The Influence of Chestnut Wood and Flubendazole on Morphology of Small Intestine and Lymphocytes of Peripheral Blood, Spleen and Jejunum in Broiler Chickens.

The study examined subpopulations of lymphocytes in peripheral blood, spleen, and jejunum including morphology of that segment in broiler chicken farm after treatment with flubendazole (Flimabend) and natural extract from chestnut wood (Farmatan). A total of 24 forty-day-old Kalimero-Super Master hybrid chickens were divided into 4 groups (n=6): the Fli group received Flimabend per os, 100 mg/g suspension in 1.43 mg of active substance/kg body weight during 7 day of experiment, Far group received Farmatan per os at 0.2 % concentration for 6 hours per day during 5 day (experimental days - from 3 to 7); the Far+Fli group received a combination of doses administered in the same way as for the first two groups; and control -C group with no active substance administration. The results demonstrated mild increase of leukocytes, lymphocytes, monocytes, leucocyte common antigen CD45, IgM+ and IgA+ cells in peripheral blood after administration of Flimabend. Similarly, subpopulations of followed lymphocytes (CD3+, CD4+, CD8+, IgM+) were increased in the jejunum after application of that drug. On the other hand, administration of Farmatan revealed opposite effect on determined immunocompetent cells what proves anti-inflammatory effect. Morphology of villi was also negatively influenced by administration of Flimabend. Administration of Farmatan suggests also its preventive administration in chickens. This tanin-containing drug as plant natural product may be used due to its antibacterial activity and as promising alternative to conventional drug with possible antihelminthic effect.

Introduction

Flubendazole is a widely used antihelmintic drug belonging to benzimidazole group. The molecular mechanism of action of flubendazole is based on its specific binding to tubulin. Microtubule-targeted drugs are highly effective for treatment of fungal and parasitic infections (Chatterji et al., 2011). They cause disruption of microtubule structure and function, also in the interference with the microtubule-mediated transport of secretory vesicles in absorptive tissues of helminths (Čaňová et al., 2017). As the drugs act by disrupting the tubulin-microtubule equilibrium in cells, this resulted to cessation of nutrient transport and eventual cell death. Flubendazole is a potent and efficacious antihelmintic for gastrointestinal nematode infections in poultry and domestic animals (Bradley et al., 1983; Tarbiat et al., 2016).

Flubendazole is usually administered orally and absorbed through the gastrointestinal tract. The drug is very poorly soluble in aqueous systems, causing its low absorption to the bloodstream and thus very low bioavailability (Michiels et al., 1982).

Over the last years, the dietary role of tannins is receiving increasing interest as they may reduce the number of gastrointestinal parasites in birds (Marzoni et al., 2005). Multiple reports suggest the efficacy of tannins or plant extracts in the control of zoonotic bacteria (Tosi et al., 2013; Redondo et al., 2014) and animal viruses (Lupini et al., 2009) in gastrointestinal tract. Tannins can have beneficial effects on the digestion when incorporated into animal diets although their primary mode of action is often not sufficiently known to explain the final in vivo effects (Redondo et al., 2014). Schiavone et al. (2008) found positive influence on growth performance, especially in young birds at the using up to 0.20 % natural extract of chestnut wood in diet. They did not observe any gross lesions at slaughtering as well as a lack of differences in intestinal length. Those results could indicate that toxicity of chestnut tannin used in their trial was low or absent. On the contrary, severe damage was reported in intestinal wall and other internal organs when doses higher than 30g/kg of tannic acid were administered to chicks (Singleton, 1981).

In the veterinary practice tannins are often use as additives with antihelmintic flubendazole for treatment of birds and to improve animal performance. We suggest that prolong application of flubendazole can be responsible for the modulation of immune response. On the other hand, moderate tannin level could improve health status of poultry.

That’s why the goal of the paper is to follow the effect of flubendazole and extract of sweet chestnut (Castanea sativa Mill.) on the immunocompetent cells in peripheral blood, spleen and jejunal mucosa. Morphological parameters of intestinal wall evaluated in duodenum and jejunum included height and total area of villi. The potential immunomodulatory and antiparasitical effects are discussed.

Materials and Methods

Chickens and diets

The experiment was conducted in a commercial broiler chicken fattening farm. The broilers were housed in four floor pens identical with the same direction and covered area (0.12 m2/broiler chicken). Wooden barriers separated the individual groups of chickens from each other. Twenty four chickens 40 days old Kalimero-Super Master hybrid in finisher rearing period were included in the trial. The chickens were weighed, labelled and randomly divided into four groups of 6 chickens each (n=6): C (control), Far (Farmatan), Fli (Flimabend) and Far + Fli (Farmatan+Flimabend).

