EFFECTS OF ZN2+- AND CR3+-MODIFIED PALYGORSKITE ON THE TREATMENT OF EIMERIA TENELLA
D. W. Yao1,2, F. M. Khand2, Y. Xu1, Q. Q. Shen2, Y. Yang2 and D. J. Yang2*
1Jiangsu Provincial Key Laboratory of Palygorskite Science and Applied Technology, Huaiyin Institute of Technology, Huaian 223003, China
2 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
Corresponding author’s: yangdeji63@sohu.com
ABSTRACT
The objective of this research was to investigate the effects of Zn2+- and Cr3+-modified palygorskite (ZCM-Pal) in an experimental E. tenella infection in chickens. ZCM-Pal was produced by cationic ion exchange with Zn2+/Cr3+ and palygorskite (Pal). The anticoccidial activity of ZCM-Pal, Diclazuril was evaluated by comparing the differences in the anticoccidial index (ACI). During the experiment, clinical signs, bloody feces, relative weight gain (RWG), survival rate (SR), cecal lesion scores (LS), and oocysts/gram of feces (OPG) were recorded in all the chickens to calculate the ACI. The results showed that the ACI of the ZCM-Pal group (10 g/kg) was 170.38, indicating moderate activity. The histopathological examination of cecal samples revealed that the ceca from the chickens medicated with ZCM-Pal were intact compared with those from chickens from the infected control. The number of inflammatory infiltrates and parasites in ZCM-Pal group was less than the other groups. The blood endotoxin concentrations were decreased with supplementation of ZCM-Pal. At the end of the experimental period, the weight gain of chickens treated with ZCM-Pal was similar to those in the uninfected control group. These results suggested that ZCM-Pal might be an aid in the control of E. tenella in chickens.
Key words: coccidiosis; palygorskite; chromium; zinc; anticoccidial activity
https://doi.org/10.36899/JAPS.2022.2.0436
Published first online August 13, 2021
INTRODUCTION
Chicken coccidiosis is one of the most economically devastating diseases in the poultry industry due to its low productivity. Economic losses in poultry worldwide exceed 3 billion US dollars annually (Dalloul and Lillehoj 2006; Blake et al. 2020). In chickens, most species that can produce coccidiosis belong to the genus Eimeria, and they infect various sites in the intestine. The infection process is rapid (4-7 days) and is characterized by parasite replication in host cells, with damage to the intestinal mucosa. The control of chicken coccidiosis principally depends on prophylactic chemotherapy by the inclusion of drugs, such as polyether ionophore antibiotics and chemically synthesized coccidiostats, in the feed or drinking water (Noack et al. 2019). Because of the development of anticoccidial resistance to these drugs, there is a need to find safe alternatives to control chicken coccidiosis (Abbas et al. 2011).
Palygorskite (Pal), formerly called attapulgite, is a hydrated magnesium aluminium silicate that exists in nature as a fibrillar silicate clay mineral with reactive OH groups on the surface (Zhou et al. 2014). The major chemical components of palygorskite are SiO2 (55.03%), Al2O3 (10.24%), MgO (10.44%), Fe2O3 (3.53%), H2O+ (8.24%) and H2O− (7.29%) (Lopez-Galindo et al. 2007). Palygorskite has some physical properties, including a high viscosity, a high specific surface area, and a high absorption as well as cation exchange abilities (Murray 2000). These properties make Pal widely useful in the animal industry as an aflatoxin adsorbent, a tannin adsorbent, and an excipient in pharmaceutical preparations and, in addition, as antacids, gastrointestinal protectors and anti-diarrhetics (Chen et al. 2020). Many researchers have suggested that exchanging montmorillonite or palygorskite with cations enhanced the antibacterial activity of Pal (Karel et al. 2015; Yao et al. 2017; Lobato-Aguilar et al. 2020).
