Dr' Fouad M' Fathy,Osama Salama and Ahmed Massoud

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Effect of Mirazid (Commiphora Molmol) on
Experimental Heterophyidiasis
By
Dr. Fouad M. Fathy,Osama Salama and Ahmed
Massoud
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Introduction Aim
Heterophyids are common parasites of fish-eating
mammals including man. Early treatment is
important to avoid the potential complication of
egg emobolization to heart or brain. Praziquantel
(PZQ), the current treatment has potential
hazards including carcinogenicity and
mutagenicity. Mirazid (MZ), an oleo-resin derived
from Myrrh, was reported in several experimental
and clinical trials-to be safe and effective
against other trematodes, namely schistosomiasis
and fascioloiasis. Therefore, this work aimed at
studying the effect of mirazid on experimental
heterophyidiasis, using praziquantel as a
therapeutic control.
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Material and methods

• Three equal groups of swiss albino mice (each
  21) infected control (I), PZQ-treated (II) and
  MZ-treated (III), were subdivided into 3
  subgroups (a, b, c) which were given 1, 2, 3
  daily doses of the drug respectively. Infection
  dose 300 encysted metacercaria drug doses MZ
  500 mg/kg/day (emulsion form), PZQ 796
  mg/kg/day drugs were given starting from day 7
  P.I. Mice were sacrificed 1 day after the end of
  decided dosage (day 8, 9 or 10).
• Assessment of anti-heterophyid drug activity
• Adult worm count in intestine.
• Adult worm length measurement.
• Intestinal histopathological changes.
• SEM of adult worms.

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Results and conclusion
Efficacy of MZ was proved by significant
reduction in adult worm count and length compared
to infected controls, with a 100 clearance of
the parasite after the triple dose (500
mg/kg/day-for 3 successive days). PZQ gave 100
cure after single dose. SEM of MZ treated
heterophyids, showed tegumental damage with
deformity and erosion of palmate scaly spines,
and loss of sensory ciliated papillae. By light
microscopy, intestinal and liver tissues were
normal in MZ-treated group. Therefore, since MZ
is an effective safe, natural and new drug (no
resistance), it is recommended for use (in
emulsion form) as an alternative to PZQ in
treatment of human heterophyidiasis.
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Efficacy of new benzothiazole dervatives against
experimental trichinosis and schistosomiasis
By Suzanne F.El-Nassery, Sonia R.Allam, Safya
M.Ali, Lobna A.El-Zawawy, Iman R.Ibrahim, Fouad
M.Fathy, and Mona M.Mahran
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Introduction Aim

Benzothiazole derivatives were reported to have
potential antihelminthic activities (e.g. in
schistosomiasis). They are the isosters of
benzimidazoles (mebendazole and albendazole)
currently used for treatment of trichinosis with
more effect on intestinal phase than on encysted
larvae. Therefore, this work aimed at synthesis
of a new series of benzothiazoles and studying
their efficacy against two important parasites,
namely, T.spiralis as a nematode model having
both intestinal and tissue phase, and S.mansoni
as a trematode model.
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Material and method

Antinematode activity Six groups of mice (I ?
VI), subdivided into (a) and (b), received 6
compounds (a, b1, b2, c1, c2, d2) on days 2 and
35 P.I. respectively, to study the effect on
adult and encysted larva. Drugs were given for 3
days (dose 20 mg/kg/day). Mice were sacrificed 3
days later (on days 7, 40). Half of subgroup (a)
was sacrificed late with subgroup (b) on day 40,
to study the effect of early drug administration
on larviposition and encystement. Non-infected
control mice were simultaneously sacrificed.
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• Antinematodal drug activity was assessed by
• Adult worm count in intestine (on day 7 P.I.).
• Larval count (on day 40 P.I.).
• Length measurement of adult ? and larva.
• Reproductive capacity index (RCI ratio of
  ingested encysted larvae).
• SEM of adult and larva.
• Antischistosomal activity
• Two groups of mice received 2 compounds (d1,d2)
  on day 35 P.I. (dose, 20 mg/kg ? for 3 days), and
  sacrificed 10 days later (day 47).
• Parameters for assessment of drug activity were
• Adult worm count.
• Tissue egg count (in intestine and liver).
• Number and size of liver granulomata (by
  histopathology).
• SEM of adult schistosomiasis.

