THE AMELIORATING EFFECT OF MENTOFIN® ON RESPIRATORY SYSTEM OF BROILER CHICKENS CHALLENGED WITH MYCOPLASMA GALLISEPTICUM STRAIN IN ASSIUT GOVERNORATE

Assiut University web-site: www.aun.edu.eg

THE AMELIORATING EFFECT OF MENTOFIN^® ON RESPIRATORY SYSTEM OF BROILER CHICKENS CHALLENGED WITH MYCOPLASMA GALLISEPTICUM STRAIN IN ASSIUT GOVERNORATE

AL SHIMAA, R.SAYED ¹; MARWA, A.AHMED ²; MOSTAFA, A. SHEHATA ³;

MOEMEN, A. MOHAMED ³; SABRY, I. EISSA ⁴ AND MARWA, M. SAFWAT ³

¹ Animal Health Research Institute, Asiut.

² Department of Pathology, Faculty of Veterinary Medicine, Aswan University, Egypt.

³ Faculty of Veterinary Medicine, Assiut University.

⁴ Chief Researcher of Mycoplasma Dept. Animal Health Research Institute Agriculture, Research Center, Doki, Giza.

Received: 31 December 2019;     Accepted: 31 January 2020

INTRODUCTION

Mycoplasma gallisepticum (MG) is the most pathogenic avian mycoplasmas that cause respiratory infection in poultry.  M. gallisepticum causes chronic respiratory disease (CRD) in chickens and have been reported to cause serious economic losses (Osman et al., 2009). The clinical signs of avian mycoplasmosis are sneezing, coughing, and respiratory rales; ocular and nasal discharge; decreased feed intake and increased morbidity. Increasing mortality in birds with MG infection caused by concurrent bacterial and/or viral infection has been widely reported (Raviv and Kleven, 2009).

Mycoplasma gallisepticum infection has tropism primarily for mucosal membranes of the respiratory tract, conjunctiva and sinuses (Levisohn and Kleven,

Corresponding author: Dr. Marwa, M. Safwat

E-mail address: merovet84@gmail.com

Present address: Faculty of Veterinary Medicine, Assiut University.

2000).The organism usually enters the host via the respiratory tract; upper airways and trachea are the preferred sites of infection for most of the strains of MG (Kleven, 1997).

Mentofin^®, a natural herbal product consisting of some essential volatile fatty acids and natural herbal essences (10%) eucalyptus oil, 10% menthol, 33% liquid builders, and 47% saponins) Rehman et al. (2013). The essential oils of Eucalyptus species possess important biological activities including antibacterial, anti-inflammatory, diaphoretic, antiseptic, analgesic effects (Cimanga et al., 2002) and antioxidant properties (Damjanović-Vratnica et al., 2011), and safely used in broiler and layer chicken production, the product was able to help in preventing respiratory disease complexes (Barbour et al., 2005), increasing performance and strengthening the immune system (Bragg, (2004 and 2006); Carli et al., 2008).

The aim of this study was to estimate the Mentofin^® effect on respiratory system of Mentofin^® treated groups following a challenge with Mycoplasma gallisepticum through gross and histopathological evaluation.

MATERIALS AND METHODS

I-Isolation and Identification of the Mycoplsama from birds suspected to be infected:

A) Sampling:

One hundred and twelve (112) samples including lung and trachea were collected from 112 freshly dead and slaughtered broiler chickens showing respiratory manifestation (watery to mucoid nasal discharge, sneezing, gasping, tracheal rales and conjunctivitis) were collected from different localities in Assiut with ages ranged from (20-35 days), weren't vaccinated against Mycoplasma gallisepticum.

B) Procedures for isolation of Mycoplasma (Sabry and Ahmed, 1975):

Samples were placed on PPLO broth and brought to the laboratory of avian and rabbit diseases Department, Assiut University for subsequent culturing  on PPLO media that containing horse serum, dextrose , yeast extract  and thallium acetate with penicillin.  Broth bottles were incubated at 37^°C for 3-4 weeks before being discarded as negative samples; however the agar plates were incubated under reduced oxygen tension in humidified candle jar then examined microscopically for Mycoplasma colonies after 48 hrs. and daily up to 7-10 days for "fried-egg shape colonies", then subjected to arginine deamination and glucose fermentation tests for biochemical characterization according to (Enro and stipkovits, 1973) .

