Investigation of a Cryptosporidiosis Outbreak Caused by Cryptosporidium parvum in a Dairy Farm

Diarrhea caused by parasitic agents is common in neonatal calves and causes signicant economic losses in cattle farms worldwide. Cryptosporidium spp. is one of the most frequently detected parasitic agents causing diarrhea in neonatal calves. Also, Giardia intestinalis is shown to cause diarrhea in calves. This study aimed to investigate the presence of Cryptosporidium spp. in calves (n:36), cows (n: 11), drinking water and two different artesian water supplies as well as in environmental swap samples (n:32) obtained from the manger, silage, bottle, and doorknob in a dairy farm which has big diarrhea problems. For this purpose, all fecal samples investigated with using direct microscopy for routine parasitological screening. Then, the presence of Cryptosporidium spp. was examined in feces samples and other environmental samples using Kinyoun acid-fast stained slides and Real-Time PCR targeting Cryptosporidium spp.. In addition, Real-Time PCR positive samples were investigated by nested PCR and subsequently by sequencing, BLAST, and phylogenetic analysis for species identication by MEGA7.

During the direct microscopic examination of fecal samples, G. intestinalis cysts were observed in 6 of the 26 (23.07%) calves younger than two months. In this group, G. intestinalis positive samples were detected in 6 calves that had diarrhea previously (n: 14; 42.85%). G. intestinalis cysts were observed in 3 calves (n: 10, 30%) older than two months without diarrhea. In addition, G. intestinalis cysts were detected in one of the 11 cows (9.09%). Among all animals examined, G. intestinalis cysts were found in 21.27% (10/47) of animals (Fig. 2B, Table 1).

Real-Time PCR
As a result of the investigation of fecal DNA samples, Cryptosporidium spp. DNA was detected in 11 of 26 (42,30%) calves younger than two months using Real-Time PCR. Among these, Cryptosporidium spp. DNA was observed in 9 of 12 (75%) calves with diarrhea, while Cryptosporidium DNA was detected in 2 of 14 (14,28%) with previous history of diarrhea. The difference between calves with diarrhea and calves with previous history of diarrhea was statistically signi cant (P < 0,001).In terms of the diagnosis of Cryptosporidium in diarrhea calves, Real-Time PCR method was found to be signi cantly sensitive than the Kinyoun acid-fast staining method (P < 0,05).
Cryptosporidium spp. DNA was not recognized by Real-Time PCR in calves older than two months and cows. Among all animal examined, Cryptosporidium spp. DNA was detected in 27.65% (13/47) of animals ( Table 1).
In addition, while positivity was not detected in paddock drinkers and artesian water 2, interestingly high positivity was found in artesian water 1 (Crossing point: 33.92). This water source was located near the city center and next to the wastewater treatment plant (Fig. 1). Also, Cryptosporidium spp. DNA was found on paddock irons (numbered 1-10, 22-30, 31-40, 61-70, 101-110), waterer, and feeders at the edges ( Table 2).

Species identi cation
As sequences belonging to Cryptosporidium spp. isolates were analyzed, all isolates three RsaI digestion regions. After RsaI digestion, obtained fragment lengths were 410, 106 and 34 bp. These fragment lengths were compatible with expected band patter for C. parvum isolates. Also, BLAST results showed high similarity rate varying 98.72-99.52% among Cyptosporidium spp. isolates and C. parvum isolates used in phylogenetic analysis (Table 3).

Phylogenetic analysis
Phylogenetic analysis of the COWP sequences belonging to Cryptosporidium isolates showed that all isolates were clustered in the same branch with reference samples of C. parvum (Fig. 3). No unexpected or mixed branches containing different Cryptospordium species were found. Nearly all branches of the tree showed high bootstrap values.

