Using Various Approaches of Design of Experiments for High Cell Density Production of the Functionally Probiotic Lactobacillus plantarum Strain RPR42 in a Cane Molasses-based Medium.

Considering the economic importance of the probiotics, industrial production of their biomass became important. Cane molasses, as an industrial byproduct, was used in this study to design a medium for biomass overproduction of a functionally probiotic strain, designated as Lactobacillus plantarum strain RPR42. The results showed that strain RPR42 can be best grown anaerobically in 22.5% cane molasses solution. Also, the findings of the single variable at a time experiments and either factorial design indicated that the optimal growth of strain RPR42 can be observed when beef extract, casein hydrolysate, and yeast extract were added into the medium. The central composite design experiments suggested a medium which was designated as cane molasses medium (CMM). Eventually, this medium contained 21.9% cane molasses, 30.72 g/L of a combined mixture of nitrogenous compounds: 0.0754% of a 1:1:1 mixture of polysorbates 20, 60, and 80, and 18.53 gr/L of the combined minerals. Such an optimized cane molasses-based medium supported a significant biomass production since a considerably high cell density, 13.8 g/L/24 h of dry biomass, of the strain was produced. Hence, cane molasses can be regarded as a promising substrate for industrial production purposes.

Probiotic characters of Bifidobacterium and Lactobacillus are a result of the ongoing gene acquisition and genome minimization evolutionary trends.

Bifidobacterium and Lactobacillus are the main probiotic genera. Collectively, these two genera harbor over 200 species among which are many strains have been introduced as probiotics. These health-promoting microbes confer health benefits upon the host and so used in food productions and as supplements. Considering the economic importance of probiotics, the biochemistry, genomics, phylogeny and physiology of such genera have been exhaustively studied. According to the genomic data, the probiotic capabilities are strain specific which may be a result of the niche-specialization of the genomes of these bacteria to certain ecological niches like gastrointestinal tract of a diverse range of animals. These microbes have a wide distribution but the culture-based studies and either genomics data suggest selective affinity of some Lactobacillus and either Bifidobacterium species to certain ecological niches. An ongoing genome degradation, which is thought to be a result of passage through an evolutionary bottleneck, is the major trend in the evolution of lactobacilli. Further, evolutionary events resulted into two categories of lactobacilli: habitat generalists and habitat specialists. In place, the main trend in the evolution of bifidobacteria tend to be the gene acquisition. However, probiotic features are the results of a co-evolutionary relationship between these bacteria and their hosts and the aforementioned evolutionary tends have driven the evolution of these probiotic genera.

Genetic diversity and effect of natural selection at apical membrane antigen-1 (AMA-1) among Iranian Plasmodium vivax isolates.

Apical membrane antigen-1 (AMA-1) of Plasmodium vivax Grassi et Feletti, 1890 is a promising malaria vaccine candidate. However, antigenic variation is a major problem to design a universal malaria vaccine. Hence, detailed understanding of the pvama-1 gene polymorphism can provide conductive information on this potential vaccine component. Therefore, this study investigated the extent of genetic polymorphisms at domain I (DI), DII and partial DIII of AMA-1 among Iranian P. vivax isolates. Out of 107 blood samples, 92 were analysed based on the quality of the sequencing data. The sequences were classified into 53 haplotypes. Amino acid changes were observed at 31 positions that 17 were located at DI, 11 were at DII and the rest of them (3 positions) were at DIII. Thus, codon polymorphisms at DI were found to be higher than DII. Also, five of these polymorphic codons (D242E, T374P, S389R, Y391F, I395F) were novel and have not been reported yet. Neutrality analysis by using the dN-dS difference (the difference between the rate of non-synonymous and synonymous mutations) showed a negative diversifying selection at DI, DII and across the length of both domains. The potential B-cell epitopes were found in 5 regions of the PvAMA-1 with 10 mutation sites (E145A, K188N, E189N/K/D, K190Q/E, P210S, E227V, D242E, R249H, G253E, K352E), whereas only one mutation (G288E) has been detected in intrinsically unstructured/disordered regions. Fixation index (Fst) estimation between Iranian and Indian isolates (0.0131) indicated a significant low genetic differentiation. Distribution of the polymorphic sites and IURs mapped on a three dimensional structure of PvAMA-1 showed that these regions were located at two opposite faces of the molecule. In conclusion, the results have significant value in the design and development of a malaria vaccine based on this antigen.

