Genetic differences among Moraxella bovis and Moraxella bovoculi isolates from infectious bovine keratoconjunctivitis (IBK) outbreaks in southern Brazil

Abstract

The objective of this study was to evaluate the genetic diversity of Moraxella bovis and Moraxella bovoculi bacteria isolated from infectious bovine keratoconjunctivitis (IBK) outbreaks in the state of Rio Grande do Sul, Brazil. The genetic diversity among Moraxella spp. was evaluated by RAPD-PCR, JWP1-JWOPA07-PCR, ERIC-PCR and by sequencing the 16S-23S intergenic regions. Based on the dendrogram, two genetically differentiated clades were observed; 14 isolates were classified as M. bovis and 17 as M. bovoculi. Genetic distances between the M. bovis samples ranged from 0.0379 to 0.4285, while for M. bovoculi the dissimilarities ranged from zero to 0.7297. Alternatively, based on sequencing analyses of the 16S-23S intergenic region, M. bovis and M. bovoculi isolates were grouped into the same two different clades, but it was not possible to differentiate between isolates within clades. PCR techniques were demonstrated to be a satisfactory tool to unravel the genetic variability among Moraxella spp., while sequencing of the 16S-23S intergenic region was only able to differentiate two species of the Moraxella genus. Despite sampling geographically close regions, we demonstrate considerable genetic diversity in M. bovis and M. bovoculi strains and genetically distinct M. bovis strains co-infecting the same animal.

Keywords:
Beef cattle; DNA sequencing; genetic diversity; RAPD

Infectious bovine keratoconjunctivitis (IBK) is the most important ocular disease in bovine production (Snowder et al., 2005). The main observed symptoms are: intense lacrimation, photophobia, conjunctival swelling, opacity in the center of the cornea and ulceration (Postma et al., 2008). Despite its low mortality, this disease is characterized by high morbidity, as it can affect up to 80% of the herd (Postma et al., 2008). It causes significant economic and productive losses due to the reduction in weight gain, decrease in milk production, expenses with repetitive treatments, commercial devaluation, and eventual discard of animals that present severe and permanent ocular sequelae (George, 1990; McConnel et al., 2007). There are no estimates of the economic impact of this disease in the last 20 years.

The etiologic agent of IBK is Moraxella bovis, a Gram-negative bacterium, widely disseminated and highly contagious (McConnel et al., 2007). Another Moraxella species, M. bovoculi, has been frequently isolated from ocular and nasal secretions of animals affected by IBK (Angelos et al., 2007; Sosa and Zunino, 2012; Karthik et al., 2018). However, the role of this species as IBK causal agent is not fully defined, since the experimental infection with this species failed to provoke the disease (Angelos, 2015).

IBK was diagnosed in most Brazilian states and is widely disseminated in the southern region of Rio Grande do Sul state (Turnes, 2001), mainly affecting taurine bovines (Conceição and Turnes, 2003). It is a seasonal disease, with a worldwide distribution and, generally, highly prevalent (McConnel et al., 2007). It affects animals of all ages, but young animals are generally more susceptible (Killinger, 1977).

Therapeutic and prophylactic conduct against IBK includes the use of antimicrobials and vaccines. However, prophylaxis is generally hampered by low vaccine efficacy (O’Connor et al., 2011; Cullen et al., 2017; O’Connor et al., 2019). This situation can be explained by the genetic and antigenic variation between M. bovis strains and the presumed presence of other microorganisms involved in this disease (Brown et al., 1998; Kowalski et al., 2017).

The development of molecular techniques allowed the characterization of genetic differences among bacterial isolates. Bacterial genetic variability can be estimated by comparing differently sized DNA fragments, generated from PCR amplification with the use of random primers (Saiki et al., 1988), or by sequencing the internal transcribed spacer (ITS) (Cieslinska et al., 2015; Tokajian et al., 2016).