Discussion
Calf health is vital for the economical production of cattle breeding, which is widespread all over the world. In order to run a cost-effective operation, it is aimed for each cow to give birth to a calf once a year (19) In a lot of studies, it has been stated that the prevalence of diarrhea in calves is related to Cryptosporidium spp. density. In addition, the more general hygiene of the farm is terrible, the more Cryptosporidium spp. related diarrhea and calf deaths can be seen (23).
Every year 9 million calves are born in the USA. More than 500,000 (6%) of calves born die before weaning. 56.4% of deaths have been determined as digestive system diseases (USDA 2018). This rate is 6.4% in Canada (24). In a study conducted in Brazil, it was determined that 8.5% of 1451 calves died before weaning and 44% of deaths were caused by diarrhea (25). Acceptable mortality rate before calving was determined as 5% in calves (DCHA Gold Standards, 2013).
The prevalence of Cryptosporidium in cattle in the world is around 30% (26). The prevalence of Cryptosporidium spp. was found to be 22.3% (81/364) in Australia in calves (27). In India, the prevalence of Cryptosporodium by PCR was 32.3% in diarrhea calves (28). In another study conducted in India, Cryptosporidium positivity was 50% (40/80) in calves with diarrhea by modi ed Ziehl-Neelsen staining (29). In another study conducted in 75 heifers in Poland, the prevalence of Cryptosporidium was determined as 30.7% by modi ed Ziehl-Nielsen staining (30). 2009). In Canada, 40.6% (203/500) of calves with diarrhea were Cryptosporidium positive by microscopy (31). In a study conducted with immuno uorescent staining in Norway, the prevalence of Cryptosporidium was determined as 12% (167/1386) in calves and 53% (72/136) in farmers (32).
The high prevalence of Cryptosporidium spp. have been shown in studies conducted in Turkey (33). Cryptosporidium oocysts were rst detected in cattle in a study conducted in 1984 in Turkey (34). Later, Cryptosporidium oocyst prevalence in calf stool samples was found to be 32.9% in Kars, 27.33% in Konya, and 35.8% in Ankara by microscopy (1,35,36).
In another study conducted in Nevşehir, Cryptosporidium DNA was found in 20.7% of stool samples of 150 calves with diarrhea using Real-Time PCR (33). In this study, Cryptosporidium spp. DNA was found in 75% of calves with diarrhea. This high prevalence value indicates that there is an outbreak of Cryptosporidium spp. in calves in the examined dairy farm. In addition, the detection of Cryptosporidium spp. DNA in one of the artesian water source has been linked to the constant infection of the daily water supplied to the calves with the parasite.
PCR and microscopy are the most commonly used diagnostic methods in the diagnosis of Cryptosporidium in calves, and studies have shown that PCR is more sensitive and speci c than microscopy (16,37). Similar to these studies, Real-Time PCR method was found to be signi cantly more sensitive (P < 0.05) than microscopy in our research. Also, PCR methods are advantageous because they do not require experienced personnel, many samples are run at the same time, and they do not require concentration methods used in microscopy (16,37).
Cryptosporidium spp. is a zoonotic infection. It can be passed from calves and cattle to man, and vice versa. Cryptosporidium spp. oocysts were found in 33.47% of human cases with diarrhea by Kinyoun acid-fast staining in İzmir. İzmir is the neighboring province of the dairy farm examined in this study (38).
For these reasons, it is crucial to follow the stool samples of the personnel working in such farms to prevent the spread of the disease.
The high incidence of Cryptosporidium infections in newborn calves results from inadequate hygiene practices. Transmission in farms can occur through direct contact with infected animals, and poor conditions in terms of medical care and water supplies contaminated with Cryptosporidium oocytes increase the prevalence of Cryptosporidium (39).
Diarrhea related to Giardia intestinalis in calves has been reported in many studies (32,40). In our study, G. intestinalis cysts were observed in 21.27% (10/47) all animals examined. It was thought that the high prevalence could be due to the Cryptosporidium spp. detected-water source.
In this study, Cryptosporidium spp. was detected in one of the artesian water sources. This drinking water used for calves is brought from this polluted water source through an individual water pipeline. It is supplied to calves and dairy cows without any ltration or disinfection. Due to the lack of sanitation, not only Cryptosporidium but also other agents that cause diarrhea can infect calves and cows. In this study, the microscopic detection of G. intestinalis cysts supports insu cient water sanitation. Adjacent paddocks facilitate the passage of diarrhea feces that are infected with Cryptosporidium oocysts. Also, relevant environmental samples that are positive for Cryptosporidium are another factor that eases the communication of Cryptosporidium oocytes among animals.
After the detection of Cryptosporidium spp. and G. intestinalis in calves, water intake from infected artesian water source was stopped and a new artesian source was opened within the boundaries of the dairy farm. Meanwhile, calves were treated with anti-parasitic drugs for Cryptosporidium and G. intestinalis. Later, when Cryptosporidium spp. was checked in water samples collected from the newly opened artesian source with 3 months intervals, DNA was not detected. As a result of these precautions, it has been reported that diarrhea cases have decreased in calves on this farm.

