Cryptosporidium from sheep herds around water tributaries of the Malakand region of Pakistan

Cryptosporidium is an intestinal protozoan parasite that infects gastrointestinal tracts and causes cryptosporidiosis in many hosts, including humans (Ryan et al. 2016). Cryptosporidium infections have been reported in more than 170 species, including cold-blooded and warm-blooded animals (Zahedi & Ryan 2020). Forty-four species and more than 120 genotypes of Cryptosporidium have been identified up to now, out of which 19 species are reported in small and large ruminants and two species, C. hominis and C. parvum, are the most widely distributed pathogens responsible for 95% of the infections (Ryan et al. 2021; Karimi et al. 2023). Cryptosporidiosis in livestock may have a significant economic impact on farmers due to the high rate of mortality and morbidity in young animals and ranks first in waterborne parasitic protozoans globally (Aniesona & Bamaiyi 2014; Efstratiou et al. 2017). Cryptosporidiosis in animals and humans results from contaminated water and food sources with oocysts, which may pollute the water supplies. According to the World Health Organization, 2.2 billion people still lack access to safely managed water services, including 1.5 billion with basic services, 292 million with limited water, 296 million who use unimproved sources, and 115 million who still collect drinking water directly from rivers, lakes, and other surface water bodies (WHO/UNICEF JMP 2023). Drinking water is a critical source of cryptosporidial infection in low-income countries with a basic foundation and infrastructure (Pal et al. 2021). Cryptosporidium infection is the leading cause of diarrheal diseases in both animals and humans. It poses a high risk to drinking water in communities of developed countries and counts as 60% of the most waterborne outbreaks reported (Aniesona & Bamaiyi 2014). Oocysts of Cryptosporidium are resistant to challenging environmental conditions, and water treatment processes facilitate their transmission to animals. The importance of cryptosporidiosis in farm animals, especially in sheep, results in diarrhea caused by this parasite alone and may occur with other pathogens affecting their health and production. Clinically, the primary sign of infection in sheep is diarrhea, although there may also be dehydration, abdominal pain, cramps, depression, listlessness, unthriftiness, and loss of body weight, leading to high morbidity and mortality globally (Mammeri et al. 2019; Santin 2020). Cryptosporidium was considered the chief cause of diarrhea in sheep and had public health significance because their zoonotic transmission can occur when contact with infected animals (Mohammad & Mohammad 2015). Cryptosporidium species affect small ruminants and raise the importance of sheep in transmitting the infection to humans and cattle. However, young animals such as lambs and calves are susceptible to infection, while adults mostly do not show symptoms (Bouzid et al. 2013). Worldwide, Cryptosporidium is responsible for 30–50% of deaths in young ages and considered as the second highest reason of diarrhea and deaths after retrovirus (Striepen 2013). This study is of significant importance in understanding the prevalence of Cryptosporidium in sheep herds located around water bodies, which are potential risk factors for humans in Pakistan. Sheep are infected with three main Cryptosporidium species, namely, Cryptosporidium parvum, C. xiaoi, and C. ubiquitum; among these three species, C. parvum is the most zoonotic species (Ozdal et al. 2009; Ryan et al. 2014). The transmission routes of infection and the role played by host animals, such as sheep and goats, in the epidemiology of human infections (Dessì et al. 2020) involve infected sheep or lambs secreting feces with high concentrations of oocysts. These oocysts can survive in cool and moist environments and may cause environmental, possibly surface water, contamination (Santin 2020). Oocysts in feces can be carried to surface water bodies by runoff specifically from high rainfall areas. Increased stream flow, bed sediments, and runoff events increase the risk of Cryptosporidium infection (Swaffer et al. 2018; Zahedi et al. 2020). The present study was conducted to investigate the prevalence of Cryptosporidium in sheep herds located around the water bodies, which have various potential risk factors for sheep in Pakistan.

