Short Communication: effect of sodium butyrate, monensin, and butyric acid on the viability of Eimeria bovis sporozoites and their degree of damage to a bovine epithelial cell line

Abstract

This study aimed to determine the viability of sporozoites from Eimeria bovis when exposed to sodium butyrate (SB), monensin (MON), or butyric acid (BA), and to determine the effects of SB on sporozoite invasion of cells in comparison to MON as measured by the damage to a bovine epithelial cell line. To determine viability, isolated sporozoites were suspended in one of four treatments: control (CON) of cell culture medium alone, SB = 0.028 mg/mL suspended in control medium, MON = 0.01 mg/mL suspended in CON, and BA = 0.18 mg/mL suspended in CON. The number of live sporozoites was less for the MON and BA treatments compared to the CON and SB treatments. The number of dead sporozoites was similar regardless of treatment. There was a trend for treatment to affect the percent sporozoite viability. Control, SB and BA treatments were similar, while MON compared to control and SB had decreased percent viability. Results for MON, when compared to BA, were similar for percent viability. Lactate dehydrogenase (LDH) release was used to determine cellular damage to Madin Darby Bovine Kidney (MDBK) cells when exposed to E. bovis sporozoites in vitro. Cells were exposed to similar numbers of sporozoites and treated with: CON, SB = 0.028 mg/mL in control medium, MON = 0.01 mg/mL in control medium. Control LDH result (with sporozoites) was greater than both the SB and MON treatments while the LDH for SB and Mon and cells not exposed to sporozoites were similar. SB and MON were both shown to decrease cellular damage to MDBK cells as determined by decreased LDH release. SB has the potential to act as an anticoccidial alternative to MON.

Sodium butyrate appears to have promise as a coccidiosis preventative. Its component part, butyric acid, is similar to monensin in reducing the number of viable sporozoites compared to untreated sporozoites.

Introduction

Coccidiosis is a common disease found on many dairy farms that can cause significant economic losses for producers from the cost of treatment as well as decreased growth rates, veterinary expenses, and reduced feed conversion. It is estimated that the global losses in dairy cattle and bison are $400 million to $700 million (USD) (Jolley and Bardsley, 2006). Mortality can also occur in severe cases. Coccidiosis is caused by the protozoan species Eimeria. Many species of Eimeria infect different varieties of livestock. Twelve species have been identified in the feces of cattle. Of the twelve, E. zuernii, E. bovis, and E. auburnensis are most commonly associated with clinical disease in dairy cattle (Constable, 2015).

From birth, dairy calves are susceptible to infection. Although symptoms and diagnosis do not occur until 15–21 d after ingestion due to the parasite’s lifecycle or prepatent period (Jolley and Bardsley, 2006). The route of transmission is fecal to oral. After the ingestion of sporulated oocysts from the environment, the life cycle begins. Oocysts can survive harsh conditions, making them present in nearly all environments (Jolley and Bardsley, 2006). Symptoms include fever, decreased appetite, weight loss, and dehydration. In the intestine, excysted sporozoites invade intestinal epithelial cells becoming merozoites. Merozoites multiply, infecting surrounding cells, and differentiate into gametes for reproduction, producing large quantities of oocysts. These oocysts are shed into the environment, allowing for the infection to reach other animals (Pié Orpí, 2020).

With the change of season, temperature fluctuations, and extreme weather events, dairy calves undergo high levels of stress (Matjila and Penzhorn, 2002). This can increase the presence of oocysts in the environment and make animals more susceptible to infection. Other times of stress throughout the life of a dairy calf, including crowding, shipping, changes in nutrition, and nutritional deficiencies can increase the chance and severity of parasite infection (Jolley and Bardsley, 2006). Coccidiosis due to stress is mediated by increased circulating corticosteroids (Eness, 1984). Subclinical infections are common where animals will appear healthy and the infection will often go undiagnosed, even though oocysts are present in the feces. Although no symptoms are seen, intestinal damage is occurring, causing feed efficiency to be reduced. Chronic infections are seen when bloody diarrhea continues for more than a week or when thin feces with shreds of epithelium and mucus are seen (Constable, 2015). Enteritis occurs in the large intestine and damage to the mucosa in the small intestine is seen when oocytes are present in the feces (Constable, 2015). The resulting intestinal damage to epithelial cells decreases nutrient absorption, hindering the growth of young dairy calves.

