- Email: [email protected]
Molecular and phenotypic analysis of Moraxella spp. associated with infectious bovine keratoconjunctivitis in Uruguay
The Veterinary Journal 193 (2012) 595–597
Contents lists available at SciVerse ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
Molecular and phenotypic analysis of Moraxella spp. associated with infectious bovine keratoconjunctivitis in Uruguay Vanessa Sosa, Pablo Zunino ⇑ Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avenida Italia 3318, Montevideo, Uruguay
a r t i c l e
i n f o
Article history: Accepted 6 January 2012
Keywords: Infectious bovine keratoconjunctivitis (IBK) Moraxella bovis Moraxella bovoculi
a b s t r a c t Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease of cattle, which is generally thought to be caused by Moraxella bovis. However, a recently characterized Moraxella, M. bovoculi, has been isolated from animals with IBK. The aim of this study was to identify and characterize strains of Moraxella spp. obtained from IBK cases in different geographic locations within Uruguay. Ribosomal gene sequencing indicated that there were two groups of isolates that showed homology with either M. bovis or M. bovoculi. Phylogenetic analysis confirmed the presence of two species as the isolates grouped in different branches of the dendrogram. Conventional biochemical characterization did not distinguish between the species; only 9/25 isolates which had genetic homology with M. bovoculi showed any differences in biochemistry. Ó 2012 Elsevier Ltd. All rights reserved.
Infectious bovine keratoconjunctivitis (IBK) is the most important ocular disease of cattle. For decades Moraxella bovis has been identified as the key etiological agent (Henson and Grumbles, 1960), although Moraxella ovis has also been isolated from cases of IBK and linked it to its pathogenesis (Elad et al., 1988; Cerny et al., 2006). In 2007, a novel species, Moraxella bovoculi, was identified from corneal ulcers of cattle in the USA (Angelos et al., 2007). M. bovoculi was initially distinguished from M. bovis and M. ovis on the basis of phenylalanine deaminase and gelatinase activity (Angelos et al., 2007), although the former was subsequently found not to be a consistent feature of M. bovoculi (Angelos and Ball, 2007). The results of partial sequence analysis of housekeeping genes, the 16S–23S rRNA gene interspacer region (ITS) and partial 23S-rRNA gene provided strong support for this novel taxon. However at present there is only limited information on M. bovoculi, all of which has come from the USA (Angelos et al., 2007, 2011; Galvão and Angelos, 2010). The aim of this work was to identify and characterize Moraxella spp. from the eyes of cattle with IBK symptoms in Uruguay and to investigate the phylogeny of these strains. Ocular secretions from IBK-affected calves were inoculated onto blood agar. Colonies exhibiting b-hemolysis were selected. Fortyeight possible isolates of Moraxella spp. obtained from several Uruguayan provinces between 1982 and 2009 were used. Six isolates from IBK cases in USA, kindly provided by Dr. Fenwick (Kansas State University), were also included. Reference strains of M. bovis, M. bovoculi and M. ovis were obtained from ATCC. Media
⇑ Corresponding author. Tel.: +598 24871616. E-mail address: [email protected] (P. Zunino). 1090-0233/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2012.01.011
were obtained from Difco Laboratories, chemicals were reagent grade (Sigma) and ovine erythrocytes from Biokey Laboratory. Gram stains were done on fresh cultures and standard biochemical tests performed (Smibert and Krieg, 1994). Genomic DNA was isolated using a genomic DNA extraction kit (Sigma). PCR was performed using 0.2 mM dNTPs, 5 lM of each primer, 2 lL DNA, 1U Taq DNA polymerase (Invitrogen). Cycling reactions were 30 cycles of 1 min to 93 °C, 20 s to 60 °C and 1 min to 72 °C. The 16S-rRNA gene was amplified using universal primers for bacteria 27f (50 -AGAGTTTGATC(AC)TGGCTCAG-30 ) and 1492r (50 -ACGG(CT)TACCTTGTTACGACTT-30 ). DNA of the first seven strains which 16S-rDNA sequences showed homology with M. bovoculi was used for additional sequencing of 16S-ITS-23S rDNA. These sequences were amplified with MorNeissDn5 (50 -GCTGCATGGCT GTCGTCAGCT-30 ) and MorRRL1R (50 -GCTTTTCCTGGAAGCAGGGTATT-30 ) (Angelos et al., 2007). DNA sequences were aligned using Clustal X2 and Neighbor-Joining distance trees were constructed using MEGA software. All isolates grew aerobically at 37 °C. Cell morphology corresponded to Gram-negative diplococci with flattened adjacent sides. All isolates showed positive test reactions for catalase and oxidase and exhibited negative reactions for growth on MacConkey, motility, indole production from tryptophan, sugar fermentation (fructose), DNAse, urease and phenylalanine deaminase activity, as expected for M. bovis. However, nine isolates were unable to hydrolyze gelatin, consistent with M. bovoculi or M. ovis but not M. bovis (Angelos et al., 2007). All isolates were phenylalanine deaminase-negative. These results obtained in this study support the finding of Angelos and Ball (2007) in that the biochemical tests do not unequivocally distinguish between these Moraxella spp.
