Detection and Genotyping of Toxoplasma Gondii in Wild Canids in Serbia
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Parasitology International 73 (2019) 101973 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint Detection and genotyping of Toxoplasma gondii in wild canids in Serbia T Aleksandra Uzelaca, Ivana Kluna,Duško Ćirovićb, Aleksandra Penezićb, Vladimir Ćirkovića, ⁎ Olgica Djurković-Djakovića, a Center of Excellence for Food- and Vector-borne Zoonoses, Institute for Medical Research, University of Belgrade, Belgrade 11129, Serbia b Department for Animal Ecology and Zoogeography, Faculty of Biology, University of Belgrade, Belgrade 11000, Serbia ARTICLE INFO ABSTRACT Keywords: To gain insight into the population structure of the protozoan parasite Toxoplasma gondii in wildlife in Serbia, Toxoplasma gondii multiplex nested PCR- RFLP was performed on T. gondii DNA positive samples of heart tissue of three wild canid Genotyping species, including red foxes (Vulpes vulpes), golden jackals (Canis aureus) and grey wolves (Canis lupus). Out of Strains 110 samples (28 red foxes, 72 golden jackals, 10 wolves), 30 (27.3%) were positive for T. gondii DNA, of which Golden jackal 24 were genotyped (nine red foxes, 13 golden jackals and two wolves). The results showed a large dominance of Red fox lineage II (n = 20, 83.4%) over lineage III (n = 4, 16.7%) strains. Lineage II comprised four archetypes, 14 Grey wolf variants and one which remained undetermined, while within lineage III there were three archetypes and one variant strain. However, a notable difference in diversity was observed among the examined canids, with eight (88.9%) of the nine strains detected in red foxes distinct, vs. six (46.1%) distinct strains of the 13 detected in golden jackals. Since anthropogenic food sources are a major reservoir of archetypal lineage II and III T. gondii strains, the results suggest that the golden jackal may be outcompeting the red fox, forcing it to hunt wild mammals and birds for sustenance, thereby increasing its chances of infection with variant strains. In addition to presenting the first data on the T. gondii population structure in wild canids in Serbia, this is the first report ever on T. gondii genotypes in golden jackals, a species with an increasing presence in Europe and another important reservoir of domestically circulating T. gondii strains. 1. Introduction fragment length polymorphism (RFLP). Strains that have identical al- leles at all loci are considered archetypes, while those displaying Toxoplasma gondii is a ubiquitous food and waterborne protozoan combinations of alleles are considered variants. The majority of strains parasite whose definitive hosts are only Felidae, but is capable of in- can be grouped by haplotype, while related haplogroups make up the fecting a remarkably wide range of species which serve as intermediate six major clades of T. gondii [5]. Most European and North American T. hosts. Sexual replication and subsequent genetic recombination occur gondii strains belong to one of the three major lineages (types I - III) only in the intestine of the cat, resulting in the production of highly which occur globally, and one minor lineage (haplotype 12) which infectious oocysts, which are shed in the faeces into the environment. In occurs in North American wildlife. South and Central America are re- intermediate hosts asexual replication yields clonal progeny which form gions with the greatest strain diversity whereas in contrast, Europe and tissue cysts that can persist for the lifetime of the host mostly in the North America are regions dominated by lineage II strains [5,6]. In muscles and the brain [1,2]. Transmission of T. gondii occurs primarily Europe, lineage I strains are extremely rare [5,7], while lineage III through ingestion of tissue cysts in contaminated meat or of oocysts via seems to be more frequent in animals in southern and south-eastern contaminated water, soil and fresh produce [3,4]. Any animal whose regions [8–11]. Isolates from humans and domestic animals tend to be diet includes meat is at risk of infection with tissue cysts, while grazers archetypal strains, whereas isolates from wildlife tend to be variants and ground feeding birds are particularly exposed to infection with and recombinants of the three major lineages, or haplotype 12 [12,13]. oocysts. Consequently, there is a dichotomy between the T. gondii strain popu- The genus Toxoplasma features only one species, of which there are lations from domestic animals and humans, and wild animals [14,15]. a number of different strains. T. gondii strains can be identified by Moreover, Jiang et al. (2018) reported that strain diversity in wildlife biallelic polymorphism at particular loci using multilocus restriction decreases as the proximity to human settlements increases, adding more ⁎ Corresponding author. E-mail address: [email protected] (O. Djurković-Djaković). https://doi.org/10.1016/j.parint.2019.101973 