Veterinary chemicals are substances that are used to prevent, diagnose, treat, change, or relieve disease or injury in animals. Prescription drugs such as antibiotics and over-the-counter products are examples of veterinary chemicals, e.g., drenches and vaccines [1-5]. The following are some of the most commonly utilized veterinary chemicals on livestock: a) Worm drenches for the intestines; b) treatments for lice, blowflies, and ticks; c) vaccines against illnesses including pulpy kidney, tetanus, and scabby mouth; d) antibiotics as drugs that are used to treat bacterial infections; and e) anti-inflammatory drugs used to treat pain [6, 7].
Pinkeye, also known as infectious bovine keratoconjunctivitis (IBK), is a highly contagious and infectious ocular illness of cattle characterized by conjunctivitis and ulcerative keratitis that affects cattle all over the world [8-11]. The sickness is also found in other livestock  and wildlife , and it is thought to be a multifactorial disease. The ocular disease maybe is not a fatal disease, but the morbidity is relatively high and combines with conjunctivitis, keratitis, congestion cornea, and sometimes abscesses . Many studies from different countries have demonstrated that keratoconjunctivitis in large and small ruminants result from various bacteria or viruses like Rickettsia, Chlamydia, Viruses, Mycoplasma spp., Neisseria catarrhalis, and Moraxella bovis .
The ocular infection may be causing temporary blindness/or permanent blindness, and the consequences could be extremally worse by turning down grazing behavior, which affects animal welfare and productivity . So, ocular infections in livestock have obviously an important role in economic deflection . However, multiple potential factors are associated with inflammation of the eye, including direct contact with lacrimal and nasal secretions contaminated with bacteria that have a significant role in spreading infections and producing corneal and conjunctival irritation. In moderate and acute infections and severe and persistent infections, ocular infections can affect one eye [unilateral] or both eyes [bilateral]. Conjunctivitis, excessive tears, photosensitivity, ocular pain, squinting of the eyelid, corneal edema, and corneal ulceration are all symptoms of ocular infection, as are corneal rupture and blindness [18, 19].
An outbreak probably occurs, especially when there is an implication of bad housing/animal management such as animal overcrowding and ignorance in maintaining of sanitation environment. Additionally, many seasonal insects such as domestic flies, insects, and stable flies have mechanical and dynamic important roles in conveying pathogenic bacteria [20, 21]. Little information is available regarding the study of bacteria pathogen-infected eyes of the livestock in Basra province. Therefore, the object of this study was to isolate and identify the occurrence of bacterial infection from cattle clinically associated with keratoconjunctivitis.
Material and methods
Sampling of animals
The current study was carried out from October 2019 to February 2020. Four regions (Al-Qurna, Al-Dayer, Al-Shenana, and Al-Nashwa) in Basrash province, selected purposively for this study. Clinical examination, including history and visual and physical examinations, has been previously done [22, 23]. According to the owner, the main complaint was eye problems in adult and young animals observed some weeks ago. A total of one hundred and twenty conjunctival swabs specimen were obtained from the cattle that showed an ocular infection symptom of infection. The age, breed, and sex were recorded from all examined animals. Swab sticks wet with sterile normal saline were used to collect the samples. The eyelid membrane was opened widely, allowing a spinning swab stick to be used on the corneal surface and conjunctiva, after which it was transferred to sterile test tubes containing 5 ml of sterile nutrition broth. Following that, all samples sent to the bacteriological laboratory were owned by Veterinary Medicine.
For 24 hours, the tubes were incubated aerobically in the incubator at 37°C. After that, it was cultivated on blood agar containing 7% sheep blood, MacConkey agar, Eosin Methylene blue, and incubated aerobically for 24 hours at 37°C. Pure colonies were saved on brain heart infusion agar for the purpose of conducting genetic identification of bacterial strains, and colonies were further sub-cultured to investigate the morphology of forming colonies and germ interactions that were taken from it to Gram stain.
Extraction of genomic DNA
The isolates' DNA was isolated using a genomic DNA purification kit. Electrophoresis on 0.8 percent Agarose revealed the result, which was visible under UV light as bands of DNA .
Identification by Polymerase Chain Reaction (PCR)
Primers for 16SrDNA F- 5' AGAGTTTGATCCTGGCTCAG-3' and R- 5' GGTTACCTTGTTACGACTT-3" were used , as well as a PCR software developed earlier  from . With minor tweaks, the amplification stages were abstracted from .
Sequencing for 16S rDNA and identification of bacterial species
The national instrumentation center for environmental management (nicem) sequenced the gene (http://nicem.snu.ac.kr/main/?en skin=index.html). For complete identification of bacterial isolates, a homology search was conducted using the Basic Local Alignment Search Tool (BLAST) program, available at the National Center Biotechnology Information (NCBI) online at (http://www.ncbi.nlm.nih.gov), and the Bio Edit program, available at the National Center Biotechnology Information (NCBI) online at (http://www.ncbi.nlm.nih.gov).
