Introduction

The neonatal period (0 to 28 days) represents the most critical phase in calf rearing (1) and directly influences future productivity parameters of the animals (2). Scours is commonly observed during this stage (3). Although its long-term consequences are not fully clarified, neonatal scours may significantly reduce the average daily weight gain of calves, particularly during the first six months of life (4). Calf scours represent a significant health challenge for dairy and beef farms globally, as they are one of the most prevalent diseases in pre-weaned calves, leading to high treatment rates and considerable mortality. Among the various pathogens implicated in neonatal calf diarrhea, Cryptosporidium spp., rotavirus, coronavirus, and Escherichia coli are the most commonly identified causative agents (5). Cryptosporidium is a zoonotic protozoan parasite that can spread from human to human or animal to animal (anthroponotic transmission) or from animal to human (zoonotic transmission) and is important for both human and animal health worldwide (6). It infects the digestive system of all types of vertebrates, causing stomach problems and significant financial losses (7,8). Cryptosporidium has been designated as a "neglected pathogen" by the World Health Organization (WHO) within its 2004 Neglected Diseases Initiative. The significance of this protozoan has become increasingly recognized (9). Cryptosporidium species have attracted more attention due to their periodic outbreaks, zoonotic potential, clinical relevance in immunodeficient individuals, and the considerable economic losses they cause, particularly in newborn calves (10). Mortality resulting from calf scours causes significant financial losses. For example, annual losses in Norway have been estimated at approximately 10 million USD (11). In Türkiye, it is estimated that at least 15% of neonatal calves are lost during this period, with economic losses reaching approximately 3.15 billion TRY in 2018 alone (12). The persistence of Cryptosporidium in the environment and its resistance to conventional water treatment methods further complicate efforts to control its spread, highlighting the need for improved public health strategies and research into effective treatment options (13). Understanding these dynamics is crucial for developing targeted interventions that can reduce transmission risks. Collaborative efforts between the veterinary and public health sectors will be essential in formulating comprehensive strategies to combat the challenges posed by Cryptosporidium (14).

Although the exact neonatal calf mortality rate in Türkiye remains unclear, it is assumed to be high. Nevertheless, data on the prevalence of Cryptosporidium infections in neonatal calves and their association with farm management practices are scarce. This lack of information hinders the implementation of effective preventive measures and limits the control of zoonotic transmission risks. Therefore, the present study aims to determine the prevalence of Cryptosporidium spp. in neonatal calves and to investigate its relationship with farm management practices. In this context, the findings are expected to contribute to the development of farm management strategies and biosecurity measures and to support national and international efforts for controlling zoonotic infections. Furthermore, although the study was conducted in a limited geographical area, it represents the first large-scale investigation in Türkiye focusing on the relationship between Cryptosporidium prevalence and farm management practices.

Materials and methods

Ethical approval

This study was approved by the Local Ethics Committee for Animal Experiments of Aydın Adnan Menderes University (Decision No: 64583101/2021/040, 2021 - Session III). The present study was conducted in 2021 as part of a doctoral thesis project, and the data collection period reflects the timeline of the original research design approved by the institutional ethics committee.

Study area and sample collection

This descriptive study was conducted on cattle farms located in the Denizli province of Türkiye (Figure 1). A total of 250 fecal samples were collected from neonatal calves aged 0-28 days. The sample size was initially calculated as 345 based on the epidemiological formula defined by Martin et al. (15) (n=4×P×Q/L²); however, it was limited to 250 due to logistical and economic constraints. The study was carried out on 26 farms selected based on herd size, hygiene practices, and calf housing conditions. Fecal samples were collected via rectal stimulation (16). Additional data were recorded regarding calf age, sex, breed, health status, timing and duration of colostrum intake, feeding programs, housing conditions, drug or vaccine administration, total herd and calf numbers, vaccination practices, and farm-level sanitation and disinfection procedures.

