Prevalence of Cryptosporidium infection and associated risk factors in calves in Egypt

Cryptosporidium is one of the causative parasitic agents that causes gastrointestinal diseases in calves. The parasite poses a zoonotic risk to immunocompromised individuals and children. Thus, this study aimed to determine the prevalence of Cryptosporidium infection in calves in three Egyptian governorates situated in Nile Delta and assess the associated risk factors. The Cryptosporidium oocysts were detected in 81 out of 430 calves (18.84%). In addition, the univariant analysis showed that age, feeding source, hygienic status, presence of diarrhea and contact with other animals were significantly (P < 0.05) associated with Cryptosporidium prevalence in calves. Furthermore, the risk factors related with Cryptosporidium prevalence were age (OR 1.96, 95%CI 0.97–3.94), feeding on milk and pasture (OR 2.07, 95%CI 1.15–3.72), poor hygienic condition (OR 2.25, 95%CI 1.28–3.94), presence of diarrhea (OR 2.47, 95%CI 1.23–4.96) and contact with other domestic animals (OR 2.08, 95%CI 1.24–3.50). In addition, the PCR assay targeting 18srRNA showed that the most prevalent species among calves was C. parvum. Although additional researches are required to understand the most effective steps that farmers and veterinary professionals should take to decrease the occurrence of Cryptosporidium infection.

Cryptosporidium is an intracellular protozoan and one of the most common enteric pathogens in claves during first two weeks of life.Four species are usually discovered infecting cattle: C. parvum, C. andersoni, C. bovis, and C. ryanae 1,2 .C. parvum is commonly associated with diarrhoea in susceptible hosts, causing sickness and even mortality, notably in neonatal calves 3 .The life cycle of this pathogen is direct, and it can grow and replicate in infected animal's gastrointestinal epithelial cells 4,5 .The infective stage of Cryptospordium's life cycle is the oocyst, which is secreted in the faces and contains four sporozoites.When the oocyst is ingested, sporozoites are released.These sporozoites enter the cells, forming oocysts with thick and thin walls.The thick-walled oocyst is discharged in the faces.The thin-walled oocysts can rupture, allowing the sporozoites to infect new host enterocytes and produce autoinfection, leading to relapses or persistent gut illness.These sporozoites go through several phases before creating new oocysts.Cell infection results in cell death, which causes intestinal villi to shrink and fuse 6 .
In addition, the parasite can be passed from person to person, animal to animal, or animal to human (zoonotic transmission) 7 .
Infections are usually transmitted through the faecal-oral route, with infective stages of expelled sporulated oocysts through direct or collateral contact 8 .The infection is known to be self-limiting in the immunocompetent hosts, but it can cause acute or severe diarrhea in young animals or in immunocompromised hosts 9 .
Even though bovine cryptosporidiosis has been identified as a significant contributor to newborn diarrhea and financial losses on dairy farms, it is frequently misdiagnosed 10 .It is characterized by anorexia, abdominal pain and diarrhea, which can cause slow growth and even death.Diarrhea usually begins 3-5 days after infection and lasts 4 to 17 days in most of infected calves 11 .Oocyst shedding begins four days after birth and peaks

Ethical statement
The ethical committee for animal research at Benha University approved the entire study's methodology and procedures.Informed consent was obtained from owners of examined claves.The Faculty of Veterinary Medicine's ethical committee ensured that all procedures were carried out in accordance with the relevant laws and guidelines.The ARRIVE criteria were followed during research procedure.
The Delta is the driest region in the country, has relatively mild temperatures, which increase to 38 °C in summer season.On average, the delta receives 100-200 mm of rain each year, with the majority of this falling during the winter months.These research areas focus mostly on agriculture, livestock husbandry, and have a high number of farms and pastures.

Sample size and sampling
A cross sectional study was conducted during October 2020 to September 2021 using simple random sampling approach to achieve the forementioned goals.Based on Thrusfield's formula 31 , the sample size was calculated using an expected prevalence of 19.2% 30 at a 95% confidence interval and a 5% precision value.Consequently, 238

Questionnaire
At the time of sampling, the farmer completed the provided questionnaire, which mostly asked about animalrelated information including breed, age, sex, and body condition.Data on management, including feed source (pasture and milk or pasture), hygienic status (good or poor), the presence of diarrhea (yes or no), and contact with other domestic animals (yes or no), were also gathered.

Sample analysis
The samples were transferred to the laboratory for further processing on the same day they were collected.After that, the samples were treated via faecal flotation in a Sheather's sugar solution 24 .Then, fecal smear was prepared and stained by modified Ziehl-Neelsen stain 32 .According to Anderson 33 , the severity of the infection was determined by counting the cryptosporidial oocysts in a field at a 1000× magnification; the levels were mild (1-5 oocysts/field), moderate (6-20 oocysts/field), and severe (more than 20 oocysts/field), Fig. 2.

