Exploring the Cause of Diarrhoea and Poor Growth in 8-11-Week-Old Pigs from an Australian Pig Herd Using Metagenomic Sequencing

Abstract

Diarrhoea and poor growth among growing pigs is responsible for significant economic losses in pig herds globally and can have a wide range of possible aetiologies. Next generation sequencing (NGS) technologies are useful for the detection and characterisation of diverse groups of viruses and bacteria and can thereby provide a better understanding of complex interactions among microorganisms potentially causing clinical disease. Here, we used a metagenomics approach to identify and characterise the possible pathogens in colon and lung samples from pigs with diarrhoea and poor growth in an Australian pig herd. We identified and characterized a wide diversity of porcine viruses including RNA viruses, in particular several picornaviruses-porcine sapelovirus (PSV), enterovirus G (EV-G), and porcine teschovirus (PTV), and a porcine astrovirus (PAstV). Single stranded DNA viruses were also detected and included parvoviruses like porcine bocavirus (PBoV) and porcine parvovirus 2 (PPV2), porcine parvovirus 7 (PPV7), porcine bufa virus (PBuV), and porcine adeno-associated virus (AAV). We also detected single stranded circular DNA viruses such as porcine circovirus type 2 (PCV2) at very low abundance and torque teno sus viruses (TTSuVk2a and TTSuVk2b). Some of the viruses detected here may have had an evolutionary past including recombination events, which may be of importance and potential involvement in clinical disease in the pigs. In addition, our metagenomics data found evidence of the presence of the bacteria Lawsonia intracellularis, Brachyspira spp., and Campylobacter spp. that may, together with these viruses, have contributed to the development of clinical disease and poor growth.

Keywords: abundance; bacteria; disease; metagenomic sequencing; phylogenetic analysis; viruses.

Figure 1

Similarity plot generated in SimPlot using the sequence MZ515502-PSV-AUS-2018 (Pig 46 Colon) as the query sequence against three other sequences using a 200 nucleotide sliding window at 20 nucleotide intervals and the F84 distance [64] model with the maximum likelihood method. Percentage identities at each analysis point were plotted on a line chart. For similarity plot analysis, the y-axis shows the percentage similarity between the reference sequences and the query sequence. Different colours are indexed for different reference sequences.

Figure 2

Phylogenetic analysis of complete nucleotide sequence of VP1 capsid region of EV-G. The nucleotide sequences were aligned and analysed using the maximum likelihood method in MEGA 7.0 [63] using the General Time Reversible (GTR + G) [70] model with a bootstrapping of 1000 replicates. The analysis involved 39 reference sequences of the VP1 capsid region of EV-Gs genome and four EV-G sequences from this study. The numbers at nodes represent bootstrap values and values above 60% are shown. Branch lengths are scaled according to the numbers of nucleotide substitutions per site. Sequences name indicated in red colour from Pig 45 colon and lung sample have been labelled with a black (⬤) and white circle (⭘), respectively while Pig 46 colon and lung sample have been labelled with a black (♦) and white rhombus (◊), respectively.

Figure 3

Phylogenetic analysis of the partial nucleotide sequences from the NS1 region of PBoV3. The nucleotide sequences were aligned and analysed using the maximum likelihood method in MEGA 7.0 [63] using the Hasegawa–Kishino–Yano (HKY + G + I) [84] model with a bootstrapping of 1000 replicates. The analysis included 21 reference sequences of the NS1 non-structural region of PBoV3 genome and two PBoV3 sequences from this study. The numbers at nodes represent bootstrap values and values above 60% are shown. Branch lengths are scaled according to the numbers of nucleotide substitutions per site. The sequence name indicated in red colour from Pig 45 lung sample has been labelled with a white circle (⭘) while the Pig 46 colon sample has been labelled with a black rhombus (♦).