Chromium is an essential element required for carbohydrate, fat, and protein metabolism (Sahin et al. 2021). Trivalent Cr is a well-known essential trace element for humans and animals. Organic and inorganic chromium are usually applied as additives to animal feeding. Many studies have shown that trivalent Cr significantly improves the function of the humoral immune system and the carcass quality of broilers under heat-stress conditions (Tian et al. 2014; Bin-Jumah et al. 2020). Zn is an essential trace element and a constituent of different enzymes involved in cell growth and development, antioxidant mechanisms, and the immune system (Mohammadi et al. 2015; Jarosz et al. 2017). Dietary zinc supplementation improved growth, intestinal morphology and intestinal microbiota in Salmonella-challenged broiler chickens (Zhang et al. 2012; Shao et al. 2014; Tang et al. 2014). The ameliorating role of copper and zinc against Eimeria acervulina-induced oxidative damage has been reported in infected chickens (Georgieva et al. 2011).
To combine gastrointestinal protectors and anti-diarrhetics with palygorskite, antioxidants, and the anti-heat stress of chromium and zinc, Zn2+- and Cr3+-modified palygorskite (ZCM-Pal) was produced by cationic ion exchange with Zn2+/Cr3+ and palygorskite. The aim of this research was to investigate the effects of ZCM-Pal on experimental E. tenella infection in chickens.
MATERIALS AND METHODS
Preparation of ZCM-Pal: ZCM-Pal was prepared with Pal, Zinc chloride and Chromium trichloride as described previously, with some modifications (Yao et al. 2017). The Pal (Jiangsu Shenlite Biotechnology Co., Ltd., Jiangsu, China) was pre-treated in a muffle furnace at 350 °C for 2 h. Then, 10.0 g of Pal was dispersed in 200 mL of 0.05 mol/L Zinc chloride and 0.05 mol/L Chromium trichloride solution. The dispersion was placed on a magnetic stirring apparatus at 55 °C and 200 r/min for 3.5 h. The ZCM-Pal was washed repeatedly with distilled water to remove the dissociative Zinc chloride and Chromium trichloride and was then dried at 105 °C to a constant weight. The ZCM-Pal was subsequently pulverized to a powder using a mortar and was filtered using a 200-mesh strainer. The concentration of Zn and Cr in the ZCM-Pal was measured by ICP-OES (PerkinElmer Optima 2100DV, USA) as described previously (Yao et al. 2017).
Parasites: The oocysts of E. tenella were kindly provided by the Laboratory of Veterinary Medicine and Immunological Parasitology, Nanjing Agricultural University, China. The oocysts were propagated in broiler chickens and collected from feces on post-infection days 8 and sporulated by placing in 2.5% potassium dichromated solution at suitable humidity and temperature (28°C). The sporulated oocysts were washed three times with PBS (pH 7.4) and counted using a McMaster Egg Slide Counting Chamber under a light microscope. The concentration of the sporulated oocysts was 5×104/mL.
Chickens: One-day-old Chinese Yellow Broiler chickens were purchased from the hatchery of Qinglongshan, Nanjing, China. The birds were reared for 14 days in coccidia-free conditions with ad libitum access to water and a standard diet without drug supplements. All the animal experiments were conducted strictly in accordance with laboratory animal guidelines. The protocol was approved by the Laboratory Animal Management Committee of Jiangsu Province (SYXK2017-0027).
Experimental design: In a total of 12 pens, 120 chicks (10/pen) were weighed and placed into 3 replicate pens for each of the 4 groups in 2 environmentally controlled rooms. The groups were as follows: A, the normal control group, chickens were not infected with E. tenella oocysts and not treated with medicine. The chickens in groups B-D were challenged with a 1 mL oral gavage containing 5×104/mL sporulated oocysts and were fed the following diets for one week: B, basal diet plus ZCM-Pal (10 g/kg); C, basal diet plus Diclazuril (1 mg/kg); D, (untreated group). During the experiment, all the chickens were monitored daily to record clinical signs, bloody feces and deceased birds.