C) Polymerase Chain Reaction (PCR) for detection of Mycoplasma gallisepticum:

The extraction of bacterial DNA was performed using QIAamp manual kit according to manufacturer's instructions. Two specific oligonucleotide primers for identification of M.gallisepticum were used for detection of MGc2 specific gene for M.gallisepticum. The sequence of primer forward (F) was (5'- CGC AATTTG GTC CTA ATC CCC AAC A-3').The sequence of primer reverse(R) was (5'-TAAACC CAC CTC CAG CTT TAT TTC C-3')(Garcia et al., 2005). Amplification was performed by heating the sample for 4 minutes at 94^oC for initial denaturation. After this step forty cycles were performed as follows: Denaturation for 20 sec. at 94^oC, annealing for 40 sec. at 58^oC and extension for 1 minute at 72^oC with the exception of final extension step was held for 7 minutes for final extension. The analysis of PCR amplified products was done by using 8μl of amplified PCR product, mixed with 2μl loading dye and electrophoresed through 1% agarose gel and DNA was visualized by UV fluorescence after ethidium bromide staining.

II- The evaluating effect of Mentofin^® on the experimentally challenged birds with isolated M. gallisepticum:

Birds: One hundred five–one day old broiler chicks Ross (308) from breeder farm were used in the present study, with an average body weight of 37-40 gm, obtained from El Qusia Assiut. Chicks were housed in a well-isolated floor pens under complete hygienic conditions. Chicks were reared in a complete block design provided with wood shaving litter, plastic feeders and waters. Chicks were fed adlibtum on commercial broiler ration.

Preparation of Challenge Strain:

Isolated field M. gallisepticum strain was morphologically, molecularly characterized and subjected to sequencing for detection of partial 16s rRNA gene with accession number in GenBank (15mgAsyut-Egypt-2016) used in preliminary study after counting using colony forming unit method according to Jett et al. (1997) for detection of the pathogenicity and evaluation of Mentofin^® as a reducing agent of histopathological lesions which caused by MG infection.

Experimental design:

One hundred and five– one day old chicks were randomly divided into three equal groups (35 in each group).Group (A) MG challenged, Group (B) was treated with Mentofin^® (EWABO - Germany) and MG challenged, Group C (control –ve group not challenged with MG and not treated with Mentofin^® ). At 1^st week of age, an intra-tracheal challenge of the birds with MG strain (containing 1 x 10⁶cfu / mL/bird) was given to (A and B) groups. Mentofin^® was given to (B) groups.

Essential oils of Mentofin^® were administered for 6 days, beginning from 8^th day to 13^th day of age.

Mentofin^® was diluted in V/V of 0.025 mL/100 mL of drinking water. Each treated bird started receiving the diluted Mentofin^®, twice a day (morning and evening), in a volume of 1mL/bird/time in the drinking water. Group C (control group not challenged with MG and not treated with Mentofin^®).

Histopathological examination

Tracheal and lung samples for histopathological examination were collected from chicken at 9^th day to 14^th day of age and at 28^th day of age. Three birds were slaughtered from each group and trachea and lung were collected and fixed in 10% buffered neutral formalin.  The  fixed  tissues  were  embedded  in paraffin,  sectioned  at  4  μm  thick  and  stained  with haematoxylin  and  eosin  H&E (Bancroft and Stevans, 1993). The slides were examined under light microscope.

RESULTS

From 112 trachea and lung pooled samples 6 were suspected to be positive for Mycoplasma gallisepticum isolation with incidence rate (5.36%). Microscopically the shape of colony was fried egg appearance as showing in (fig.1), biochemically glucose fermentation test was positive and negative to arginine. M. gallisepticum strain detected by PCR using MGc2 gene gave a characteristic common band at 300 bp fragment (fig. 2).

Fig. (1): colonial appearance of Mycoplasma gallisepticum isolates by dissecting microscope (fried egg appearance).

Fig. (2): Mycoplasma gallisepticum detected by PCR using mgc2 gene Positive samples produce band (300 bp), Lane M: 100 bp DNA

Ladder, Lane 1, 2, 3, 4, 5, 6 and 7 were positive samples

Produce band (300 bp).

