Lizards can serve as the reservoir for large number of disease-causing agents including protozoa, helminths, pentastomids and arthropods and can be involved directly or indirectly in their transmission to usual as well as new hosts (Swei et al. 2011). Despite having diverse reptilian fauna, wild lizards from Pakistan have never been screened, by using traditional as well as molecular tools, for the presence of blood borne parasites in general and for apicomplexan protozoa parasites specifically.
During the present investigation, 3% of screened blood samples from wild lizards were found infected with Hepatozoon ophisauri (Table 1). A number of wild reptile species are known to be infected with Hepatozoon parasites and these parasites have the potential to be transmitted onwards by a wide range of possible vectors (e.g. mosquitoes, ticks, mites and leeches) (Maia et al. 2011; Er-Rguibi et al. 2023). Maia et al (2012) had captured 133 lizards including Algyroides marchi, Podarcis bocagei, Podarcis hispanica and Podarcis lilfordi from different areas in Spain and Portugal and found 58% of them infected with Hepatozoon spp. Godfrey et al. (2011) trapped 208 lizards from Stephens Island in New Zealand and found that 16.8% of them were infected with Hepatozoon tuatara. Zechmeisterová et al. (2022) had reported that 7.7% of fresh water turtles enrolled from Viet Nam was infected with Hepatozoon cuorae. Maia et al. (2014) had found that 6% of chameleons (Furcifer sp.) blood samples collected from Madagascar were Hepatozoon domerguei positive. In another study, Maia et al. (2011) had reported 5% infection rate of Hepatozoon spp. in lizard blood samples that were trapped from North Africa. Infected reptiles belonged to genera Chalcides, Eumeces, Lacertidae, Atlantolacerta, Timon, Podarcis, Scelarcis,. Quedenfeldtia and Tarentola. Er-Rguibi et al. (2023) Timon screened tangitanus, Atlantolacerta andreanskyi, Podarcis vaucheri, Scelarcis perspicillata, Acanthodactylus dumerilli, Tarentola mauritanica and Quedenfeldtia trachyblepharus lizard species from Morocco and found that 1.26% of them were infected with Hepatozoon spp. Harris et al. (2012) reported that two endemic species: Mabuya wrightii (lizard) and Lycognathophis (snake), trapped from the Seychelles Island in east Africa were infected with Hepatozoon spp. but the parasite prevalence was low. This limited information, especially regarding the prevalence of Hepatozoon spp. in Pakistani as well as wild lizards across the globe reflects the importance of this least explored research avenue and highlights the importance of screening wild hosts from various geo climatic regions to learn about the genetic diversity of these parasite that will confirm not only their zoonotic potential but will also pave the way for their effective control.
Genetic diversity of Hepatozoon spp. has never been reported from Pakistani reptiles before. So, our data is the first report regarding the genetic diversity of Hepatozoon ophisauri based on their partial 18S rRNA gene sequences. The sequences amplified in this study resembled the 18S rRNA sequences of Hepatozoon sp. isolated from snake in North Africa (Accession number KC696569, Tomé et al. 2013), Hepatozoon bashtari in painted saw-scaled viper from Saudi Arabia (Accession number MN497412, unpublished data), Hepatozoon sp. (Accession numbers MZ412878, unpublished data), Hepatozoon colubri (Accession number MN723844, unpublished data) and Hepatozoon ophisauri (Accession number MN723845, unpublished data) in snakes from Iran, Hepatozoon sp. in snake from Chaina (Accession number KF939627, unpublished data), Hepatozoon griseisciuri in Ontario eastern gray squirrels from Canada (Accession number MK452252, Léveillé et al. 2020), Hepatozoon annularis in Tarentola annularis (white spotted wall gecko) from Egypt Accession number MZ851987, Abdel-Baki et al. 2022), Hepatozoon sp. in Monito del Monte (marsupial) from Chile (Accession numbers FJ719816, Merino et al. 2009), Hepatozoon musa in Philodryas nattereri Steindachner (snake) (Accession numbers KX880079, Borges-Nojosa et al. 2017) and Hepatozoon caimani in Caiman crocodilus (Accession number KT274178, unpublished data) from Brazil, Hepatozoon ayorgbor in Apodemus sylvaticus (rodent) (Accession numbers MF435047 and MF435048, unpublished data) from Crotia, Hepatozoon ayorgbor in tick (Accession number MZ475989, unpublished data) from Portugal, Hepatozoon domerguei in chameleons (Accession numbers KM234648 and KM234649, Maia et al. 2014) from Madagascar, Hepatozoon cuorae in fresh water turtles (Accession numbers MW514213 and MW514214, Zechmeisterová et al. 2022) from Viet Nam, Hepatozoon ewingi in ticks (Accession numbers MG593275 and MZ502171, Greay et al. 2018) from Australia, Hepatozoon felis in Asiatic lion (Panthera leo persica) (Accession number HQ829439, unpublished data) from India and Hepatozoon felis in wildcats (Accession number AB771570, unpublished data) from Japan (Fig. 2). This limited and mostly unpublished data indicates that more and more reptilian/lizard samples should be collected from different geo climatic regions in Pakistan as well as worldwide and must be analyzed for the genetic diversity of Hepatozoon sp. to get more information about their proper taxonomic identification and to develop therapeutic approach against their effective control.
