Jourrlul of Gmrrcil Microhiologj~( I985), 131, 2627-2635.
2627
Pritlted in Great Britain
Interaction between Human Polymorphonuclear Leucocytes and
Chlamydia trachomatis Elementary Bodies: Electron Microscopy
and Chemiluminescent Response
By M E N A C H E M Z V I L L I C H A N D I S R A E L S A R O V *
VirologjaUnit, Faculty qf' Health Sciences and Soroka Universitj?Hospital, Ben Gurioii Uriiwrsitj*
qf the Negei., Beer Shesa 84 105, Israel
(Receiwd 26 Noremher 1984 :reiiyecl 12 April 1985)
Incubation of human polymorphonuclear leucocytes ( H P M N ) with highly purified Chlamydia
trachovliutis serotype L2/434/Bu elementary bodies (EB), in the presence and absence of specific
antibody, resulted in a 103-fold reduction of viable count after 24 h incubation. Electron
microscopy observations indicated activation of the H P M N by the EB. Attachment of the EB to
the HPMN cell membrane, formation of a cytoplasmic cup and EB-containing vacuoles were
observed. In addition, two types of phagocytic vacuoles were observed after 30 min incubation;
in one type, a single EB was tightly surrounded by the vacuolar membrane, while the other type
was enlarged and held one or more intact EB or degenerated EB or both. A fuzzy coat was
observed on EB located in the H P M N vacuoles only in the presence of specific antibody. Empty
vacuoles containing degenerated EB were observed in the H P M N after 24 h incubation. H P M N
exposed to EB of C. trachornatis produced a marked chemiluminescent response with a peak 14
times greater than the peak value of the control. A second stimulation with phorbol 12-myristate
13-acetate and zymosan was achieved. The chemiluminescent peak value in the presence of
heat-treated EB (56 "C, 20 min) was 5006 of that obtained in the presence of untreated EB. The
significance of the chemiluminescent response in the killing mechanism of C. trachomutis EB by
H P M N is discussed.
INTRODUCTION
The role of human polymorphonuclear leucocytes ( H P M N ) in host defence against microorganisms is well established (Quie & Mills, 1978; Walach et a/., 1982). Their microbicidal
activity is facilitated by a number of antimicrobial systems, some dependent on oxygen and
others operative in its absence (Gabig & Babior, 1981). Phagocytosis and killing of some microorganisms by H P M N requires opsonization of the invading pathogens, while others are ingested
without being coated by serum opsonins (Danley & Hilger, 1981). In addition to the initiation of
phagocytosis, the H P M N response to stimulation by some micro-organisms is characterized by
an increase in oxidative metabolism, and the production of highly reactive, antimicrobial
substances (Quie rt al., 1977). The metabolic processes can be detected in several ways. Among
these is light emission stimulated by the oxygen radicals being generated, which can be detected
in a chemiluminescence assay such as that described originally by Allen et a/. (1972).
Chlamrdia trachomutis is an obligate intracellular Gram-negative bacterium with a genome of
660 x loh Dal (Sarov & Becker, 1968, 1969; Moulder, 1982; Ward, 1983). The multiplication
cycle of C. trachomatis involves two distinct forms of micro-organisms: the elementary body
(EB) which is the infectious extracellular form and the reticulate body which is the metabolically
active, highly permeable intracellular form (Moulder, 1982; Ward, 1983). A total of 15 serotypes
Ahhrrriutibns: EB. elementary bodies; HBSS, Hanks' balanced
polymorphonuclear leucocytes; P M A , phorbol 12-myristate 13-acetate.
0001-2340
salt solution;
0 1985 SGM
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H P M N , human
2628
M . ZVILLICH A N D I . SAROV
of C . trachomatis have been described. The A, B, Ba and C serotypes are the cause of
hyperendemic trachoma in developing countries (Dunlop, 1983). Strains D to K are a major
cause of sexually transmitted infections, non-gonococcal urethritis (Oriel, 1983), cervicitis,
endometritis (Hare & Thin, 1983), salpingitis (Westrom & Mardh, 1983), conjunctivitis and
pneumonia (MacFarlane & Macrae, 1983). Subclinical infections by C. trachomatis appear to be
a major cause of mechanical infertility in women (Westrom & Mardh, 1982). The three serotypes
L , , L2 and L3 are the causes of lymphogranuloma venereum, a sexually transmitted systemic
disease (Schachter & Caldwell, 1980). Current knowledge of the humoral and cellular immune
responses generated during chlamydial infections and how these control or exacerbate disease is
limited (Yong et a/., 1982). It has been demonstrated that chlamydial antigens classified as group
antigens can be detected on the membranes of infected host cells (Richmond & Stirling, 1981).
