INTRODUCTION
Cryptosporidium parvum is an enteric coccidian commonly isolated from calves with neonatal calf diarrhea and a potential zoonotic agent [1]. Cryptosporidium spp. belong to the phylum Apicomplexa (Sporozoa) and possess an apical complex. Members of family Cryptosporidiidae have four naked sporozoites within the oocysts [2]. To date, 45 Cryptosporidium spp. have been identified molecularly and >100 genotypes exist [3].
Cryptosporidiosis is common in neonatal calves, usually at 1–2 weeks of age with a peak at 11 d. Most calves are exposed and infected, but not all calves develop diarrhea [4]. C. parvum-associated diarrhea rarely occurs after 3 months of age [5]. After infection, diarrhea may be intermittent and last from 4–17 d with a peak at 3–5 d [6]. Depression and anorexia follow the profuse yellow-to-brown diarrhea, which contains mucus and occasionally blood streaks [7].
C. parvum oocysts are shed in huge numbers (107 oocysts per gram of feces) as early as 3 d of age, peaking at 2 weeks of age, and continuing into adulthood [6]. Oocysts shed from calves are already fully sporulated when excreted in feces and are therefore immediately infectious for animals and humans [8]. Contaminated water as well as vegetables planted on manure-fertilized soil may be a possible source of infection for humans [8]. The oocysts can survive in the environment under favorable conditions (e.g., high temperatures and moisture with low UV radiation) and resist most disinfectants [9].
In humans signs of cryptosporidiosis range from a self-limiting gastrointestinal infection in immunocompetent individuals to chronic in immunocompromised individuals (e.g., acquired immunodeficiency syndrome) or a fulminant illness with fatal consequences [10,11]. In developing countries Cryptosporidium spp. are a major cause of a diarrheal disease, nutritional deficiencies, and growth impairment in children [12,13].
Diagnosis of cryptosporidiosis requires more sensitive methods because the oocyst morphology does not permit differentiation at the species level. The current method of choice for the diagnosis of cryptosporidiosis is PCR, which is a highly sensitive tool that allows detection of a small number of oocysts. To increase sensitivity, nested PCR assays are recommended [14]. Molecular diagnostics have permitted the identification and subtyping of Cryptosporidium spp., as reported by Morgan and Thompson [15].
This study aimed to determine the prevalence and distribution of Cryptosporidium spp. infections and associated risk variables in diarrheic calves, as well as molecular identification of the circulating Cryptosporidium spp. among calves in different localities in Egypt.
MATERIALS AND METHODS
Ethical consideration
The study protocol was approved by the Animal Research Ethics Committee of the Faculty of Veterinary Medicine (Beni-Suef University, Beni Suef, Egypt) with a letter for the approval and credibility of the work (No. 022-403), in accordance with the international guidelines for animal research.
Area of study and animals
A total of 222 diarrheic calves (180 cattle and 42 buffaloes) located at 3 governorates (Al-Behira, Alexandria, and Beni Suef) were used in this study (Fig 1). These areas were selected to clarify the effect of different management systems and the spatial distribution of the disease in Al-Behira and Alexandria, which are located in Lower Egypt (North) and the Beni Suef governorate, which is located in Upper Egypt (Mid; Table 1). The study was conducted from October 2021 to March 2023, during which the animals were examined clinically and epidemiologically. Animals were examined for the suckling reflex, septicemia, degree of dehydration, and body temperature. The consistency of feces, odor, color, and presence or absence of mucus and blood were checked according to the methods described by Constable et al. [16]. Individual fecal samples were collected in sterile plastic containers, tightly sealed, properly labeled, and immediately sent to the laboratory at 4°C.