Ionophores (polyether antibiotics) were introduced to the broiler industry for the prevention and control of coccidiosis (McGuffey, 2017). In the 1970s, the dairy industry began the use of ionophores such as monensin. Monensin (MON) is commonly used on U.S. dairy farms today for coccidiosis control as a feed additive to promote growth, reduce coccidiosis, and improve feed efficiency in lactating cows. The antibiotic effect of MON is due to its mechanism of action, by which it reduces the number of gram-positive bacteria and thereby enhances gram-negative bacteria and the production of propionic acid. Monensin causes a large uptake of Na⁺ ions into the cell and the inhibition of the Na⁺-K⁺-ATPase pump (Smith and Galloway, 1983). This increases intracellular osmotic pressure and causes water to flow in and resulting in cellular lysis. The use of ionophores will not prevent infection or eradicate the parasite, but rather will reduce infections to subclinical levels. Anticoccidials are widely used in the poultry industry, and some species of Eimeria have developed resistance (Jolley and Bardsley, 2006). Recently, more pressure has come from consumers to reduce antibiotic usage due to the potential development of antibiotic resistance. The European Union banned the use of ionophores in livestock feeds (European Commission, 2005). This has led researchers to seek alternative feed additives that can ameliorate the effects of coccidiosis.

Recent studies with sodium butyrate (SB) have been shown to decrease coccidial infections (Rice et al., 2019; Stahl et al., 2020). Heifers starting at 12 wk of age were supplemented with differing concentrations (0, 0.25, 0.50, 0.75 g/kg of body weight (BW)) of SB and were seen to have an increase in BW (P = 0.04), a tendency for greater feed efficiency (P = 0.08) and final BW (P = 0.07), and a reduction of coccidial oocysts in feces (P = 0.03. Stahl et al. (2020) supplemented diets with 0.75 g/kg BW SB, MON (1 mg/kg BW), or a combination of SB and MON, to evaluate if SB-fed heifers responded similarly to MON-fed heifers. When fed either additive or the combination, heifers tended to have greater average BW (P = 0.10), increased dry matter intake (P = 0.03), and a reduction in the prevalence of coccidian oocysts (P = 0.03) compared to animals not fed an additive. The rate of diarrhea has also been shown to be decreased with the supplementation of different forms of SB. Wu et al. (2022) supplemented free or fat-coated SB to calves in starter grain and observed the diarrhea rate to drop in the free SB and rapid-release SB treatments (P < 0.05). Using sheep, Gorka et al. (2018) observed that feeding exogenous butyrate at approximately 0.99 g/kg BW resulted in an increased concentration in the reticuloruminal digesta (20.7 mmol/L vs. 11.7 mmol/L) and abomasal digesta (2.03 vs. 1.31 mmol/L) compared to sheep receiving no supplemental butyrate (P ≤ 0.01). They observed that the concentration of butyrate in the proximal intestine digesta was greater than three times that of the control sheep (0.68 vs. 0.22 mmol/L; P = 0.05). This feeding rate is similar to the rate of Stahl et al. (2020) and the highest rate fed by Rice et al. (2019) to dairy heifers (0.75 g/kg BW). These data indicate that supplemental butyrate can flow out of the rumen and affect the small intestine and its contents.

