Volume 8, Number 10, October 2014 (Serial Number 78)

1. Introduction  Mycoplasma infections of avian species are both transmitted vertically, by egg transmission, and

Avian mycoplasmosis is caused by members of horizontally, by direct or indirect contact [2]. Mycoplasmataceae family [1]. Mycoplasma Currently, MG infections are rare except for multi-age gallisepticum (MG) and Mycoplasma synoviae (MS) egg production sites, whereas MS is considered one of are considered the most pathogenic strains. the most frequent pathogenic avian mycoplasmas,

because of its emergence in several countries [3, 4]. It Corresponding author: Nassik Saâdia, Ph.D., research field:

avian pathology. E-mail: snassik@yahoo.com. is an important pathogenic agent of chickens, causing

816 Detection of Mycoplasma synoviae Infection in Broiler Breeder Farms of Morocco Using Serological

Assays and Real Time PCR

air sacculitis and synovitis [5]. The economic losses in principal area of poultry production in Morocco were intensive production are caused by reduced growth

sampled (Fig. 1).

rates, weight loss, condemnation at slaughter because Serological screening and molecular diagnosis were of chronic respiratory disease in broilers and a

conducted as follows:

suboptimal production of egg in layers [6]. First, 20 to 60 one day old chicks imported

Screening of poultry farms for pathogenic from Europe were sampled, before being placed in mycoplasma infections is usually accomplished with

the farms. Then, every selected farm was visited at 8, serological tests, especially Rapid Slide Agglutination

16, 32 and 56 weeks from introduction to collect (RSA) and enzyme-linked immunosorbent assay

60 blood samples and 60 tracheal swabs. The (ELISA), which can detect specific antibodies. sampling of the chickens was carried out from a single However, serological tests may be insensitive or may

randomly selected house of each farm. During this give false-positive reactions. In fact, nonspecific

investigation, all birds were apparently healthy and not reactions (false positive) were frequently observed by

vaccinated against MS; Birds were bled from the RSA, especially in flocks vaccinated with cutanea ulnae (wing) vein. Samples (blood and tracheal oil-emulsion vaccines (of tissue culture origin) against

swabs) were transported under cold conditions for various agents [7]. Commercial ELISA kits are

analysis in the Laboratory of Avian Pathology Unit at available and are often used to detect antibodies to

the Agronomy and veterinary Institute Hassan II in MG and MS. ELISA detects basically Ig-Y class

Rabat, Morocco.

antibodies, whereas RSA detects IgM. In general, After coagulation during 2 h at room temperature, ELISA tests are slightly less sensitive but more

sera were collected and sterile aliquots were specific than RSA [8]. Moreover, certain commercial

maintained at 4 °C until use within the following 48 h. indirect ELISA kits give false positive reactions with

The tracheal swabs were stored at -20 °C until analysis. sera from chickens vaccinated with oil-emulsion

2.2 Serology

vaccines, probably due to the presence of Serum Albumin in ELISA antigens [9].

Antibodies to MS were detected by RSA and Isolation and identification of mycoplasmas Indirect ELISA (iELISA):

antigens is still the gold standard method for diagnosis

2.2.1 Rapid Slide Agglutination Test and PCR is a rapid and sensitive test. Nevertheless, it

Sera were tested according to the OIE protocol [11], is possible to find some nonspecific reactions with

using BIOVAC antigens©, MS (strain WVU-1853 PCR, i.e., contamination with DNA resulting with

colorful).

false negative reactions due to inhibitors in clinical To avoid the false positive results caused by samples examined by PCR [10].

cross-reactions with other pathogenic mycoplasmas [7, The aim of this study was to evaluate the

12] or non-specific reactions induced by some vaccines, prevalence of MS infection in broiler breeder flocks in

the positive sera were diluted to 1/5 and inactivated at Morocco at different ages and to compare serological

56 °C during 30 min [11].

tests and molecular diagnosis.

2.2.2 ELISA

2. Materials and Methods

Sera were analyzed using the iELISA kit manufactured by IDEXX© “IDEXX MS Ab test”,

2.1 Sampling Procedure according to the manufacturer’s instructions.

The study was conducted from October 2011 to Following the French measures the threshold for April 2013. Eleven broiler breeder flocks located in the

seropositivity was set at 5% [13].