Molecular identification of cryptosporidiosis
The QIAamp DNA stool Mini Kit (QIAGEN, Hilden, Germany) was used to extract direct DNA from 220 mg of faeces samples according to the manufacturer's instructions.The Cryptosporidium small subunit (18S) rRNA gene was amplified with the oligonucleotide primers CRP-DIAG1 forward (5-TTC TAG AGC TAA TAC ATG CG-30) and CRP-DIAG1 reverse (50-CAT TTC CTT CGA AAC AGG A-30).The PCR assay was performed in total volume of 25 µl including 1 µl of each primer of (20 pmol), 12.5 µl of PCR Master Mix (EmeraldAmp Max, Takara, Japan), 5.5 µl of water, and 5 µl of DNA template.The PCR Conditions was performed as described by Paul et al. 34 .
For the secondary/nested PCR, 1 µl of the primary PCR products was utilized as a template and amplified using the primers CRP-DIAG2 forward (50-GGA AGG GTT ATT TAT TAG ATA AAG -30) and CRP-DIAG2 reverse (50-AAG GAG TAA GGA ACA ACC TCCA-30).The reaction mixture was initially denaturated at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for one min, annealing at 57 °C for 1 min, elongation at 72 °C for 1 min, and final elongation at 72 °C for 10 min as described by Paul et al. 34 .The amplified products were identified using 1.5% agarose gel electrophoresis and ethidium bromide staining.

Data analysis
The statistical SPSS software programme, Version 24.0 (IBM, USA), was used for all data analyses.To ascertain the relationship between predicted risk variables and the occurrence of Cryptosporidium infection, the univariate logistic regression approach was applied.When the P value is less than 0.05, the findings are considered statistically significant.A multivariable analysis comprised factors that were significantly (P < 0.05) related to the outcome variable in the univariable analysis 20,[35][36][37][38][39][40] .A test for multicollinearity was also conducted to determine confounding factors and assess the fit of the multivariate model.

Results
Cryptosporidium oocysts were detected in 81 (18.84%) of the 430 examined calves and the highest prevalence rate (24.67%) was observed in Kafr ElSheikh governorate, but the lowest rate (14.29%) found in Qalyubia governorate, Fig. 1.PCR analysis of all positive samples with microscopic examination revealed predicted bands for Cryptosporidium spp. at 1,325 (Fig. 3) in primary PCR which confirm presence of C. parvum in all samples in secondary PCR at 835 bp (Fig. 4).
By using univariate logistic regression analysis, five variables had a substantial impact on the prevalence of Cryptosporidium infection in calves.The prevalence of the disease was similar between the sex (P = 0.764), between breeds (P = 0.076), across geographic regions (P = 0.064), and in terms of body condition (P = 0.785), Table 1.
Table 2 showed the results of multivariate logistic regression analysis on significant factors (P < 0.05) in univariate analysis, which were age, feed source, sanitary state, presence of diarrhea, and contact with other domestic animals.The prevalence of Cryptosporidium infection increased significantly with age, older calves were two times (OR 1.96, 95%CI 0.97-3.94)more likely to contract the Cryptosporidium infection as compared to young calves.Farms had poor hygiene condition and pasture and milk as source of feeding increased the risk of Cryptosporidium infection by two folds (OR 2.25, 95%CI 1.28-3.94)and (OR 2.07, 95%CI 1.15-3.72),respectively.Animals with diarrhea were 2.47 times (OR 2.47, 95%CI 1.23-4.96)more likely to acquire Cryptosporidium infection than normal calves.Moreover, the risk of Cryptosporidium infection increased two times (OR 2.08, 95%CI 1.24-3.50)more among calves in contact with domestic animals than other.

