Ante-mortem and Post-mortem Diagnosis Modalities and Phylogenetic Analysis of Rabies Virus in Domestic and Wild Animals of Gujarat, India.

In the present study, total of 32 ante-mortem (AM) samples (saliva = 18 and corneal smears = 14) from six animal species (cattle = 5; camel = 1; goat = 1; horse = 1; buffalo = 4; dog = 6) and 28 post-mortem (PM) samples of domestic (cattle = 6; camel = 1; goat = 1; buffalo = 5; dog = 7) and wild animals (lion = 4, mongoose = 2; bear = 1; leopard = 1) were examined for rabies diagnosis in Gujarat, India. Direct fluorescent antibody test (dFAT) and reverse transcriptase polymerase chain reaction (RT-PCR) were applied on AM samples, whereas along with dFAT and RT-PCR, histopathological examination, immunohistochemistry (IHC) and real time PCR (qPCR) were used for PM diagnosis. Nucleotide sequencing of full nucleoprotein (N) and glycoprotein (G) genes were carried out upon representative amplicons. In AM examination, 7/18 saliva and 5/14 corneal impressions samples were found positive in dFAT and 8/18 saliva samples were found positive in RT-PCR. In PM examination, 14/28 samples showed positive results in dFAT and IHC with unusual large fluorescent foci in two samples. In histopathology, 11/28 samples showed appreciable lesion and Negri bodies were visible in 6 samples, only. Out of 23 brain samples examined. 12 samples were found positive in N gene RT-PCR and qPCR, and 10 samples in G gene RT-PCR. Phylogenetic analysis of N gene revealed that test isolates (except sample ID: lion-1; lion, Gir) form a close group with sequence ID, KM099393.1 (Mongoose, Hyderabad) and KF660246.1 (Water Buffalo, Hyderabad) which was far from some south Indian and Sri Lankan isolates but similar to Indian isolates from rest of India and neighboring countries. In G gene analysis, the test isolates form a close group with sequence ID, KP019943.1. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01126-0.

Antibiotic Susceptibility Pattern of Canine Coagulase Positive and Coagulase Negative Staphylococcus spp. in a Hot and Dry Region of India.

The emergence of antimicrobial resistance among Staphylococcus spp. isolated from clinical cases of canines should be continuously monitored hence the present study was formulated to ascertain the antibiotypes and methicillin resistance in coagulase positive and coagulase negative staphylococci of canine skin and associated mucous membrane affections from a hot and dry region of India. A total of 165 clinical samples were collected and Staphylococcus aureus was identified by conventional bacteriological methods and PCR. Antibiotic susceptibility test was done against commercially available antibiotic impregnated discs as per Clinical and Laboratory Standards Institute (CLSI) method. Methicillin resistance was determined by plate methods and then via PCR of mecA gene. These 165 samples yielded, 88 (53.33%) isolates of genus Staphylococcus and 46 S. aureus and 51/88 (57.95%) isolates were coagulase positive staphylococci. Total 55 (62.5%) isolates showed susceptibility to Ceftriaxone/Sulbactum, 37 (42.05%) to Ciprofloxacin, 26 (29.55%) to Oxacillin, 24 (27.27%) to Penicillin, and 10 (11.36%) to Gentamicin. Total 21 methicillin-resistant S. aureus (MRSA) and 12 methicillin-resistant coagulase negative staphylococci (MRCoNS) were found on phenotypic basis whereas the mecA gene was detected in 6/21 MRSA and 2/12 MRCoNS isolates. Staphylococcus spp. showed increased level of resistance against commonly used antibiotics. The higher prevalence of methicillin resistance found with phenotypic methods than to mecA PCR indicates toward additional mechanisms responsible for emergence of MRS, especially in CoNS.

VP2 gene sequencing based Geno-grouping of infectious bursal disease viruses isolated from Gujarat and Maharashtra state (India).

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), has recently been reported in chickens vaccinated with classical or intermediate types of vaccines from various regions of India due to the emergence of novel very virulent strains of infectious bursal disease virus (vvIBDV). In the present study, suspected samples of IBD were collected from poultry flocks of districts of Gujarat and Nagpur (Maharashtra), identified using PCR and grouped as per traditional and new genogrouping pattern. Out of 54 bursa samples, 21 (38.89%) yielded the expected amplicon of 743 bp (701-1444 bp), and were found positive for IBDV. Among these 21 positive flocks, 11 (52.38%) were already vaccinated. Upon nucleotide sequencing of amplicon and its deduction into amino acids, it was found that all the sequences of present study were related to vvIBDV according to old classification pattern. Considering the new genogrouping pattern, nine and four sequences of this study fell within G3a and G3b lineage, respectively. These sequences revealed important differences at key amino acid positions with respect to classical (G1 genogroup), variant (G2 genogroup) type of IBDV and classical vaccines. Further divergence from prototypic vvIBDV strains was revealed as, D-N at 212 position (N = 9) and 279 position (N = 1). In sequences from Maharashtra (group 2 of G3a lineage), occurrence of V instead of P/T/A at 222 position was recorded as a novel and conspicuous substitution in the immunodominant peak A of VP2 hypervariable region. Additional changes at 270 (3 sequences) and 272 positions (4 sequences) could be attributed to reverse mutation or recombination with vaccine strains. In conclusion, both point mutation and genetic reassortment with intermediate type of vaccines were found to be responsible for generation of novel vvIBDV strains in this area which belonged to G3a and G3b genogroups.

Evaluation of the oncolytic potential of R2B Mukteshwar vaccine strain of Newcastle disease virus (NDV) in a colon cancer cell line (SW-620).

Virotherapy is emerging as an alternative treatment of cancer. Among the candidate oncolytic viruses (OVs), Newcastle disease virus (NDV) has emerged as a promising non-engineered OV. In the present communication, we explored the oncolytic potential of R2B Mukteshwar strain of NDV using SW-620 colon cancer cells. SW-620 cells were xenografted in nude mice and after evaluation of the safety profile, 1 x 107 plaque forming units (PFU) of NDV were inoculated as virotherapeutic agent via the intratumoral (I/T) and intravenous (I/V) route. Tumor growth inhibition was compared with their respective control groups by gross volume and histopathological evaluation. Antibody titer and virus survival were measured by hemagglutination inhibition (HI)/serum neutralization test (SNT) and real-time PCR, respectively. During the safety trial, the test strain did not produce any abnormal symptoms nor weight loss in BALB/c mice. Significant tumor lytic activity was evident when viruses were injected via the I/T route. There was a 43 and 57% tumor growth inhibition on absolute and relative tumor volume basis, respectively, compared with mock control. On the same basis, the I/V route treatment resulted in 40 and 16% of inhibition, respectively. Histopathological examination revealed that the virus caused apoptosis, followed by necrosis, but immune cell infiltration was not remarkable. The virus survived in 2/2 mice until day 10 and in 3/6 mice by day 19, with both routes of administration. Anti-NDV antibodies were generated at moderate level and the titer reached a maximum of 1:32 and 1:64 via the I/T and I/V routes, respectively. In conclusion, the test NDV strain was found to be safe and showed oncolytic activity against the SW-620 cell line in mice.