Received 5 July 2019; Received in revised form 30 July 2019; Accepted 8 August 2019 Available online 09 August 2019 1383-5769/ © 2019 Elsevier B.V. All rights reserved. A. Uzelac, et al. Parasitology International 73 (2019) 101973 evidence to the proposed impact of mankind on the geographical dis- 2.2. Sample preparation and DNA extraction tribution and perhaps even the evolution of genetic diversity and virulence of this parasite [16]. Total nucleic acids were extracted from a roughly 2 cm by 1 cm strip Our group has previously genotyped a number of strains from hu- of heart tissue. The tissues were cut using sterile surgical scissors and mans and domestic animals in Serbia [9,11,17–19]. However, our placed into 2 ml microfuge tubes pre-filled with 1.4 mm diameter knowledge of genotypes from non-domestic animals is limited to only ceramic beads (Omni International, Kennesaw, GA, USA) and 1 ml of three T. gondii isolates from feral pigeons from the city of Belgrade, two Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The of which were lineage II archetypes and one a lineage III variant [9]. To tissues were homogenized using a bead beater instrument (Omni in- gain insight into the population structure of T. gondii in wildlife in ternational, Kennesaw, GA, USA), set to the maximum (number 5) for Serbia, we genotyped T. gondii DNA detected in three canid species, 3 min. The level of homogenization was monitored visually. Samples including the mesopredators golden jackal (Canis aureus) and red fox which were insufficiently broken down were subjected to an additional (Vulpes vulpes) and the apex predator grey wolf (Canis lupus). The dis- homogenization cycle using identical settings. All samples were suc- tribution ranges of golden jackals and red foxes in Serbia overlap to a cessfully homogenized after two cycles. Total DNA was extracted ac- great extent across a variety of different habitats, including those close cording to the Trizol reagent manufacturer's protocol for DNA extrac- to human settlements. In contrast, grey wolves inhabit remote, densely tion. forested mountainous areas of the country. Therefore, these wild canids exploit different resources of the food-web which allows them to be- 2.3. Detection of T. gondii DNA by real time qPCR come reservoirs of T. gondii strains occurring both in the domestic and in the sylvatic cycles. The T. gondii 529 bp repeat element (AF146527) was detected as previously described [19 ,20]. Briefly, each PCR reaction contained 10 μl of Taqman Universal Master Mix (Applied Biosystems, Foster City, 2. Materials and methods CA, USA), 0.25 mM of each forward and reverse primers (5′-AGA GAC ACC GGA ATG CGA TCT-3′;3’-CCC TCT TCT CCA CTC TTC AAT TCT- 2.1. Study area and samples 5′), 0.10 mM of the specific TaqMan probe FAM-ACG CTT TCC TCG TGG TGA TGG CG-TAMRA (Invitrogen, Life Technologies, Carlsbad, In collaboration with local hunting organizations, hearts of 72 CA, USA) and 3 μl of extracted gDNA as template in a final reaction golden jackals, 28 red foxes and 10 grey wolves (n = 110) were ob- volume of 20 μl. The thermal cycling program consisted of the following tained. All animals were legally hunted in Serbia between 2013 and steps: 5 min at 95 °C for initial denaturation, followed by 45 cycles of 2016. Heart sections were kept frozen at −20 °C until DNA extraction. 15 s at 95 °C for denaturation and 60 s at 60 °C for annealing/extension. The locations of the hunting areas from which the animals originated Amplification detection occurred at the end of the 60 °C annealing/ where grouped into five geographical regions (R1-R5, Fig. 1). Thirty- extension step. Amplification and detection were performed in a Ste- seven samples originated from R1, 43 from R2, 18 from R3, 10 from R4 pOnePlus Real Time PCR System (Applied Biosystems, Foster City, CA, and five from R5. In terms of human population density, R1 is the most USA). highly populated, since it includes the capital city of Belgrade, followed by R2 and R3, which are similarly populated, while R4 and R5 have the lowest population density. 2.4. Genotyping Genotyping was performed using multiplex nested RFLP-PCR (Mn- PCR-RFLP) with markers including alt. SAG2, GRA6, BTUB, C-22, PK1, Fig. 1. Map of Serbia showing the hunting localities in the different regions (R1-R5). 2 A. Uzelac, et al. Parasitology International 73 (2019) 101973 CS3, L358, C29–2 and APICO, and reaction mixtures prepared as pre- viously described [21,22]. Briefly, the multiplex PCR reaction con- tained 10 μl of 2× PCR MasterMix (Thermo Fischer Scientific, Wal- 0 tham, MA, USA), 0.15 μM of the external forward and reverse primers 0 for each marker and 2 μl of gDNA template. The nested PCR reaction + Genotyped was performed for each marker separately with the same final reaction volume, but using 0.3 μM of each internal forward and reverse primer and 2–4 μl of the multiplex reaction as template.