Figure 1: Distribution of calf samples by location from farms in Denizli province, Türkiye. Location of the study area.                                                                                                                                                             

Laboratory analyses

For microscopic examination, smears prepared from fecal samples were fixed with methanol, stained using Kinyoun’s acid-fast staining method, and examined at 100× magnification with an Olympus BX51 microscope (17). Images were captured using a DP70 camera. DNA extraction was performed using the Exgene™ Stool DNA kit according to the manufacturer’s protocol, and samples were stored at −20 °C. PCR amplification targeted a 1325 bp fragment of the SSU-rRNA gene using primers Crypto F1 and R1 (18). Nested PCR was subsequently conducted using primers specific to the 826-864 bp region (19). Amplifications were carried out in 30 µl volumes using a SimpliAmp Thermal Cycler. Final products were separated by agarose gel electrophoresis and visualized under UV light with the UVP EC3 Chemi HR410 Imaging System.

Statistical analysis

Statistical analysis was performed using the IBM SPSS Statistics software (version 25). Descriptive statistics for categorical variables were presented as counts and percentages. The relationships between diagnostic results and farm management variables were analyzed using the Pearson chi-square test. A 95% confidence level was used, and p-values less than 5% were considered statistically significant.

Results

Cryptosporidium was detected in 171 out of 250 samples examined, resulting in an overall prevalence of 68.4% based on nested PCR results (Figure 2). A statistically significant association was observed between microscopic staining and molecular analysis (p=0.000; p<0.05), as all microscopically positive samples were confirmed by molecular methods, while 74 microscopically negative samples were identified as positive by nested PCR (Table 1). Among the host-related variables, only calf age, sex, and E. coli hyperimmune serum administration were found to be significantly associated with infection (p < 0.05) (Table 2). In contrast, no significant associations were observed between Cryptosporidium positivity and the non-host-related factors such as herd size, season, feeding practices, housing conditions, or sanitation measures (Table 3).

Figure 2: Agarose gel electrophoresis image of nested PCR products (original). M: DNA marker (100 bp); P: known positive control; N: known negative control.

Table 1: Distribution of the rates of kinyoun’s acid-fast staining results and nested PCR data

Table 2: Distribution of Cryptosporidium spp. positivity in neonatal calves according to host-related variables (n=250; based on nested PCR result

Table 3: Distribution of Cryptosporidium spp. positivity in neonatal calves according to non-host-related variables (n=250; based on nested PCR result).

Discussion

Compared to microscopic techniques, molecular methods are known to have higher sensitivity for detecting Cryptosporidium oocysts (19). In our study, every sample identified as positive by microscopic examination was also confirmed positive by molecular analysis. Additionally, Cryptosporidium spp. was detected exclusively by molecular methods in 74 samples. This finding confirms the ability of PCR to detect low oocyst concentrations.

The lack of a statistically significant relationship between diarrhea and Cryptosporidium reflects the conflicting findings in the literature. While some studies have reported a strong association between diarrhea and Cryptosporidium infection (20), others have identified high prevalence even in clinically healthy calves (21). In our study, the prevalence of Cryptosporidium was found to be equal in both diarrheic and non-diarrheic neonatal calves (Table 2). These results suggest that, although Cryptosporidium spp. may be an important pathogen responsible for diarrhea, stool consistency cannot reliably be used as an indicator of its presence.

Herd size is considered another potential risk factor for Cryptosporidium infection (22). Some studies have suggested that calves in small-scale farms may shed more oocysts compared to those in larger operations (23) or that the increased contact among calves in large herds elevates the risk of infection (24). However, other studies have found no association between larger herd size and increased infection risk (25). Similarly, in our study, no direct relationship was identified between herd size and infection rate; the highest infection rate was observed in medium-sized herds (51-100 head) at 76.9% (Table 3). Therefore, the influence of herd size on infection risk cannot be evaluated in isolation and should be considered alongside other factors such as farm management practices, animal density, nutrition, water sources, and age.

