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Moraxella bovoculi sp. nov., isolated from calves with infectious bovine keratoconjunctivitis

¹ Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
² Section of Evolution and Ecology, University of California, Davis, CA 95616, USA

Correspondence
John A. Angelos
jaangelos{at}ucdavis.edu

	ABSTRACT

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Eighteen isolates of a Gram-negative coccus (strain 237^T) were cultured from the eyes of dairy and beef calves affected with infectious bovine keratoconjunctivitis (IBK; ‘pinkeye’) in northern California, USA, during summer 2002. These isolates had near full-length (1397 bp) 16S rRNA gene sequences that clustered into three groups with 99.9 % sequence similarity. On the basis of 16S rRNA gene sequence, the isolates were most closely associated with Moraxella bovis and Moraxella ovis in clade I of the classical moraxellae. Biochemically, the novel isolates could be distinguished from the other members of the genus Moraxella isolated from animals on the basis of phenylalanine deaminase activity. The results of partial sequence analysis of six housekeeping genes, the 16S–23S rRNA gene interspacer region and partial 23S rRNA gene provide strong support for the inclusion of these isolates in a novel taxon, for which the name Moraxella bovoculi sp. nov. is proposed. The type strain is strain 237^T (=ATCC BAA-1259^T=CCUG 52049^T).

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Moraxella bovoculi sp. nov. isolates 2467, 2473, 6170, 212, 371, 4624, 4773, 4787, 237^T, 317, 4794, 8342, 2471-2, 2470-1, 380, 376, 4786 and 4785 are DQ153081–DQ153098, respectively. Those for Moraxella bovis Tifton I, Moraxella ovis ATCC 33078^T, Moraxella boevrei ATCC 700022^T and Moraxella caprae ATCC 700019^T are DQ647927–DQ647928 and DQ156147–DQ156148, respectively. Further GenBank accession numbers for sequences determined in this study are given in Supplementary Table S1 in IJSEM Online.

The GenBank/EMBL/DDBJ accession numbers for the partial sequences from the six housekeeping genes of the Moraxella bovoculi sp. nov. isolates, M. ovis ATCC 33078^T and M. bovis Tifton I determined in this study are available as a supplementary table in IJSEM online.

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Infectious bovine keratoconjunctivitis (IBK; ‘pinkeye’) is the most common ocular disease of cattle and manifests as corneal ulceration and oedema, ocular pain, photophobia and lacrimation. Animals that recover develop varying degrees of corneal scarring, while permanent blindness follows corneal rupture in severe cases. The only organism for which Koch's postulates have been fulfilled with respect to IBK is Moraxella bovis (Henson & Grumbles, 1960). Nevertheless, other bacteria have been isolated in the absence of M. bovis from cattle with IBK. In 1966, Fairlie first reported the isolation of haemolytic Neisseria spp. from calves with severe keratitis and corneal ulceration (Fairlie, 1966). In Australia, Spradbrow isolated Neisseria spp. in 24 of 25 outbreaks of IBK; M. bovis was isolated in only two of those outbreaks (Spradbrow, 1967). In 1970, Wilcox reported Neisseria (Branhamella) catarrhalis from nearly 45 % of IBK cases while M. bovis was isolated from only 28 % of IBK cases (Wilcox, 1970). M. bovis and bacteria classified as species of the genus Neisseria have also been cultured from normal eyes of cattle (Barber, 1984; Barber et al., 1986). Moraxella ovis and Neisseria ovis were reported from IBK-affected cattle in Israel (Elad et al., 1988; Yeruham et al., 2001). Pederson investigated the pathogenicity of N. ovis inoculated into the eyes of experimental calves, however, lesions typical for IBK did not occur despite previous corneal irradiation (Pedersen, 1972). Haemolytic and cytolytic activity from culture filtrates of M. ovis isolated from cattle with IBK has been reported recently and this suggests a possible role for Gram-negative cocci in the pathogenesis of IBK (Cerny et al., 2006).

During an antibiotic efficacy trial in beef and dairy calves in northern California during summer 2002, swabs from 138 corneal ulcers were cultured from 3- to 9-month-old calves with clinical IBK. Haemolytic Gram-negative cocci were isolated from 68 ulcers; M. bovis was isolated from 29 ulcers. To characterize the haemolytic Gram-negative cocci and determine if they were related to known members of the genus Moraxella, biochemical characterizations and ribosomal/housekeeping gene sequence analyses were performed.

