nader pestehchian

Leishmaniasis is one of the endemic diseases in Iran and Isfahan province. This study was conducted to investigate the trend of cutaneous leishmaniasis in 6 main centers of Isfahan province during the years 2007 to 2018.

This research is a cross-sectional descriptive-analytical study that was conducted based on the data available in the Zoonosis Diseases Unit located in the Isfahan Health Vice-Chancellor. The data related to the new cases of cutaneous leishmaniasis that were reported by the detection laboratories as well as the data related to the control measures and the fight against the reservoir were collected. The trend of disease incidence was analyzed separately by foci, year and the relationship between disease incidence and control measures.

The results showed that during this 11-year period, the incidence of leishmaniasis is decreasing, but some centers, such as the number 1 Isfahan region, have had severe fluctuations in some years. The relationship between the average incidence of the disease and the total number of poisons consumed using the Pearson Correlation statistical test was linear and significant. In other words, in the centers where the highest amount of poison has been consumed, the incidence rate of leishmaniasis is also reported to be higher.

Considering the fluctuations of the disease in some years, and the number 1 Isfahan region has the highest rate of incidence despite the highest amount of poisons consumed compared to the Shahin-shahr region, the effectiveness of this type of intervention in the long term is doubtful.

Toxocariasis is a parasitic disease caused by the larval stage of Toxocara canis and Toxocara cati. Infective stage of this parasite for human develops on soil. So, in this work contamination of the soil of public environments in five geographical areas of Isfahan province of Iran has been investigated.

In this descriptive study, 355 soil samples were collected from parks, children’s playgrounds, student dormitories, and university environments, and examined by Flotation method. The samples were then inspected using microscopic and molecular methods.

From the 355 examined soil samples in 77 (21.69%), and 87 (24.50%) cases Toxocara eggs were detected by microscopic and molecular methods, respectively. In the molecular method, 31 (8.70%) cases of T. cati and 44 (12.39%) cases of T. canis were identified.

Toxocara eggs were identified in all areas of Isfahan province, although contamination rate was higher in Fereydun Shahr and Semirum counties.

Toxoplasma gondii (T. gondii) is a pathogenic and zoonotic parasite, which felines implicate as definitive hosts; intermediate hosts are warm-blooded vertebrates. The protozoa can cause serious symptoms in humans, while in poultry is usually asymptomatic. The prevalence of Toxoplasma gondii in poultry due to the way poultry are fed is an important indicator of the distribution of oocysts in the environment; in addition, consumption of raw or under cooked meat of chickens can cause infection in human and other animals. Therefore, in this study, the prevalence rate of infection to toxoplasma gondii in domestic and industrial breeding poultry in Isfahan City, Iran, was assessed.

From three groups of domestic breeding, broiler, and laying eggs poultry, 60 blood clot samples were collected. On collected serums, serological modified agglutination test (MAT) was performed. Toxoplasma gondii-specific antibodies were assayed by this test. Then, the obtained results were analyzed.

By performing the MAT serological test, 20, 15, and 30 samples were positive in domestic breeding, industrial broiler, and in laying eggs samples, respectively. Therefore, relative frequency was 33.3, 25.0, and 50.0 percent in domestic breeding, industrial broiler, and industrial laying eggs, respectively, which by performing chi-square test and calculating the P < 0.050 between the three groups, a significant difference was observed serologically.

A considerable percent of domestic and industrial poultry was infected with Toxoplasma gondii. Therefore, preventive measures should be conducted to provide safe foods for vertebrate animals and human. </div

Leishmaniasis is one of the most important vector-born zoonosis diseases that transmits to human by sandfly species. Cutaneous leishmaniasis is one of the most important public health problems in majority areas of Iran. The current study was carried out to identify species of sand flies in Dezful District, Khuzestan Province, Iran.

Sandfly specimens were collected by using sticky traps from indoors and outdoors of different parts of Dezful City during the years 2018-2019. All sandflies were identified by valid identification keys based on external and internal morphological characters of the head and genitalia of both males and females.

2132 sandflies were collected. 8 species of sandflies from the Psychodidae family and the Phlebotomine subfamily were identified. Phlebotomus papatasi (46%) was the dominant species and Sergentomia squamiplaris (1%) was the non-dominant species in the area. Phlebotomus papatasi with 27.67% of mosquitoes caught as the dominant species indoors, and Sergentomia tibriadis was the dominant species in outdoors with 21.43% of mosquitoes caught. The peak activity of sandflies was in August, September, and October.