Intra-hippocampal injection of lipopolysaccharide inhibits kindled seizures and retards kindling rate in adult rats.

Neuroinflammation facilitates seizure acquisition and epileptogenesis in developing brain. Yet, the studies on impact of neuroinflammation on mature brain epileptogenesis have led to inconsistent results. Hippocampus is particularly vulnerable to damage caused by ischemia, hypoxia and trauma, and the consequent neuroinflammation, which can lead in turn to epilepsy. Lipopolysaccharide (LPS) is extensively used in experimental studies to induce neuroinflammation. In this study, effect of acute and chronic intra-CA1 infusion of LPS on amygdala-kindled seizures and epileptogenesis was examined in mature rats. LPS (5 µg/rat) inhibited evoked amygdala afterdischarges and behavioral seizures. Anticonvulsant effect of LPS was observed 0.5 h after administration and continued up to 24 h. This effect was accompanied by intra-hippocampal elevation of nitric oxide (NO), interleukin1-ß, and tumor necrosis factor-α and was prevented by microglia inhibitor, naloxone, NO synthase inhibitor, Nω-nitro-L-arginine methyl ester, cyclooxygenase inhibitor, piroxicam, and interleukin1-ß receptor antagonist, interleukin1-ra. Moreover, daily intra-hippocampal injection of LPS significantly retarded kindling rate. In order to further elucidate the effect of LPS on synaptic transmission and short-term plasticity, changes in field excitatory postsynaptic potentials and population spikes were measured in stratum radiatum and stratum pyramidale of LPS-treated kindled rats. LPS impaired baseline synaptic transmission in hippocampal Schaffer collateral-CA1 synapse and reduced the magnitude of paired-pulse facilitation. Our results suggest that direct suppression of presynaptic mechanisms in Schaffer collateral-CA1 synapses, as well as the inflammatory mediators released by LPS in the hippocampus, is involved in antiepileptic effect of LPS.

Evaluation of anti-leishmanial effects of killed Leishmania vaccine with BCG adjuvant in BALB/c mice infected with Leishmania major MRHO/IR/75/ER.

There are many strategies to control leishmaniasis, but majority of them are inadequate. Killed Leishmania vaccine (KLV) has been applied for its immunogenicity in human and mouse model. Bacillus Calmette-Guerin (BCG) as adjuvant is an immune-modulator inducing humoral and cellular immune responses during zoonotic cutaneous leishmaniasis (ZCL). Both KLV and BCG have been applied for their immune responses in hosts for controlling leishmaniasis. In this study, KLV and BCG were applied to inhibit replication and visceralization of Leishmania major in BALB/c mice. Mice were injected with KLV and BCG, followed by infection with promastigotes of L. major. Six weeks after infection, a small nodule appeared, which was followed by development of a large lesion and visceralization. Effects of KLV and BCG, physiopathological changes, lesion size, delay of lesion formation, proliferation of amastigotes inside macrophages and detection of amastigotes in target organs were studied. Results showed that the KLV had anti-leishmanial activity by reducing lesion size on late infection. In KLV and BCG group, the average number of amastigotes in macrophages was lower than in other groups. Significant reductions in number of amastigotes in both spleen and lymph node were observed, indicating lower visceralization of Leishmania parasites in these target organs. No significant changes were presented in body weights, survival rates and degrees of splenomegaly in test group. It can be concluded that application of KLV and BCG had acceptable efficacy in reduction of skin lesions size and proliferation of parasites, even though a few side-effects were observed. It is indicated that KLV/BSG may have ability to modulate host immune responses against Leishmania parasites and to reduce pathophysiology of the disease during infection.

Comparison of antimalarial activity of Artemisia turanica extract with current drugs in vivo.

