ROLE OF ESSENTIAL OIL FOR CONTROL OF AVIAN ASPERGILLOSIS IN EXPERIMENTALLY INFECTED CHICKENS

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

ROLE OF ESSENTIAL OIL FOR CONTROL OF AVIAN ASPERGILLOSIS IN EXPERIMENTALLY INFECTED CHICKENS

YASMIN SADIEK; T.Y. ABD EL MOTELIB and OMAR AMEN

Department of Avian and Rabbit Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt

Received: 4 August 2019;     Accepted: 10 September 2019

INTRODUCTION

Aspergillosis is the most common opportunistic mycotic infection of respiratory tract in birds causing high morbidity and mortality thus inducing significant economic losses especially in poultry (Tell, 2005).

Aspergillosis can be acute or chronic form. Acute aspergillosis generally occurs in young birds and resulting in high morbidity and mortality. The chronic form is sporadic and it causes lesser mortality and generally affects older birds, especially a compromised immune system due to poor husbandry condition. Inhalation of A. fumigatus asexual spores (conidia) can cause a spectrum of clinical manifestations depending on the immunological status of the host (Ben-Ami et al., 2010; McCormick et al., 2010).

Although aspergillosis is predominantly a disease of the respiratory tract, all organs can be infected, leading to a variety of manifestations ranging from acute to chronic infections. It is trusted that impaired immunity and the inhalation of a considerable amount of spores are important causative factors (Beernaert et al., 2010).

Corresponding author: Dr. Yasmin Sadiek

E-mail address: yasminali24@yahoo.com

Present address: Department of Avian and Rabbit Diseases, Faculty of Veterinary Medicine, Assiut University, Egypt

Aspergillosis in poultry is caused by a fungal species under the genus Aspergillus. Organisms cultured from affected organ in decreasing frequency are: Aspergillus fumigates, A. flavus, A. niger, A. glaucus and A. terreus. From these spp, A. fumigatus is a common cause of the disease (salem and ali, 2014).

Treating fungal infections is a challenging task due to the limited number of effective antifungal drugs, the emergence of drug resistance and elevated renal and liver toxicities. Current treatments include the synthetic azoles (e.g. fluconazole and flucytosine) or the natural polyene amphotericin B (AmB). However usage is becoming limited by resistance development to the azoles, and acute toxicity of AmB. Also, the burden of antifungal resistance is becoming a major concern, and has thus intensified the search for new, safer, and more efficacious agents to combat serious fungal infections (Shreaz   et al., 2016).

Essential oils can represent one of the most promising natural products for fungal inhibition, many kinds of EOs obtained from different plants or herbs exhibited intense antifungal properties (Nazzaro et al., 2017).

EOs’ antifungal activity may be due to the inhibition of ergosterol biosynthesis and the disruption of membrane integrity and cell wall, which prompted the morphological deformations, collapse and deterioration of the conidia and/or hyphae (Pianalto and Alspaugh, 2016).

The fungitoxic properties of essential oil extracted from Cinnamon (Cinnamomum zeylanicum Breyn) bark have been investigated against five species of Aspergillus spp A. flavus, A. fumigatus Fresenius, A. nidulans, A. niger and A. terreus (Srivastava, 2015).

Cinnamon oil strongly inhibited the radial mycelial growth of Aspergillus species and associated with morphological changes as decreased condition, leakage of cytoplasm, loss of pigmentation and disrupted cell structure indicating fungal wall degeneration (Carmo et al., 2008).

To our knowledge, no published data exist for examining Cinnamon oil treatment efficacy for avian aspergillosis in experimentally infected chickens. Therefore, the objectives of this study were (i) evaluation of the in vitro antifungal activity of EOs and antifungal drugs against A. fumigatus, flavus, niger (ii) assessment of cinnamon prophylaxis and treatment efficacy in an experimental avian aspergillosis model.

MATERIALS AND METHODS

Experimental inoculum

18 isolated strains (6 A.fumigatus, 6 A.niger, 6 A.flavus) were used for antifungal assay, all 18 strains of avian species with clinical aspergillosis (lungs and air sacs).

Spore suspensions of A. fumigatus, A.flavus, A.niger were prepared in sterile 0.85% NaCl with Tween 20 from fresh colonies grown on Sabouraud dextrose agar at 35 °C for 5 days, Cell concentration was adjusted to final concentrations of 0.4 x 10⁴ to 5 x 10⁴. These suspensions were used directly for the inoculation (Espinel-Ingroff et al., 2007).