Investigated herds in different localities.
| Animal population | Locality | No. of examined calves | No. of diarrheic calves | No. of diarrheic sampled calves |
|---|---|---|---|---|
| Herd 1 | El-Nobaria (Al-Behira) | 170 | 139 | 24 cattle calves |
| Herd 2 | El-Nobaria (Al-Behira) | 617 | 146 | 5 cattle calves |
| Herd 3 | Abo-El-matamer (Al-Behira) | 350 | 90 | 6 buffalo calves |
| 22 cattle calves | ||||
| Herd 4 | Al-Ameria (Alexandria) | 45 | 15 | 6 cattle calves |
| Herd 5 | Beni Suef governorate | 56 | 40 | 35 buffalo calves |
| 5 cattle calves | ||||
| Herd 6 | Beni Suef governorate | 17 | 14 | 14 cattle calves |
| Individual calves | Beni Suef governorate | 105 | 105 | 1 buffalo calves |
| 104 cattle calves | ||||
| 180 cattle calves | ||||
| Total | 1360 | 549 | 42 buffalo calves | |
| Total 222 |
Microscopy screening
Detection of Cryptosporidium spp. oocysts was performed using the modified Ziehl-Neelsen staining technique as described by Casemore et al. [17]. Briefly, after concentration of oocysts in the collected feces using the Sheather’s sugar flotation technique [18], fecal smears were prepared on a microscope slide, air-dried, and fixed with methanol for 5 min. Fixed smears were stained with concentrated carbol fuchsin for 3–5 min and rinsed in tap water. Smears were decolorized using sulphoric acid (10.0%), then counterstained with malachite green solution (0.4%) for 1 min. Smear slides were air-dried and examined under the microscope at 100x magnifications. Cryptosporidium spp. oocysts appear as pink-to-red spherical-to-ovoid bodies against a pale green background. Samples were considered positive if at least one morphologically distinct Cryptosporidium oocyst was observed. Microscopy-positive samples were stored at −20°C without any preservatives until additional molecular analyses were performed.
Molecular characterization of Cryptosporidium spp.
DNA extraction
Fecal samples that were positive based on the modified Ziehl-Neelsen staining technique were subjected to 10 freeze-thaw cycles by freezing in liquid nitrogen for 5 min, followed by thawing in a boiling water bath for 5 min to disrupt the oocyst walls and release the target DNA, as described before [19]. DNA extraction was carried out according to the instruction manual of the gSync TM DNA extraction kit (Geneaid; New Taipei City, Taiwan).
DNA Amplification
Amplification of the gp60 gene was carried out in two steps (nested PCR) [20] in a final volume of 25 μl containing 12.5 μl of 2x Master Mix (Applied Biotechnology, city, Egypt), 1 μl of forward primer, 1 μl of reverse primer (Table 2), 4 μl of DNA template, and 6.5 μl of nuclease-free water. The thermo-cycling parameters consisted of an initial denaturation cycle at 95°C for 5 min followed by 35 cycles of the following program: 94°C for 45 sec; annealing temperature of 50°C for 45 sec for each primer; and 72°C for 45 sec followed by a final extension step at 72°C for 7 min. Amplifications were carried out in 0.2 mL tubes using Labnet® Multigene Gradient thermal cycler (catalog no. TC9600-G-230V; Labnet International, Inc., Edison, NJ, USA). Secondary PCR products were separated by electrophoresis on a 1.5% agarose gel for 30 min and visualized using a UV transilluminator.
Primer sets for PCR and nested-PCR specific to Cryptosporidium genes.
| Target gene | Primer | Nucleotide sequence (5′-3′) | Amplicon size (bp) | Ref. |
|---|---|---|---|---|
| Glycoprotein 60KDa (Gp60) | Gp60-F Gp60-R | ATAGTCTCCGCTGTATTC GCAGAGGAACCAGCATC | 950-1000 | [20] |
| Nest Gp60-F Nest Gp60-R | TCCGCTGTATTCTCAGCC GAGATATATCTTGGTGCG | 450 |
Gene sequencing and sequence analysis
PCR products were purified using a PCR purification Kit® (Thermo, Germany) [21]. All purification steps were run in accordance with the manufacturer’s instructions using reagents provided in the kit, then sequenced. Sequencing was performed by Macrogen Inc. (Seoul, Korea) for both forward and reverse directions using the same primer sets that have been used for amplification of each gene.