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• ?The benzothiazole derivative c2 (active moiety,
  R 5-acetoxymethyl furane) was the most
  effective cpd against early and late T.spiralis
  infection, as evidenced by significant reduction
  in adult count and length, larval count and
  length and RCI. The drug was more effective
  against larvae when given early. ( r 84
  early, 66 late). SEM also showed loss of adult
  folding pattern and cuticular erosion in larvae.
• ?Compound d1 (R bromophenyl), showed the
  highest antischistosomal activity as proved by
  maximal significant reduction in adult count,
  tissue egg count in intestine and liver, and in
  number and size of liver granulomata SEM of adult
  tegument revealed tubercle spacing, erosion and
  loss of spines.
• ? Compound c2 has satisfactory potency against
  both enteral and parenteral stages of T.spiralis,
  particularly when given early in infection,
  therefore, it can be a suitable alternative to
  albendazde. Compound d1 could be valuable in
  schistosomiasis if widespread resistance to
  praziquantel emerges.

Result and conclusion

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The Adjuvant Effects of IL-12 and BCG on
Autoclaved Leishmania major Vaccine in
Experimental Cutaneous Leishmaniasis
By
Magda Y. Michel, Fouad M. Fathy, Eman. H.
Hegazy, Eman D. Hussein, Maha M. Eissa and Doaa
E. Said.
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Introduction aim
Cutaneous leishmaniasis is a globally
distributed disease which causes skin ulceration,
scarring and deformity. Difficulties in control
of vector and reservoirs, together with known
drug resistance and toxicities, suggest that a
reliable vaccine remains to be the most practical
tool for control of the disease. L.amastigostes
multiply intracellularly in macrophages provoking
a cell-mediated type of immune response. IL-12 is
the central cytokine of CMI, produced by
sensitized macrophages, stimulates Th1 cell and
NK cell to secrete IFN-? which enhance the
intracellular killing of amastigotes via
increasing ROIs. Bacterial adjuvants which
enhance Th1 response are believed to act by
stimulating IL-12 secretion from macrophages.
Therefore, this work aimed at studying the
adjuvant effect of IL-12 itself on autoclaved
L.major vaccine (ALM) in experimental cutaneous
leishmaniasis, compared to the effect of BCG.
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Material and methods

• Swiss albino, mice were divided into 2 main
  groups. The test group (II) received (ALM
  IL-12) and the control group (I) divided into 5
  equal subgroups normal (Ia), infected (Ib), and
  3 other subgroups infected after receiving BCG
  (Ic), IL-12 (Id) and ALM BCG (Ie). Two doses of
  vaccine or adjuvant were given (2 weeks apart)
  and mice were infected after another 2 weeks.
  Leishmania strain (MHOM/IQ/81/Sukkar I)
  mass-cultivated on El-On's medium for vaccine
  preparation and infection. ALM L-promastigotes,
  killed by autoclaving (15 min. at 120C, pressure
  15 Ib). Dosages ALM 2X107/ 0.1 ml killed
  promostigotes. BCG 5 X 105 CFU/0.1 ml. IL-12
  0.1µgm/0.1ml. All injected intradermal over the
  sternum. Infection dose 2 X 107 viable
  promastigotes given intradermal in the right hind
  footpad
• Assessment of vaccine efficacy was done by
• Evaluation of cutaneous lesion development (16
  weeks).
• Measurement of footpad thickness.
• Parasite density (by impression smear).
• IFN-? in blood (by sandwich ELIZA- 4 weeks after
  infection).

