INTRODUCTION: Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic. Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529-base pair (bp) repetitive element (TgREP-529) is of utmost diagnostic importance for polymerase chain reaction (PCR) diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529). METHODS: Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species was used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon gene knockout (GKO) mice at varying time points post-infection. RESULTS: Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than one oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent, suggesting 100- to 10,000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post-infection. DISCUSSION: The use of the 529-bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay, because this repeat probably exists about 200 times in the genome of a single organism, like its counterpart in T. gondii. Although there were enough sequence data available, only a few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529-bp repetitive element of H. hammondi. CONCLUSIONS: The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell biological studies on H. hammondi and related parasites.

Animal Parasitic Diseases Laboratory Beltsville Agricultural Research Center Agriculture Research Service United States Department of Agriculture Building 1001 Beltsville MD 20705 2350 USA

Central European Institute of Technology University of Veterinary and Pharmaceutical Sciences Palackého tř 1946 1 Brno 612 42 Czech Republic

Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Palackého tř 1946 1 Brno 612 42 Czech Republic

IDEXX Laboratories Humboldtstraße 2 Kornwestheim 70806 Germany

National Reference Laboratory for Toxoplasmosis Institute of Epidemiology Friedrich Loeffler Institut Federal Research Institute for Animal Health Südufer 10 17493 Greifswald Insel Riems Germany

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Walzer KA, Adomako-Ankomah Y, Dam RA, Herrmann DC, Schares G, Dubey JP, et al. Hammondia hammondi, an avirulent relative of Toxoplasma gondii, has functional orthologs of known T. gondii virulence genes. Proc Natl Acad Sci USA. 2013;110:7446–51. doi: 10.1073/pnas.1304322110. PubMed DOI PMC

Walzer KA, Wier GM, Dam RA, Srinivasan AR, Borges AL, English ED, et al. Hammondia hammondi harbors functional orthologs of the host-modulating effectors GRA15 and ROP16 but is distinguished from Toxoplasma gondii by a unique transcriptional profile. Eukaryot Cell. 2014;13:1507–1518. doi: 10.1128/EC.00215-14. PubMed DOI PMC

Dubey JP, Sreekumar C. Redescription of Hammondia hammondi and its differentiation from Toxoplasma gondii. Int J Parasitol. 2003;33:1437–1453. doi: 10.1016/S0020-7519(03)00141-3. PubMed DOI

Schares G, Vrhovec MG, Pantchev N, Herrmann DC, Conraths FJ. Occurrence of Toxoplasma gondii and Hammondia hammondi oocysts in the faeces of cats from Germany and other European countries. Vet Parasitol. 2008;152:34–45. doi: 10.1016/j.vetpar.2007.12.004. PubMed DOI

Dubey JP, Tilahun G, Boyle JP, Schares G, Verma SK, Ferreira LR, et al. Molecular and biological characterization of first isolates of Hammondia hammondi from cats from Ethiopia. J Parasitol. 2013;99:614–618. doi: 10.1645/12-51.1. PubMed DOI

Schares G, Herrmann DC, Beckert A, Schares S, Hosseininejad M, Pantchev N, et al. Characterization of a repetitive DNA fragment in Hammondia hammondi and its utility for the specific differentiation of H. hammondi from Toxoplasma gondii by PCR. Mol Cell Probes. 2008;22:244–51. doi: 10.1016/j.mcp.2008.04.003. PubMed DOI

Schares G, Ziller M, Herrmann DC, Globokar MV, Pantchev N, Conraths FJ. Seasonality in the proportions of domestic cats shedding Toxoplasma gondii or Hammondia hammondi oocysts is associated with climatic factors. Int J Parasitol. 2016;46:263–273. doi: 10.1016/j.ijpara.2015.12.006. PubMed DOI

Eydelloth M. Experimentelle Untersuchungen ueber das Wirtspektrum von Hammondia hammondi. Dissertation, Faculty of Veterinary Medicine, Munich, Germany, 1977.

Mason RW. The detection of Hammondia hammondi in Australia and the identification of a free-living intermediate host. Z Parasitenkd. 1978;57:101–106. doi: 10.1007/BF00927150. PubMed DOI

Frenkel JK, Dubey JP. Hammondia hammondi: a new coccidium of cats producing cysts in muscle of other mammals. Science. 1975;189:222–224. doi: 10.1126/science.806116. PubMed DOI

Frenkel JK, Dubey JP. Hammondia hammondi gen nov., sp. nov., from domestic cats, a new coccidian related to Toxoplasma and Sarcocystis. Z Parasitenkd. 1975;46:3–12. doi: 10.1007/BF00383662. PubMed DOI

Christie E, Dubey JP. Cross-immunity between Hammondia and Toxoplasma infections in mice and hamsters. Infect Immun. 1977;18:412–415. doi: 10.1128/iai.18.2.412-415.1977. PubMed DOI PMC

