Molecular Confirmation of Moraxella Bovis Associated Infectious Keratoconjunctivitis in Ruminants

 
Moraxella are Gram negative diplococci, non-motile, catalase and oxidase positive and the infection were easily transmitted between the animals by direct contact with nasal and ocular discharges and by flies as mechanical transmitter (Ojo et al., 2009 and Athira et al., 2018). Moraxella ovis produces heat-labile exotoxins causing both hemolysis for bovine RBCs and cytotoxic activities against corneal tissues leads to permanent blindness (Cerny et al., 2006). Moraxella spp form biofilm in vivo but the biofilm easily broken by the action of lysozyme naturally present in tears (Ely et al., 2018). The prevalence rates of Moraxella associated infectious keratoconjunctivitis have been observed by several authors (Van Halderen et al., 1994; Naglić et al., 2000 and Karthik et al., 2017). The variations in results of antimicrobials susceptibility were noticed between Moraxella spp isolates of different regions. This may refer to differences in genotypic characters of isolates (Loy and Brodersen, 2014). The Cure rate of IKC was 100% by using various types of antibiotics locally and systemically by injection along with topical antibiotic eye preparations (Pandey, 2018).

 
The suspected samples were inoculated in nutrient broth and incubated at 37^oC for 24 hr in nutrient broth. A loopful of culture from each tube was streaked onto 5% blood agar, BHI, Mac Conkey and SDA agar (Himedia -India) plate and incubated at 37^oC for 48 h with daily examination for any bacterial and fungal growth. The Staphylococcus, Strptococcus, Pseudonmoas, Klebsiella  Trichophyton and Moraxella were isolated from the samples (Table 2). The suspected Moraxella colonies were picked-up, subcultured and subjected to Gram’s staining and lactophenol cotton blue staining followed by catalase test, indole production test, oxidase test, motility test, sugar-fermentation test, Simmon’s citrate test. The suspected colonies genomic DNA were extracted by Phenol Chloroform and Isoamyl alcohol and 0.2 M NAOH method. The purity and concentration of DNA were assessed by using nano drop. The PCR amplification was performed by using Moraxell aspecific primers F-5'GTGAAGTCGTAAC AAGGTAGCCGT-3' and reverse primer R-3'ACCGACGC TTATCGCAGGCTATCA-5' with cyclic condition of 95^oC for 5 min, 95^oC for 30 sec, 55^oC for 45 sec, 72^oC for 30 sec and 72^oC for 7 min for 30 cycles. The gel electrophoresis was performed with 1% agarose gel. The positive samples showed amplification size of 650 bp. The amplified PCR product were purified with Quiagen gel purification kit and subjected to partial gene sequencing. The antibiotic sensitivity test was performed with  Mueller-Hinton agar (Himedia-India), all the Moraxella isolates were tested for their antimicrobials sensitivity based on the method prescribed  by  Bauer et al. (1966) using commercial antimicrobial discs (Himedia - India). Ampicillin plus Sulbactam 20 μg, Vancomycin 30 μg, Ciprofloxacin 30 μg, Gentamicin 10 μg, Oxytetracycline 30 μg, Sulfamethoxazole 30 μg, Endrofloxacin 30 μg, metronidazole 30 μg Gatifloxacin 30 μg and Tylosin 30 μg were used.  The affected eye washed with 2% Boric acid lotion, topical eye ointment (Terramycin®), eye drops contains Gentamicin, Chloramphenicol and Microflox-DX were applied 3 times daily and systemic antibiotic and antiinflammatory and vit ADE3 supplement were suggested for the positive animals for 5 successive days.

 
India is the largest animal population and many farmers depends on animal husbandry practice for their livelihood. The cattle, sheep and goats occupied source of Meat and milk production and important source of incomes to the country. The infectious keratoconjunctivitis representing a considerable health problem for cattle, sheep and goat flocks leads to greater economic loss (Hidson and Winter, 2008). The observed clinical findings of suspected cases strongly refer to infectious keratoconjunctivitis. Similar clinical findings were reported previously (Ojo et al., 2009 and Karthik et al., 2017). Various type of microorganisms were the causative agents of infectious keratoconjunctivitis in small ruminant, Moraxella and Mycoplasma appear to be the most important one causing infectious keratoconjunctivitis in small ruminants (Åkerstedt and Hofshagen, 2004). In the present study Moraxella was successively isolated from 9 cases of 138 examined animals. The low isolation rate of Moraxella may give an indication that infectious keratoconjunctivitis is a multi-complexes and Moraxella is not the only pathogen responsible for infectious keratoconjunctivitis in ruminants and caused by different microorganisms and the presence of risk factors is crucial (Van Halderen et al., 1994; Jansen et al., 2006; Åkerstedt and Hofshagen, 2004 and Pandey, 2018).
 