A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) was performed for each sequence. Evolutionary analyses and sequence alignments were performed using MEGA X software [22]. A tree was generated using the maximum likelihood method with 1000 bootstrapped data sets. The Kimura 2-parameter was used as a model and the tree was initially obtained using Neighbor-Join and BioNJ algorithms [23]. The maximum composite likelihood (MCL) was estimated as a matrix of pairwise distances. Nucleotide and amino acid identities were determined using Geneious® 7.1.3 (build 2014-03-17, Java version 1.7, Copyright© 2005-2014 Biomatters Ltd.).
Subtype identification using the gp60 gene
The recovered subtypes of sequence analysis were determined on the basis of the BLAST search in the GenBank® in addition to the protocol previously described [24] based on the numbers of serine coding trinucleotide repeats as, follows: TCA (designated by the letter A); TCG (designated by the letter G); TCT (designated by the letter T); and ACATCA (designated by the letter R) repeats in the microsatellite region.
RESULTS AND DISCUSSION
A total of 222 diarrheic fecal samples were collected from calves located in three governorates (Al-Behira, Alexandria, and Beni Suef) that were selected to clarify the effect of different management systems as well as the spatial distribution of the disease (Al-Behira and Alexandria are located in lower Egypt [North] and the Beni Suef governorate is located in upper Egypt [Mid]). Fecal samples were stained using the modified Ziehl-Neelsen technique to quantify the prevalence of Cryptosporidium spp. infection under different epizootiologic circumstances and different clinical alterations. Cryptosporidium spp. were identified in fecal smears stained with modified Ziehl-Neelsen stain (Fig 2).
Fecal sample stained with modified Ziehl-Neelsen stain; pink-to-red ovoid or spheroid acid-fast stained Cryptosporidium parvum oocyst of 4–5 μm in diameter.
The prevalence of Cryptosporidium spp. was 75 (33.78%) with 71 (39.44%) in cattle calves and 4 (9.52%) in buffalo calves (Table 3).
Prevalence of Cryptosporidium species infection in relation to different epidemiologic variables.
| Parameter | Animals (222) | Microscopy-positive samples (n=75 [33.78%]) | PCR-positive samples (n=63 [84%]) | * P value |
|---|---|---|---|---|
| Species | Cattle calves (180) | 71 (39.44%) | 59 (83.1%) | 0.001 |
| Buffalo calves (42) | 4 (9.52%) | 4 (100%) | ||
| Age | Birth to 15 days (123) | 56 (45.53%) | 47 (83.93%) | 0.004 |
| 16 to30 days (75) | 17 (22.67%) | 14 (82.35%) | 0.01 | |
| 31to 45 days (24) | 2 (8.33%) | 2 (100%) | 0.298 | |
| Sex | Male (121) | 38 (31.4%) | 34 (89.47%) | 0.412 |
| Females (101) | 37 (36.63%) | 29 (78.38%) | ||
| Breed | Native cross breed (150) | 29 (19.33%) | 23 (79.31%) | 0 |
| Foreign breed (72%) | 46 (63.89%) | 40 (86.96%) | ||
| Rearing system | Individual boxes (63) | 39 (61.9%) | 35 (89.74%) | 0.295 |
| Calf rearing unit (14) | 11 (78.57%) | 9 (81.82%) | 0 | |
| Mixed with other animals (145) | 25 (17.24%) | 19 (76%) | 0 | |
| Area of Study | Beni Suef (159) | 36 (22.64%) | 29 (80.56%) | |
| Al-Behira (57) | 33 (57.89%) | 28 (84.85%) | ||
| Alexandria (6) | 6 (100%) | 6 (100%) | ||
| Method of colostrum feeding | Natural (145) | 25 (17.24%) | 19 (76%) | 0 |
| Artificial (77) | 50 (64.94%) | 44 (88%) | ||
| Dam party | 1st (98) | 48 (52.74%) | 43 (89.58%) | 0.767 |
| 2nd (36) | 18 (41.86%) | 14 (77.78%) | 0.011 | |
| 3rd (30) | 5 (16.67%) | 3 (60%) | 0.015 | |
| 4th (22) | 2 (9.09%) | 2 (100%) | 0.001 | |
| 5th and more (36) | 2 (5.56%) | 1 (50%) | 0.175 0.101 0.013 0.937 0.631 0.986 |
*P<0.05 were considered statistically significant.
