Characterization of Cockroach Allergens as A factor of Dermatitis in Egypt

The Journal of Medical Entomology and Parasitology is one of the series issued quarterly by the Egyptian Academic Journal of Biological Sciences. It is an important specialist journal covering the latest advances in that subject. It publishes original research and review papers on all aspects of basic and applied medical entomology, parasitology and host-parasite relationships, including the latest discoveries in parasite biochemistry, molecular biology, genetics, ecology and epidemiology in the content of the biological, medical entomology and veterinary sciences. In addition to that, the journal promotes research on the impact of living organisms on their environment with emphasis on subjects such a resource, depletion, pollution, biodiversity, ecosystem.....etc.

Several districts of Egypt are highly infected by cockroaches and inhabiting by patients who suffered from sensitivity to such insect, El-Gamal et al., (1995).
In Egypt, two different species of the common native cockroaches are the German cockroach, Blatella germanica (L.) and the American one, Periplaneta americana (L.) which infest houses, schools, hospitals and other large buildings, Tara (1998).However, sustained removal of cockroach allergen from homes may be difficult to achieve.Cockroach extermination needs to be done in all rooms and should be coupled with efforts to prevent reinfestation to achieve effective control of allergen exposure in the household type, Leaderer et al., (2002).Cockroaches are among the most undesirable insect intruders in the home.They are associated with unsanitary conditions, although they occasionally invade the bestkept homes, McConnell et al., (2005).
The insects also produce a secretion that has a repulsive odor and can affect the flavor of food.Cockroaches can cause allergic reactions when sensitive people come into contact with contaminated food or house dust, Hansel et al., (2006).
For nearly a half century, cockroaches have been recognized as a major cause of asthma morbidity in the urban, inner-city environment.
Several cockroach-produced allergens have been identified and characterized, and a few have been produced as recombinant proteins.Gore et al., (2007), reviewed that the current understanding of cockroach allergen biology and the demographics associated with human exposure and sensitization.They also critically evaluate allergen mitigation studies from an entomological perspective, highlighting disparities between successful and failed attempts to lessen the cockroach allergen burden in homes.
This study highlights the characterization and allergenicity of the extracted proteins from cockroach to investigate clinical significance and study of the immune response against cockroach allergens (whole body) of the common native cockroaches which are the German cockroach, Blatella germanica (L.) and the American cockroach, Periplaneta americana (L.), using hamsters as animal model.
This goal is achieved through seasonal sampling of cockroaches representing different ages were collected from low-cost public housing, extraction of cockroach allergens from the whole body and fragments of the collected cockroaches, determination of total IgE-Ab in sera for patients suffering from sensitivity to cockroach allergen, who live at exposure risk to cockroaches.Identification of the local prepared cockroach allergen extracts, purification, characterization of the major allergen, protein concentration was assayed and detection of protein using PAGE and HPLC techniques.

Collection of cockroach samples:
The American cockroaches, Periplaneta americana (L.) and the German cockroach, Blatella germanica (L.)are the most common in Egypt, whole bodies, cast skins, egg shells and feces were considered as dust material during this study.They are most commonly found in restaurants, grocery stores, bakeries, pet shops and other establishments where food is prepared or stored.They can be transported into homes and apartments in boxes from infested establishments.Cockroach index was estimated in many household, the chosen household where the highest cockroach index number which was collected by trapped bags in low socioeconomic levels with highly cockroach infestation.Six households in low-cost public housing studied, all had visible cockroach infestation representing different districts from Egypt.
The collected cockroaches were killed by freezing and then lyophilized to get dry powdered samples for preservation.

Extraction of cockroach allergen:
Crushed cockroach samples were suspended in dulbeco's phosphate-buffered saline (polymethyl sulfonyl fluoride-2 m mol/L, Ethylene diaminete, tetra acetic acid-pH:7-20m mol/L).Crushed bodies were treated three times with six volume of diethyl ether for defatting, Carsten et al. (1990).The resulted mixture was stirred overnight at 4°C and was then centrifuged at 14,000 r.p.m. for 30 minutes at 4°C.Supernatants were filtered through a 0.45 mm Millipore filter, an aliquot of one mg/ml was stored at 4°C and kept for further investigations as prepared crude cockroach allergens extract.
A commercial crude extract of American & German cockroach allergen was obtained from (Allergopharma) U.S.A manufactures, it was marked for skin prick testing in solution staining glycerin and phenol, Pollart et al., (1991-A), and used for comparison.