Due to the suitable climate, prevalence and species diversity of sanflies in Dezful is high. Phlebotomus papatasi sandfly is as the dominant vector species of cutaneous leishmaniasis in this region.

Isfahan province is one of the major foci of cutaneous leishmaniasis in Iran, and Ardestan City with 93 foci is one of the infected areas. This study aimed to examine the disease trend in the last seven years considering the environmental and ecological changes in Ardestan.

The present study was a retrospective and census type. 1278 cases enrolled in the study who were diagnosed with the acute form of leishmaniasis in Ardestan during the years 2012-2019, and also had a file in Ardestan health care centers. Patients information about age, sex, residency, occupation, place of work, the month of incidence of illness, duration of illness, number of wounds, nationality and date of incidence were entered in the related checklists.

The highest prevalence of leishmaniasis was observed among young Ardestanian with 20-29 years old and with a rate of 38.4%, and the lowest prevalence rate was observed in Zavareh villages with the rate of 5.0%. Moreover, the incidence of disease was significantly higher in men, natives infected compared to immigrants, and patients who live in urban areas compared to rural areas. In terms of occupation, "housewife/unemployed" had the highest prevalence. Besides, children, adolescents, and young people have the highest frequency. The incidence rate in Ardestan was also estimated 1.2 per thousand people

Although, the rate of incidence has been decreasing during 2012-2018 due to reasons such as carrying out programs to control vectors and reservoirs, but increasing rate of incidence of disease in 2019 in the area shows the importance of public health problem of this endemic disease.

We aimed at genotyping and evaluating the predominance of G. duodenalis assemblages isolated from patients referred to medical laboratories in Khorramabad, Iran from Nov 2015 to Sep 2016. Hence, the development of a cost-effective HRM approach to determine genotypes of G. duodenalis based on the triosephosphate isomerase (tpi) gene was examined and the genotyping results with and without diarrhea was compared.

Seventy G. duodenalis positive fecal samples were collected. A microscopic confirmation for the presence of Giardia spp. was performed, cysts of 70 Giardia spp. positive specimens were concentrated using sucrose flotation technique and sucrose solution PCR amplification was performed on 69 of 70 (98.5%) samples, and High Resolution Melting (HRM) analysis was performed using a software.

The results showed two distinct genotypes (assemblages A and B) of G. duodenalis but infections with mixture of both assemblages were not detected. The genotypes of G. duodenalis showed that the sub assemblage AI, BIII and BIV were present in a proportion of 68.1%, 20.3% and 11.6% respectively in samples. Assemblage AI was significantly (P<0.05) more frequently found in patients with diarrhea.

The sub-assemblage AI, BIII, and BIV are more zoonotic potential. According to the comparison of the results of this study with the results of previous studies in this area and around of it, as well as the way people live and keep pets. This pattern established in Khorramabad city. HRM can be an ideal technique to detect and genotyping of G. duodenalis in clinical samples.

Human toxocariasis is caused by Toxocara canis and cati, which are the main ascarids of dogs and cats, respectively. Most infected humans are asymptomatic and our understanding of human disease burden is limited by lack of epidemiological studies and insufficient clinical awareness. There is no precise report on the seroprevalence of toxocariasis in Ilam children. Therefore, this study found an opportunity to investigate this subject. In this cross-sectional study, 300 sera of children aged between 2 to 15 years old were collected during March 2016 to February 2017 in urban and rural areas. Demographic variables were filled for each person in accordance with risk factors along with sampling. Some hematological parameters were measured. The sera were examined for anti Toxocara canis and cati antibodies, according to the ELISA kit protocol. Among a total of 300 serum samples, 35 (11.7%) were positive for anti-Toxocara IgG. The infection rates were 5.3% and 6.3% in female and male, respectively. There was no significant relationship between gender and Toxocara infection rates (P = 0.59). A total of 26 (17.3%) of the 150 rural children, and nine (6.0%) of the 150 urban children were positive. There was a significant relationship between place of living with ELISA tests results (P = 0.004). Hematologic parameters showed a significant increase in the numbers of peripheral eosinophil in the sample of patients whose sera were positive (P = 0.037). High prevalence of toxocariasis among Ilam children in the west of Iran can be considered as a public health problem. The evaluating infection control programs in dog and cats are necessary for controlling the disease in this region.

