S.D. Narnaware

Phylogenetic analysis of immunomodulatory protein genes of camelpoxvirus obtained from India

The haemagglutinin (HA) encoding gene and genes encoding for immunomodulatory proteins i.e., schlafen-like protein, epidermal growth factor and golgi anti apoptotic protein of camelpoxvirus (CMLV) obtained from Indian dromedarian camels were cloned and characterized. In this study, the size of the HA encoding gene obtained from the Indian CMLV is 941 bp which is only partial. Sequence analysis of schlafen-like protein gene revealed that CMLV obtained from India shared 99.6% identity with CMLV-Iran and CMLV-Kazakhstan strains both at nucleotide and amino acid level. The size of epidermal growth factor (EGF) gene of Indian CMLV obtained in this study was 418 bp, which was due to the addition of one cytosine residue position 132 of EGF gene of Indian CMLV. Sequence analysis revealed that the Golgi anti-apoptotic protein (GAAP) of Indian CMLV shared 99.5% sequence identity both at the nucleotide and amino acid level with CMLV-Kazakhstan. Based on the nucleotide and amino acid sequence identities and phylogenetic analyses of these genes, it is found that CMLV-India is forming a cluster with Kazakhstan and Iranian CMLV isolates.

Sequence analysis of topoisomerase gene of pseudocowpoxvirus isolates from camels (Camelus dromedarius)

Topoisomerase gene of pseudocowposvirus from Indian dromedarian camel was amplified by PCR using the primers of PCPV from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of amino acid identity revealed that Indian PCPV of camel shared 95.9-96.8 with PCPV of reindeer, 96.2-96.5 with ORFV and 87.5 with BPSV.

Comparison of virokine from camel pseudocowpoxvirus (PCPV) with Interleukin 10 of the Dromedary camel (Camelus dromedarius)

Cellular interleukin-10 (IL-10) gene from the peripheral blood mononuclear cells of the healthy Dromedary camel (Camelus dromedarius) and viral IL-10 (vIL-10) from the skin scabs of the Dromedary camels infected with contagious ecthyma (a parapoxviral infection in the camels) were amplified by polymerase chain reaction, cloned and characterized. Sequence analysis revealed that the open reading frame (ORF) of dromedarian camel IL-10 is 537 bp in length, encoding 178 amino acid polypeptide while open reading frame of vIL-10 from camel is 561 bp, encoding 187 amino acid polypeptide. The Dromedary camel IL-10 exhibited 62.6% and 68.5% sequence identity at the nucleotide and amino acid level, respectively, with vIL-10 from camel. Sequence analysis also revealed that the Dromedary camel IL-10 shared 99.4% and 98.3% identity at the nucleotide and amino acid level, respectively, with the Bactrian camel (Camelus bactrianus). But vIL-10 from camel shared 84.7% and 83.4% sequence identity at the nucleotide and amino acid level, respectively, with vIL-10 from reindeer (Rangifer tarandus), which is a ruminant species belonging to the order Artiodactyla. The present study was conducted to evaluate the evolutionary origin of the camel parapoxvirus with parapoxviruses of cattle and sheep and the resultant sequence analysis revealed that camel parapoxvirus is closely related to cattle parapoxvirus than sheep parapoxvirus (Orf virus).

Pathological and diagnostic investigations of abortions and neonatal mortality associated with natural infection of Brucella abortus in dromedary camels

Brucellosis is a significant zoonotic disease that is emerging or reemerging in many parts of the world. This study was carried out to diagnose and investigate the pathological lesions associated with Brucella abortus in the fetuses and placenta of naturally infected dromedary camels. For diagnosis, the blood samples from infected dams were subjected to Rose Bengal Plate Test (RBPT) and blood PCR. The placental and fetal lung tissue samples were subjected to pathological examination and PCR. Nine cases of abortions and one case of early neonatal mortality was reported in the infected camels. The hematological parameters in infected dams revealed anemia and leukocytosis. The important pathological lesions in the infected fetuses were subcutaneous edema, moderate amount of serohemorrhagic fluid in the body cavities, interstitial pneumonia, degenerative changes in the liver, severe congestion with mononuclear infiltration in the kidney, and congestion in other visceral organs. The placentas were thickened, edematous, and showed necrosis along with mononuclear infiltration in histopathology. The RBPT and PCR for placental and fetal lung tissues detected all ten cases positive for B. abortus infection. However, blood PCR was positive only in two pregnant camels. The findings of the study indicated that B. abortus infection in pregnant dromedary camels causes necrotizing placentitis and fetal pneumonia resulting into abortion during mid to last trimester of pregnancy. The RBPT was found to play an important role in early serological diagnosis, whereas PCR was useful in confirmatory diagnosis of brucellosis from placental and fetal lung tissue samples.

Molecular characterization of Camelpox virus isolates from Bikaner, India: Evidence of its endemicity

Camelpox is an important viral disease of camels, which may produce mild skin lesions or severe systemic infections. Camelpox virus (CMLV) isolates retrieved from an incidence of camelpox in camels at Bikaner, India were characterized on the basis of genotype and pathotype. Histopathological examination of the CMLV scab revealed intracytoplasmic-eosinophilic inclusion bodies. The phylogenetic analysis of all eight CMLV isolates for C18L gene nucleotide sequence revealed its clustering with its strains M-96 from Kazakhstan and CMS from Iran. The study will help to understand the transmission chain, pathobiology, and epidemiology of circulating CMLV strains. The full genome sequencing of some of the exemplary samples of CMLV is recommended in order to plan and implement a suitable control strategy.

