Evaluation of Chitosan Nanoparticle Antimicrobial Effect on Isolated Listeria monocytogenes Bacteria from Pregnant Women and L. monocytogenes ATCC 7644.

Background: Listeria monocytogenes is a gram positive, facultative intracellular bacteria and it is a causative agent of listeriosis. Abortion is one the most important side effect of listeriosis. Nano chitosan is widely used as nanomaterials considered due to its characteristics such as bactericidal and nontoxicity activity. The aim of this study was isolation of L. monocytogenes bacteria from pregnant women vaginal samples and evaluation of chitosan nanoparticles effects against them. Methods: Overall, 100 vaginal specimens were collected from pregnant women with and without a history of abortion referred to Tehran's Hospitals from Sep 2019 to Jul 2020 with questionnaires. Then, using biochemical methods, L. monocytogenes bacteria were isolated and identified. Isolated L. monocytogenes strains were confirmed using PCR and evaluation of prfA gene, which is the main gene for identification of this bacterium. The effect of chitosan nanoparticles was evaluated in comparison with the antibiotics used based CSLI guideline on isolated bacteria by well diffusion method. MIC and MBC were determined for nanoparticle at the end. Results: Five strains of L. monocytogenes that were confirmed by PCR method. Moreover, a statistically significant relationship was observed between the isolated strains and the samples taken from women with a history of abortion. MIC and MBC for L. monocytogenes ATCC 7644 were 156.25 and 312.5 µg/ml and for 5 isolated strains were 78.12 and 158.25 µg/ml, respectively. Conclusion: L. monocytogenes could be a causative agent of abortion in pregnant women. Concerning the results, Nano chitosan has acceptable antibacterial activity against L. monocytogenes.

Investigating the Efficiency of Recombinant FliC-Loaded Bacillus subtilis Spores in Mice Immunization against Salmonella enterica Serovar Typhi.

BACKGROUND: Bacterial spores are among the most efficient vaccine delivery vehicles. Because of their safety and efficacy, Bacillus subtilis spores are increasingly used in this regard. The negatively charged surfaces of the spores allow antigens to be adsorbed onto these structures. In this study, a candidate vaccine against Salmonella enterica serovar Typhi was adsorbed onto B. subtilis spores and the immunogenicity of the formulation was investigated in BALB/c mice. METHODS: This work was performed during 2018-2019 in Islamic Azad University of Lahijan. FliC protein was recombinantly expressed in E. coli BL21 (DE3) cells and purified by affinity chromatography. On the other hand, B. subtilis strain PY79 (ATCC1609) was cultured in DSM medium and after the sporulation, FliC protein was adsorbed onto the spores in three different pH values (4, 7 and 10) and the adsorption was verified using dot-blot assay. FliC-adsorbed spores were then administered to BALB/c mice through the subcutaneous route. Mice immunization was evaluated by serum IgG assessment and challenge study. RESULTS: FliC protein was successfully expressed and purified. Sporulation was controlled by phase-contrast microscopy. Serum IgG assay showed significant stimulation of the mice's humoral immune system. Immunized mice were able to resist bacterial infection. CONCLUSION: The results showed the efficiency of spores as natural adjuvants for the stimulation of mice immune system. The formulation can be exploited for the delivery of recombinant vaccines against bacterial pathogens.

Two new rapid PCR-based methods for identification of Acinetobacter baumannii isolated from clinical samples.

The accurate identification of Acinetobacter spp. is challenging due to their high phenotypic and biochemical similarities. Because clinical relevance and antibiotic susceptibility are significantly different among different genomic species of Acinetobacter, the exact identification of A. baumannii is necessary and it can help us prevent inappropriate antibiotic use and inferior clinical care. This project employed a sequence-specific PCR assay for the rpoB region in A. baumannii to distinguish it from non-Acinetobacter baumannii Acinetobacter species. Moreover, a duplex PCR assay was used to detect blaOXA-51-like and gluconolactonase genes as a second identification method. In this study, 210 isolates of Acinetobacter spp. were considered and identified by PCR-sequencing of rpoB gene as a reference test. PCR-sequencing of rpoB revealed that 179 isolates were A. baumannii and 31 were non- A. baumannii Acinetobacter strains. PCR amplification targeting the rpoB gene as the first method, detected 182 isolates of A. baumannii, while duplex PCR assay confirmed 163 isolates as A. baumannii. Data analysis indicated that the sensitivities of sequence-specific PCR of the rpoB gene and duplex PCR assay were 100% and 91.06%, respectively, while specificities were 91.18% and 100%, respectively. Given the data, it was revealed that these two methods showed a reasonable potential for the accurate identification of A. baumannnii from non- A. baumannii species. Sequence-specific PCR assay for the rpoB gene and duplex PCR assay for blaOXA-51-like and gluconolactonase genes are rapid, reliable and cost-effective methods which can be used in clinical laboratories for the accurate identification of A. baumannii.