BACKGROUND & OBJECTIVES: The purpose of this study was to compare antimalarial activity of Artemisia turanica Krasch as Iranian flora with current antimalarial drugs against Plasmodium berghei in vivo in mice. METHODS: Air-dried aerial parts of Iranian flora A. turanica were collected from Khorasan, northeastern Iran, extracted with Et2O/MeOH/Petrol and defatted. Toxicity of herbal extracts was assessed on male NMRI mice, and their antimalarial efficacy was compared with antimalarial drugs [artemether, chloroquine and sulfadoxinepyrimethamine (Fansidar)] on infected P. berghei animals. All the groups were investigated for parasitaemia, body weight, hepatomegaly, splenomegaly and anemia. The significance of differences was determined by Analysis of Variances (ANOVA) and Student's t-test using Graph Pad Prism software. RESULTS: The inhibitory effects of A. turanica extract on early decline of P. berghei parasitaemia highlights its antimalarial activity, however, this effect no longer can be observed in the late infection. This may be due to the metabolic process of A. turanica crude extract by mice and reduction of its concentration in the body. Crude extract of A. turanica represented its antisymptomatic effects by stabilization of body, liver and spleen weights. CONCLUSION: This study confirmed antimalarial effects of A. turanica extracts against murine malaria in vivo during early infection, however, there are more benefits on pathophysiological symptoms by this medication.

Leishmania major: secreted antigens of Leishmania major promastigotes shift the immune response of the C57BL/6 mice toward Th2 in vitro.

BALB/c mice are sensitive to Leishmaniamajor infection, while C57BL/6 mice are resistant and able to mount an effective immune response against the parasite. Since the secreted antigens of L. major suppress the proliferation of BALB/c mice lymphocytes in vitro, we analyzed their effects on the immune system of resistant C57BL/6 mice. Secreted antigens were semi-purified and two fractions with immunosuppressive activity were isolated. 15 µg/ml of fraction could suppress 60% of lymphocyte proliferation and prevent the stimulated lymphocytes entering from G1 phase into the S phase of the cell cycle. These fractions decreased the production of IFN-γ, increased IL-4 level in the lymphocyte culture and down-regulated the nitric oxide production by activated macrophages. These results may suggest that L. major parasite by secreting immunosuppressive factors could down-regulate the immune system of both sensitive and resistant mice for own survival advantage.

Screening for efficient nitrogen sources for overproduction of the biomass of the functionally probiotic L. plantarum strain RPR42 in a cane molasses-based medium.

Nitrogen source has a vital role for the efficient growth of lactobacilli. The effects of cheese whey, corn steep liquor, and wheat germ extract on the growth of L. plantarum strain RPR42 in cane molasses-based media was evaluated using various approaches of design of experiments. Our results showed that such protein-rich agricultural by-products significantly increase the biomass production of the strain RPR42 in cane molasses-based media. The most affecting nitrogenous material was cheese whey followed by CSL and the minor effect was reported for wheat germ extract as revealed in factorial and Box-Behnken design experiments. The replacement of costly beef extract and yeast extract with a defined mixtures of the above nitrogenous agricultural by-products in cane molasses-based medium led to production of up to 12.64 g/L/24 h of dry biomass of strain RPR42. A detectable cell density of strain RPR42 (~ 9.81 × 109 CFU/mL 24 h) which was observed in such an economic medium showed that the large-scale production of the strain RPR42 tend to be feasible at significantly low costs.

Evaluation of anti-leishmanial activity by induction of nitric oxide and inhibition of prostaglandin in Balb/c mice infected with Leishmania major.

Cutaneous leishmaniasis is still one of the health problems in Iran and in the region. Nitric oxide (NO) has a key mechanism in the elimination of parasite from the body by its anti-leishmanial activity. Prostaglandin (PG) is a critical inhibitory factor of infected macrophage to decrease their anti-leishmanial activity. This study was designed to induce NO by L-arginine (L-Arg) precursor and inhibit PG production by anti-inflammatory Indomethacin (INDO) in Leishmania major infected Balb/c mice, in order to evaluate the effects of NO and PG on delay of lesion formation, size of lesion and proliferation of amastigotes inside macrophages. Liver, spleen and lymph nodes were also studied as target organs to detect amastigotes. Serum, liver and spleen suspensions were investigated for NO induction by using Griess microassay and serum PG was determined by ELISA. The results indicated that NO production was inhibited by Leishmania in infected Balb/c mice as compared with naive animals. Serum NO was inhibited by a combination therapy of L-Arg and INDO. Although NO was decreased in the liver by L-Arg, however it increased in the spleen after L-Arg and INDO application. A significant decline was observed in lesion size from Week 6 after infection by INDO. Both L-Arg and INDO had significant inhibitory effects on visceralization of leishmania in target organs. Only L-Arg decreased proliferation of promastigotes in macrophages. Pathophysiological signs including hepatomegaly, splenomegaly, survival rate and body weight all were affected in this experiment. Statistical analysis of data revealed an association between NO induction and PG inhibition in leishmaniasis. These data may indicate a possible candidatory for L-Arg and INDO as novel drugs for the treatment of leishmaniasis in mouse model.