Screening for antifungal activity of antifungal drugs and EOs in vitro

Disc diffusion test

M 44-A (CLSI/NCCLS 2004); (Espinel-Ingroff    et al., 2007)

A sterile swab was dipped in the suspension and the excess fluid drained by rotating the swab against the inside of the container, Potato dextrose plates was inoculated from the same suspension, with a fresh swab with a rotary movement. The plates were incubated at 35°C for 18–24 h, read, and then incubated for a further 18–24 h to determine the optimal incubation time. Zone sizes were measured in millimeters with calibers.

 The 18 isolated strains (6 A.fumigatus, 6 A.niger, 6 A.flavus) were tested against 5 anti-fungal discs of commonly used chemotherapeutic agents from Oxoid® Hampshire, UK: Nystatin (NY) 100 I.U, Amphotericin B (AMP) 20 μg, Ketoconazole (KTC) 10 μg, Itraconazole (ITR) 10 μg and Fluconazole (FLU) 25 μg. The following tentative zone diameter categories were assigned: susceptible, ≥17 mm (azoles and nystatin) and ≥15 mm (amphotericin B (AP)); intermediate, 14–16 mm (azoles and nystatin) and 13–14 mm (AP); resistant, ≤13 mm (azoles and nystatin) and ≤12 mm (AP).

Agar well diffusion testBalouiri et al. (2016) and Tartor and Hassan (2017)

Garlic, thyme, cinnamon, tea tree, clove and chamomile oils (ELCaptain Company and rose monde company) were screened for antifungal activity using agar well diffusion method.

The agar plate surface is inoculated by spreading 1ml of the microbial inoculum (5 x 10⁴) over the entire agar surface, then, a hole with a diameter of 6 to 8mm is punched aseptically with a sterile cork borer or a tip, and a volume of 50 µl of each essential oils dissolved in DMSO is introduced into the well, then the agar plates are incubated for 48 hour at 37 °C. Zone sizes were measured in millimeters with calibers. Its considered positive when ≥ 10 mm.

Evaluation of cinnamon oil efficacy against A.fumigatus experimentally

A total of 65-0ne day old chicks were used, 5 killed at the beginning of the experiment (pre-inoculation control) lung sections were cultured on SDA plates incubated at 37 °C and checked for A.fumigatus, the remaining chicks were randomly assigned into 4 equal separated groups.

Chicks in groups 1, 2 (at 3-day-old) were inoculated intratracheally with 1ml A.fumigatus spore (2,7×10⁶) detected by spectrophotometer (Hereba   et al., 2016), chickens in group 2 were treated with (1 ml / kg diet)cinnamon oil (the most effective in vitro sensitivity) and treatment started at the day of infection and continued for 15 days. Group 3, chicks were taken cinnamon oil (1ml / kg diet) for 15 day. Group 4, served as uninfected untreated control (negative control). All clinical signs, postmortem lesions and mortalities were recorded. At the end of the experiment, the remaining chicks were sacrificed and subjected to post mortem and mycological examination.

RESULTS

Table 1: Sensitivity of A. fumigatus strains to antifungal drugs.

A.Fumigatus isolates showed 100 % resistant to Fluconazole, 67% resistant to both Nystatin and Amphotericin B while 50 % resistant to Itraconazole and only sensitive to Ketoconazole with 67%

Figure (1):  Degree of sensitivity of Aspergillus fumigatus to antifungal drugs – disk diffusion method

Table 2: Sensitivity of A. flavus strains to antifungal drugs.

A.flavus isolates showed 100 %resistant to Fluconazole, Itraconazole, Nystatin and Amphotericin B while 100% susceptible to Ketoconazole.

Figure (2):  Degree of sensitivity of Aspergillus flavus to antifungal drugs – disk diffusion method.

Table 3: Sensitivity Susceptibilities of A. niger strains to antifungal drugs.

A.niger strains isolates showed 100 %resistant to Fluconazole and Itraconazole while 50 % resistance to Nystatin and Amphotericin B while only susceptible to ketoconazole with 83%.

Figure (3): Degree of sensitivity of Aspergillus niger to antifungal drugs – disk diffusion method.

