Journal of Veterinary Diagnostic Investigation Vol. 18 Issue 6, 594-596
Copyright © 2006 by the American Association of Veterinary Laboratory Diagnosticians
Isolation of Vibrio cholerae from the brain of a feedlot heifer with meningoencephalitis
Jamie M. Bush1,
Doreene R. Hyatt,
Denise Bolte and
Karamjeet Pandher
Correspondence: 1Corresponding Author: Jamie M Bush, Colorado State University Veterinary Diagnostic Laboratory, 300 W Drake Ave, Fort Collins, CO 80524
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Abstract
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A 700-pound, 9-month-old Angus heifer from a feedlot presented with acute neurologic signs, characterized by circling, posterior weakness, and nonresponsiveness, followed by death. Histologically, the frontal lobe and the thalamus contained multiple foci of liquefaction that contained numerous degenerative neutrophils and foamy macrophages. Some of these foci were centered on blood vessels that contained fibrin thrombi and exhibited varying degrees of fibrinoid necrosis of the vessel wall. There was adjacent axonal degeneration and neuronal necrosis characterized by pronounced cytoplasmic eosinophilia, peripheralization of the nuclei, and loss of Nissl substance. Aerobic culture of the brain yielded moderate growth of Vibrio species, which was determined to be Vibrio cholerae by polymerase chain reaction analysis of a 438-base pair fragment of the 16 S ribosomal RNA gene. V. cholerae are motile, gram-negative, curved rod-shaped bacteria. Some strains of V. cholerae are important food- and water-borne bacterial pathogens that produce an often fatal diarrhea in humans. This is the first known case report of V. cholerae meningoencephalitis and cerebral abscessation in a bovine.
Key Words: Cattle • cerebral abscess • meningoencephalitis • Vibrio cholerae
There are a variety of infectious and noninfectious causes of neurologic disease in cattle, including locoweed and solanum toxicosis, nitrite poisoning, trauma, polioencephalomalacia, lead poisoning, Histophilus somni septicemia, Listeria monocytogenes infection, sporadic bovine encephalomyelitis, bovine spongiform encephalopathy, and neoplasia.5 A 700-pound, 9-month-old Angus heifer from a feedlot in the central midwestern USA presented for acute onset of circling, posterior weakness, and nonresponsiveness, which quickly progressed to recumbency with minimal paddling.
The heifer was treated unsuccessfully with thiamine, oxytetracycline, and tylosin (Tylan) and soon died. Necropsy was performed by the referring veterinarian 15 to 18 hours after death. No significant gross lesions were identified. Tissue samples of the liver, spleen, heart, and intestinal tract, as well as the head, were submitted to the Colorado State University Diagnostic Laboratory. The brain was removed and upon gross examination revealed marked congestion of the meningeal vessels. Histologic evaluation was performed on hematoxylin and eosin (HE)-stained sections. The cerebrum (frontal lobe) and the thalamus contained multiple foci of liquefaction and microabscessation. Some of these foci were centered on blood vessels that contained fibrin thrombi and exhibited varying degrees of mural fibrinoid necrosis (Fig. 1). There was axonal degeneration characterized by spheroid formation and neuronal degeneration and necrosis characterized by pronounced cytoplasmic eosinophilia, loss of Nissl substance, peripheralization, and pyknosis of the nuclei. There was moderate perivascular cuffing of lymphocytes and plasma cells, with fewer neutrophils within the meninges and cerebral parenchyma. In addition, the hepatic sinusoids contained increased numbers of neutrophils, many of which were hypersegmented (sinusoidal leukocytosis). The intestinal tract demonstrated marked superficial autolysis. A primary histologic diagnosis of subacute multifocal necrosuppurative meningoencephalitis with fibrinoid necrosis of vessels and thrombosis was made. The histopathology and the lesion distribution were compatible with an acute bacterial septicemia.
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Figure 1 Brain. Venous thrombosis with associated cerebral parenchymal liquefaction and increased numbers of foamy macrophages (gitter cells), necrotic neurons, degenerative neutrophils and cellular debris. HE, Bar = 200 µm.