Protein concentration and profile:-
Protein concentration was assayed by total protein colorimetric method [Diamond Diagnostics] using Specord 200 photometer to read the absorbance of the local prepared crude cockroach allergens extract and the commercial crude cockroach allergen extracts, standardized against reagent blank, Henry et al., (1964).Sodium dodecyl sulfatepolyacrylamide gel electrophoresis was carried, using cockroach allergens as samples, according to the method described by Laemmli et al., (1970).

Amino Acids Analysis:-
Amino acid standard mixture was evaluated by Epp Biotronik, LC.3000, amino acid analyzer (Beckman Instrument) , using LC 3000 standard H1, ready-made buffers HI (4-buffer system), column type H 125x4 mm, pre-column type H 60x4 mm were used for identification of the prepared cockroach allergen extract compared with the commercial crude cockroach extracts.
High Performance Liquid Chromatography (HPLC):-HPLC apparatus Beckman system Gold, dual pump, module 125 was used.The purified allergen samples (the prepared cockroach allergen extract and the commercial crude cockroach extract) were passed through the apparatus to fractionate the crude cockroach allergens, according to Susan et al., (1991).

Determination of antigenic property of cockroach allergen extracts with human serum antibodies:-
Antigen-antibody reaction test was carried out for confirmation antigenic property of the local prepared crude cockroach allergen extracts compared with the commercial allergen extracts using ELISA technique.The sera were used as known antibody samples from persons had highly detectable IgE-Ab compared with normal persons as control samples had normal ranges of total IgE-Ab analysis.Microtiter strip ELIZA plate 96 sterile non coated wells was used in this test.

Statistical analysis:-
The data were analyzed on a personal computer for parametric IgE-Ab using student "t" test.

Characterization of cockroach allergens:
In this study, one thousand, two hundred and forty six cockroaches were trapped from different households for the preparation of the crude allergens, Fig.

(1).
In each household, cockroach samples were collected from houses, occupied by patient with asthma.The patients have detectable serum total IgE-Ab.

Determination of antigenic property of cockroach allergen with human serum antibodies:
The human sera were used as known antibody samples.
Antigen-Antibody reaction revealed positive reaction to both mixture of equal crude whole body extracts from American & German cockroaches (CRa-M) local prepared and commercial allergen as antigen with the serum samples which were taken from persons had highly detectable total IgE-Ab (ranged from 980-2100 IU/ml), which in case of serum samples were taken from normal persons as a control had normal T.IgE level (ranged from 17-100 IU/ml) the normal is less than 150 IU/ml.The result showed in

Measuring protein concentration and SDS PAGE analysis:-
Protein concentration was assayed by total protein colorimetric method [Diamond Diagnostics] using Specord 200 photometer absorbance; the readings were between 2.8-3.0 mg/dl.Protein analysis:-SDS-PAGE analysis (using Image Master analyzer) of crude cockroach, whole body, cast skin and egg shells extracts revealed that the molecular weights estimated to be (190, 139, 132, 106, 90, and Tables (3&4).The two crude cockroach allergen extracts were very closely related in the presence of glycine amino acid at the same time (21 minutes).The most notable differences were found in other amino acids, this is due to the commercial CRa-M mixed with phenol and glycerol that made the analysis impossible, on the other hand the difference types of amino acids of the prepared CRa-M of cockroach allergens were found with 49%concentration as ammonium sulphate followed by Cystine 9%, Alanine 6.2%, Histidine 5.2%, Leucine 5%, Glycine & Glutamic Acid4.6%,Aspartic Acid 3.1%.Other types of amino acid were found in low percent as Threonine, Serine, Valine, Methionine, Tyrosine, Phenylalanine and Lysine.