To date, no in vitro experiments have been performed to investigate the effects of SB on isolated coccidia sporozoites. The objective of this study was to determine the viability of sporozoites in vitro when SB, MON, or butyric acid (BA) was present and to determine the effects of SB on sporozoite damage to bovine epithelial cells in comparison to MON. It was hypothesized that SB would reduce the viability of sporozoites and decrease cell damage.

Materials and Methods

This experiment was reviewed and approved by the University of New Hampshire (Durham) Animal Care and Use Committee (Protocol No. 210201).

Cell culture and reagents

Madin Darby Bovine Kidney (MDBK) cells were obtained from the American Type Culture Collection (ATCC CCL-22, Manassas, VA). The cell line was cultured in Roswell Park Memorial Institute 1640 (RPMI-1640; Sigma-Aldrich, St. Louis, MO) medium with 10% Fetal Bovine Serum (FBS; JRH Biosciences, Lenexa, KS), in a humidified incubator at 37 °C with a 5% CO₂ atmosphere. Treatments (SB, MON, and BA) were purchased from Sigma-Aldrich. Treatments were dissolved in differing concentrations in RPMI with 10% FBS. A concentrated stock solution of MON was first dissolved in 100% ethanol as an intermediate solvent due to its low solubility in water.

For each treatment, a dose–response using MDBK cells and a viability determination using trypan blue vital dye were conducted to determine the dose of treatment. All selected concentrations were seen to be effective without cellular harm. Concentrations from 0 mg/mL to 0.898 mg/mL SB were tested with 0.028 mg/mL SB as the chosen concentration. Monensin concentrations ranged from 0 mg/mL to 0.08 mg/mL with 0.01 mg/mL MON as the chosen concentration. Concentrations from 0 mg/mL to 1.42 mg/mL BA were tested with 0.18 mg/mL BA as the selected concentration.

Coccidia oocyst isolation

Fecal samples were obtained from 12 to 24-wk-old Holstein heifers from the Fairchild Dairy Teaching and Research Center at the University of New Hampshire in Durham, NH. Briefly, heifers were group-housed in a naturally ventilated free-stall barn with mattresses bedded with kiln-dried sawdust. Two adjacent pens (pen 1: 5.46 × 4.75 m; pen 2: 5.54 × 4.88 m) were used, pen 1 having the capacity to hold 6 heifers, and pen 2 having the capacity to hold 8 heifers. Heifers had unlimited access to water through automatically refilling water troughs and no competition for stall space. Heifers were fed a TMR once daily. Feces were taken from heifers every other Monday via gloved hand at 1330 h. The modified Wisconsin sugar fecal worm egg flotation method was performed on fecal samples to determine oocysts/gram of feces (Bliss and Kvasnicka, 1997). Fecal samples were then diluted with tap water and passed through a series of metal sieves with pore sizes of 850, 400, 149, 88, and 49 μm to separate oocysts from debris. The diluted and filtered sample was mixed 1:1 volumetrically with a saturated sucrose solution. The saturated sucrose solution was made by dissolving 454 g sucrose in 355 mL deionized water with constant stirring on a hot plate and was cooled to room temperature before use. The diluted sample was then transferred to a 23 × 33 cm shallow glass dish and covered with a plastic sheet that was in contact with the solution. Every 2–10 h the plastic sheet was washed with a squirt bottle filled with deionized water into a large beaker. This was performed for 48 h with washing happening every 2 h for the first 12 h and then every 9 h thereafter, the wash was centrifuged at 600 × g for 12 min to collect oocysts. The remaining pellet was resuspended in 2% potassium dichromate in water. The suspension was covered and placed into a water bath at 28 °C under constant aeration for 48 h. After, the suspension was centrifuged at 600 × g for 12 min and resuspended in 2% potassium dichromate, and stored at 4 °C for future use. The counters were trained to identify Eimeria bovis by an individual who was trained to speciate coccidia through the New Hampshire Veterinary Diagnostic Laboratory.

