Cloning and expression of P67 protein of Mycoplasma leachii

Research (Published online: 21-09-2017)

15. Cloning and expression of P67 protein of Mycoplasma leachii

Sabarinath Thankappan, Rajneesh Rana, Arun Thachappully Remesh, Valsala Rekha, Viswas Konasagara Nagaleekar and Bhavani Puvvala

Veterinary World, 10(9): 1108-1113

ABSTRACT

Aim: The present study was undertaken to clone, express and study the immunogenicity of P67 protein of Mycoplasma leachii.

Materials and Methods: P67 gene was amplified from genomic DNA of M. leachii. The polymerase chain reaction (PCR) product was inserted in pRham N-His SUMO Kan vector and was used to transform competent Escherichia cloni 10G cells. Recombinant protein expression was done by inducing cells with 0.2% Rhamnose. Purification was done using nickel nitrilotriacetic acid affinity chromatography. Western blot and dot blot analysis were performed to assess the immunoreactivity of P67 protein.

Results: PCR amplicon size of P67 gene was found to be 1500 base pair. The size of the fusion protein with SUMO tag was 79 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The recombinant P67 fusion protein expressed in pRham N-His SUMO Kan vector was found to be immunogenic in both western blot and dot blot analysis.

Conclusion: Western blot and dot blot analysis of P67 protein of M. leachii revealed that the protein is immunogenic. Further work is needed to evaluate the role of P67 antigen of M. leachii as an immunodiagnostic agent.

Keywords: cloning, dot blot, expression, Mycoplasma leachii, P67 protein, western blot.

Cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of Clostridium chauvoei

Research (Published online: 21-09-2017)

14. Cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of Clostridium chauvoei

Saroj K. Dangi, Pavan Kumar Yadav, Aakanksha Tiwari and Viswas Konasagara Nagaleekar

Veterinary World, 10(9): 1104-1107

ABSTRACT

Aim: Blackleg disease is caused by Clostridium chauvoei in ruminants. Although virulence factors such as C. chauvoei toxin A, sialidase, and flagellin are well characterized, hyaluronidases of C. chauvoei are not characterized. The present study was aimed at cloning and sequence analysis of hyaluronoglucosaminidase (nagH) gene of C. chauvoei.

Materials and Methods: C. chauvoei strain ATCC 10092 was grown in ATCC 2107 media and confirmed by polymerase chain reaction (PCR) using the primers specific for 16-23S rDNA spacer region. nagH gene of C. chauvoei was amplified and cloned into pRham-SUMO vector and transformed into Escherichia cloni 10G cells. The construct was then transformed into E. cloni cells. Colony PCR was carried out to screen the colonies followed by sequencing of nagH gene in the construct.

Results: PCR amplification yielded nagH gene of 1143 bp product, which was cloned in prokaryotic expression system. Colony PCR, as well as sequencing of nagH gene, confirmed the presence of insert. Sequence was then subjected to BLAST analysis of NCBI, which confirmed that the sequence was indeed of nagH gene of C. chauvoei. Phylogenetic analysis of the sequence showed that it is closely related to Clostridium perfringensand Clostridium paraputrificum.

Conclusion: The gene for virulence factor nagH was cloned into a prokaryotic expression vector and confirmed by sequencing.

Keywords: black quarter, Clostridium chauvoei, hyaluronoglucosaminidase.

Immunological and histopathological changes in sheep affected with cutaneous squamous cell carcinoma and treated immunotherapeutically

Research (Published online: 20-09-2017)

13. Immunological and histopathological changes in sheep affected with cutaneous squamous cell carcinoma and treated immunotherapeutically

Faten A. M. Abo-Aziza, A. A. Zaki, A. El-Shemy, Sahar S. Abd Elhalem and Amany S. Amer

Veterinary World, 10(9): 1094-1103

ABSTRACT

Background and Aim: Recently, it has been recorded unexpected percentage of cutaneous squamous cell carcinoma (cSCC) in sheep. Despite the improvement in surgical treatment, the outcome of animals remains limited by metastatic relapse. Although antibodies for cancer treatment have been practiced for many decades, the use of this methodology in animals is deficient. This study aimed to establish cSCC therapy by tumor cell protein antibody (Ab1) or secondary antibody (Ab2) raised by two series of immunization in the same strain of rabbits.

