—Notice the severe heterophilic and lymphocytic necrotizing inflammation with the presence of bacilli (asterisk) in the vitreous humor. H&E stain; bar = 50 μm. F—The bacilli were gram negative (asterisk). Brown and Hopps stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 260, 1; 10.2460/javma.19.05.0253

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Severe panophthalmitis (Figure 2) consisting of heterophilic, mononuclear, and lymphocytic infiltrates with intralesional gram-negative bacterial colonies, fibrin, and edema was observed. Similar infiltration was present throughout multiple components of the eye, causing severe necrotizing iridocyclitis, chorioretinitis, hyalitis, and keratitis. Severe necrotizing inflammation of the pecten, corneal erosion, destruction of the annular pads, lens degeneration with the presence of morgagnian globules, and optic nerve disruption were also present.

Aerobic bacteriologic culture of specimens of the pullets’ brains, yolk sacs, areas of femoral head necrosis, bone marrow, eyes, and hearts yielded Pseudomonas aeruginosa. Escherichia coli was isolated from the brains, tarsal joints (hocks), yolk sacs, and hearts of several pullets. Pseudomonas aeruginosa is considered the primary disease-causing agent, as the clinical signs observed were consistent with P aeruginosa infection; E coli was thought to be a secondary opportunistic pathogen or contaminant.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: severe necrotizing heterophilic and lymphohistiocytic panophthalmitis with gram-negative bacilli and focal ulcerative keratitis and severe multifocal necrotizing fibrinoheterophilic and lymphohistiocytic meningoencephalitis with gram-negative bacilli.

Case summary: P aeruginosa infection in 4-day old broiler breeder pullets.

Comments

Pseudomonas spp are gram-negative, strictly aerobic bacilli associated with soil, water, or liquid systems and humid environments. It is an opportunistic organism, causing diseases in young, stressed, or immunodeficient poultry.¹ The most common Pseudomonas sp that causes disease in young poultry is P aeruginosa; infections begin as omphalitis and can spread systemically. In young birds, bacterial infections, such as those with P aeruginosa, can lead to elevated mortality rates,^1,2 as observed for the case described in the present report. Clinical signs include diarrhea, dehydration, lameness, ataxia and incoordination, conjunctivitis, and swelling of the head, wattles, sinuses, hocks, and other joints. Following infection of young birds with P aeruginosa, the mortality rate can be 100% within a 72-hour period.¹ Severe P aeruginosa infection can lead to septicemia, with bacteria replicating around vessels in the brain and perforating the cornea of the eye, as observed in affected birds of the present report.

Infection with P aeruginosa results in histopathologic lesions similar to those observed in some other poultry submitted to the Poultry Diagnostic and Research Center in Athens, Georgia. In those previous cases, infections with other gram-negative organisms such as E coli and Salmonella enterica subsp arizonae were identified as the causative agents. In the cerebrum and cerebellum, infection with any of these 3 bacterial organisms leads to extensive disseminated necrosis with intralesional bacteria, perivascular heterophilic and lymphohistiocytic inflammation, and perivascular cuffing. Chronic infection can result in granulomatous ventriculitis.³ Primary infection with Aspergillus fumigatus can also result in necrotizing encephalitis, meningoencephalitis, heterophilic granulomas, gliosis, and perivascular lymphocytic cuffing, with the presence of dichotomous branching hyphae that can be seen after H&E staining of tissue sections. Ocular lesions associated with these gram-negative bacterial and fungal organisms include fibrinoheterophilic inflammation of the anterior chamber, posterior chamber, vitreous humor, pecten, ciliary body, iris, retina, and cornea and corneal perforation.⁴ Infection also results in the presence of intralesional bacteria or hyphae, depending on the organism.

Isolation of the infective agent is crucial because of the microscopic similarities of the lesions caused by various organisms. Sterile swabs of yolk sac, bone marrow, and brain should be submitted for bacteriologic culture, and tissue specimens (including the eyes and brain) should be submitted for histologic examination. In the case described in the present report, Salmonella arizonae and A fumigatus were not isolated from any lesions in any pullets. Although both P aeruginosa and E coli were detected in pullets at the Poultry Diagnostic and Research Center, E coli was isolated from fewer types of tissue and fewer birds, and the isolated E coli was not characterized as pathogenic or nonpathogenic. Their presence was likely due to contamination. The primary agent causing disease in case described in the present report was P aeruginosa.

The first step in prevention of Pseudomonas infection in poultry is to locate and eliminate the source of the organism. Good hygiene practices among workers, cleaning and disinfecting of equipment, and use of sterile technique are invaluable in preventing infection at the hatchery level and in chicken houses. Normal hatchery procedure is to change all tubing every day on both the in ovo and day-of-age vaccine machines. Samples are collected from the bags of diluent and vaccine every 4 hours throughout the vaccination event to test for presence of bacteria. For the case described in the present report, water was collected from all pullet houses from the point at which it entered the house, prior to going through the medicator. The total bacterial count was < 100 CFUs/100 mL of water in all houses. The most probable number of total coliforms in 100 mL of water was < 1 in all except 1 house, in which it was 25.4. The most probable number of fecal coliforms in 100 mL of water was < 1 in all except 1 house, in which it was 3.1. In commercial poultry house drinking water, optimal levels of total bacteria, total coliforms, and fecal coliforms are 0 CFUs/mL; maximum acceptable levels of total bacteria, total coliforms, and fecal coliforms are 1,000 CFUs/mL, 50 CFUs/mL, and 0 CFUs/mL, respectively.⁵ No Pseudomonas organisms were isolated in the water samples collected from the pullet houses that housed the flock of the present report. Identification of the source of the organism that caused the infection among the birds was diligently sought; however, the source was not determined.

Pseudomonas spp have resilience against common disinfectants; therefore, the disinfectant used is crucial. Disinfectants such as a broad-spectrum quaternary ammonium compound blend with EDTA-Tris (Biosentry 904; Neogen Corp) have been found to be effective at killing P aeruginosa.^6,7 Although antimicrobials can be used to treat P aeruginosa infections in poultry, death of infected birds occurs suddenly and P aeruginosa has high antimicrobial resistance.¹ In the case described in the present report, the isolates were intermediately susceptible to tetracycline and ceftiofur. Clinicians should be aware that although E coli infection is a common cause of increases in mortality rates among young chicks, Pseudomonas infection should be considered as a differential diagnosis in such situations.