A non-significant increase in the prevalence of Cryptosporidium spp. in males was 38 (31.4%) compared to 37 (36.63%) in females. There was a significant increase in prevalence of Cryptosporidium spp. in calves from birth to 15 d of age 56 (45.53%; P=0.004) and a significant increase (17 [22.67%]) from 16-30 d of age at a P=0.01 compared to 2 (8.33%) from 31–45 d as non-significant at a P=0.298 (Table 3). Age was considered by Ogendo et al. [25] as an important risk factor affecting the prevalence of cryptosporidiosis in calves. Moreover, it was reported that cryptosporidiosis usually occurs in neonatal calves at 1–2 weeks of age with a peak at 11 d of age [19]. Most calves become exposed and infected but not necessarily all calves develop diarrhea. The high prevalence and the rapid transmission among calves has been attributed to the high number of Cryptosporidium oocysts (107/g of feces) contaminating the environment as early as 3 d of age, which peaks at 2 weeks of age [6,26]. Moreover, these oocysts are fully sporulated when excreted in feces and are therefore immediately infectious for both animals and humans, as discussed previously [8].
Crossbred calves were shown to have a significant increase in the prevalence of calf cryptosporidiosis (46 [63.89%] vs. 29 [19.33%] in native bred (P=0; Table 3). This finding indicates that the animal breed is an important host determinant influencing the immune response and disease severity [27]. Similarly, it was previously reported that calf diseases are significantly higher in crossbred than native animals [13].
The highest prevalence of cryptosporidiosis was observed in calves derived from dams of the first parity compared to calves derived from dams of the 2nd parity (P=0.767), 3rd parity (P=0.011), and 4th parity (P=0.015). This finding can be attributed to the IgG concentration, which increases as the parity increased, as previously reported [28].
The prevalence of cryptosporidiosis in calves was 36 (22.64%), 33 (57.89%), and 6 (100%) in Beni Suef, Al-Behira, and Alexandria, respectively (Table 3). This finding highlights the enzootic nature of this disease in different localities in Egypt and clarifies the significant clinical impact and economic losses in calves as well as the possibility of exposure and infection of humans in contact with these animals. The results also demonstrate the possibility of survival of the causative agent as an important component in the epidemic triangle of this disease, which can be discussed on the basis that the Cryptosporidium oocysts can survive in the environment under favorable conditions in Egypt, including high temperature and moisture with low UV radiation as well as resistance to most disinfectants, as reported before [9].
In this study the prevalence of cryptosporidiosis in calves was 39 (61.9%) in calves reared in individual boxes, 11 (78.57%) in calves reared in calf rearing units, and 25 (17.24%) in calves reared mixed with other animals (Table 3). This finding indicates the high spread and enzootic nature of the disease and lack of hygienic measures in the populations under study. The prevalence of cryptosporidiosis was 25 (17.24%) in naturally suckled calves and 50 (64.94%) in artificially suckled calves, which was significantly higher (P=0) and may be related to contamination of milk in artificially suckled calves.
It is important to point out that of clinically affected calves, 39 (52.7%) had severe dehydration (Table 4) and 49 (7.12%) had an increase rise in body temperature to 39.1–40oC. Of the clinically affected calves, 34 (40%) had no suckling activity, 29 (32.22%) had watery diarrhea, 43 (34.96%) had mucoid diarrhea (Fig 3), and 3 (33.33%) had bloody diarrhea. The results were in agreement with Kumaresan [7], who reported that profuse yellow-to-brown diarrhea, which contains mucus and occasionally blood streaks that commonly occur in Cryptosporidium spp.-infected calves [7].
