A novel species of the genus Moraxella was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species was Moraxella bovis (99.59 % nucleotide identity). Average nucleotide identity was calculated using a draft whole genome sequence of this strain compared with type strains of closely related Moraxella species and results established that it represents a new species. The genome size was 2 006 474 nucleotides and the G+C content was 42.51 mol%. The species could not be identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry using a commercial database, confirming the novelty of the strain. We propose the name Moraxella oculi sp. nov. for this new species. The type strain is Tifton1T and has been deposited into the American Type Culture Collection (TSD-373T) and the National Collection of Type Cultures (NCTC), UK Health Security Agency (NCTC 14942T).

Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria Moraxella bovis and Moraxella bovoculi are its main etiological agents. Antimicrobial therapy against IBK is often difficult in beef and dairy herds and, although vaccines are commercially available, their efficacy is variable and dependent on local strains. The aim of this study was to analyze for the first time the genomes of Uruguayan clinical isolates of M. bovis and M. bovoculi. The genomes were de novo assembled and annotated; the genetic basis of fimbrial synthesis was analyzed and virulence factors were identified. A 94% coverage in the reference genomes of both species, and more than 80% similarity to the reference genomes were observed. The mechanism of fimbrial phase variation in M. bovis was detected, and the tfpQ orientation of these genes confirmed, in an inversion region of approximately 2.18kb. No phase variation was determined in the fimbrial gene of M. bovoculi. When virulence factors were compared between strains, it was observed that fimbrial genes have 36.2% sequence similarity. In contrast, the TonB-dependent lactoferrin/transferrin receptor exhibited the highest percentage of amino acid similarity (97.7%) between strains, followed by cytotoxins MbxA/MbvA and the ferric uptake regulator. The role of these virulence factors in the pathogenesis of IBK and their potential as vaccine components should be explored.

Artificial intelligence (AI) was developed to distinguish cattle by their muzzle patterns and identify early cases of disease, including infectious bovine keratoconjunctivitis (IBK). It was tested on 870 cattle in four locations, with 170 developing IBK. The AI identified 169 of the 170 cases prior to their identification by veterinarians, and another 17 cases that remained free of IBK signs (sensitivity = 99.4%, specificity = 97.6%). These results indicate the AI can detect emerging IBK cases by muzzle images very early in the disease process and be used as an intervention tool in the prevention of IBK outbreaks.

Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

BACKGROUND: Infectious bovine keratoconjunctivitis (IBK) is a common cause of morbidity in cattle, resulting in significant economic losses. This study aimed to characterize the bovine bacterial ocular surface microbiome (OSM) through conjunctival swab samples from Normal eyes and eyes with naturally acquired, active IBK across populations of cattle using a three-part approach, including bacterial culture, relative abundance (RA, 16 S rRNA gene sequencing), and semi-quantitative random forest modeling (real-time polymerase chain reaction (RT-PCR)).
RESULTS: Conjunctival swab samples were obtained from eyes individually classified as Normal (n = 376) or IBK (n = 228) based on clinical signs. Cattle unaffected by IBK and the unaffected eye in cattle with contralateral IBK were used to obtain Normal eye samples. Moraxella bovis was cultured from similar proportions of IBK (7/228, 3.07%) and Normal eyes (1/159, 0.63%) (p = 0.1481). Moraxella bovoculi was cultured more frequently (p < 0.0001) in IBK (59/228, 25.88%) than Normal (7/159, 4.40%) eyes. RA (via 16 S rRNA gene sequencing) of Actinobacteriota was significantly higher in Normal eyes (p = 0.0045). Corynebacterium variabile and Corynebacterium stationis (Actinobacteriota) were detected at significantly higher RA (p = 0.0008, p = 0.0025 respectively) in Normal eyes. Rothia nasimurium (Actinobacteriota) was detected at significantly higher RA in IBK eyes (p < 0.0001). Alpha-diversity index was not significantly different between IBK and Normal eyes (p > 0.05). Alpha-diversity indices for geographic location (p < 0.001), age (p < 0.0001), sex (p < 0.05) and breed (p < 0.01) and beta-diversity indices for geographic location (p < 0.001), disease status (p < 0.01), age (p < 0.001), sex (p < 0.001) and breed (p < 0.001) were significantly different between groups. Modeling of RT-PCR values reliably categorized the microbiome of IBK and Normal eyes; primers for Moraxella bovoculi, Moraxella bovis, and Staphylococcus spp. were consistently the most significant canonical variables in these models.
CONCLUSIONS: The results provide further evidence that multiple elements of the bovine bacterial OSM are altered in the context of IBK, indicating the involvement of a variety of bacteria in addition to Moraxella bovis, including Moraxella bovoculi and R. nasimurium, among others. Actinobacteriota RA is altered in IBK, providing possible opportunities for novel therapeutic interventions. While RT-PCR modeling provided limited further support for the involvement of Moraxella bovis in IBK, this was not overtly reflected in culture or RA results. Results also highlight the influence of geographic location and breed type (dairy or beef) on the bovine bacterial OSM. RT-PCR modeling reliably categorized samples as IBK or Normal.