Ocular secretions from IBK-affected calves at two separate calf-rearing facilities (one beef cattle ranch and one dry-lot dairy) in northern California were collected from the inferior cul-de-sac onto sterile Dacron swabs. The swabs were used to inoculate 5 % sheep blood agar plates which were incubated at 35 °C for 24 h. Colonies exhibiting -haemolysis were subcultured for further study; one -haemolytic isolate (2470-1) that exhibited colony morphology similar to the -haemolytic isolates was also selected and subcultured. Isolates were stored frozen in 50 % skim milk–glycerol media at –80 °C for subsequent biochemical and molecular characterizations. The eighteen isolates were designated 212, 237^T, 2467, 2470-1, 2471-2, 2473, 317, 371, 376, 380, 4624, 4773, 4785, 4786, 4787, 4794, 6170, 8342 and were from the ocular secretions of different calves. M. bovis was isolated concurrently with isolate 2471-2 from one calf. The isolates were propagated on 5 % sheep blood agar plates and grown in either Luria–Bertani broth or brain heart infusion (BHI) broth supplemented with 1.5 mM calcium chloride.

Moraxella caprae (ATCC 700019^T), Moraxella boevrei (ATCC 700022^T) and M. ovis (ATCC 33078^T) were obtained from the ATCC. M. bovis Tifton I is a strain of M. bovis that was originally isolated from a beef cow with IBK in Georgia, USA. Genomic DNA was isolated by the use of commercial DNA extraction kits (DNEasy kit; Qiagen). The 16S rRNA gene was amplified using primers NewEcoliDn1 (5'-GTTTGATCATGGCTCAGATTG-3') and EcoliUp4 (5'-TTCCCCTACGGTTACCTTGTT-3'). Ribosomal DNA from the end of the 16S rRNA gene, the 16S–23S interspacer region and a portion of the 23S rRNA genes was amplified with primers MorNeissDn5 (5'-GCTGCATGGCTGTCGTCAGCT-3') and MorRRL1R (5'-GCTTTTCCTGGAAGCAGGGTATT-3'). The PCR conditions consisted of 30 cycles of the following steps: 95 °C for 60 s, 60 °C for 45 s and 72 °C for 60 s. The PCR amplification products were sequenced directly by the UC Davis DNA Sequencing Laboratory or by Davis Sequencing, Inc. Additional primers were developed as needed for the sequencing of both strands. The final complete sequences were determined for both strands from overlapping fragments using DNA sequence analysis software (Sequencher 4.5, Gene Codes; Vector NTI 9.0, Invitrogen). The initial 16S rRNA gene sequence dataset was composed of up to 1556 bp for 27 taxa. Of the sequences generated for the initial analysis, all were similar in length, except for the sequence generated for M. caprae which contained a 103 nt insert (with respect to all other sequences) that was excluded from the phylogenetic analysis.

The oligonucleotide primers and PCR conditions that were used for the amplification of the six housekeeping genes are shown in Table 1. The housekeeping genes used were selected on the basis of previously described bacterial housekeeping genes (Christensen et al., 2004; Lim et al., 2003; Noller et al., 2003). In some instances, the deduced amino acid sequences of the amplicons generated using published housekeeping gene primers did not correspond to the expected protein sequences obtained from BLAST searches of GenBank (Altschul et al., 1997). In these cases, additional primers were designed from the aligned DNA sequence data and used for amplification and resequencing; subsequent BLAST searches of the resequenced amplicons matched the target proteins and were used to represent the housekeeping genes. The five-taxon housekeeping gene dataset was composed of up to 7121 nt. Despite extensive PCR optimizations, four housekeeping genes did not amplify from M. bovis Tifton I genomic DNA.

