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Brief Communications |
Correspondence: 1Corresponding Author: John A. Angelos, Department of Medicine and Epidemiology, School of Veterinary Medicine, 2108 Tupper Hall, Davis, CA 95616, e-mail: jaangelos{at}ucdavis.edu
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Abstract |
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Key Words: Infectious bovine keratoconjunctivitis, IBK • Moraxella bovis • Moraxella bovoculi sp. nov., Moraxella ovis
Infectious bovine keratoconjunctivitis (IBK; pinkeye) is the most common ocular disease of cattle; affected animals exhibit corneal ulceration leading to corneal scarring of varying severity. Koch's postulates have been satisfied for Moraxella bovis and IBK.12 Pathogenic proteins of M. bovis include pilin that enable the organism to attach to corneal epithelial cells4,16,18 and a pore-forming cytotoxin9 (cytolysin; hemolysin) that lyses corneal epithelial cells7 and host neutrophils resulting in a release of neutrophil-derived degradative enzymes into corneal stroma.14,15,20 The M. bovis cytotoxin is an RTX (repeats in the structural toxin) toxin designated MbxA encoded by an RTXA gene (mbxA)1 that is contained within an RTX operon (mbx operon)2 that resides on a pathogenicity island in M. bovis.13
Along with M. bovis, other gram-negative bacteria have been identified in ocular fluid from the eyes of normal and IBK-affected cattle. These bacteria have variously been designated hemolytic diplococci,19 hemolytic Neisseria,11 Neisseria spp.,5,6,21 Neisseria (Branhamella) catarrhalis,22 or Moraxella ovis.8,10 During an IBK antibiotic efficacy trial conducted in Northern California during the summer of 2002, hemolytic gram-negative cocci isolated from the ulcerated eyes of dairy and beef calves were further characterized. DNA sequence analyses and biochemical testing demonstrated that 18 characterized isolates were distinct from M. bovis and M. ovis and as such represented a novel species that was named Moraxella bovoculi sp. nov., within the genus Moraxella.3 M. bovoculi sp. nov. could be differentiated from M. bovis and M. ovis as well as other moraxellae isolated from animals on the basis of positive phenylalanine deaminase (PADase) activity.
During an IBK vaccine trial conducted during the summer of 2005, ulcerated eyes of calves were cultured, and a total of 44 additional isolates of hemolytic gram-negative cocci were characterized. Forty-two were PADase positive and were identified as M. bovoculi sp. nov.; however, 2 PADase-negative isolates (120L and 159R) could not be readily differentiated from M. ovis on the basis of PADase activity. To facilitate the molecular identification of PADase-positive or -negative hemolytic gram-negative coccoid moraxellae, a polymerase chain reaction (PCR) coupled with restriction enzyme analysis was developed.
Moraxella bovoculi sp. nov. (44 isolates), M. ovis,a M. bovis Tifton I,b Moraxella caprae,c and Moraxella boevreid were propagated on 5% sheep blood agare at 35°C; strains were stored frozen in 10% skim milk–50% glycerol at –80°C. To prepare genomic DNA, 1 loopful of bacteria was scraped from plates and resuspended in 500 µl of sterile deionized water, and genomic DNA was harvested according to the manufacturer's instructions.f Primers for amplifying the 16S-23S intergenic spacer region (ISR) were developed from the DNA sequence of the 16S rDNA gene, the 16S-23S ISR, and a portion of the 23S rDNA gene from M. bovis, M. ovis, and M. bovoculi sp. nov.3 The PCR primers used were ISRdown (5'-GTG AAG TCG TAA CAA GGT AGC CGT-3') and ISRup (5'-ACC GAC GCT TAT CGC AGG CTA TCA-3'). The PCR reactions were made up to a final volume of 50 µl with ultra pure waterg and contained 3 µl of genomic DNA template, 0.4 µl (2U) Taq DNA polymerase,h 5 µl of 10x PCR buffer (the final 1x buffer was 1.5 mM MgCl2, 1.5 mM MgSO4, 10 mM KCl, 8 mM [NH4]2SO4, 10 mM Tris-HCl [pH 9.0], and 0.05% NP-40), 