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Aerobic and microaerophilic culture of the brain was performed by using tryptic soy agar with 5% sheep blood, MacConkey agar, and Columbia agar with 5% sheep blood in 5% CO2.a Aerobic culture yielded multiple bacteria, including moderate growth of a Vibrio spp. Moderate growth is defined as bacterial growth within 2 quadrants of the plate. This culture was not established as a quantitative culture thus colony forming units were not determined. Given the extremely low incidence of Vibrio cholerae identified at the Colorado State University Veterinary Diagnostic Laboratory and the observation that V. cholerae is not a frequently recognized contaminant in this laboratory, the chance of laboratory contamination is minimal. In addition, there were no other samples that were positive for V. cholerae in the months before or after this sample was received. Other isolates, including Actinobacter spp., Escherichia coli, and Proteus vulgaris, were interpreted to be contaminants because of the mixed culture, the species identified, and the observation that these organisms are common contaminants within this diagnostic laboratory. Escherichia coli is implicated in sporadic cases of septicemia, which may lead to the formation of cerebral abscesses, but such cases are often restricted to neonates.1 Listeria monocytogenes was not isolated after inoculation on Oxford agar. Cultures for H. somni were also negative. Negative cultures for H. somni, especially in treated animals, are possible, however, other lesions frequently associated with H. somni, including necrotizing laryngotracheitis, fibrinous bronchopneumonia, myocarditis, polyarthritis, retinal hemorrhage, and widespread serosal and skeletal muscle petechiation, were not identified.5 A panel of biochemical tests was performed to identify the isolate as Vibrio spp. The isolate was gram negative based on the potassium hydroxide method, positive for catalase, indole, and oxidase, and gram negative on a Brown-Hopps modified Gram stain. An API 20 E systemb was used to obtain a presumptive identification (99.9% accuracy) of the isolate as V. cholerae. In addition, V. cholerae is positive for glucose fermentation, which helps exclude Pseudomonas spp. and other nonfermenters. The chromosomal DNA was extracted by using a Qiagen QIAamp DNA Mini Kit.c A PCR was performed to amplify a 438-base pair fragment of the 16 S ribosomal RNA gene from base pair 59 to base pair 496 (primers F: 5'CGG GAT CCC AGG CCC GGG AAC GTA TTC AC-3' and R: 5' GGA ATT CCC GCA CAA GCG GTG GAG CA 3').11 Briefly, the extracted DNA was eluted into 100 µl buffer AE. The PCR reaction used 5 µl eluate as a template, 5 µl 10x reaction buffer, 1.5 µl magnesium chloride (50 mM), 2 µl forward and reverse EubacII primer set,d 0.5 µl Taq polymerase, and 30 µl sterile water. The PCR amplification was performed in a MJ Research (MJR) thermocycler,e with a cycle that contained incubations at 94°C, 65°C, and 72°C for 1 minute each for 30 cycles, followed by an extension step of 5 minutes at 72°C. Sequencing of the amplified isolate was performed by Macromolecular Resources, Colorado State University, by using the ABI 3100 Genetic Analyzer.f The sequence was analyzed for homology to known sequences by using the Web-based program Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/BLAST/). The National Center for Biotechnology Information (NCBI) BLAST result aligned with the PCR results. Upon analysis, there was a 99% sequencing homology between the partially sequenced amplified isolate and V. cholerae. Serotyping and further analysis could not be performed, because repeated attempts at a later date to propagate the primary isolate from agar plates and from the brain tissue were unsuccessful. All cultures were saved for 2 weeks on their original culture media (Columbia agar plate with 5% sheep blood) and then placed in a box and held at 5°C.a The isolate was sent to a reference laboratory on a Copan swaba for confirmation but was not subcultured promptly. Upon later attempts to subculture by the referring laboratory, the culture was no longer viable. Approximately 1 month after the original culture attempts to reculture the isolate from tissues stored at –70°C were unsuccessful.