Detection of Mycoplasma synoviae Infection in Broiler Breeder Farms of Morocco Using Serological

Assays and Real Time PCR

Fig. 1 Map of Morocco showing farm’s localization.

Molecular Biology Analysis: DNA from five technologies™ (Life Technologies™, Foster city, CA) pooled tracheal swabs was extracted using the Pure

according to the manufacture’s protocol. Link® Genomic DNA Mini Kit (Invitrogen™ by Life

3. Results

technologies™, Foster City, CA) according to manufacturer’s instructions. Real time PCR reactions

3.1 Serology

were conducted on a 7500 Fast Real Time PCR

3.1.1 RSA

system (Applied Biosystems ® by Life Technologies™, One day old chicks were negative to MS by

Foster City, CA) using VetMax TM —Plus qPCR master RSA (Table 1). From the 8th week of age, the

mix with TaqMan Probe and primers designed by Life seroprevalence found by RSA was variable. In fact, 6

818 Detection of Mycoplasma synoviae Infection in Broiler Breeder Farms of Morocco Using Serological

Assays and Real Time PCR

Table 1 Prevalence of Mycoplasma synoviae infection estimated by RSA, iELISA and real time PCR.

Breeder farm Test

56th week RSA (%)

One day old

8th week

16th week

32nd week

0 10 41.6 51.5 63.3 A iELISA (%)

75 50 34 B iELISA 75

36.6 64.7 41.4 43.7 PCR 0 0

0 8.3 8.3 RSA 0 0

26.8 100 67.8 C iELISA 12.5 91.6

21.6 58.2 90 D iELISA 60.6 63.3

23.3 72.5 78.9 E iELISA 16

56.6 53.3 12 100 PCR 0 0

0 100 100 RSA 0 0

0 73.3 15 F iELISA 53.3 40

35.2 18 94.3 PCR 0 0 8.3 8.3 100 RSA 0 6.6

100 14 G

iELISA 82.5 100 97 100 Not tested PCR 0 8.3

100 100 RSA 0 15

38.3 66.6 98.3 H iELISA 85.7 13.3

17.3 100 100 PCR 0 0

0 100 100 RSA 0 28.3 36.6 83.3 63.6 I iELISA 16.6 96.6

iELISA 91.5 71.6 41.6 100 Not tested PCR 0 0

0 91.7 RSA 0 0

10 86.4 98.3 K

iELISA 48.3 40 38 15 100 PCR 0 25

8.3 0 100 RSA: Rapid Serum Agglutination iELISA: Inderict Enzyme-Linked ImmunoSorbent Assay

farms became seropositive at this time. The infection

3.1.2 iELISA

rate was situated between 6.6% and 28.3%. At the Variable levels of (Ig-Y) antibodies were detected 16th week, IgM class antibodies were detected in 10

by iELISA in one day old chicks, with seroprevalence farms. At the 32nd week, all farms became positive

varying between 12.5% and 91.5%. (Table 1). All farms with a seroprevalence over 50%. The prevalence noted were positive by iELISA from 8 to 56 weeks (Table 1).

by RSA increased with the age and followed an The average of Ig-Y’s titer recorded by iELISA was exponential trend in 6 farms; however it decreased in

2540 for all flocks on one day old chicks to reach

5 others (Table 1). 9447 at 56 weeks of age (Fig. 2).

Detection of Mycoplasma synoviae Infection in Broiler Breeder Farms of Morocco Using Serological

Assays and Real Time PCR

RSA, PCR and iELISA results

0% One days old

8th week

16th week

32nd week

56th week

RSA

PCR

Average of Ig Y's titers (iELISA)

Fig. 2 Percentage of positive farms and antibody titers recorded by iELISA by age group.