Anticoccidial index analysis: The faeces were collected starting from 6-day post infection (dpi) till the end of the experiment (8 dpi). The complete cecum contents of each group were also collected and then put together with faeces of each group. Oocysts were counted from 1 g of the contents (oocysts per gram, OPG) using McMaster method. Ratio of oocysts was determined by comparing the oocysts/gram of faeces (OPG) in the treated groups with that in the untreated group by a previously described method. Then ratio of oocysts can be transform to the corresponding assigned oocyst index (McManus et al. 1968).
On day 8 dpi, fifteen birds of each group were humanely killed for the grading of cecal lesions on a scale of 0 to 4, based on the general macroscopic appearance of epithelial cells, petechiae, thick of shrunken cecal walls and bloody cecal content (McManus et al. 1968). Cecal lesion score (LS) of 0 (no lesions), 1 (mild lesions), 2 (moderate lesions), 3 (severe lesions), or 4 (extremely severe lesions or death due to coccidiosis) was recorded for each chicken. The relative weight gain (RWG) was the weight gain (WG) in the treatment groups compared with that in Group A. The anticoccidial efficacy was evaluated using the survival rate (SR) and the anticoccidial index (ACI). ACI = RWG + SR – 10LS − oocyst index, and an ACI greater or equal to 180 indicated excellent activity, an ACI from 160 to 179 indicated moderate activity, an ACI from 120 to 159 indicated limited activity, and an ACI less than 120 indicated lack of efficacy (McManus et al. 1968).
Histopathological examination: The caeca were excised and immediately fixed in 10% formalin solution, dehydrated with an ethanol and toluene series, and embedded in paraffin wax via a routine process. All the sections were stained with haematoxylin and eosin (H&E) and were observed using an optical microscope to examine the morphological changes in the caeca.
Serum endotoxin levels detection: Blood samples were collected from fifteen birds of each group at day 8 dpi. Serum samples (100 μL) were added into endotoxin-free tubes for measuring endotoxin levels using ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit (GenScript, China) according to the manufacture introduction (Jeong et al. 2014). Endotoxin levels of samples were calculated with the standard curve obtained by standard solution.
Statistical analysis: The data in the tables are presented as the arithmetic mean ± standard deviation (SD). The statistical analysis was performed by one-way analysis of variance (ANOVA) using Predictive Analytics Software 18.0. Duncan’s multiple-range test was used, with differences considered to be significant at P<0.05.
RESULTS
Characterization of ZCM-Pal: ZCM-Pal was produced by ion exchange processes between Zn2+/Cr3+ and the Pal cation. The Zn and Cr concentrations in the ZCM-Pal were 18.19±0.24 mg/g and 19.43±0.23 mg/g, respectively, as measured by ICP-OES.
Anticoccidial activity: All the chickens in group D infected with E. tenella exhibited significant clinical symptoms, such as depression, ruffled feathers, closed eyes, inappetence, diarrhoea, and bloody feces compared to the uninfected normal control group. Four deaths occurred in group D during the experiment. The clinical symptoms of the groups B and C were slight compared to group D, and there were no deaths in those groups. The WG was significantly decreased after E. tenella infection (Table 1). The WG in groups B and C medicated with ZCM-Pal and Diclazuril was significantly higher than in group D infected with E. tenella (P<0.05). The lesion scores in the treated groups were significantly lower than the untreated group (P<0.05) (Table 1). The rest of the chickens were allowed free access to food and water after 8 days of infection. In the one-month-old chickens, the weight gain was calculated and is shown in Table 2. The WG of group B administered ZCM-Pal (10 g/kg) was similar to the normal group. The untreated group D had the lowest WG among those groups (P<0.05).
After the chickens were killed, it was found that there was no significant difference in cecal lesions between the groups medicated with ZCM-Pal and Diclazuril (Figure 1a-b). The caeca in group D were dilated to 1.5 times the normal diameter (Figure 1c). The lumen of the distended caeca was filled with blood clots.