Molecular weight analysis as protein marker
Molecular weights for commercial crude cockroach allergen Molecular weights for the local prepared crude cockroach allergen (0.4 mg/ml of protein) Molecular weights for the local prepared crude cockroach allergen (0.5 mg/ml of protein) Molecular weights for the prepared crude American cockroach allergen (0.5 mg/ml of protein) Molecular weights for the prepared crude German cockroach allergen (0.5 mg/ml of protein) Fig. 3: Molecular weight analysis of cockroach allergent proteins.The purified allergen samples were analyzed to detect fractionation of the crude cockroach allergens CRa-M.The purified allergens of the commercial &the local prepared crude cockroach extracts were illustrated in Figs.(5,6 & 7) and Table (5).
The HPLC analysis revealed many distinct peaks which corresponding to the molecular weights calculated from of SDS-PAGE analysis.In comparing between the purified allergens of the commercial & the local prepared crude cockroach extracts, the results revealed that there were many peaks closely the same in retention time as shown in Fig. (7) and Table (5

DISCUSSION
In some patient clinic of allergy & Immunology Centers, in Egypt, it was observed that many patients suffering from allergic diseases (atopic dermatitis) and with relatively high titers of serum total IgE-Ab showed positive cockroach skin prick tests, Merdan et al., (2015).
This observation attracted our attention that cockroaches might be the source of allerginicity and also it is an increasingly important public health source of allergens.This assumption may be supported by Bennet et al., (1993), Williams et al., (1999), andYeh (2006).
Since the cockroach is a popular insect in houses, we set a plane to prove that cockroach allergens induce atopy.
To achieve this goal, the collected cockroach (whole body) both sexes were killed by freezing and then lyophilized to get dry powder samples for the preparation of the local crude cockroach allergens.This procedure was followed according toZwick et al., (1990) and Zwick et al., (1990).
During the present work, the purified cockroach allergens and the commercial cockroach allergens were examined for the determination of total protein profile by photometric assay.The results revealed similarity between both allergens (2.8 -3.0 mg/dl).This result may assure that the local prepared allergen is similar to the already manufactured commercial one.It also proved that our preparation is on the right track.Our results ran parallel with those reported by Gore et al., (2007).
Several concentrations of protein extracts for both types of allergens were tested until we reached the suitable electrophoretic analysis by SDS-PAGE of the purified allergens.Total protein were carried out using dilution (0.5 mg/dl) for both types of allergens which gave the best results with SDS-PAGE and showed several protein bands ranging from 190 -35 KD.These results are in accordance with Wu et al., (2005), who identified the characteristics of major cockroach allergens with molecular weights ranging from 6 to 120 KD.
No significant difference in SDS-PAGE pattern between the local prepared CRa extracts and the commercial one.This result indicates that the responsible protein fractions for induction of allergy are identical in both tested WBE of locally prepared and commercial one.Our result ran parallel with those reported by Gore et al., (2007) andWU et al., (1998).
The protein concentration (0.5 mg/dl) of the CRa extracts identified at least six different bands on SDS-PAGE.The most dense stained band ranged between 75-35 KD.Similar protein fractions particularly at 35 and 75 KD were reported by Pollart et al., (1991-A).
This finding is slightly different from those determined by Stankus et al., (1990).
This concentration in specifying may be due to cockroach species strains and method of preparation of the crude extracts.The similarities and differences between the SDS patterns in this study and those reported by other authors may be related to some extent to the differentiation of SDS analysis of each individual cockroach species.It was found that the protein extracted from whole body of Blatella germanica alone was different from M.W of those produced from Periplaneta americana WBE.
These differences were detected by Helm et al., (1996), andWU et al., (1998), who noticed several allergens to which there was a strong band at 75 KD, while the first author restricted the band at 36 KD.
For subsequent analysis two mixtures were prepared; one is a locally prepared mixture of American & German cockroaches and the other mixture is a commercial allergen from both species.
Antigenic property of cockroach allergen extracts with human serum antibodies was tested by ELISA technique.The two mixtures (allergens) were used as antigens against the human serum samples which were used as antibodies.Antigen-Antibody reaction revealed positive reaction to both locally prepared mixture and the commercial one, in sera sample taken from patients who had a highly detectable total serum IgE-Ab.While negative reactions in sera sample taken from people with normal total serum IgE-Ab.This result may clarify that our prepared allergen is active antigens.Information about the IGE binding epitope of cockroach allergens may help in designing diagnostic and therapeutic approach to cockroach allergy.Lee et al., (2015).
The results of amino acids analysis for the local cockroach allergens and commercial allergens revealed that the two crude cockroach allergen extracts (local and commercial) were similar in presence of glycine amino acid at the same time (21 minutes).The most notable differences found in other amino acids were due to the phenol and glycerol mixed with the commercial CRa which made the analysis impossible.The different types of amino acids of the local prepared cockroach allergens were found with 49 % concentration as ammonium sulphate followed by cystine 9%, alanine 6.2%, histidine 5.2%, leucine 5%, glycine & glutamic acid4.6%,aspartic acid 3.1%.Other types of amino acid were found in low percent as threonine, serine, valine, methionine, tyrosine, phenylalanine and lysine.Our findings were almost identical to results of amino acids analysis made by Carsten et al., (1990), while Jeon et al., (2014 ), found that using Elisa technique and amino acid analysis investigate allergenicity of German cockroach.They also found that the amino acid sequencing of German cockroach's chemotrypsin allergen showed 32.7 to43.1%identity to dust mite chemotrypsin.On applying another analysis, High Performance Liquid Chromatography (HPLC) analyzer used to determine the fractionation of the purified locally prepared allergen, compared with the commercial formulated one, many peaks were found.The purified allergens demonstrated two overlapping peaks, eluting at 26 to 35 minutes with the sharpest peak eluting at 30 to 34 minutes.The use of this fraction for chromate-focusing studies on whole body extracts of cockroach allergens confirmed the presence of significant antigens.Similar results were reported by Stankus et al., (1990), Carsten et al., (1990), and Gore et al., (2007).
These results highlight the antigenic properties of cockroaches and more investigations are going on to correlate such antigens with the children allergy and may improve their immunotherapy forms.