The concurrent infection of trichophyton verocussum, Aspergillus and Moraxella in affected flocks will be confirmed by culture isolation and identification. Mycotic keratitis is caused by filamentous fungi and occurs in conjunction with trauma to the cornea.  Eye trauma is the cause of fungal keratitis in temperate areas by Fusarium, Alternaria and Aspergillus (Aristimuno et al., 1993). Keratitis caused by yeasts such as the Candida spp. almost always occur in previously abnormal eyes, like dry eye, chronic corneal ulceration or corneal scarring.
 
In this study the prevalence of Moraxella sp. among the cattle, sheep and goats was confirmed by PCR. The Moraxella ovis was isolated from 85.7% of affected sheep with IKC (Saðlam et al., 2018). Moraxella was isolated from 100% of diseased goats (N=8) (Ojo et al., 2009). Variation in prevalence rates depends upon differences in the number of examined animals and distinction in managemental and environmental conditions in different localities where the studies were conducted. The different environmental predisposing factors are greatly influence for Moraxella and the presence of corneal hurt by mechanical factors or ultraviolet irradiation is important for occurrence of the disease (Dubay et al., 2000). However, Moraxella was isolated as a single pathogen from goats with characteristic signs of IKC (Athira et al., 2018). The corneal dryness and corneal abrasion caused by aero-sandy particles carried by wind, as well as exposure to high percentage of ultraviolet rays which may predispose for Moraxella colonization during dry and summer season.
 
The therapeutic management of affected population with right antibiotic will provide significant results usage of Oxytetracycline systemically and topically come in accordance with the results of antimicrobial sensitivity test as the sensitivity of Moraxella to Oxytetracycline was intermediate. The susceptibility rate of Moraxella of small and large ruminants to Oxytetracycline ranged from 80% to 91% and the haphazard use of Oxytetracycline in veterinary practice contributed to the emergence of resistant strains (Maboni et al., 2015). Using of Oxytetracycline topically as eye ointment and systemically by injection gives better results with a rare relapse rate. Currently, Moraxella isolates were 100% sensitive to Tylosin and Gentamicin and Moraxella were resistant to Penicillin, Ampicillin, Cloxacillin and Chloramphenicol while being 100% susceptible to Gentamicin and Ofloxacin by disk diffusion technique (Ojo et al., 2009).
 
In our study, Moraxella isolates were 100% sensitive towards Chloramphenicol, Ciprofloxacin, Gentamicin, Tylosin and 82% sensitive to Oxytetracycline, Endrofloxacin, Gatifloxacin,Metronidazole. All tested isolates were resistant to Norfloxacin, Vancomycin, Ampicillin Sulbactam, Methicillin and Sulphadiazine. The antibiotic sensitivity test will vary according to the geographical location and strain of pathogens.
 
Bovine alphaherpes virus-¹ (BoHV-¹) is a highly contagious virus is responsible for causes conjunctivitis of one or both the eye, upper respiratory tract infection known as infectious bovine rhinotracheitis (IBR), reproductive tract lesions in cows, infectious pustular vulvovaginitis (IPV) and in bulls infectious balanoposthitis (IBP) and new-born systemic infection. The  Glycoprotein D is significantly immunogenic and subunit vaccine candidate give protection against BoVH-¹ and also BoVH-⁵. The gE-live or killed marker vaccine has proven to be promising in inducing protective immune response and in such animals g^E based ELISA are used to differentiate infected animals from vaccinated animals . Analysis of genes of g^B/ g^C/ g^D and g^E by PCR plays an important role in diagnosis and cost effective epidemiological studies of the virus (Rashmi et al., 2023).

The antioxidant and micronutrients are plays important roles for body functions especially during transitional period in ruminants and improve feed intake, cell functions, carbohydrates, protein and metabolism. The animal production, reproduction were improved by increasing the immunity. The antioxidant micronutrients change feed intake, nutrient digestibility, rumen fermentation and energy production in ruminants leads to prevent bacterial, viral and fungal infection (Mohammed et al., 2024).
 

Infectious keratoconjunctivitis is a serious problem of ruminants located in hilly areas particulary dry summer season. Moraxella infection was confirmed both culture isolation and identification and PCR for the pathogen causing infectious keratoconjunctivitis. In this present study, the prevalence of Morexalla species among the cattle, sheep and goats was confirmed by PCR. However, further indepth detailed study needed for developing antibiogram and vaccine for infectious keratoconjunctivitis and further clinical studies needed for selecting systemic and topical antimicrobials agents for complete recovery of affected animals.

All authors declared that there is no conflict of interest.