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ThesisItem Open Access MOLECULAR DETECTION AND CHARACTERIZATION OF FOOT AND MOUTH DISEASE VIRUS (FMDV) AND STUDY OF CYTOKINE EXPRESSION IN NATURALLY INFECTED LOCAL/CROSSBRED CATTLE FROM ASSAM(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2021-09) BRAHMA, DERHASAR; Sharma, K.Foot and mouth disease (FMD) is a transboundary and the most contagious disease of cloven-hoofed animals including domestic and wild ruminants and pig, and has a great potential for causing severe economic loss due to loss of production and deprivation from international trade of animal products to FMD free countries. FMDV may occur in all the secretions and excretions of acutely infected animals, including expired air. Following recovery from the acute stage of infection, infectious virus may persist in the oropharynx of some ruminants (carriers), where live virus or viral RNA may continue to be recovered from oropharyngeal fluids and cells for upto 6 months or more. In this study, besides Sandwich ELISA, molecular detection and typing of FMDV was done using multiplex Reverse Transcription Polymerase Chain Reaction (mRTPCR), Reverse Transcription Loopmediated Isothermal Amplification (RT-LAMP) and SYBR Green real-time PCR targeting 3D gene. Isolation and molecular characterization of FMDV by sequencing was done. Also, study of expression of cytokines like interferon (IFN-α, IFN-β, IFN-) as well as certain interleukins (IL-1α, IL-1β, IL-2, IL-6, IL-10 and IL-12) and tumour necrosis factor (TNF-α) was estimated at mRNA level by SYBR Green real-time PCR from whole blood (White Blood cells) samples during the natural infection and during the period of persistence. This study was carried out in a total of 129 animals, comprising of 93 crossbred (vaccinated) and 36 local (non-vaccinated) cattle and additionally 12 healthy in-contact animals were taken as control animals. For carrying out this study, Tissue (n=29), whole blood (n=36) and oropharyngeal fluid (n=190) samples were collected as per standard procedure in 50% glycerol, EDTA and 0.8 M PBS/transport media, respectively. OP fluid was collected from recovered animals until complete recovery (i.e. 1st, 3rd, 6thand 9thmonth) from FMD infection. All the RNA extractions were done using Qiagen RNA extraction kit. We found that, out of 29 tissue samples, 20 samples were positive for serotype O, 9 were positive for serotype A and none of the samples was positive for Asia-1 by the multiplex RT-PCR as well as RT-LAMP. FMDV could be detected in 86.21%, 100%, 100% and 100% of tissue samples by sandwich-ELISA, mRT-PCR, RT-LAMP and SYBR Green real-time PCR respectively. Sensitivity test was run using 10 fold serial dilution of RNA extracted from FMDV antigen and found that, the real-time PCR was more rapid and highly sensitive technique of all, secondly the RT-LAMP, followed by the mRT-PCR. From the follow-up cases of the FMD recovered cattle, 38 (23.75%), 47 (29.38%) and 49 (30.63%) OPF samples (n=178) were found to be positive for FMDV by the multiplex RT-PCR, RT-LAMP and SYBER Green real-time PCR respectively, indicating persistence (carriers).The SYBR green real-time PCR was very much useful for detection of persistence from the OPF samples.However, OPF (n=12) and blood (n=12) samples from all the healthy controls and blood (n=12) from persistent animals were negative for FMDV. All blood samples (100%, n=12) from the clinically FMD infected cattle were positive for FMDV. The persistence of FMDV in oropharyngeal region of cattle lasted for upto 3 to 4 months in most of the FMD infected cattle. Persistence in crossbred (vaccinated) cattle didn’t last for more than 4 months. Only 2 Local non-vaccinated cattle (1.6%) was found to have persistence upto 6-7 months after infection. The overall number of persistent animals and the rate of persistence in cattle (n=129) at 1st month, 3rd month and 6th month were 32 (24.81%), 15 (11.26%) and 2 (1.6%) respectively, and was slightly higher in the local non-vaccinated compared to the crossbred vaccinated cattle. No statistical significance was observed between the two groups as the P value was found to be 0.23 (>0.05) and the Chi-square value was 5.57. The sequencing results showed that the Serotype O sequence (MZ501211-G-02- 19, MZ501212-G-03-19 and MZ501213 Op) shared 98.81% identity with Pakistan isolate MN953620, 96.43% identity with India isolate KY579948.1 (Nagaland, submitted by RRC Assam) and 94.05% identity with India complete genome isolate MN983158.1; and theSerotype A sequence (MZ501214-Mg/01/19) shared 95.29% identity with Indian isolate HQ832583.1 and 94.24% identity with Bangladesh isolate KT982204. The identity range was 98.81%-96.43% and 95.29%-92.22% for type O and A respectively, based on the nucleotide sequence Blast search in NCBI. The multiple sequence alignment showed that there are some minor changes in the nucleotide sequences with the consensus sequences. There were nucleotide insertions in the 3953 and 3954 positions in two of the query sequences of FMDV type O. Whereas, in FMDV type A, there were nucleotide insertions at 3807, 3813-3815 and 3841 positions and deletions at 3771 and 3874 positions of the nucleotide sequences. The result from this study shows that cytokine genes had general trend of upregulation during acute infection and decreased level of expression or down regulation during persistence. Cytokines in blood were generally upregulated in both acute infection and persistence, but compared to acute, there was decreased mRNA level of expression of cytokines during persistence except the down regulation of IFN-β, IL-2 and IL-6, whereas, all but IFN-α and IL-1α were down regulated in OPF during persistence. These cytokines may have certain role in persistence of FMDV by suppression of immune response and also by having anti-inflammatory or immunomodulatory response in carrier cattle. Thus, from this study, we can conclude that, molecular detection techniques are the most sensitive and specific techniques for detection of FMDV and particularly for diagnosis of persistence from OPF samples. Persistence occurred in 32 cattle (25%) after 1st month of the FMDV infection, out of which the proportion of local non-vaccinated cattle was slightly higher. And that cytokines may have a role in persistence of FMDV in cattle.
