Introduction

The Cryptosporidium parasite is a vital protozoan parasite, and over 150 species of mammals have been identified as hosts of nearly 20 genotypes of C. parvum and also considered a worldwide problem in humans and animals (1,2). The parasite is considered a significant cause of neonatal profuse watery diarrhea due to its life cycle of C. parvum is done in the epithelial cells in gut canal (3). Infection by C. parvum results in intercellular colonization of the extra-cytoplasmic microvillus of the small intestine (4) and leads to production losses and a high mortality rate in pre-weaning calves during the first 28 days of life (5). Cryptosporidium parvum is easily transmitted to humans, especially immune-compromised individuals, and is therefore considered an anthroponotic zoonotic disease (6(. Different methods were used in the diagnosis of apicomplexan protozoal infection included cryptosporidiosis. A direct microscopic method is obtained using morphological identification with a 100× oil immersion lens. It uses different staining techniques such as hot or cold modified Ziehl-Neelsen (MZN) and auramine phenol to detect the oocyst containing four mature sporozoites (7). The microscopical method is an excellent tool and highly economical but requires expertise and experienced diagnostic personnel to reduce false-positive results and thus can be time-consuming and also parasites species and multiple infections the microscopic examination cannot detected these issues (8,9). Indirect serological methods, like latex agglutination test (LAT), modified agglutination test (MAT), ELISA enzyme-linked immunosorbent assay which, depend on coproantigen detection. Coproantigen detection assays are rapid and useful for screening large numbers of specimens simultaneously but do not provide details concerning the species of apicomplexan protozoal included Cryptosporidium detected (10,11). Molecular methods, e. g. conventional polymerase chain reaction (PCR), are used to detect parasite DNA but needed amplification before PCR test to overcome eliminate or inactivation inhibitors (12,13). PCR is a sensitive method compared to microscopical and serological diagnosis in humans and animals for detecting Cryptosporidium and is used to differentiate the species of helminthes but incurs a high cost in developing counties (14). The main advantage of the PCR method is detecting various Cryptosporidium species at the species, genotype, and subtype levels. Therefore, choosing the most effective diagnostic technique relies on multiple factors, including accessible resources, trained personnel, available time, and the number of specimens tested. Each technique also has different levels of specificity and sensitivity (15).

In Saudi Arabia, no studies guide the diagnosis of Cryptosporidium species in calves. Therefore, this study aimed to detect C. parvum using microscopic (hot MZN staining), ELISA, and PCR methods and subsequently estimate the diagnostic accuracy of each method.

Materials and methods

Ethical approve

The study is not an animal experiment, but a diagnostic study, using common sampling methods for diagnostic purposes. The methods were performed in accordance with the guidelines of ethical committee of ministry of environment, water, and agriculture, KSA, the authors confirm the study was carried out in compliance with the ARRIVE guidelines. Also All respective animal protocols were reviewed by state ethics commission and have been approved by competent authority (Ethical committee of Alexandria university, Egypt, serial number (0305796) at 20-10-2022, FWA No: 00018699 and IRB No: 00012098). Write the name of scientific or institutional board that give the ethical approve to conduct this scientific work and give the approval issue number and date.

Sample collection

One hundred fifty fecal samples from different private farms were collected from calves (> 3 months old). All 150 calves were clinically examined for body temperature, mucus membranes, and degree of dehydration. The calves were suffering from profuse watery diarrhea. Fecal samples were collected from profuse watery diarrheic calves. Each sample was divided into two parts: one part was preserved in 10% formalin prepared via the formol ether concentration technique for later staining with the hot MZN method (16). The second part was preserved in laboratory tubes at −20°C for further investigation.

Hot MZN staining

The reagents needed to perform hot MZN staining were Carbol-Fuchsin (10 gm basic fuchsin, 100ml absolute ethanol, 50 gm carbol, and one-liter distilled water), sulfuric acid-ethanol solution (3ml concentrated sulfuric acid and 100 ml methanol 95%) was put in a glass stopper. (sulfuric1%) Moreover, methylene blue (100ml glycerol, 1ml 3%aqueous Mb, 100ml distilled water). Furthermore, the procedure of hot MZN staining was done through three steps: carbol fuchsin, decolorization, and counter-stain. Respectively in between each step rinsed, the slides with tap water were drained and air drying, so the practical procedures were done as follows; the first step was a thin fecal smear was made from the sediment of the centrifuged formalized specimen and was allowed to air dry, then the slide was placed on a staining rack and flooded with carbol fuchsin for five minutes. The slide was heated gently with a Bunsen burner. Then the slide was rinsed with tap water. The second step was decolorization with 1% sulfuric acid ethanol solution for about 2 minutes, then rinsing with tap water, draining, and air drying. The final third step was the slide was flooded with methylene blue (counter stain) for one minute, and then rinsed with tap water; draining and air drying was done. The smear was examined microscopically using a high-power magnification to detect oocysts and oil immersion objective to identify them as Cryptosporidium oocyst retained a red/pink color due to Cryptosporidium was acid-fast versus blue or clear background(17).