To determine whether the representative isolates from each of the three groups were significantly different from other recognized species of the genus Moraxella, the following strategy was employed: (i) a BLAST search of GenBank (Altschul et al., 1997) using the 16S rRNA gene sequence from isolate 237^T was performed to identify and select a set of 16S rRNA gene sequences that were most similar to the representative isolates; (ii) phylogenetic analysis was performed on this dataset and M. bovis and M. ovis were identified as the most closely related taxa to the representative isolates, and (iii) a five-taxon dataset composed of the representative isolates of each of the three groups (based on the near full-length 16S rRNA gene sequence), M. bovis and M. ovis was subjected to phylogenetic analysis using the 16S rRNA gene, the 16S–23S interspacer region and a portion of the 23S rRNA gene plus six housekeeping genes (Table 1). For both datasets, sequences were aligned in MacClade 4.06 (Maddison & Maddison, 2003). Missing data were coded as ‘?’ and gaps were coded as ‘–’. Phylogenetic trees were estimated with neighbour-joining (NJ) and maximum-likelihood (ML) using PAUP* 4.0b10 (Swofford, 2002). For the NJ distance metric, uncorrected P distances were used. Maximum-likelihood searches employed tree bisection reconnection (TBR) branch swapping with ten random-stepwise heuristic searches. Model parameter values were estimated using MODELTEST v3.06 PPC (Posada & Crandall, 1998) and selected under the Akaike information criteria. ML parameters conformed to the GTR+I+ model of sequence evolution. The statistical reliability of the NJ and ML trees was assessed using non-parametric bootstrapping (Felsenstein, 1985) with 1000 pseudoreplicates. The BLAST search identified 219 16S rRNA gene sequences, of which only the first 39 were downloaded. Redundant sequences were identified using MacClade 4.06 and these were excluded from the phylogenetic analysis. ‘Psychrobacter meningitidis’ SGHMS (GenBank accession no. AY057116) and Psychrobacter faecalis Iso-46^T (AJ421528) were selected in the BLAST search and included as outgroups. The 16S rRNA gene sequences for Moraxella cuniculi CCUG 2154^T (AF005188), Moraxella osloensis 5873 (AF005190), Moraxella atlantae 1922 (AF005191) and Moraxella phenylpyruvica 752/52 (AF005192) were not identified by the BLAST search and were downloaded from GenBank. The final 16S rRNA gene dataset was composed of 1453 nt from 27 taxa (TreeBASE accession no. S1634).

Based on the 16S rRNA gene sequence analysis, the three representatives of the groups of novel isolates were found to be most closely related to M. ovis and M. bovis; the gene sequence divergence between the novel strains and M. ovis was between 2.2 % and 2.4 %. The gene sequence divergence between the representative novel strains and M. bovis was between 2.1 % and 2.4 % (see Table 2). The ML searches recovered two equally likely trees, but the differences between the topologies were trivial. The NJ tree was not significantly different from the ML tree (Shimodaira-Hasegawa test P=0.376), thus, Fig. 1(a) presents the ML tree with the NJ and ML bootstrap proportions as indicated. The results of phylogenetic analysis were congruent with previously defined phylogroups (Pettersson et al., 1998). The three groups of novel isolates formed a distinct, well-supported monophyletic unit that was most closely related to M. ovis and M. bovis and the novel isolates are therefore members of the M. bovis cluster. Uncorrected P sequence divergence between the novel strains and M. ovis/M. bovis was 1.75 % (Table 3). A closest neighbour (based on near full-length 16S rRNA gene sequence) dataset was composed of up to 7121 nt for five taxa. ML parameters conformed to the TrN+I model of sequence evolution. The ML searches recovered a single tree in which the novel strains were monophyletic with respect to M. bovis and M. ovis with strong support (Fig. 1b) and had >4 % gene sequence divergence from M. bovis/M. ovis (Table 3). In addition, NJ searches of the coding data converted to amino acids recovered the same topology, also with strong support. Based on these results, the 18 novel isolates are considered to represent a novel taxon within the genus Moraxella, for which the name Moraxella bovoculi sp. nov. is proposed.

View larger version (203K): [in this window] [in a new window]   Fig. 2. Transmission electron micrograph of a broth culture-derived bacterial cell pellet of M. bovoculi sp. nov. 237T. The positively stained ultrathin section was prepared following standard fixation/epoxy resin embedding protocols. Bar, 1 µm.