0.5 µl of 10 mM dNTP,i and 2.5 µl of each primer (10 pmole/µl). The reactions were run in a thermocyclerj using an initial denaturation at 95°C for 1 minute, followed by 35 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds. Fifteen microliters of the resulting PCR reaction was mixed with 0.5 µl (4U) of AfaIk and incubated at 37°C for 1 hour. Following incubation, 5 µl of the original PCR reaction and 5 µl of the AfaI digested PCR reaction were electrophoresed on a 2% agarose gell containing 0.25 µg/ml of ethidium bromidem in 1x TAE buffer (0.04 M Tris-acetate; 0.001 M EDTA). The gel was visualized on an ultraviolet lightboxn and photographed.o The ISRdown-ISRup amplicon generated from genomic DNA was sequenced directly with primers ISRdown and ISRup.p
ISRdown-ISRup amplified DNA exhibited a single band of 
600 bp for M. bovoculi sp. nov. and M. ovis and 650 bp for M. bovis, M. boevrei, and M. caprae (Fig. 1). AfaI digestion of amplified DNA cut the 600 bp amplicon from M. bovoculi sp. nov. at 1 site, resulting in 2 bands of 450 and 150 bp. The original amplicons generated for M. bovis, M. ovis, M. boevrei, and M. caprae were not cut by AfaI and remained as a single band (Fig. 1). For the 44 isolates examined, the DNA sequence of the 16S–23S ISR amplified by the ISRdown-ISRup primers matched the previously reported 16S–23S intergenic spacer sequence for M. bovoculi sp. nov. (Genbank accession DQ153085).3
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During the original characterization of M. bovoculi sp. nov., the M. ovis chosen for comparison was a type strain of M. ovis (ATCC 33078) that was originally cultured from a sheep with keratoconjunctivitis.17 Other species of gram-negative cocci have been isolated from cattle with IBK, and these have historically been referred to as Moraxella, Branhamella, or Neisseria spp./ovis.5,6,8,10,11,21,22 It is possible that some agents historically identified as M. ovis might have fit the description of M. bovoculi sp. nov., and as such, future descriptions of bacteria identified as M. ovis or M. bovoculi sp. nov. should conclusively demonstrate speciation of these isolates. While biochemical testing has traditionally been used to differentiate moraxellae, a determination of the 16S–23S ISR DNA sequence may help to speciate the coccoid moraxellae, especially PADase-negative coccoid moraxellae isolated from cattle with IBK. Future studies on more geographically diverse isolates of M. bovoculi sp. nov. should help elucidate whether additional genetic and biochemical variability exists among isolates of this species. Until additional M. bovoculi sp. nov. are characterized at the molecular level, it may be most informative to use a combination of morphologic, biochemical, and molecular analyses to positively identify Moraxella bovoculi sp. nov.
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Acknowledgments |
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a. ATCC 33078, ATCC 19575, and ATCC 19576, American Type Culture Collection, Manassas, VA.
b. Provided by Lisle W. George, UC Davis, Davis, CA.
c. ATCC 700019, American Type Culture Collection, Manassas, VA.
d. ATCC 700022, American Type Culture Collection, Manassas, VA.
e. Veterinary Medicine Biological Media Services, UC Davis, CA.
f. DNEasy kit, Qiagen, Valencia, CA.
g. Gibco UltraPure molecular biology grade water, Invitrogen, Carlsbad, CA.
h. Choice Taq DNA Polymerase, Denville Scientific Inc., South Plainfield, NJ.
i. 100 mM dNTP Set, Eppendorf, Westbury, NY.
j. PTC-200 Peltier Thermal Cycler, MJ Research, Inc., Watertown, MA.
k. AfaI (RsaI), Amersham Biosciences, Piscataway, NJ.
l. GenePure LE Quick Dissolve Agarose, ISC BioExpress, Kaysville, UT.
n. Dual Light Transilluminator FB-DLT-88A, Fisher Scientific, Pittsburgh, PA.
o. Photodocumentation Handheld Camera, Fisher Scientific, Pittsburgh, PA.
p. Davis Sequencing, Inc., Davis, CA.
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