The materials for the proposed study consisted of tissue samples of lungs and upper respiratory tract collected from various slaughter houses, field mortalities, private piggery farms located in and around Guwahati and from postmortem examinations carried out at Department of Pathology, College of Veterinary Science, A.A.U., Khanapara, Guwahati, Assam for a period of one year
i.e. from March, 2020 to February, 2021. A total of 180 pigs irrespective of age, sex were subjected to detailed post mortem examination. Based on gross examination, 127 pneumonic lungs samples were collected aseptically and subjected for bacteriological investigation.
Bacteriological studies
For isolation of
Streptococci, the samples (n=127) were streaked onto Brain Heart Infusion (BHI) agar plate for primary isolation of bacteria and the inoculated plates were incubated aerobically at 37°C for 24 hours. The morphological, cultural and biochemical characters were studied (Fig 1 and Fig 2) (
Cowan and Steel, 1993;
Tarradas et al., 1994).
The primary isolation of
Pasteurella spp. was done on Brain Heart Infusion agar plates and Blood Agar Base medium and incubated at 37°C for 24 hours
(Jabeen et al., 2013). On Gram’s staining of suspected colonies, small, non-motile, gram negative coccobacillus was visible. On further methylene blue staining, bipolar characteristics were visible.
For primary isolation of
Staphylococcus spp., the samples were streaked onto Mannitol Salt Agar (MSA) plates. The plates were then incubated aerobically at 37°C for 24 hours giving yellow colonies. On Gram’s staining purplish spherical and grape like clusters were observed.
For isolation of
Escherichia coli,
Klebsiella spp and
Salmonella spp., the samples were directly inoculated onto MacConkey Lactose Agar (MLA) plates for primary isolation. The plates were then incubated aerobically at 37°C for 24 hours. IMViC test was further performed for differentiation.
Molecular detection by polymerase chain reaction (PCR)
Confirmation of
Streptococcus suis was made on detection of
gdh by simplex Polymerase Chain Reaction (PCR). The PCR was performed in a thermocycler using oligonucleotide primers (Table 1) as per the reported methods of
Okwumabua et al., (2003).
For extraction of bacterial DNA BHI Agar plate grown
Streptococci cultures were enriched in 2 ml Todd Hewitt Broth by incubation at 37°C for 24 hours and then centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded and the pellet was washed with 150 µl of tris-EDTA (TE) buffer with pH 8 by centrifuging at 3000 rpm for 5 minutes and discarding the supernatant twice. To the pellet, 100 µl of sterile nuclease free water was added, mixed properly and transferred to 1.5 ml microcentrifuge tubes. The suspension was boiled for 15 to 20 minutes, snap chilled on ice for 10 minutes and then centrifuged at 12,000 rpm for 10 minutes. The supernatant was used as templates and stored at -20°C for further use. The extracted DNA concentration was measured in Nanodrop Spectrophotometer (Thermofisher scientific, USA).
Amplification of the target gene was carried out in 25 µl reaction volume containing 2× Dream Taq master mix (Thermo scientific, K1081) containing 0.05 units/µl Taq DNA polymerase in reaction buffer, 4.0 mM MgCl
2, 0.4 mM each of dNTP (dATP, dCTP, dGTP and dTTP), 4.0 µl (100-150ng) of template DNA and 0.5 µl each of the forward and reverse primers (10 pmol conc.) of
gdh gene. PCR was performed in a Thermal cycler (Applied Biosystems) with previously reported thermo cycling condition
(Okwumabua et al., 2003). The amplified products were confirmed by agarose gel electrophoresis, using 2% agarose containing ethidium bromide in 1× Tris-Acetic acid- EDTA (TAE) buffer at 80-100 V for 1-2 hrs. The gel was visualized under UV light in Gel Doc System (BioRad, USA) and images were captured by Imagelab software. DNA lysate brought from ICAR-NRC on pig, Rani was taken as positive control. A non-template control (NTC) was considered as negative control in the present PCR based studies.
Molecular characterization
For sequencing, two amplified PCR products were purified using the QIAquick PCR purification kit (Qiagen) following the manufacturer’s instructions. Phylogenetic analysis was carried out using 688 bp region encoding
gdh gene corresponding to the two
Streptococcus suis positive sequences along with 10 reference sequences retrieved from the NCBI GenBank nucleotide database. Phylogenetic and molecular evolutionary analyses were conducted using DNASTAR software with the neighbour-joining method.