Results of experimental infection

Clinical findings in infected group (A) (challenged group), Chicks in infected group were dull, depressed with ruffled feather, coughing and sneezing, nasal discharges and open mouth breathing with moist rales.

Gross Pathology of infected group (A (, trachea showed the evidence of congestion and hemorrhages. Lung revealed dark red color appearance and congestion (fig.3), air sacculitis was observed in air sacs, these become cloudy thickened and covered with caseous exudates. Gross pathology of group (B), trachea showed no evidence of congestion and hemorrhages. Lung revealed normal color and no congestion. Air sacs appear normal as thin-walled and no cloudness.

Fig. (3): showing gross pathology of Mycoplasma gallisepticum on trachea and lung which represented in congestion and hemorrhages on both tissues

Histopathological evaluation:

The histopathology of the tracheal sections in birds of the challenged group (A) showed mucosal hyperplasia. This microscopic lesion of mucosal hyperplasia was reduced in group (B) Mentofin^® treated and MG challenged as shown in (fig 4a-5a).

Tracheal sections  with mucus accumulation in bird challenged group (A) versus clear tissue in tracheal sections of birds treated with Mentofin^® and MG challenged group (B) are shown in (figs 4b-5b).

Tracheal deciliated tissue from a MG challenged bird deprived of Mentofin^® treatment in group (A). There was an apparent reduction in tracheal deciliation in MG challenged and Mentofin^® treated (group B) as shown in (fig 4c-5c).

The inflammatory cells infiltration in lamina propria of tracheal mucosa was reduced in MG challenged and Mentofin^® treated group (B) in comparison to group A- challenged bird deprived of Mentofin^® treatment as shown in (fig 4d-5d).

Trachea with goblet cell-hyperplasia was observed in group A, (MG challenged and deprived of Mentofin^®) which was absent in Group B, (MG challenged and Mentofin^® treated) as trachea appeared normal with normal goblet cells (fig 4e-5e).

Normal tracheal tissue was showed in group C (control group, not challenged and not treated).

Histopathological examination of the lung revealed marked congestion and pneumonic foci in birds of the challenged group (A), Mentofin^® caused relieve somewhat histological lesions in lung from mild to no congestion and no pneumonic foci in MG challenged and Mentofin^® treatment group (B) as in (fig 4f-5f).

Normal lung tissue was showed in group C (control group, not challenged and not treated)

Fig. (4a): Tracheal section showing mucosal hyperplasia in group A (MG challenged and Mentofin deprived).

Fig. (4b): Tracheal section showing accumulations of mucus in tracheal lumen in group A (MG challenged and Mentofin deprived).

Fig. (4c): Tracheal section showing deciliation in group A (MG challenged and Mentofin deprived).

Fig. (4d): Tracheal section showing high inflammatory cells infiltration in group A (MG challenged and Mentofin deprived).

Fig. (4e): Tracheal section showing hyperplasia in goblet cells in group A (MG challenged and Mentofin deprived).

Fig (4f): Lung section showing multiple pneumonic foci and congestion in group A (MG challenged and Mentofin deprived).

Fig. (5a): Tracheal section showing absence of mucosal hyperplasia in group B (MG challenged and Mentofin treated).

Fig. (5b): Tracheal section showing clear of mucus in tracheal lumen in group B (MG challenged and Mentofin treated).

Fig. (5c): Tracheal section showing normal ciliated in group B (MG challenged and Mentofin treated).

Fig. (5d): Tracheal section showing low inflammatory cells infiltration in group B (MG challenged and Mentofin treated).

Fig. (5e): Tracheal section showing goblet cells within normal in group B (MG challenged and Mentofin treated).

Fig. (5f): Lung section showing no pneumonic foci and no to mild congestion in group B (MG challenged and Mentofin treated)

DISCUSSION

In this study the percentage of isolation and identification of Mycoplasma gallisepticum of collected samples (112 lung and trachea) was 5.36 % (6 out of 112samples). These results accepted with those results of Shaker (1995) who found that the overall isolation rate was 4.04% for M.gallisepticum, On the other hand our results do not go hand by hand with those of Stalkencht et al. (1998) they could isolate M.gallisepticum from naturally infected chickens through the classical methods at rate of 30%. This confusion may be due to several causes such as number of birds, media used, cultivation and age of birds.

