PCR Methods for Detecting Bovine Respiratory Pathogens

Introduction

Bovine Respiratory Disease (BRD) is an important disease in cattle production, causing serious economic losses world widely [1]. The occurrence of BRD is a combination of multiple factors and may be associated with one or more pathogens [2]. Among them, Mycobacterium bovis, Mycoplasma bovis, and Klebsiella pneumoniae are three important pathogens. Mycobacterium bovis can infect many kinds of animals. Besides cattle, there are 50 kinds of vertebrates such as humans. Sick animals showed a gradual loss of body weight, anemia and cough. Cattle with active tuberculosis are the main source of infection. Their respiratory tract carrys bacteria, which are excreted from coughing and sneezing [3]. Mycoplasma bovis is one of the main pathogens involved in cattle pneumonia. It was found that 5.5% of the nasal swabs from cattle with respiratory symptoms were positive for Mycoplasma bovis [4]. Klebsiella pneumoniae, an important conditional pathogen, mainly exists in the intestine, respiratory tract and urogenital tract [5]. The incidence of respiratory and urinary tract is the highest. Aslan et al. isolated bacteria from bovine upper respiratory tract infections and found that Klebsiella pneumoniae accounted for 20% [6].

PCR Detection

Polymerase Chain Reaction

PCR technology is a molecular biotechnology in which DNA of pathogenic microorganisms is expanded to conventional detectable levels in vitro. Quan Z et al. designed a multiplex PCR with primers targeting the 16S rRNA, Rv3873 and a 12.7-kb fragment in the genomes of a Mycobacterium tuberculosis complex to differentiate Mycobacterium bovis from Mycobacterium tuberculosis and NTM species [7]. Gioia et al. developed and validated a multitarget PCR assay that can discriminate between Acholeplasma and Mycoplasma and identify Mycoplasma bovis [8]. Turton et al. identified and typed Klebsiella pneumoniae by PCR using capsular type-specific, variable number tandem repeat and virulence gene targets [9]. Fonseca et al. established a one-step multiplex PCR to identify klebsiella pneumoniae, klebsiella variicola and klebsiella quasipneumoniae in the clinical routine [10].

Quantitative Real-Time PCR

Quantitative Real-time PCR technology can achieve quantitative analysis, and it is more specific and sensitive than conventional PCR. Choi Y et al. developed a real-time PCR targeting 16S ribosomal RNA for the detection of Mycobacterium tuberculosis complex [11]. Sales et al. developed and validated two real-time PCRs targeting the PE-PGRS 20 gene and the region of difference 4 (RD 4) for the characterization of Mycobacterium bovis isolates. The qPCR for PE-PGRS 20 had 91% efficiency and a detection limit of 0.32 ng. The qPCR for RD4 had 100% efficiency, and a detection limit of 4 pg [12]. Cezar et al. developed a qPCR targeting the region of RD4, which showed that 0.25% milk and 2% blood samples were positive for Mycobacterium bovis [13]. Fu-Xiang et al. developed a TaqMan real-time PCR for detection of Klebsiella pneumoniae, which could be applied for early diagnosis of Klebsiella pneumoniae infection [14]. We developed a TaqMan-based multiplex real-time PCR assay primer and TaqMan probes were designed based on the specific 229 bp sequence of Mycobacterium bovis, the uvrC gene of Mycoplasma bovis and the khe gene of Klebsiella pneumoniae. The assay sensitivity was 10 copies/μL. 37 bovine nasal swabs collected from cattle were identified, of which 21.62% (8/37) was Mycoplasma bovis-positive, 18.92% (7/37) was Klebsiella pneumoniae-positive, none (0/37) was Mycobacterium bovispositive. However, Mycobacterium bovis was detected in nasal swabs of cattle with symptoms of respiratory disease.

Summary

PCR detection technology is a sensitive, specific and fast method for the detection of BRD. We believe that the establishment of a TaqMan-based multiplex real-time PCR for the simultaneous detection of Mycobacterium bovis, Mycoplasma bovis, and Klebsiella pneumoniae can contribute to the early diagnosis and control of BRD.