Seasonal variation is considered an influential factor in the spread of Cryptosporidium infections (22). Several studies have reported a higher incidence of calf diarrhea during the winter and spring months (26). However, it has also been suggested that this increase may be due to the overlap between calving periods and specific seasons (27). In farms with year-round calving, the infection is more prevalent during the summer months, which is thought to be associated with elevated temperatures and humidity (28). Some studies have linked the frequency of infection not directly to seasonality, but rather to the amount of precipitation (29). In the present study, the highest positivity rate was observed in the autumn months (76.6%) (Table 3); however, during the sampling period, average temperatures were high, and precipitation was low. Consequently, this study, in line with similar findings in the literature, suggests that Cryptosporidium infection can be prevalent throughout the year and that seasonal variability may not play a decisive role (30).

As reported in various studies, no significant association was found between calf breed and Cryptosporidium infection rate in this study (31). However, some studies have indicated that pure breeds may be at higher risk compared to crossbreeds (32) and that Holstein calves may have a greater likelihood of being infected with C. parvum (33). In this study, although the infection rate among Montbeliarde calves appeared to be 100%, only two calves of this breed were included. The other breeds showed comparable levels of infection risk (Table 2).

In our study, the infection rate in female calves (87.4%) was notably higher compared to male calves (Table 2). The literature contains conflicting findings regarding the relationship between sex and Cryptosporidium infection; some studies have reported higher prevalence in male calves (34), while others have found no significant association (35). These discrepancies may be attributed to various factors, including sample size, characteristics of the calf population, methodologies employed, potential differences in immune responses of female calves, or differences in management practices (e.g., housing, feeding).

The most significant risk of mortality in calves occurs during the first 21 days of life (36). According to the United States National Animal Health Monitoring System, 57% of calf deaths are attributed to diarrhea (37). In cases where C. parvum is identified as the sole pathogen, high mortality rates have been observed in association with prolonged and persistent diarrhea (38). In our study, six calves died during the neonatal period, and four of them were found to be infected with Cryptosporidium spp., exhibiting severe diarrhea.

There are conflicting findings in the literature regarding the protective efficacy of E. coli vaccination against infection (24). While one study by Trotz-Williams et al. (39) reported that E. coli vaccination reduced C. parvum oocyst shedding, another study found that it increased the risk of disease (39), and yet another reported no effect at all (40). In our study, a lower infection rate (46.2%) was observed in calves that received E. coli hyperimmune serum immediately after birth (Table 2). This protective effect may be associated with the prevention of potential secondary infections caused by E. coli, the absence of additional infections that cause damage to the intestinal epithelium, or general support of the immune system.

Prophylactic vitamin supplementation is recommended for calves (41). However, some studies suggest that the administration of vitamin E, selenium, antibiotics, additives, and antimicrobials through liquid diets does not significantly affect disease risk (42). In our study, no oocysts were detected in calves that received fluid therapy, multivitamins, tetracycline, and Setifour (Table 2), which is a noteworthy finding. Nevertheless, due to the limited number of such cases, further research is needed to generalize this relationship.

 Paromomycin, used against C. parvum infection, is recommended both therapeutically and prophylactically due to its effect in reducing oocyst shedding and shortening the duration of diarrhea (43). “Parofor Crypto” should be administered at a dose of 2.5 ml per 10 kg body weight for 7 consecutive days, only to animals with a confirmed Cryptosporidium diagnosis and before the onset of diarrhea (44). In our study, infection was detected in 4 out of 5 calves that received “Parofor Crypto” treatment (Table 2); however, it was reported that the treatment was discontinued after only three days upon improvement of clinical signs. Therefore, no definitive conclusion can be drawn regarding its effectiveness.

Halofuginone lactate acts on the merozoite and sporozoite stages of C. parvum, reducing the severity of cryptosporidiosis, delaying the onset of infection, and decreasing oocyst shedding. It is therefore recommended as both a therapeutic and prophylactic agent for calves (45). It has also been reported to reduce mortality (46,47). In our study, the infection rate was found to be 54.5% in a farm where halofuginone lactate was routinely administered to neonatal calves between days 3 and 8 postpartum (Table 2). However, the success of this application depends not only on pharmacological intervention but also on the integrated management of individual housing, hygiene, and environmental factors (48).

