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Biological characterization of the fungus Aspergillus sp found

in the seeds ofCaesalpinia coriaria (Nacascol) and its impact

on the health of the Environment. El Salvador.

Author : ANTONIO VASQUEZ HIDALGO

Teaching Department of Microbiology

Faculty of Medicine

University of El Salvador

Address: final 25 av north University City. San Salvador

Tel (503) 2273-0771 or (503) 2225-1500 ext 4246

E-Mail [email protected]

Summaryobjective: To characterize and identify fungus found in the seeds of Nacascol and its

impact on the health of the environment. Methodology. We proceeded in three phases:

First phase in the collection of seeds nacascol in different areas of the country; Second

phase of tannin extraction and processing of the seed and fungus; Third phase of

laboratory tests that were to spread inoculum of the fungus present in the seed tubes

agar sabouroud microscopy and examine the sample and its relation to lung infections

and allergies in human .Result : From the seeds of nacascol was found on board a

fungus Aspergillus sp influencing acute respiratory infections. We found a new species

differs from otherAspergillus gener. Conclusion. The environmental impact caused by

spores of the fungus Aspergillus contributes to the disability employment and public

spending on health in treating acute respiratory infections. We found a new species of

Aspergillus in El Salvador.

Key words.Aspergillus sp, Nacascol.

INTRODUCTION

In our environment Health is a value-added human capital, not only involves identifying

the environmental risk, but also help solve the problems found, which is a task for

everyone to work together to minimize harm to the environment.


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This time is investigated on a natural plant well known in our area at the National

known as the seed of Nacascol or Nacascolo widely used in leather tanning and pottery

in the area north of the country.

This seed has a particularity required by the inert presence of a fungusAspergillus sp of

the genre, so you can stain along with the properties of mud stained black, the pots and

tanning the skin of cattle. Besides the pots give you the special characteristic of black

mud, which give it a colonial look and beauty to the pots.

JUSTIFICATION. But due to ignorance of science, has found that an apparent injury

to health from the presence of the fungus, and which by negligence or lack of education

ignored the "danger" or risk that holds claim to the spores during the preparation of the

product. The research aims primarily to develop educational levels in future to the

people who handled during the extraction of the product, to protect the environment and

health of the inhabitants engaged in manufacturing work. In many cases this is

diagnosed as acute bronchitis or bronchial asthma is a pulmonary fungal infection, so

that treatment must be an antifungal and not an antibiotic.

Material and Methods

We proceeded in three phases: First phase in the collection of seeds nacascol in different

areas of the country in the northern part of Chalatenango and Morazan. Second phase of

tannin extraction and processing of the seed and fungus; Phase III tests which consisted


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of planting the inoculums of the fungus present in the seed tubes agar sabouroud

microscopy and examine the sample.

Results

Fig 1 the seeds NACASCOL called the tree

whose scientific name is Caeselpinia coriaria,

family Caesalpnieaceae, the genre Caesalpinia,

leguminous plant with stems of 3 to 11 meters in height, with leaves in pairs pinnae 5 to

10 cm long, each with more 10 leaflets from 4 to 8 mm in length and 2 mm thick,

rounded apex, seeds brown-looking black, which grows like a fungus which is the

Aspergillus niger, seen as opportunistic pathogen for humans, whose morphology is not

equal, and whose spores vary with the previous one, but in this case are arranged in the

form of separate spicules across the spore that are coalescing multiple pigmented and

other clear, small diameter, which are black to brown as pigmentation and are not flat

and rough like the kind of niger. To cultivate in a particular place in stain black pigment

production in the tube in the region later in the tubes before 24 o'clock to add to the

medium Agar saboroud more oxidizing substances. Preliminary phytochemical analysis

of the tree Caeselpinia coriaria contains tannins, triterpenes, and flavonoid glycosides.

The fungus is played exclusively on the seeds of the tree Nacascol, which is preparing

for the extraction and preparation of the dye and then in vitro cultivation for the

production of the dye.