596
V. Sosa, P. Zunino / The Veterinary Journal 193 (2012) 595–597
Table 1 Molecular identification of isolates by 16S-ITS-23S rDNA sequencing. Isolates
GenBank accession number
Description (strain)
Score
Query coverage (%)
Maximum identification (%)
2419 2358 3346-II BM3B SES SJ03A TT3
DQ153089.1 DQ153089.1 DQ153089.1 DQ153089.1 DQ153089.1 DQ153089.1 DQ153089.1
M. M. M. M. M. M. M.
1386 1369 1380 1373 1384 1386 1386
100 99 100 100 100 100 100
100 99 99 99 100 100 100
bovoculi bovoculi bovoculi bovoculi bovoculi bovoculi bovoculi
(3709) (3709) (3709) (3709) (3709) (3709) (3709)
Fig. 1. Molecular phylogenetic analysis using the Maximum Likelihood method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the phylogenetic relationships of the taxa analyzed.
V. Sosa, P. Zunino / The Veterinary Journal 193 (2012) 595–597
Using primers 27f and 1492r, an approximately 1500 bp product was amplified. These products were sequenced and the initial sequence identification was done by BLAST1 (Supplementary Table S1). The sequencing of 16S-rDNA indicated that 23 sequences were similar to published sequences of M. bovis while 25 showed homology with M. bovoculi sequences. The nine isolates that could not hydrolyze gelatin all showed homology with M. bovoculi. No isolate showed homology with M. ovis. These results were confirmed by sequencing ribosomal DNA from 16S-ITS-23S, as recommended by Angelos and Ball (2007) (Table 1). All strains corresponded to individual animals except isolates BM3A and BM3B which were recovered from different eyes of the same animal. A phylogenetic tree based on the 16S-rDNA gene sequence homologies was constructed (Fig. 1). M. bovis and M. bovoculi isolates showed differences between their sequences and grouped in different branches of the dendrogram, both were clearly differentiable from the M. ovis reference strain. The samples used in this study were obtained and submitted by veterinarians at different clinical stages of the disease. No relationship between reported clinical signs and species was observed, nor was there any association with the number of infected animals or evolution of the disease. This is the first report of M. bovoculi isolation from IBK cases outside of the USA. As the species was identified across Uruguay from isolates collected as long ago as 1983, it is likely that this species has been circulating in Uruguayan cattle for many years but been misidentified, consistent with the suggestion made by Angelos (2010) for the USA. Our study s also demonstrated that M. bovoculi may be an important cause of IBK in Uruguayan cattle, and confirmed that sequencing ribosomal genes is necessary to differentiate M. bovis from M. bovoculi. Further research is required to establish the impact that these findings will have on IBK control, particularly vaccination. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
1
See: http://blast.ncbi.nlm.nih.gov/.