The animals had free access to feed and water. The diet corresponded to commercial diet for broiler chickens referred to Feeding Norms for Poultry in Slovakia (code of laws and decrees No. 440/2006). The diet included premix Tekro-finischer, extracted soya starch, wheat, maize, mineral additive. Composition of the diet (g.kg-1) was next: Crude protein: 240.84, Crude fat: 52.01, Crude fibre: 35.16; Crude ash: 62.12, Starch: 463.84, Total sugar: 58.13, Reducing sugar: 12.62; Calcium: 8.87; Phosphorus: 9.28; Sodium: 2.58; Methionine: 4.71; Lysine: 13.42; Cystine: 2.90. Chickens were vaccinated against coccidiosis with Livacox Q on 5 day of age.

Administration of preparations and sampling

Extract of sweet chestnut (Castanea sativa Mill.) was added into water (Farmatan liquid, Tanin Sevnica d.d., Slovenia) of Far group chickens in 0.2 % concentration for 6 hours per day (8.00 – 14.00) during 5 days (from 3 to 7 experimental day). Chickens of Fli group received individually per os antihelminticum flubendazol (Flimabend, 100 mg/g suspension, KRKA d.d., Slovenia) in 1.43 mg of active substance/kg body weight during 7 days of experiment. Group Far+Fli received Farmatan liquid and Flimabend in the similar way and for the same period as groups Fli and Far. Group C served as a negative control and fed with diet without supplementary Farmatan or Flimabend.

Two days after the administration of Farmatan and Flimabend all chickens were euthanized by intraabdominal injection of xylazine (Rometar 2 %, SPOFA, Czech Republic) and ketamine (Narkamon 5 %, SPOFA, Czech Republic) at doses 0.6 and 0.7 ml/kg body weight, respectively. The samples of peripheral blood were collected before euthanasia by wing vein bleeding from subcutaneous ulnar vein into Heparin (10-20 U/ml PBS, Zentiva, Czech Republic). Remaining samples were collected during the necropsy. Spleen and intestines for flow cytometry and immunohistochemistry were put into phosphate buffered saline (PBS, Sigma, Germany) and for morphometry into 10 % neutral buffered formalin (NBF, Formaldehyd p.a., Centralchem, Slovakia).

White blood cell count

Leukocytes were counted in a haemocytometer using Fried-Lukačová solution (475 μl of solution plus 25 μl of blood). Differential cell counts of 100 cells per slide were done by light microscopy at 1000 magnification using blood smears stained with Hemacolor (Merck, Germany). The total numbers of different subtypes of white blood cells was then calculated: total leukocytes count x proportion of differential cells counted ( %) / 100.

Flow cytometry

Measurement and analysis of stained cells was performed on FACS system (Becton Dickinson, Germany) provided with a 15 mV argon ion laser. The analysis examined a dot plot of the leukocytes obtained by the forward and side scattering of the physical character of the lymphocyte population. Gates were drawn around lymphocytes based on 90° and forward-angle light scatter. The fluorescence data were collected on at least 10,000 lymphocytes using the Becton Dickinson CellQuest programme. The results are therefore expressed as the relative percentage of the lymphocyte subpopulation which was positive for a specific MoAb. Counting to absolute values was next: absolute count of lymphocytes x relative percentage of subpopulation’s lymphocytes/100.

Immunohistochemistry

The jejuna were collected into phosphate-buffered saline (PBS; pH 7.6) and then frozen and cut at –20 °C with Thermo Scientific Cryotome E (Shandon, USA). Frozen 4 μm sections fixed in cold acetone and rinsed in PBS were stained in Shandon Coverplate Technology system (BU Thermo Shandon, Germany). A streptavidin-biotin amplified peroxidase detection system (VECTASTAIN Elite ABC kit, Mouse IgG, PK 6102, Vector Laboratories, USA) was used to detect CD3, CD4, CD8, CD45, IgM and IgA positive lymphocytes. Unlabelled primary mouse anti-chicken monoclonal antibodies (Southern Biotech, USA) for staining CD3 (CD3-UNLB, Clone CT-3, Cat. No. 8200-01), CD4 (CD4-UNLB, Clone CT-4, Cat. No. 8210-01), CD8 (CD8α-UNLB, Clone CT-8, Cat. No. 8220-01),IgA (IgA-UNLB, Clone A-1, Cat. No.8330-01), IgM (IgM-UN-LB, Clone M-1, Cat. No. 8300-01) were used in 1:10 dilution with PBS. Mouse IgG1-UNLB antibody (Clone 15H6, Cat. No. 0102-01, Southern Biotech, USA) was used as negative control. All incubations were done at room temperature according to the manual instructions. The sections were rinsed three times with PBS between the two consecutive incubations. The specific colour reaction was developed for 5 min with 3.5 mmol/L 3,3’-diaminobenzidine (DAB, Sigma, Germany), and 30 ppm hydrogen peroxide in200 mmol/L Tris-HCl (pH 7.6). Subsequently the sections were counterstained with haematoxylin and mounted into Pertex (Histolab AB, Swedish). Quantification of labelled lymphocytes was performed under light microscope (NIKON Labophot 2, Germany) at a magnification of × 200 and by using of NIS-Elements version 3.0 software (Laboratory Imaging, Czech Republic). The photos from three jejunal sections at one slide were done and positive staining lymphocytes in 10 randomly chosen areas (a=60 000 μm2) were counted. Calculation to 1 mm2 was done as follows: 1 000 000/60 000 x cell numbers.