Conclusion
As a result, controlling calf deaths is crucial for a sustainable farm economy. Therefore, it is necessary to take measures against diarrhea, which is the most important cause of calf deaths. Also, even if death does not occur, intestinal damage resulting from pathogens that cause diarrhea is a signi cant cause of economic loss. Cryptosporidium spp. is quite common in neonatal calves. Filtration or disinfection applications applied to drink water used for calves are very important in protecting against Cryptosporidium and other waterborne pathogens. In addition, the routine parasitological examination of animals and pathogen-speci c treatment of animals will play an essential role in controlling the disease and reducing drug costs.

Properties of the farm
The dairy farm is located on the Izmir-Ankara road and 1.5 hours away from Izmir. There are about 600 cows and 150 calves in the farm. In this farm, cows which are in peri-parturient period, are taken to a separate paddock for delivery. After birth, the colostrum is given to calves by a bottle, and the calves are transferred to single paddocks in which calves are fed until two months old. At the end of the two months, calves are placed in paddocks with a capacity of 10-15 animals.
Drinking water for all animals in the farm is provided from two different artesian water sources (Fig. 1). The artesian water source 1 coming to the dairy farm is close to the city center, and there is a wastewater treatment plant nearby. The artesian water source 2 is located on a hill away from the city center. The artesian water is coming to the farm with an individual pipeline from both sources and collected in a central tank and delivered to the calves and cattle without any ltration or disinfection. Two veterinarians work in the farm 24 hours a day, seven days a week. Veterinarians helped the project staff to collect feces from animals with diarrhea and from animal with previous history of diarrhea.

Samples
During the study, 47 stool samples were collected from calves (n: 36) and cows (n: 11). Of the 26 calves younger than two months, 12 had clinical diarrhea, and the remaining 14 had diarrhea previously.
Antibiotic treatment was applied empirically to all of these calves. The remaining ten calves without diarrhea were older than two months (These samples were called D1-D47, Table 1).
Two samples with a volume of 8 liters were collected from two different artesian water sources (one of these examples is close to the city called SK1, and the other is called SK2, Table 2). Water sample with a volume of 50 ml was collected from each paddock with calves younger than two months, and 20 samples were pooled, and as a results six pool samples consisting of one-liter water were generated (These samples were called SK3-SK8, Table 2). Also, two-liter water sample pool was created from 6 different paddocks with calves older than 2 months (SK9, Table 2). In addition, one-liter of water sample was taken from the delivery paddocks (SK10, Table 2) (11).
The swab samples were obtained from various surfaces in the farm. Swabs were taken into 1 ml saline in 50 ml sterile tube using sterile cotton swabs. Swaps were collected from calf paddocks, waterer and mangers by making pools (each pool covers ten paddocks, these pooled samples were called S1-S18, Table 2). In addition, the swap samples from the inside (S19, Table 2) and outside (S20, Table 2) part of the calf bottle, inside (S21, Table 2) and outside (S22, Table 2) of milk heating tank, the inside (S23, Table 2) and outside (S24, Table 2) of cabinet containing colostrum, towels (S25, Table 2), the inside (S26, Table 2) and outside (S27, Table 2) of milk warmer in the deliver paddock, the inside (S28, Table 2) and outside (S29, Table 2) of medicine cabinet, the medicine cabinet door (S30, Table 2) and colostrum bottle (S31, Table 2) were collected to investigate Cryptosporidium spp. by PCR .