597
Acknowledgements This work was supported by Laboratorios Santa Elena, DICYT (Jóvenes en el Sector Productivo 02/06, Uruguay), PEDECIBA (Uruguay) and Agencia Nacional de Investigación e Innovación (ANII, Uruguay). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tvjl.2012.01.011. References Angelos, J.A., 2010. Moraxella bovoculi and infectious bovine keratoconjunctivitis: Cause or coincidence? Veterinary Clinics of North America – Food Animal Practice 26, 73–78. Angelos, J.A., Ball, L.M., 2007. Differentiation of Moraxella bovoculi sp. nov. from other coccoid Moraxella by the use of polymerase chain reaction and restriction endonuclease analysis of amplified DNA. Journal of Veterinary Diagnostic Investigation 19, 532–534. Angelos, J.A., Spinks, P.Q., Ball, L.M., George, L.W., 2007. Moraxella bovoculi sp. nov., isolated from calves with infectious bovine keratoconjunctivitis. International Journal of Systematic and Evolutionary Microbiology 57, 789–795. Angelos, J.A., Ball, L.M., Byrne, B.A., 2011. Minimum inhibitory concentrations of selected antimicrobial agents for Moraxella bovoculi associated with infectious bovine keratoconjunctivitis. Journal of Veterinary Diagnostic Investigation 23, 552–555. Cerny, H.E., Rogers, D.G., Gray, J.T., Smith, D.R., Hinkley, S., 2006. Effects of Moraxella (Branhamella) ovis culture filtrates on bovine erythrocytes, peripheral mononuclear cells, and corneal epithelial cells. Journal of Clinical Microbiology 44, 772–776. Elad, D., Yeruham, I., Bernstein, M., 1988. Moraxella ovis in cases of infectious bovine keratoconjunctivitis (IBK) in Israel. Zentralblatt für Veterinärmedizin 35, 431– 434. Galvão, K.N., Angelos, J.A., 2010. Ulcerative blepharitis and conjunctivitis in adult dairy cows and association with Moraxella bovoculi. Canadian Veterinary Journal 51, 400–402. Henson, J.B., Grumbles, L.C., 1960. Infectious bovine keratoconjunctivitis. II. Susceptibility of laboratory animals to Moraxella (Hemophilus) bovis. Cornell Veterinarian 50, 445–458. Smibert, R.M., Krieg, N.R., 1994. Phenotypic characterization. In: Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. (Eds.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, DC, USA, pp. 607–654.
Contents lists available at SciVerse ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short Communication
Molecular and phenotypic analysis of Moraxella spp. associated with infectious bovine keratoconjunctivitis in Uruguay Vanessa Sosa, Pablo Zunino ⇑ Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avenida Italia 3318, Montevideo, Uruguay
a r t i c l e
i n f o
Article history: Accepted 6 January 2012
Keywords: Infectious bovine keratoconjunctivitis (IBK) Moraxella bovis Moraxella bovoculi
a b s t r a c t Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease of cattle, which is generally thought to be caused by Moraxella bovis. However, a recently characterized Moraxella, M. bovoculi, has been isolated from animals with IBK. The aim of this study was to identify and characterize strains of Moraxella spp. obtained from IBK cases in different geographic locations within Uruguay. Ribosomal gene sequencing indicated that there were two groups of isolates that showed homology with either M. bovis or M. bovoculi. Phylogenetic analysis confirmed the presence of two species as the isolates grouped in different branches of the dendrogram. Conventional biochemical characterization did not distinguish between the species; only 9/25 isolates which had genetic homology with M. bovoculi showed any differences in biochemistry. Ó 2012 Elsevier Ltd. All rights reserved.