3.1.3 Real Time PCR age of birds (Fig. 2). Despite the disagreement noted The age of detection of MS infection by PCR was

between RSA and real time PCR at 8 (6 variable and increased following the age of birds. In

disagreements), 16 (5 disagreements) and 32 weeks (2 fact, all samples of one day old chicks were negative

disagreements), a perfect agreement was noted in the to MS by real time PCR, but at 8 weeks of age 4 farms

one day old chicks and at 56 weeks of age. became positive and the infection rate was situated

3.2.1 Discussion

between 8.3% and 33.3%. At the 16th week the RSA and iELISA used for serological screening in number of positive farms had increased to reach 7

this study are most commonly used for the diagnosis farms, with a variable rate of infection (8.3% to

of avian mycoplasmosis. They are generally 100%). At the 32nd week of age 9 farms became

recommended for monitoring flocks rather than testing positive and at the end of this survey (56th week) all

individual birds [11] because of lack of specificity farms were positive by PCR, with rates of infection

and/or sensitivity [7]. Results obtained show that one exceeding 90% except on farm with a low rate

day old chicks were negative to MS by RSA, with a infection (8.3%) (Table 1).

maternal stock of antibodies detected by iELISA. The disagreement between serological assays may be due

3.2 Concordance Between RSA and PCR Results to the fact that these serological techniques recognize

The results obtained by RSA and PCR showed that two different immunoglobulin isotypes: IgM, which is the number of positives farm to MS increased with the

detected by the RSA, appears within 2 to 3 d after

820 Detection of Mycoplasma synoviae Infection in Broiler Breeder Farms of Morocco Using Serological

Assays and Real Time PCR

infection [14]. The concentrations of IgM and IgA in infectious bursal disease virus (IBDV) vaccine, their the albumen of chicken and turkey eggs are much

sera reacted in immunoblots with calf serum albumin lower than those of Ig-Y in the yolk [15]. Ig-Y, which

(SA), swine and horse serum and sometimes with is detected by iELISA, appears later than IgM and it

Ig-Y from swine and horse sera. Certain indirect penetrates easily through the epithelium of the oviduct

ELISA kits were found to yield false positive to accumulate in the yolk forming the bulk of

reactions, probably due to the presence of SA in maternally derived antibodies in infected or ELISA reagents. vaccinated chickens [16, 17].

Ig-Y titers increase with age (Fig. 2), confirming PCR analysis showed that one day old chicks were

the persistence of MS infection in the investigated negative to MS. This result confirms the study

farms.

conducted by Bencina et al. [18]. In fact, maternal The present study has revealed that the prevalence antibodies can have an important protective role,

of Mycoplasma synoviae infection in Moroccan because of their presence in compartments that can be

broiler breeder flocks is very high (100% at the age of invaded by vertically transmitted M. synoviae and M.

56th week) in comparison with results obtained by galisepticum [18].

Amaqdouf in Morocco in 2005 [21] (66.7 %). At 8 weeks of age MS infection was detected in 55%

The prevalence in other countries seems to be lower. of the farms by RSA test, and it reached 100% at 32

Actually, the prevalence reported in the Netherlands is weeks. The same profile was found by PCR with

25% [22], in Algeria (20%) [23]. These results may be some discordance. In fact, at 8 and 32 weeks only 36% explained by the absence of vaccination against MS and 82% of farms were positive respectively by PCR.

and the lack of specific measures to control this The important disagreement noted between RSA

infection in broiler breeder flocks in Morocco.

and PCR at 8 and 16 weeks may due to false-positive 4. Conclusions

reactions observed by RSA, because of cross In conclusion, MS infections become very frequent

reactivity reported for M. gallisepticum (MG) and M. and widespread among poultry flocks in Morocco.

synoviae (MS) antigens as described by Kleven et al. Serological assays are useful for a regular monitoring,

[19], or to the false-negative because of lack of but must be combined with direct detection by PCR

sensitivity of Real time PCR, to detect MS DNA due for confirmation. The identification of the risk factors to the presence of inhibitory in tracheal swabs [20]. related to the propagation of MS infection in breeder At 56 weeks, all farms became positive to MS. This flocks will be essential for the development of may due to the lack of biosafety measures and hygiene

appropriate control programs.

at the end of the production period. Results obtained by iELISA confirm the infection