Vibrio cholerae is a motile, gram-negative, curved rod that belongs to the family Vibrionaceae and is considered an important food- and water-borne bacterial pathogen.2 There are approximately 200 recognized O serogroups, of which serogroups O1 and O139 are associated with cholera pandemics in humans.12 Vibrio cholerae causes fatal diarrhea in humans, primarily through the action of a secreted cholera toxin, which does not play a role in nonepidemic enteric and septicemic forms of the disease.3,12 Vibrio cholerae typically is isolated from aquatic environments.12 There have been rare reports of brain abscesses in people secondary to Vibrio infection, often after neurosurgery.10 More specifically, there have been human case reports of intracerebral abscesses and meningitis associated with non-O1 V. cholerae.4,6,8,9,13,14 From 1988 to 1998, V. cholerae non-O1 has been found in association with diseases of farm animals 15 times in the Netherlands, including a goat with enterotoxicosis, a bull with abomasitis and enteritis, a heifer with a hemorrhagic diarrhea, and a calf with diarrhea.15 There have been 2 reports implicating V. cholerae non-O1/non-O139 in lamb diarrheal outbreaks in the Netherlands, though no brain lesions were identified in these situations.15,16 In 1 report, there were 5 cases of sudden death in 2- to 4-day-old lambs with severe watery diarrhea.15 These lambs died within 24 hours of the onset of clinical signs.15 At necropsy, intestines were slightly hemorrhagic, with increased watery contents.15 Investigations of additional outbreaks involving 17 lambs that died from a flock of 150 ewes and two 1-year-old animals that died from another flock yielded positive cultures of V. cholerae from a barn water tank and surface water locations, respectively.15 In addition, a case series of stranded beluga whales in Canada reported a single case of non-O:1 V. cholerae septicemia in a juvenile beluga whale.7
To the authors' knowledge, the present case is unique in that it demonstrates meningoencephalitis and cerebral abscessation in a bovine associated with the isolation of V. cholerae. Given the epidemiology of previous cases of exposure of farm animals to V. cholerae, the source of exposure in this case is likely to be contaminated water.15 Though other organisms such as H. somni and E. coli cannot be entirely excluded as the cause of disease in this bovine, circumstantial evidence points toward this being a case of V. cholerae induced disease. Vibrio cholerae is not commonly reported as a cause of cerebral abscesses and encephalitis in domestic animals, which may reflect a low incidence or possibly a lack of extensive diagnostic pursuit. Firstly, V. cholerae is not a commonly isolated contaminant in our large animal diagnostic service. Indeed, the reason this case is interesting was the isolation of this unique organism. After isolation of a Vibrio spp., the decision was made to classify it further by biochemical and molecular methods that identified the organism as V. cholerae. Unfortunately, further characterization of the organism could not be performed, because culture could neither be revived by the Colorado State Veterinary Diagnostic Laboratory nor the referency laboratory, which is proficient in V. cholerae procedures. Secondly, there is precedence of non-O1/O139 V. cholerae causing cerebral lesions in other species4,6,8,9,13,14 and severe gastrointestinal disease in lambs,15,16 as mentioned above.
A compromised gastrointestinal mucosal barrier opens the possibility of subsequent septicemia. Although the lesions are histologically compatible with H. somni infection, the fact that it was not isolated from this case precludes its direct association with this particular case. Conventional wisdom holds that many cases of H. somni infection yield negative results upon bacterial culture after antibiotic treatment, however, this hypothesis is inherently not provable. In such cases, alternate etiologies for bacterial encephalitis should be pursued. In this particular case, such pursuit yielded a unique organism that is isolated less than once a year in this laboratory.
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Sources and manufacturers
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From the Colorado State University Veterinary Diagnostic Laboratory, Fort Collins, CO 80524 
a. BBL, Sparks, MD.
b. API Biomerieux, Marcy l'Etolle, France.
c. Qiagen, Valencia, CA.
d. Integrated DNA Technologies, Inc., Coralville, IA.
e. MJ Research, Waltham, MA.
f. Applied Biosystems, Foster City, CA.
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References
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