Diversity of Moraxella Spp. Strains Recovered from Infectious Bovine Keratoconjunctivitis Cases in Uruguay

Background: Infectious bovine keratoconjunctivitis (IBK) is the most common ocular disease that affects cattle throughout the world and it has a very significant economic impact. IBK is caused by members of the genus Moraxella and therapeutic and preventive measures have shown limited success. Vaccines, most of them chemically inactivated bacterins, generally induce a limited protection. Methodology: In this study, the genetic diversity of Uruguayan clinical Moraxella bovis and Moraxella bovoculi isolates was assessed by RAPD-PCR, ERIC-PCR and BOX-PCR fingerprinting. Also, antibiotic resistance of the Moraxella spp. isolates was assessed utilizing the disk diffusion method. Results: When interspecific molecular diversity was assessed, different bands patterns were observed even within a single outbreak of IBK, showing the coexistence of different genotypes of Moraxella spp. The high genetic diversity within M. bovis and M. bovoculi isolates did not permit to correlate isolates DNA fingerprints with geographical origins, dates or even with both different Moraxella species. Antibiotics resistance patterns showed significant differences between M. bovis and M. bovoculi. Conclusions: This is the first study of diversity that includes M. bovis and M. bovoculi associated to IBK cases. Genetic diversity did not allow to correlate DNA fingerprints of the isolates with geographical origins, isolation dates or even both different Moraxella species. Antibiotics resistance patterns showed differences between M. bovis and M. bovoculi. This remarkable variation within isolates could explain the partial protection induced by commercial vaccines. All these findings could be important for the design of prevention or treatment strategies against IBK.


Introduction
Infectious bovine keratoconjunctivitis (IBK), commonly known as pink eye, is the most common ocular disease of cattle including symptoms like corneal ulceration and edema, blepharospasm, photophobia and lacrimation.Its economic impact is significant since prevention and treatment strategies are often ineffective [1].
Moraxella bovis has been traditionally recognized as the etiologic agent of IBK.However, the recently characterized species Moraxella bovoculi, has been recovered from corneal ulcers of beef and dairy calves in USA [2] and in Uruguay [3].
Many vaccines available nowadays contain mainly different strains of M. bovis.Although these vaccines have shown partial protection, they are not completely effective.This fact may be due to intraespecific diversity of M. bovis [4].This irregular protection conferred by vaccines against IBK has led several authors to investigate the biology of the etiologic agents associated with the disease.Several features related to the biology of M. bovis have been studied, including analyses of outer membrane proteins (OMP), lipopolysaccharides (LPS), pili and cytotoxin or antibiotic susceptibility [4][5][6][7].
Phenotypic typing based on expression of cellular characteristics may vary according to culture or experimental conditions; moreover, these methods involve time-consuming processes [8][9][10].The relatively recent development of molecular techniques to assess bacterial genetic diversity has introduced the possibility to characterize genetic differences among isolates.However, reports regarding genetic typing of M. bovis and M. bovoculi collections are scarce.
Rep-PCR is a molecular biology based method suitable for the grouping of microorganisms.This technique is based on DNA amplification and has been usually considered as an extremely reliable, reproducible, rapid and highly discriminatory method [11].Rep-PCR genomic fingerprinting is based on the use of DNA primers complementary to naturally occurring, highly conserved, repetitive DNA sequences, present in multiple copies in the genomes of most microorganisms [12].Three families of repetitive sequences have been identified, including the enterobacterial repetitive intergenic consensus (ERIC) sequence, and the BOX element [11].The repetitive elements may be present in both orientations, and oligonucleotide primers have been designed to prime DNA synthesis outward from the inverted repeats in ERIC, and from the boxA subunit of BOX, in the PCR [11].The use of these primers and PCR lead to the selective amplification of distinct genomic regions located between ERIC or BOX elements.
Randomly Amplified Polymorphic DNA (RAPD) has been also frequently used to assess genetic diversity among isolates.Only one primer is used in RAPD analysis, its sequence is arbitrarily chosen and sequence knowledge of the genomic DNA is not required.This results in a number of anonymous, not previously determined, but reproducibly amplified fragments [13].
So far, RAPD and ERIC fingerprint have been used to study differences between small numbers of only M. bovis strains in studies focused on IBK epidemiology [4,14].
The main objective of the present study was to evaluate the genetic diversity of a wide collection of M. bovis and M. bovoculi isolates recovered from outbreaks of IBK between 1983 and 2009 in Uruguay using RAPD-PCR, ERIC-PCR and BOX-PCR.Also, antibiotic susceptibility of the strains was assessed in order to add information about diversity of Moraxella spp.

Bacterial strains, media and growth conditions
Fifty four Moraxella spp.strains comprising forty five Uruguayan field isolates ( 1).
All isolates had been identified and characterized in a previous study [3].
Eight μl of each PCR reaction mixture were analyzed by gel electrophoresis in a 0.8 % agarose gel and stained with ethidium bromide.A 1 kb DNA ladder (Fermentas Thermo Scientific, Waltham, USA) was used to determine molecular size.The gels were photographed under UV light record the results.

Analysis of genetic diversity
The DNA fingerprints obtained by RAPD, ERIC and BOX-PCR reactions were firstly visually analyzed.A positive response (a score '1') was defined as the presence of a visible band of a given size, while a negative response (a score '0') was defined as the absence of any band of the same size.These scores were then merged in a Microsoft Excel spreadsheet and then inserted in the PAST Version 2.1 computer software for the construction of a dendogram using Paired Group to determine the relatedness of the Moraxella spp.isolates.Fingerprinting pattern types were considered to be distinct if they differed by more than one band.