Neutralizing antibodies present in colostrum have been reported to reduce the infectivity of Cryptosporidium by preventing the adhesion of sporozoites to host cells and may even exhibit direct cytotoxic effects (49). Calves fed with colostrum from dams possessing high titers of specific antibodies are partially protected against infection (50). Therefore, feeding with high-quality colostrum and ensuring passive transfer is considered a critical management practice to minimize protozoal infections (51). Moreover, Cryptosporidium infections have been reported to be more severe in calves with failure of passive transfer (52).

On the other hand, some studies have shown that colostral immunoglobulins have limited effectiveness in preventing infection (39). Although there is weak evidence suggesting that colostrum may have a protective role, colostrum intake has not yet been effectively tested as either a risk or protective factor (24). In the present study, high rates of oocyst detection were also observed in calves that had received colostrum, indicating no significant association between colostrum intake and infection (Table 2). This finding may be explained by the intracellular nature of C. parvum, which allows it to evade antibody-mediated effects within the intestinal epithelial cells (43).

Pooling colostrum from multiple cows is generally thought to reduce overall colostrum quality and increase the risk of disease transmission (51). However, there is no consistent evidence regarding the impact of colostrum feeding methods on infection risk (24). Trotz-Williams et al. (39) and Silverlas et al. (53) reported that bottle feeding had no significant effect on the risk of infection. Nevertheless, milk contamination is possible through contaminated bottles or during milking (54). In our study, the infection rate among calves fed with a bottle was 66.8%, whereas it was 81.8% among those allowed to nurse directly from the dam (Table 2). This may be explained by the direct exposure to a high number of oocysts through contact with teats contaminated by feces. The use of nipple-less buckets to feed calves is thought to increase the risk of oocyst shedding, as it fails to satisfy the calves' natural suckling behavior (55). Although all calves fed with buckets in our study were found to be oocyst-positive, only two calves were fed this way; thus, the results are not generalizable (Table 2).

The source and handling of feed can influence Cryptosporidium infection risk (6). Feeding starter grains in addition to milk has been linked to higher oocyst shedding than milk replacers alone (56). In contrast, contamination of improperly prepared replacers may also contribute to infection (57). Poor-quality replacers can impair immune function and increase susceptibility (58). However, evidence regarding milk replacer use remains inconsistent (40,42), and further investigation is warranted (24). In our study, no significant association was observed between the use of supplemental feeds and the prevalence of Cryptosporidium infection. However, the lowest infection rate was detected in calves that were fed with cold milk treated with formic acid and calf starter feed. It is important to note that this feeding practice was implemented in only one farm (Table 3).

Disinfectants containing hydrogen peroxide and quicklime can reduce oocyst counts (52); however, in this study, high infection rates (69.4%) persisted even on farms practicing routine disinfection, and no significant difference was observed compared with farms where disinfection was not applied (Table 3). This supports previous findings that many commercial disinfectants are ineffective against oocysts at recommended concentrations. At the same time, higher doses may be toxic (24). The ineffectiveness likely results from inadequate cleaning before disinfection, as organic residues may protect oocysts within underlying layers (7).

Hygiene is a fundamental element in the control of Cryptosporidium infections (59). In order to strengthen the calf’s immune system and reduce the risk of contamination, it is recommended to change bedding regularly, apply quicklime to dried floors, and minimize contact with contaminated feces (60).

 It has been reported that calves kept under unhygienic conditions have a threefold higher infection rate (6). Poor hygiene creates favorable conditions for oocyst survival and increases the rate of infection (61).

The association between cleaning methods and disease risk has also been linked to the type of barn flooring (e.g., soil, concrete) (25). Changing bedding more than 12 times a year may increase the risk of disease, as personnel and equipment can act as fomites in spreading infections (62). Deep and dry straw bedding may provide protection by reducing contact with feces (24).