The significance of this finding regarding cell mediated immunity needs further investigation.
Polymorphonuclear leucocytes are the predominant inflammatory cells which infiltrate the
sites of early infection (Monnickendam & Pearce, 1983) and interaction of these cells with C.
trachornatis is currently being investigated (Soderlund et a/., 1984).
In the present study we examined the interactions between HPMN and highly purified EB of
the L,/Bu/434 biotype of C . trachomatis. The one step growth curve technique was used to
measure loss of infectivity of the EB ingested by the HPMN. Preparations of HPMN incubated
with C. trachomatis EB, in the presence and absence of specific antibody, were examined by
electron microscopy. The ability of the EB to induce HPMN to produce potentially
chlamydicidal oxidative metabolites was tested by measuring the chemiluminescent response of
the HPMN.
METHODS
Orgunisms und grorr,th conditions. Ckluniyciiu trachomutis biotype lymphogranuloma venereum (L2/434/Bu)was
grown in MA-104 (embryonic rhesus monkey kidney) cell cultures (Cevenini rt a/., 1983). The cells were grown in
RPMI-1640 medium supplemented with 10% (v/v) foetal calf serum, 100 U penicillin ml-I, 100 pg streptomycin
mI-l7 100 U mycostatin ml-1 and 2 mM-L-glutamine (Bio-Lab, Jerusalem, Israel) and were passed every 4 d.
Before infection. the cells were passed and the medium was replaced by maintenance medium which contained
minimum essential medium supplemented with 5 % foetal calf serum, 1 % (w/v) glucose, 0.15% (w/v) sodium
bicarbonate, 100 pg streptomycin ml-l, 10 pg gentamicin ml-I and 10 pg Fungizone ml-I. After 2 d the cells were
infected and the medium was replaced with maintenance medium supplemented with 1 pg cycloheximide ml-I
(growth medium).
Purificution c?f'chlamy/iar.Elementary bodies (EB) of C. truchonratis were purified 48-72 h post-infection from
MA-104 monolayers grown in 175 cm2 polystyrene culture flasks (Nunc Inc., Denmark) by a modification of the
method described by Caldwell Pt ul. ( I 98 1). The medium was poured off and the cells were removed with 4 mm
glass beads into 10 ml cold Hanks' balanced salt solution (HBSS; Bio-Lab, Jerusalem, Israel). The cell suspensions
were pooled from the flasks, and ruptured by vortexing for 2 min. The resulting suspension was centrifuged at
500 g for 15 min at 4 "C. The supernate was layered over 8 ml of a 35% (v/v) Urografin solution (diatrizoate
meglumine and diatrizoate sodium 76% for injection; Schering AG Berlin/Bergkamen, FRG) in 0.01 M-HEPES
I then centrifuged at 43000g for 1 h at 4 "C in a Beckman SW 27 rotor. The pellets
containing 0.15 M - N ~ Cand
were suspended in 0.01 M-sodium phosphate (pH 7.2) containing 0.25 M-Sucrose and 5 mM-L-glutamic acid (SPG),
pooled and layered over discontinuous Urografin gradients [ 13 ml400,/,,8 ml44% and 5 ml52% (v/v) Urografin].
These gradients were centrifuged at 43000g for I h at 4 "C in an SW 27 rotor. EB bands located at the 44/52%
Urografin interface were collected, diluted with SPG and then centrifuged at 43000g for 45 min. The EB pellets
were suspended in SPG and stored at -70°C. For the chemiluminescence assay, EB were washed by
centrifugation (43000g, 45 min in an SW 27 rotor) and resuspended in HBSS.
