High Prevalence of Porcine Circovirus 3 in Hungarian Pig Herds: Results of a Systematic Sampling Protocol

Abstract

Porcine circovirus type 3 (PCV3) is an emerging pathogen that has been reported worldwide in all ages of healthy and clinically ill pigs. The presence of this virus in Hungary has been confirmed in a commercial farm experiencing reproductive failures, but there were no data on the circulation of PCV3 in the country. Here we report the prevalence and the genetic diversity of PCV3 in Hungarian herds. To estimate the prevalence, 1855 serum samples, 176 oral fluid and 97 processing fluid samples were collected in a systematic, cross-sectional method from 20 large scale swineherds and tested by real-time qPCR. PCV3 was present in at least one type of diagnostic matrix in 19 out of the 20 (95%) pig farms. The highest detection rates were observed in the processing fluid samples (61%), but 41% of the oral fluid and 23% of the serum samples were positive. The virus was found in all age groups, and slightly more adult animals were infected than growing pigs, but the viral burden was lower amongst them. Phylogenetic analysis of nine complete genomes, obtained from either the sampled herds or organ samples of PCV3-positive carcasses, showed high nucleotide identity between the detected sequences, which all belonged to the PCV3a genotype. Our results indicate that PCV3 is widespread in Hungary, but in most cases, the virus seems to circulate subclinically, infecting all age groups and production phases without the presence of apparent clinical disease.

Keywords: PCV3; oral fluid; phylogeny; prevalence; processing fluid; whole genome.

Figure 1

Map of Hungary showing the geographic location of the sampled farms (red/green) and the origin of PCV3-positive cases (blue) used for genome sequencing.

Figure 2

Boxplots representing the Ct values of the PCV3-positive samples of the three different sample types. The whiskers show the minimum and the maximum, and the “+” signs represent the average. The horizontal lines of the box show the first quartile, the median and the third quartile. The statistical comparison of the Ct values was performed using the Mann–Whitney test. The asterisks above the boxes represent the statistically significant differences (***: p < 0.001).

Figure 3

Percentages of PCV3-positive processing fluids and serum sample pools of different age groups. The statistical comparison of the PCV3 prevalence of each age group was performed by Fisher’s exact test. The asterisk above the columns represents the statistically significant difference (*: p < 0.05).

Figure 4

Boxplots representing the Ct values of the PCV3-positive processing fluids and serum sample pools of different age groups. The whiskers show the minimum and the maximum, and the “+” signs represent the average. The horizontal lines of the box show the first quartile, the median and the third quartile. The statistical comparison of the Ct values was performed using the Mann–Whitney test. The asterisks above the boxes represent the statistically significant differences (*: p < 0.05; **: p < 0.01).

Figure 5

(A–C) Percentages of PCV3-positive processing fluid and serum samples of different age groups from Farm K (A), Farm JA (B) and Farm SZ (C). (D–F) Boxplots representing the Ct values of the PCV3-positive processing fluid and serum samples of different age groups from Farm K (D), Farm JA (E) and Farm SZ (F). The whiskers show the minimum and the maximum, and the ”+” signs represent the average. The horizontal lines of the box show the first quartile, the median and the third quartile.

Figure 6

Phylogenetic analysis of PCV3 full-genome sequences. The phylogenetic tree was constructed using MEGAX [35] with the Maximum Likelihood method performing 1000 replicates of bootstrap analysis. A total of 41 reference sequences listed by Franzo et al. [33] were aligned against the sequences detected in this study. The sequences obtained from serum samples collected on the examined farms are presented by red dots. The blue dots mark the sequences obtained from PCV3-positive clinical cases.