Effect of the Administration of Bacteriophages to Calves Infected with Cryptosporidium spp. on the Health Status, Nutrition and Survival Rate

Enteric and respiratory diseases are calves’ leading causes of morbidity and mortality (Tora et al., 2021; Diao et al., 2017). A frequent origin of these diseases is due to Cryptosporidium spp. infection (Thomson et al., 2017), a parasite that damages the epithelial cells of the gastrointestinal tract, causing diarrhea but with low mortality (Santin, 2020). However, when it is associated with bacteria, the disease becomes more severe and could cause death (Santin, 2013). To treat this enteric disease, antiparasitic and antimicrobial compounds are used. Nevertheless, antibiotics have ceased to be efficient in the last decade due to the increase in bacterial resistance (Grami et al., 2023; Hommerich et al., 2019). Therefore, new environmentally friendly alternatives are being searched for. An option is the use of bacteriophages, also known as phages. They are viruses that infect and lysate specific bacteria, even if those bacteria are resistant to antibiotics (Gaborieau and Debarbieux, 2023; Squires, 2018). In previous studies, the effectiveness of phages in controlling bacteria in the digestive system has been proven (Jeong et al., 2021; Schmoeller et al., 2021; Xie et al., 2021). In addition, it has been found that the same strain could sometimes lysate other varieties and even other bacterial species, which makes its range of action broader (Dueñas et al., 2017; Moreno-Switt et al., 2013). Hence, this study aims to evaluate the use of bacteriophages in calves infected with Cryptosporidium spp. upon their health, nutritional status and survival rate.  

The present study was conducted on a dairy farm with the prevalence of Cryptosporidium spp. Animal management and experimental protocols were reviewed and approved by the Committee of Animal Research Ethics of the Antonio Narro Autonomous Agrarian University (approval number 38111-425502002).
 
Farm selection
 
Feces samples were taken randomly from six different farms to diagnose Cryptosporidium spp. by the Ziehl Neelsen technique (Rekha et al., 2016); A farm with claves that presented some degree of infection (+, ++, +++, ++++) was chosen.  During the experiment, all the calves received 2.5 mg/Kg of oral decoquinate as a prophylactic and the collection of samples continued at random to monitor the protozoan. On this farm, calves were kept in individual cages from birth to weaning and fed twice daily. The bedding was a mixture of dirt and sand that was not changed between each cycle; it was turned over. In addition, the cages were not periodically disinfected. Calves were vaccinated against respiratory (Bovine Infectious Rhinotracheitis, Bovine Viral Diarrhea type I and II, Bovine Respiratory Syncytial Virus and Parainfluenza 3) and gastrointestinal (rotavirus, coronavirus) diseases. The dams were not immunized to protect the calves against gastrointestinal diseases.
 
Experimental scheme and treatment
 
A random study was performed on a dairy farm in Torreón, Mexico, from February to June of 2021 (winter to spring). Seventy-two pre-weaned female Holstein-Friesian calves (37.5±4.3 kg body weight) were used from birth (d 1) to weaning (d 60). They were divided into two groups (control and phage) with 36 animals each, homogeneous in body weight. The groups were assigned randomly at birth.
       
To the phage group (treated), 5 g (5x10⁶ PFU) of bacteriophages (eXolution ® CTCBIO, Inc., Seúl, Corea) were given per day, targeting Salmonella typhimurium, Salmonella enteritidis, Salmonella dublin, Salmonella derby, Staphylococcus aureus, Escherichia coli k99 and f41 and Clostridium perfringens of types A and C. The phages were added daily to the recipient, where milk was given to each calf. The control group didn’t receive phages.
 
Accommodation and feeding
 
At birth, the calves were subjected to a physical exam; they were weighted, received 4 L of colostrum of the same quality and were kept in individual cages until weaning. The calves were fed with whole milk and balanced solid food (Iniciador supra®, Nuplen S.A. de C.V.). The balanced food contained protein (21.5%), fiber (10%), fat (2%) and ashes (14%). The calves had access to water ad libitum and their feeding protocol (milk and balanced solid food) was adjusted daily according to age (Table 1).
 

