Mammal-related Cryptosporidium infections in endemic reptiles of New Zealand

New Zealand’s endemic reptile fauna is highly threatened and pathogens causing infectious diseases may be a significant risk to already endangered species. Here, we investigate Cryptosporidium infection in captive endemic New Zealand reptiles. We found two mammal-related Cryptosporidium species (C. hominis and C. parvum) and six subtypes from three gp60 families (Ib, Ig and IIa) in 12 individuals of captive endemic Tuatara, Otago and Grand skinks, and Jewelled and Rough geckos. Cryptosporidium serpentis was identified in two Jewelled geckos using 18S. In New Zealand, C. hominis and C. parvum are associated with infections in humans and introduced domestic animals but have also been recently found in wildlife. Our finding of Cryptosporidium infection in endemic reptiles can help inform strategies to monitor the conservation of species and manage potential introductions of pathogens to in-situ and ex-situ populations. Supplementary Information The online version contains supplementary material available at 10.1007/s00436-023-07824-4.


Introduction
New Zealand has a diverse endemic terrestrial reptile fauna consisting of about 124 described species of lizards (geckos and skinks) and the Tuatara (Chapple et al. 2009;Hay et al. 2010;Hitchmough et al. 2021;Nielsen et al. 2011;O'Neill et al. 2008;Tingley et al. 2013;Towns et al. 2001). A significant majority of New Zealand's reptile taxa are threatened with extinction (Hitchmough et al. 2010). Introduced mammal predators are the major driver of species declines and endangerment (Doherty et al. 2016;Tingley et al. 2013). However, there are other factors, including diseases, that can contribute to increasing the risk of extinction.
Gastrointestinal tract infections by protozoans can cause diarrhea, emaciation, anorexia, weight loss, and even death in some reptiles (Alley and Gartrell 2019;Gartrell 2016;Gartrell and Hare 2005;Scullion and Scullion 2009;Terrell et al. 2003). Cryptosporidiosis, for instance, can be chronic and sometimes lethal. Chronic cases show regurgitation, anorexia, and weight loss (Fayer et al. 1997;Fayer and Xiao 2008;Koudela and Modrý 1998). Cryptosporidiosis is caused by Cryptosporidium species, protozoal parasites that were first confirmed to infect reptiles in the 1970s (Brownstein et al. 1977) and currently recognized as a cause of gastrointestinal disease in a wide range of reptiles (Kváč et al. 2014;O'Donoghue 1995;Upton et al. 1989;Xiao et al. 2004).
The modes of transmission of Cryptosporidium in reptiles are the faecal-oral route including via direct contact between animals or through contact with contaminated objects (Graczyk et al. 1997;Xiao et al. 2004). The two most common species infecting reptiles are C. varanii (syn. C. saurophilum) (Pavlasek and Ryan 2008) and C. serpentis, both found in snakes and lizards (Fayer et al. 2000;Morgan et al. 1999;Ryan et al. 2021b;Xiao et al. 2004). Reptiles infected with C. serpentis may show symptoms of mild to severe gastritis with frequent regurgitation, particularly after feeding, while C. varanii causes enteritis and diarrhea. Other species, C. ducismarci, and C. testudines have been identified causing intestinal disease in tortoises (Ježková et al. 2016;Traversa 2010). A few other Cryptosporidium species and undescribed genotypes (e.g., mouse and tortoise 1 3 genotypes) reported in reptiles were likely ingested through infected prey (Alves et al. 2005;Pedraza-Díaz et al. 2009;Richter et al. 2011;Rinaldi et al. 2012;Traversa et al. 2008;Xiao et al. 2004), including species associated with mammalian hosts, such as C. parvum, C. tyzzeri, and C. muris (Zahedi et al. 2016b).
The identification of Cryptosporidium species, and especially species associated with mammal infections, in endemic New Zealand reptiles is unknown. Here, we carry out a molecular epidemiological investigation to identify the species and subtypes infecting captive endemic reptiles of New Zealand.

Sampling
Between November 2018 and July 2021, we received 22 samples (faeces, intestinal tissue, and gastric or cloacal washes) from the Auckland Zoo (n = 17), Wellington Zoo (n = 2), the Wildbase at Massey University (n = 2) and Invercargill City Council (n = 1) from five New Zealand endemic reptile species including Otago skink (n = 9), Grand skink (n = 5), Jewelled gecko (n = 4), Tuatara (n = 3) and Rough gecko (n = 1) (Table S1). Samples were sent to the m EpiLab at Hopkirk Research Institute (Massey University) for DNA extraction, PCR amplification, and sequencing. Histology diagnosis by a veterinary pathology laboratory (Gribbles Veterinary, Auckland) in tissues collected from only two clinically ill and dead Jewelled geckos in the Auckland Zoo (Lab IDs 16,911 and 16,912) suggested that gastritis and/or stomach inflammation was likely caused by Cryptosporidium ( Fig. 1). All other samples were taken from animals that had no clinical signs of disease.