Infectious bovine keratoconjunctivitis (IBK) is the most important ocular disease of cattle. For decades Moraxella bovis has been identified as the key etiological agent (Henson and Grumbles, 1960), although Moraxella ovis has also been isolated from cases of IBK and linked it to its pathogenesis (Elad et al., 1988; Cerny et al., 2006). In 2007, a novel species, Moraxella bovoculi, was identified from corneal ulcers of cattle in the USA (Angelos et al., 2007). M. bovoculi was initially distinguished from M. bovis and M. ovis on the basis of phenylalanine deaminase and gelatinase activity (Angelos et al., 2007), although the former was subsequently found not to be a consistent feature of M. bovoculi (Angelos and Ball, 2007). The results of partial sequence analysis of housekeeping genes, the 16S–23S rRNA gene interspacer region (ITS) and partial 23S-rRNA gene provided strong support for this novel taxon. However at present there is only limited information on M. bovoculi, all of which has come from the USA (Angelos et al., 2007, 2011; Galvão and Angelos, 2010). The aim of this work was to identify and characterize Moraxella spp. from the eyes of cattle with IBK symptoms in Uruguay and to investigate the phylogeny of these strains. Ocular secretions from IBK-affected calves were inoculated onto blood agar. Colonies exhibiting b-hemolysis were selected. Fortyeight possible isolates of Moraxella spp. obtained from several Uruguayan provinces between 1982 and 2009 were used. Six isolates from IBK cases in USA, kindly provided by Dr. Fenwick (Kansas State University), were also included. Reference strains of M. bovis, M. bovoculi and M. ovis were obtained from ATCC. Media
⇑ Corresponding author. Tel.: +598 24871616. E-mail address: [email protected] (P. Zunino). 1090-0233/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2012.01.011
were obtained from Difco Laboratories, chemicals were reagent grade (Sigma) and ovine erythrocytes from Biokey Laboratory. Gram stains were done on fresh cultures and standard biochemical tests performed (Smibert and Krieg, 1994). Genomic DNA was isolated using a genomic DNA extraction kit (Sigma). PCR was performed using 0.2 mM dNTPs, 5 lM of each primer, 2 lL DNA, 1U Taq DNA polymerase (Invitrogen). Cycling reactions were 30 cycles of 1 min to 93 °C, 20 s to 60 °C and 1 min to 72 °C. The 16S-rRNA gene was amplified using universal primers for bacteria 27f (50 -AGAGTTTGATC(AC)TGGCTCAG-30 ) and 1492r (50 -ACGG(CT)TACCTTGTTACGACTT-30 ). DNA of the first seven strains which 16S-rDNA sequences showed homology with M. bovoculi was used for additional sequencing of 16S-ITS-23S rDNA. These sequences were amplified with MorNeissDn5 (50 -GCTGCATGGCT GTCGTCAGCT-30 ) and MorRRL1R (50 -GCTTTTCCTGGAAGCAGGGTATT-30 ) (Angelos et al., 2007). DNA sequences were aligned using Clustal X2 and Neighbor-Joining distance trees were constructed using MEGA software. All isolates grew aerobically at 37 °C. Cell morphology corresponded to Gram-negative diplococci with flattened adjacent sides. All isolates showed positive test reactions for catalase and oxidase and exhibited negative reactions for growth on MacConkey, motility, indole production from tryptophan, sugar fermentation (fructose), DNAse, urease and phenylalanine deaminase activity, as expected for M. bovis. However, nine isolates were unable to hydrolyze gelatin, consistent with M. bovoculi or M. ovis but not M. bovis (Angelos et al., 2007). All isolates were phenylalanine deaminase-negative. These results obtained in this study support the finding of Angelos and Ball (2007) in that the biochemical tests do not unequivocally distinguish between these Moraxella spp.
596
V. Sosa, P. Zunino / The Veterinary Journal 193 (2012) 595–597
Table 1 Molecular identification of isolates by 16S-ITS-23S rDNA sequencing. Isolates
GenBank accession number
Description (strain)
Score
Query coverage (%)
Maximum identification (%)
2419 2358 3346-II BM3B SES SJ03A TT3
DQ153089.1 DQ153089.1 DQ153089.1 DQ153089.1 DQ153089.1 DQ153089.1 DQ153089.1
M. M. M. M. M. M. M.
1386 1369 1380 1373 1384 1386 1386
100 99 100 100 100 100 100
100 99 99 99 100 100 100
bovoculi bovoculi bovoculi bovoculi bovoculi bovoculi bovoculi
(3709) (3709) (3709) (3709) (3709) (3709) (3709)
Fig. 1. Molecular phylogenetic analysis using the Maximum Likelihood method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the phylogenetic relationships of the taxa analyzed.