Histology and morphometry of duodenum and jejunum

Routine histological method with haematoxylin-eosin staining was used. Height and surface area of the villi in duodenal and jejunal samples collected from five chickens were analysed. The histological samples were microphotographed (Nikon LABOPHOT 2 with a camera adapter DS Camera Control Unit DS_U2, 4x) and then the NIS-Elements version 3.0 software (Laboratory Imaging, Czech Republic) was used. The heights of the villi were measured from the basal region, which corresponded to the higher section of the crypts, to the apex (μm). Total cutting surface area of separate intestinal segments included length and breadth of villi (μm2). The data were finally exported to MS Excel and subsequently statistically analysed.

Statistical analysis

Statistical analysis of obtained data was done by one-way analysis of variance (ANOVA) with the post hoc Tukey multiple comparison test using GraphPad Software, statistical version 5.0 (USA). The differences between the mean values for the groups of chickens were considered significant when P < 0.05. Values were expressed as means ± standard deviation (SD).

Ethical Approval and/or Informed Consent

All applicable national and institutional guidelines for the care and use of animals were followed.

Results

Peripheral white blood cells

Increase in total number of leukocytes (Table 1) was found in Fli and Far+Fli groups when compared with Far group (P<0.05). Similarly, the number of lymhocytes was higher in Fli and Far+Fli groups than in Far group (P<0.001). Monocytes also demonstrated improved values in Fli and Far+Fli groups in comparison with Far group, but with significance only in Far+Fli group (P<0.01).

Table 1

Absolute count of white blood cells (WBC; G.L–1= 109. L–1) in the peripheral blood of chickens (mean ± SD).

WBC	C	Far	Fli	Far+Fli
Leukocytes	13.72 ± 2.71	8.85 ± 2.92a	14.58 ± 3.02b	14.40 ± 3.46b
Lymphocytes	6.68 ± 0.35	5.44 ± 1.57a	8.63 ± 1.53d	8.37 ± 0.72d
Heterophiles	6.23 ± 2.44	2.65 ± 1.41	4.91 ± 2.39	4.63 ± 3.18
Eosinophiles	0.53 ± 0.23	0.47 ± 0.19	0.61 ± 0.34	0.88 ± 0.34
Monocytes	0.43 ± 0.06	0.29 ± 0.07a	0.44 ± 0.14	0.46 ± 0.12b

Specific superscripts in row indicate significant differences – abP<0.05; adP<0.001

Phenotyping of lymphocytes in blood and spleen

In the peripheral blood the highest values in all determinated subpopulation of lymphocytes (Table 2) were achieved in Fli group. Its levels outnumbered the counts in C and Far groups with exception to CD4+ cells in combined Far+Fli group. Density of CD3+ and CD4+ (T cells) showed no significant improvement in Fli and Far+-Fli groups. Determination of B cells showed higher values of IgM+ cells in both Fli and Far+Fli groups when compared to Far group, but were significant (P<0.01) only for Fli group. IgA+ subpopulation demonstrated highest values (P<0.01) not only to Far but also to Far+Fli group. Density of leucocyte common antigen CD45 was increased in Fli and Far+Fli groups when compared with Far group (P<0.001).

Table 2

Subpopulations of lymphocytes in the peripheral blood (total counts = G.L–1) and spleen (relative percentage)(mean ± SD).