Design, Expression and Purification of Strongyloides stercoralis IgG4 Immunoreactive Protein (NIE) in Escherichia coli.

BACKGROUND: Strongyloidiasis is a public health concern in northern regions of Iran, caused by Strongyloides stercoralis. Auto-infection cycle can be resulted in high parasitic load, especially in immunocompromised hosts. Because of low sensitivity of stool culture and stool-based microscopy techniques, detection of antibodies in patient's sera can be an alternative diagnostic technique for detection of the nematode. In the present study, as the first step of the development of an ELISA kit for the detection of antibodies against the nematode, IgG4 immunoreactive protein (NIE) was expressed in Escherichia coli expression system, purified and verified. METHODS: The NIE gene sequence was retrieved from the GenBank. This sequence was codon-optimized for the expression in E. coli BL21 (DE3). The sequence was inserted into the expression vector pET-30b (+). The recombinant vector was then transferred into competent E. coli BL21 (DE3). Transformed colonies were selected and verified by colony PCR. NIE gene expression was induced with IPTG induction. The protein production was evaluated by SDS-PAGE and verified using Western blotting. RESULTS: The codon-optimized NIE gene had required parameters for expression in E. coli. NIE protein was proved and verified by SDS-PAGE and Western blotting. CONCLUSION: NIE recombinant protein was successfully expressed in E. coli expression system in appropriate amounts. The recombinant protein can be used for developing ELISA kit in diagnosis of S. stercoralis.

Effect of Chitosan Nanoparticle from Penaeus semisulcatus Shrimp on Salmonella typhi and Listeria monocytogenes.

BACKGROUND: After cellulose, chitin is one of the most important polymers in crustaceans, insects, and fungi. Chitosan is one of the most important derivatives of chitin, which has important characteristics including degradability, non-toxicity, and biocompatibility antimicrobial and antioxidant properties. METHODS: Chitosan was extracted from Penaeus semisulcatus shrimp using chemical methods and the degree of its austenitization was determined using a sub-red spectrophotometer and XRD. The nanoparticles were then synthesized using the ionic gelation method and analyzed through SEM. The antimicrobial effects of nanoparticles were also evaluated using antimicrobial tests on Listeria monocytogenes and Salmonella typhi. RESULTS: Nanoparticles have antimicrobial activity and can inhibit bacterial growth at different concentrations. CONCLUSION: Chitosan nanoparticles have an inhibitory effect on Listeria monocytogenes, which is a gram-positive bacterium.

ZnO Q-dots as a potent therapeutic nanomedicine for in vitro cytotoxicity evaluation of mouth KB44, breast MCF7, colon HT29 and HeLa cancer cell lines, mouse ear swelling tests in vivo and its side effects using the animal model.

Nanoformulations derived from fine porous ZnO quantum dot nanoparticles (QD NPs) can offer strong potential medical applications; especially in cancer therapy. ZnO QD NPs was synthesized by sol-gel hydrothermal process, fast cold quenching and further smart surface functionalization methods to obtain ultrasmall size (1-4 nm) NPs. ZnO nanopolymer, a wetting agent, PEG co-solvent and water/oil emulsion stabilizer were considered in our nanofluid formulation. The resulting nanofluid was characterized by SEM, FTIR, photoluminescence, band gap energy, zeta potential and UV-Vis spectroscopy. The cytotoxic effects on the growth of four cancer cell lines were evaluated by MTT assay. The IC50 (µg/ml) values of 30, 41, 40 and 35 for KB44, MCF-7, HT29 and HeLa cells, respectively, after 48 h of nanoformulation treatment suggested the cytotoxic effect of this nanoformulation on these cell lines in a concentration-dependent manner (p < .05). ZnO nanofluid destroyed cancer cell lines more efficiently than the normal HFF-2 (IC50 = 105 µg/ml). The reduction in cell viability in response to ZnO nanofluid treatment induced apoptosis in the cultured cells. Skin sensitization test plus antibacterial activity were also measured. Side effect tests on 70 white mice in vivo resulted in only 3-4 abnormal situations in hepatic tissue section possibly due to the idiosyncratic drug reactions.