The fungus discovered found in the seeds is described like this:

Fig1 . Seeds Nacascol


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Esterigmas. The head of the conidia is black, conidiophores smooth over 1 to 4 mm

with conidia spores or moderate internal, 1 to 3

microns, colored brown to black.

Macroscopic characteristics: Agar colony in

saboraud culture is white then switched to

green in appearance and then radiated is black,

the reverse is yellow or black colours pigment as oxidizing agent, mycelia low altitude,

and

aspect of the colony is dusty colours black.

Microscopic characteristics: smooth conidial head of a round wall, ready to form radial

stipe of smooth and thin wall pronounced, collared brown to black, there is no sign of

gallbladder or columella, there is abundant conidia off of his head, has a row of fialides.

The sporangium is a simple structure globosa peridial of esterigma is black.

Figure 3 The conidiophores are smooth, rough; the spore is in

the form of a racket with spicules. The conidia mature brown

are spherical with projections in the form of triangular peaks

throughout the periphery, abundant and the plants are scarce

forming spicules triangular number greater than 10, the conidia

looks spherical star with spicules that emerge from the ends

filaments arranged in the form of linear chains emerging from the bumps, the whole

structure forms a solid shell. Immature asexual spores are spherical thin wall colorless

large, which is then filled by forming masses of internal brown. The long conidiophores

Fig 3. spore Aspergillus

Fig. 2 . Aspergillus sp. in culture.

40 x.


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is smooth with abundant spores. There are other differences that conidia of the former

because they have a heart to the inside of regular size of a large light blue color that

takes the edge coloring with hyaline single wall that are smaller than the previous ones,

without bumps rough-looking plane number no more than 10, no form spicules.

In general the spore is spherical in shape, aseptic, ameropora, and dark colored type

feospora, large size, and diameter of 1 to 3 microns septet hyphae are continuing with

mycelium. Growing at room temperature or 37 C.

The fig 4 .Growing saboraud Agar plate and

microcultive was found abundant colonies of

white to green radiated that changes color to

black, arranged in radially from the center

outwards, the back of a yellow pigmented black

as use of oxidants.

This strain of Aspergillus found, it is suggested that given their morphological

characterization is another species found in El Salvador deducted by the International

taxonomic classification does not correspond to the species niger.

The remark was made in the procedure of extracting the tannin they occupy in dyeing,

black pottery, finding interviews by the extraction procedure lasts one week, then the

next week the villagers remain prostrate in bed with febrile process, dry cough ,

Lassitude and anorexia, for the handling and aspiration of gases from the burning being

done to fix the dye.

Fig. 4 . Microcultive.


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In Fig. 6 and Fig 7 Image gathered picture is

observed with scanning electron micro photo:

gallbladder and spores of the fungus (6500 x).Can

be noted as reporting CENSALUD "filamentous

fungus, which protrudes from the mycelium

conidial head, rounded. These heads are made up

of a vesicle surrounded by a crown of fialides in the form of a bottle, on whose end is

fixed spores, in this case only shows a not a bonding. Other differences are that the

rough is conidiophores without septa, the spores are in the form of cactus or deck with

triangular spicules dispersed. The gallbladder is round where emerging linear chain of

spores in the form of mallet or bottle with multiple spicules with projections, the

conidiophores are smooth, rough. Phenotypically corresponds to another species of the

genus Aspergillus. For their morphological characteristics.

Fig. 6 Image obtained with

a scanning electron

microphoto: gallbladder

and spores of the fungus

(6500 x). (Courtesy of Dr

Vianey Abrego. Taken

CENSALUD. UES in February

2008.)

Fig. 7. Image obtained with a scanning electron

microphoto: gallbladder and spores of the fungus (10000

x). (Courtesy of Dr Vianey Abrego. TakenCENSALUD.

UES in February 2008.)


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For genotypic studies were found that the genere is presenting the followingAspergillus

DNA sequencing in which the sequence of bases genotypically purics and piridimidics

differentiate between the various species of the genre, as is the chromosome map.

Discussion

Impact on Health

The direct contact of seeds with the presence of the fungus with the human

consequences brings harm to health and outlay of public expenditure in the care of acute

respiratory infections, at the level of care I, II and III of the network of the Ministry of

Health. So that a general fall in the diagnosis of acute respiratory infections without

explaining the cause of their origin.