Peripheral blood
Groups	Subpopulations of lymphocytes (mean ± SD)
	CD3	CD4	CD8	IgM	IgA	CD45
C	2.33 ± 0.79	1.32 ± 0.44	0.80 ± 0.20	0.85 ± 0.24	0.29 ± 0.26	4.32 ± 0.62
Far	2.37 ± 1.08	1.45 ± 0.63	0.72 ± 0.37	0.58 ± 0.23a	0.13 ± 0.11c	2.89 ± 1.01a
Fli	3.12 ± 0.90	1.82 ± 0.71	0.84 ± 0.19	1.20 ± 0.32c	0.70 ± 0.39a	5.42 ± 1.05d
Far+Fli	3.05 ± 0.73	1.99 ± 0.69	0.75 ± 0.18	0.95 ± 0.22	0.26 ± 0.12c	5.18 ± 0.84d

Specific superscripts in columns indicate significant differences – abP<0.05; acP<0.01; adP<0.001

In spleen (Table 2) the T cell subpopulations (CD3+, CD4+, CD8+) did not changed. Regarding the B cells an increase in IgM+ in Fli group was seen when compared to the control broilers (P<0.05). Density of IgA+ in experimental groups was higher. However, not significant when compared with controls.

Phenotyping of lymphocytes in jejunal mucosa

Jejunal mucosa in Far group (Table 3) showed lower number of CD3+ lymphocytes than seen in Fli, Far+Fli and C broilers (P<0.001). In similar way, the numbers of CD4+ and CD8+ lymphocytes were lower in Far group when compared to Fli, Far+Fli and C groups (P<0.001). Moreover, CD4+ cells were increased in Fli group contrasting to Far+Fli group (P<0.01).

Table 3

Subpopulations of lymphocytes in jejunal mucosa (mm2).

Subpopulations	C	Far	Fli	Far+Fli
CD3	598.90 ± 204.20d	312.00 ± 105.10a	680.10 ± 186.80d	672.30 ± 252.00d
CD4	614.40 ± 187.00d	282.00 ± 94.69a	641.70 ± 185.40da	547.10 ± 151.70dc
CD8	770.10 ± 213.40d	388.60 ± 159.50a	794.20 ± 257.30d	743.20 ± 220.20d
IgM	203.90 ± 122.30a	146.70 ± 91.80c	216.00 ± 88.14a	149.60 ± 73.22bc
IgA	331.10 ± 162.40	250.80 ± 180.40	294.10 ± 200.20	274.60 ± 160.40

Specific superscripts in row indicate significant differences – abP<0.05; acP<0.01; adP<0.001

IgM+ cells were found to be lower in Far (P<0.01) and Far+Fli (P<0.05) groups than in C group. In contrast, decrease of IgM+ lymphocytes was found in Far+Fli group in comparison to Fli (P<0.01) group. The IgA+ subpopulation was downregulated in experimental group when compared to control broilers.

Morphometry of duodenum and jejunum

In duodenum the Far group showed decrease in height of villi (Fig. 1) as compared with Fli and Far+Fli (P<0.001). Similarly, significant decrease was found between Fli and Far+Fli groups when compared with control group (P<0.001). The highest differences were detected in duodenum (Fig. 5). In comparison to control the cutting surface of duodenal villi (Fig. 2) was lower in Far, Far+ Fli groups (P<0.001) and Fli group (P<0.05).

Fig. 1

Height of duodenal villi (mean±SD, adP>0.001).

Fig. 5

Histological pictures from duodenum of different chicken’ groups (HE stain).

Fig. 2

Cutting surface of duodenal villi (mean±SD, abP>0.05, adP>0.001).

In jejunum Far and C groups showed increase in height of villi (Fig. 3, Fig. 6) when compared to Fli and Far+Fli groups (P<0.001). However, the Far group outnumbered C group (P<0.05). Cutting surface of jejunal villi (Fig. 4) was lowest in Far+ Fli group in comparison to Fli (P<0.001) and Far groups (P<0.05). Values of Far group were merely higher than values detected in C group (P<0.05).

Fig. 3

Height of jejunal villi (mean±SD, abP<0.05,*adP<0.001).

Fig. 6

Histological pictures from jejunum of different chicken’ groups (HE stain).

Fig. 4

Cutting surface of jejunal villi (mean±SD, *abP<0.05, adP<0.001).

Discussion

Flubendazole, one of the benzimidazole antihelmintics, is widely used for treatment and prevention of endoparasitic infections in poultry (Baliharová et al., 2004). Commercially available flubendazole-based products are used mainly against helminth parasites of chickens as Ascaridia galli, Heterakis gallinarum and Capillaria spp. (Squires et al., 2012). On the other hand, tannins included in low concentration of the diet have positive influence on the growth performance (Redondo et al., 2014), reduce the spread/occurrence gastrointestinal parasites in birds (Marzoni et al., 2005) and was proven to posess antimicrobial activity (Elizondo et al., 2010). Beneficial properties of tannins prompted us to test this substance during administration of flubendazole.