The main DNA fingerprinting techniques to assess genetic variability among bacteria are PCR-based ones, such as random amplification of polymorphic DNA (RAPD), and repetitive enterobacterial intergenic consensus (ERIC)-PCR. The PCR-based techniques require no specific knowledge of the DNA sequence of the target organism, resulting in several anonymous, not previously determined, but reproducibly amplified fragments (Bowditch et al., 1993). RAPD is based on randomic amplification of DNA with decamer primers (Shekhawat et al., 2019). ERIC-PCR is similar to RAPD, but with larger primers (Hulton et al., 1991). These techniques were successfully used to verify the genetic variability between M. bovis and M. bovoculi strains in studies focused on the epidemiology of IBK (Prieto et al., 1999; Conceição et al., 2004; Sosa and Zunino, 2013). On the other hand, DNA sequencing of ITS is valuable for species identification (Cieslinska et al., 2015; Tokajian et al., 2016), due to the high variability in this region. Additionally, it can be used to trace inter- and intraspecific genetic variability (Spacov et al., 2006).

The objective of the present study was to characterize the genetic diversity of M. bovis and M. bovoculi isolates from IBK outbreaks between 2015 and 2017 from five municipalities in Rio Grande do Sul, Brazil, by RAPD-PCR, JWP1-JWOPA07-PCR, ERIC-PCR, and sequencing the ITS.

The study was approved by Ethics Committee on Animal Experimentation of the Federal University of Pelotas (approval number 2328-2017, approved at 02 October 2017). Five non-vaccinated herds, with most of the cows displaying signs of IBK at the time of collection were enrolled in the study. Around 18 animals per herd were sampled, although the number of animals in each herd was 120 on average. Four samples per animal were collected, totalizing 360 samples, from 90 cattle with initial clinical symptoms of IBK. Swabs from both eyes and nostrils were collected, immediately seeded on blood agar, incubated at 37 °C, and 24 h later, small, white and β-hemolytic colonies were re-seed on blood agar for further characterization. From 360 samples, 28 Moraxella spp. were isolated and characterized as M. bovis and M. bovoculi by PCR-RFLP, according to Angelos and Ball (2007). Additionally, three other strains, previously isolated and identified as M. bovis and M. bovoculi (Taim-1; Taim-2 and Taim-3) were added to the study. All samples were collected in Rio Grande do Sul state, Brazil, totalizing 31 samples (Table 1).

Table 1
Molecular characteristics of Moraxella spp. isolated from IBK, in five municipalities of Rio Grande do Sul state, Brazil.

N°	Isolates	Origin	Surge	Homology
1	151 OE	Alegrete	2016	M. bovis
2	Taim – 2	Rio Grande	2016	M. bovis
3	171 OD	Alegrete	2016	M. bovis
4	171 OE	Alegrete	2016	M. bovis
5	324 OE	Alegrete	2016	M. bovis
6	6213 OD	Dom Pedrito	2016	M. bovis
7	391 OD	Alegrete	2017	M. bovis
8	391 OE	Alegrete	2017	M. bovis
9	976 OD	Alegrete	2016	M. bovis
10	128 OE	Alegrete	2016	M. bovis
11	6587 ND	Dom Pedrito	2017	M. bovis
12	6587 OD	Dom Pedrito	2017	M. bovis
13	6219 OD	Dom Pedrito	2016	M. bovis
14	6456 OE	Dom Pedrito	2016	M. bovis
15	666 OD	Alegrete	2016	M. bovoculi
16	Taim – 1	Rio Grande	2016	M. bovoculi
17	Taim – 3	Santa Vitória do Palmar	2016	M. bovoculi
18	6052 OE	Dom Pedrito	2015	M. bovoculi
19	1368 OD	Dom Pedrito	2016	M. bovoculi
20	1138 OE	Dom Pedrito	2017	M. bovoculi
21	1192 OD	Dom Pedrito	2017	M. bovoculi
22	281 OD	Uruguaiana	2017	M. bovoculi
23	5623 OE	Dom Pedrito	2015	M. bovoculi
24	330 NE	Alegrete	2016	M. bovoculi
25	259 OD	Dom Pedrito	2016	M. bovoculi
26	29 OE	Alegrete	2016	M. bovoculi
27	1295 OD	Dom Pedrito	2016	M. bovoculi
28	1362 OE	Dom Pedrito	2016	M. bovoculi
29	1213 NE	Dom Pedrito	2017	M. bovoculi
30	120 ND	Uruguaiana	2015	M. bovoculi
31	120 OD	Uruguaiana	2015	M. bovoculi

• OD = Right eye / OE = Left eye / ND= Right nostril and NE = Left nostril.