The ACI of group B given ZCM-Pal (10 g/kg) was close to 180. It was greater than other groups, which indicated moderate activity (Table 1). It suggested that ZCM-Pal had excellent anticoccidial activity. The ACT in Diclazuril groups was only 152.93 in this research, which indicated limited activity.
Table 1 Anticoccidial activity of Diclazuril and ZCM-Pal
Groups
|
WG (n=30)
|
RWG %
|
SR %
|
LS (n=15)
|
Ratio of oocyst %
|
Oocyst index
|
ACI
|
A
|
92.15±9.26a
|
100
|
100
|
0
|
0
|
0
|
/
|
B
|
85.99±3.46b
|
93.38
|
100
|
1.8±0.78 b
|
8.3
|
5
|
170.38
|
C
|
78.59±6.05c
|
81.93
|
100
|
1.9±0.95 b
|
27.3
|
10
|
152.93
|
D
|
67.44±5.42d
|
72.55
|
86.7
|
3.6±0.69 a
|
100
|
40
|
/
|
Group A: normal control group. Group B: ZCM-Pal 10 g/kg. Group C: Diclazuril 1 mg/kg. D: untreated group.WG: weight gain. RWG: relative weight gain. LS: lesion score. SR: survival rate. ACI: anticoccidial index. Different letters within a column indicate statistically significant difference (P<0.05).
Table 2 Weight gain of the chickens on one-month-old
Groups
|
WG
|
A (n=15)
|
212.67±9.21a
|
B (n=15)
|
204.72±8.87a
|
C (n=15)
|
187.94±8.80 b
|
D (n=11)
|
164.768±11.97 c
|
Group A: normal control group. Group B: ZCM-Pal 10 g/kg. Group C: Diclazuril 1 mg/kg. D: untreated group. WG: weight gain.
Different letters within a column indicate statistically significant difference (P<0.05).
Histopathological examination: Microscopically, the lesions induced by E. tenella were characterized principally by severe villous atrophy and disintegration abscission (Figure 1ca). The enteric cavity was filled with degenerated and necrotic epithelial cells, lymphocytes, phagocytes, and red blood cells as well as different phases of parasites. The inflammatory infiltrate and the different phases of parasites were present in the submucosa. The four-layer structures of the caeca were intact in the Diclazuril-treated group, although a part of the villous was destroyed (Figure 1b). The mucosa of the caeca in group B medicated with ZCM-Pal was improved compared to group C (Figure 1a). There were also large numbers of intracellular parasites in various developmental stages, and lymphocytes and red blood cells were present in the enteric cavity, in groups B and C. The numbers of inflammatory infiltrate and parasites in the various developmental stages in groups B and C were less than those of group D.
Fig.1 Gross pathology and histological photomicrographs (400×, HE Staining) of ceca of group B-D at 8 days following with E. tenella.
The 14-day-old chickens were infected with E. tenella oocysts using a 1 mL oral gavage containing 5×104/mL sporulated oocysts. The ceca were collected after 8 days infection. a, group B, ZCM-Pal (10 g/kg). The four-layer structures of the caeca were intact. b, group C, Diclazuril (1 mg/kg). A part of the villous was destroyed.c, group D, untreated group, the chickens were not treated, distended and club-shaped ceca, the lumen of the distended ceca is filled with blood clots. The enteric cavity was filled with degenerated and necrotic epithelial cells, lymphocytes, phagocytes, and red blood cells as well as different phases of parasites.
Serum endotoxin levels detection: The serum endotoxin concentrations in studied birds are presented in Figure 2. The data showed a statistically significant increase of endotoxin concentrations in chicken infected with E. tenella. The levels of endotoxin in group B and C treated with ZCM-Pal and Diclazuril were significantly reduced, compared to untreated group (P<0.05). The endotoxin concentrations in group B were found to be significantly different in comparison to the group C or group A (P<0.05).