Oocyst excystation

Oocyst samples stored in 2% potassium dichromate were centrifuged at 2,000 × g for 10 min. The supernatant was aspirated off. Samples were then washed in 1% sodium hypochlorite on ice for 20 min, centrifuged at 2,000 × g for 10 min and the supernatant was removed. Samples were further washed five times by repeated centrifugation for 10 min at 2,000 × g in sterile deionized water. Oocysts were then suspended in 0.02 M l-cysteine HCl–H₂0 (Sigma-Aldrich)/0.2 M NaHCO₃ (Sigma-Aldrich) solution and incubated for 20 h at 37 °C in a 100% CO₂ atmosphere. To remove the solution, the sample was centrifuged for 10 min at 2,000 × g and the supernatant was removed. Samples were suspended in excystation solution (Hanks Balanced Salt Solution (HBSS)) with 0.4% trypsin (Sigma-Aldrich) and 8% bovine bile (Sigma-Aldrich)) and incubated for 2 h at 37 °C in a 5% CO₂ atmosphere in a vented T75 cell culture flask (Corning, Inc. Elmira, NY). The sample was then washed until clear with sterile phosphate-buffered saline (PBS) by centrifugation at 2,000 × g for 10 min. Samples were suspended in Minimum Essential Medium (MEM; Sigma-Aldrich). Due to the presence of bacterial contamination, samples were suspended in a gentamicin solution (100 μg/mL; Sigma-Aldrich) for 1 h inside a vented T75 cell culture flask at 37 °C in a 5% CO₂ atmosphere (Schafer et al., 1971). Sterile sporozoites were then suspended in RPMI-1640 with 10% FBS. Sterility tests performed for contamination were negative.

Sporozoite viability

Sporozoites were suspended in different sterile treatments and control to determine their effects on sporozoite viability. A 24-well sterile cell culture plate (Corning, Inc.) was used with each well containing 0.25 mL CON medium with sporozoites suspended in solution. Treatments were replicated in four wells for each treatment. Treatments were as follows: CON, SB (0.028 mg/mL), MON (0.01 mg/mL), and BA (0.18 mg/mL). Dose–response curves were conducted to determine the lowest possible concentration used. Each well received 1 mL of control or treatment medium with 0.25 mL medium with approximately 13 sporozoites suspended in solution resulting in a total of 1.25 mL per well. The multi-well plate was incubated at 37°C in a 5% CO₂ atmosphere for 48 h. After incubation, a dye exclusion test using trypan blue was used to determine sporozoite viability (Nakai and Ogimoto 1983; Khalafalla et al. 2011). Trypan blue is a vital dye that is taken up by dead or dying cells that have cell membrane damage, 0.1 mL trypan blue (0.4%) was added to 0.8 mL of medium from each well (n = 4). Glass slides were prepared by pipetting the sporozoite-containing medium with dye onto a glass slide and covering with a coverslip. Sporozoites were visualized using bright field microscopy at 200× magnification to distinguish living and dead sporozoites. Counts were performed to determine the percent of viable sporozoites. Five random sections on each slide were counted by two different individuals and recorded. The two individual counters were trained in cell enumeration and viability determination by a cell culture researcher (Co-author T.F.). The two individual counters counted the same random sections on each slide at approximately the same time (in sequence). The number of viable and non-viable sporozoites was recorded. To calculate the percent viable, the number of live sporozoites was divided by the total number of sporozoites, dead and alive.

Lactate dehydrogenase (LDH) assay

An LDH assay was used to investigate potential cellular damage by sporozoites to healthy bovine epithelial cells in vitro. MDBK cells were seeded into a 24-well plate (Corning, Inc., Elmira, NY ) at 50,000 cells per well and grown to approximately 85% confluency. To each well, 0.25 mL CON medium (after vortexing) was added with sporozoites suspended in solution. Treatments were replicated with n = 4. Treatments were as follows, SB (0.028 mg/mL), MON (0.01 mg/mL), sporozoites alone, and CON containing no sporozoites or SB and MON. To each well, 1.25 mL of the given treatment was added, and the plate was incubated at 37 °C in a 5% CO₂ atmosphere for 48 h. The medium from each well was placed into separate labeled microcentrifuge tubes and centrifuged for 5 min at 1500 × g to ensure no cells were present in the medium that was assayed for LDH release. The supernatants from each tube were then stored frozen at −20 °C until analysis.