V. Sosa, P. Zunino / The Veterinary Journal 193 (2012) 595–597
Using primers 27f and 1492r, an approximately 1500 bp product was amplified. These products were sequenced and the initial sequence identification was done by BLAST1 (Supplementary Table S1). The sequencing of 16S-rDNA indicated that 23 sequences were similar to published sequences of M. bovis while 25 showed homology with M. bovoculi sequences. The nine isolates that could not hydrolyze gelatin all showed homology with M. bovoculi. No isolate showed homology with M. ovis. These results were confirmed by sequencing ribosomal DNA from 16S-ITS-23S, as recommended by Angelos and Ball (2007) (Table 1). All strains corresponded to individual animals except isolates BM3A and BM3B which were recovered from different eyes of the same animal. A phylogenetic tree based on the 16S-rDNA gene sequence homologies was constructed (Fig. 1). M. bovis and M. bovoculi isolates showed differences between their sequences and grouped in different branches of the dendrogram, both were clearly differentiable from the M. ovis reference strain. The samples used in this study were obtained and submitted by veterinarians at different clinical stages of the disease. No relationship between reported clinical signs and species was observed, nor was there any association with the number of infected animals or evolution of the disease. This is the first report of M. bovoculi isolation from IBK cases outside of the USA. As the species was identified across Uruguay from isolates collected as long ago as 1983, it is likely that this species has been circulating in Uruguayan cattle for many years but been misidentified, consistent with the suggestion made by Angelos (2010) for the USA. Our study s also demonstrated that M. bovoculi may be an important cause of IBK in Uruguayan cattle, and confirmed that sequencing ribosomal genes is necessary to differentiate M. bovis from M. bovoculi. Further research is required to establish the impact that these findings will have on IBK control, particularly vaccination. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
1
See: http://blast.ncbi.nlm.nih.gov/.
597
Acknowledgements This work was supported by Laboratorios Santa Elena, DICYT (Jóvenes en el Sector Productivo 02/06, Uruguay), PEDECIBA (Uruguay) and Agencia Nacional de Investigación e Innovación (ANII, Uruguay). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tvjl.2012.01.011. References Angelos, J.A., 2010. Moraxella bovoculi and infectious bovine keratoconjunctivitis: Cause or coincidence? Veterinary Clinics of North America – Food Animal Practice 26, 73–78. Angelos, J.A., Ball, L.M., 2007. Differentiation of Moraxella bovoculi sp. nov. from other coccoid Moraxella by the use of polymerase chain reaction and restriction endonuclease analysis of amplified DNA. Journal of Veterinary Diagnostic Investigation 19, 532–534. Angelos, J.A., Spinks, P.Q., Ball, L.M., George, L.W., 2007. Moraxella bovoculi sp. nov., isolated from calves with infectious bovine keratoconjunctivitis. International Journal of Systematic and Evolutionary Microbiology 57, 789–795. Angelos, J.A., Ball, L.M., Byrne, B.A., 2011. Minimum inhibitory concentrations of selected antimicrobial agents for Moraxella bovoculi associated with infectious bovine keratoconjunctivitis. Journal of Veterinary Diagnostic Investigation 23, 552–555. Cerny, H.E., Rogers, D.G., Gray, J.T., Smith, D.R., Hinkley, S., 2006. Effects of Moraxella (Branhamella) ovis culture filtrates on bovine erythrocytes, peripheral mononuclear cells, and corneal epithelial cells. Journal of Clinical Microbiology 44, 772–776. Elad, D., Yeruham, I., Bernstein, M., 1988. Moraxella ovis in cases of infectious bovine keratoconjunctivitis (IBK) in Israel. Zentralblatt für Veterinärmedizin 35, 431– 434. Galvão, K.N., Angelos, J.A., 2010. Ulcerative blepharitis and conjunctivitis in adult dairy cows and association with Moraxella bovoculi. Canadian Veterinary Journal 51, 400–402. Henson, J.B., Grumbles, L.C., 1960. Infectious bovine keratoconjunctivitis. II. Susceptibility of laboratory animals to Moraxella (Hemophilus) bovis. Cornell Veterinarian 50, 445–458. Smibert, R.M., Krieg, N.R., 1994. Phenotypic characterization. In: Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. (Eds.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, DC, USA, pp. 607–654.