Molecular epidemiology of Pneumocystis carinii f. sp. suis

Pneumocystis (P.) spp. are a group of highly diversified opportunistic fungi which are adapted to the lungs of a large variety of mammals. High numbers of Pneumocystis carriers have been described in rats and humans, but also in preliminary studies of pigs prevalences between 51% in pigs with pneumonia and 73% in pigs without respiratory disorders were observed. In humans, Pneumocystis colonization has several potentially important clinical effects as development of acute pneumonia, transmission to other hosts, or involvement in the progression of certain lung diseases as cofactor. As different age classes of pigs may be kept together in a single facility and thus share the same microbiological environment, these animals are predisposed to polymicrobially caused diseases and in modern pig production systems respiratory disorders pose one of the main health problems. Preliminary data have shown that Pneumocystis can essentially contribute to polymicrobially caused respiratory diseases and in case of infections especially of young pigs the economic impact can be quite high, because developmental deficits early in life can hardly be compensated for. Currently, the genomic landscape of P. carinii f. sp. suis (referred as P. suis in this proposal) is almost completely unknown. Whole genome sequencing of P. suis will reveal its genome content and structure, and metabolic and other biological pathways, and provide insights into its unique characteristics compared to other Pneumocystis species. This work will contribute to a better understanding of the origination and development of Pneumocystis pneumonia in pigs, and potentially guide the development of new strategies for diagnosis, treatment and prevention. The examination of samples from different farms will provide an excellent opportunity to examine if different P. suis genotypes exist and if they are associated with P. suis organism loads, pigs ages, clinical signs and co-infections and to study the P. suis epidemiology in Austrian pig herds.

Pneumocystis species are a group of highly diversified opportunistic fungi which are adapted to the lungs of a large variety of mammals. The genome landscape of Pneumocystis (P.) carinii f. sp. suis (referred as P. suis in this abstract) was completely unknown before we initiated this project. Hence, our first aim was to sequence the genome of P. suis and use comparative genomics to highlight genetic features that allow the fungus to infect pigs. We encountered two significant roadblocks during the initial phase of the project. Infected pig samples did not contain enough of P. suis organisms for sequencing. Furthermore, all the pigs were infected by multiple genetically distinct populations which made impossible to construct a homogenous reference genome. Our first genome assembly attempt based on Illumina sequencing produced a chimeric genome assembly. We applied a different approach based on Nanopore technology and ad hoc haplotype phasing to obtain a reference genome sequence for multiple P. suis populations at once. Preliminary phylogenetic analysis of the current data suggested the presence of multiple highly divergent P. suis species. Our second aim was to establish a P. suis genotyping system to facilitate the investigation of P. suis epidemiology. We genotyped the samples using six genomic loci. We observed co-infections in all samples with an intrasample genetic heterogenicity up to 20%. Additionally, in some genes single nucleotides differed and even subvariants could be defined. Genotype variation was also observed within samples obtained on the same day from the same farm. The third aim of this study was to investigate the P. suis epidemiology in pigs in connection with other relevant respiratory pathogens in Austrian farms. We longitudinally quantified P. suis and 13 other pulmonary pathogens in a cohort of 50 pigs from five Austrian farms with a history of respiratory disease at five time points between the first week and the fourth month of life. The fungus was present as early as the suckling period, albeit in low loads. Over time, both the organism load and prevalence (up to 88% of positive animals in the third month) increased in each farm. The relative prevalence of various co-infection patterns was significantly different over time. Sequencing and subsequent genotyping results of P. suis indicate a heterogeneous organism, which may also complicate the already complex respiratory co-infection scenario of the pig.