It has been determined that patients with allergic bronchopulmonary aspergillosis its

causative agent of the genre include species of Aspergillus fumigatus, niger, terreus,

flavus, nidulans. In our environment is the most common genre of the species

Aspergillus niger. (1)

There are several forms of aspergillosis that sick man, among the best known are:

Pulmonary Aspergillosis bronco pulmonary allergic-type: it is due to an allergic

reaction to the fungus by aspiration, which is being developed with bronchial asthma.

Aspergilloma: it is a tumor that develops in a lung lobe, such as tuberculosis or lung

abscess.


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invasive pulmonary aspergillosis: is a serious infection with pneumonic spreads to

other parts of the body. The infection occurs in people with weakened immune systems

due to cancer, AIDS, leukemia, organ transplants, chemotherapy and others. (2,3,5)

Reviewing the literature, we found that the Aspergilloma formed when the fungus

Aspergillus grows as a body cavity in pulmonary (lung) or when the organism invades

previously healthy tissue, causing an abscess. (4,6-9)

Aspergillus, in high concentrations can cause aspergillosis, invasive species cause lung

disorders. The disease appears more often in farmers, as they inhale dust from the

fungus more easily. (4)

The way of playingAspergillus of its spores, which with a size of 2 to 3.5 cm fall easily

into the airways. When these spores germinate, and introduced into a susceptible

individual (atypical asthma, cystic fibrosis) form hyphen that colonize the bronchi. The

hyphen are 3 to 7 cm. in diameter, and are characterized by their branches in 45-degree

angle. (10-13, 1)

The inoculums size is variable; the number of spores inhaled is an important factor in

the production of Aspergillosis, Asthma (atopy), cystic fibrosis and asthma are among

the most frequent outpatient visits. (14)

In general, the fungus Aspergillus is a group of filamentous fungi Deuteromycetes or

imperfect microscopic appearance is typical and is characterized by structures

esporferas or breeding calls conidial head. These heads are made up of a vesicle


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surrounded by a crown of filides in the form of a bottle, in which extreme form chains

of spores in a linear fashion. (15.10)

Rapper Fennell and the classified into 18 groups based on their macroscopic appearance

and the morphological characteristics of the conidiophores and filides; of them, 12 are

related to human disease, the most important:Aspergillus fumigatus, A. flavus niger. (16,

18.)

Other authors believe that theAspergillus is a genus of about 200 fungi. It can exist in

two basic forms: yeast and hyphae. Aspergillus is filamentous (composed of chains of

cells, called hyphae, the opposite type of fungi to yeast, which consist of a single cell

round). (18)

The Aspergillus nigerhas the mycelium wooly white - yellow changing to black, the

reverse is yellowish white, conidiophores long, smooth and filides biseriate completely

covering the gallbladder. (10)

The Aspergillus can cause multiple pathological processes. Among the factors

pathogenicity of the fungus are: For its small size of their conidia permits that are

vacuum cleaners, causing infection in the lungs and sinuses; their ability to grow

quickly to 37 C which makes it ideal to affect human; Its ability to adhere to surfaces

and possibly epithelial and endothelial its strong tendency to invade blood vessels,

producing a large number of extracellular products toxic to cells. (9)

In people who have contact with the Aspergillus, that by its nature is "opportunistic


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pathogen", ie often affects patients with compromised defense mechanisms so that his

injury is very serious.

Among the main symptoms upon Aspergillus, they may be: Chest pain, expectoration

with blood (can be seen by up to 75% of patients), fever, respiratory failure, weight loss,

wheezing, hacking cough. (18)

Among the complications related, are: Difficulty breathing progressive pulmonary

bleeding spread of the infection among others. Aspergillosis is the most common

opportunistic fungal infection of the lung, (5) as in AIDS patients is fulminating. (19)

In Fig. 8 is observed by chest X-ray simple chest of a

patient who presents nodular patterns characteristic of

patients that have been contaminated with the fungus

Aspergillus sp, with clinical manifestations of a

pulmonary aspergillosis and in other cases Aspergilloma

lung.