Toxoplasma gondii can infect a wide range of mammalians, especially humans. It controls several intracellular signals for the inhibition of apoptosis. This study aimed to investigate the apoptogenic effect of cisplatin and sodium azide on T. gondii infected HeLa cells and isolate apoptotic bodies (blebs) as a potent stimulator of the immune system. The cytotoxic properties of cisplatin and sodium azide (NaN3) on HeLa cells were evaluated by MTT assay. Moreover, the apoptogenic activity of cisplatin and NaN3 was studied using flow cytometry (Annexin V/PI double staining) and scanning electron microscopy (SEM). Finally, apoptotic bodies were separated by centrifugation. MTT assay data showed that the survival rate of cells treated with different concentration of NaN3 was significantly reduced, compared to negative control groups. Concerning cisplatin, only concentration of 20 μM had not a significant impact on the cell viability; however, the other concentration of cisplatin significantly reduced cell viability, compared to negative control groups. The level of early apoptosis in uninfected HeLa cells was higher compared to infected HeLa cells treated with cisplatin and NaN3. Finally, apoptotic bodies were separated from T. gondii infected HeLa cells treated with cisplatin. Apoptosis was induced in both uninfected and infected HeLa cells with T. gondii and apoptotic bodies were isolated from infected cells. Therefore, further studies on apoptotic bodies are required in order to find a proper candidate for vaccine preparation against T. gondii infections.

A novel series of antimony (V) complexes with the hydroxypyranone and hydroxypyridinone ligands were synthesized and characterized by 1HNMR, FT-IR and electron spin ionization mass spectroscopic (ESI-MS) techniques. The synthesis process involved protection of hydroxyl group followed by the reaction of the intermediate with primary amines and finally deprotection. All compounds were evaluated for in vitro activities against the amastigote and promastigote forms of Leishmania major. Most of the synthesized compounds exhibited good antileishmanial activity against both forms of L. major. IC50 values of the most active compounds; 9d, 9d and 9e, after 24, 48 and 72 h against amastigote model were 15, 12.5 and 5.5 µg/mL, respectively. 9e, 11 and 9e inhibited the promastigote form of parasite after 24, 48 and 72 h with IC50 values of 10, 2 and 1 µg/mL, respectively.

Soil contamination with parasites is a serious risk for parasitic infections in humans and animals. Because of the importance of soil, as an important source for transmission of parasites to humans and animals, this study was carried out to determine the prevalence of various forms of parasite oocytes and helminthes ova.
A total of 150 soil samples were collected from 150 sites in public places of Isfahan City, Iran, in 2016. Isolation of parasites from the soil was performed by saturated solution of sodium nitrate. Then, the samples were observed under microscope and positive isolates to sporulation and better recognize, were poured in 3% potassium dichromate. All the isolates were studied for further investigations with acid-fast specific staining.
Findings: from 150 samples, 84 were positive and the rest were free of any contamination. 33 samples had multiple simultaneous contaminations. 2 samples were positive for Toxoplasma gondii oocytes, 12 samples for Isospora sp. oocytes, 6 samples for Eimeria sp. oocytes, 10 samples for Cryptosporidium sp. oocytes, 13 samples for Toxocara canis oocytes, 6 samples for Toxocara cati oocytes, 4 samples for Toxascaris leonina oocytes, and 65 samples for larvae of soil nematodes.
Soil is considered as an important potential source in transmission of parasitic infections. Environmental hygiene is required for prevention of soil pollution and control of soil-borne parasitic infections especially. According to our study, the prevalence of parasites in the soil is decreased.