All 18 isolates grow on 5 % sheep blood agar at 25 and 35 °C under aerobic conditions; 13 of 18 isolates grow on 5 % sheep blood agar incubated at 42 °C (type strain 237^T is negative). Seven of 18 isolates grow on nutrient agar with 0 % NaCl (strain 237^T is positive). Bacteria grown on 5 % sheep blood agar form 1 mm diameter colonies that are white to off-white, circular and convex with an entire margin and a moist-looking surface. Seventeen of 18 isolates have a zone of -haemolysis around or under (when picked) each colony (strain 237^T is -haemolytic); isolate 2470-1 exhibits -haemolysis. Gram-negative diplococci with occasional cocci; the cell diameters are 0.7–1.3 µm (Fig. 2) and adjacent sides appear flattened. Growth does not occur on MacConkey agar, Salmonella–Shigella agar, Simmons' citrate agar, sodium acetate agar or nutrient agar with 6 % NaCl. Sixteen of 18 isolates are nitrate-reduction positive (strain 237^T positive) and 9 of 18 isolates show proteolysis of Löffler medium (strain 237^T negative). Fourteen of 18 isolates are positive for leucine arylamidase activity (strain 237^T positive); 9 of 18 isolates are positive for DNase activity (methyl green agar; strain 237^T negative). Seventeen of 18 isolates are positive for hydrolysis of Tween 80 (96 h result; strain 237^T positive). Seventeen of 18 isolates were negative for alkaline phosphatase (strain 237^T negative). All 18 isolates have positive test reactions for the following: penicillin sensitivity, oxidase, lipase (hydrolysis of 5-bromo-3-indoxyl-caprate), catalase, phenylalanine deaminase, C4 esterase (hydrolysis of 2-naphthyl butyrate) and C8 esterase lipase (hydrolysis of 2-naphthyl caprylate). All 18 isolates have negative reactions for motility, Litmus milk reaction, hydrogen sulfide production, indole production from tryptophan, sugar fermentation (glucose, xylose, mannitol, lactose, fructose, maltose and sucrose), aesculin hydrolysis, gelatin hydrolysis, -galactosidase, tests for carbon assimilation (glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, potassium gluconate, caproic acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid), arginine dihydrolase, urease, ornithine decarboxylase, proline arylamidase, -glutamyl transferase, C-14 lipase (hydrolysis of 2-naphthyl myristate), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase.

View larger version (32K): [in this window] [in a new window]   Fig. 1. (a) ML reconstruction of 27 taxa and up to 1453 bp of the 16S rRNA gene. Estimated model parameter values conform to the GTR+I+ model of sequence evolution. –lnL=5513.85. Base frequencies were A=0.26, C=0.21, G=0.31 and T=0.22. Parameters for the substitution rate matrix were A–C=0.7086, A–G=2.7186, A–T=1.5815, C–G=0.6792, C–T=4.0333 and G–T=1. Proportion of invariable sites (I)=0.6757 and -shape parameter=0.5321. GenBank accession numbers are given in parentheses. Bar, 0.01 substitutions per site. (b) Multi-gene analysis of the 16S rRNA gene, 16S–23S interspacer region, partial 23S rRNA gene and six housekeeping genes. ML reconstruction of the 5-taxon 7121 nt dataset. Estimated model parameter values conform to the TrN+I model of sequence evolution. –lnL=12202.0126. Base frequencies were A=0.28, C=0.21, G=0.26 and T=0.25. Parameters for the substitution rate matrix were A–C=1, A–G=3.2058, A–T=1, C–G=1, C–T=6.2479 and G–T=1. Proportion of invariable sites (I)=0.7197. Numbers above branches are ML bootstrap proportions while those below branches are NJ bootstrap proportions. Bar, 0.01 substitutions per site. The GenBank accession numbers for all the sequences generated for this analysis are DQ156147–DQ156148, DQ153081–DQ153098 and DQ647927–DQ647954 (see Supplementary Table S1 in IJSEM Online).

	ACKNOWLEDGEMENTS

 
We gratefully acknowledge the technical assistance of Natalie Tankersley in performing biochemical testing and PCR/DNA sequencing. Funding for completion of this work was provided by a University of California New Faculty Research Grant, the University of California School of Veterinary Medicine Dean's Office Start Up Funds for New Faculty and USDA Formula Funds.

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