Molecular techniques, such as polymerase chain reaction (PCR), are established and are sensitive, fast and highly specific methods for the detection of Mycoplasma (Garcia et al., 2005). Several PCR primers targeting different genes of MG were described previously (Collett et al., 2005) in which MGc2 one is preferable.

The percentage of samples detected by using MGc2gene was (46.88%) 15 out of 32 samples. This result in agreement extent with Gondal et al. (2015) who detected of the nucleic acid of MG by using PCR from tracheal tissue 42.47% by using specific primer of MG. On other hand this result confuses with Eissa et al. (2008) who used PCR identification of the obtained isolates of 5 MG isolates from broiler chickens (14.25%).

In an examined trial for testing the effect of Mentofin^® in reduction of the lesions of MG infection, the challenged group (A) exhibited  dullness, depression  with ruffled feather and various respiratory signs and there were serious involvement of trachea, lungs, air sacs in postmortem examination in opposite to the control group C (not challenged and not treated) that did not exhibit any clinical and pathological changes  and similar results reported by Islam et al. (2011).

The histopathological changes of trachea in the challenged (group A), were in the form of deciliation, mucosal and goblet Cell hyperplasia, mucus accumulation, and inflammatory cells infiltration. Our results are in concordance with Barbour et al. (2006); Khairy et al. (2012) and Stipkovits et al. (2012), and the pulmonary histopathological changes, were in the form of congestion, pneumonic areas, the obtained results are in agreement with those mentioned by Eissa, (2001); Dardeer et al. (2004) and Ahmed (2005). Furthermore, Stipkovits, (1995) mentioned that the Mycoplasma enters the respiratory tract and attaches to the cilia and the surface of the epithelial cell lining of the respiratory tract. The Mycoplasma produces various toxic metabolites, besides the depletion of the amino acids, fatty acids and DNA precursors. Such products disturb the normal function of the epithelial cells of the mucous membrane of the respiratory tract. A decrease of the excretion of mucus and motility of the cilia or even their destruction can be observed. These factors help the Mycoplasma to move down to the lungs and air sacs causing their damage and Volatile oils that existed in Mentofin could be recommended as an alternative natural safe way to the drugs in controlling MG infection to overcome the problems of drug resistance and the common drug tissue residues under our field condition  (El-Ghany, 2008).

The mechanism by which the volatile oils can relief the respiratory sings was explained by Page, (2004); Zakay-Rones et al. (2004) and Salari et al. (2006). Those authors suggested that the active ingredients of Eucalyptus spp can protect the first line of defense in the poultry host through the thinning of the mucus in the respiratory tract which could help in its outward flow, pushing with it the microorganisms, preventing their colonization, and thus protecting the cilia from consequent damage.

Concerning to the histopathological findings in group B (which challenged and treated with Mentofin^®), revealed the effectiveness of Mentofin^® in comparison to group A. Improving in cilia and decreasing hyperplasia in mucosa and goblet cell and clear mucus and low inflammatory cells infiltration were observed of trachea. These results are in accordance with Barbour et al. (2006). Who evaluated the histopathological changes of eucalyptus and peppermint oils treated versus deprived broilers subjected to three different natures of challenges (MG, H9N2, and a combination of MG/H9N2) they found that this treatment resulted in significant decrease in tracheal deciliation in MG and MG/H9N2-challenged birds, significant decrease in tracheal goblet cells degeneration in MG-and MG/H9N2-challenged birds, significant decrease in tracheal mucus accumulation in MG-challenged birds, and significant decrease in heterophil infiltration in MG/H9N2- challenged birds. The previously mentioned authors detected that there is synergism among the active ingredients of Eucalyptus spp. And peppermint for providing protection of the goblet cells in the upper respiratory system.

The  results concerning to group B in comparison to group A may be presumed due to the action of volatile oils in liquefaction and loosening of the respiratory thick sticky exudates which could be expelled by the birds and consequently reduced the hypoxia, improved breathing and finally increased the feed intake and body weights. In the present study we found that Mentofin^® in group B caused relieve to some extent tohistopathological lesion in lung.

ACKNOWLEDGMENT

The authors would like to thank Prof. Dr/ Awad Abd El-Hafez Ibrahim, Department of Avian and Rabbit Diseases, Faculty of Veterinary Medicine, Assiut University, for his sincere revision of the paper.

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