Molecular prevalence, phylogeny and hematological impact of Toxoplasma gondii and Plasmodium spp. in common quails from Punjab, Pakistan

Abstract

This study investigates the molecular prevalence and phylogenetic characteristics of two prominent blood-borne pathogens, Toxoplasma gondii (T. gondii) and Plasmodium spp., in common quails (Coturnix coturnix) sampled from both wild (N = 236) and farmed (N = 197) populations across four districts (Layyah, Dera Ghazi Khan, Lahore, and Multan) in Punjab, Pakistan, during the hunting seasons from 2021 to 2023. Additionally, the impact of these pathogens on the complete blood count (CBC) of the hosts is examined. Out of 433 quails tested, 25 (5.8%) exhibited amplification of the internal transcribed spacer (ITS-1) gene for T. gondii, while 15 (3.5%) showed amplification of the Cytochrome b gene for Plasmodium spp. A risk factor analysis indicated that the prevalence of both pathogens was not confined to specific sampling sites or bird sexes (P > 0.05). District-wise analysis highlighted that hens were more susceptible to both T. gondii and Plasmodium spp. infections than cocks. Wild quails exhibited a higher susceptibility to T. gondii compared to farmed birds. Significant CBC variations were recorded in infected birds as compared to uninfected ones. BLAST analysis of generated sequences has confirmed the identity of recovered PCR amplicons as T. gondii and Plasmodium relictum. Phylogenetic analysis revealed that Pakistani isolates clustered with those reported from various countries globally. This study provides the first documentation of T. gondii and Plasmodium sp. infections in Pakistani quails, underscoring the need for detailed investigations across different regions to enhance our understanding of infection rates and the zoonotic potential of these parasites.

Fig 1. Map of Pakistan with highlighted sampling districts.

Magnified map is showing the districts and sites from where the common Quail samples were collected during present study.

Fig 2. Representative maximum likelihood tree based on multiple sequence alignment of the partial ITS-1 nucleotide sequences (266 bp) of Pakistani Toxoplasma gondii isolates infecting common quails (Coturnix coturnix) with those isolated from wild and domestic animals from several worldwide countries published in GenBank.

Numbers in nodes represent the percentage of 1,000 bootstrap iterations supporting the nodes (only percentages greater than 70% are shown). The host, the strain, isolate, voucher or clone identification, country of origin, and GenBank accession number are indicated in the tree for each sequence. Sequences newly obtained in this study are highlighted in bold and marked with asterisks. One Hammondia hammondi ITS-1 partial sequences was added as an out-group.

Fig 3. Phylogenetic tree of Plasmodium spp. inferred with marker genomic sequences (317 bp) using the Maximum likelihood method showing the position of revealed sequence isolated from Plasmodium relictum infecting a common quail (Coturnix coturnix) in Pakistan.

Isolate from this study represented in bold and marked with an asterisk. Numbers associated with nodes represent the percentage of 1,000 bootstrap iterations supporting the nodes (only percentages greater than 70% were represented). The host, the strain, isolate or clone name, the country of origin and the GenBank accession number are indicated. One Haemoproteus columbae marker genomic sequence was added as an out-group.