Malaria is caused by a parasitic protozoan called Plasmodium with an estimated mortality rate of one million people annually and it is estimated that half of the world''s population are at risk for this disease.
The current study aimed to compare the effect of hydroxychloroquine and pyrimethamine with sulfadoxine-pyrimethamine to treat Plasmodium berghei infection in mice. Pyrimethamine, hydroxychloroquine and sulfadoxine-pyrimethamine were used in the current study.
Four groups of mice each including nine mice were selected. The mice were infected by intraperitoneally injection of Plasmodium berghei infected red blood cells. One group was considered as a positive control and three other ones received each one of the drugs. On days 4, 7, 14, 21 and 28, a blood smear was prepared from each mouse, stained with Giemsa and the parasitemia rate was calculated.
Parasitemia in the positive control group showed an increase on days four and seven at a level of 5.9% and 23.7%, respectively, which was statistically significant compared with the other groups (P The results of the article suggested that malaria treatment strategy shifts completely from the monotherapy; therefore, the combination drugs should be used for this purpose, especially in endemic areas.

Toxoplasma gondii infection is usually detected by using immunoglobulin IgG and IgM antibodies in different populations. This parasite is implicated in the pathogenesis of many autoimmune diseases such as celiac disease (CD). In the present study the prevalence of Toxoplasmosis and the level of IL8 in patients with Celiac disease were evaluated.
In this case-control study in 2013, 150 patients with CD diagnosis were selected by using questionnaire and the presence of antibodies specific for Toxoplasma gondii (IgG, IgM) was detected by ELISA. On the other hand the level of IL8 was detected using available commercial kit.
patients (n=59, 39.9%) were positive for anti-toxoplasma IgG. On the other hand, only 2 of them were positive for IgM antibodies, which did not show statistical correlation to the disease (1.4%, P = 0.65). The mean level of IL8 was 176.38 (SD=191.79) in 150 CD patients, which did not show a statistical significant correlation between the mean level of IL8 and celiac disease (P=0.001).
Like other studies showing that intestinal infection can help to develop celiac disease in susceptible individuals, our results also revealed that Toxoplasma gondii infection can increase the risk of developing celiac disease.

The aim of this study was to determine the prevalence of Cryptosporidium infection in patients with multiple sclerosis compared to control group in Isfahan city, Iran.
In a case-control study, that was done in Alzahra hospital in Isfahan during 2015-2016, 50 multiple sclerotic and 50 healthy people were selected and the prevalence of Cryptosporidium in the two groups were detected by direct methods and concentration and compared between the two groups.
Findings: Only 3 cases out of 100 were positive for Cryptosporidium SPP where one case was from control and 2 cases were from the case group (2 versus 4 percent). But, prevalence of Cryptosporidium in the two groups was not statistically different (P = 0.56). Prevalence of this parasite in residence and domestic animal keeping had significant difference. 3 cases of positive were in rural areas and kept domestic animals on their houses (P In this study, there was significant difference between this parasite and residence and keeping domestic animal.

Diagnosis of cryptosporidiosis is based on the use of the routine microscopic methods, but these methods possess poor sensitivity and also need an expert technician. The aim of the present study was to determine sensitivity and specificity of dot blot method for diagnosis of bovine cryptosporidiosis.
In this study 131 fecal samples were collected from suckling calves during summer 2014. The collected samples were concentrated using formalin ether method. The samples were then examined by modified-Ziehl-Neelsen (mZN) and finally all samples were examined by dot blot methods.
Findings: Oocysts of Cryptosporidium spp. were found in 40 (30.5%) out of 131 fecal samples using mZN staining method but 49 (37.4%) of the samples were positive for Cryptosporidium antigen using dot blot method. No significant differences were observed among the results of the two methods and the sensitivity and specificity of dot blot calculated as 87 % and 86%, respectively.
Based on the results of the present study, dot blot method showed rather equal performance as modified-Ziehl-Neelsen method.

Patients with diabetes mellitus (DM) are at increased risk of certain infections; however, little is known about the prevalence of intestinal parasitic infections in them. The aim of this study was to assess the risk of intestinal parasitic infections in patients with DM in comparison with a healthy control group.
This case-control study was conducted on 118 patients with DM and 118 healthy people as control group from April to September 2014. Oral glucose tolerance test (OGTT) and hemoglobin A1c level were checked, and checklists including risk factors for parasitic infections were filed for all participants. Three stool samples and one scotch tape were obtained. Samples were examined by direct wet smear, formol-ether concentration, Kinyoun acid-fast staining, and modified trichrome stain. Data were analyzed using chi-square and logistic regression tests.
The rate of parasitic infection was significantly more in the patients (26.3%) than the controls (6.8%) (P Patients with DM might be at an increased risk of infection with intestinal parasites specifically B. hominis as an opportunistic infection, and routine stool examination should be considered for them.