All the research study, including experiments and procedures/designed protocols, was performed with the approval taken from the Research Ethics Committee at the Laboratory of Parasitology and Faculty of Veterinary Medicine, College of Veterinary Sciences and Animal Husbandry Abdul Wali Khan University Mardan, Pakistan. Several samples and data were recorded from suspected animals (lambs and sheep), herds, and farms using a pre-tested questionnaire (see Appendix I) by following instructions from the Animal Genetics and Parasitology Laboratory, and permission was also taken from farms and sheep owners before data recording and sample collection.

All the collected samples and experimental protocols were processed in the Animal Genetics and Parasitology Laboratory at the College of Veterinary Sciences and Animal Husbandry Abdul Wali Khan University Mardan, Pakistan.

During sampling, 5–10 g of fecal pellets were collected from the rectum of sheep into plastic, sterile, wide-mouthed, labeled, germ-free containers. These samples were transported to the laboratory within 48 h, and preserved in 10% formalin for microscope analysis and 75% ethanol for molecular detection, stored at 4 °C in the refrigerator for further processing. All the data from sheep were recorded during sampling with the help of a pre-tested questionnaire proforma, including locality, breeds, sex, age, water source, management, feeding, environmental and body conditions, physical health, and diarrhea.

All the collected samples were weighed and processed using a modified centrifuge-flotation technique described by Connelly et al. (2008), and solid samples were liquefied in bottles containing distilled water to make a homogenized solution. Then, this solution containing 3 g of the sample was centrifuged at 1,500 rpm for 3–5 min, supernatants were discarded, and sediments were stained using the modified Ziehl–Neelsen (MZN) staining method to confirm the presence of Cryptosporidium oocysts (Lebbad et al. 2021).

For molecular detection, all the microscope-positive Cryptosporidium samples were preserved in ethanol and refrigerated at −60 °C. According to Elsafi et al. (2013), the samples were subjected to polymerase chain reaction (PCR) after DNA extraction.

Deoxyribonucleic acid (DNA) was extracted using a commercial kit from Invitrogen, the Thermo Fisher PureLink™ Microbiome DNA Purification Kit (Waltham, USA, C. No. A-29790). The kit standard protocol was used at 20–25 °C according to the manufacturer's instructions with some minor modifications.

The extracted DNA from all the positive samples was subjected to the thermocycler (Kyratec, made in Australia, Model: SC300G-R2) using species-specific primers designed through bioinformatics tools such as Vector NTI and GenBank accession number (L16996).

Forword – AWA722F 3′-AGTGCTTAAAGCAGGCAACTG-5′

Reverse – AWA1235R 5′-CGTTAACGGAATTAACCAGAC-3′

They were used to amplify the 18S rRNA gene (556-bp) of C. parvum in sheep. The manufacturer obtained and synthesized the same designed primer sequence (Macrogen, Korea). For dilution, 300 μL of distilled water was added to each forward and reverse primer, vortexed to reconstitute the primer, and stored at −20 °C. A total of 20 μL volume of the PCR was performed, comprising 10 μL master mix, 1 μL genomic DNA from C. parvum, 2 μL forward and reversed primers, and 7 μL PCR grade water, which was added into PCR tubes and subjected to the PCR assay. The initial denaturation was performed at 95 °C for 5 min, denaturation at 96 °C for 5 s and 35 cycles, annealing for 5 s at 55 °C, extension at 68 °C for 17 s, and final extension for 1 min at 72 °C. After that, the amplified DNA sample was properly labeled and stored at −20 °C until further processing. A 1% (w/v) agarose gel was prepared, the DNA samples were visualized using ethidium bromide, and photographs were taken in the gel documentation system.

Statistical analysis of all the recorded data and experiments was analyzed using XL formulae and the IBM SPSS Statistics 20 version. All the risk factors and variables were tested using the chi-square test (x²), and the values at p < 0.05 were considered significant. Moreover, the sensitivity and specificity of the experimental data were subjected to online software (WinEpi-Veterinaria, Universidad de Zaragoza 2010). Adobe Photoshop 8.0 was used for all figures, including cropping, editing, and graphical presentation of the data.