Acknowledgment

of broiler breeder flocks by MS, during the present This present research was supported by FISA survey. However, these results may partly be due to (Federation Interprofessionnelle du Secteur Avicole). false positive reactions caused by cross-reacting The authors are thankful to Dr. DUCROTOY.M.J

pathogens or vaccines; especially as noted that during from University of Edinburg for reading the the first 8 weeks, all the broiler breeders flocks

manuscript.

sampled in this study had been vaccinated against infectious bursal disease virus (IBVB). In fact,

References

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Diseases of Poultry, edited by Saif, Y. M., Barnes, H. J.,

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Assays and Real Time PCR

Glisson, J. R., Fadly, A. M., McDougald, L. R., and couver, Ministère de l’agriculture et de la pêche, Swayne, D. E. Ames: Iowa State Press, 756-766.

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Drug-treated Chickens: Comparison of Diagnosis H., and Markham, P F. 2000. Report for the Rural

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[4] Vienot, E. 2008. “Pathologies Résurgentes et Nouveaux [15] Dohms, J. E., Saif, Y. M., and Bacon, W. L. 1978. Tests PCR.” Filières Avicoles 706: 76-79.

“Metabolism and Passive Transfer of Immunoglobulins [5] Lockaby, S. B., Hoerr, F. J., Lauerman, L. H., and Kleven,

in the Turkey Hen.” American Journal of Veterinary S. H. 1998. “Pathogenicity of Mycoplasma synoviae in

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“Immunoglobulin Classes in the Hen’s Egg: Their Poultry Mycoplasma Workshop-Histopathology.

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Palmipèdes: Approche Moléculaire Appliquée au Gallisepticum and Potential Causes of False Positive

Diagnostic et L’épidémiologie du Goose Hemprrhagic Reactions in Avian Mycoplasma serology.” Zentralblatt

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Thèse de Doctorat de l’université de Toulouse. [8] Ley, D. H. 2003. “Mycoplasma gallisepticum Infection.”

[18] Bencina, D., Mojca, N., Bidovec, A., and Rojs, O. Z. In Diseases of Poultry, edited by Saif, Y. M., Barnes, H.

2005. “Transfer of Maternal Immunoglobulins and J., Glisson, J. R., Fadly, A. M., McDougald, L. R., and

Antibodies to Mycoplasma gallisepticum and Mycoplasma Swayne, D. E. Ames: Iowa State Press, 722-744.

synoviae to the Allantoic and Amniotic Fluid of Chicken [9] Avakian, A. P., and Kleven, S. H. b. 1990. “The Humoral

Embryos.” Avian Pathology 34 (6): 463-472. Immune Response of Chickens to Mycoplasma

[19] Kleven, S. H., Jordan, F. T. W., and Brabury, J. M. 2000. gallisepticum and Mycoplasma synoviae Studied by

“Avian Mycoplasmosis (Mycoplasma gallisepticum).” In Immunoblotting.” Vet. Micro. 24: 155-169.

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Paris: Office International des Epizooties. Diagnosis and Epidemiological Investigations of Avian

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[11] Manuel Terrestre de L.oie. 2008. “Avian Mycoplasmosis [21] Amaqdouf, K. 2005. “Enquête Sérologique (ARL) et (Mycoplasma gallisepticum, Mycoplasma synoviae).” In

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Journal of Life Sciences 8 (2014) 822-827

doi: 10.17265/1934-7391/2014.10.005

DAVID PUBLISHING

Prebiotic Isomalto-oligosaccharide Production from Thai Rice

Premsuda Saman, Achara Chaiongkarn, Somporn Moonmangmee and Chantra Poonsiri Bioscience Department, Thailand Institute of Scientific and Technological Research, 35 M3 Technopolis, Khlong 5, Khlong Luang, Pathum Thani 12120, Thailand

Received: May 27, 2014 / Accepted: August 27, 2014 / Published: October 30, 2014.