Result and Disscution
Out of 120 samples involved in this study, 30 were identified as having keratoconjunctivitis with an estimated prevalence of (25%). The results of the study showed that eye infections in cows were mostly unilateral, in one eye (69%), and less than bilateral infections in both eyes (30.9%), as showed in Table 1. The highest ocular infection in the examined animals was found during the winter (from January to February) comparing with other times (Table 2).
Infected animals showed conjunctival hyperemia (15.8%), (Figure 1), serious, mucoid and/or purulent ocular secretions (20.8%) (Figure 2) photophobia (24.1%) (Figure 3), lacrimation (15%) (Figure 4), congregation of flies on the eyes (12.5%) (Figure 5), and corneal opacity (11.6%) (Figure 6), Table 3.
Bacterial identification by 16S rDNA
A genomic DNA purification kit was used to recover DNA from twenty different strains. The result revealed under UV light after electrophoresis on 0.8 percent Agarose, and the isolated DNA from each isolate was submitted to PCR for amplifying 16S rDNA (figure 7). By comparing the gene's unique band to the typical molecular DNA ladder, 1250 bp was determined (100bp).
Figure 7: Amplification of 16S rDNA
Sequencing for 16S rDNA and identification of bacterial species
Table 4 shows the results of 16S rDNA nucleotide sequencing for 20 isolates. Isolates were identified to the strain level. Table 5 shows the number and proportion of bacteria recovered from inflammatory smears, while Figures 8 and 9 illustrate the phylogenetic tree of some of the isolates.
Figure 8: Phylogenetic tree of Enterobacter
Phylogenetic tree analysis of Enterobacter showed five distinct groups of local strains. S1, S2, S3 & S4, S6, and S9. S3 & S4 have a similarity with those previously isolated from Malaysia and India. S9 has a similarity with a strain isolated from Chile. The other three strains are far away from previously compared strains.
Figure 9: phylogenetic tree of Pseudomonas aeruginosa
Phylogenetic tree analysis of Pseudomonas aeroginosa showed two related strains S5 and S7. Those strains have a high similarity with Pseudomonas aeroginosa isolated in China.
Infectious bovine keratoconjunctivitis (IBK) is an infectious ocular illness that affects cattle, particularly those of the dairy breeds. Gram-negative bacteria, primarily Moraxella Bovis, cause the disease, and it is treated by parenteral or systemic antibiotic therapy. In most cases, M. Bovis infections are treated topically with antibiotic-containing formulations, especially when clinical symptoms are present. The antibiotic benzathine cloxacillin is known to be effective in the treatment of M. Bovis infections, and it has been frequently suggested for topical use. Due to an extremely painful ailment affecting beef and dairy cattle worldwide [29, 30], infectious bovine keratoconjunctivitis generates major economic losses resulting from lower weight gain in beef breeds and loss of milk production, short-term disruption of breeding plans, and treatment expenditures .
IBK is a set of organisms and predisposing factors that cause ocular alterations that favor bacterial colonization of the eye, similar to many other disorders. When dealing with less aggressive gram-negative and positive bacteria strains, predisposing factors may be an essential component of the IBK ocular illness complex [32, 34]. In our study, out of 120 samples, 24 were identified to have keratoconjunctivitis with an estimated prevalence by 25%. The results of the study showed that eye infections in cows were mostly unilateral, in one eye (69%), and less than bilateral infections in both eyes (30.9%). Infected animals showed conjunctival hyperemia (15.8%), serious, mucoid and/or purulent ocular secretions (20.8%), photophobia (24.1%), lacrimation (15%), the congregation of flies on the eyes (12.5%), and corneal opacity (11.6%). Because the 16S rDNA exists in this region in all bacteria, an amplified region of around 1250bp was found using the PCR technique, which agreed with the finding of previous studies [26, 35]. The isolates identified to the level of strain with the percentage of bacteria isolated from inflammatory smears were Escherichia coli by 20%, Bacillus subtilis by 10%, Enterobacter cancerogenus by 10%, Enterobacter hormaechei by 10%, Enterobacter cloacae by 15%, Klebsiella pneumonia by 15% and Pseudomonas aeruginosa by 20%, which corresponded to past research [36–39].
Phylogenetic tree analysis of Enterobacter showed five distinct groups of local strains. Some have a similarity with those previously isolated from Malaysia and India. Other has a similarity with a strain isolated from Chile. Furthermore, three strains are far away from previously compared strains. On the other hand, Phylogenetic tree analysis of Pseudomonas aeroginosa showed a high similarity with Pseudomonas aeroginosa isolated in China.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
All authors contributed toward data analysis, drafting and revising the paper and agreed to be responsible for all the aspects of this work.
Conflict of Interest
The authors declare no conflicts of interest.
Nawres N. Jaber
Noor Amjad Kazem
HOW TO CITE THIS ARTICLE
Nawres N. Jaber, Moaed H. Sayhood, Nada Salih Hadi, Basil A. Abbas, Noor Amjad Kazem. Genetic Detection of Some Bacterial Species Associated with Bovine Keratoconjunctivitis Infections in Basra Governorate, J. Med. Chem. Sci., 2022, 5(1) 19-26