Cryptosporidium infections were confirmed through a rigorous process of molecular detection using the PCR. A comprehensive assessment of risk factors such as diarrhea, age, gender, species, breed, season, and area was conducted to determine the prevalence rate of Cryptosporidium infection in sheep.

The cryptosporidial infection was recorded in 26 sheep herds in different areas of the Malakand region, where 23.88% of the highest prevalence rate was observed in Meherdi at 40%, followed by Mahajar camp at 37.25%, Wartair at 34.37%, Palai at 27.5%, Sakhakot at 24.32%, Dargai at 25.80%, Jaban at 21.27%, and the lowest prevalence was in Khar at 17.85%, Heroshah at 17.5%, Kot at 15.55%, Kharki at 15%, and Gharri UK, which is the area with no infection, as shown in Table 1.

The infection rate was also recorded in different sheep breeds. The highest prevalence was recorded in the Australian breed at 8.25%, followed by Hashtnagri at 4.67%, Balkhi at 6.47%, and the lowest prevalence was recorded in Afghani sheep at 3.57%, as shown in Table 2.

Around 448 sheep were sampled and microscopically examined. Out of 448 sheep, 190 were diarrheic, and 258 were non-diarrheic. The high prevalence rate was in diarrheic sheep, 41, and 12% in non-diarrheic sheep and lambs, as presented in Table 3.

Cryptosporidium infection was observed to be high in diarrheic sheep. Thus, sheep with diarrheal conditions have a greater risk of being positive for Cryptosporidium infection than healthy sheep. Statistical analysis shows a significant difference (p-value <0.001) among diarrheic and non-diarrheic sheep. The results showed that Cryptosporidium infection prevalence rates decreased with fecal consistency, and the highest percent of oocyst counts were in diarrheic feces.

Collected data from 448 investigate sheep were divided into three age groups: pre-weaned, post-weaned, and adult sheep. The results showed that the highest prevalence observed in pre-weaned lambs was 33.33%, followed by post-weaned 25.17 and 16% in adults as presented in Table 4. Statistical analysis showed a non-significant difference between p-values (p > 0.76) of different age groups. The prevalence found in pre-weaned lambs was significantly higher than that found in groups; thus, sheep aged 1–6 months were more likely to be exposed to Cryptosporidium infection.

The meteorological data, such as temperature and average rainfall, were also analyzed in different seasons throughout the year. The Cryptosporidium infection rate was investigated in all four seasons of the study area. The highest prevalence ratio was recorded in the summer season, 36.65%, and 18.51% in the spring season, 18.75% in autumn, while the lowest prevalence was 11.60% in the winter season, as shown in Table 5. A statistical significance (p < 0.51) was observed among different seasons.

The study also examined the prevalence of Cryptosporidium infection in male and female sheep. Of the 448 sheep studied, 218 were males, with a prevalence rate of 28.44%, and 230 were females, with a prevalence rate of 19.56%, as shown in Table 6. The study's conclusion that males are more susceptible to Cryptosporidium infection than females is a significant finding to takeaway. Although a statistically non-significant difference (p > 0.27) was recorded, the gender-based prevalence of the infection is a key finding that could guide future research and management practices.

The evaluation of microscope analysis is less predictable and inexpensive when finding Cryptosporidium oocysts, as given in Table 7. At the same time, PCR is more accurate and specific for identifying species and diagnosing Cryptosporidium.

The present study was conducted for the first time on diarrheic and non-diarrheic sheep in herds rearing around the water tributaries in the Malakand region, which are considered an essential reservoir for diseases caused by Cryptosporidium infections in animals and humans, carrying veterinary and public health significance. C. parvum is mainly responsible for causing infections and transmission through the drinking of surface water (streams, lakes, rivers, and springs) or recreational water contaminated with oocysts, which are key sources of infection among animals and humans.