UNASSIGNED: Infectious bovine keratoconjunctivitis (IBK) causes a significant economic loss to cattle industries in many countries, including Kazakhstan. Although Moraxella bovis is recognized as an etiologic agent of IBK, other bacterial and viral agents have been suspected to play a role in the pathogenesis of this disease. This study aimed to evaluate samples collected from the eyes of IBK-affected cattle in Eastern Kazakhstan at different stages of IBK for the presence of Mor. bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi, and Bovine Herpes Virus Type 1 (BHV-1) and to characterize Mor. bovoculi pilA gene sequence diversity from Mor. bovoculi positive samples.
UNASSIGNED: Individual ocular swabs (n = 168) were collected from cattle that had clinical signs of IBK during the summer of 2022 on farms in the Abay region of Kazakhstan. Eye lesion scores (1, 2, and 3) were assigned depending on the degree of ocular damage. Infectious bovine keratoconjunctivitis-associated organisms were detected using a multiplex real-time polymerase chain reaction assay. The Mor. bovoculi pilA gene was sequenced from Mor. bovoculi positive samples.
UNASSIGNED: Mycoplasma bovis and BHV-1 were not detected in any of the collected samples. Mycoplasma bovoculi was identified in the majority of samples overall, usually in mixed infection with Moraxella spp. Moraxella bovoculi was detected in 76.2% of animals and predominated in animals with eye lesion scores 2 and 3. Mycoplasma bovoculi was detected only in association with Mor. bovis and/or Mor. bovoculi in animals with eye lesion scores 2 and 3. Moraxella bovis was found in 57.7% of animals and was always identified in association with another organism. Sequencing of the pilA gene in 96 samples from Mor. bovoculi positive samples identified five PilA groups. The majority belonged to PilA group A. However, three new PilA groups were identified and designated PilA groups N, O, and P.
UNASSIGNED: The results indicate a high prevalence of Myc. bovoculi and Mor. bovoculi in eyes of cattle with IBK on livestock farms in Eastern Kazakhstan. Additional novel Mor. bovoculi PilA groups were identified.

UNASSIGNED: Infectious bovine keratoconjunctivitis (IBK; pinkeye) is generally considered to be caused by corneal infections with Moraxella bovis. Previous studies demonstrated that M. bovis cytotoxin-specific mucosal immune responses in the bovine eye can be stimulated by intranasal vaccination with a recombinant M. bovis cytotoxin subunit adjuvanted with polyacrylic acid.
UNASSIGNED: A randomized controlled field trial (two-arm parallel design with blinding) was conducted in beef steers in Northern California to determine if this vaccine could prevent naturally occurring IBK and/or reduce morbidity rates associated with this disease. Beef steers were vaccinated intranasally on days 0 and 21 with either a recombinant M. bovis cytotoxin subunit adjuvanted with polyacrylic acid (Vaccine group) or adjuvant alone (Control group). Eye examinations were performed on all steers every 7 days for 16 weeks to document the occurrence of IBK and to determine sizes of corneal ulcers. Serum and tear samples were collected on days 0, 42, and 112 from a subset of animals to measure changes in systemic and ocular immune responses to M. bovis cytotoxin.
UNASSIGNED: The cumulative proportion of steers that developed IBK after 16 weeks did not differ between groups. Variables related to disease severity were numerically lower in steers that received the experimental vaccine. IBK-affected Vaccine group steers had a significantly lower number of observation weeks with severe ulcers versus Control group steers. Cytotoxin-specific tear IgA was significantly higher in Vaccine group compared to Control group steers on day 112. Conclusion: Although the proportion of animals that developed corneal ulcers associated with IBK did not differ between groups, the lowered metrics of disease severity in vaccinated steers suggests that intranasal vaccination with recombinant M. bovis cytotoxin can reduce the severity of IBK in cattle.