Anti-leishmanial effects of trinitroglycerin in BALB/C mice infected with Leishmania major via nitric oxide pathway.

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.

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.

Assessment of Recombinant A2-Latex Agglutination Test (RA2-LAT) and RA2-ELISA for Detection of Canine Visceral Leishmaniasis: A Comparative Field Study with Direct Agglutination Test in Northwestern Iran.

BACKGROUND: This study aimed to set-up latex agglutination test (LAT) and ELISA based on recombinant A2 from Iranian strain of Leishmania (L.) infantum (rA2-Ag) and evaluated for detection of anti-Leishmania antibodies in dogs compared to standard direct agglutination test (DAT). METHODS: The rA2-Ag was synthesized under a part of the A2 gene sequences which contain immune dominant sequences and less number of repetitive sequences. Latex beads, 0.8 µm (Sigma, USA) were sensitized with rA2-Ag. The tests were carried out on sera collected from 350 ownership dogs including symptomatic (n=67), asymptomatic (n=230) canine visceral leishmaniasis (CVL), and (n=53) uninfected domestic dogs as control group. RESULTS: Anti-leishmanial antibodies were detected in 97 (27.7%), 96 (27.4%) and 29 (%9) of the serum samples by using DAT, rA2-ELISA, and rA2-latex, respectively with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A combined sensitivity of 52% and specificity of 82.40% for rA2-ELISA and 23.8% and specificity 95.38%, respectively were found with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. The concordance between rA2-ELISA and rA2 latex compared with DAT as a gold standard serological test for VL were found 73.7% and 77.5%, respectively. CONCLUSION: A good degree of agreement was found between rA2-ELISA and DAT (73.7%). rA2-ELISA could detect more seropositive serum samples than rA2-LAT and it may be recommended as an alternative tool for the diagnosis of CVL.

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.

Epidemiological Distribution of Rodents as Potent Reservoirs for Infectious Diseases in the Provinces of Mazandaran, Gilan and Golestan, Northern Iran.

Rodents are mammals that comprise more than 2000 species and approximately 30 families. There are many morphological and ecological differences among them as variations in their shape, size, weight and habitat. In addition to significant economic losses, rodents have a major role in the dissemination of infectious diseases caused by viruses, bacteria, parasites or other micro-organisms. Rodents are important reservoirs of diseases which have been observed in many cities of Iran provinces especially along Caspian Sea border to Alborz Mountain. The aim of this study is to assess the geographical distribution of rodents in three provinces of northern part of Iran as reservoir of potential endemic infectious diseases. Rodents in 10 major parts of each of the three provinces of Mazandaran, Gilan and Golestan, northern Iran were collected and a total of 404 rodents were trapped alive. They were determined by the key characteristics such as gender, genus, species, different locations and topological situation. Statistical analysis was performed to characterize the study sample and to correlate all variables and parameters. The distribution frequencies of three, five and six genera of rodents were identified in Mazandaran, Gilan and Golestan provinces respectively. The overall distribution frequency of eight genera of rodents in the three provinces were identified as Rattus (R.) norvegicus (67.3%), R. rattus (13.6%), Apodemus sylvaticus (13.9%), Arvicola (1%), Mus musculus (0.3%), Nesokia indica (2.5%), Cricetulus migrates (0.7%) and Rhombomys opimus (0.7%). The results of this study determined the geographic distribution of the rodents in the three northern provinces of Iran. It is indicated the association of various distribution and diversity of rodents with provincial location. The overall distribution frequency of eight genera of rodents was recognized in the above three provinces geographical locations. This study confirms epidemiological distribution of various rodents as potent reservoirs for infectious diseases, such as leptospirosis, salmonellosis, tularemia, leishmaniasis, etc. in the three provinces.

Simultaneous increases in immune-competent cells and nitric oxide in the spleen during Plasmodium berghei infection in mice.