Camelpox: A brief review on its epidemiology, current status and challenges.

Camelpox caused by a Camelpox virus (CMLV) is a very important host specific viral disease of camel. It is highly contagious in nature and causes serious impact on health even mortality of camels and economic losses to the camel owners. It manifests itself either in the local/mild or generalized/severe form. Various outbreaks of different pathogenicity have been reported from camel dwelling areas of the world. CMLV has been characterized in embryonated chicken eggs with the production of characteristic pock lesions and in various cell lines with the capacity to induce giant cells. Being of Poxviridae family, CMLV employs various strategies to impede host immune system and facilitates its own pathogenesis. Both live and attenuated vaccine has been found effective against CMLV infection. The present review gives a comprehensive overview of camelpox disease with respect to its transmission, epidemiology, virion characteristics, viral life cycle, host interaction and its immune modulation.

Comparative sequence analysis of double stranded RNA binding protein encoding gene of parapoxviruses from Indian camels

The dsRNA binding protein (RBP) encoding gene of parapoxviruses (PPVs) from the Dromedary camels, inhabitating different geographical region of Rajasthan, India were amplified by polymerase chain reaction using the primers of pseudocowpoxvirus (PCPV) from Finnish reindeer and cloned into pGEM-T for sequence analysis. Analysis of RBP encoding gene revealed that PPV DNA from Bikaner shared 98.3% and 76.6% sequence identity at the amino acid level, with Pali and Udaipur PPV DNA, respectively. Reference strains of Bovine papular stomatitis virus (BPSV) and PCPV (reindeer PCPV and human PCPV) shared 52.8% and 86.9% amino acid identity with RBP gene of camel PPVs from Bikaner, respectively. But different strains of orf virus (ORFV) from different geographical areas of the world shared 69.5–71.7% amino acid identity with RBP gene of camel PPVs from Bikaner. These findings indicate that the camel PPVs described are closely related to bovine PPV (PCPV) in comparison to caprine and ovine PPV (ORFV).

Sequence analysis of the Toll-like receptor 2 gene of old world camels.

The Toll-like receptor 2 (TLR2) gene of old world camels (Camelus dromedarius and Camelus bactrianus) was cloned and sequenced. The TLR2 gene of the dromedary camel had the highest nucleotide and amino acid identity with pig, i.e., 66.8% and 59.6%, respectively. Similarly, the TLR2 gene of the Bactrian camel also had the highest nucleotide and amino acid identity with pig, i.e., 85.7% and 81.4%, respectively. Dromedary and Bactrian camels shared 77.9% nucleotide and 73.6% amino acid identity with each other. Interestingly, the amidation motif is present in camel (Dromedary and Bactrian) TLR2 only, and the TIR domain is absent in Dromedary camel TLR2. This is the first report of the TLR2 gene sequence of Dromedary and Bactrian camels.

Vertical transmission of Trypanosoma evansi in dromedary camels and studies on fetal pathology, diagnosis and treatment.

In the present study, a total of 14 (73.68%) cases of abortions and two (100%) cases of still births were detected positive for Trypanosoma evansi infection by wet and dry blood smear examination and fetal tissue PCR in camels of an organized farm. The abortions in infected dams were recorded from 8 to 11.5 month of gestation, however majority occurred during 9th to 10th month. The important laboratorial findings in infected dams were anemia, hypoglycemia, hyperproteinemia and leukocytosis. At necropsy the T. evansi infected aborted and still born fetuses showed subcutaneous edema, presence of moderate amount of dark red hemolysed blood in thoracic and abdominal cavity, bronchopneumonia, hepatic necrosis and acute congestion in all vital organs. Microscopically, there was severe congestion, thickening of bronchial and alveolar wall and mononuclear infiltration in the fetal lung, necrotic and degenerative changes in the liver, nephritis along with severe congestion and tubular necrosis in the kidneys and necrotic and degenerative changes and congestion of capillaries in the brain. The T. evansi DNA was detected by PCR from blood, lung, spleen, liver, kidney and brain of all the infected aborted and still born fetuses. The results of the study indicated that T. evansi can cross placental barrier and cause pathological events in the fetus resulting into abortion or still birth in pregnant camels.

Development and Utilization of VHH Antibodies Derived from Camelus Dromedarius Against Foot-and-Mouth Disease Virus

ABSTRACT Foot-and-mouth disease (FMD) is an acute, highly contagious, and economically devastating viral disease of domestic and wildlife species. For effective implementation of FMD control program, there is an imperative need for developing a rapid, sensitive, and specific diagnostics which help in the identification of serotypes involved in the outbreaks. The humoral immune response of the Camelidae is unique since in these animals 75% of circulating antibodies are constituted by heavy-chain antibodies and 25% are conventional immunoglobulin with two identical heavy chains. In the present study, we developed and characterized FMD virus-specific single-domain heavy-chain antibodies (VHHs) against inactivated whole-virus antigens of FMDV serotypes O (INDR2/1975), A (IND40/2000), and Asia 1 (IND63/1972) vaccine strains. After six rounds of panning and enrichment, these VHHs were stably expressed in Escherichia coli cells. The VHHs directed against outer capsid proteins of FMD virus were successfully utilized as the capture antibody in liquid-phase blocking ELISA (LPBE) thus replacing rabbit coating antibodies. Our study demonstrated the utility of FMD virus-specific VHHs as potential candidates in FMD research and diagnostic application.