Detection of porcine parvovirus in domestic pigs in north Kerala #

During the period from 2019 to 2021, a total of 45 tissue samples were collected from pigs in Kerala and tested for the presence of porcine parvovirus (PPV) by polymerase chain reaction (PCR) targeting NS1 gene of the virus. Of the samples tested, 4 (8.89 per cent) were found to be positive which was higher than the positivity reported for the virus in Kerala in 2016. Sequence analysis of the amplicons revealed a high degree of similarity to PPV sequences previously reported from India. Biosecurity measures should be adopted to control the spread of viral diseases.

Pigs are prolific breeders and have one of the highest feed conversion efficiencies making pig rearing lucrative.The second-most popular meat consumed worldwide is pork (Shahbandeh, 2022).The occurrence of infectious diseases can significantly lower the profitability of pig farming.Since pigs are prolific breeders, diseases causing reproductive dysfunction can have an adverse impact on the economics of pig production.Porcine parvovirus (PPV) (currently known as Ungulate protoparvovirus 1) is a single-stranded DNA virus classified under the family Parvoviridae (ICTV, 2021).The virus is one of the causative agents of stillbirth, mummification, embryonic death and infertility (SMEDI) syndrome, the other viral agents being porcine teschovirus and reproductive and respiratory syndrome virus (PRRSV) (Ehrenfeld et al., 2010).The most prominent and only clinical indication of PPV infection that has been well-established is maternal reproductive failure.In Kerala, there have been reports of reproductive dysfunction in pigs.Hence a study was undertaken to detect the presence of PPV in cases of reproductive dysfunction in pigs in Kerala.
A total of 45 tissue samples (tonsil, mesenteric lymph node, lung and spleen) were collected from pigs during the period from 2019 to 2021.The pigs were from farms with a history of reproductive disorders.From the tissues, total DNA was extracted by Qiagen DNeasy Blood and Tissue Kit as per the manufacturer's protocol.The presence of PPV genome in the clinical samples was detected by polymerase chain reaction (PCR) using specific primers for NS1 of the virus as described by Xu et al. (2012) with slight modifications.The primer sequences used were PPV FP 5' AGTTAGAATAGGATGCGAGGAA3' and PPV RP 5' AGAGTCTGTTGGTGTATTTATTGG 3' corresponding to nucleotide positions 1761-1782 and 2026-2002 respectively of NADL-2 strain of PPV (GenBank Accession number NC001718).The 25 µL reaction mixture of the PCR comprised of 12.5 μL 2X EmeraldAmp GT PCR master mix (2X) (Takara), 1 µL each of forward and reverse primers (10 pmol), 2 μL of DNA and the NFW to make up the volume.The cycling conditions were 95 o C for 2 min (initial denaturation), 35 cycles of 95 o C for 15 sec (denaturation), 54°C for 30 sec (annealing) and 72°C for 30 sec (polymerisation) followed by a single cycle at 72°C for 2 min (final extension).A no-template control, which did not contain the sample DNA, was also kept.The PCR products were resolved in agarose gels containing ethidium bromide and viewed using a gel documentation system.Specific amplicons were purified using Gene JET Gel Extraction Kit (Thermo Fisher Scientific, USA) and sent to M/s AgriGenome Lab Private Limited, Cochin, India for sequencing.BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST) of the sequence data obtained was done to confirm the presence of porcine parvovirus.
Out of the 45 samples tested, four (8.89 per cent) were positive.Three positive samples were obtained from Wayanad while one positive was from Kozhikode district (Table 1).Specific amplicons of 265 bp were observed in the positive samples (Fig. 1).This is higher than the percentage positivity reported by Aishwarya et al. (2016) wherein two out of the 38 samples (5.26 per cent) tested in Kerala were positive.This indicates that the virus is slowly spreading among pigs in the State.Reports on the detection of PPV in India are scarce and reports are mainly on the basis of detection of antibodies against the virus.In Punjab, seroprevalence ranging from 39.10 to 41.1 per cent has been reported and in the north-eastern states, a lesser prevalence ranging from 1.24 to 10.15 per cent have been reported (Kaur et al., 2016;Pegu et al., 2017;Deka et al., 2021).
On BLAST analysis, the nucleotide sequences generated in the study were found to have 98.50 to 99.50% similarity with isolates from Uttar Pradesh and 98.99% similarity with isolates from Madhya Pradesh (Table 2).
In farms, PPV spread horizontally between acutely infected and in-contact naive pigs by touch, swallowing or breathing virusladen excretions and secretions.Oronasal and transplacental infections are, respectively, the most typical entry points for postnatal and prenatal pigs (Mengeling et al., 2000) and infected pigs presumably act as important PPV reservoirs (Brown, 1981;Mengeling, 1999).However, there is no proof to support often and continuous shedding of PPV and it is more likely that a contaminated environment served as the main virus reservoir (Mengeling and Paul, 1986).
Since infection with PPV does not produce any specific clinical signs in pigs other than decreased reproductive rates, diagnosis of the condition has to be carried out by molecular methods.However, in farms with a high incidence of reproductive disorders with SMEDI, porcine parvoviral infection could be suspected.Since SMEDI could be caused by other viruses such as PRRS and porcine teschoviruses, diagnosis by molecular techniques will reveal the specific cause of the disease.In order to prevent the  spread of the virus, strict biosecurity measures must be implemented and periodic assessment of the prevalence of the disease condition has to be carried out.

table 1 .
Results and details of samples tested for PPV by PCR

table 2 .
Nucleotide identity of NS1 gene sequences of PPV with other Indian PPV sequences