Wallace GD. Observations on a feline coccidium with some characteristics of Toxoplasma and Sarcocystis. Z Parasitenkd. 1975;46:167–178. doi: 10.1007/BF00389874. PubMed DOI

Dubey JP. Protective immunity against clinical toxoplasmosis in dairy goats vaccinated with Hammondia hammondi and Hammondia heydorni. Am J Vet Res. 1981;42:2068–2070. PubMed

Dubey JP, Wong M. Experimental Hammondia hammondi infection in monkeys. J Parasitol. 1978;64:551–552. doi: 10.2307/3279809. PubMed DOI

Dubey JP, Streitel RH. Further studies on the transmission of Hammondia hammondi in cats. J Parasitol. 1976;62:548–551. doi: 10.2307/3279410. PubMed DOI

Burg JL, Grover CM, Pouletty P, Boothroyd JC. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbiol. 1989;27:1787–1792. doi: 10.1128/jcm.27.8.1787-1792.1989. PubMed DOI PMC

Hurtado A, Aduriz G, Moreno B, Barandika J, Garcia-Perez AL. Single tube nested PCR for the detection of Toxoplasma gondii in fetal tissues from naturally aborted ewes. Vet Parasitol. 2001;102:17–27. doi: 10.1016/S0304-4017(01)00526-X. PubMed DOI

Homan WL, Vercammen M, De Braekeleer J, Verschueren H. Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int J Parasitol. 2000;30:69–75. doi: 10.1016/S0020-7519(99)00170-8. PubMed DOI

Reischl U, Bretagne S, Kruger D, Ernault P, Costa JM. Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect Dis. 2003;3:7. doi: 10.1186/1471-2334-3-7. PubMed DOI PMC

Sreekumar C, Vianna MC, Hill DE, Miska KB, Lindquist A, Dubey JP. Differential detection of Hammondia hammondi from Toxoplasma gondii using polymerase chain reaction. Parasitol Int. 2005;54:267–269. doi: 10.1016/j.parint.2005.06.008. PubMed DOI

Xia J, Venkat A, Le Roch K, Ay F, Boyle JP. Third generation sequencing revises the molecular karyotype for Toxoplasma gondii and identifies emerging copy number variants in sexual recombinants. bioRxiv. 2020 doi: 10.1101/2020.03.10.985549. PubMed DOI PMC

Herrmann DC, Pantchev N, Vrhovec MG, Barutzki D, Wilking H, Fröhlich A, et al. Atypical Toxoplasma gondii genotypes identified in oocysts shed by cats in Germany. Int J Parasitol. 2010;40:285–292. doi: 10.1016/j.ijpara.2009.08.001. PubMed DOI

Schares G, Pantchev N, Barutzki D, Heydorn AO, Bauer C, Conraths FJ. Oocysts of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and Hammondia hammondi in faeces collected from dogs in Germany. Int J Parasitol. 2005;35:1525–1537. doi: 10.1016/j.ijpara.2005.08.008. PubMed DOI

Ho MSY, Barr BC, Marsh AE, Anderson ML, Rowe JD, Tarantal AF, et al. Identification of bovine Neospora parasites by PCR amplification and specific small-subunit rRNA sequence probe hybridization. J Clin Microbiol. 1996;34:1203–1208. doi: 10.1128/jcm.34.5.1203-1208.1996. PubMed DOI PMC

Schares G, Dubey JP, Rosenthal B, Tuschy M, Bärwald A, Conraths FJ. Sensitive, quantitative detection of Besnoitia darlingi and related parasites in intermediate hosts and to assess felids as definitive hosts for known and as-yet undescribed related parasite species. Int J Parasitol Parasites Wildl. 2020;11:114–119. doi: 10.1016/j.ijppaw.2020.01.011. PubMed DOI PMC

More G, Schares S, Maksimov A, Conraths FJ, Venturini MC, Schares G. Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle. Vet Parasitol. 2013;197:85–94. doi: 10.1016/j.vetpar.2013.04.024. PubMed DOI

Ghosh S, Debnath A, Sil A, De S, Chattopadhyay DJ, Das P. PCR detection of Giardia lamblia in stool: targeting intergenic spacer region of multicopy rRNA gene. Mol Cell Probes. 2000;14:181–189. doi: 10.1006/mcpr.2000.0302. PubMed DOI

Felleisen RS. Comparative sequence analysis of 5.8S rRNA genes and internal transcribed spacer (ITS) regions of trichomonadid protozoa. Parasitology. 1997;115:111–9. doi: 10.1017/S0031182097001212. PubMed DOI

Hoffmann B, Depner K, Schirrmeier H, Beer M. A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses. J Virol Methods. 2006;136:200–209. doi: 10.1016/j.jviromet.2006.05.020. PubMed DOI

Talabani H, Asseraf M, Yera H, Delair E, Ancelle T, Thulliez P, et al. Contributions of immunoblotting, real-time PCR, and the Goldmann-Witmer coefficient to diagnosis of atypical toxoplasmic retinochoroiditis. J Clin Microbiol. 2009;47:2131–2135. doi: 10.1128/JCM.00128-09. PubMed DOI PMC