Abstract: This study aimed to evaluate the potential use of Thai rice for the production of prebiotic isomalto-oligosaccharides through fungal fermentation. Solid-state fermentations of two rice varieties, waxy rice RD6 and non-waxy rice KDM 105 with Aspergillus oryzae TISTR 3108 were compared. The effects of the main parameters such as incubation time, temperature, pH, fungal spore inoculum size and moisture content were also studied individually to maximise the isomalto-oligosaccharides yield. Results showed that the maximum values of amylolytic activity and total reducing sugar were observed when using rice in SSF with initial

moisture content of 70% and inoculated with the inoculum size of 10 7 spores/g. The optimal conditions of SSF were performed at initial pH 6 and 30 °C for 5 d. SSF of waxy rice RD6 with Aspergillus oryzae produced highest concentrations of isomalto-oligosaccharides which consisted of isomaltose, panose and isomaltotriose. After fermentation, mashing was used to further hydrolyse the remaining starch in rice slurry. The subsequent rice syrup contained high amounts of isomaltose, panose and isomaltotriose with the values of 44, 10 and 7 g/L respectively.

Key words: Rice, solid-state fermentation, prebiotic, isomalto-oligosaccharides.

1. Introduction  synthesized from starch [4, 5]. The specific amylolytic

enzyme, α-glucosidase, has been found to possess the Solid-state fermentations (SSF) has been applied in activity of transglucosylation. This enzyme can catalyse the production of many fermented cereal foods. both the hydrolysis of α-D-gluco-oligosaccharides and Various fungi have been used in order to produce transfer of the glucosyl group to 6-OH of other amylolytic enzymes for starch degradation. Possessing glucosyl residues resulting in the synthesis of

a high amylolytic enzyme activity and

isomalto-oligosaccharides [6].

transglucosylation, Aspergillus oryzae has been Isomalto-oligosaccharides have a great potential to determined to be a suitable culture for food and improve the physiochemical quality of many foods as beverages products. A. oryzae biomass is abundant in anti-fading agent for food pigments, as food protein and does not produce aflatoxins or any other antioxidant and as a sweetener. In addition, these carcinogenic metabolites [1]. Furthermore, A. oryzae oligosaccharides have physiological functions such as has been recommended as one of the potential improving of intestinal microflora based on the probiotics [2, 3]. selective proliferation of bifidobacteria stimulation [7, Several commercial oligosaccharides are produced 8]. They are also associated with a lower risk of using fungal amylolytic enzymes. infections and diarrhea, and an improvement of the Isomalto-oligosaccharides which are known as

immune system response [9].

prebiotic branched-oligosaccharides have been This study was designed to investigate the potential

use of rice to produce isomalto-oligosaccharides using Corresponding author: Premsuda Saman, Ph.D., research

field: bioscience. E-mail: Premsuda@tistr.or.th. the process of fungal fermentation. The effects of the

Prebiotic Isomalto-oligosaccharide Production from Thai Rice

main parameters such as rice variety, incubation time,

2.5 Measure of Total Reducing Sugar (TRS) and Free temperature, pH, fungal spore inoculum size and

Amino Nitrogen (FAN)

moisture content were studied individually to The samples of fermented rice were diluted with

maximise the isomalto-oligosaccharides yield. distilled water and analysed for TRS and FAN

2. Materials and Methods

following the methods of Miller [11] and Lie [12] respectively.

2.1 Preparation of Fungal Spore Inoculum

2.6 Enzyme Activity

Spore suspension of Aspergillus oryzae TISTR 3108 was prepared using 0.85% NaCl solution. Each

Crude enzyme from the fermented mass was spore suspension was used as the master suspension,

extracted and measured for amylolytic using the which was appropriately diluted to required density.

Terashima method [13]. The α-amylase activity was Spore concentration in the inoculum was estimated by

measured following the increase of reducing sugars

a haemacytometer. with time [14]. The α-glucosidase activity was

determined using a modified method of McCue and

2.2 Substrate Preparation

Shetty [15].

2.7 Sugar and Oligosaccharide Analysis non-waxy rice KDM 105 was weighed separately into

300 g (on a dry basis) of waxy rice RD6 and

a 2-l Erlenmeyer flasks and distilled water containing The samples of sugars and oligosaccharides were diluted and analyzed by HPLC with ELSD using an

10% (v/v) of supplementing salt solution was added and adjusted to 70% moisture level. The contents of

Inertsil NH 2 column (5 µm, 250 × 4.6 mm, Shimadzu, Japan) maintained at 40 °C. The injection volume was

the flasks were mixed thoroughly and autoclaved at 121 °C for 15 min.

