Guru Angad Dev Veterinary and Animal Sciences University

Aim: The present study deals with molecular technique Nested RT-PCR for detection of rabies viral RNA from biological fluid samples (Saliva, Milk and Urine) collected from animal suspected for rabies and to compare the sensitivity of Nested RT-PCR applied for ante mortem diagnosis of rabies with conventional technique (immunofluorescence) applied on neural tissue. Molecular technique Materials and Methods: Nested RT-PCR was applied on 62 biological fluid specimens collected from rabies suspected animals. First round amplification with nested set of primers (RabN1 and RabN5) yielded 1477 bp product while amplification with second round primers (RabNfor and RabNrev) yielded 762 bp product. Sensitivity of the technique was compared in accordance with WHO recommended gold standard test viz. Immunofluorescence (FAT) applied on brain samples. Results: By Nested RT-PCR, viral RNA could be detected in 9/24 (37.50%) saliva samples, 2/17 (11.76%) milk samples and 6/21 (28.57%) urine samples. Confirmatory diagnosis by Immunofluorescence performed on brain sample revealed 18 true positive cases. Overall, Sensitivity of Nested RT-PCR technique employed on fluid samples was 69.23% when compared with immunofluorescence performed on brain samples. Conclusions: Early reliable ante mortem diagnosis of rabies can be obtained from biological fluid samples of animals suspected to be rabid when tested with Nested RT-PCR technique. Key-words: milk,nested RT-PCR, rabies, saliva, urine


Introduction
minutes a life is taken away by rabies.A national multicentric rabies survey conducted by APCRI in Rabies though a dreaded disease unfortunately India in collaboration with WHO revealed an remains as a neglected disease in majority of countries incidence of 20,565 human deaths per year due to particularly in Asia [1].Rabies virus belongs to the rabies in India [3].Although the loss of livestock due genus Lyssavirus of the family Rhabdoviridae.The to rabies is significant, there are few publications on susceptibility to rabies infection depends on the virus estimates of the incidence of rabies in livestock [4].strain, genetic makeup of the host, concentration of the Rabies is a neurological disease, but the rabies neurotransmitter receptors at the site of bite, virus spread to several organs outside the central inoculums size and very importantly proximity of the nervous system (CNS).The rabies virus antigen or bite to the central nervous system of the host.Rabies RNA has been identified from the salivary glands, has been a continuing problem in several countries lungs, kidneys, heart and liver [5].Infection of across the globe and posed a challenge in front of salivary glands (and thus viral excretion) depends on researchers [2].
centrifugal neural spread of virus from the central Rabies is a major health problem in India too and nervous system (CNS) and transfer of virus from is responsible for extensive morbidity and mortality.axons to glandular epithelial cells.In addition to the Animals as well as human cases are reported from all salivary glands, the adrenal medulla, nasal mucosa, over the country.It is estimated that in India, every 30 cornea and epidermis are frequently infected via spread in peripheral nerves [6].With the help of high-capacity cDNA reverse transcription kit (Applied advance molecular approaches rabies virus RNA can Biosystems, USA).be detected in a range of biological fluids and tissue Reverse transcriptase (Applied Biosystems, samples e.g.saliva, CSF, tears, skin biopsy sample and USA) mix was prepared and subjected to conditions urine [7].
25°C for 10 min, 37°C for 2 h, 85°C for 5 min and chilling Thus the present study was envisaged to on ice for 5 min in a thermal cycler (Eppendorf).RNA evaluate the importance of Nested RT-PCR technique and cDNA concentration was measured using Nano for ante mortem diagnosis of rabies from biological Drop Spectrophotometer (Nanodrop Technologies, fluid samples and to compare the sensitivity of Nested CA) in ng/µl and quality was checked as a ratio of OD RT-PCR assay with immunofluorescence.260/280.