Materials and Methods: A total of 19 Ossimi sheep were used (14 sheep suffered from cSCC and 5 were apparently healthy). Each animal from control healthy group (n=5) and control cSCC (n=4) group was treated with a course of eight injections of normal globulins. Animals in the third (n=5) and the last (n=5) groups received a course of eight injections of Ab1and Ab2, respectively. Each tumor was measured before and after treatment. The eight injections were applied at 1st, 3rd, 5th, 7th, and 9th week and the remaining three injections were at 1 week interval. Tissue specimens and blood samples were taken for histological and immunological studies.

Results: The obtained results revealed that injection of Ab1 might prevent the bad prognostic picture of polymorph infiltration without any criteria of regression % of tumor. Treatment with Ab2 showed regression of tumor size ranged between minimum of 8.99% and maximum of 78.12%, however, the measurements in most cases reached the maximum regression after the past two injections. In additions, infiltration of lymphocytes to tumor site, normalization of leukocytes picture and also increase of antibody titer were observed.

Conclusion: This profile might confirm that Ab2 could act as an antigen and encourage us to use it as a tumor vaccine. Extensive studies are needed to isolate the idiotypic portion of Ab1 for raising Ab2 as an anti-idiotypic antibody to be used as tumor vaccine. The question of how lymphocyte traffic to the tumor site as a result of Ab2 injection needs further investigation.

Keywords: antibody, cutaneous squamous cell carcinoma, histopathology, immunotherapy, sheep.

Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry as a reliable proteomic method for characterization of Escherichia coli and Salmonella isolates

Research (Published online: 19-09-2017)

12. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry as a reliable proteomic method for characterization of Escherichia coli and Salmonella isolates

Waleed S. Shell, Mahmoud Lotfy Sayed, Fatma Mohamed Gad Allah, Fatma Elzahraa Mohamed Gamal, Afaf Ahmed Khder, A. A. Samy and Abde Hakam M. Ali

Veterinary World, 10(9): 1083-1093

ABSTRACT

Aim: Identification of pathogenic clinical bacterial isolates is mainly dependent on phenotypic and genotypic characteristics of the microorganisms. These conventional methods are costive, time-consuming, and need special skills and training. An alternative, mass spectral (proteomics) analysis method for identification of clinical bacterial isolates has been recognized as a rapid, reliable, and economical method for identification. This study was aimed to evaluate and compare the performance, sensitivity and reliability of traditional bacteriology, phenotypic methods and matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) in the identification of clinical Escherichia coli and Salmonella isolates recovered from chickens.

Materials and Methods: A total of 110 samples (cloacal, liver, spleen, and/or gall bladder) were collected from apparently healthy and diseased chickens showing clinical signs as white chalky diarrhea, pasty vent, and decrease egg production as well as freshly dead chickens which showing postmortem lesions as enlarged liver with congestion and enlarged gall bladder from different poultry farms.

Results: Depending on colonial characteristics and morphological characteristics, E. coli and Salmonella isolates were recovered and detected in only 42 and 35 samples, respectively. Biochemical identification using API 20E identification system revealed that the suspected E. coli isolates were 33 out of 42 of colonial and morphological identified E. coli isolates where Salmonella isolates were represented by 26 out of 35 of colonial and morphological identified Salmonella isolates. Serological identification of isolates revealed that the most predominant E. coli serotypes were O1 and O78 while the most predominant Salmonella serotype of Salmonellawas Salmonella Pullorum. All E. coli and Salmonella isolates were examined using MALDI-TOF MS. In agreement with traditional identification, MADI-TOF MS identified all clinical bacterial samples with valid scores as E. coli and Salmonella isolates except two E. coli isolates recovered from apparently healthy and diseased birds, respectively, with recovery rate of 93.9% and 2 Salmonella isolates recovered from apparently healthy and dead birds, respectively, with recovery rate of 92.3%.

Conclusion: Our study demonstrated that Bruker MALDI-TOF MS Biotyper is a reliable rapid and economic tool for the identification of Gram-negative bacteria especially E. coli and Salmonella which could be used as an alternative diagnostic tool for routine identification and differentiation of clinical isolates in the bacteriological laboratory. MALDI-TOF MS need more validation and verification and more study on the performance of direct colony and extraction methods to detect the most sensitive one and also need using more samples to detect sensitivity, reliability, and performance of this type of bacterial identification.

Keywords: ABI, Bruker Daltonics, colibacillosis, Escherichia coli, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Salmonella, Salmonella pullorum.