The Resistance to Plague Infection among Meriones persicus from Endemic and Non-endemic Regions in Iran: The Role of Gut Microbiota.

BACKGROUND: The present study was conducted approximately 40 years ago, but its results have not been released. At the time of this study, the importance of the gut microbiota was not fully understood. METHODS: Meriones persicus rodents, as one of the major reservoirs of Yersinia pestis bacterium in Iran, were compared in a disease endemic area (Akanlu, Hamadan, western Iran) and a non-endemic zone (Telo, Tehran, Iran) from 1977 to 1981. RESULTS: This study was able to transmit the resistance to Y. pestis to other rodents creatively by using and transferring gut microbiota. CONCLUSION: The study indicated for the first time that the gut microbiota could affect the sensitivity to plague in Meriones in Telo.

Combined Evaluation of AFP, CA15-3, CA125, CA19-9, and CEA Tumor Markers in Patients with Hepatitis B and C.

BACKGROUND: This study aimed to determine the role of tumor markers AFP, CA15-3, CA125, CA19-9 and CEA in patients with hepatitis B and C. METHODS: This descriptive cross-sectional study was performed from Oct 2012 to Oct 2014. Serum samples of 129 patients with hepatitis B and C referred to Guilan Liver and Digestive Disease Research Center in Rasht, Iran were collected and checked for the existence of the listed tumor markers by ELISA. RESULTS: No increase in serum levels of tumor marker CA19-9, CEA and CA15-3 were seen in patients with hepatitis (P>0.05). In patients with hepatitis B, increase in CA125 were observed (P=0.03). In hepatitis C patients, there was an increase in AFP levels (P=0.03). CONCLUSION: The levels of AFP and CA125 markers were high in hepatitis C and hepatitis B, respectively. However, the increased levels were not seen is malignancy. Due to the small sample size, further study is necessary to find the reasons of the increase.

Culture of Borrelia persica and its flagellar antigen in vitro.

Borrelia persica is a strain seen only in the Middle East and responsible for relapsing fever. These spirochetes are notable for multiphasic antigenic variation of polymorphic outer membrane lipoproteins, a phenomenon responsible for immune evasion. Diagnosis of the disease is a problem and requires a fixed antigen like the flagellar antigen. In vitro culture of B. persica was carried out for the first time and flagellar antigen was purified from culture. 10% SDS was added to the mixture to dissolve the cell wall and then the solution was sheared in an Omni mixer. Electron microscopy confirmed the purity of a 42 KDa periplasmic antigen as revealed by SDS-PAGE. Indirect haemagglutination kits were designed using the pure flagella and tested for cross reactivity with another relapsing fever spirochaete Borrelia microtii positive serum. The kit showed 98% sensitivity and 95% specificity.

Use of dense granule antigen GRA6 in an immunoglobulin G avidity test to exclude acute Toxoplasma gondii infection during pregnancy.

The usefulness of a specific immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) based on recombinant GRA6 antigen for distinguishing between acute and chronic Toxoplasma infection was investigated. Two sets of serum samples obtained from pregnant women with acute, chronic, or no Toxoplasma infection collected in France and Iran were used. Among the French subjects, 19 of 20 (95%) women who experienced seroconversion during the past 4 months before sampling displayed low-avidity IgG antibodies against GRA6, while all 17 (100%) women with chronic infection had high-avidity antibodies. When the Euroimmun IgG avidity ELISA was used, 15 of 19 (78.9%) recently infected women had low-avidity antibodies, and 20 of 22 (90.9%) women with chronic infection displayed high-avidity antibodies. The results suggested better performance of the GRA6 avidity ELISA than the Euroimmun avidity ELISA for exclusion of a recent infection occurring less than 4 months previously. Similarly, all 35 Iranian women with acute Toxoplasma infection had low-avidity antibodies against GRA6, whereas all 34 women with chronic infection displayed IgG antibodies of high avidity, indicating the value of GRA6 avidity testing for ruling out a recent infection. Avidity tests based on lysed whole-cell Toxoplasma gondii antigen are currently used to exclude recently acquired infections; however, the use of recombinant antigen(s) might improve the diagnostic performance of avidity tests and facilitate the development of more standardized assays.

