Respiratory diseases are one of the most leading problems in animals worldwide. Especially in cattle, they damage cattle industry and national economies by decreasing feeding efficiency and weight gain, and causing death in severe cases. Rapid and accurate diagnosis of the disease is thus critical to ensure animal health and welfare, reduce losses, and enable effective treatment and positive prognosis
19. Studies of bacterial isolation from slaughterhouse material
1,20 and nasal cavities of healthy and ill cattle have been conducted in Turkey before
21-23. However, our study is the first to obtain data using nasal swabs obtained from cows with pneumonia using the MALDITOF MS.
In the present study, we performed clinical examinations of both the animals showing pneumonia symptoms and control-healthy animals. The clinical measurements (respiratory rate, heart rate, and rectal temperature) were higher in cows and calves showing pneumonia symptoms than control group animals and reference values 24. Hanzlicek et al. endoscopically infected calves with Mannheimia haemolytica and also detected changes from physical examination. While clinical measurements (respiratory rate, heart rate, and rectal temperature) varied in the following days, there were a general increase compared to reference values 25. Similarly, Van Donkersgoed et al. conducted an epidemiological study of enzootic pneumonia in calves. They reported an increase in respiratory rate and rectal temperature in pneumonia cases 26.
MALDI-TOF MS has gained popularity in recent years because it enables precise and rapid (i.e. on the same-day), species-level identification of microorganisms isolated from blood, urine, peritoneal, synovial, bronchoalveolar lavage fluid, body fluids like cerebrospinal, and from many sources (e.g., slaughterhouse material) 27. Van Driessche et al. used MALDI-TOF MS to determine the etiological spectrum of bacteria isolated from bronchoalveolar lavage fluid. They identified 45 different bacterial species from 100 samples. Furthermore, compared to traditional bacterial culturing to detect pathogens in the lower respiratory tract, they reported that this method reduced the diagnostic time from 24-48 hours to an average of 6.5 hours and gave more accurate results 28. Choudhary et al. applied MALDI-TOF to nasal swabs, tracheal swabs, and lung tissue samples from cattle and buffalo. They identified major pneumonia pathogens, such as Pasteurella spp, Stenotrophomonas maltophilia (3.1%), S. sciuri (0.79%), E. coli (20.63%), Pseudomonas aeruginosa (6.3%), and Enterobacter cloacae (3.1%) while 96% of samples were polymicrobial 29. Using the same method, our study isolated 19 different bacterial species from 103 samples. The most frequent bacterial species were E. coli (37.8%), S. lentus (33%), Mycoplasma spp. (28.1%), S. sciuri (16.5%), E. cloacae (10.6%), and S. maltophilia (2.91%). Polymicrobial isolation was observed in 60% of samples.
Because the bovine respiratory tract acts as a reservoir for pathogenic microorganisms, poor hygiene conditions, stress factors, and climate changes may trigger mixed infections in the lungs. In particular, some Staphylococcus spp. and E. coli induced pneumonias are thought to develop in this way 30. Our study detected high rates of S. lentus, S. sciuri, and E. coli. The first two of these are generally isolated from domestic, farm, and wild animals, and foods of animal origin. Hay et al. were the first to isolate S. lentus from the sinonasal cavity as a sinusitis agent, although it is under reported in humans 31. In humans, this bacterium causes serious infections, such as endocarditis, peritonitis, septic shock, endophthalmitis, pelvic inflammatory diseases, and wound infections 32. The fact that this bacterium was isolated at high rates in our study suggests that the sampled animals had sinusitis in addition to pneumonia symptoms.
In recent years, it has been reported that caseonecrotic bronchopneumonia, a type of pneumonia characterized by chronic mycoplasma infection, is caused by M. bovis, which is more virulent than other mycoplasma species 33. In bovine respiratory system diseases, more than 70% of Mycoplasma spp pneumonia cases are generally seen as mixed infections, with only 20% reported as solitary 34,35. Booker et al. identified microbiological agents in respiratory system diseases of beef cattle and investigated the relationship between these agents and pathological processes. It was found that 12/15 (80%) of animals positive for Histophilus somni were also positive for M. bovis 34. The prevalence of M. bovis in Denmark increased from 0.6% in 1983 36 to 2% in 1999 37 and 24% in the 2000s 38. Kusiluka et al. investigated the prevalence of mycoplasmas in pneumonic cattle lungs. They found that the most dominant (72.0%) mycoplasma species and Ureaplasma spp while 18.6% of samples had mixed M. bovis Ureaplasma infection 38. Soehnlen et al. reported a positivity rate for M. bovis as 40% from nasal swabs 39. Our study detected M. bovis in 23% of 103 animals while all samples with M. bovis also contained other bacteria. No bacterial growth was observed in the isolation performed on blood agar from samples containing the five Mycoplasma spp. Thus, the probability that suspected animals have mixed infections with M. bovis is around 80%. Bacterial identification using the isolation method from lower respiratory tract samples is the gold standard for diagnosing Mycoplasma induced pneumonia. Thomas et al. compared the isolation rates in different samples collected from cattle with clinical signs and reported much higher isolation rates from BAL fluids than nasal swabs 40. We could not take lower respiratory tract samples becuase the method is invasive, requires skilled extra labor and equipment, and, more importantly, is not acceptable by the animal owners. Karahan et al. examined a total of 148 samples (3 lung, 4 eye swab, 51 nasal swab, 90 milk) and found 23.5% positivity for M. bovis in nasal swabs 11. Akan et al. found 16 (12.5%) of 127 nasal swabs positive for M. bovis by PCR 41. In our study, the isolation rate for Mycoplasma spp. was 9.7% (10/103), which was quite low compared to the 28.1% (29/103) positivity rate according to the PCR analysis. Possible causes include the lengthy transport of samples after collection, freezing until processing, and using nasal swabs. This may also explain why five Mycoplasma spp. could not be typed due to the presence of contaminant bacterial and yeast DNA in their extracted DNA.
In conclusion, pneumonia is a common problem in dairy cattle and calves that harms animal welfare and causes significant economic losses every year. It has been a universal problem since the beginning of farm animal husbandry. Accurate diagnosis and treatment are as important as prophylactic measures for this disease. In our study, the samples were collected and the bacteria detected accurately and quickly using MALDI-TOF MS. This method is easy to apply in the field and causes minimal discomfort to the animals. The method successfully demonstrated in our study can contribute to preventing antibiotic resistance in both animals and humans by enabling the correct use of antibiotics and will contribute positively to the field.
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