Multiple Sclerosis (MS) is a chronic demyelinating disease of the central nervous system which has been identifies more prevalent in economically developed countries than in the developing countries. Low prevalence of parasitic infections (which can activate immune response and prevent or modulate damage to host antigens) in these areas is among the possible responsible factors for such a difference. In this study we aimed to compare frequency of blood-tissue parasitic infections in patients with MS, as compared to their healthy family members. This study was conducted on 50 relapsing remitting MS patients and 50 family members attending MS clinic at Alzahra Hospital. IgM and IgG anti-Toxoplasma gondii were measured. Given the high prevalence of cutaneous leishmaniasis in Isfahan, all the participants were also examined for protozoan leishmania microscopically. Furthermore malaria parasite was investigated. Eighteen patients and 24 healthy family members had positive test in IgG Toxoplasma gondii(P = 0.09). In both groups, there was no positive IgM Toxoplasma gondii. In investigating leishmania, only 3 participants in the case group and 2 in the control tested positive (P = 0.25). No case of malaria was found among the participants. Our results showed a mismatch with hygiene hypotheses examined. However, considering that the prevalence of parasites varies with time, and depends on numerous epidemiological factors; these results do not discredit the theory investigated.

Cutaneous leishmaniasis(CL) continues to be an increasing public health problem in Iran. The dominant etiologic agents of CL in the Old World are Leishmania major and Leishmania tropica. One of the important endemic foci of CL in Iran is Yazd. Recently, previous studies showed the equal prevalence of L. major and L. tropica as the agents of cutaneous leishmaniasis in this area. This prompted us to identify the genotype of L. major isolates obtained from patients with cutaneous leishmaniasis.

After completing a clinical/epidemiologic data questionnaire for 218 patients with suspected skin lesions, scraping samples were collected, and each specimen was examined using both direct microscopy and molecular assay of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Results showed that of the 218 samples, Leishman body was observed in 77 by direct smear and 104 by PCR assay. Molecular assay indicated 50 cases as L. major, 52 cases as L. tropica, and two cases as unknown. Molecular characterization of L. major isolates showed four patterns, named LmA1, LmA2, LmA3, and LmA4.

Our study is the first report for molecular characterization of L. major from one of the important central province of Iran that could affect the control strategies in this field.

Giardia duodenalis is one of the most prevalent intestinal parasites of human. It also infects a wide range of mammals. Two genotype of G.duodenalis (A and B) were commonly reported among humans with different frequency of distribution in different geographical locations. This work was conducted to discriminate genotypes of Giardia duodenalis human isolates in Isfahan city using PCR- RFLP. This is the first molecular study on human isolates of G.duodenalis in the area.

Samples were collected from different health centers of Isfahan city during June 2011 and February 2012. From 175 Giardia positive stool samples 67 specimens were selected randomly. Cysts of Giardia positive samples were concentrated by flotation sucrose. Extraction of genomic DNA from trophozoite and cysts was performed using QIAamp Stool Mini kit with a modified protocol. PCR- RFLP method was used to amplify a fragment of 458bp at the glutamate dehydrogenase locus, and restriction enzymes BspLI and RsaI differentiated human genotypes A and B and their subgroups.

PCR – RFLP assay of 67 isolates showed 40 (59.7%) isolates as Genotype A group II, 23 (34.32%) samples as Genotype B Group III and two (2.98%) sample as Genotype B group IV. Mixed genotype of (AII and B) was detected only in two isolates (2.98%).

PCR – RFLP assay targeting gdh locus is a sensitive tool and discriminates genotypes, sub genotypes and mixed type of G.duodenalis. Results of our study suggest both anthroponotic and zoonotic origins for the infections respectively.