Ascaridia galli is a common parasite in small intestine of chickens. After ingestion of the embryonated eggs, these are transported until they reach the duodenum and/or jejunoileum, where they hatch; larvae are released and penetrate the epithelium (Luna-Olivares et al., 2015). From these reasons, the evaluation of immunocompetent cells and morphological parameters in intestine – jejunum – was selected.

Seven days administration of antihelmintic drug caused mild increase in the number of peripheral blood leukocytes, lymphocytes and monocytes in groups with flubendazole. Similarly, flubendazole modulated the level of IgM+, IgA+ cells and CD45 in peripheral blood, and IgM+ cells in spleen. Observed shift in immunocompetent cells suggests systemic immune response to antihelmintic drug. Our results are in accordance with results done in our laboratory (Karaffová et al., 2019) where the upregulation of pro-inflammatory cytokines (IL-1β and IL-18) in Fli group was demonstrated. CD3+, CD4+, CD8+, IgM+ increase in chicken intestine after seven days flubendazole administration support the mild inflammatory role of drug in the chicken duodenum. On the other hand, five days administration of farmatan decreased the number of immunocompetent cells in chickens what suggest on tannins anti-inflammatory effect. Recently, many studies demonstrated the anti-inflammatory effect of tannins. Araújo-Neto et al. (2010) have shown the anti-inflammatory effect of tannins with Sideroxylon obtusifolium on paw oedema induced by carrageenan. Another study exploiting a wound model and skin healing in rats showed its topical anti-inflammatory and healing activity (Leite et al., 2015). Finally, anti-inflammatory properties were found in ethyl acetate phase of Anacardium occidentale rich in tannins (de Araújo Vilar et al., 2016). Recently, Williams et al. (2014) provided clear evidence of the direct antihelmintic effects of tannins against Ascaris. On the other hand, our working group in another paper (Karaffova et al., 2019) demonstrated the upregulation of MUC-2 and IgA gene in the duodenum of chickens after administration of tannin. IgA forms the first line of defence to limit epithelial contact with and penetration by intestinal microbiota and other potentially dangerous antigens (Zhang et al., 2015). MUC2 expression increases from anterior to posterior through the gastrointestinal tract (Jiang et al., 2013), and protective role of mucins against parasites include the demonstration of trapping of worms in the mucus as well inhibition of parasite motility and feeding capacity (Khan et al., 2008). Consistent increase of T cell subpopulations and MUC2 gene upregulation suggest subsequent mucus production in our experiment (Theodoropoulos et al. 2001).

Height of villi and cut surface of villi decreased in experimental groups comparing to control. This phenomenon can be explained by lower number of immunocompetent cells in the mucous including villi in Far group and negative effect of benzimidazoles on proliferation of enterocytes. It is known that benzimidazoles can affect also host tubulin (Mackenzie and Geary, 2011) and what can be connected with the decrease the height of villi in groups with benzimidazoles. Increased thickness of depth of crypts in Fli group also suggests mild inflammatory process with increased number of immunocompetent cells.

A normal morphology and intestinal permeability of the small intestine is important to prevent bacteria translocation from the intestinal tract to the body as well as for digestion and absorption of nutrients (Quinteiro-Filho et al., 2010; Awad et al., 2017). An increase of immunocompetent cells in the peripheral blood and jejunal mucosa in our trial is likely to be indicative of a possible intestinal mucosa barrier dysfunction (Beatty et al., 2017) and, consequently bacterial infection. Inflammatory infiltrate is suggests to be contributed to the production of proinflammatory cytokines observed in that experiment (Karaffová et al., 2019) and affect on the intestinal epithelium’s tight junctions, in turn increasing the mucous permeability to pathogenic bacteria.

In conclusion, the results in our study demonstrated mild inflammatory effect on leukocytes, lymphocytes, monocytes, leucocyte common antigen CD45, IgM+ and IgA+ cells in peripheral blood after administration of Flimabend. Similarly, subpopulations of followed lymphocytes (CD3+, CD4+, CD8+, IgM+) were increased in the intestine after application of that drug. On the other hand, administration of Farmatan revealed the opposite effect on immunocompetent cells what proves to have an anti-inflammatory effect. Morphology of villi and depth of crypts was negatively influenced by administration of Flimabend. Results obtained also suggest the utilisation of Farmatan as preventive – immunomodulatory substance reducing inflammation as well as the adjuvant in treatment with antihelmintics.