Infectious bovine keratoconjunctivitis (IBK) or pink eye is the most important ocular disease affecting cattle worldwide. It is a contagious bacterial infection caused by Genus Moraxella. The affected animals show clinical signs of conjunctivitis, conjunctival swelling, corneal opacity, corneal edema, ulcer, blepharospasm, photophobia and mucoid or purulent lachrymal discharge¹. The severity of the cases varies from mild ocular signs to severe clinical cases showing profuse lacrimation, conjunctival swelling, corneal opacity, and ulceration. Even though the mortality is low, the infection causes high morbidity resulting in considerable economic loss due to ocular damage, injuries, weight loss and emaciation, production loss, and cost of treatment. Earlier Moraxella bovis was thought to be the sole pathogen associated with IBK. Further reports identified another pathogen M. bovoculi being more frequently isolated from IBK than M. bovis²’³. Researchers have reported two distinct genotypes of M. bovoculi viz. Genotype 1, associated with IBK in catle, and genotype 2, associated with asymptomatic infection in cattle^4,5. The manifestation of IBK is influenced by host factors like breed, age, immune status, and environmental factors like ultraviolet (UV) light exposure, face fly population, concurrent pathogens, and climate and pasture conditions⁶. Outbreaks are reported more inthe summer months, however,rarely occurin other seasons^7,8. This study reports the isolation and confirmation of M. bovoculi from an outbreak of IBK in a dairy farm in Goa.

A disease condition was reported in a Goshala located at North Goa district of Goa State, wherein a herd of bulls was suffering from conjunctivitis, corneal opacity, and blindness. The farm has more than 600 cattle of indigenous breeds like Gir, Shweta Kapila, and nondescript cattle of different age and sex groups, and the disease was observed only in a herd consisting of more than 60 bulls of Shweta Kapila and nondescript catle maintained in a separate enclosure. On examination, most of the animals showed emaciation (Fig. 1) and blindness and they were reluctant to move and feed. More than 25 animals had conjunctivitis, corneal opacity (Fig. 2) in one or both the eyes, and excessive lachrymation. The shed and premises had a high population of flies of different species. Swabs were collected aseptically from the eyes of 12 severely affected cows and transported in ice to the laboratory.

Swabs were inoculated on 5% blood agar, Nutrient agar, and MacConkey agar and were incubated at 37°C overnight. DNA isolation was carried out from colonies on blood agar using QiagenDNeasy blood and tissue kit and multiplex PCR was carried out targeting 16srRNA gene and 16S-23S intergenic spacer region to detect the presence of M. bovis, M. bovoculi, and M. ovis as per the method of Shen et al.⁹ Two out of the five PCR products were sequenced by Sanger’s method using forward and reverse primers at Eurofins Genomics India, Bangalore, India. Obtained sequences were aligned using DNASTAR software and NCBI BLAST search was used to find the matching sequences available with the data bank. Phylogenetic analysisof the 16S-23S intergenic spacer region sequences was carried by the neighbor-joining (NJ) method in the MEGA X program¹⁰using reference sequences Moraxella spp. available from NCBI data bank to find the relatedness of the present isolate with global strains.

Streaked blood agar showed small colonies with a narrow zone of complete hemolysis in five samples out of the 10 samples collected. Growth was also noticed on nutrient agar from the same five samples whereas no growth was observed on MacConkey agar plates. On microscopy, the bacteria were found to be gram-negative coccoid with diplococci arrangement. The multiplex PCR using primers for differentiation of Moraxella bovis, M. bovoculi, and M. ovis amplified a product of approximately 1800bp size (Fig. 3), suggestive of M. bovoculi, and ruled out any mixed infection with the other two pathogens. The aligned nucleotide sequence on NCBI BLAST analysis showed 100% identity with M. bovoculi strains from India and United States. Phylogenetic analysis showed clustering of the isolate with M. bovoculi strains from India (Fig. 4). In India, M. bovoculi was reported only once in an outbreak in Tamil Nadu¹¹, while M. bovis was reported in an outbreak in Uttar Pradesh¹² and was also isolated from frozen semen¹³. Several factors affect the occurrence of this disease. Previous study show a higher prevalence of IBK in calves and yearlings as compared to adults¹⁴. In the present outbreak, the disease was noticed only in adult bulls and no disease was noticed in younger animals in separate enclosures on the same farm. Also earlier studies show a higher susceptibility of Bos taurus breeds than the Bos indicus⁶, whereas in the present case severe disease was observed in indigenous breeds and nondescripts of Bos indicus. Exposure to adverse environments like excessive sunlight, dust, and presence of fly population, or any mechanical factors increases the susceptibility of the catle to IBK infection⁶. This was true in the present case as the affected herd was placed in a non-covered enclosure with no flooring, where the animals were exposed to harsh sunlight, dust, and flies, whereas, other groups of catle including milking, dry females, and calves, which were maintained in pucca sheds with proper roof covering did not show the disease.

The affected animals were treated with ciprofloxacin dexamethasone eye drops and intramuscular injection of Dicrysticin for 7 days. The animals were also given oral vitamin A, D, E, and B12 supplements, which showed improvement after treatment and completely recovered after 21 days. The farm manager was advised to follow fly control measures in the farm and to apply ectoparasiticide Flumethrin 10mg/ml pour-on solution.

In conclusion, the present report confirms the prevalence of M. bovoculi in Indian dairy farms. In the present case, the disease was noticed only in the bull herd exposed to the harsh environment and not in other cattle groups maintained in the pucca shed, which shows that harsh environmental conditions are an important predisposing factor in India for the disease.

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