The extraction of genomic DNA was based on the protocol developed by Ausubel et al. (2003). The concentration and purity of DNA samples were evaluated by nano-spectrophotometry (NanoDrop ND-2000), and DNA integrity by 1% agarose gel electrophoresis.

Initially, the genetic diversity was evaluated by RAPD. For primer selection, a set of 20 decamer primers with arbitrary sequence (OPA-01 to OPA-20 – Operon Technologies, Alameda, USA) was tested with two M. bovis and two M. bovoculi samples. The primers that produced a higher number of analyzable amplicons, i.e. producing thick and strong bands, were chosen for the reproducibility of the technique. Amplification reactions used the parameters of Domingues et al. (2011). DNA fragments from PCR amplification were loaded on a 1.5% agarose gel and submitted to electrophoresis in TBE buffer, stained with ethidium bromide, and photo-documented on UV light using the Imaging Systems (UVITEC). This initial step allowed to choose seven primers that were then used to analyze all the samples.

The degree of genetic diversity among Moraxella spp. was also investigated with primers the JWP1-JWOPA07 and ERIC, according to Sosa and Zunino (2013), who had used them to assess the genotypic diversity of the isolates of M. bovis and M. bovoculi, from Kansas (USA) and Uruguay. The primers used for amplification were: JWP1 (5’-GCACTGAAGTGACCAAGCGG-3’) and JWOPA7 (5’-GAAACGGGTG-3’), ERIC-2 (5’-AA GTAGTGACTGGGGTGAGCG-3’) and ERIC-1R (5’-ATGTAAGCTCCTGGGGATTCAC-3’) (Sosa and Zunino, 2013). For ERIC-PCR we used amplification conditions slightly different from Sosa and Zunino (2013), including an initial denaturation step at 94 °C for 5 min, followed by 45 cycles of 1 min at 94 °C, 1 min at 52 °C and 2 min at 72 °C, with a final extension at 72 °C for 10 min. The products of the reactions were analyzed by agarose gel electrophoresis, as previously explained.

To analyze the amplicons, a matrix with binary data (presence = 1 or absence = 0) was constructed for the quantification of genetic diversity using the GENES software (Cruz, 2008). Estimates of genetic similarity between each pair of lineages were obtained by means of the Dice similarity coefficient (Reif et al., 2005), observing the interval of occurrence between one (putatively clones) to zero (highly divergent). A dendrogram was constructed using the matrix of dissimilarity estimated by unweighted paired group method of cluster analysis using arithmetic averages (UPGMA) (Cruz and Carneiro, 2003).

For ITS sequencing, after amplification of the 16S-23S intergenic regions (Angelos et al., 2007), the amplicons were purified with kit MinElute® (Qiagen®). For Sanger sequencing, the reactions were carried out with kit BigDye Terminator v3.1 Cycle Sequencing, following the conditions laid down by the manufacturers. After the reaction, the generated fragments were subjected to capillary electrophoresis in a ABI 3500 Genetic Analyzer (Applied Biosystems). Consensus sequences were generated for each sample in DNA Baser (v.5.15) and aligned with sequences from GenBank (accesses: Moraxella bovis – CP030241.1; DQ647927.1; EU014535.1; EU014547.1 and EU014575.1, and Moraxella bovoculi – DQ153085.1; DQ153089.1 and DQ153093.1), and a phylogenetic tree was constructed by the Maximum Likelihood method using MEGA (v7.0.26).

In our study, the bacteria were isolated from around 10% of the herd, only from ill animals. Bacteria isolation prior to identification allows only viable bacteria to be detected. In addition, we focused only on hemolytic colonies, since the cytotoxin β-hemolysin is a known virulence factor to M. bovis (Postma et al., 2008). We identified 13/28 M. bovis, and 15/28 M. bovoculi, from 90 animals, contradicting previous works that more frequently isolated M. bovis (O’Connor et al., 2012). In our study, no M. ovis was isolated, while Schnee et al. (2015), who worked with Moraxella identification in pre-IBK, post-IBK, and acute IBK phase herds, observed M. ovis more frequently, but concluded that only M. bovoculi was directly related to the disease, since it was the only species with increased prevalence only in the acute IBK phase herd.