Isolurion y f ' H P M N . This was done by the method of Boyum ( I 968). Heparinized whole blood was obtained from
healthy human donors, diluted twofold, layered onto Ficoll-Hypaque (1.077 g cm-? at 25 "C) and centrifuged at
400g for 30 min. The pellet was resuspended in an equal volume of 6% (w/v) dextran in minimum essential
medium. The erythrocytes were allowed to sediment at 37 "C for 30 min, and the HPMN-enriched supernate was
removed and centrifuged for 5 min at 400g. Contaminating erythrocytes were removed by osmotic shock. The
HPMN cells (990i pure) were washed three times in minimum essential medium and resuspended in RPMI-1640
+ 109; foetal calf serum. For the chemiluminescence assay, HPMN were suspended in HBSS without phenol
red. The viability was 997; as determined by 17; Trypan blue staining. After 50 h incubation, viability of the
HPMN was 800;.
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Chlamydia trachomatis interactioiis with H P M N
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/nucrir-ufion t c w . H P M N (lo-) and 10' inclusion forming units (IFU) of C. truchomutis EB were incubated [ I h,
37 "C in 5"; (viv) C 0 2 ]in 1-0ml RPMI-1640 containing 5 7 , heat-inactivated foetal calf serum. 50 pg streptomycin
ml-', 5 pg gentamicin ml-l, 5 pg Fungizone ml-I, with or without human serum diluted 1 : 10. The human serum
had a chlamydia-specific IgC titre of 5 12 determined by immunoperoxidasc assay, as described by Cevenini et ui.
(1983). The cells were washed twice with RPMI-1640 medium ( 100g, 5 min) and further incubated at 37 'T in an
atmosphere of 5"" CO,, in a volume of I ml of the same medium. At 0, 4, 24 and 50 h post-infection, samples
(200 pl) were taken out and sonicated for 20 s in a Bransonic-12 sonifier.
MA-104 cells as a control (loh cells m1-I) were infected at an m.0.i. (multiplicity of infection) of 1 IFU per cell
with I h adsorption at 37 T. The cells were washed twice with RPMI-1640 medium (30Og, 5 rnin), diluted with
the same medium as the H P M N and seeded in a 96 microwell plate (2 x loJ cells per well). At 0 , 4 . 24 and 50 h
post-infection, the cells were scraped into the medium inside the well. Triplicate wells were pooled (300 pl) and the
sample was sonicated for 20 s. Viable organisms in the H P M N and control cell lysate were titrated on MA-104
cells by a modified procedure of Kimmel el ul. (1984). MA-104 cells were seeded ( 5 x loJ cells per well) in a 96
microwell plate (Nunc). After 48 h the cells were infected, in duplicate with 10-fold dilutions of the control and
H P M N lysates. At 48 h post-infection the cells were fixed with ethanol (100qJ and subjected to an immunoperoxidase assay (Haikin & Sarov, 1982). The final results of the titration were expressed as IFU ml-I.
E k t r o n microscopy. Samples were prepared according to the method of Biberfeld (1971 ). Pellets of H P M N were
fixed in 2"" ( v i v ) glutaraldehyde in cacodylate buffer (pH 7.2,O.l M) for 90 rnin and washed twice with the same
buffer. After fixation samples were treated with 1 (w/v) OsO, for 60 min, dehydrated in alcohol, washed three
times with propylene oxide iind embedded in Araldite. The blocks were sectioned and stained with lead citrate and
uranyl acetate. Electron micrographs of thin sections were taken with a Philips 201C transmission electron
microscope.
Cheniiluniinesceirce ussay. Luminol-enhanced chemiluminescence was measured by a modified procedure of
Trush c't ul. ( I 978), in a liquid scintillation counter (Packard, Tri-Carb liquid scintillation spectrometer, model
3310) kept at ambient temperature (approx. 23 & 1 "C) and set 'out of coincidence' in the tritium mode. Samples
for the chemiluminescence assay were obtained by adding 0.8 ml HBSS without phenol red and containing 2 VMluminol (Sigma), 0.1 ml cell suspension ( l o h H P M N ) and 0.1 ml purified EB, to sterile. disposable 4 m l
polypropylene tubes (38 x 12.5 mm; Nunc). The tubes (dark adapted for 24 h before the assay) were put into glass
vials. placed in the scintillation counter and the light emission was recorded for 0.1 rnin at intervals of 6-10 min.
