Detection of Mycoplasma bovis in Pneumonic Calves

Mycoplasma bovis is a major pathogen in respiratory diseases of calves and cause an excessive economic loses. The current study was a goal to diagnoses bovine Mycoplasma and chiefly M. bovis from an outbreak of pneumonia in calves that occurred in Mosul city and mainly in Gogjaly village. Forty-two lung samples were collected from slaughtered and dead pneumonic calves in seven herds of imported calves. Extraction and amplification for DNA were conduct from all samples for diagnosis of Mycoplasma and M. bovis by PCR technique. The results have recorded the presence of Mycoplasma in 88.1% of examined lungs and M. bovis was diagnosed in 86.5% of the positive Mycoplasma samples. Finally the present study is the first local study at the moment which diagnoses Mycoplasma in general and mainly M. bovis from pneumonic calves, also according to the results it recommended the use of molecular techniques and principally PCR for the diagnosis of Mycoplasma and M. bovis.


INTRODUCTION
Pneumonia is one of the famous diseases of cattle and calves. It causes severe economic losses, including weight loss, feed loss, significant morbidity and mortality, neglected and underdeveloped animals, as well as the costs of treatment and early unwanted exclusion (such as death, euthanasia or slaughter) [1][2][3][4][5] .
Respiratory diseases and mainly pneumonia are caused by different agents including biological, chemical, and physical agents. The biological agents include viruses, bacteria, Mycoplasma, fungi, protozoa and parasites 2,4 .
Mycoplasmas are one of the principal causes of diseases in the respiratory system and other organs, and include lots of species Mycoplasma bovis is a principal pathogen of cattle and calves, and cause many infections in calves including pneumonia, arthritis, , and conjunctivitis 1,7-10 . So the targeted of the current study was diagnosis Mycoplasma mainly M. bovis from an outbreak of pneumonia in calves.

MATERIALS AND METHODS Samples
Lung samples were collected from fortytwo slaughtered and dead pneumonic calves during an outbreak that occurred in Mosul city and mainly in Gogjaly village during the period of January-February/2019. The diseased calves were distributed in seven herds, the calves' numbers in these herds were ranged between (27-62 calves). The clinical signs and necropsy findings were recorded. The samples under cooling condition were transported to Microbiology and PCR laboratories at department of Microbiology-College of Veterinary Medicine/ University of Mosul, and saved under refrigeration till used. All the samples were undergone to extraction and amplification.

DNA extraction and Amplification
The DNA extraction was performed according to manufacturer instructions (gSYNC™ Geneaid extraction kit): Lung samples were collected and prepared as described by 11 . They were collected in the plastic container and stored at -80°C until use. Lung tissue (25mg) was macerated in a 1.5 ml microcentrifuge tube with a pestle. To each sample, both of 200µl GST buffer solution and 20 µl of Proteinase-K were added. Samples were vortex thoroughly for 10 seconds and incubated at 60°C overnight. Dissolved samples were centrifuged at 16000 xg for 2 min, the supernatant was collected in new 1.5 ml tube, then 200 µl of GSB was added to the supernatant, again it was a vortex for 10 sec. 200µl absolute ethanol was added to the lysate sample and mixed well via vortex. All mixtures were transferred to GS columns and centrifuged at 16000 xg for 1min., then both W1(400µl) and W2 (600µl) buffers were added respectively to GS column with centrifugation. Finally 100µl of preheated elution buffer was added to each tube to elute the purified DNA, and stored at -20°C until used.
Two pairs of primers Table 1 were synthesized by BIONEER Co. (Korea) according to [12][13] for detecting the targeted genus Mycoplasma and species M. bovis. PCR reaction was done in 25µl as in Table 2. The amplification program was performed depending on the instructions as in Table 3. All PCR products were analyzed by gel electrophoresis 2% agarose (Biometra, Germany), containing 0.8µl ethidium bromide in TBE buffer. DNA bands were visualized over a UV transilluminator.