Clinical abnormalities observed in Cryptosporidium species-positive diarrheic calves.
| Signs | Finding | Microscopy-positive samples (n=75 [33.78%]) | PCR-positive samples (n=63 [84%]) |
|---|---|---|---|
| Body Temperature °C | ≤ 37 (10) | 0 (0%) | 0 (0) |
| 37.1 to 38 (18) | 5 (27.8%) | 5 (100%) | |
| 38.1 to 39 (78) | 21 (26.92%) | 14 (66.67%) | |
| 39.1 to 40 (104) | 49 (47.12%) | 44 (89.9%) | |
| > 40 (12) | 0 (0%) | 0 | |
| Suckling ability | Positive (96) | 29 (30.21%) | 26 (89.66%) |
| Relative (41) | 12 (29.26%) | 10 (83.33%) | |
| Negative (85) | 34 (40%) | 27 (79.41%) | |
| Dehydration | Normal (73) | 5 (6.85%) | 5 (100%) |
| Mild (34) | 12 (35.29%) | 10 (83.33%) | |
| Moderate (41) | 19 (46.34%) | 14 (73.68%) | |
| Severe (74) | 39 (52.7%) | 33 (84.62%) | |
| Attitude | Standing (143) | 36 (25.17%) | 31 (86.11%) |
| Recumbent (79) | 39 (49.37%) | 32 (82.05%) | |
| Condition of feces | Watery (90) | 29 (32.22%) | 23 (79.31%) |
| Mucoid (123) | 43 (34.96%) | 38 (88.37%) | |
| Bloody (9) | 3 (33.33%) | 2 (66.67%) |
The occurrence of diarrhea in Cryptosporidium infections can be attributed to pathogen invasion into enterocytes, which induces changes in intestinal structures that leads to villous atrophy in calves [29]. Crypt hyperplasia also follows to substitute the damaged epithelial cells but disturbance of the epithelial barrier and secondary infections can occur [30,31]. The combined loss of the microvillus border and villus atrophy, as well as loss of membrane-bound digestive enzymes, lead to fermentation of undigested milk in the intestinal lumen, which promotes a malabsorptive diarrhea [9,32].
DNA extraction was performed on 75 Cryptosporidium spp.-positive fecal samples based on modified Ziehl-Neelsen staining. Owing to the Cryptosporidium spp. DNA that is contained in oocysts with a strong wall that is difficult to lysis, fecal samples were subjected to 10 freeze-thaw cycles [1].
Amplification of the two-step nested PCR for the gp60 gene was performed with detection in 59 (83.1%) cattle calves and 4 (100%) buffalo calves.
Unsuccessful PCR amplification from all DNA extracted from microscopy-positive samples might be explained by the low concentration of oocysts in the fecal samples. The difficulty in molecular characterization of Cryptosporidium spp. may be attributed to the existence of large amounts of host DNA in the feces from sloughed intestinal cells, intestinal microflora, and other pathogens. Moreover, the presence of heme, bilirubin, bile salts, and carbohydrates in feces may deteriorate oocyst lysis, degrade DNA, and/or hinder polymerase activity.
The purified nested PCR amplicons of the gp60 gene in Cryptosporidium isolates were sequenced and the nucleotide sequences were deposited in the GenBank database under the following accession numbers: PP262546; PP262547; PP262549; PP262550; and PP262551.
Due to the heterogeneity of the sequences and its relevance to the biology of the parasite in the identification of genotypes, subtypes, or lineages require sequencing of highly polymorphic regions, such as the gp60 gene. Analysis of the gp60 gene is commonly used in Cryptosporidium spp. subtyping [20]. Alignment of the obtained sequences with reference sequences downloaded from GenBank showed that isolates obtained from cattle belonged to the C. parvum subtype family IIa. C. parvum isolates in this study belong to uncommon C. parvum subtype, especially in ACATCA repeats, as demonstrated in Tables 5 and 6. Chen et al. [19] reported that the IIa subtype family is globally prevalent and found in Asia, Europe, and Africa.
