Development of a qPCR molecular diagnostic assay for the detection of kiwi Eimeria species and its application to determine tissue-specificity of species causing coccidiosis in North Island brown kiwi (Apteryx mantelli)

Abstract

Juvenile kiwi (Apteryx spp.) within captive-rearing programmes commonly suffer from coccidiosis, which primarily affects the intestine but can also impact other organs, such as the kidneys, liver, lung, and spleen (Morgan et al. Avian Pathol 42:137-146 2013). In some immune-compromised birds, disease causes significant morbidity and, occasionally, mortality (Morgan et al. NZVJ 62:315-320 2014); however, understanding of the biology of disease-causing Eimeria species in kiwi is limited. A probe-based qPCR assay targeting a 115-bp fragment of the Eimeria mitochondrial cytochrome c oxidase I (CO1) gene was developed to identify three distinct kiwi Eimeria species: the two species most commonly recovered from faeces, Eimeria kiwii and Eimeria apteryxii, as well as the newly described species, Eimeria koka (Scheltema et al. Syst Parasitol 102:30 2025). The qPCR assay was then applied to retrospectively analyse formalin-fixed paraffin-embedded intestine, kidney, liver, lung, and spleen tissues from ten historic post-mortem cases from kiwi diagnosed with extraintestinal coccidiosis. This novel assay detected infection more often (33/47 tissues) than manual histopathological identification (25/47 tissues). Only one species, E. koka, was detected in extraintestinal tissues with the highest prevalence (9/10) in kidney tissues. In contrast, E. kiwii was reliably detected in 8/9 intestinal tissues but was not detected in the other tissues tested. E. apteryxii was not detected in any of the tissues analysed. These findings suggest that kiwi are infected by at least one intestinal and one renal-specific species, the latter of which is suspected to disseminate under certain conditions to other organs of the body.

Keywords: Eimeria; CO1; Coccidiosis; Extraintestinal infection; qPCR.

Fig. 1

Alignment of primers (Eimeria TM-F and TM-R1) and species-specific hydrolysis probes (EK-FAM, EA-560 and Tissue-FAM) designed for the present study, with kiwi Eimeria spp. CO1 consensus sequence, Eimeria spp. specific reference sequences and Isospora sp.(GenBank PQ609360) isolated from kiwi faeces. Numbers at each end of the sequence indicate base position on Eimeria CO1 gene

Fig. 2

qPCR amplification curves from qPCR screening of representative Eimeria DNA samples (n = 7) across all assays. A Eimeria generic assay; B Eimeria kiwii assay; C Eimeria apteryxii assay and D Eimeria koka assay. Sample key indicates if DNA is of faecal (F) or tissue (T) origin and, if faecal, whether it contains a single or multiple species

Fig. 3

Examples of intracellular coccidia stages from North Island brown kiwi (Apteryx mantelli) observed on histological examination of H&E slides in this study. A intestinal tissue with coccidial meronts within crypt epithelial cells (circled) (PM accession # 51506); B intestinal tissue with extracellular coccidial oocysts admixed with sloughed autolysed mucosa (58596); C coccidia meronts within epithelial cells of branches of the ureter (51506); D early oval-shaped oocyst stages within epithelial cells of collecting ducts of the kidney (peripheral cortex) (arrow heads) (51506); E kidney tissue with arrows indicating (i) immature coccidial meronts, (ii) gametocytes and (iii) formation of early oocysts in the epithelial cells of collecting ducts (49058); F kidney tissue with coccidial meronts within collecting tubules (circled)—arrows indicating formation of merozoites within meronts (51484); G individual oocysts (arrow heads) observed scattered within the lung interstitium in a single case (49058); and H oocysts observed forming in the lumen of the gall bladder of a single case (circled) (51506), with indeterminate origins. Scale bars = 50 µm