The kit used for this study was Anigen, Rapid Rabies Ag Test Kit produced by Bionote, Inc. (Hwaseong-si, Korea). Brain tissue (brainstem and hippocampus) collected through the foramen magnum approach was homogenized and directly diluted at a 1:10 ratio in the assay buffer provided with the diagnostic kit. The test device was removed from the foil pouch and placed on a flat, dry surface. The sample was taken from the specimen tube using a disposable dropper provided in the kit. Four drops of the sample were added into the sample hole using the disposable dropper. The result of the test was observed within 5–10 min. The presence of two bands in the result window at position “T” (Test sample) and “C” (Control) indicated the presence of the virus (15).

An impression smear (~10 mm diameter) was prepared from two brain regions (brainstem and cerebellum) on clean, dry, pre-rinsed glass slides. Control slides were similarly prepared using a normal dog brain (negative control) and rabies-infected brain samples confirmed by DFA and RT-PCR (positive control). The smears were air-dried (10–15 min) at room temperature, fixed in chilled acetone (−80 °C, 1 h), and air-dried again. Each smear was then incubated with 40 μL of FITC-labeled anti-rabies nucleoprotein conjugate (1,100 in 1x PBS, pH 7.4) (Merck Millipore, Temecula, CA, USA) at 37 °C for 60 min in a high-humidity chamber. After staining, excess conjugate was drained, and slides were rinsed and soaked in PBS (3–5 min). Observation was made under an inverted fluorescent microscope (400 nm) (Carl Zeiss AG, Göttingen, Germany) within 2 h, confirming rabies positivity by the presence of granular intra-cytoplasmic apple-green fluorescence of aggregated nucleocapsids (3).

Impressions of suspected brain tissues, from the brainstem due to high antigen concentration, were made on labeled glass slides. All steps were conducted at room temperature. Smears were air-dried (5 min), fixed in 10% buffered formalin (10 min), and rinsed in Tween 20 and PBS (TPBS) to remove excess fixative. They were then immersed in 3% hydrogen peroxide (10 min) for cleaning, followed by another TPBS rinse. Excess buffer was shaken off, and the area around the smear was blotted dry. Slides were placed in a humid chamber, and biotinylated anti-rabies mAb was applied to the impressions, incubated for 10 min, then rinsed in TPBS. Streptavidin-peroxidase complex was added and incubated (10 min), followed by another TPBS rinse. A freshly prepared amino ethyl carbazole solution was applied (10 min), and slides were rinsed in distilled water. After blotting dry, smears were counterstained with 1:2 diluted hematoxylin (2 min), thoroughly washed, and mounted with a water-soluble medium under a coverslip. Observations under a light microscope at 20x magnification for scanning and 40x for detailed inspection revealed rabies virus antigen as red inclusions against a blue neuronal background (3).

Total RNA was extracted from brain tissues using the HiPurA® Viral RNA Purification Kit (HiMedia) with slight modifications. The RT-PCR was carried out directly from isolated RNA samples using the QIAGEN One-step RT-PCR, and the amplified PCR products were separated and visualized by gel electrophoresis in a 1.5% agarose gel. The primer used in the study is given in Table 1.

Out of the 25 brain samples tested, 19 (76%) were found to be positive. A positive outcome was indicated by the presence of a red line at both the ‘C’ and ‘T’ positions on the device. Conversely, a red line solely at the ‘C’ position signified a negative result. The positive and negative results are shown in Figure 3.

All 25 samples were assessed for the presence of viral inclusions using DFA, where 19 brain impressions (76%) exhibited a distinct apple green fluorescence, indicating the presence of rabies viral inclusions (Figure 4).

Out of the 25 samples, 19 (76%) tested positive for rabies by dRIT. The impressions showing red inclusions against a blue neuronal background were considered positive. The positive and negative results are shown in Figure 5.

All the brain samples were subjected to one-step RT-PCR for the confirmation of rabies by targeting the N-gene of RABV. The PCR results confirmed that 19 (76%) of the suspected brain samples were positive for rabies. Agarose gel image visualized under UV gel documentation system, showing RABV is indicated in Figure 6.

Lateral Flow Assay is a pen-side test following the principle of immunochromatography. In the present study, of the 25 brain samples screened for rabies, 19 were positive and had a 100% correlation with DFA. This assay also demonstrated high sensitivity and specificity in detecting rabies from brain samples.

In agreement with the present study, a study in South Africa also reported 100% sensitivity and 100% specificity of immunochromatographic tests using virus isolates representative of all African genotypes (10) and various studies conducted using different species of brain samples collected from various states in India revealed that there is 100% correlation between DFA and LFA and also has 100% sensitivity and 100% specificity (28–30).

LFA has a lot of practical utility when it comes to diagnosing rabies at the field level. The LFA can also be used for the diagnosis using saliva, serum and blood (31). Their simplicity, portability and affordability make them ideal for use in the field without any extensive lab infrastructure. However, certain concerns surround the quality of LFA. The reliability of LFA may be compromised in diagnosing rabies in degraded animal brain samples or corneal smears, underscoring the significance of maintaining optimal sample conditions. While LFA produces rapid results, its sensitivity may vary, emphasizing the need for meticulous evaluation to ensure accurate diagnosis in different scenarios (32).