Sequence diversity of the C-terminal region of Plasmodium falciparum merozoite surface protein 1 in southern Iran.

The C-terminal region of the merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a strong vaccine candidate as it is associated with immunity to the parasite. This corresponds approximately to the conserved 17th block of the gene and is composed of two EGF- like domains. These domains exhibit only four single amino acid substitutions which show several potential variants in this region of the gene. As the variations might be important for a regional vaccine design, a study was carried out to determine the variations present in P. falciparum isolates from southern Iran. Besides the usual E-T-S-R-L and the Q-K-N-G-F types, we found Q-T-S-R-L, E-K-N-G-F, E-T-S-G-L, Z-T-S-G-L and Z-T-S-R-L types, where Z was E or Q signifying the presence of mixed clones in single isolates.

PCR detection and sequencing of parasite ITS-rDNA gene from reservoirs host of zoonotic cutaneous leishmaniasis in central Iran.

Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis (ZCL) in which gerbils are the reservoir host. ZCL is of great public health importance in Iran. In the current investigation, nested polymerase chain reaction (PCR) protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene). The PCR assays detected L. major in three rodent species: Rhombomis opimus, Meriones lybicus and, for first time, Meriones persicus. L. major parasite was found in Natanz, Isfahan Province in the center of Iran in a focus of rural zoonotic cutaneous leishmaniasis. Four L. major infections were detected in R. opimus species, three in M. Lybicus, and two in M. persicus. All nine rodent infections of L. major were found to be the same haplotype based on the PCR detection and sequencing of parasite ITS-ribosomal DNA gene. In addition, also for the first time, the nested PCR assays detected Leishmania tropica only in one M. persicus. Allied to studies in country, the new findings mean that past conclusions about the reservoir of L. major in Iran must be treated with caution. Finding two Leishmania species in different rodent species as reservoir in Iran, therefore, careful molecular eco-epidemiological investigations will be an essential part of modeling the roles of different gerbil species in maintaining and spreading ZCL foci.

Patterns of co-association of C-reactive protein and nitric oxide in malaria in endemic areas of Iran.

In addition to numerous immune factors, C-reactive protein (CRP) and nitric oxide (NO) are believed to be molecules of malaria immunopathology. The objective of this study was to detect CRP and NO inductions by agglutination latex test and Griess microassay respectively in both control and malaria groups from endemic areas of Iran, including Southeastern (SE) (Sistan & Balouchestan, Hormozgan, Kerman) and Northwestern (NW) provinces (Ardabil). The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In addition, more CRP and NO positive cases were observed amongst malaria patients in comparison with those in control group. A variable co-association of CRP/NO production were detected between control and malaria groups, which depended upon the malaria endemic areas and the type of plasmodia infection. The percentage of CRP/NO positive cases was observed to be lower in NW compare to SE region, which may be due to the different type of plasmodium in the NW (Plasmodium vivax) with SE area (P. vivax, Plasmodium falciparum, mixed infection). The fluctuations in CRP/NO induction may be consistent with genetic background of patients. Although, CRP/NO may play important role in malaria, their actual function and interaction in clinical forms of disease remains unclear.

Cryptosporidiosis in immunocompromised patients in the Islamic Republic of Iran.

BACKGROUND AND PURPOSE: Cryptosporidiosis is a parasitic zoonosis, which is prevalent all over the world. The manifestation of the disease is either self-limiting acute diarrhea in immunocompetent individuals, or potentially fatal chronic diarrhea in immunocompromised patients. METHODS: In this study, which was conducted in Tehran, 214 patients from ten health centers were investigated. Stool samples were collected, fixed and examined by three methods: acid-fast staining, auramin phenol fluorescence and direct fluorescence using monoclonal antibody. RESULTS: Overall, 1.4% of all patients and 6.3% of diarrheal patients were infected by Cryptosporidium. The results revealed three cases of cryptosporidiosis, including two cases of acquired immunodeficiency syndrome (AIDS) and one of acute myeloid leukemia (AML). The prevalence of infection in subjects with AIDS or AML who were suffering from diarrhea was 33.4% and 11.1%, respectively. The duration of disease in infected patients lasted for weeks, and was terminated by death in two AIDS patients. In the patient with AML, diarrhea lasted for 18 days, and stopped after discontinuation of immunosuppressive therapy. CONCLUSIONS: Immunosuppressed people are at a significant risk of severe or even fatal Cryptosporidium infections.