Zymosan (Sigma)and PMA (phorbol 12-myristate 13-acetate: Sigma) were used in the second chemiluminescence
stimulation assay. Zymosan was prepared by boiling 100 mg in 2 ml saline for 15 rnin in a water bath and
centrif'uging at 600 g for 5 min. The pellet was suspended in 2 ml HBSS. Zymosan was used at 2.5 mg ml-' in the
assay. PMA was dissolved in a drop of DMSO and further diluted with HBSS to a concentration of 1 pg ml-I. The
final concentration of PMA added to the vials was 0.1 pg ml-I.
RESULTS
Inactivation of' C. trachomatis EB by H P M N
The ability of HPMN to inactivate C. trachomatis EB (L2/434/Buserotype) is shown in Fig. 1.
Incubation of HPMN and the EB at a ratio of 1 : 1 resulted in a 103-fold reduction in infectivity
after 24 h incubation in the presence and absence of specific antibody. The control consisted of
MA-104 cells incubated with C. trachomatis EB at a ratio of 1 : 1 in the same conditions as the
HPMN. In the control, the infectivity titre assay, using the modified immunoperoxidase assay
for detection of C. trachomatis IFU in infected cell samples, showed a decrease in infectivity 4 to
24 h post-infection followed by a 103-fold increase in IFU ml-l 50 h post-infection (Fig. 1). This
L2 serotype growth pattern in the control is in accordance with that typically observed by other
investigators (Ward, 1983).
EBect of' HPMN cells oil C. truchomutis EB : ekctron microscopy obsertations
Several steps in the phagocytosis of the EB were observed. Fig. 2 ( a ) shows an EB attached to
the HPMN membrane and formation of a phagocytic cup can be seen in Fig. 2(b).Activation of
the HPM N, characterized by the presence of large phagocytic, chlamydiae-containing vacuoles
was observed (Fig. 2 4 . Two types of EB-containing phagosomes were observed after 30 rnin
incubation. One type contained single EB in close contact with the phagosome membrane (Fig.
3a). The other type was enlarged and contained single or a number of intact EB (data not shown)
or both intact degenerated EB inside the phagosome (Fig. 2c). A fuzzy coat was observed on EB
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M . Z V I L L I C H A N D I . SAROV
10 20 30 40 50
Time (h)
Fig. I . Kineticsof C. truchomatis inactivation by H P M N . The titre of C. trachomariswas measured after
incubation for the periods indicated with H P M N at a chlamydia-to-HPMN ratioof 1 : 1 in the presence
(0)
and absence
of specific antibody. The control (A)consisted of MA-104 cells infected at the
same ratio and incubated under the same conditions as the H P M N . The results represent one of three
experiments.
(e)
inside phagocytic vacuoles only in the presence of specific antibody (Fig. 36). After 24 h
incubation, only empty and degenerated EB-containing vacuoles were observed (Figs 2e and
2.f'). These results were consistently observed in several experiments.
Eflkct of' C. trachomatis EB on the luminol-enhanced chemiluminescent response o j HPMN
Incubation of HPMN with EB of C. trachomatis induced a marked chemiluminescent
response. The peak activity was recorded after about 50 min incubation and increased with an
increase in the number of IFU per cell in a dose-response relationship (Fig. 4a). Addition of
heat-treated EB of C. trachoniatis (56"C, 20min) resulted in a decrease of 50% in the
chemiluminescence peak value (Fig. 46). The capability of the EB-sensitized HPMN to be
further stimulated was investigated using PMA, a phagocytosis-independent stimulant, and
zymosan, a phagocytosis-dependent stimulant (Quie et al., 1977). Addition of 0.1 pg PMA ml-1
(Fig. 56) and 2.5 mg zymosan ml-' (Fig. 5 a ) (approx. 3 x lo8 particles) after 40 min incubation
in the presence of C. trachomatis EB, resulted in an increased chemiluminescent response, with a
peak value similar to that obtained when PMA and zymosan were added to the control. Thus,
the response of the H P M N to C. trachomatis EB, at the multiplicity described above, indicated
that exposure of HPMN to C. frachomatis EB did not reduce the capability of the cells to be
further stimulated by PMA and zymosan to produce a second chemiluminescent response.
