Isolation, Identification and Antimicrobial Activity of Unprecedented Lactic Acid Bacterial Isolates from Honeybees.

BACKGROUND AND OBJECTIVE: Lactic acid bacteria are generally recognized as safe that could be beneficial for several uses in food industry to get their health benefits. The present study was focused on the isolation and identification of some new lactic acid bacteria that might be naturally occurred in the honeybees stomach and tried to explore their benefits. MATERIALS AND METHODS: Twenty five isolates of lactic acid bacteria were isolated from the stomach of three different types of Egyptian bees (Apis mellifera lamarckii ), Carniolan bees (A.m. carnica) and hybrid Carniolan bees. Identification of isolates was carried out based on phenotypical tests and carbohydrate assimilation using API50 CHL and 16S rDNA sequencing. RESULTS: In the present study, the results emphasized Lactobacillus plantarum to be the predominant species (62.5%), other strains were identified as Pediococcus pentosaceus (12.5%), Lactobacillus pentosus (12.5%) and Lactobacillus sakei (12.5%). Eight of 25 isolates showed a potential antibacterial activity especially against Salmonella senftenberg strain. The novel isolates (HBMSS1, HBMSS3, HBMSS4, HBMSS5, HBMSS6 and HBMSS8) showed a significant antimicrobial activity against C. botulinum, E. coli, S. Senftenberg and S. epidermidis as food borne pathogens and P. larvae and M. plutonius as honeybee pathogens. CONCLUSION: These promising findings might be beneficial for discovering novel preservatives in food industry and substitution of antibiotic drugs used in the treatment of honeybees' infection.

Nutritional, Physicochemical, and Sensorial Evaluation of Flat Bread Supplemented with Quinoa Flour.

The purpose of the present research was to develop novel flat bread supplemented with quinoa flour to raise its nutritional quality and functional properties. Furthermore, evaluation of the quality of developed bread was realized with blends at 5, 10, 15, 20, 25, and 30% of quinoa flour. Chemical composition of supplemented flat bread was determined. Several properties on dough (water absorption, dough development time, stability time, elasticity, and extensibility) and their corresponding characteristics (loaf specific volume, baking loss, roundness, height, baking time, hardness, cohesiveness, springiness, resilience, gumminess, and chewiness) were then evaluated. The protein content in bread-based quinoa blends was significantly increased gradually with increasing the percentage of quinoa flour from 12.12±0.63% in control to 15.85±0.065% in 30% quinoa flour. Also, the amino acids content was increased with increasing the percentage of quinoa flour. Mineral contents in 30% quinoa flour blend such as sodium, potassium, magnesium, calcium, iron, copper, manganese, and zinc were higher than other ratios and control bread (100% wheat flour). Rheological properties of supplemented bread such as specific volume, appearance, crust and crumb texture, aroma-odor, and colour were evaluated and found to be excellent. Physicosensory characteristics of the bread fortified with quinoa flour were evaluated and the most of panelists accepted and preferred the bread supplemented with quinoa flour more than control. The obtained unique nutritional, physicochemical, and organoleptic characteristics of quinoa flour-based flat bread open a new promising prospect for utilization of quinoa flour in an industrial scale for treatment and/or prevention of malnutrition in developing counties.

Reduced longevity and fecundity in Leishmania-infected sand flies.

Phlebotomus papatasi and P. langeroni were infected with Leishmania major and L. infantum by membrane feeding. Each sand fly ingested approximately 200 parasites per blood meal. Higher mortality in both sand fly species was seen with mixed infections than with a single parasite species. There was no significant difference between infections with either L. major or L. infantum in their natural vectors or experimental hosts. Infection significantly depressed the mean number of eggs laid per female.

Dot-ELISA, a potential immunoassay for the detection of Plasmodium falciparum antibodies.

The dot enzyme-linked immunosorbent assay (dot-ELISA) was compared to the indirect immunofluorescent assay (IIFA) and radioimmunoassay (RIA) for the detection of malaria antibodies. Of 281 sera tested by dot-ELISA, 220 were from Ethiopia, 11 from the Sudan, and 50 from Egypt. A close correlation between the dot-ELISA and RIA results was observed in 92% of the 220 Ethiopian cases. Of the remainder, 6% gave positive RIA and negative dot-ELISA results, and 2% gave positive dot-ELISA and negative RIA results. Antibody titres determined by dot-ELISA and RIA were positively correlated in 10 of the 11 Sudanese cases tested by direct microscopical examination. The eleventh case was positive by dot-ELISA at 1:1000 dilution, but negative by RIA and direct examination. With the 50 Egyptian sera, the dot-ELISA results showed close correspondence to the IIFA results, but the dot-ELISA was 20-40 fold more sensitive than the IIFA. To test specificity, 62 samples from patients with 11 different diseases and conditions were examined by dot-ELISA. No malaria antibodies were detected in any of these or in sera from healthy controls. Dot-ELISA is a potentially useful method for sero-epidemiological studies of malaria.

Leishmania infantum MON-98 isolated from naturally infected Phlebotomus langeroni (Diptera: Psychodidae) in El Agamy, Egypt.

Leishmania infantum zymodeme MON-98 was isolated in El Agamy, Egypt. A total of 15 (1.07%) Leishmania-like infections in the anterior midgut and in the head was found in 1,405 Phlebotomus langeroni (Nitzulescu); none of 1,785 Phlebotomus papatasi (Scopoli) was found infected. Four of the 15 cultures (26.7%) were indistinguishable from a reference L. infantum MON-98 strain using cellulose acetate electrophoresis. The isolation and identification of L. infantum MON-98 from naturally infected P. langeroni confirms that this species of sand fly is the vector of visceral leishmaniasis in El Agamy.

