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.

UNASSIGNED: Infectious bovine keratoconjunctivitis (IBK) is a prevalent ocular disease that affects livestock, leading to substantial economic losses due to reduced production and culling of infected animals. Moraxella spp. is common bacterial pathogens that can cause keratoconjunctivitis in livestock. Therefore, rapid and accurate diagnosis is crucial for effective treatment and disease control. This study aimed to develop a multiplex real-time polymerase chain reaction (mRT-PCR) assay for the detection and differentiation of Moraxella bovoculi, Moraxella ovis, and Moraxella bovis.
UNASSIGNED: Three reference strains of Moraxella as positive controls and 36 lacrimal swab samples collected from cattle were used to evaluate the developed mRT-PCR assay DNA extraction that was performed using the RIBO-sorb DNA/RNA extraction kit. Primers and probes were designed using the SpeciesPrimer pipeline. The annealing temperature, primer and probe concentrations, and sensitivity and specificity of the assay were optimized.
UNASSIGNED: An mRT-PCR assay was developed to detect pathogens associated with IBK in cattle on the basis of optimized parameters. The specificity and sensitivity of this assay were confirmed using samples containing individual pathogens (O - M. ovis, B - M. bovis, and BO - M. bovoculi), combinations of two pathogens (O-B, B-BO, and O-BO), and when the DNA of all three pathogens was present in a single reaction (O-B-BO). The analytical sensitivity of mRT-PCR for detecting M. ovis and M. bovoculi DNA was 21 copies or 50 fg per reaction, whereas that for M. bovis was 210 copies or 500 fg per reaction. In addition, this assay has been tested on samples isolated from the affected eyes of cattle in the Akmola region of the Republic of Kazakhstan.
UNASSIGNED: For the first time in the Republic of Kazakhstan, the proposed mRT-PCR assay for the simultaneous detection of three Moraxella spp. pathogens has been developed. This assay exhibits the required specificity and high sensitivity for m RT-PCR, facilitating the timely implementation of effective measures for disease control and the prevention of economic losses. These losses are linked to a reduction in livestock breeding value, a reduction in meat and milk production, a reduction in the reproductive performance of heifers, resulting in fewer offspring, as well as costs related to the treatment of affected animals.

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.

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.

Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live-cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

Moraxella bovis and Moraxella bovoculi both associate with infectious bovine keratoconjunctivitis (IBK), an economically significant and painful ocular disease that affects cattle worldwide. There are two genotypes of M. bovoculi (genotypes 1 and 2) that differ in their gene content and potential virulence factors, although neither have been experimentally shown to cause IBK. M. bovis is a causative IBK agent, however, not all strains carry a complete assortment of known virulence factors. The goals of this study were to determine the population structure and depth of M. bovis genomic diversity, and to compare core and accessory genes and predicted outer membrane protein profiles both within and between M. bovis and M. bovoculi.
Phylogenetic trees and bioinformatic analyses of 36 M. bovis chromosomes sequenced in this study and additional available chromosomes of M. bovis and both genotype 1 and 2 M. bovoculi, showed there are two genotypes (1 and 2) of M. bovis. The two M. bovis genotypes share a core of 2015 genes, with 121 and 186 genes specific to genotype 1 and 2, respectively. The two genotypes differ by their chromosome size and prophage content, encoded protein variants of the virulence factor hemolysin, and by their affiliation with different plasmids. Eight plasmid types were identified in this study, with types 1 and 6 observed in 88 and 56% of genotype 2 strains, respectively, and absent from genotype 1 strains. Only type 1 plasmids contained one or two gene copies encoding filamentous haemagglutinin-like proteins potentially involved with adhesion. A core of 1403 genes was shared between the genotype 1 and 2 strains of both M. bovis and M. bovoculi, which encoded a total of nine predicted outer membrane proteins.
There are two genotypes of M. bovis that differ in both chromosome content and plasmid profiles and thus may not equally associate with IBK. Immunological reagents specifically targeting select genotypes of M. bovis, or all genotypes of M. bovis and M. bovoculi together could be designed from the outer membrane proteins identified in this study.

A randomized control trial was performed over a five-year period to assess the efficacy and antibody response induced by autogenous and commercial vaccine formulations against infectious bovine keratoconjunctivitis (IBK). Calves were randomly assigned each year to one of three arms: an autogenous vaccine treatment that included Moraxella bovis (M. bovis), Moraxella bovoculi, and Mycoplasma bovoculi antigens, a commercial M. bovis vaccine treatment, or a sham vaccine treatment that consisted only of adjuvant. A total of 1198 calves were enrolled in the study. Calves were administered the respective vaccines approximately 21 days apart, just prior to turnout on summer pastures. Treatment effects were analyzed for IBK incidence, retreatment incidence, 205-day adjusted weaning weights, and antibody response to the type IV pilus protein (pili) of M. bovis as measured by a novel indirect enzyme-linked immunosorbent screening assay (ELISA). Calves vaccinated with the autogenous formulation experienced a decreased cumulative incidence of IBK over the entire study compared to those vaccinated with the commercial and sham formulations (24.5% vs. 30.06% vs. 30.3%, respectively, p = 0.25), and had less IBK cases that required retreatment compared to the commercial and sham formulations (21.4% vs. 27.9% vs. 34.3%, respectively, p = 0.15), but these differences were not significant. The autogenous formulation induced a significantly stronger antibody response than the commercial (p = 0.022) and sham formulations (p = 0.001), but antibody levels were not significantly correlated with IBK protection (p = 0.37).

Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease in cattle that causes economic losses to producers and negatively impacts animal welfare. In a 2016 survey of cow-calf producers in California, IBK was identified as the disease for which antimicrobials are most frequently used. The presented scoping review examined the available literature for methods to prevent IBK and for alternatives to antimicrobials to treat the disease that can be applied in cow-calf operations. Online databases were searched for publications about IBK in cattle populations that were reported from 1950 to 2020. Citations were systematically evaluated in a multi-stage approach using commercial software and summarized in a scoping review format. For the studies included in the review, most research (n = 50) has focused on the development of vaccines for the prevention of IBK. Although the quality of publications has improved over time, there is a lack of consistent evidence for vaccine efficacy against IBK in post-2000 experimental and conventional vaccine trials. A systematic analysis of vaccine studies is warranted. A limited number (n = 6) of studies evaluated the prevention of IBK through fly control, where most have found efficacy of this control measure. Several treatment options (n = 5) that do not include the use of antimicrobials have been investigated but remain at the preliminary stage of testing. Differences in breed susceptibility has been demonstrated with breeds belonging to the Bos indicus subspecies less frequently affected compared to those belonging to the Bos taurus subspecies. Hereford cattle and those lacking pigmentation around the eyelid margin are more frequently affected than other breeds. At present, there are few evidence-based measures that producers can utilize to reduce the burden of IBK in their herds and more research into the efficacy of fly control measures, non-antimicrobial treatment options, the continued search for a viable vaccine, as well as identifying genetic markers associated with traits that confer resistance to the disease are needed.