BACKGROUND AND PURPOSE: Nitric oxide and other reactive nitrogen intermediates (RNI) are thought to be important mediators of both immunological and pathological responses of the vertebrate host to malaria infection. The role of RNI has been studied most often by assay of stable RNI metabolites (nitrites, nitrates) in blood. This study evaluated the nature of the RNI response of mice to malaria by analyzing the subsets of immune-competent cells within the organ displaying increased RNI in vivo. METHODS: We measured RNI production indirectly, as stable metabolites of nitric oxide activity in tissue homogenates (brain, liver, spleen) from mice infected with Plasmodium berghei. Only spleen exhibited an RNI concentration response during rising parasitemia. Subsets of immune-competent cells (B cells, CD19+), macrophages/monocytes (MOMA2+) and T cells (CD4+, CD8+) in the spleen were assayed by fluorescence activated cell scan flow cytometry. RESULTS: The spleen was confirmed as a major source of RNI during mid-phase P. berghei infection. Significant increases in CD19+ and MOMA2+ spleen cells were evident during the mid-phase of P. berghei infection in MF1 mice when RNI are maximally elevated. CONCLUSIONS: The time courses of the cellular and RNI responses indicate that CD19+ and MOMA2+ cells may be responsible for the increase in RNI in the spleen. However, experiments in vitro are needed to make a definitive identification of the cell type(s) responsible for the increase in RNI in the mouse spleen during P. berghei infection.

Immune effector mechanisms of the nitric oxide pathway in malaria: cytotoxicity versus cytoprotection.

Nitric oxide (NO) is thought to be an important mediator and critical signaling molecule for malaria immunopathology; it is also a target for therapy and for vaccine. Inducible nitric oxide synthase (iNOS) is synthesized by a number of cell types under inflammatory conditions. The most relevant known triggers for its expression are endotoxins and cytokines. To date, there have been conflicting reports concerning the clinical significance of NO in malaria. Some researchers have proposed that NO contributes to the development of severe and complicated malaria, while others have argued that NO has a protective role. Infection with parasites resistant to the microbicidal action of NO may result in high levels of NO being generated, which could then damage the host, instead of controlling parasitemia. Consequently, the host-parasite interaction is a determining factor for whether the parasite is capable of stimulating NO production; the role of NO in resistance to malaria appears to be strain specific. It is known that NO and/or its related molecules are involved in malaria, but their involvement is not independent of other immune events. NO is an important, but possibly not an essential contributor to the control of acute-phase malaria infection. The protective immune responses against malaria parasite are multifactorial; however, they necessarily involve final effector molecules, including NO, iNOS and RNI.

Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs.

Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

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.

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran.

INTRODUCTION: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity. METHODS: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran. RESULTS: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%. CONCLUSIONS: This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs.

Effect of trinitroglycerin therapy on serum zinc and copper levels and liver enzyme activities in BALB/c mice infected with Leishmania major MRHO/IR/75/ER.

OBJECTIVES: To evaluate the effect of trinitroglycerin (TNG) as nitric oxide donor agent on serum copper (Cu) and zinc (Zn) levels and liver enzymes in BALB/c mice infected with Leishmania major (L. major) MRHO/IR/75/ER. MATERIALS AND METHODS: Inbred female mice were divided into three groups: healthy group (uninfected naive mice), control group (infected with L. major), and test group (L. major infected mice treated with TNG). TNG (200 µg/µl) was inoculated subcutaneously into the mice of the test group. Serum Cu and Zn levels and liver enzymes activities were then evaluated by atomic absorption spectrophometer and colorimetric methods, respectively. RESULTS: Serum Cu levels were significantly higher in the test group than in the control and naive groups (P-value <0.05), while Zn levels were higher in the test group than in the control group with no significant difference. Serum glutamicoxaloacetic transaminase concentrations in the test group were significantly lower than those in other groups (P-value <0.05), while serum glutamate pyruvic transaminase concentrations were significantly higher in test compared with those in other groups (P-value <0.05). Moreover, alkaline phosphatase in the control and test groups were significantly lower than that in the naive group (P-value <0.05). CONCLUSION: TNG treatment increased Zn and Cu levels and thus increased resistance to Leishmania because of the role of Zn and Cu; therefore, TNG therapy will be useful for treating cutaneous leishmania. In addition, the decrease of serum glutamicoxaloacetic transaminase activity can be an index of therapeutic process of TNG.