Microscopy
For microscopic examination, stool samples were diluted with saline and examined under direct light microscope using 40 × magni cation (12). Then, smears of stool samples were stained by Kinyoun acidfast dye. For this, rstly, tap water was added to the average of 5-10 g stool samples in 50 ml tubes until the upper level of the stool and incubated for 2 hours at room temperature. Then tap water was drained, and the stool was emulsi ed with a spatula by adding 10 ml sucrose solution (53 g sucrose, 100 ml water). The resulting mixture was ltered through two layers of gauze and centrifuged at 400 × g for 10 minutes (13). Subsequently, the upper liquid collected from the top of each tube was spread on slides and xed with methanol after drying in the air. The slides were then stained with the Kinyoun acid-fast dye method according to the manufacturer's protocol (RTA, Turkey). Brie y, slides were kept in the Kinyoun dye for 2-3 minutes and washed with tap water. Then, slides were kept in acid-alcohol for 5-10 seconds, rinsed in tap water, and kept in methylene blue for 20-30 seconds. Finally, the slides were washed with tap water air dried and examined under light microscope using 100 × magni cation with immersion oil (14).
DNA extraction DNA extraction from fecal samples was performed by stool DNA isolation kit (RTA Labs, Turkey) according to the manufacturer's protocol. During DNA isolation, 100 µg fecal sample was used, and DNA was eluted with 100 µl elution buffer. DNA isolation from water samples and environmental samples were performed as described with the QIAamp Mini kit (Qiagen, USA). Before DNA isolation, water samples were initially passed through 0.45 µm lters (Corning, USA). Later, the lters were removed, cut with a sterile scalpel and put into 50 ml sterile tube containing 500 µg zirconia beads, 125 µg glass beads, 20 µl proteinase K, 1000 µl saline and 1000 µl buffer AL (Qiagen, USA). Thereafter, the tubes were incubated for 4 hours in a shaker at 37 °C and 350 rpm. After incubation, the tubes were centrifuged at 3000 rpm for 10 minutes, and the supernatants obtained from each tube were collected and 500 µl absolute ethyl alcohol was added to the supernatants. Following this step, the routine DNA isolation method was applied. Swap samples were initially incubated for 1 hour at 37 °C and 350 rpm before DNA isolation. Later, 20 µl proteinase K and 1000 µl buffer AL were added and incubated for 10 min at 70 °C.
After this incubation, routine DNA isolation method was applied (15). The PCR reaction with a 20 µl nal volume included 5 µl DNA template, 4 µl LightCycler Taqman 10XMaster mix with 5 mM MgCl 2 , 0.5 µM from each primer and 0.1 µM probe. The ampli cation reaction was performed as follows: 10 min initial denaturation step at 95ºC, followed by 45 cycles of 10 seconds at 95ºC, 15 seconds at 55ºC, and 15 seconds at 72ºC and10 seconds at 40 ºC for cooling (16).
Nested PCR All Real-Time PCR positive samples were also investigated by nested PCR for species identi cation of Cryptosporidium spp.as described (Spano et al., 1997;Pedraza-Díaz et al., 2001). In the initial reaction, BCOWPF (5'-ACCGCTTCTCAACAACCATCTTGTCCTC-3') and BCOWPR (5'-CGCACCTGTTCCCACTCAATGTAAACCC-3') primers were used to amplify the 769-bp fragment of the COWP gene. In the second reaction, a 553-bp gene fragment was ampli ed from the initial reaction product by cry-15 (5'-GTAGATAATGGAAGAGATTGTG-3') and cry-9 (5'-GGACTGAAATACAGGCATTATCTTG-3') primers. In initial reaction of nested PCR, 25 µl ampli cation reaction included 2.5 µl template DNA, 1 µl primers (0.4 mM each), and 5 µl PCR Master Mix (5×, GeneMark. In the second step of nested PCR, 2.5 µl template from the rst reaction product was used like the rst reaction. The nested PCR was performed using the following protocol for both steps: 3 min initial denaturation step at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 65 °C, and 1 min at 72 °C, and a nal extension of 10 min at 72 °C. Species identi cation PCR products of 553 bp fragments belonging to Cryptosporidium spp. positive samples were sequenced by ABI3730XL. Generated sequences were edited and aligned by MEGA 7.0 software to nd the RsaI digestion regions that are used for identi cation of Cryptosporidium spp. (17,18). Also, a BLAST analysis was performed to compare with reference Cryptosporidium samples in National Center for Biotechnology Information (NCBI).

Phylogenetic analysis
The phylogenetic analysis was performed by MEGA 7.0 software. The phylogenetic tree based on COWP sequences belonging to Cryptosporidium isolates was constructed by MEGA7.0 software according to the Neighbour Joining/Maximum Likelihood method using Kimura 2 Gamma distribution (K2 + G) model with 500 Bootstrap replications.

Statistical analysis
The data obtained during the study were processed with Microsoft Excel 2010 program, and statistical analysis was performed with Graphpad Prism 3 program (GraphPad, San Diego, CA). Among the calves with diarrhea and previous history of diarrhea, the presence of Cryptosporidium spp. was compared with a two-tailed unpaired t test. P < 0.05 was considered statistically signi cant unless otherwise stated.

Declarations
Ethics approval and consent to participate Phylogenetic analysis of the COWP sequences belonging Phylogenetic tree showing the association of Cryptosporidium isolates with C. parvum isolates.