DNA extraction, PCR, and sequencing
DNA was extracted as previously described (Garcia-R et al. , 2020b using the Isolate faecal DNA (Zymo) kit following the manufacturer's instruction. DNA extractions from reptile samples were carried out separately from human samples that our laboratory receives regularly. DNA extraction required physical disruption of the oocyst using a beadbeater (Tissue Lyser II, Qiagen) at 30 Hz for 5 min. The species and subtype of the isolates were identified by nested PCRs of the gp60 and 18S using a combination of external and internal primers (Table S2) (Glaberman et al. 2002;Johnson et al. 1995;Learmonth et al. 2004;Waldron et al. 2009;Xiao et al. 2000;Xiao et al. 1999) and sequencing of the secondary PCR products in both directions on an ABI 3730XL automated DNA sequencer (Applied Biosystems). Positive (consisting of human-derived C. parvum) and negative (consisting of all reagents minus template, which was replaced by nuclease free water) controls were included in each PCR run. All our PCR positive and negative controls were positive and negative, respectively. Consensus sequences were assembled from forward and reverse reads and edited manually using Geneious v.10.1.3 (Kearse et al. 2012). The sequences derived were used to identify species and subtypes by aligning to sequence entries in nucleotide databases using the program BLAST (http:// www. ncbi. nlm. nih. gov/ blast/; last accessed October 25, 2022) and checked by their corresponding subtype by maximum % identity. The sequences of the partial gp60 and 18S genes were deposited in the GenBank database under accession numbers OP778244-OP778255 and OQ457495-OQ457496, respectively.

Results
Sequence analysis of the gp60 gene identified C. parvum and C. hominis subtypes in 12 of the 22 samples (Table 1). The remaining ten samples were gp60 PCR negative. Most reptiles were found with both Cryptosporidium species. The sequences obtained from isolates in endemic captive reptiles were found with a > 99% identity and an e-value of 0.0 to sequence data reported in GenBank. The subtypes are common in human infections (IbA10G2, IgA17, and IIaA18G3R1), however, we found subtypes of C. parvum with shorter (IIaA17G3R1) or similar (21 short tandem repeat region) but uncommon (IIaA17G4R1) repeats of the serine-coding trinucleotide. Only two samples from Jewelled geckos were successfully amplified using 18S primers (Table 1). These samples were ~ 99% identical to C. serpentis (accession number AF093499). One of these geckos was also C. hominis positive by gp60 PCR (Table 1). Three other samples were amplified using 18S primers, but sequencing results ruled them out as fungi (~ 99% identity to Tremellomycetes or Basidiomycota).

Discussion
Cryptosporidium infections have been recorded in several reptile species (Carmel and Groves 1993;Graczyk et al. 1997;Jacobson 2007;Ladds 2009;Orós et al. 1998;Upton et al. 1989) but mammal-related Cryptosporidium species infecting reptiles are rare (Xiao et al. 2004). Our results indicate that mammal-related Cryptosporidium species, the main causative agents of disease in humans, non-human primates, and livestock in New Zealand (Garcia-R et al. , 2020b can infect captive reptiles. The clinical relevance of these findings is unknown. New Zealand has a long history of isolation (Valente et al. 2019) and absence of native mammals (with exception of two bat species) may suggest that endemic reptiles have not been previously exposed to these pathogens. Cryptosporidium species adapted to mammals found infecting endemic reptiles may trigger clinical disease due to the recent coexistence and host-parasite relationship (Garcia-R et al. 2020a;Garcia-R and Hayman 2016). Importantly, these infections can act together with other stressors (e.g., habitat fragmentation or invasive species) and increase reptile mortality and extinction risk (Fey et al. 2015;Smith et al. 2009) of threatened taxa.
Cryptosporidium parvum and C. hominis have been previously reported in livestock and humans in New Zealand . However, there is an increase in the detection of these pathogens in animal hosts worldwide.
For instance, evidence of C. hominis in wildlife (kangaroos and other marsupials) and livestock (cattle and deer) residing in water catchments following its introduction by humans has been reported in Australia (Koehler et al. 2016;Ng et al. 2011;Zahedi et al. 2016a, 2016b, Zahedi et al. 2018. Cryptosporidium hominis is also widely recognised in equine populations in South America, Africa and Asia (Widmer et al. 2020). Likewise, C. parvum has been recently found in a wide variety of hosts including wildlife (Hailu et al. 2022;Karim et al. 2014;Ryan et al. 2021a).
To our knowledge there are no published cases of Cryptosporidium infections in endemic reptiles from New Zealand and this is the first report in captive endemic reptiles. We were careful to avoid cross-contamination and aimed to confirm all results by multiple methods. However, we were only able to amplify gp60 products from 12 animals and only two from 18S. We think that this may be due to the low concentration of oocyst/sporozoites and the possibility of primers amplifying numerous stretches of other organisms (including fungi or 16S rRNA bacteria) leading to reduced specificity (Xiao et al. 2000). Future studies should aim to confirm our findings and determine the source of infection and potential transmission pathways.
Reporting infections in endemic reptiles caused by Cryptosporidium has several implications for the health and conservation of wild native and endemic fauna of New Zealand. First, the reservoirs for C. hominis and C. parvum include widespread hosts, such as people and domestic animals, making the risk of infection more frequent through direct and indirect contact (Garcia-R et al. , 2020b. Second, Cryptosporidium oocysts are resilient and ubiquitous in the environment (Phiri et al. 2020) generating more opportunities for infections. And third, captive animals as part of breeding programmes must be carefully managed and screened before being Author contributions All authors contributed to the study conception and design. Material preparation, sample and data collection and analysis were performed by Juan C. Garcia-R, Anthony Pita, Niluka Velathanthiri and An Pas. The first draft of the manuscript was written by Juan C. Garcia-R and An Pas and David Hayman commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding Open Access funding enabled and organized by CAUL and its Member Institutions
Data availability The nucleotide sequences of the partial gp60 and 18S genes were deposited in the GenBank database under accession numbers OP778244-OP778255 and OQ457495-OQ457496, respectively. The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethics approval was needed.

Conflict of interest
The authors declare that they have no conflict of interest.
Consent to participate All authors consent to participate in this work.

Consent for publication All authors consent to publish this work.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.