Overall lateral flow device combined with the foramen magnum method of brain sampling can potentially provide great opportunities to diagnose animal rabies in resource-limited settings and also obtain rapid results. The reliability of LFA may be compromised in degraded animal brain samples, and cost-effectiveness analysis was not performed in this study. While LFA produces rapid results, a gold standard technique is a must for confirmation; validation in larger sample sizes is needed to confirm these findings.

It is considered the gold standard test for routine veterinary and human laboratory diagnosis of rabies (33). In this study, we assessed 25 brain samples for the presence of viral inclusions using the DFA, out of which 19 showed a distinct apple green fluorescence, suggesting the presence of rabies viral inclusions.

In India, several researchers have reported varying rates of overall positivity in their respective studies, reflecting regional and methodological differences. Chandranaik et al. (34) reported a positivity rate of 32.85%, while Mundas et al. (35) and Prabhu et al. (3) found higher rates of 67.54 and 64.98%, respectively. Yale et al. (15) observed a positivity of 55.56%, Chaudhary (36) reported 78%, Ghouse (28) noted 93%, Tilak (29) recorded 100%, and Govindaiah et al. (30) documented 80.65%. These findings demonstrate a broad range of positivity rates, from 32.85 to 100%. The 76% positivity rate observed in the present study aligns with this range, indicating consistency with previously documented trends across different regions and periods in India.

Collectively, these studies support the utility and reliability of DFA in detecting rabies virus in brain samples, but the constraints of this test include the requirement of an expensive fluorescent microscope, well-trained personnel, and to rule out the background autofluorescence that is produced. Moreover, the test could be subjective, suggesting that two independent readers may be necessary for routine diagnosis (37).

It is a modified immune peroxidase test developed by the Centers for Disease Control and Prevention (CDC), Atlanta, USA, to detect the RABV antigen (38). This test uses highly concentrated and purified biotinylated anti-nucleocapsid monoclonal antibodies produced in vitro in a direct staining approach and allows a diagnosis to be made in less than 1 hour using a simple microscope. As of now, dRIT has been tested in some countries, like Africa and India (11), reporting that the sensitivity and specificity of dRIT are equivalent to those of DFA.

In the present study, of the 25 brain samples screened for rabies, 19 samples were found to be positive by dRIT. It showed a 100% correlation with DFA, indicating perfect agreement. Additionally, the dRIT demonstrated 100% sensitivity and 100% specificity, showcasing its accuracy in detecting rabies. These results are in agreement with previous studies that have evaluated the efficacy of dRIT in rabies diagnosis. A study in India conducted by Madhusudana et al. (11) observed complete agreement between dRIT and DFA in 400 brain samples. Prabhu et al. (3) tested 257 brain samples and confirmed 100% corroboration between DFA and dRIT. Ghouse (28) observed 100% agreement between dRIT and DFA in 46 brain samples from different species.

Collectively, these studies endorse dRIT as a reliable rabies diagnostic and as an alternative to DFA. It is a simple and cost-effective method that utilizes a light microscope, making it accessible in various settings, particularly in developing countries with limited resources. Additionally, dRIT provides rapid results, aiding in timely diagnosis (39).

The Nucleoprotein (N) gene of the rabies virus is a highly conserved gene and hence is targeted for most immune and molecular diagnostic purposes. The present study aimed at partial N gene-based PCR. Initially, RNA was extracted from each sample and partial N gene PCR was taken up separately. Subsequently, agarose gel electrophoresis revealed amplified DNA at 605 bp.

In the present study, of the 25 brain samples screened for rabies, 19 were positive and had a 100% correlation with DFA. Additionally, the RT-PCR demonstrated 100% sensitivity and 100% specificity, showcasing its accuracy in detecting rabies. These findings are consistent with previous research on the diagnosis of rabies using molecular techniques. Araujo et al. (40) confirmed RT-PCR’s 100% accuracy in detecting rabies virus, matching DFA’s gold standard. Dandale et al. (41) reported RT-PCR and TaqMan real-time PCR had similar sensitivitiescompared to DFA. Dettinger et al. (42) in the USA further validated RT-PCR’s efficacy in confirming rabies in animal brain samples and early-stage infections, with a 100% correlation to DFA.

In India, Prabhu et al. (3) tested 257 samples and confirmed 100% corroboration between DFA and RT-PCR. Chaudhary (36) observed 100% agreement between RT-PCR and DFA in 140 samples.

Collectively, these studies endorse RT-PCR as a reliable rabies diagnostic tool, suggesting its potential as an alternative to DFA.

In the present study, the distribution of rabies cases based on the sex of the animals revealed that rabies is almost equally shared between both sexes, with females at 52.6% (10/19) compared to males at 47.4% (9/19). Contrary to the present findings made by Widdowson et al. (43) in Santa Cruz, Bolivia, Gunaseelan et al. (44), Yale et al. (45) and Bharathy and Gunaseelan (46) in Chennai found that the males were the major affected sex due to rabies in all the studies. In the present study, rabies positives were encountered by non-descript dogs (100%). The percentage of non-descriptive dogs was less in the studies conducted by Gunaseelan et al. (44) (71.2%) and 76.4% by Yale et al. (45) in Chennai. Non-descript stray dogs contributed greatly to the prevalence of rabies in other dogs and humans (46).