Patterns of co-association of C-reactive protein and nitric oxide in malaria in endemic areas of Iran

In addition to numerous immune factors, C-reactive protein (CRP) and nitric oxide (NO) are believed to be molecules of malaria immunopathology. The objective of this study was to detect CRP and NO inductions by agglutination latex test and Griess microassay respectively in both control and malaria groups from endemic areas of Iran, including Southeastern (SE) (Sistan & Balouchestan, Hormozgan, Kerman) and Northwestern (NW) provinces (Ardabil). The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In addition, more CRP and NO positive cases were observed amongst malaria patients in comparison with those in control group. A variable co-association of CRP/NO production were detected between control and malaria groups, which depended upon the malaria endemic areas and the type of plasmodia infection. The percentage of CRP/NO positive cases was observed to be lower in NW compare to SE region, which may be due to the different type of plasmodium in the NW (Plasmodium vivax) with SE area (P. vivax, Plasmodium falciparum, mixed infection). The fluctuations in CRP/NO induction may be consistent with genetic background of patients. Although, CRP/NO may play important role in malaria, their actual function and interaction in clinical forms of disease remains unclear.

Evaluation of protective effect of recombinant dense granule antigens GRA2 and GRA6 formulated in monophosphoryl lipid A (MPL) adjuvant against Toxoplasma chronic infection in mice.

To investigate the vaccine potential of both the Toxoplasma GRA2 and GRA6 antigens, the full length recombinant proteins were produced in Escherichia coli, formulated in MPL adjuvant, and used alone and in combination ("mix"), to immunize CBA/J mice. Although high ratios of specific IgG2a/IgG1 were measured against both proteins, only spleen cells from GRA2-immunized mice and mix-immunized mice produced high amounts of both IFN-gamma and IL-2 upon induction with Toxoplasma gondii Excretory-Secretory Antigens. Intra peritoneal challenge with Toxoplasma cysts resulted in significant reduction of brain cysts in GRA2- and in mix-vaccinated mice only. This study shows the protective efficacy of recombinant GRA2 against chronic infection by T. gondii and confirms the utility of MPL adjuvant in enabling a vaccine candidate to induce a protective Th1 immune response.

Cryptosporidiosis among immunocompetent patients with gastroenteritis in Iran: a comparison with other enteropathogenic parasites.

BACKGROUND AND PURPOSE: Cryptosporidium is a protozoan parasite that reproduces within the epithelial cells of several organs of vertebrate hosts. The manifestation of the disease is either self-limiting acute diarrhea in immunocompetent patients, or fatal chronic diarrhea in immunocompromised patients. Common clinical symptoms include watery diarrhea, abdominal pain, and weight loss. METHODS: This randomized pilot study conducted in Tehran, Iran, included 104 children and adult patients with gastroenteritis referred to the Children's Hospital Centre and Pasteur Institute of Iran. Control samples from healthy individuals (36 children and adults) were also collected; the entire test group had diarrhea and the control group had formed stool consistency. Stool samples were primarily examined by the direct method, then fixed and tested by 3 assays including acid-fast staining, auramine phenol fluorescence, and direct fluorescence using monoclonal antibody. RESULTS: The study revealed that 2.9% of the patients were infected by Cryptosporidium spp. Other parasites observed included Giardia lamblia (5.8%), Ascaris lumbricoides (1.9%), and Entamoeba histolytica (0.96%). Formed stool samples showed no oocysts of Cryptosporidia. CONCLUSIONS: In addition to common enteropathogenic organisms, Cryptosporidium is indicated as a key causative agent of diarrhea in humans. Although cryptosporidiosis may, in many cases, be terminated by self-limiting mechanisms, it could cause pathologies requiring preventive and therapeutic policies.