Samples were thawed at room temperature. An LDH assay kit (CyQUANT™ LDH Cytotoxicity Assay, Thermofisher Scientific, Waltham, MA) was used to determine levels of LDH release in each well. Each sample was assayed in duplicate. Samples were analyzed colorimetrically using a microplate reader (Epoch Bio Tek Instruments, Inc., Winooski, VT) set at wavelengths of 490 and 680 nm for two readings of each sample. Absorbance values were then calculated by subtracting the 680 nm absorbance value from the 490 nm absorbance value to remove the background signal. Lactate dehydrogenase is an important enzyme of the anerobic metabolic pathway and is found within the cytoplasm of many cells. It is constitutively released from cells in low quantities; however, release above normal control values is indicative of cell membrane damage. The control treatment acted as the “normal” levels of LDH that would be released from MDBK cells. The function of this enzyme is to catalyze the reversible conversion of lactate to pyruvate with the reduction of NAD+ to NADH and vice versa.

Statistical analysis

Sporozoite viability including total, dead, and alive counts and percent viable sporozoites were analyzed using the GLIMMIX according to the link-log procedure described by Davis (2018) using SAS (SAS Institute Inc., Cary, NC, USA) because data were not normally distributed. The value 1 was added to all data in case of any 0 values and data were corrected after statistical analyses were conducted. Both Poisson and negative binomial models were evaluated using the Quad procedure. The model selected had a Pearson Chi-square estimate was approximately 1 (Davis, 2018). All data were less than 1 when the negative binomial model was used.

The LDH assay results were analyzed using the mixed procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA) on data that were transformed to the inverse of the absorption difference which resulted in a normal distribution. The following model was used: Y_i = µ + Trt_i + E_i, where Y_i is the dependent variable; µ is the overall mean; Trt_i is the fixed effect of the ith treatment (i = CON, SB, MON, sporozoites); E_i is the residual error ~ N (0, σ2 e). The least-square means were separated using the P.DIFF function of SAS 9.4.

Treatment effects were deemed significant when P ≤ 0.05 and trends at 0.05 < P ≤ 0.10. All data points greater or lesser than 3 SD away from the mean were considered outliers and removed from the dataset.

Results

Concentrations of treatments of SB, MON, and BA were used that would allow for MDBK cell survival in culture and still be effective.

Sporozoite viability and results are presented in Table 1. No statistical differences were observed between treatments for total sporozoites per well. The total number of sporozoites per well was approximately equal to 13. Treatment also did not affect the number of dead or dying sporozoites as indicated by trypan blue. However, MON and BA were similar and reduced the number of viable sporozoites compared to the CON and SB treatments (P = 0.009). There was a treatment by counter interaction primarily due to differences in the counts of dead sporozoites in the BA treatment with one counter averaging 3.75 dead sporozoites and the other averaging 8.5 dead sporozoites (P = 0.002). A trend was observed for treatment to decrease sporozoite percent viability (P = 0.10). Control, SB, and BA treatments were all similar while MON and BA were similar and reduced the percent viable sporozoites. BA was intermediate falling between CON and MON. Because of the difference in the dead counts percent viability, there was a treatment by counter effect for percent viable sporozoites (P = 0.04).

Table 1.