Using the PCR-derived techniques, the selected primers for RAPD (OPA-02, OPA-03, OPA-04, OPA-07, OPA-09, OPA-11 and OPA-13 – Table S1), JWP1-JWOPA07 and ERIC-PCR (PCR-derived techniques) generated together 107 analyzable amplicons ranging from six (OPA-07) to 19 (OPA-03) (Figure 1). Analyzable amplicons were used to generate a dendrogram (Figure 2) from a unique diversity matrix. Two main genetically differentiable groups (clades) were observed. As expected, the 14 M. bovis isolates remained in one clade, whereas the 17 M. bovoculi remained in the other (Figure 2). According to the calculated bootstrap values, the two clades have great stability, reaching values of 76.15% and 92.35% for M. bovis and M. bovoculi, respectively.

Figure 1
Example of RAPD technique using a single primer (OPA-03). After RAPD-PCR, ten samples of M. bovis (left side) and nine samples of M. bovoculi (right side) were run in an agarose gel and stained with ethidium bromide.

Figure 2
Dendrogram representing the genetic relationships between M. bovis and M. bovoculi isolates by the UPGMA method. Bootstrap values represented in the tree (2000 bootstrap repetitions).

Genetic distances between M. bovis samples ranged from 0.0379 to 0.4285, with a mean of 0.1970. The lowest values of genetic dissimilarity were recorded among the 391 OD and 171 OD isolates, collected from two distinct animals, but belonging to the same herd. On the other hand, samples 6213 OD and 391 OE were the genetically most distant, both collected from distinct animals of different municipalities.

The values of dissimilarities between M. bovoculi ranged from zero to 0.7297, with a mean of 0.3625. Among the samples studied, two are possibly clones (120 OD and 120 ND), what is not surprising, since they were sampled from the same animal (Figure 2). The highest index of genetic divergence was verified between the samples Taim-3 and 1213 NE, these being samples collected from different animals belonging to two farms located in different municipalities. Despite the low number of samples and the geographic proximity between collection areas, genetic diversity analyzed by PCR-derived techniques proved to be a satisfactory tool to reveal the existence of genetic variability/similarity among Moraxella spp. Except for one herd, we always isolated for both, M. bovis and M. bovoculi, strains with different genetic profiles from animals coexisting in the same area (data not shown), corroborating previous work that demonstrated genetic variability of Moraxella isolates within the herds (Conceição et al., 2004).

By means of PCR-derived techniques, several previous studies have demonstrated genetic differences between isolates of M. bovis (Prieto et al., 1999; Conceição et al., 2004) or M. bovis and M. bovoculi (Sosa and Zunino, 2013) in Brazil, Argentina, and Uruguay, regions geographically related to the sampling of our study. The only previous study considering Brazilian isolates visualized a considerable higher number of possible clones than we did, possible due to the smaller number of analyzed amplicons (Conceição et al., 2004).

We also constructed a phylogenetic tree based on the homology of the ITS sequence of the Moraxella spp. isolates (Figure 3). M. bovis and M. bovoculi were grouped into two different clades, corroborating with the dendrogram analysis (Figure 2). This technique was able to satisfactorily identify/differentiate the two species of the Moraxella genus; however it was less informative than PCR-derived techniques to reveal genetic differences between isolates. It produced a less branched tree, and the concordance between the different intraspecific clades (Figure 3) with those obtained by PCR techniques (Figure 2), was not comprehensive. This apparent inconsistency, nonetheless, could be due to differences in evolutionary pressure in different parts of genome. ITS sequencing was less effective in demonstrating genetic variation, probably because regions with significant interspecies variations but low intraspecific polymorphisms were sequenced, corroborating a previous study (Wang et al., 2008). The higher discriminatory power of RAPD markers can be explained by the greater coverage of these markers throughout the whole genome. An alternative for increasing the discriminatory power of sequencing is to target additional regions of the bacterial genome within the same analysis.