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Result and conclusion
The best protection was conferred by the
combined vaccine (ALM IL-12) as evidenced by
significant reduction in footpad thickness,
compared to infected control ( late onset of
small healing ulcers without gangrene), most
significant low parasite density, and the highest
significant level of IFN-?. Good protection was
also provided by (ALM BCG) vaccine, where the
difference between the two combined vaccines was
significant in parasite density and IFN-? level,
but not in footpad thickness. Since IL-12 is a
safe natural cytokine with proved superior
adjuvant effect, and ALM is a safe killed vaccine
easily prepared with minimal protein loss,
therefore, the combined vaccine (ALM IL-12) is
recommended for use in immunoprophylaxis of human
cutaneous leishmaniasis.
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Mean footpad thickness of all studied groupsat
the different durations
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Mean grade of parasite density in footpads of
studied groups at the different durations
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The Utility of Lactoferrin in Differentiating
Parasitic from Bacterial Infections
By Safia M. A, Lobna A. El. Zawawy, Doaa E.
Said, Fouad M. Fathy, and Osama N. Mohamed.
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Introduction aim
Lactoferrin (LF) is an iron-binding glycoprotein
found in the 2ry granules of PMNc.s. It mediates
a defensive antibacterial function at mucosal
surfaces. Its secreted amount is considered a
marker of the activity and number of inflammatory
polymorphs which are liable to breakdown if
unfresh or preserved samples are used.
Lactorferrin level can, therefore, replace PMNc
counting in differentiating pathogens responsible
for diseases which differ in the degree of
inflammation. It was used for this purpose in
urine, CSF and middle ear effusion. Therefore,
this work aimed at studying the usefulness of
measuring lactoferrin titre-via lactoferrin latex
agglutination test (LFLA) test- in
differentiating some parasitic and bacterial
infections which share clinical manifestations
but differ in the degree of inflammation, namely
amoebic from bacillary dysentery and
S.haematobium from UTI infection, in stool and
urine respectively.
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Material and methods
Stool 3 equal groups of stool samples (each 20)
derived from true cases of amoebic dysentry
(confirmed by trophozoite in direct smear),
bacillary dysentery (by positive stool culture),
and normal free samples. Urine 3 groups of
urine samples (each 20), represent true cases of
S.haematobiuum (confirmed by eggs on microscopy),
UTI (bacterial cystitis-diagnosed by urine
culture) and normal free samples. LF titre was
measured in serial dilutions of stool and urine
samples. Counting of PMNc.s was performed by
microscopy in all samples. Occult blood and
microhaematuria strip tests were done in stool
and urine respectively. Blood TLC was done in
dysenteric cases.
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Results and conclusion
Significant high levels of LF titre, were
obtained in cases of bacillary dysentery and
bacterial UTI infections in stool and urine
respectively, contrary to cases of amoebic
dysentery and S.haematobium (characterized by a
lower degree of inflammation), where LF titre was
significantly lower. A positive significant
correlation between LF titre and the number of
PMNc.s was obtained in all groups, which confirm
the marker role of LF. Sensitivity of LFLA test
was satisfactory when used alone and increased
when combined with occult blood test in amoebic
dysentery (75 ? 95), with TLC in bacillary
dysentery (80 ? 100) and with microhaematuria
strip test in S.h. cases (80 ? ?90). LFLA test
is a simple and reliable screening test capable
of differentiating some parasitic from bacterial
infections. It saves performance of tedious,
