# Development and evaluation of a multiplex polymerase chain reaction in real-time for differential diagnosis of Moraxella-induced keratoconjunctivitis in livestock

## TL;DR

This paper describes a new real-time PCR test to quickly and accurately detect three Moraxella species causing eye disease in cattle, helping reduce economic losses in livestock.

## Contribution

The first multiplex real-time PCR assay for simultaneous detection of three Moraxella species in Kazakhstan is developed and validated.

## Key findings

- The mRT-PCR assay can detect as few as 21-210 DNA copies of Moraxella species per reaction.
- The assay was tested successfully on clinical samples from cattle in Kazakhstan.
- The method shows high specificity and sensitivity for differential diagnosis of Moraxella-induced keratoconjunctivitis.

## Abstract

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.

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.

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.

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.