Figure 3
Phylogenetic tree of the ITS region of the M. bovis and M. bovoculi isolates inferred by the maximum likelihood algorithm.

Interestingly, three M. bovis pairs, each one isolated from the same animal (171 OD – 171 OE; 391 OD – 391 OE and 6587 OD – 6587 ND) were not genetically identical, when analyzed by PCR-derived techniques. These pairs showed genetics distance of 0.1304; 0.3333 and 0.1034, respectively (Figure 2). On the other hand, according to ITS sequencing, only the pair isolated from animal 391 diverged at 7 bp of the 656 bp analyzed (1.07%). This pair presented the highest distance when analyzed by PCR-derived techniques (0.3333). Considering only samples from animal 391, the 391 OD was more similar with the others collected in this work and with accesses EU014535.1 and EU014575.1 from GenBank, whereas the 391 OE sample was more similar with the GenBank samples EU014547.1 and DQ647927.1 (Figure 3). Consequently, samples 391 OD and 391 OE stayed in separate clades.

The co-infection of single animals with genetically distinct strains has not yet been documented for M. bovis, although for other bacteria this phenomenon was described (Ogle and Vasil, 1991; Gaspar et al., 2011). This can be a complicating factor in the treatment of infections and in the efficacy of vaccines (Brown et al., 1998; Cullen et al., 2017), because different strains may have different antigenic and genetic properties, as well as distinct susceptibility profiles to antimicrobial (Gaspar et al., 2011). We previously tested the antimicrobials susceptibility in these isolates and found no difference, however (Comin et al., 2017).

Although there is circumstantial evidence of M. bovoculi involvement in the pathogenesis of IBK, it has not yet been possible to prove the participation in the etiology of the disease (Gould et al., 2013). In our study, M. bovoculi was frequently isolated from animals with clinical cases, corroborating several previous studies (Angelos et al., 2010; Libardoni et al., 2012; Karthik et al., 2018), and in some cases it was the only Moraxella species isolated from the herd. We also demonstrated a higher genetic difference for M. bovoculi, in comparison to M. bovis, which agrees with the recent findings that demonstrate, by sequencing, high genetic variability in this species (Dickey et al., 2016).

Even with those evidences, currently the only commercial vaccine in Brazil includes only one strain of M. bovoculi among the antigens, while in the US, the vaccine contains eight serotypes. Studies showing correlation between genetic and antigenic variability are scarce. Conceição et al. (2004) showed low correspondence between these two parameters, for M. bovis. However, most recent studies involving the sequencing of cytotoxin from M. bovis, M. bovoculi, and M ovis showed genetic variability in the aminoacid sequence for this virulence factor, depending on the period of isolation (Farias et al., 2015). Moreover, for M. catharralis, the head domains of UspA2/2H promotes a general evasion of the host immune system throughout the extensive sequence polymorphism in this protein (Su et al., 2013). However, these two works did not correlate those differences with serological variability, nor with genetic diversity. Taken together, these findings suggest a possible variability in antigenic composition, in addition to genetic variation. Thus, we postulate that the high genetic variability can be translated as an antigenic variability, hampering the universality of vaccines.

Despite sampling geographically close regions, we demonstrated considerable genetic diversity in M. bovis and M. bovoculi strains, indicating that the species must present a corresponding antigenic diversity, which can negatively affect therapeutic choice, control measures, and mainly, vaccine efficiency. Moreover, we demonstrated for the first time that M. bovis genetically distinct strains can co-infecting the same animal, which can limit therapeutic and vaccine efficiency, even within a single farm.

Acknowledgments

This research was funded by Embrapa (project number 02.13.10.002.00). The authors acknowledge the contribution of Hereford breeders: Paulo Fleck, Valter José Pötter, Patrícia Wolf and Eduardo Einchenberg. The authors also thank funding agencies CAPES (Finance code 001) and CNPq, as well as the Federal University of Pelotas.

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Supplementary material

The following online material is available for this article:

Table S1

RAPD primers.

• Associate Editor: Célia Maria de Almeida Soares

Publication Dates

• Publication in this collection
  29 May 2020
• Date of issue
  2020

History

• Received
  16 Jan 2019
• Accepted
  28 June 2019