Results of sensitivity of fungiisolates to different essential oils (n=6):

Sensitivity of A. fumigatus strains to essential oils

Cinnamon oil, tea tree oil and clove oil displayed antifungal activity for A.fumigatus isolates, with the highest efficacy by Cinnamon oil. Cinnamon oil showed the highest inhibition zone diameter (27 ±2 mm) followed by tea tree oil (13 ±2 mm), and clove (12.5 ±1 mm). Garlic oil has moderate activity (10 ±1 mm), thyme oil and chamomile oil did not exhibit any antifungal sensitivity for A.fumigatus isolates. Cinnamon oil was chosen for experimental trial in vivo based on the obtained antifungal activity results.

Sensitivity of A. flavus strains to essential oils

Cinnamon oil, garlic oil and clove oil displayed antifungal activity for A.flavus isolates, with the highest efficacy by Cinnamon oil, which showed the highest inhibition zone diameter (27.8 ±3 mm) followed by garlic oil(12.3±2 mm), and clove oil (10 ±2 mm), chamomile oil, thyme oil and tea tree oil did not exhibit any antifungal sensitivity for A.flavus  isolates.

Sensitivity of A. niger strains to essential oils

Cinnamon oil, clove oil and garlic oil displayed antifungal activity for A.niger isolates, with the highest efficacy by Cinnamon oil, which showed the highest inhibition zone diameter (26.8 ±1 mm) followed by clove oil (14.5 ±1.5 mm), and garlic oil (11.8±2 mm), chamomile oil has weak activity (9.7±2 mm), thyme oil and tea tree oil did not exhibit any antifungal sensitivity.

Figure(4): Agar well diffusion test showing susceptability of A.niger to cinnamon oil and clove oil.

Results of Experimental design:

Clinical signs: (Group 1): Two days after the inoculation, general and respiratory signs of aspergillosis were observed. Clinical signs included ruffled feathers, gasping, dyspnea, dullness, greenish watery diarrhea, loss of weight, cloudy eye and anorexia. Deaths were recorded starting from the third day of the inoculation. All chicks in infected untreated group (group 1) died within 15 days post the inoculation. While in infected and treated group (group 2), 10 chicks (66.7%) survived and no deaths or clinical signs were occurred after day 8 post inoculation.

(Groups 3, 4): Neither death nor clinical signs of aspergillosis were observed in both treated and control groups.

Post mortem: The birds in all groups were necropsied and the most common gross findings were noticed in Group (1). There were whitish cheesy materials of fungal growth appeared dispersed in the air sacs and lung tissue. Marked congestion of the lungs and thickening of the air sac membranes occur were observed. Miliary necrotic foci develop in lungs around the sites of fungal growth and result in the formation of nodules. These nodules were discrete, circumscribed, and white in color and randomly distributed throughout the lung tissue and air sac, myocardium, thoracic wall and abdominal serosa, some cases showed congested kidney. (Group 2): This lesion observed in only 5 dead birds from treated group and disappeared after 8 day post infection (day 10). (Group 3, 4): no post mortem lesions were observed.

In (Group 1): the inoculated A.fumigatus was re-isolated from the lungs, air sacs from all infected chicks, while in (Group 2): the inoculated A.fumigatus was re-isolated from the lungs, air sacs from the only five dead birds and re-isolation was unsuccessful in the survivor chicks.

Table 4: Mortality percent in the four groups.

Figure (5): Air sacs are thickened and lung tissues contain a lot of Miliary necrotic foci around the sites of fungal growth.

Figure (6): lung showing discrete, circumscribed, white in color and randomly distributed nodules throughout the lung tissues.

Figure (7): Air sacs are thickened and coverd with small whitish cheesy materials of fungal growth.

DISCUSSION

In recent years, a fungal infection have emerged as a world-wide health problem and has become an important cause of respiratory infection in poultry owing to extensive use of broad spectrum antibiotics, corticosteroids and increasing population of terminally ill and debilitated patients which trigger an interest to examine the source and reservoir of such fungi. Among the infectious diseases fungal diseases have their own importance and seem to be one of the great obstacles for the poultry farmers in the form of high morbidity, mortality and production losses Asfaw and Dawit (2017).

In vitro sensitivity of our fungal isolates to 5 antifungal drugs showed that A.Fumigatus isolates showed 100 %resistant to Fluconazole, 67% resistant to both Nystatin and Amphotericin B while 50 % resistant to Itraconazole and only sensitive to Ketoconazole with 67%. Our results agreed to some extend with those reported by Ziółkowska et al. (2014) and Tartor  and Hassan (2017) in case of Fluconazole, Amphotericin B and Itraconazole, except in case of ketoconazole who found that most strain of A.fumigatus was resistant to it.