20 μL, and the flow rate 1.2 mL/min. The elution of sugars was carried out with 75% acetronitrile.

2.3 Solid-state Fermentation

3. Results and Discussion

The sterilised solid substrate was inoculated with

3.1 Optimal Conditions of SSF

1% of the prepared inoculum. The contents were mixed thoroughly and incubated at 30 °C. Samples of

Solid-state fermentations of Aspergillus oryzae with triplicate flasks were withdrawn after desired two rice verities, RD6 and KDM 105 as raw materials, incubation. The effects of the main parameters such as

were demonstrated to optimise the time-course of rice variety, incubation time, temperature, pH, fungal

incubation. Initial moisture content of each substrate spore inoculum size and moisture content were

was 70%. All SSF were inoculated with 1% of the studied individually to maximise the prepared inoculum having 10 8 spores/mL and isomalto-oligosaccharides yield.

maintained at 30 °C. Results in Fig. 1 showed that the highest concentration of 639 mg/g TRS was obtained

2.4 Mashing from SSF of RD6 at day 5 while the highest

The fermented mass was mixed into water to form concentration of FAN was obtained from SSF of the slurry of 30% w/v (on a dry basis). 1 L of the

KDM 105. The initial pH decreased from 6.5 to 4.2 in

slurry was added with 0.03 g of CaCl 2 and adjusted to

both SSF through a 7-day period.

pH 6 by using 0.1 M lactic acid. Mashing was carried During fermentation, starch was degraded by

out by following the method of Okafor and Iwouno amylolytic enzymes which produced from fungi to [10].

release smaller sugars and oligosaccharides.

Prebiotic Isomalto-oligosaccharide Production from Thai Rice

RD6

600 120 (U/g )

KDM 105

y g/g

100 it iv

m 400

80 ic act

60 TRS (

40 ylolyt

20 Am

50 ) /g

/g) 20

40 U

g (m 15

30 lase ( N

y FA 10

20 -Am 

2.5 ) g

2.0 pH 5.0

1.5 cosidase(U/

1.0 u -Gl

Time (days)

Time (days)

Fig. 1 Evolution of TRS, FAN, pH, amylolytic activity, α-amylase and α-glucosidase during solid-state fermentations of waxy rice RD6 and non-waxy rice KDM 105 with Aspergillus oryzae at 30 °C for 7 d.

Comparing between two substrates, waxy rice RD6 rice RD6 has a higher potential to be used as the and non-waxy rice KDM 105, significant difference in

substrate of SSF. Besides, the 5-day period was the the levels of amylolytic enzyme and α-glucosidase

optimum incubation time for SSF. The next were observed after fermentation. Results show the

experiment was designed to investigate the other highest concentrations of amylolytic enzyme, optimal conditions of SSF to obtain the maximal α-amylase and α-glucosidase were obtained in SSF of

yields of enzyme activities.

RD6 with the value of 136 U/g, 52 U/g and 2.3 U/g After selecting waxy rice as the substrate, the respectively at the 5th day.

optimal conditions of SSF were investigated for the From these results, it might be concluded that waxy

best yield of enzyme activities. The fungus showed its

Prebiotic Isomalto-oligosaccharide Production from Thai Rice

best performance with the highest amylolytic activity selected and applied in further studies The maximum at the initial moisture content of 70% (Fig. 2a). The

value of amylolytic enzyme was found at pH 6 (Fig. optimal inoculum size was showed at 10 7 and 10 8 2c). The incubation condition was maintained at 30 °C

spores/g (Fig. 2b). Therefore, the 10 7 spores/g was

Fig. 2 Effect of different parameters on amylolytic activity; (a): moisture content, (b): inoculum size and (c): pH.

Prebiotic Isomalto-oligosaccharide Production from Thai Rice

120 /l) (g 100

80 atio tr n 60

ce o n 40

C 20

se

se

se

se

se

se

uco

lto

to al

trio

no

rio

Gl

Ma

lto

Pa

ot

Iso

alt

Ma

om Is