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Cerebral Metastases as First Clinical Manifestation of Ovarian/Fallopian Tube Carcinoma

Introduction

Recent knowledge on histopathology and genetics have induced a paradigm shift that peritoneal, ovarian and fallopian tube carcinomas are considered as a spectrum of disease originating from the Müllerian duct remnant [1,2]. Intraperitoneal dissemination is the most commonly encountered pattern and initial distant metastasis beyond the liver or lung, including brain, is very rare [3]. Central nervous system involvement occurs as a late manifestation of the disease developing after the initial diagnosis of primary tumor; less than 5 % are of the carcinoma [4-6]. The multimodal treatment with a combination of surgery, chemotherapy and radiotherapy may improve survival but also increase the incidence of brain metastases [4-6]. Diagnosis of brain metastases which precede the diagnosis of ovarian/fallopian tube origin rarely occurs. In such cases, the carcinoma may be remarkable for the small size of the occult primary tumor and the early metastatic spread to the brain, which is ultimately responsible for the patient’s presenting symptoms. Considering the brain as a potential site of metastasis even early in the primary lesion may give a new insight to identify the factors, both host and tumor based, that could make the brain prone to metastasis so early in the disease process.

Literature Review

In a literature review we found 5 single case reports in which brain metastases were present on initial presentation of ovarian/fallopian tube carcinomas before or at the time of diagnosis of primary cancer (Table 1). The poorly differentiated adenocarcinoma accounts for 40%, and the characterized as endometrioid adenocarcinoma, clear cell carcinoma and serous adenocarcinoma only I one case. Metastases prefer the parenchyma rather than the leptomeninges in all cases as previously reported [7,8], usually as a single lesion [9-11], but also multiple in 2 cases [7,12].

Upon analyzing 12 previous articles on the clinical course of brain involvement in patients without a known primary tumor [13-24], 60-70% of total patients eventually had a primary cancer identified. The most commonly identified source was lung cancer. Other frequently discovered primary tumors include gastrointestinal tumors and melanoma, in approximately 10 to 15% of cases. Primary origin cancers remained unidentified in approximately 30 % of all cases; ovarian/fallopian tube carcinoma was not mentioned in these articles. Brain metastasis may occur via the unique cerebrospinal venous system named as Batson’s plexus. This system consists of veins, sinuses and venous plexuses [25], communicating with the veins around the spinal column, the segmental veins of thoracoabdominal wall, the plexuses and the azygous system of veins of pelvic organ. The main characteristics of the Batson plexus is lack of venous valves. This lack of venous valves causes bidirectional blood flow, and it may play as a route for rostral metastatic spread of tumor, infection or emboli from pelvic organs [26,27].

The lack of spread into the pelvic, intraabdominal or thoracic organs except for local lymph node metastasis suggests that the brain metastases probably occurred by tumor cell dissemination hematogenous through the Batson plexus. Ovarian/fallopian tube carcinoma is common but metastasizes rarely to the brain. The brain involvement is traditionally considered as a late manifestation of the diseases and generally occur in patients with prolonged survival. The metastatic brain involvement of brain as early presentation of ovarian/fallopian tube carcinoma is extremely unusual, but we suggest the importance of considering the brain as a potential site of metastasis, even early in the disease course of ovarian/fallopian tube carcinoma. There is some evidence that patients with only solitary brain metastases may have an improved prognosis. The possibility of the occult ovarian/fallopian tube carcinoma should be always considered in females with brain involvements.

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The #Importance of Basic and #Applied Natural Sciences in Service of #Medicine by Hatil Hashim EL-Kamali in BJSTR

The enormous diversity of #microorganisms in the environment offers an almost unlimited gene pool for new enzymes and metabolic pathways. The discovery of new natural products continues to be of great interested for the production of #pharmaceuticals. An exciting approach to generating new products is to evolve novel metabolic pathways in a microbial cell by means of the technique of metabolic engineering. Cell culture is playing an increasingly more significant role in drug discovery. Animal cell biotechnology constitutes an important field in #Biotechnology nowadays. Animal cells produce many biological of great value involving enzymes, hormones, growth factors, viral vaccines, or #monoclonal antibodies, and many therapeutic proteins require #post-translational modifications which can be only fully performed by animal cells. Insect virus, immune-regulators or whole cells, which are used for toxicological testing, can be obtained from animal.

 https://biomedres.us/abstracts/BJSTR.MS.ID.000705.php

https://biomedres.us/pdfs/BJSTR.MS.ID.000705.pdf 

https://biomedres.us/fulltexts/BJSTR.MS.ID.000705.php 

 The #Importance of Basic and #Applied Natural Sciences in Service of #Medicine by Hatil Hashim EL-Kamali in BJSTR