DISC USSIO N
This study describes the results of experiments showing that HPMN inactivate C. trachomatis
EB (L2/434/Bu biotype) in both the presence and absence of specific antibody (Fig. 1). This
finding is in agreement with that of Yong et al. (1982) who found that two biotypes of C.
rrachomatis, TW-5 and L2/434,were killed by HPMN. Electron micrographs of HPMN exposed
to C . trachomatis EB (Fig. 26) showed EB within phagocyte vacuoles in the HPMN cytoplasm
after 30 min incubation. Two types of EB-containing phagosomes were observed. One appeared
as a tightly enclosed vacuole containing a single EB (Fig. 3a) while the other was enlarged and
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Chlamydia trachomatis interactions with H P M N
263 I
Fig. 2. Transmission electron micrographs of H P M N infected with C. rrachornatis EB. Cells were fixed
30min after chlamydia1 challenge. ( u ) Attachment of the EB to the H P M N cell membrane; ( h )
formation of a phagocytic cup; (c) intact and degenerated EB inside a large vacuole; ((1) activated
H P M N with phagocytic EB-containing vacuoles; ( e ) H P M N with empty vacuoles and with (.I )
degenerated EB-containing vacuoles after 24 h incubation. Bar markers: ( u ) 0.2 p m ; ( h ) 0.4 pm; (c)
0.4 pm; ( e ) 2 p m ; (,/') 2 pm.
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M. ZVILLICH AND I . SAROV
Fig. 3 . Transmission electron micrographs of H P M N infected with C. trachomariv. Cells were fixed 2 h
after chlamydia1 challenge. ( u ) EB inside a tightly enclosed phagocytic vacuole; ( h )an EB-containing
vacuole in the presence of specific antibody. The human serum had a chlamydia-specific IgG titre of
256 determined by the immunoperoxidase assay described by C'evenini ('1 d.(1983). A fuzzy coat can be
observed on the EB inside the phagocytic vacuole. Bar markers: ( ( 1 ) 0.2 p m ; ( h ) 0.1 pm.
contained a single or a number of intact EB, degenerated EB, or both (Fig. 2c). These two types
of vacuoles could represent various stages of digestion of the C. trnchornatis EB or may indicate
the existence of two different mechanisms of entry of the infectious particles into the H P M N .
Rikihisa & Ito (1980), using electron-dense tracers during entry of rickettsia into HPMN,
characterized two types of vacuoles. Silva et a/. (1982), studying the mechanism of entry of
To.uoplasrna gondii into mouse macrophages, observed the existence of two types of cytoplasmic
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Chlamydia trachomatis interactions with HPMN
2633
g 100
c)
(/1
5
0
W
X
90
b
320
8
E
8
80
280
.-2 70
240
.- 60
2
2
160
40
g 120
30
80
20
40
10
v
E
2E 200
E
u
50
10 20 30 40 50 60 70 80
Time (min)
10 20 30 40 50 60 70 80
Time (min)
Fig. 4
Fig. 5
Fig. 4. Luminol-enhanced chemiluminescent response of IllhH P M N incubated ( a )in the presence of C.
Irrrclzomafis EB at a m.0.i. of 1 (A),10 ( O ) ,and 50 (0)
(i.e. 1, 10 or 50 I F U per cell) and (h) in the
presence of heat-treated (56 "C, 20 min) EB (0).
Controls in ( N )and ( h )consisted of H P M N incubated
with medium alone ( 0 ) The
. results represent one of three experiments.
Fig. 5 . Luminol-enhanced chemiluminescent response o f 1 Oh H P M N which were initially stimulated
with 3 x 10' I F U C. rrcic.lron?ctti.s EB mi-' (0).