Zoonotic diseases have always threatened humans. They are common between human and many animals. Large numbers of dogs, especially stray dogs, are born every year. In addition, they inhabit around the cities and people's residences. Therefore, they have always been considered as threats for human health and environment. Identification of parasites in stray dogs around Isfahan, Iran would have economic, medical, veterinary, and particularly zoonosis benefits. In this study, 96 killed stray dogs were collected from several regions of Isfahan. After autopsy in place, the two ends of animal's bowel were closed and the intestines were moved to the laboratory. The intestines were opened longitudinally, washed with water, and examined for the presence of worms. The worms isolated from water were then transferred to a 10% formaldehyde solution. After temporary and permanent staining and transparency of isolates, the appropriate isolates were mounted and prepared for taxonomic study. The shapes of all samples were drawn by a drawing tube. They were then identified by diagnostic keys such as length of large hook, shape of proglottid, and characteristics of mature worms like cirrus sucks and the positions of testis. Nematode body length, the cuticle, cephalic and caudal fins, number of lips, morphology of esophagus, and position of rectum and vagina were also evaluated as crucial properties in taxonomic study. All isolates were identified at genus and species levels. In this study, 96 dogs were examined. Overall, 60.4% intestinal samples of stray dogs were infected with at least one isolated worm. The isolates included a total of seven species of cestodes and three species of nematodes. The identified cestodes consisted of 24% Taenia ovis, 13.54% T. hydatigena, 5.21% T. pisiformis, 5.21% T. multiceps, 27.1% Echinococcus granulosus, 22.92% Dipylidium caninum, 5.21% Mesocestoides lineatus. The nematodes were categorized as 21.9% Toxascaris leonina, 6.25% Toxocara canis, 3.13% Rictularia affini Taenia pisiformis was reported for the first time in the province of Isfahan. Our findings reveal the role of parasites in medical and veterinary aspects. Pet dogs and sheepdogs are thus required to be treated properly. On the other hand, stray dogs should be killed and the infected bodies need to be destroyed.

Differentiation between Entamoeba histolytica and Entamoeba dispar is very important for both clinical therapy and epidemiological studies. Although these two species are morphologically identical, they have differences in genetic, chemical specifications and pathogenicity. This study was carried out to differentiate E. histolytica from E. dispar and also to find out prevalence of the two species. Fecal samples were collected three times from 655 patients with gastrointestinal complaints (47.3% male), who were referred to the primary health care centers of Chelgerd, Chaharmahal and Bakhtiary province. Samples were examined microscopically with direct smear, formalin-ethyl-acetate concentration and trichrom staining methods to distinguish E. histolytica from E. dispar complex and differentiate them from non-pathogenic intestinal amoeba. Genomic DNA was extracted from microscopy positive isolates and polymerase chain reaction (PCR) was carried out to different the two morphologically identical Entamoeba isolates. Among the 655 recruited patients, eleven subjects with E. histolytica / E. dispar isolates (1.7%) were identified by microscopy methods. Ten of the positive isolates (90.9%) were identified as E. histolytica by PCR and one isolate (9.09 %) was positive for E. dispar. This study revealed that E. histolytica was more prevalent than E. dispar in the studied area. This result was different from the previously reported data in other parts of Iran.

Trichostrongyliases as zoonotic diseases are of major public health and socio-economic importance. Accurate identification of these parasites is the first step in strategic planning for the prevention,control and treatment of the parasitic disease. The aim of this study is using PCR-RFLP technique for genotyping. samples were collected from elementary tract content of 70 sheep in Isfahan and Khorasgan slaughterhouses. Identification of Trichostrongylus was determined based on morphological character and key identification after direct and flotation methods. For genotyping studies, DNA was extracted and the ITS-2 of ribosomal DNA of each species with specific primers was amplified by PCR and restricted with the endonucleases DraI, RsaI, HinfI. The profile was visualized in agarose gel under ultraviolet transillumination. The eggs of most species are morphologically indistinguishable at the generic level. rDNA-ITS2 fragment size of all species were the same size about 330bp, however, there were differences among species in their PCR-RFLP patterns. The two fragments produced with RsaI in all species were the same size about 138bp,190bp. The PCR product T.probulurus with DraI remained unrestricted. Restriction with DraI produced two fragments in the PCR product of T.axei and T.colubriformis about 110bp,215bp, however, the larger of the two fragments in T.vitrinus is smaller in size than in T.axei and T.colubriformis had a size about145bp,185bp.The two fragments produced with HinfI in T.colubriformis had a size about 90bp,238bp, By contrast other Trichostrongylus remained unrestricted. Based of our results from genotyping studies using PCR-RFLP technique, it can be concluded that four species had been identified. This study provides a pattern to distinguish Trichostrongylus species in Isfahan region using the DraI, RsaI, HinfI restriction endonucleases.