Fig. 1 :
Fig. 1: Total number of the trapped cockroaches / month of the year which were collected from different households.
75 and 35 KD) and separated protein bands of the crude cockroaches together (lane 3،4) or separated (lane A، G) and the commercial one are measured and represented in Fig. (2).

Fig. 2 :
Fig. 2: A photograph of SDS-PAGE of separated components of crude cockroach extracts.Lane 1:-The high molecular-weight protein marker.Lane 2:-The commercial crude cockroach extract.Lane 3:-The local crude cockroaches extract contained 0.4 mg/ml of protein.Lane 4:-The local prepared crude cockroach extract contained 0.5 mg/ml of protein.Lane A:-The local prepared crude American cockroach extract,Whole body, cast skin and egg shells contained 0.5 mg/ml of protein.Lane G: -The local prepared crude German cockroach extract,Whole body, cast skin and egg shells contained 0.5 mg/ml of protein.
Many bands with MWt ranging from (35 to 190 KD) were separated from the local prepared CRa-C extracts (lane 3&4) and commercial CRa-M extract (lane 2) compared with wide range molecular-weight gel marker (lane 1) proved the purity in preparation of the local prepared CRa-M extracts.At least six antigenic bands with MWt ranging from (35 to 190 KD) of the crude cockroach extracts were visible after staining.The American crude cockroach allergen extracts was almost sharing in many bands (lane A) with the German CRa extracts (lane G) with total protein concentration ranging between 0.4-0.5 mg/ml.No significant difference of SDS-PAGE patterns was detected between the local prepared extract and the commercial crude cockroach allergen extracts that were manufactured in U.S.A.Amino Acids Analysis:-The amino acid analysis of the purified allergens [commercial and prepared crude cockroach extracts allergen] is illustrated in Figs.(3&4)

Fig. 5 :
Fig. 5: Purification of the commercial crude cockroach allergen extracts by HPLC gel filtration and fractions were analyzed for protein content.

Fig. 6 :
Fig. 6: Purification of the local prepared crude cockroach allergen extracts by HPLC gel filtration and fractions were analyzed for protein content

Fig. 7 :
Fig. 7: Comparison between Retention time of local prepared and commercial crude (*) cockroach extracts with the percentage of peak area by HPLC.

Table 1 :
Table (1).There is a high significant difference between human serum antibodies compared with human serum total IgE-Ab, as shown in Table (2).Antigenic property of cockroach allergen extracts with human serum antibodies compared with human serum total IgE-Ab.

Table 2 :
Comparison between antigenic properties of the allergens with normal human Serum antibodies compared with patient serum total IgE-Ab.

Table 3 :
Amino acids analysis of the commercial crude cockroach allergen extracts Peak No.

Table 4 :
Amino acids type related to different retention times and concentrations of the local prepared crude cockroach allergen extracts.

Table 5 :
Purification of the local prepared crude cockroach allergen extracts and the commercial (*) HPLC gel filtration and fractions were analyzed for the protein content.