As this is the first report regarding the prevalence of Hepatozoon ophisauri in Pakistani lizards, so no comparable data regarding the risk factors associated with this parasite is available in literature. During the present investigation, we have observed that Hepatozoon ophisauri infection was not restricted to a particular lizard species or the sampling sites (Tables 1 and 2). We selected the sampling sites having different altitude ranging from 1200 m to 2250 m above sea level (asl). Interestingly, all the three parasite positive lizards were captured from high altitude: one infected Laudakia tuberculata was captured from 1900 m while the other two were captured from 2250 m asl but the difference in parasite prevalence with altitude did not reach statistical significance (Table 3) (Lizard sex and presence of ectoparasites on lizard were also not associated with the detected parasite (Table 4) Contrary to our observations, Wozniak and Telford (1991) had reported that the prevalence of Hepatozoon parasites in lizards varied between the species and similar to our observations they also found that parasite prevalence varied between the various geographical locations from where the samples were collected. Similar to our observations, Moreira (2013) had reported that Hepatozoon spp. prevalence in lizards varied with the sampling sites in Morocco. Maia et al. (2012) had also reported that Hepatozoon spp. prevalence varied between the host species and geographical locations but age, sex and nature of the habitat had no association with Hepatozoon spp. prevalence. Godfrey et al. (2011) had reported that Hepatozoon tuatarae (Apicomplexa) infection pattern varied predominantly with the host size and tick infestation during their study that was conducted in Stephens Islands, New Zealand.
The present investigation is the first report from Pakistan regarding the presence of Toxoplasma gondii among the wild lizards. We found a very high infection rate (45%) among the screened lizard species. The parasite was detected in four (Laudakia agrorensis, Abblepharus pannonicus, Laudakia pakistanica and Laudakia tuberculate) out of seven lizard species that were trapped during present investigation (Table 1). There are few reports from across the globe reporting the presence of Toxoplasma gondii among the reptiles. Nasiri et al. (2016) screened five snake species from Iran and found that 81% screened snakes were infected with Toxoplasma gondii and the parasite was detected in all five snake species, Feitosa et al. (2017) had detected anti Toxoplasma gondii antibodies among 33% reptiles including Caiman crocodilus (spectacled caiman), Chelonoidis carbonaria (red-footed tortoise) and Paleosuchus palpebrosus (Cuvier’s dwarf caiman) that were screened in Brazil. Aziz Anah and Aziz Anah (2023) had found Toxoplasma gondii infection in 11.9% tortoises (Testudo graeca) that were screened in Iran. These findings suggest that reptiles have a role in the transmission of Toxoplasma gondii and they are believed to acquire this infection by feeding on small invertebrate animals or plants contaminated with the oocysts of the parasite or by consuming the fecal material of infected canines (Nasiri et al. 2016)). On the other hand Sroka et al. (2019) did not find Toxoplasma gondii infection among the grass snake (Natrix natrix) that was captured for screening from Poland. Costa Viegas de Lima et al. (2019) did not also detect this parasite in 21 tegu lizard (Salvator merianae) that was trapped from the Island of Fernando de Noronha in Brazil. These few reports available in literature regarding the presence of Toxoplasma gondii among the reptiles from the whole world demands more extensive research on this topic in order to extend our knowledge regarding the reptilian species hosting this one of the most common Protozoan parasite and to learn that interactions of Toxoplasma gondii with their diverse reptilian hosts.
Phylogenetic studies are important as they provide new insights into the parasite population structure, explains their evolutionary relationships, transmission between different hosts and also delimits the species in morphologically challenging groups (Hasegawa, 1999). Nuclear internal transcribed spacer (ITS) regions, ITS1 and ITS2, are widely used in phylogenetic studies due to their relatively high variability and facility of amplification (Tippery and Les, 2008). The Toxoplasma gondii haplotypes detected in this study very showed genetic diversity as they resided in different clads indicating endemic diversity within this parasite (Fig. 3). The two haplotypes amplified from Pakistani lizards resembled the ITS-1 gene sequences of Toxoplasma gondii deposited in GenBank from soft ticks in Pakistan (Accession numbers PP033151 and PP033152, unpublished data) and large ruminants (Accession numbers MH793503 and MH793505), sparrows (Accession number GQ160468, Gondim et al. 2010) and free ranging chicken (Accession number JF810951, Gonçalves et al. 2012) in Brazil, humans in Iraq (Accession numbers OR672854, OR672855 and OR672856, unpublished data) and straw cats in China (Accession number JX456456, unpublished data). This data suggested that although the haplotypes generated from Pakistani lizards were distinct from each other but phylogenetically they are related to known Toxoplasma gondii species found in various animal species
Risk factor analysis revealed as significant variation in Toxoplasma gondii prevalence among the screened lizard species (Table 1) but there was no effect of sampling sites, lizard sex or presence of the ecto-parsites on the lizard on the parasite’s prevalence (Table 2 and4). An interesting trend was observed when the prevalence of Toxoplasma gondii was compared with the altitude of the sampling sites. It was observed that the parasite infection rate was increasing with the altitude as the minimum infection rate (25%) was observed in lizards that were trapped from 1200 m asl while maximum parasite prevalence (50%) was observed in lizards enrolled from 2250 m asl but the difference in parasite prevalence with increasing altitude of the sampling did not reach statistical significance (Table 3). Our results can be compared to those of Nasiri et al. (2016) as they had observed that Toxoplasma gondii infection was not limited to any of the five snake species that were screened in Iran. More epidemiological survey must be conducted globally to get more information regarding the risk factors that are associated with Toxoplasma gondii infection among reptiles in general and among lizards specifically.