Clarifies that this diagnosis gives a priori similarities of

acute bronchitis, but that ignorance prescribed to patients in the north are treated with

antibiotics, which really should be an antifungal.

The Aspergillus fungus is widespread in nature that develops in decaying vegetables,

cereal grain, hay, cotton and wool, feathers, to be its ideal, the rooms dark, wet and

Fig. 8. Chest radiograph.

Observed bilateral nodular

opacities characteristics ofinvasive pulmonary

aspergillosis. (20)


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closed. Aspergillus spores can be found in the deposits of wheat, in buildings under

construction, in stationary air conditioning and moldy foods.

Aspergillus is a filamentous fungus, ubiquitous and cosmopolitan found in nature and in

homes. You can insulate the ground, ventilation systems, and water. (4)

The aspergilos reproduce easily at higher temperatures and are frequently found in

cereals, fodder, cotton, some birds such as pigeons, abound in organic material. The

spores spread through the air (conidia) and are inhaled. (5)

The spores can survive in the right conditions, for thousands of years. Recent studies

have shown that the spores of Aspergillus kept intact their ability invasive, and even

seems to increase its potential allergenicity after thousand years. Spores have been

found inA. nigerandflavus in food, clothes, flowers and other objects from the tombs

of the pharaohs of ancient Egypt. in mummies and the sarcophagus of Ramses II (13)

The rapid spread of Aspergillus in environments filled with dust and through the air

conditioning systems, can be the source of outbreaks of aspergillosis that occur in

hospitals and other buildings after renovation or construction works. (4)

Aspergillus species are thermostable, and are able to grow at temperatures between 15

and 53 degrees centigrade. The spores are present throughout the year, but dominated in

autumn and winter, recovering from straw, manure, wood, decaying plants, fertilizer

land, sewers, and depositions of birds, moldy hay and atmospheric air. (2-5)


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Health prevention in order to resolve the problem identified

.

The research is a new contribution of scientific knowledge in the environmental impact

on health of our country, not only in identifying and locating the fungus, but also help in

health education in the area of environmental education in the preparation and

extraction of the product by the inhabitants, as well as training for health personnel on

the discovery and understanding of microbiological aspects in the health prevention of

the disease.

It has been found that by using a simple method of protection for the use of mask and

gloves, protects and reduces the spread of contamination and susceptible to contracting

the fungus as well as protect the post burned in other areas sterile.

CONCLUSION: We found a new species of the genera Aspergillus fungus found in soil

and seed nacascol El Salvador in the north of the country, which he called a future

scientific name: Aspergillus uessalvadoriensis , the genre Aspergillus, ues because it

was discovered at the University of El Salvador and salvadoriensis because in this

territory of El Salvador; or in the case binomial scientific name: Aspergillus ues

salvadoriensis A Vasquez.

THANKS: Dr Vianney Abrego for the courtesy of the electronic scanning

microphotographs taken CENTER ON HEALTH RESEARCH at the University of El

Salvador.

Reference Book


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5. Gassiot C. et al. Pulmonary Aspergillosis: a new approach in the reemergence. Acta Medica. 2000 9 (1-2): 67-72

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13. The Aspergillus Website. Fungical dries trust. 2007.

14. Arenas. R. Medical Mycology Illustrated. 2003. 2nd edition, Inter. Mc Graw Hill. Mexico.

15. Rippon. JW. Treaty of Medical Mycology. 1990. 3rd edition Inter. Mc Graw Hill Mexico.16. Negroni R. Bronchopulmonary mycosis of the adult and child. 1981. 2nd edition. Edit. Beta SRL Buenos Aires

.17. Negroni R. Mycosis skin and visceral. 1984. 8th edition. Lopez Publishers libero SRL Buenos Aires.

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19. Torres-Rodriguez JM, Brunet MI. Systemic aspergillosis. Clinical Infectious

Diseases paper. Systemic fungal infection. Doyma. 1991; Cap. 9: 59-69.

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