However, the existing literature and findings from this study indicate that while hygiene is essential, it is not sufficient on its own for effective infection control. Considering the environmental resilience of C. parvum, cleaning and disinfection practices must be supported by more comprehensive management strategies (63).

Environmental and housing conditions are key determinants of Cryptosporidium transmission (64). Because young calves are highly susceptible, grouping animals by age is recommended to limit contact with older, asymptomatic shedders (24,65). Studies on dam-calf contact have yielded inconsistent results- some report reduced risk with brief postnatal contact, while others found no effect (25,53,66). Contamination of calving areas remains a significant source of exposure within the first hours after birth (16), as supported by the detection of positivity in a 3-day-old calf in this study.

The influence of housing and flooring on Cryptosporidium infection remains controversial. Some studies have suggested that individual housing provides partial protection, whereas others reported no difference between individual and group systems (24, 40,48). Sharing pens may increase exposure (32). Concrete floors have been linked to lower prevalence than soil, though results are inconsistent (25,66,67). Similarly, bedding type shows variable effects-straw may elevate risk, while deep, dry straw or slatted floors could be protective (24,31,42). High humidity and poor drainage can prolong oocyst survival (68). In this study, no significant association was found between housing conditions and infection rates (Table 3), highlighting the need to evaluate these variables together with other management factors.

Due to an underdeveloped immune system, C. parvum infection occurs predominantly in calves younger than four weeks, with prevalence decreasing as age increases (69,70). The infection usually manifests as watery diarrhea between 1 and 4 weeks of age, and the development of acquired immunity with age enhances resistance (71). Oocyst shedding is most intense during the first week of life and declines after day 21 (72,73). In the present study, the highest prevalence (84%) occurred in calves aged 1-10 days, supporting the inverse relationship between age and susceptibility (53). The high infection rate in neonates likely increases environmental contamination, particularly in large herds where calf density is high, thereby perpetuating transmission (23,38). Moreover, C. parvum shedding by clinically healthy adult cattle can serve as an initial infection source for newborns (74). Detection of positivity in a 3-day-old calf in this study, together with the reported 2-3 day prepatent period (53), suggests that infection may occur within the first hours after birth (75).

Conclusion

In conclusion, the prevalence of Cryptosporidium spp. in neonatal calves in the province of Denizli was determined to be 68.4%. The infection rate decreased with age, was higher in female calves compared to males, and was found at a lower rate in calves that received hyperimmune serum (E. coli) immediately after birth. Furthermore, molecular methods demonstrated higher sensitivity compared to microscopic examination. Cryptosporidium spp. is a common pathogen responsible for diarrheal syndrome in neonatal calves. It causes significant economic losses globally in the livestock industry through calf mortality and treatment costs. The high prevalence emphasizes the need for improved biosecurity and farm management practices, as well as the widespread adoption of preventive strategies. Its zoonotic potential increases the risk to public health. In this context, animal and human health should be considered as a whole, and the "One Health" approach should be adopted. Although limited in geographic scope and sample size, this study helps fill a gap in national data. It provides a foundation for future epidemiological research. More comprehensive studies are needed to clarify the transmission dynamics and to develop effective control strategies.

Acknowledgements

This article is derived from the doctoral thesis of Dr. Zeynep Toprak Cınar, completed in the Department of Parasitology, Faculty of Veterinary Medicine, Aydın Adnan Menderes University. The authors gratefully acknowledge the valuable support of all faculty members of the Department of Parasitology. Special thanks are extended to Dr. Selin Hacilarlıoglu and Dr. Metin Pekagirbas for their contributions to the laboratory analyses, and to Dr. Ahmet Fatih Demirel (Department of Animal Science, Faculty of Agriculture, Van Yüzüncü Yıl University) for his assistance with statistical analysis. This study was supported by the Scientific Research Projects Unit of Aydın Adnan Menderes University under project number VTF-21023.

Conflict of interest

The authors declare that there are no conflicts of interest regarding the publication and/or funding of this manuscript.