Contagious Bovine Pleuropneumonia (CBPP) is a severe respiratory disease affecting cattle, caused by Mycoplasma mycoides subsp. mycoides (Mmm). Accurate and rapid detection is essential for effective disease control and management. This study evaluates the performance of the AffiVET PCR Kit for the detection of CBPP, focusing on its sensitivity, specificity, and practical application in the field.
Contagious Bovine Pleuropneumonia (CBPP) is a highly contagious bacterial disease that affects cattle, leading to severe economic losses in affected regions. The disease is caused by Mycoplasma mycoides subsp. mycoides (Mmm) and is characterized by fever, respiratory distress, and pleuropneumonia. Traditional diagnostic methods, including bacteriological culture and serological tests, often require several days to weeks to yield results and may lack sensitivity and specificity. The development of molecular diagnostic techniques, such as Polymerase Chain Reaction (PCR), has provided a faster and more accurate alternative. The AffiVET PCR Kit is specifically designed for the detection of Mmm and offers the potential for rapid diagnosis and control of CBPP outbreaks.
Materials and Methods
Sample Collection
A total of 150 samples were collected from cattle exhibiting clinical signs of CBPP, such as coughing, nasal discharge, and labored breathing. Samples included nasal swabs, pleural fluid, and lung tissue collected post-mortem.
Control samples were collected from 50 healthy cattle with no history of CBPP exposure and 20 known positive CBPP cases confirmed by culture and serology.
DNA Extraction
DNA was extracted from all samples using the AffiVET DNA Extraction Kit. The extraction protocol involved lysing the bacterial cells, binding the DNA to a silica column, washing away contaminants, and eluting the purified DNA.
The concentration and purity of the extracted DNA were assessed using a Nanodrop spectrophotometer, measuring absorbance at 260 nm and 280 nm.
PCR Amplification
The AffiVET PCR Kit includes primers and probes specifically designed to target the Mmm genome. PCR reactions were prepared in a final volume of 25 µL containing 2 µL of template DNA, 12.5 µL of 2x PCR Master Mix, 0.5 µL of each primer (10 µM), 0.5 µL of probe (10 µM), and nuclease-free water.
Thermal cycling conditions were optimized for the kit: initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes.
Detection
Amplified products were analyzed by gel electrophoresis on a 2% agarose gel stained with ethidium bromide. Bands were visualized under UV light, and the presence of the target sequence was confirmed by comparing band sizes to a DNA ladder.
Real-time PCR analysis was also performed to quantify the bacterial load in positive samples, using a fluorescence-based detection system.
Results
The AffiVET PCR Kit demonstrated high sensitivity, detecting as few as 10 copies of Mmm DNA in spiked samples.
Specificity was confirmed by the absence of amplification in negative control samples and non-target bacterial species, including other Mycoplasma species commonly found in cattle.
Field application showed reliable performance, with 95% of clinical samples from suspected cases testing positive by the AffiVET PCR Kit. These results were in agreement with traditional diagnostic methods, which showed 93% concordance.
Real-time PCR analysis provided quantitative data on the bacterial load, correlating with the severity of clinical symptoms observed in the cattle.
Discussion
The AffiVET PCR Kit offers several advantages over traditional diagnostic methods for CBPP. Its high sensitivity allows for the detection of low levels of Mmm, making it suitable for early diagnosis and surveillance. The rapid turnaround time, with results available within 4 hours from sample collection, enables timely interventions and reduces the risk of disease spread. The kit's high specificity minimizes false positives, ensuring accurate identification of CBPP cases. Field application demonstrated the kit's robustness and reliability in various sample types, including nasal swabs and pleural fluid.
The use of real-time PCR not only confirms the presence of Mmm but also provides quantitative data, which can be useful for monitoring the progression of the disease and evaluating the effectiveness of treatment strategies. The ability to rapidly and accurately diagnose CBPP using the AffiVET PCR Kit can enhance disease control programs, improve cattle health, and mitigate economic losses associated with CBPP outbreaks.
The AffiVET PCR Kit is a valuable tool for the rapid detection of CBPP, providing high sensitivity and specificity. Its implementation in veterinary diagnostics can improve the management and control of CBPP outbreaks, ultimately protecting cattle health and productivity. Further studies are recommended to evaluate the kit's performance in large-scale field trials and its potential integration into routine surveillance programs.