Pharmacological evaluation of anti-leishmanial activity by in vivo nitric oxide modulation in Balb/c mice infected with Leishmania major MRHO/IR/75/ER: an Iranian strain of cutaneous leishmaniasis.

Cutaneous leishmaniasis with a variation in its clinical signs is still one of the health problems in the world, region and Iran. Immune responses against leishmania consist of cytokines, immune cells and mediators. Macrophages participate actively in the inflammatory response by releasing chemokines and mediators including nitric oxide (NO) and reactive oxygen and nitrogen intermediates. This study investigates whether NO had anti-leishmanial effects and/or mediated pathology in mice infected with Leishmania major MRHO/IR/75/ER (IR/75). NO inducer lipopolysaccharide (LPS) and NO donor S-nitrosoglutathione (SNOG) were used for their ability to increase RNI and to modify leishmania infection in susceptible Balb/c mice, in order to evaluate the effects of NO production on size and lesion macroscopy, delay of lesion formation and proliferation of amastigotes inside macrophages. Liver, spleen and lymph nodes were also studied as target organs to detect amastigotes. In addition to plasma, liver and spleen suspensions were investigated for NO induction by using Griess microassay. Statistical analysis of data revealed an association between increases in NO level with the pathology of disease in Balb/c mice infected with L. major IR/75. The survival of leishmania parasite inside the macrophages and its proliferation was affected by LPS and SNOG-treatments. An inconsistent relationship was evident between the NO modulation and pathological changes in treated Balb/c mice infected with L. major IR/75.

Reactive nitrogen intermediate production and tolerance variability in different mouse strains after in vivo treatment with lipopolysaccharide from Salmonella abortus equi.

Lipopolysaccharide (LPS) stimulation in animal models generates a large number of immune factors including cytokines and mediators. It also acts as a potent inducer of macrophage reactive oxygen intermediates and reactive nitrogen intermediates (RNI). RNI as stable metabolites of nitric oxide (NO) are produced by cells stimulated with LPS and cytokines. In this study, LPS from Salmonella abortus equi was investigated as an inducer of RNI in untreated controls and test groups of white Naval Medical Research Institute (NMRI) mice. Animals were humanely killed at 30, 60, 120 and 180 min after LPS injection, and plasma RNI was measured by Griess microassay. In a further experiment, host tolerance against bacterial LPS was evaluated by sequential intravenous injection of LPS concentrations of 4, 1 and 0.5 mg/kg at 24 h intervals in NMRI and with the same schedule but via subcutaneous injection in Balb/c mice. Statistical analysis of RNI values using analysis of variance test indicated that in vivo LPS stimulation induced high levels of NO in murine hosts (p<0.001). Comparison of RNI levels at different times after administration revealed the largest amount of RNI at 180 min after inoculation. Analysis of the time course until maximum RNI induction indicated that NMRI mice had the longest delay, suggesting a difference in tolerance of NMRI and Balb/c mice to LPS stimulation dependent on LPS concentration, dose, and route of inoculation.

Immunochemical characterization of alkaline phosphatase from the fluid of sterile and fertile Echinococcus granulosus cysts.

Alkaline phosphatase (ALP) from hydatid cyst fluid (HCF) was purified and characterized for comparison between fertile and sterile HCF. Samples were obtained from slaughtered sheep and then sterile and fertile cysts were separated. ALP was purified from aspirated cyst fluid and biochemical parameters were determined. Sera from patients with hydatid disease (15 samples) and patients with other parasitic diseases including fascioliasis (2 samples), taeniasis ( Taenia saginata, 5 samples) and also sera from uninfected controls (15 samples), were collected and used in immunoblotting experiments with ALP from sterile and fertile HCF as antigen. Our results showed that ALP activity in fertile HCF [10.75+/-3.78 (SD) U/ml) was significantly more than in sterile HCF (6.25+/-2.43 U/ml). There were also some differences between the kinetic parameters and biochemical characteristics of ALP in fertile and sterile HCF. Immunoreactive bands were clearly observed when sera from hydatid infected patients were tested with ALP from fertile HCF as the antigen. However, this method revealed no cross-reaction between purified ALP from sterile HCF and sheep liver tissue. These findings suggest that there is some variation in the immunochemical characteristics of ALP from fertile and sterile HCF.