 
Evaluated variables
 
The presence of diarrhea and antibiotic treatment was evaluated according to the methodology used by Chamorro et al., (2017). Those calves that presented some nasal discharge (mucous, serous, or muco-serous) were considered positive for pneumonia. These evaluations were performed every day in the mornings and afternoons. On day 60, the survival rate was evaluated. When a calf presented diarrhea, a sample of the feces was taken from the rectum and then taken to the laboratory of the Universidad Autónoma Agraria Antonio Navarro to determine the growth of Salmonella spp. (Cho and Yoon, 2014) and Cryptosporidium spp. In the same laboratory, the diagnosis of the death of the calves was made.
       
The body weight was evaluated with a digital scale on the day of birth and after that, every 15 days until weaning. The average daily gain (ADG) was assessed by dividing the body weight of the period in evaluation by the 15 last days. At the same time, the height at withers was measured. These evaluations were carried out in the mornings before the morning feeding.
 
Use of antibiotics
 
The antimicrobial treatment of infectious diarrheas consisted of gentamicin sulfate and penicillin G procaine. For pneumonia, the calves were given a product containing florfenicol, paracetamol and bromhexine. The treatments used, including the decoquinate, were established by the farm.
 
Statistical analysis
 
The data were analyzed with the statistical package SPSS ver. 25.0 IBM Corp. (2017). For the assumption of normality, the Kolmogorov-Smirnov test was used. The verification of homoscedasticity was made with the Levene test. The survival test was calculated by a chi-square test. For each group’s total days of diarrhea, a univariate General Linear Model (GLM) was used, where the dependent variable was the duration of diarrhea in days and the independent variable was the presence of diarrhea. Latency until the day of the presence of diarrhea was analyzed through a Mann-Whitney U test. The probability of receiving antibiotic therapy or developing pneumonia among the groups was calculated with Odds Ratio; if any cell was lower than 5, Fisher’s exact value was considered. For the analysis among the groups of weight, ADG and height at weaning, a t-student was used. It was considered statistically different with a value of p≤0.05.

The infection by Cryptosporidium spp. is frequent and produces chronic diarrhea, which brings high economic losses (Shaw et al., 2020). To our knowledge, this is the first study that evaluates bacteriophages in Holstein-Friesian pre-weaned calves infected with Cryptosporidium spp.
       
The diarrheas were not wholly eradicated during the study period, probably due to Cryptosporidium spp. (Santin, 2013), which was tried to be controlled by administrating decoquinate. Nevertheless, some moderate to severe (++, +++) infections were found in the random sampling. This drug reduces the number of oocysts, but sometimes it is not enough to avoid the appearance of diarrheas (Åberg et al., 2020). The origin of diarrheas is multifactorial and we cannot attribute them only to this protozoan.
 
Survival rate
 
A significant difference was observed between the two groups regarding the survival rate of the calves (p=0.05). In the group that received phages, 100% survived, while in the control group, only 86.11%. Most deaths were due to gastroenteric problems. This survival is similar to the 99% of Schmoeller et al., (2021), although there was no cryptosporidiosis in their study. The deaths in the control group could be caused by enteropathogens such as E. coli, Salmonella spp. and C. perfringens (Thomson et al., 2017). Not having mortality in the phage group complies with the most crucial indicator of animal well-being (Uetake, 2013). Also, the diarrheas in the control group were treated with antibiotics; therefore, not having deaths in the phage group would indicate that these are better at controlling bacterial populations than the antibiotic.
 