Eimeria bovis sporozoite viability in the presence of no treatment (CON), sodium butyrate (SB), monensin (MON), or butyric acid (BA)

Treatment1					P 2
Item	Con	SB	MON	BA	SEM3	TRT4	Counter5	TRT × Counter6
Alive	8.98a	8.38a	5.15b	5.59b	1.23	0.01	0.77	0.28
Dead	5.01	5.28	6.43	5.72	0.88	0.62	0.57	<0.01
Total	13.75	13.57	11.61	11.39	1.41	0.43	0.88	0.27
Percent viable	60.51a	61.00a	42.75a,b	47.73,b	5.22	0.07	0.44	0.11

^a,b Denotes differences in least mean squares (P ≤ 0.05).

¹ Treatment CON = RPMI-1640 with 10% FBS, SB = 0.028 mg/mL sodium butyrate, MON = 0.01 mg/mL monensin, BA = 0.18 mg/mL butyric acid, Sporozoites = RPMI-1640 with 10% FBS w/sporozoites.

² P-value significant if <0.05; trend if <0.10.

³ Standard error of the mean.

⁴ TRT = treatment.

⁵Counter = the difference between the observers who counted the sporozoites.

⁶TRT × Counter = the interaction between treatment and individual counter.

Results for the degree of potential damage to MDBK cells by sporozoites in vitro were based on the release of LDH as determined by LDH activity (Figure 1). Treatment had a significant effect (P < 0.001) on LDH levels. Lower LDH values indicated less cell membrane damage. The control treatment (no sporozoites) LDH release was similar to both the SB and MON treatments. While the sporozoite treatment was greater than the SB and MON treatments (P < 0.001), cells constitutively release LDH, with additional LDH release occurring during cellular damage. The sporozoite exposure had the highest LDH release.

Figure 1.

Lactate dehydrogenase release from MDBK cells with no sporozoites (Control), sporozoites with no treatment (Sporozoites), sporozoites with sodium butyrate (SB), and sporozoites with monensin (MON)^1,2. ^a,bDenotes differences in least mean squares (P ≤ 0.0001). ¹ Treatment Control = RPMI-1640 with 10% FBS, Sporozoites = RPMI-1640 with 10% FBS with sporozoites, SB = 0.028 mg/mL SB with sporozoites, MON = 0.01 mg/mL monensin with sporozoites, BA = 0.18 mg/mL butyric acid with sporozoites. ²LDH = Lactate dehydrogenase absorbance difference (680 nm–490 nm). Data were transformed to the inverse of the absorbance difference to allow for normal distribution before statistical analyses. The results presented are data transformed back to the actual absorbance difference. The SE is presented as the inverse of the absorbance.

Discussion

In vitro, MON was shown to affect the sporozoite life stage of Eimeria tenella. This was due to the large uptake of Na⁺ ions and the inhibition of Na⁺ ions out of the cell through the Na⁺-K⁺-ATPase pump (Smith and Galloway, 1983). Previous research showed that MON causes merozoites to swell and burst resulting in sporozoites being released and coming into contact with the antibiotic (Mehlhorn et al., 1983). Thus, the MON treatment produced a lower percentage of living sporozoites.