Our isolates of A.flavus showed 100 % resistant to Fluconazole, Itraconazole, Nystatin and Amphotericin B, while 100% susceptible to Ketoconazole. These results were totally in agreement with Kumar et al. (2010) in case of Fluconazole and Ketoconazole but totally disagreed in case of Itraconazole and Amphotericin B, this may be due to the difference in countries.

Aspergillus.niger strains isolates showed 100 % resistant to Fluconazole, Itraconazole, 50 % resistance to Nystatin and Amphotericin B while only susceptible to ketoconazole with 83%. These results accepted with those mentioned by Tokarzewski et al. (2012). It can be assumed that as in the case of other Aspergillus species, the effectiveness of antifungal agents may depend not only on the type of fungus, but also on the site of isolation (environment, patient), and even the species of the host (human, animal, bird) (Tokarzewski et al., 2012).

In vitro sensitivity of our fungal isolates to 6 EOs showed that in case of A.fumigatus: cinnamon oil, tea tree oil and clove oil displayed antifungal activity for A.fumigatus isolates, with the highest efficacy by cinnamon oil, which showed the highest inhibition zone diameter (27 ±2 mm) followed by tea tree oil (13 ±2 mm), and clove (12.5 ±1 mm). Garlic oil has moderate activity (10 ±1 mm), thyme oil and chamomile oil did not exhibit any antifungal sensitivity for A.fumigatus isolates. These results were in agreement with that reported by Singh et al. (1995); Inouye et al. (2000); Carmo et al. (2008); Rana et al. (2011); Srivastava (2015) and Tartor and Hassan (2017).

Concerning to in vitro sensitivity of A. Flavus to tested EOs: cinnamon oil, garlic oil and clove oil displayed antifungal activity for A.flavus isolates, with the highest efficacy by cinnamon oil, which showed the highest inhibition zone diameter (27.8 ±3 mm) followed by garlic oil (12.3±2 mm), and clove oil (10 ±2 mm), while chamomile oil, thyme oil and tea tree oil did not exhibit any antifungal sensitivity for A.flavus isolates.

These results were similar to that mentioned by Singh et al. (1995); Carmo et al. (2008); Gutiérrez  et al. (2010); Rana et al. (2011) and Srivastava (2015).

Our result in vitro sensitivity of A. niger to tested EOs: cinnamon oil, clove oil and garlic oil displayed antifungal activity for A.niger isolates with the highest efficacy by cinnamon oil, which showed the highest inhibition zone diameter (26.8 ±1 mm) followed by clove oil(14.5 ±1.5 mm), and garlic oil (11.8±2 mm). Chamomile oil has weak activity (9.7±2 mm) while thyme oil and tea tree oil did not exhibit any antifungal sensitivity. These result were in agreement with that illustrated by Tansey and Appleton, (1975); Singh et al. (1995); Carmo et al. (2008); Rana et al. (2011) and Srivastava (2015).

General and respiratory signs of aspergillosis in experimentally infected birds were observed two days after the inoculation in group 1 and in some chicks in experimentally infected and treated birds in group 2. Clinical signs included ruffled feathers, gasping, dyspnea, dullness, green watery diarrhea, loss of weight, cloudy eye and anorexia. These signs were similar to those described by Atasever and Gȕmȕssoy, (2004); Gȕmȕssoy et al. (2004). All chicks in infected untreated group (group 1) died within 15 days after the inoculation, this was similar to that found by Fadl Elmula et al. (1984) and agreed to certain degree with Peden and Rhoades, (1992); Le Loc’h et al. (2006). While in infected treated birds (group 2), 10 chicks (66.7%) survived and no death or clinical signs occurred after day 8 post inoculation ,so the cinnamon oil reduced the mortality rate between the chicks, while in only treated birds (group 3) or control groups (group 4) neither death nor clinical signs of aspergillosis were observed. These was going hand by hand with that mentioned by Symeon et al. (2014) who found that cinnamon oil at the selected concentrations (0.5 and 1 ml/kg feed) may not have the potential to influence mortality rates and Atasever and Gȕmȕssoy (2004) who found that the controled group don’t have any clinical sign or mortality.

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