ELISA method

The method was performed using a multiscreen Antigen ELISA kit for antigenic detection of Cryptosporidium in feces (Bio-X Diagnostics S.A., Belgium) according to the manufacturer’s instructions. The microtitration plate was coated with specific antibodies. Fecal samples were diluted and incubated in the coated wells. After 1 h incubation at 21°C +/- 3°C, tetramethylbenzidine was added, and the absorbance was read at 450 nm using a microplate ELISA reader (Thermo Scientific, USA) (18).

Conventional PCR method

Specific primers for C. parvum were as follows: forward primer 5`-GCCCACCTGGATATACACTTTC-3`; reverse primer 5`-TCCCCCTCTCTAGTACCAACAGGA-3`. Amplified DNA was separated using agarose gel electrophoresis and was visualized using a U.V. transilluminator (320 nm) (19).

Isolation of C. parvum DNA from fecal samples:

Fecal specimens were collected from the rectum of all selected animals and were prepared according to Johnson et al. Fecal samples stored in 2.5% potassium dichromate were washed several times (4 times) with PCR buffer (10 mM Tris, 50 mM K Cl, 3.5 mM Mg Cl2) by centrifugation. A 20% Chelex 100 (Bio-Rad Lab., Calif.) was mixed with fecal samples (20 µl of chelex stock to 100µl of samples). Then, samples were subjected to six cycles of freezing and thawing to release the DNA from oocysts using a dry ice ethanol bath for freezing and a water bath (98 oC) for thawing with incubation time from 1 to 2 minutes in each bath. In the next step, samples were centrifuged, and 50 µl of supernatant was used as the template in the PCR assay (20).

DNA amplification

The primer set described by Laberge et al. was used in PCR which is specific for Cryptosporidium parvum. Sequences of primers were as follows: forward - 5'GCC CAC CTG GAT ATA CAC TTT C3`; reverse - 5'TCC CCC TCT CTA GTA CCA ACA GGA 3`. The size of the amplified product was 358 bp. (Figure 1) The PCR mixture contained PCR reaction buffer (10 mM Tris-HCl, 1.5 mM MgCl, 50 mM KCl, pH 8.3) and contained 1.0 mM each of forward and reverse primers, 0.2 mM each dATP, dGTP, dCTP and dTTP, 100 mg/ml BSA and 2.5 U Taq DNA polymerase (Boehringer Mannheim Canada, Laval, Que´bec, Canada). 10 µl of template DNA was added in the case of fecal samples and 30 µl in the case of filtered environmental water pellet suspensions. Reaction mixtures were initially denaturated at 94ºC for 1 min and then subjected to 40 cycles of denaturation at 94 ^oC for 15 s, annealing at 50oC for 1 min, and extension at 72ºC for 1 min, with an additional 7-min extension at 72 ^oC (21).

Figure 1: Sensitivity of the PCR assay for detecting Cryptosporidium parvum as determined by 2 % agarose gel electrophoresis. M: 100 bp ladder, 358 PB positive samples lines 1-9, line 10 negative.

Statistical analysis

Data in a Microsoft Excel® (Microsoft® office 2013) spreadsheet were recorded and analyzed using SPSS (version 22, IBM Corp., Armonk, NY). The Kappa test (cross-tabulation) measured the agreement values between microscopic and ELISA methods and PCR. Diagnostic accuracy of any diagnostic procedure or a test gives discriminates between certain two conditions of interest (healthy or negative and disease or positive cases). This discriminative ability can be quantified by the measures of sensitivity, specificity, positive and negative predictive values (PPV, NPV), likelihood ratio, diagnostic efficiency%, and discrimination ability. Receiver operating characteristic (ROC) curve values were calculated using the area under the curve (AUC) as a diagnostic accuracy test to validate the prediction of cryptosporidiosis; a level of 95% was considered statistically significant.

Results

The current study reported infection rate percentages based on the hot MZN staining method, ELISA, and conventional PCR at 26.66%, 40%, and 52%, respectively. In this study, the ELISA technique diagnosed more positive samples (25 true positive samples) than those found via microscopic examination (15 true positive samples) (Tables 1 and 2); however, microscopic examination diagnosed more negative samples (47 true negative samples) more than the ELISA technique did (37 true negative samples). Analysis of the two screening techniques was performed via kappa testing. This revealed no agreement between screening tests and PCR, with a kappa value of -0.152 for the hot MZN staining method and -0.164 for ELISA, although there was a significant difference between the screening test results and that for PCR (Table 3). AUC values of 0.577 and 0.5833 (Table 4) were obtained for the microscopy and ELISA, respectively (Figure 2). These values of less than 0.6 indicated that both screening techniques were unsatisfactory diagnostic tests compared with the gold standard PCR technique.

Table 1: Correlation of Ziehl-Nielsen staining results and conventional PCR results for detection of C. parvum from fecal samples investigated in the present study

Table 2: Correlation of ELISA results and conventional PCR results for detecting C. parvum from calf fecal samples

Table 3: Measure of the agreement by kappa test between both screening techniques compared with conventional PCR as the gold standard for C. parvum diagnosis in calf fecal matter