Performance of diagnostic assays used to detect Cryptosporidium oocysts in faecal samples of cattle in Kuwait and genotyping of Cryptosporidium species

Abstract

Backgroud: Cryptosporidium species are zoonotic protozoan parasites responsible for gastroenteritis in various animals and humans. The diagnosis of Cryptosporidium presents many challenges. This research attempted to match the diagnostic efficiency of the modified Ziehl-Neelsen technique (mZN), immunochromatographic assays (IC), and enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium in faecal samples of cattle in Kuwait. In addition, polymerase chain reaction (PCR) was utilised to determine the predominant species infecting cattle in Kuwait and correlating the detected species with the results of different diagnostic tests used, the presence or absence of clinical signs, and the age group of the infected cattle.

Results: Of 400 analysed faecal samples, Cryptosporidium positive samples were 23%, 15.25%, and 14% using IC, ELISA, and mZN. IC had the highest sensitivity (74.07%), and mZN had the highest specificity (98.29%) using a composite reference standard (CRS) as a gold standard. The rapid IC test results in high false-positive results of cryptosporidiosis, whereas using mZN alone is insufficient to declare a negative faecal sample. Only 74.5% (35/47) of Cryptosporidium-positive samples by the three assays could be amplified by PCR. This study was the first to genotype Cryptosporidium in Kuwait. Cryptosporidium parvum (n = 26) was the dominant species detected from cattle samples, followed by C. andersoni (n = 6), C. bovis (n = 2), and C. raynae (n = 1). The findings showed a statistically relevant relationship between diarrhoea and the detection of Cryptosporidium spp. in faecal samples of cattle (p-value = 0.0003). Pre-weaned calves were the most vulnerable age group to Cryptosporidium spp. infection (p-value = 0.0007).

Conclusion: For screening of Cryptosporidium infection in faecal samples, antigen detection or PCR methods combined with one of the microscopy techniques should be used. Cryptosporidium parvum was the prepoderant Cryptosporidium spp. recovered from cattle samples in Kuwait followed by C. andersoni. Cryptosporidium parvum is a significant risk factor for diarrhoea in pre-weaned calves. However, further study is needed as many other causes of diarrhoea in calves must be ruled out before a diagnosis of Cryptosporidium diarrhoea can be made.

Keywords: Cattle; Cryptosporidium spp.; ELISA; Immunochromatography; Kuwait; PCR; mZN.

Fig. 1

Phylogenetic sequence relationship of recovered Cryptosporidium spp. to other species in Genbank; C. andersoni isolated from cattle in China (KC580754), C. parvum; strain Bovine C. parvum genotype (BOH6) isolated from calf in Ohio (AF093490), C. bovis; Bovis 2622 isolated from cattle (AY120911), C. ryanae isolated from calves in China (HQ009807), and C. parvum_2_Sheep_Kuwait isolated from sheep in Kuwait

Fig. 2

A mZN stained faecal smear, Cryptosporidium oocyst (arrow) pink spherical body against purple background

Fig. 3

Bovid-4 kit device showed a positive result for Cryptosporidium antigen and negative for Rotavirus, coronavirus, and E. coli antigens. Diluted sample added into the sample hole, the results were interpreted after 5–10 min; the sample was considered negative if only the control line “C” appeared and positive if both “C” and “T” lines appeared

Fig. 4

A sandwich, double wells ELISA plate showed positive samples for Cryptosporidium antigen. Rows A, C, E, G were sensitized with the anti-Cryptosporidium-specific antibody and rows B, D, F, H coated nonspecific antibodies. Control positive added in wells A1 and B1. The diluted samples added to the wells as follows: sample 1 in wells C1 and D1, sample 2 in wells E1 and F1, sample 3 G1 and H1, etc., till sample 47 in wells G12 and H12. Validation of the test; positive control optical density net (Delta) value < 1.151. Positive samples were numbers 25, 26, 42, 44 and 47 their S/P% (Delta optical density of sample/ Delta optical density of positive control *100) were > 7%

Fig. 5

RFLP analysis identifying the four species of Cryptosporidium isolated from cattle samples. M: Markers; 100-bp molecular markers. Lanes 1–3: C. andersoni SspI products 448, 397 bp, and lanes 14–16: C. andersoni MboII products 769, 76 bp. Lane 4: C. ryanae SspI products 432, 267,103, 33 bp, and lane 17: C. ryanae MboII products 574, 185, 76 bp. Lane 5: C. bovis SspI products 432, 267,103, 33 bp, and lane18: C. bovis MboII products 412, 185, 162, 76 bp. Lanes 6–11 C. parvum SspI products 449, 267, 397, 12, 11 bp, and lanes 19–24: C. parvum MboII products 771, 76 bp. Lanes 12 and 25: C. baileyi (control sample)