After 5 0 min incubation (arrows) zymosan (o)and PMA
( h )were added at concentrations of2.5 pg ml-I and 0.1 pg ml-I, respectively. Thecontrol (.)consisted
of H P M N incubated with medium alone. The control for EB-stimulated H P M N treated with zymosan
or PMA was stimulated cells without addition of zymosan or PMA. For clarity of the experimental
curve this control is not shown. The results represent one of three experiments.
vacuoles. Both groups suggested that their findings could be related to a dual mechanism of entry
of the parasites. EB of chlamydiae enter phagocytes such as mouse macrophages by two
different endocytic pathways. Infectious EB enter via a parasite-specified pathway, whereas
heat-inactivated EB are taken in by a host-specified route (Byrne & Moulder, 1978; Zeichner,
1983). The involvement of these two mechanisms in the entry of C. rruchoniatis EB to H P M N is
a subject for further investigations.
A fuzzy coat was observed only on EB treated with specific antibody, in the H P M N
phagosome (Fig. 3h). A fuzzy coat was also observed on rickettsia located in phagosomes of the
H P M N , in the presence of specific antibody (Rikihisa & Ito, 1983). The role of this coat is
unknown but i t may serve as a marker for the entry of opsoniLed chlamydiae to the cells.
It is well recognized that activation of H P M N by some micro-organisms leads to the
production of highly reactive molecules in these cells. Superoxide anions, singlet oxygen and
hydroxyl radicals are produced as a result of respiratory burst activation. The production of
these highly reactive molecules by H P M N can be measured as light emission, termed
chemiluminescence (Trush cr al., 1978). As shown in Fig. 4(a) the highly purified EB of C .
truchomatis induced a chemiluminescent response with a maximum peak calue after 40-50 mill
of incubation, 14 times greater than the peak value of the control. The H P M N retained activity
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M . ZVILLICH A N D I. SAROV
since a second stimulation by PMA or zymosan could be achieved. Recently, Soderlund et al.
(1984) also found that serotypes E and L, of C. trachomatis induced a chemiluminescent
response in HPMN which was increased by type-specific antibody. The lower chemiluminescent production observed in the presence of heat-treated (56 "C, 20 min) EB may be related to
modification of some EB surfice molecules, possibly responsible for the stimulation of these
chemiluminescent responses generated by the HPMN. Bose & Paul (1982) observed a reduced
rate of association of heat-treated EB (L2/434/Buserotype) to HeLa 229 cells and related this to
the modulation of ligands (adhesins) present on the surface of the EB.
Yong et al. (1982) suggested that oxygen-dependent antimicrobial systems are not essential for
the chlamydicidal activity of HPMN. This conclusion was based on the fact that the peroxidase
inhibitors azide and cyanide had little or no effect on the inactivation of C. trachomatis by
normal HPMN. However, HPMN lacking a respiratory burst and with myeloperoxidase
(MPO) deficiency obtained from patients with chronic granulomatous disease inactivated C.
trachornatis serotype B less efficiently than they did an L2 serotype. Thus, the findings of Yong et
al. (1 982) do not necessarily exclude the involvement of the MPO system in the chlamydicidal
activity of the HPMN, and it is possible that some C. trachomatis serotypes are killed by oxygendependent systems while others are sensitive to other powerful antimicrobial systems present in
the HPMN (Gabig & Babior, 1981). It should be noted that activation of the respiratory
metabolism does not necessarily lead to engulfment and subsequent phagocytosis and killing
(Henson et al., 1972). For example, cytochalasin B-treated HPMN, interacting with aggregated
IgG or with opsonized zymosan, respond with enhanced superoxide generation and production
of chemiluminescence, even though particles remain on the outer surface of the cells (Goldstein
e t a / . , 1975). H 2 0 2and free radicals at the site of the infection might have a direct effect on the
tissue damage caused by C. trachomatis in acute and chronic infections and may even have some
role in the pathogenesis of cervical carcinoma (Henry-Suchet et al., 1981; Paavonen et al., 1979;
Schachter et al., 1982). Clearly, further studies are required to understand the mechanism of
killing and the significance of the C. trachomatis EB-induced chemiluminescent response by
HPMN.
We are grateful to Ms Kol and M. Amir for assistance with electron microscopy, to Mr J . Ruhan, Soroka
Medical Center, for photographic assistance and to D r M. G. Friedman for her enlightening remarks. This study
was supported i n part by the Middle East Eye Research Institute (MEERI).
This study is part of the P h D thesis of M. Zvillich in the Department of Biology and the Faculty of Health
Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
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