These contaminated water bodies may therefore represent an important source of lamb-to-lamb or sheep-to-sheep transmission for grazing animals and into humans. The role of drinking water in the transmission of Cryptosporidium spp. has not been well described. Therefore, the current study was designated to determine the prevalence of Cryptosporidium infection in sheep herds rearing around water bodies. Potential risk factors, including drinking sources, herd management, age, gender, seasons, and symptoms of diseases like diarrhea, were considered.

In total, 448 samples were examined under the microscope, and 107 were detected as positive by the MZN technique, with a prevalence rate of 23.88%. This rate of cryptosporidiosis is higher in comparison with previous studies reported by Kabir et al. (2020) who reported a 19.4% prevalence from Turkey, and Shafiq et al. (2015) from Lahore Pakistan, with a 21.33% prevalence in sheep. More studies have reported the following prevalence rate in sheep.

Qi et al. (2019) reported a 6.3% prevalence by PCR in grazing China; Holsback et al. (2018) reported 12.2% from Brazil; Al-Saeed et al. (2019) reported 22.12% from Iraq; Chikweto et al. (2019) reported a 19.5% prevalence rate from West India in sheep; Berhanu et al. (2022) reported 14% from Ethiopia; Khan et al. (2022) reported 18.80% from Pakistan; Dessì et al. (2020) reported 34.4% from Italy; Papanikolopoulou et al. (2022) found 29.54% from Greece; Baroudi et al. (2018) reported a study from Algeria on the occurrence of Cryptosporidium in lambs with diarrhea that was 70%; Kaupke et al. (2017) detected a 19.5% prevalence in lambs from Poland; Rabee et al. (2020) found a 24.26% prevalence in sheep and diarrheic lambs from Iraq. Mammeri et al. (2019) reported 100% positivity in diarrheal lambs from France using microscopy and immunological and molecular methods. Bhat et al. (2019) found a 54.05% prevalence in sheep from India through the MZN staining method. Similarly, Pinto et al. (2021) reported a comprehensive study on cattle from the Netherlands, France, and Belgium, with an overall prevalence of 93% of farms being positive and the highest prevalence in Belgium at 25.7%, followed by France at 24.9%, and the Netherlands at 20.8%, correspondingly. These differences in prevalence may be due to various factors such as environmental problems, including sources of drinking water and food hygiene geographical region, sample size, climate conditions, management practices, age of infected animals, diagnostic techniques used and breed.

The positive samples (n = 107) were also assessed through molecular detection using the conventional PCR. Out of 107 samples, 77 were found positive for Cryptosporidium with a prevalence rate of 17.18%. The results suggest that the PCR is 100% sensitive and highly specific for the molecular detection and identification of Cryptosporidium compared to microscopy. The present result aligns with the agreement of the study reported by Dessì et al. (2020) in 61 sheep farms in Italy. Similar results were also found by Pal et al. (2021), who used the most common methods (MZN technique and modified Koster). Microscopy is time-consuming and less effective and has low specificity and sensitivity. For detection and diagnosis of Cryptosporidium in animals and humans, stained slides by microscopy, various serological tests, and molecular techniques such as real-time PCR, multiplex PCR, nested PCR, and conventional or other modified PCR methods are most reliable for rapid diagnosis and accurate procedures for exact identification up to species level.

Various risk factors such as sheep breeds, diarrheic and non-diarrheic conditions, age groups, seasons, and animal gender associated with Cryptosporidium infection in sheep show that this highest prevalence in the study area is probably due to variations in these factors. Infected sheep shed many oocysts in feces, which can cause tremendous environmental contamination of water bodies that are essential sources of infection. Direct and indirect contact between humans and animals infected with Cryptosporidium is the primary cause of cryptosporidiosis. Increases and decreases in Cryptosporidium infection rates are primarily associated with different risk factors and significantly influence the epidemiology, occurrence, and transmission of cryptosporidiosis. Animal kids, such as lambs, goat kids, and calves, are more infected than adults, and researchers from different countries in both animals and humans have reported a higher prevalence ratio. Moreover, climatic factors like temperature, humidity, and precipitation also play an essential role in spreading and transmitting cryptosporidiosis.