Data of selected gp60 gene sequences used in comparative analysis.
| Accession No. | Country/Year | Subtypes | Species |
|---|---|---|---|
| MG516785.1 | Australia/2017 | IIaA19G2R1 | Cattle |
| OL598539.1 | Sweden/2021 | IIaA22G1R1 | Homo sapiens |
| MT010362.1 | Uruguay | IIaA22G1R1 | Bos Taurus |
| PP262546 | Beni Suef/Egypt2021 | IIaA9G1R7 | Calf |
| PP262547 | Beni Suef/Egypt2021 | IIaA9R8 | Calf |
| PP262548 | Beni-Suef/Egypt2021 | IIaA9G1R7 | Sheep |
| PP262549 | El-Beheira/Egypt2021 | IIaA7G1R8 | Calf |
| PP262550 | Beni Suef/Egypt2021 | IIaA6G2R8 | Calf |
| PP262551 | Beni Suef/Egypt2021 | Calf | |
| PP262552 | Beni Suef/Egypt2021 | IIaA11G1R6 | Sheep |
| KM085026.1 | UK/2014 | IIaA19G1R2 | Homo sapiens |
| JX575586.1 | USA/2012 | IIaA16G2R3 | Homo sapiens |
| OP132400.1 | Egypt/2022 | A9-R11 | Homo sapiens |
| OP132399.1 | Egypt/2022 | A9-R11 | Homo sapiens |
| OP132398.1 | Egypt/2022 | A9-R11 | Homo sapiens |
| OP132397.1 | Egypt/2022 | A9-R11 | Homo sapiens |
| OP750035.1 | Al-Sharkia3/Egypt2022 | Calf |
Red color; sequences obtained in the current study.
Xiao et al. [33] reported that with respect to the subtype family C. parvum IIa, there are few genotypes that possess two copies of the ACATCA sequence just before the trinucleotide repeat. These genotypes are represented as “R2” (R1 represents many subtypes). Interestingly, the current study proved that all genotypes possess more than two copies of the ACATCA sequence. These results were in agreement with that reported in another study that detected C. parvum isolates in samples collected from patients with gastrointestinal cancer in Egypt [18].
Phylogenetic analysis of the gp60 gene clustered 100.0% of sequenced isolates in the current study into 1 cluster, which included the C. parvum IIa subtype family.
The tree showed three clusters (C. parvum IIa subtype family, C. parvum IId subtype family, and C. hominis subtypes; Fig 4). The C. parvum IIa subtype family falls into two subgroups (A and B). Isolates in the current study (C. parvum isolate Egy/BSU/2022-1: 5 [PP262546, PP262547, PP262549, PP262550, and PP262551]) were identified in subgroup B with C. parvum isolates (OP132397.1 and OP132400.1) that were detected in samples collected from patients with gastrointestinal cancer in Egypt. C. parvum isolates in this group belonged to an uncommon C. parvum subtype family, especially in ACATCA repeats.
Phylogenetic analysis of glycoprotein 60 (gp60) of Cryptosporidium spp.
The tree was generated using the MEGA X program by the neighbor-joining analysis. Bootstrap confidence values were calculated on 1000 replicates according to the maximum likelihood approach. Cattle sequences obtained in this study are labeled red.
CONCLUSION
The high prevalence and the enzootic nature of cryptosporidiosis in calves have been proved in this study in different localities in Egypt associated with lack of good hygienic measures. Different important risk factors were shown to influence the prevalence of cryptosporidiosis. Isolates of Cryptosporidium spp. obtained from cattle belonged to the C. parvum subtype family IIa that possess zoonotic importance. C. parvum isolates in this study belong to an uncommon C. parvum subtype family, especially in ACATCA repeats.