SB is composed of Na⁺ ionically bound to butyrate. For SB to function in the gastrointestinal tract, the Na ion is removed, and as the pH drops the butyrate ion will be dissociated to BA (Ahsan et al., 2016). The equilibrium equation indicates that at pH = 4.82 half of the molecule will be in the dissociated state (BA) as the pH is reduced more of the molecule will be in the dissociated state while when the pH is > 4.82 more of the molecule will be in the undissociated state (butyrate). Therefore, the low pH of the abomasum shifts the equilibrium to the production of associated butyrate or BA. This may explain why BA treatments caused a decline in the number of viable sporozoites per well while the SB treatment did not alter the number of live sporozoites. However, it is not known if butyrate can reduce sporozoites present in the reticulorumen. BA and its derivatives have been used in research associated with colon cancer in humans. Pattayil and Balakrishnan-Saraswathi (2019) performed a study looking at the morphological assessment of apoptotic cells with the inclusion of butyrate derivatives. One of the derivatives used here was SB. Researchers found that when human colorectal carcinoma cells (HCT116) were treated with BA derivatives, it induced apoptosis (Pattayil and Balakrishnan-Saraswathi, 2019). SB has been supplemented with milk replacer and starter grain of preweaned calves (Górka et al. 2011a, b, 2014). Researchers observed an increase in mitotic indices and a decrease in apoptotic indices of small intestine enterocytes suggesting an enhanced maturation of mucosal cells. In chickens, Eimeria alters the gut microbiota, more importantly, short-chain fatty acids reducing their prevalence, including butyrate (Leung et al., 2018). Butyrate is known to reduce inflammation in the gut (Andoh et al., 1999; Segain et al., 2000; Song et al., 2006; Elce et al., 2017). Therefore, increasing levels of BA in the gut can help to reduce inflammation and the severity of coccidiosis. Our data are consistent with the in vivo observations of reduced oocyst shedding in heifers fed SB (Rice et al., 2019; Stahl et al., 2020). Rice et al. (2019) observed a decrease in oocyst shedding at 0.25 g/kg BW while Stahl et al (2020) found comparable results to monensin (1 mg/kg BW) when they fed 0.75 g/kg BW. Zhou et al. (2017) showed that microencapsulated SB decreased the abundance of Bacteroides abundance in E. tenella infected chickens indicating that SB can alter the microflora of the intestine of infected birds. Bortoluzzi et al. (2018) observed that SB modulated the diversity of the microbiota present in the intestinal tract of chickens challenged with E. maxima reducing the negative impact of necrotic enteritis. Using tributyrin, Wang et al. (2021) observed that broiler chicks exposed to a mix of Eimeria and fed tributyrin had greater villus heights and widths and reduced oocyst shedding between d 19 and 26 (P < 0.05). However, it is not clear from these studies if the response was due to the anticoccidial effects of SB or the effect of SB on the intestinal epithelium, or both.

Results from the LDH assay are supported by results from Rice et al. (2019) and Stahl et al. (2020) when SB was supplemented in vivo to heifers. Rice et al. (2019) saw a quadratic effect on SB reducing the prevalence of coccidia in feces. Stahl et al. (2020) supplemented heifer diets with SB, MON, or a combination (SB + MON). A decrease in coccidia in the feces was seen when animals were supplemented with an additive (SB, MON, or combination). Their research supports the present findings on LDH absorbance difference in the SB and MON treatments being lower than the sporozoite treatment constitutive release. Dead or dying sporozoites are not viable to invade and damage the MDBK cells, thus decreasing cellular damage and the quantity of LDH released. As described above, MON causes sporozoites to lyse protecting the host cell from sporozoite invasion and subsequent damage. It is not clear why SB was effective in reducing the amount of LDH released. Possibly the removal of the Na ion resulting in butyrate was effective in killing the sporozoites. While it is common to use MDBK cells in these types of studies, it is not clear if these results would be as effective in gastrointestinal cells.

BA and MON had similar anticoccidial activity (lesser number of live sporozoites) as compared to SB and CON. SB and MON caused a decrease in LDH compared to the sporozoite treatment indicating that fewer sporozoites in these wells damaged the MDBK cells potentially due to the lower viability of sporozoites in these treatments. These data indicate that SB may be an effective anticoccidial under ad libitum feeding situations. It appears that SB is probably most effective against coccidial sporozoites after it is converted to BA in the gut. Further research is needed to see the exact mechanism of how BA and SB affect sporozoites both in vitro and in vivo.

Glossary

Abbreviations

ATCC

American type cell collection

ATPase

adenosine triphosphatase

BW

body weight

HCT116

human colorectal carcinoma cells

LDH

lactate dehydrogenase

MDBK

Madin Darby bovine kidney

MEM

minimum essential medium

RPMI

Roswell Park Memorial Institute

USD

United States Dollars

Conflict of Interest Statement

The authors declare that there is no conflict of interest.