Diarrhea seems to be a prominent symptom of cryptosporidial infections in sheep, as reported by previous researchers. This study also determined the association between diarrheic and non-diarrheic sheep. The highest prevalence was found in diarrheic sheep (n = 90), with a prevalence rate of 41%, and in non-diarrheic sheep (n = 258), with a prevalence rate of 12%, respectively. Cryptosporidium prevalence is higher in diarrheic sheep than in non-diarrheic.

Based on the data, the highest prevalence was in the summer season, 36.65%, 18.51% in the spring season, 18.75% in autumn, and the lowest prevalence was 11.60% in the winter season. A similar report was provided by Ryan et al. (2003). Despite Mi et al. (2018) reporting higher cryptosporidial infection rates in sheep during the autumn season, Hijjawi et al. (2016) reported a higher prevalence in the spring period in Jordan. Chikweto et al. (2019) observed the highest prevalence in India during winter. These seasonal variations in cryptosporidial infections may be probably due to rainfall, temperature, swimming, water intake, and indoor and outdoor activities of the animals.

The infection rate in the current study was different between different age groups; the highest prevalence observed in pre-weaned lambs was 33.33%, followed by post-weaned 25.17% and 16% in adults, respectively. This agrees with Berhanu et al. (2022) who found that the infection rate was higher in sheep below 6 months; our results also align with the findings of Dessì et al. (2020). The result is also similar to Mulunda et al. (2020), who reported higher cases of diarrhea among the 1–4 year age groups; Dankwa et al. (2021) reported a higher prevalence in those under 12 months (12.3%) followed by 12–24 months (27.3%) and those below 24 months (27.0%). Other researchers have documented that cryptosporidial infection occurs more in young animals than adults (Ayinmode et al. 2012; Dessì et al. 2020).

Cryptosporidium primarily affects young animals, causing diarrhea and death; thus, the infection rate decreases with the increasing age of the animals. Young animals such as goat kids, calves, and lambs are known to have reduced immunity and are susceptible to infections; this may lead to diarrhea and other chronic complications. Some age-related associations of four Cryptosporidium species found in cattle, namely C. andersoni, were reported in older animals. At the same time, C. ryanae, C. parvum, and C. bovis were predominant in younger animals (Santín et al. 2008; Brook et al. 2009).

Our results regarding gender showed that the highest prevalence was found in male sheep, 218 (28.44%), while the lowest prevalence was observed in female sheep, 230 (19.56%). Based on the present result, Cryptosporidium infection occurs more in males than females in sheep and lambs. Similar observations were reported by Jafri et al. (2013) and Gharekhani et al. (2014), who recorded the highest prevalence in males (14.3%), than in females (10.3%).

These differences in prevalence between genders may be due to the exact number of male and female sheep in the stock or herd, sample size, and other physiological parameters (poor immune status, pregnancy, hormonal changes, stress, etc.); cryptosporidial infection is probably sex independent. Parasites have the same ability to infect both male and female (Mulunda et al. 2020) animals.

The present study concluded a high prevalence of cryptosporidial infections in Malakand sheep herds around the water supplies. The possible contamination of these water resources with oocysts excreted by the sheep poses a significant risk of infection to animals and humans. To control the contamination of these water bodies and spreading of this enteric protozoan, educational programs and farm management-based One Health control should be implemented for prevention.

Humans and animals are at high risk of infection, especially young animals and humans. Adult or asymptomatic sheep excrete large numbers of oocysts into the external environment and cause environmental contamination, so the isolation of young animals or single-infected animals from the herd decreases the transmission and spread of this zoonotic infection. Understanding the spread of Cryptosporidium types, modes of transmission, and sources of infection is essential for the prevention and control of the disease. Properly following methods include drinking treated water, treating swimming pool water with cleaning agents, not allowing animals with diarrhea to swim, washing hands after visiting toilets or changing diapers for children, and wearing protective gloves, shoes, and clothes, which is essential when handling animals and may reduce the risk of infection.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.