Materials and Methods
Nested RT-PCR Assay: The procedure used for the nested RT-PCR based on N (Nucleoprotein) gene was In the present study, samples were collected for that used earlier [8-10] with minor modifications.diagnosis of rabies from 25 animals suspected for Briefly 12 µl of cDNA was subjected to a first round rabies presented to the Veterinary Clinics, amplification using RabN1 and RabN5 primers (30 GADVASU, Ludhiana, Punjab and Civil Veterinary pmol/µl) (Table 1), dNTP's and Taq DNA polymerase Hospital from different districts of Punjab over a for 95°C for 2 min followed by 35 cycles of 95°C for 1 period of seventeen months from December 2010 to min, 55°C for 1 min, 72°C for 1 min 30 s and a final May 2012.Out of 25 animals, saliva samples were extension step at 72°C for 5 min.obtained from 24 (14 buffaloes, 4 cows, 3 cow calves For the second round, 5 µl of first round PCR and 3 dogs), milk samples from 17 (14 buffaloes and 3 product was amplified using RabNfor and RabNrev cows) and urine samples from 21 (14 buffaloes, 1 cow, (Table 1) and subjected to thermocycling conditions as 3 cow calves and 3 dogs) animals suspected for rabies.
first round except annealing at 55°C and extension for Thus the total of 62 biological fluid samples served as 1 min.The amplified PCR products were loaded on a basis for current investigation.
agarose gels along with positive control, negative Soon after the clinical diagnosis was made, control and DNA ladder (100 base pair plus, Fermentas).saliva (collected directly or by syringe aspiration), The agarose gels were visualized under Geldoc (Biomilk (strip milking), urine samples (direct while Rad).urinating or urethral catheterization) were collected in sterilized vials from the animals suspected to be rabid.Results The vials were closed tightly to avoid any contami-An attempt was made to detect rabies viral RNA nation and stored at -80ºC until further processing of from secretions (saliva, milk) and excretion (urine samples.General data about the animals, such as samples) by Nested RT-PCR technique (Table 2).9 out estimated age, sex, clinical signs, ownership status, of total 24 saliva samples collected were detected sample details etc. were recorded.Saliva, milk and positive for rabies by Nested RT-PCR with a urine samples obtained from two healthy animals sensitivity of 68%.In milk rabies viral RNA was served as negative controls.Rabies positive brain detected in 2 out of total 17 milk samples collected homogenate was used as positive control.
with a sensitivity of 54.54% while 6 out of 21 urine Total RNA from all fluid samples, positive and samples were found positive for rabies with a negative controls was extracted using Qiazol (Qiagen, sensitivity of 62.50%.USA) according to the manufacturer's instructions.
Overall, Sensitivity of Nested RT-PCR technique The RNA was subjected to cDNA synthesis employed on fluid samples was 69.23% when using a primer RabN1 (30 pmol/µl) and subjected to compared with immunofluorescence performed on 65ºC for 10 min and was later snap cooled on ice and brain samples.briefly spun down.cDNA synthesis was done using

Discussion
confirmatory ante mortem diagnosis of rabies from body secretions and excretions within a short span of More sensitive molecular technique such as time.conventional PCR assay has produced satisfactory results for detection of rabies virus RNA from brain Author' s contribution tissue and saliva [9,11].Real time PCR assay on saliva CKS: Substantial contribution to conception and samples was more sensitive than conventional RTdesign, MD: Acquisition of data, CKS, VR, DD, BSS, PCR assay (sensitivity 75% versus 37%) [11].NKS: Analysis and Interpretation, MD, KB: Drafting Positivity of 17.6% (6/34) was obtained with the use of the article, CKS, VR, DD, BSS, NKS: revising it heminested RT-PCR on urine samples [12] whereas critically for important intellectual content.All urine and saliva testing using nucleic acid base authors read and approved the final manuscript.

Table - 1
. Primers used for Nested RT-PCR PCR for ante mortem diagnosis of rabies from body secretion/excretion of animalsTable-2.Details of results of Nested RT-PCR on secretions and excretion