RESULTS
All infected calves were suffered from pneumonic signs that included labored breathing, fever, tachypnea, frothy salivation. Morbidity rates ranged between (8-15%) and the mortality rates were about (15-34%). In necropsy findings, the main lesions recorded were congestion, disseminating caseous nodules on the surface of lungs, marble appearance, hepatization (Fig. 1), and in some slaughtered calves, the lungs had a putrefied bad odor with ulceration on surface. The results of amplification appeared that 37(88.1%) samples were positive for Mycoplasma ( Table  4,

DISCUSSION
Mycoplasma bovis is a principal pathogen of respiratory diseases and mainly pneumonia in calves, and causes heavy economic losses in the cattle industry which may reach up to the third of losses that correlated to respiratory infections 1,9,14 .
The present study was targeted to diagnose the Mycoplasma, principally M. bovis from an outbreak of pneumonia in calves. So the results of the study recorded an excessive presence of Mycoplasma (88.1%) in samples of pneumonic calves, and M. bovis has appeared in 86.5% of these positive samples. The Mycoplasma rates and especially M. bovis excessive and terrible, and should care about especially when it a companied by considerable Morbidity (8-15%) and Mortality rates (15-34%) with the knowledge that it appears as a resistant pathogen for several antimicrobials [15][16][17][18][19] , where one study reported that the increase in deaths of calves due to respiratory disease from an average 9.7% per year to 36.5% per year communicated with the isolation of M. bovis from the lungs 20 .
Although there were no local studies about Mycoplasma and M. bovis in pneumonic cases for comparing, lots of studies worldwide diagnosed and/or isolated these microorganisms in high or considerable rates from pneumonia in calves. One of these studies which concurred the current results is a Turkish study 21 revealed that 80.9% of examined herds were positive for Mycoplasma infection and 87.6% of isolates were M. bovis. Another research in France 22 was diagnosed M. bovis in 78.5% of feedlot calves that were suffered from respiratory signs, while in more recently French study 23 the rate of isolation of M. bovis from cases of bovine respiratory disease was about 12-18% and it constituted about 55% of all isolated Mycoplasma from different pathogenic cases in cattle, whearas thorough investigation in feedlots in France 24 , one M. bovis strain turns out to be prevalent via the fattening stage and was accountable for shrill epidemics of bovine respiratory disease (BRD) with excessive insidegroup pervasiveness. Whilst in the Netherlands M. bovis was found in 20% of pneumonic calves in fattening flocks, though it found in very little rate in healthy calves (3%) [25][26] , and in a recent Dutch study 17 the lung isolates from M. bovis accounted about 58.5% of all M. bovis isolates diagnosed from a different infection during 2008-2014. Also, a study in Denmark was reported that 86% of pneumonic lungs found infected with mycoplasmas, and M. bovis appeared in 24% of these lungs 27 . Whereas Tschopp et al.,   The results of current study showed diagnosis of M. bovis from 86.5% of the positive lungs for Mycoplasma, which means the presence of other Mycoplasma species (13.5%) that may be playing a role in the occurrence of pneumonia in calves, and there are many universal studies that confirmed the presence of other Mycoplasma species excluding M. bovis in pneumonic calves lungs, as well as they may be isolated or were diagnosed in an excessive rates and in some times in a rates higher than M. bovis 23,[26][27]36 . While other studies reviewed no diagnosis of M. bovis from pneumonic infections in calves [37][38] .
Depending on the obtained results, the PCR technique was appeared as an excellent directly method in the diagnosis of Mycoplasma and principally M. bovis from pneumonic lungs, without the demands for culturing microorganism, which is accurate method and will reduce the consuming time and efforts. Many global studies supported the use of PCR instead of the culturing or together for diagnosis of Mycoplasma and particularly M. bovis [39][40][41][42][43][44][45] .
In conclusion, this study is the first local research at the moment to diagnose Mycoplasma in general and in particular M. bovis of pulmonary calves using molecular techniques. Also, according to the current results it recommends the use of PCR in the direct diagnosis of M. bovis from pneumonic lungs, and detect the other Mycoplasma species in pneumonic calves.