N-terminal-pro brain natriuretic peptides in dogs and cats: A technical and clinical review

Review (Published online: 18-09-2017)

11. N-terminal-pro brain natriuretic peptides in dogs and cats: A technical and clinical review

Gabriela Vieira de Lima and Felipp da Silveira Ferreira

Veterinary World, 10(9): 1072-1082

ABSTRACT

Biomarkers are quantitative indicators of biological processes performed by an organ or system. In recent years, natriuretic peptides (NPs) have emerged as important tools in the diagnosis and therapeutic monitoring of heart diseases. Research has shown that serum and plasma levels of N-terminal pro brain NP (NT-proBNP) in dogs and cats are the only biomarkers that afford to diagnose and monitor congestive processes and, indirectly, the myocardial function of small animals. The present review discusses the peer-reviewed specialized literature about NT-proBNP and presents and compares the potential clinical applications of this NP in veterinary medicine of small animals, considering diagnosis, follow-up, and prognosis of myocardial or systemic diseases. The relevance of NT-proBNP is associated with sample stability, easy determination in laboratory, sensitivity, accuracy, and the possibility to analyze myocardial function. These advantages are specially important when NT-proBNP is compared with other cardiac biomarkers, mostly those that indicate the integrity of the myocardial cell. Fast NT-proBNP assays are marketed today and may be used in association with complementary tests. Together, these methods are an important source of information in differential diagnosis of heart and lung diseases as well in the early diagnosis of cardiopathy in dogs and cats, proving valuable tools in treatment and prognosis.

Keywords: cardiac biomarkers, cats, congestive heart failure, dogs, NT-proBPN.

Pathology and immunohistochemistry study of Newcastle disease field case in chicken in Indonesia

Research (Published online: 13-09-2017)

10. Pathology and immunohistochemistry study of Newcastle disease field case in chicken in Indonesia

Etriwati, Dewi Ratih, Ekowati Handharyani and Surachmi Setiyaningsih

Veterinary World, 10(9): 1066-1071

ABSTRACT

Aim: The aim of the study was to examine pathology and the distribution pattern of Newcastle disease virus (NDV) in internal organs of chickens from a field case using immunohistochemical staining.

Materials and Methods: 10 groups of broiler, layer, and domestic chicken were collected from necropsy room Division of Pathology, Bogor Agricultural University. These chickens were originated from West Java and collected based on pathologist diagnosis as suspect of Newcastle disease (ND). They were subsequently confirmed positive of ND with real-time-reverse transcription polymerase chain reaction assay. The respiratory, circulatory, digestive, lymphoreticular and central nervous systems were collected for histopathology examination.

Results: The gross pathology and histopathology changes were tracheitis, pneumonia, pericarditis, myocarditis, catarrhal proventriculitis, catarrhal enteritis, typhlitis, perihepatitis, pancreatitis, nephritis interstitial, splenitis, atrophy of Bursa Fabricius, and encephalitis.

Conclusion: The distribution pattern of NDV in internal organs of chickens from a field case in this study is similar with a previous reported pattern in systemic cases of the internal chicken organs. High intensity of immunohistochemistry stain result was detected in trachea, lung, proventriculus, duodenum, cecal tonsil, kidney, and brain.

Keywords: broiler, domestic chicken, immunohistochemistry, layer, Newcastle disease.

Detection of anti-Toxoplasma gondii antibodies in chronic myeloid leukemia and acute myeloid leukemia patients

Research (Published online: 13-09-2017)

9. Detection of anti-Toxoplasma gondii antibodies in chronic myeloid leukemia and acute myeloid leukemia patients

Mohammad Javad Gharavi, Mona Roozbehani and Zienat Mandeh

Veterinary World, 10(9): 1063-1065

ABSTRACT

Background and Aim: Infection of Toxoplasma gondii is a worldwide distribution. Toxoplasmosis in patients who are immunocompromised by virtue of underlying leukemia disease has received relatively little attention. This study was aimed to evaluate IgG and IgM antibodies of T. gondii and to minimize the role of T. gondii and opportunistic infection complication at the early stage of infection in leukemia patients.

Materials and Methods: The purpose of this assay was to measure anti-T. gondii IgG and IgM antibodies by enzyme-linked immunosorbent assay (ELISA) technique in leukemia patients.

Results: IgG antibodies against T. gondii were detected by ELISA in 96 (56.4%) leukemia patients and 72 (42.4%) control group. IgM antibodies were found in 10 patients (5.9%) with leukemia and 3 (1.8%) in the corresponding.

Conclusion: Our finding indicated that leukemia patients under immunosuppressive condition should not be neglected. Toxoplasmosis in leukemia patients as a main risk factor is considered, meanwhile in some patients, due to possibility of the presence of secondary infection that leads to severe toxoplasmosis.

Keywords: acute myeloid leukemia, chronic myeloid leukemia, Toxoplasma gondii.