Discussion
Cryptosporidiosis in animals is considered a zoonotic risk to humans, due to the release of large numbers of resistant oocysts, which contaminate surface water.The Veterinary researchers were interested in cryptosporidiosis because, in addition to its zoonotic significance, it may develop into a dangerous, difficult-to-control disease in many farm animals and cause large economic losses.The present study aimed to evaluate the prevalence of Cryptosporidium infection and asses the associated risk factors in calves.www.nature.com/scientificreports/ In the present study, the total Cryptosporidium prevalence in calves was found to be 18.84% (81/430).This corresponds to the findings of Ayele et al. 41 , who reported an infection rate of 18.6% in dairy calves in northwest Ethiopia.In addition, the prevalence rate in this study is consistent with the previously reported rate (19.2%) for bovine calves in Ismailia governorates in Egypt 30 .Similar prevalence rate (17.9%) was found in dairy calves from France 42 .This study's prevalence rate for Cryptosporidium infection was lower than the reported rates in eastern Ethiopia 27.8% by Regassa et al. 43 , USA 35.5% by Santın et al. 44 , Vietnam 44.3% by Nguyen et al. 45 and in UK 27.9% by Brook et al. 46 but higher than 7.8% in, 13.6%, and 15.8% which reported by Wegayehu et al. 47 , Ayana and Alemu 48 , and Wegayehu et al. 49 in Ethiopia, respectively.
Furthermore, the detectable species in examined calves was C. parvum which come in accordance with the findings of Singh et al. 50who reported 79.41% of young dairy calves in Punjab infected by C. parvum.Also, other studies reported the more prevalent Cryptosporidium species in calves in Ethiopia and Egypt is C. parvum with prevalence rate of 18.6% and 32.2%, respectively 30,51 .
The differences in overall Cryptosporidium prevalence between studies could be attributed to differences in ecology, research design, seasons, management system, age, herd size, and laboratory tests used 23,28,46,48,[52][53][54][55][56][57][58] .The diagnostic procedures used in this survey are less sensitive and can produce misleading negative results.This could potentially be the explanation for report variation 59 .
The sex had no effect on the prevalence of Cryptosporidium infection in the current study, which come in agreement with previous findings of Paul et al. 34 .In contrast, other studies reported significant role for sex on prevalence of Cryptosporidium in calves 32,60 .
The significant effect of age on Cryptosporidium prevalence in calve in this study was consistent with previous findings of Wegayehu et al. 49 in Ethiopia, they found higher prevalence in calves older than 3 months and stated infection was age related and 92.1% were infected with C. andrsoni which infect older age calves.In contrast, Geurden et al. 61 , Ayele et al. 41 and Venu et al. 59 stated that infection rate decreased with the increase of age.Similarly, the effect of age on prevalence of Cryptosporidium infection in calve was observed in other studies 16,41 .This might be due to lower tolerance of young calves as a result of immature immune system.Calves under four months of age are more susceptible to contracting Cryptosporidium infection 46 .This findings is consistent with the findings of Kváč et al. 62 , who observed that infection resistance can evolve with age due to immunological development over time.
Additionally, a significant correlation was found between the hygienic condition of the farm and the occurrence of Cryptosporidium infection in calves.The current result is confirmed by the findings of Abebe et al. 63 , who found a significant association between Cryptosporidium infection and the hygiene status of the farms.In addition, a similar results were reported by El-Khodery and Osman 24 and Castro-Hermida et al. 64 , they confirmed that poor hygiene enhances the infection rate and dissemination of Cryptosporidium species in animals.This could be attributed to the fact that muddy or dirty farm could probably establish a favourable condition for the presence or existence of Cryptosporidium oocysts in the surround environment of animals.This can be increasing the risk of contamination of food and water, hence increase the risk of Cryptosporidium infection in calves 41,52,[65][66][67][68][69][70] .
The present findings are directly in line with previous findings of Ayele et al. 41 , who observed that the prevalence of Cryptosporidium increased significantly among calves feeding on pasture and milk.This may be due to pasture being contaminated with infectious oocysts, and switching between pasture and milk may produce digestive disturbances that increase the prevalence of cryptosporidiosis.
Cryptosporidiosis causes sever watery diarrhea in calves.The findings of the present study revealed strong association between presence of diarrhea and prevalence of Cryptosporidium in calves.This was explained by the fact that diarrheal animals had a higher rate of oocyst shedding than calves with regular faeces.This is consistent with those has been found in previous studies of El-Khodery and Osman 24 and Abebe et al. 63 .This could be as a result of the infection causing villous atrophy and crypt hyperplasia, which reduces the intestine's surface area available for absorption 71 .Consequently, interfere with absorption of water, glucose and sodium leading to diarrhea 72 .Additionally, the parasite may be able to decrease the activity of the enzyme disaccharidase, which would reduce the amount of sugars broken down.This would lead to bacterial growth, the production of volatile fatty acids, and changes in osmotic pressure, which would then cause severe watery diarrhea 73 .
Different animal species and human are susceptible to Cryptosporidium infection and the infection can be transmitted by direct or indirect routes through fecal-oral route 32 .Consequently, mixing different animals species could facilitate the transmission of the disease 42 .Similarly, Mohammed et al. 74 observed that keeping animals separately or avoiding close contact with animals of various species can reduce the risk of Cryptosporidium infection.

Conclusion
The prevalence of Cryptosporidium infection was widely spreading among calves in the studied governorates with rate of 18.84%.Based on the present findings, age, feed source, farm hygiene, occurrence of diarrhea, and interaction with various domestic animals were all risk factors for Cryptosporidium infection.It is essential to raise awareness of risk factors, sources of infection, and modes of transmission to control and reduce the disease transmission between animals and human.Further molecular researches in different areas of the country are recommended to determine species allocation and the disease's national impact.

Table 1 .
The prevalence of Cryptosporidium infection in calves in relation to different factors.*The result considered significant if P < 0.05.

Table 2 .
Risk factors associated with Cryptosporidium prevalence in calves.B Logistic regression coefficient, SE Standard error, OR Odds ratio, CI Confidence interval.