costly confirmatory tests in both types of
disease. It can be done by paramedical staff
under field situations and in control programs.
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Measurement of respiratory burst of TNF and
IL-1 cytokine-activated murine peritoneal
macrophages challenged with Toxoplasma gondii
By Nibal A. Hammouda, Eman Ali Rashwan,
Eman. D. hussien,Eman Abo El-Naga and Fouad M.
Fathy.
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Introduction aim
Toxoplasma gondii is considered the most
successful protozoan, which prevails because it
adapts to its host keeping him alive and mostly
asymptomatic. T.tachyzoites survive in
macrophages in modified phagocytic vacuoles that
fail to trigger reactive oxygen intermediates
(ROI) formation and resist fusion with lysosomes.
Activation of parasitized macrophage with
cytokine is required to overcome the infection.
TNF and IL-1 share common biological activities
and were reported to protect mice against lethal
Toxoplasma infection their synergistic effect
inhibited the intracellular proliferation of the
parasite, possibly by increasing ROI (like H2O2
and OH). Therefore this work aimed at measuring
the respiratory burst of oxidative metabolism of
murine peritoneal macrophages, activated by TNF
and IL-1 and challenged with T. gondii
tachyzoites.
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Material and methods
Toxoplasma tachyzoites (virulent RH strain) were
harvested form peritoneal exudates of infected
mice, (after 3 days), filtered (polycarbonate
filter 3 µm mesh) and centrifuged (300 g ? 5
min). Murine peritoneal macrophages were
collected from normal mice (5 ml saline injected
intraperitoneal for 5 min ? withdrawn) and
adherent cells were cultured in plastic tissue
culture plates. Macrophage monolayer cultures
(each 5 X 105 cells) were incubated with each
test cytokine for 24 hr, before challenge with
Toxoplasma tachyzoites (5 X 105 organism/culture)
for 1 hr. Respiratory burst of challenged
macrophages was measured by chemiluminescence
index (c.p.m) in activated and non-activated
cultures. (after addition of zyomsan and luminol,
in liquid scintillation counter).
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Result and conclusion
Activation of murine peritoneal macrophages by
either TNF-? or IL-1 (prior to challenge)
produced significant increase of
chemiluminescence after challenge. Therefore,
these two cytokines are believed to mediate
intracellular killing of Toxoplasma tachyzoites
via increasing respiratory burst with production
of more ROIs which are directly toxic to the
parasite. A biotherapeutic role for TNF and IL-1
- through this mechanism - is justified in human
infection.
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The Influence of BCG as an Adjuvant on
Toxoplasma gondii Vaccination
By Mervat Z. El Azzouni, Fouad M. Fathy, Nahed
M. Baddour2 and Maha M. Eissa
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Introduction aim
Toxoplasma gondii is one of the most prevalent
protozoan parasites on the earth affecting a wide
range of warm blooded vertebrates. In man benign
infection may reactivate under conditions of
immunosuppression resulting in fatal
encephalitis. Abortion or fatal neonatal
malformations complicate primary infection during
pregnancy. An effective vaccine could protect man
and domestic animal or prevent shedding of
oocysts in cats. Autoclaved Toxoplasma vaccine
(ATV), is safe and easily prepared with minimal
loss of proteins and no autodegradation, however
being a type of killed vaccine it requires
adjuvant to increase its immunogenicity. Since
Toxoplasma tachyzoites multiply intracellularly,
an adjuvant which enhance Th1 response is
considered appropriate. Therefore, this work
aimed at studying the adjuvant effect of BCG
(Bacille Calmette-Guerin) on autoclaved
Toxoplasma vaccine (ATV).
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Material and method

