Open Access
Research (Published online: 28-12-2023)
19. Development and evaluation of a multiplex polymerase chain reaction in real-time for differential diagnosis of Moraxella-induced keratoconjunctivitis in livestock
Vitaliy Strochkov, Rano Sattarova, Karlygash Boranbayeva, Flyura Bakiyeva, Kuandyk Shynybayev, Batyrbek Aitzhanov, and Markhabat Kassenov
Veterinary World, 16(12): 2526-2532

Vitaliy Strochkov: Laboratory of Bacteriology, LLP “Kazakh Scientific Research Veterinary Institute”, Almaty 050016, Kazakhstan; Kazakhstan-Japan Innovation Centre, Kazakh National Agrarian Research University, 050010 Almaty, Kazakhstan.
Rano Sattarova: Laboratory of Bacteriology, LLP “Kazakh Scientific Research Veterinary Institute”, Almaty 050016, Kazakhstan.
Karlygash Boranbayeva: Laboratory of Bacteriology, LLP “Kazakh Scientific Research Veterinary Institute”, Almaty 050016, Kazakhstan.
Flyura Bakiyeva: Laboratory of Bacteriology, LLP “Kazakh Scientific Research Veterinary Institute”, Almaty 050016, Kazakhstan.
Kuandyk Shynybayev: Laboratory of Bacteriology, LLP “Kazakh Scientific Research Veterinary Institute”, Almaty 050016, Kazakhstan.
Batyrbek Aitzhanov: Laboratory of Bacteriology, LLP “Kazakh Scientific Research Veterinary Institute”, Almaty 050016, Kazakhstan.
Markhabat Kassenov: Laboratory of Bacteriology, LLP “Kazakh Scientific Research Veterinary Institute”, Almaty 050016, Kazakhstan.

doi: 10.14202/vetworld.2023.2526-2532

Article history: Received: 22-08-2023, Accepted: 30-11-2023, Published online: 28-12-2023

Corresponding author: Vitaliy Strochkov

E-mail: vitaliy.strochkov@kaznaru.edu.kz

Citation: Strochkov V, Sattarova R, Boranbayeva K, Bakiyeva F, Shynybayev K, Aitzhanov B, and Kassenov M (2023) Development and evaluation of a multiplex polymerase chain reaction in real-time for differential diagnosis of Moraxella-induced keratoconjunctivitis in livestock, Veterinary World, 16(12): 2526-2532.
Abstract

Background and Aim: 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.

Materials and Methods: 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.

Results: 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.

Conclusion: 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 mRT-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.

Keywords: Moraxella bovis, Moraxella bovoculi, Moraxella ovis, Moraxella spp., multiplex real-time polymerase chain reaction, Pinkeye.