Diarrhea, antibiotic therapy and pneumonia
 
The total of days with diarrhea was lower in the phage group than in the control group (4.69 ± 2.29 and 13±4.88, respectively, p=0.04) (Fig 1). A significant difference was observed between the groups regarding the beginning of diarrheas (p=0.03). In the control group, they appeared faster (13 d) than in the bacteriophages group (18 d) where, in addition, the range of appearance was broader than in the control group (Fig 2); the phage group was the healthiest presenting fewer days with diarrheas and delaying their appearance even with the prevalence of Cryptosporidium spp. Similar results have been obtained in other studies, although they did not report infection by protozoans (Alomari et al., 2021; Schmoeller et al., 2021). This explains why bacteriophages are effective controllers for bacteria that cause diarrheas (Xie et al., 2021; Dueñas et al., 2017; Moreno-Switt et al., 2013) and oral administration is helpful despite the pH of the abomasum (Hong et al., 2016). This control was observed in the phage group, where there was no growth of Salmonella spp.; this did not happen in the control group. Cryptosporidium spp. opens the opportunity to other agents (Santin, 2020), but the diarrheas decrease if the enteropathogen bacteria are controlled.
 

 

       
Additionally, calves of the treated group tended to have a lower probability of receiving antibiotics treatment than the control group (OR = 0.05, p = 0.005). Furthermore, it was observed that the treated group had a lower risk of developing pneumonia (OR = 0.4; p = 0.32) (Table 2). Other bacteriophage therapies have been tried, with favorable results (Alomari et al., 2021; Schmoeller et al., 2021; Kwiatek et al., 2020). The phages are very effective for the lysate of bacteria, so technically, there is no need to use an antibiotic (Nobrega et al., 2018); this also avoids the destruction of intestinal microbiota (Low et al., 2021). Hence bacteriophages are an excellent alternative for addressing antimicrobial resistance despite phage resistance (Gaborieau and Debarbieux, 2023; Grami et al., 2019; Hommerich et al., 2019). If the diarrheas are eliminated, pneumonia could also be diminished (Pardon et al., 2015). This study obtained a preventive effect of the phages on the appearance of pneumonia but without an acceptable statistical significance. Some complementary studies are required to evaluate this effect in the absence of Cryptosporidium spp.  
 

 
ADG, weight and height
 
In the results at weaning, it was observed a significant difference in the ADG, weight (77.35±1.47 vs 71.38±1.62 kg) and height (92.21±0.8 vs 89.2±0.59 cm). The phage group had the highest mean (p=0.03, 0.01 and 0.009, respectively) (Table 3 and Fig 3). Although the difference in ADG was not significant when being evaluated every 15 days, it was during the entire trial period (60 d); the phage group gained more weight. These results agree with Jeong et al., (2021) but disagree with Schmoeller et al., (2021), who obtained statistical differences in all the evaluated periods. Regarding body weight, there was a difference only in the second half of the study, including the weaning. Some studies reported differences (Schmoeller et al., 2021), but others showed no difference (Jeong et al., 2021). The statistical equality in the first month could be because it is the period where the highest infection of Cryptosporidium spp. (Åberg et al., 2020). Otherwise, the difference could be observed since day 11 (Alomari et al., 2021). The most evident effect was in height, where in the whole period of study, there was a statistical difference except in birth, similar to that obtained by Schmoeller et al., (2021). This indicates that although weights vary, height growth is relatively constant. The positive effects on the parameters of nutrition could be explained by the low morbidity in the phage group, which allowed better nutrition (Tora et al., 2021). Hence, bacteriophages improve health and, consequently, enhance nutritional efficiency.
 

 

Cryptosporidium spp. causes diarrheas in pre-weaned calves which could complicate by opportunistic bacteria, increasing the mortality rate. However, if bacteriophages are administered, the survival rate is improved, days of diarrhea are decreased and the onset of diarrhea is delayed. Additionally, the use of antibiotics is reduced, contributing to the strategy of mitigating bacterial resistance to these compounds. While improving the health of calves, the nutritional state is also enhanced.

Author Jung-Jin Lee is listed as the inventor on patent applications filed by CTCBIO Inc. All other authors declare no conflict of interest.