• Mice were divided into 4 equal groups (I, II,
  III, IV-each 20) normal, infected, BCG-infected
  and combined vaccine (ATV BCG) infected group.
  Three doses of vaccine or adjuvant were given at
  days 1, 10, 30. Dosages ATV 8 x 106/0.1 ml,
  autoclave-killed T. tachyzoites (15 min at 120C,
  15Ib,). BCG 5 x 105 CFU/0.1 ml. Both given
  subcutaneously over the sternum. Mice were
  challenged 1 month later with viable Toxoplasma
  tachyzoites (virulent RH strain). Half the mice
  in infected groups was sacrificed on day 3 P.I.
  and the rest were observed for survival rate.
• Parameters for assessment of vaccine or adjuvant
  efficacy
• Mice survival rate.
• Parasite-load in tissue (liver, spleen, lung,
  brain-by impression smear).
• Histopathological study of liver.
• CD8 percentage in spleen (by flowcytometry).

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Result and conclusion
The combined vaccine (ATV BCG) conferred
evident protection against Toxoplasma challenge
infection as proved by significant increase in
mice survival rate (14 days), marked reduction of
parasite density in tissue (liver, spleen, lung,
brain) and significant increase in percentage of
splenic CD8 cells (17.9). BCG proved to be an
effective adjuvant capable of enhancing the
immunogenicity and protection induced by a
special type of safe killed vaccine, namely,
autoclaved Toxoplasma vaccine. The combined
vaccine (ATV BCG) is, therefore, recommended
for use in man and domestic animals.
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The survival rate of the different studied groups
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Study on Human Ophthalmomyiasis Externa caused by
Oestrus ovis Larva, in Sirte-Libya Parasite
Features, Clinical Presentation and Management
By Fouad M Fathy, Adam El-Barghathi, Abdalla
El-Ahwal and Shaban El-Bakar
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Introduction aim
Oestrus ovis (sheep botfly) is-by far- the
commonest cause of human ophthalmomyiasis. The
disease is common in Mediterranean region
including north Africa. The larva usually limits
its activity to the conjunctiva and cornea,
however, penetration of the globe is a potential
complication. Misdiagnosis is common because the
larva is easily overlooked during routine
ophthalmologic examination. Therefore, the
present work aimed at studying the parasite
structural features, highlighting its damaging
capacity together with the related clinical
manifestations and treatment strategy.
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Material and methods
Material included 20 cases of human ophthalmic
oestrosis (attending university hospital in
Sirte-Libya, in 1 year). Personal, past and
present history of complaint were recorded.
Ophthalmologic examination was done using
slit-lamp. Treatment and follow up of patients
were undertaken. The larva examined in situ by
slit-lamp, isolated intact and examined by light
microscopy, both in living and preserved
preparations.
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Result and conclusion
History data showed that the condition was
commoner in adult ? shepherds or farmers in rural
area in worm seasons. The clinical presentation
was that of acute catarrhal conjunctivitis.
Conjunctival hyperemia was found in all cases,
focal haemorrhage in 50, keratitis and corneal
ulceration in 20 and 5 respectively. By slit
lamp, the larva was small (1 mm), fusiform,
translucent and fast-moving avoiding the light
beam and therefore easily overlooked it could be
isolated intact by a swab-mounting technique.
Basic effective treatment consisted of mechanical
removal, saline washout and occlusive-suffocation
of any residual larvae by using liberal amount of
antibiotic ointment. Improvement was rapid
(mostly within 2 days). The larva was identified,
as the 1st stage instar of O.ovis, by the
T-shaped cephalophyaryngeal skeleton and 2
posterior respiratory spiracles opening ventrally
on the 8th abdominal segment. Detailed
microscopic examination of larval surface
structures, revealed a multicomponent
attachment apparatus consisting of backward
curved pair of oral hooks, intersegmental spines,
forward curved caudal spines and a multilayered
spiny thoracic complex described for the first
time.
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In Conclusion

• ?The condition is not uncommon and should be
  born in mind in diagnosis of acute catarrhal
  conjunctivitis. Important criteria for diagnosis
  include sudden mobile foreign body sensation with
  abrupt itching and lacrimation, occurring in
  endemic area in worm season even without recalled
  history of fly strike. These signs justify
  careful eye examination using local anaesthetic
  and fluorescin stain drops to diminish larval
  movement and trace its track.
• ?The injurious observed attachment structures
  are believed be responsible for observed
  haemorrhage and ulceration and even potential
  invasion-if time is given-as these signs were
  more obvious in neglected cases (gt 1 week).
• ?The disease should be considered as
  occupational hazard among shepherds and farmers.
• ?Treatment of natural I.H.s blocks insect life
  cycle and should protect man and save economic
  losses.

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Dr. Fouad
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