Legnani S, Pantchev N, Forlani A, Zini E, Schares G, Balzer J, et al. Emergence of cutaneous neosporosis in a dog receiving immunosuppressive therapy: molecular identification and management. Vet Dermatol. 2016;27:49–e14. doi: 10.1111/vde.12273. PubMed DOI

Galal L, Schares G, Stragier C, Vignoles P, Brouat C, Cuny T, et al. Diversity of Toxoplasma gondii strains shaped by commensal communities of small mammals. Int J Parasitol. 2019;49:267–275. doi: 10.1016/j.ijpara.2018.11.004. PubMed DOI

Sokol SL, Primack AS, Nair SC, Wong ZS, Tembo M, Verma SK, et al. Dissection of the in vitro developmental program of Hammondia hammondi reveals a link between stress sensitivity and life cycle flexibility in Toxoplasma gondii. ELife. 2018;7:e36491. doi: 10.7554/eLife.36491. PubMed DOI PMC

Alfonso Y, Fraga J, Gonzalez Z, Jiménez N, Borrero Y, Cox R, et al. Multiplex PCR to detect T. gondii infection based on B1 gene and 529 bp repetitive element. J AIDS Clin Res. 2015;6:1–5. doi: 10.4172/2155-6113.1000435. PubMed DOI

Wahab T, Edvinsson B, Palm D, Lindh J. Comparison of the AF146527 and B1 repeated elements, two real-time PCR targets used for detection of Toxoplasma gondii. J Clin Microbiol. 2010;48:591–592. doi: 10.1128/JCM.01113-09. PubMed DOI PMC

Kornacka A, Cybulska A, Moskwa B. Comparison of sensitivity of two primer sets for the detection of Toxoplasma gondii DNA in wildlife. Acta Parasitol. 2018;63:634–639. doi: 10.1515/ap-2018-0072. PubMed DOI

Gomez CA, Sahoo MK, Kahn GY, Zhong L, Montoya JG, Pinsky BA, et al. Dual-target, real-time PCR for the diagnosis of intraocular Toxoplasma gondii infections. Br J Ophthalmol. 2019;103:569. doi: 10.1136/bjophthalmol-2018-313064. PubMed DOI PMC

Belaz S, Gangneux JP, Dupretz P, Guiguen C, Robert-Gangneux F. A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR. J Clin Microbiol. 2015;53:1294–1300. doi: 10.1128/JCM.02900-14. PubMed DOI PMC

Veronesi F, Santoro A, Milardi GL, Diaferia M, Branciari R, Miraglia D, et al. Comparison of PCR assays targeting the multi-copy targets B1 gene and 529 bp repetitive element for detection of Toxoplasma gondii in swine muscle. Food Microbiol. 2017;63:213–216. doi: 10.1016/j.fm.2016.11.022. PubMed DOI

Chemoh W, Sawangjaroen N, Nissapatorn V, Sermwittayawong N. Molecular investigation on the occurrence of Toxoplasma gondii oocysts in cat feces using TOX-element and ITS-1 region targets. Vet J. 2016;215:118–122. doi: 10.1016/j.tvjl.2016.05.018. PubMed DOI

Veronesi F, Santoro A, Milardi GL, Diaferia M, Morganti G, Ranucci D, et al. Detection of Toxoplasma gondii in faeces of privately owned cats using two PCR assays targeting the B1 gene and the 529-bp repetitive element. Parasitol Res. 2017;116:1063–1069. doi: 10.1007/s00436-017-5388-z. PubMed DOI

Costa JM, Bretagne S. Variation of B1 gene and AF146527 repeat element copy numbers according to Toxoplasma gondii strains assessed using real-time quantitative PCR. J Clin Microbiol. 2012;50:1452–1454. doi: 10.1128/JCM.06514-11. PubMed DOI PMC

Salant H, Markovics A, Spira DT, Hamburger J. The development of a molecular approach for coprodiagnosis of Toxoplasma gondii. Vet Parasitol. 2007;146:214–220. doi: 10.1016/j.vetpar.2007.02.022. PubMed DOI

Salant H, Spira DT, Hamburger J. A comparative analysis of coprologic diagnostic methods for detection of Toxoplama gondii in cats. Am J Trop Med Hyg. 2010;82:865–870. doi: 10.4269/ajtmh.2010.09-0635. PubMed DOI PMC

Rothe J, McDonald PJ, Johnson AM. Detection of Toxoplasma cysts and oocysts in an urban environment in a developed country. Pathology. 1985;17:497–499. doi: 10.3109/00313028509105508. PubMed DOI

Opsteegh M, Maas M, Schares G, van der Giessen J. Relationship between seroprevalence in the main livestock species and presence of Toxoplasma gondii in meat (GP/EFSA/BIOHAZ/2013/01). An extensive literature review. Final report. 2016;13:996E.