Fig.2 Serum endotoxin concentrations in each groupat 8 days following with E. tenella.
Group A: normal control group. Group B: ZCM-Pal 10 g/kg. Group C: Diclazuril 1 mg/kg. Group D: untreated group. Different letters within a column indicate statistically significant difference (P<0.05).
DISCUSSION
Chicken coccidiosis is characterized by enteric lesions of variable extents and severities, which reduce the absorptive function of the intestinal mucosa and lead to weight loss, diarrhoea, and higher mortality in the affected flocks. The addition of Pal to the diet positively protects the intestinal mucosa and reduces the concentrations of endotoxin in the plasma (Guarino et al. 2009; Yao et al. 2017). In this study, the histopathological examination found that the mucosa of the caeca in group B medicated with ZCM-Pal was improved compared to groups C and D. These results suggest that ZCM-Pal could protect the enteric mucosa.
Endotoxin is a unique cell wall component of Gram-negative bacteria. Under normal circumstances, no endotoxin can pass through the epithelial mucosa, and their presence in blood is an indicator of intestinal permeability (Luk et al. 1983). In this study, it was found that the levels of endotoxin were increased in the chickens infected with infected with E. tenella. This result was different with previously report. Baba et al had reported that endotoxin levels in the plasma of E. tenella-infected and control chickens were not significantly different (Baba et al. 1990). This research results also showed that the endotoxin concentrations were decreased with supplementation of ZCM-Pal. It has been reported that the dietary supplementation with Pal may protect the gut mucosa and decrease the blood concentrations of endotoxin(Zhang et al. 2013). In the present study, the features of intestinal morphology also indicated that the ZCM-Pal could protect the enteric mucosa.
Antioxidant compounds may hold promise for the control of Eimeria infections due to the association of coccidial infection with lipid peroxidation of the intestinal mucosa (Masood et al. 2013). In a recent experimental study, Georgieva et al. find an antioxidant role of Zn by improving the activity of antioxidant enzymes in infected birds (Georgieva et al. 2011). Organic and inorganic chromium added to animal feed significantly improves the function of the humoural immune system and the carcass quality of broilers under heat-stress conditions (Moeini et al. 2011; Perai et al. 2014; Tian et al. 2014). In this study, the anticoccidial activity of Pal was improved by the ion exchange processes between Zn2+/Cr3+ and the Pal cation (data not show).
Diclazuril, a broad-spectrum anticoccidial agent, is highly efficacious against both the asexual and sexual stages of E. tenella (Zhou et al. 2015). Owing to the widespread use of this drug in the poultry industry, the development of resistant pathogenic strains and drug residues in animal products has become a major concern (Abbas et al. 2011). Highly efficacious against coccidiosis, the ACI of Diclazuril should be more than 180. However, the ACI in Diclazuril groups was only 152.93 in this research, which indicated limited activity. Those results suggested that the E. tenella used in this research might be resistant to Diclazuril. The anticoccidial activity of ZCM-Pal was better than that of Diclazuril.
The growth of the chickens was seriously affected by challenge with E. tenella, although most chickens survived E. tenella infection. The chickens in the untreated group had the lowest WG compared with the other groups. The WG of chickens administered ZCM-Pal (10 g/kg) was similar to the normal group. These results show that ZCM-Pal could improve the growth of chickens challenged by E. tenella.
In conclusion, the present study is the first to describe the anticoccidial activity of ZCM-Pal in chickens. These research results demonstrated that ZCM-Pal had a better effect on the treatment of chicken coccidiosis. It could protect the enteric mucosa, decrease the blood concentrations of endotoxin and improve the growth performance of chickens.
Acknowledgments: This study was supported by a project funded by the Foundation of Key Laboratory for Palygorskite Science and Applied Technology of Jiangsu Province (HPK201506) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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