Infectious bovine keratoconjunctivitis (IBK), commonly known as pinkeye, has a marked negative impact on the economy of the cattle industry. Moraxella species, including Mor. bovis and Mor. bovoculi, which have been associated with this disease, colonize clinically healthy eyes as well, suggesting that there are intrinsic changes that may occur to the ocular microbiota or the involvement of additional unrecognized organisms that contribute to IBK. To evaluate this, 104 ocular swabs collected from eyes with IBK or clinically healthy eyes from 16 different cattle herds were subjected to 16 S rRNA gene PCR and next generation sequencing (NGS) analysis. Organisms detected were similar across the herds and there was no difference in the total number of bacterial groups detected among IBK cases and controls. However, the percentages of the different organisms detected varied between the two groups, including Moraxella spp., with more Moraxella spp. in eyes with IBK than controls. Further, using culture and whole genome NGS, a new species of Moraxella (suggested name Mor. oculobovii) was detected from the eyes of cattle from two farms. This strain is non-hemolytic on blood agar, is missing the RTX operon, and is likely a non-pathogenic strain of the bovine ocular microbiome. Alteration of the ocular microbiota composition may have a predisposing role, enhancing bacterial infection and the occurrence of clinical IBK. Future studies are required to evaluate if these changes are permanent or if there is a shift in the microbiome following recovery from the infection and how antibiotics might affect the microbiome.

Moraxella bovis (M. bovis) is regarded as a causative agent of infectious bovine keratoconjunctivitis (IBK), the most common ocular disease of cattle. Recently, whole genome sequencing identified the presence of two distinct genotypes within M. bovis that differ in chromosome content, potential virulence factors, as well as prophage and plasmid profiles. It is unclear if the genotypes equally associate with IBK or if one is more likely to be isolated from IBK lesions. We utilized 39 strains of M. bovis that had previously undergone whole genome sequencing and genotype classification to determine the utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) to accurately genotype M. bovis strains. We successfully developed two biomarker models that accurately classified strains according to genotype with an overall accuracy of 85.8-100% depending upon the model and sample preparation method used. These models provide a practical tool to enable studies of genotype associations with disease, allow for epidemiological studies at the sub-species level, and can be used to enhance disease prevention strategies.

Pinkeye (a generic term to describe infectious bovine keratoconjunctivitis) is a significant disease of cattle worldwide, impacting productivity and animal welfare. One commercial pinkeye vaccine, a systematically administered Moraxella bovis bacterin, has been available in Australia since 2007. This is the first field trial of the effectiveness of this vaccine for the prevention of naturally occurring disease in Australia. Extensively run beef herds in southwest Queensland that regularly experienced pinkeye were enrolled in the trial and animals were randomly allocated to vaccinated and control groups in different proportions in each herd. The subsequent incidence of clinical pinkeye between the two groups was compared for animals less than one-year-old. Data were analysed from 649 cattle from five herds over two pinkeye seasons: three herds of 390 calves from 1st November 2019 to 20th January 2020 and two herds of 259 calves from 23rd September 2020 to 21st April 2021. Pinkeye was common with 24% of all calves (156/649) contracting the disease during the trial. Univariable and multivariable binary logistic mixed-effect models were fitted to account for clustered data and potential residual confounding due to sex, weight, breed, coat colour, and periocular pigmentation. The incidence of pinkeye was not significantly different between vaccinated and control groups, both alone (p = 0.67) and after adjusting for sex and weight differences (p = 0.69). The vaccine was not protective against naturally occurring pinkeye under the field conditions.