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resistance against antimicrobial drugs, Moraxella spp.strains were just considered resistant when growth was not at all inhibited.To assess differences in antibiotic resistance associated with M. bovis and M. bovoculi the chisquare test was used.

DNA-fingerprints of Moraxella spp. strains
The DNA fingerprints of Moraxella spp.isolates generated by RAPD, ERIC and BOX-PCR were complex.In general, fingerprint patterns obtained with RAPD, ERIC and BOX-PCR were very diverse among the collection of isolates.As a representative example, Figure 1 shows the ERIC-PCR fingerprinting patterns of the strains used in this study.
The number of polymorphic bands went from 1 to 12 bands in fingerprints obtained by RAPD, ranging between 5000 and 250 bp for primer JWP1 and 6000 to 250 pb for primer JWOPA7.
In the case of ERIC-PCR, the numbers of generated bands were among 3 and 20 ranging between about 250 bp and up to 5000 bp.Patterns of bands obtained by BOX-PCR 5 and 21 bands ranging from approximately 250 bp up to 6000 bp.
DNA fingerprint for M. bovis and M. bovoculi isolates generated with two RAPD primers (JWP1 and JWOPA7) showed different patterns between isolates except a few that had the same profile.In the case of primer JWOPA7 the isolates that showed the same profile were Fs327/Fs328 and EV121/EV345, obtained from the same IBK outbreak, respectively.When the diversity was analyzed using the JWP1 primer, CANIIIA and CANIIIB also obtained from the same IBK outbreak, showed an identical bands pattern.
SJ01 and SJ02 were the only isolates that showed the same ERIC-PCR bands patterns.When BOX-  PCR was used, the only isolates the showed the same pattern were EV345 and EV366.

Genetic diversity
The genetic relationships among electrophoretic types were evaluated with the unweighted-pair group method using average linkages (UPGMA) were represented by a dendogram.The discriminatory index was calculated using the Rho coefficient (Figure 2).The results showed that exist a high degree of genotypic diversity among Moraxella spp.isolates recovered from IBK cases.

Antimicrobial resistance
Resistance to different antimicrobial agents was assessed using the agar disk diffusion technique (Table 2).As it was stated above, an isolate was considered resistant when growth was not at all inhibited around the disks containing the drug.
Most of the isolates were non resistant to the majority of the antibiotics used.However, significant differences were observed between M. bovis and M. bovoculi in the pattern of resistance to cloxacillin, cotrimoxazole and tetracycline (P = 6.6x10 -4 , P = 0.008 and P = 0.008, respectively).In all cases, M. bovis showed the higher number of resistant isolates compared to M. bovoculi.

Discussion
The diversity of a wide group of Moraxella spp.strains collected since 1983 to 2009 from different geographical locations of Uruguay was analyzed by DNA fingerprint.Resistance against a series of commonly used antibiotics was also assessed.
When the genetic diversity of the isolates was analyzed using RAPD-PCR, a high heterogeneity of bands patterns was observed.This result was similar to that obtained by Conceição et al. [14] when a limited collection of clinical M. bovis isolates obtained from Argentina, Brazil and Uruguay was analyzed by the same technique.
In this study, we found that both ERIC-and BOX-like sequences are present in the genomes of M. bovis and M. bovoculi.When our collection of isolates was analyzed by ERIC-PCR, results were similar to those obtained by Prieto et al. [4] who evaluated intra-specific diversity within a limited number of Argentinian M. bovis strains using ERIC-PCR and obtaining a very high degree of intraspecific heterogeneity.
In our study, BOX-PCR was used for the first time for the typing of M. bovis and M. bovoculi strains.Our results indicated that BOX-PCR allowed the typing of the different isolates although intraspecific heterogeneity was remarkably high so it was not possible to correlate bands profiles and characteristic of isolates.This result was similar to those obtained in the cases of RAPD-and ERIC-PCR.
In general, our results indicated that there were no obvious relationships between geographical and/or temporal origin or even species of isolates and fingerprint profiles.It can be stated that in general, RAPD, ERIC and BOX-PCR produced complex DNA fingerprint patterns for all of the isolates tested showing a high discriminatory power for the typing of Moraxella spp.isolates.This is the first report that describes the genotypic diversity of M. bovoculi strains associated to IBK using DNA fingerprints.In a recent study, we were able to differentiate M. bovis and M. bovoculi using the sequences of ribosomal RNA genes [3].However, in the present study we were unable to differentiate both species at a genetic level through fingerprint patterns.
Results obtained in this study showed that most of the strains were susceptible to the antibiotics used in the treatment of IBK [20].Another interesting finding was that the antibiotic resistance profile differed between M. bovis and M. bovoculi.M. bovis strains presented a significant higher resistance against cloxacillin, cotrimoxazole and tetracycline compared to M. bovoculi.Although Angelos et al. [21] found a 3.5% of resistant M. bovoculi to oxytetracycline, in our study all the isolates were sensitive against this antibiotic.The difference observed in resistant pattern against antibiotics between M. bovis and M. bovoculi may have implications in the pathogenicity and the treatment of IBK.

Conclusions
Results of this study confirm the high genotypic diversity within M. bovis and M. bovoculi isolates, which can be a serious problem for the design of effective vaccines against IBK.Additional research is required to evaluate the diversity within both species of Moraxella and its relevance for the control of IBK.

Figure 1 .
Figure 1.ERIC-PCR fingerprints of Moraxella spp.isolates used in the present study.

Table 1 .
Moraxella spp.strains used in the present study.