Enzyme polymorphism and genetic variability of one colonized and several field populations of Phlebotomus papatasi (Diptera: Psychodidae).

The Alexandria laboratory colony and five field populations of Phlebotomus papatasi (Scopoli) from Egypt were analyzed for genetic variation at 17 enzyme loci. The laboratory colony was characterized by a low level of genetic variation as measured by the average number of alleles per locus (A = 1.70 +/- 0.16) and the average expected heterozygosity (He = 0.06 +/- 0.02). Polymorphism was observed at 23.5% of the examined loci, and genotype frequencies at two loci (PGM, AK-2) were found to deviate slightly from the Hardy-Weinberg equilibrium. In contrast, the average number of alleles per locus for field populations ranged from A = 2.35 +/- 0.20 to 2.76 +/- 0.10, and He ranged from 0.15 +/- 0.03 to 0.21 +/- 0.05. All loci of field populations exhibited polymorphism, ranging from 47.0% to 76.5%, and four to seven loci in each population were found to deviate from the Hardy-Weinberg equilibrium. Deviations in both colonized and field populations were caused by heterozygote deficiency. Despite geographic isolation and some individual deviations from the Hardy-Weinberg equilibrium, no evidence of significant genetic difference was obtained for any of the populations sampled. Calculated indices of genetic distance and genetic identity for the five field populations showed minor variation but were collectively representative of a single, genetically uniform population.

Electrophoretic comparison of the Leishmania vectors Phlebotomus papatasi and P. langeroni (Diptera: Psychodidae).

Cellulose acetate electrophoresis was employed to detect 22 different enzyme systems in laboratory-reared populations of the sympatric Leishmania vectors, Phlebotomus papatasi (Scopoli) and P. langeroni Nitzulescu. Electrophoretic conditions sensitive enough to permit as many as eight separate enzyme assays to be performed on individual specimens were developed. Under these conditions, 18 enzymes were detected with high resolution and regularity. Evidence was obtained which suggested that a number of enzymes in both species are under multilocus genetic control. Polymorphism was observed in 14 of 25 (56%) loci detected in P. papatasi and in eight of 24 (33%) loci detected in P. langeroni. Differences in electrophoretic profiles of malic enzyme, 6-phosphogluconate dehydrogenase, and fumarate hydratase were considered to be genetically fixed in both sexes of P. papatasi and P. langeroni. Their detection may permit an accurate and more rapid separation of these vectors in field collections.

Enzyme electrophoresis as an alternative method for separating the sympatric Leishmania vectors Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae).

Trials were conducted to determine the accuracy of separating the sympatric sand fly species Phlebotomus papatasi (Scopoli) and P. langeroni Nitzulescu by means of cellulose acetate enzyme electrophoresis. Malic enzyme, phosphoglucomutase, 6-phosphogluconate dehydrogenase, and fumarate hydratase were each evaluated in laboratory-reared and field-collected populations of the two species. Each of the four enzyme-based identifications was highly sensitive (greater than 97%) and specific (greater than 93%). Identifications based upon fumarate hydratase were in perfect agreement with morphological identifications, and evidence was obtained which indicates that this enzyme may be the most stable of the four enzymes tested. The application of enzyme-based vector identification is discussed in relation to classical and novel survey procedures for Leishmania promastigote detection in sand flies.

Investigation of sampling methods for the study of Phlebotomine sandflies in Egypt.

Different sampling field techniques were investigated for the study of the natural behaviour of the sand flies in Egypt (South Sinai and Alexandria). Sampling methods were divided into two groups: the first group is techniques for catching alive flies [Active search (Aspirator), CDC-light trap; funnel trap; Fan trap and catches off bait] and the second group is techniques for collecting dead specimens [sticky traps, and illuminated sticky traps (chemical light sticky trap)]. Comparison between the efficiency of the different trapping methods for collecting sandflies indicated that the CDC light trap was the most productive type for species of the genus phlebotomus followed by the illuminated paper traps and then the sticky paper traps. Members of the Sergentomyia group showed to be more attracted to illuminated/sticky paper followed by the sticky paper and then the CDC light traps. In general, sticky paper traps are the most widely used technique for sand fly outdoor collection as it is easy to be used, unexpensive and convenient for the purpose of sandfly surveys.

Development of an enzyme linked-immunosorbent assay (ELISA) for the sand fly fever viruses detection.

An Enzyme linked-immunosorbent assay (ELISA) was recently established for the detection of sandfly Naples and Sicilian viruses (SFN and SFS) from laboratory infected as well as wild caught sandflies in Egypt. Optimal dilutions of the reactants including the coating antibodies (Rabbit antiserum), detecting antibodies (Mouse antiserum), conjugate and the time used for incubation of the substrate (ABTS) were determined for both SFN and SFS viruses. The ELISA test showed to be highly specific and sensitive except for the SFN virus which cross reacted with Toscana virus. A total of 1582 sandflies, forming 54 pools (each consisted of 2-109 flies) were collected from different governorates in Egypt (North Sinai, South Sinai, Alexandria, Giza, Qualubyia, Sharkiya and Aswan) in the years 1986-1987 and 1988, and tested for virus detection by the SFN-ELISA and SFS-ELISA. Only one pool (from Giza governorate) revirus was detected. Tests were repeated two times and confirmed by the complement fixation and plaque neutralization tests. The established ELISA technique hold great promise as a routine surveillance tool, permitting rapid, simple, sensitive, specific and inexpensive assay for the detection of the sandfly fever viruses.

A simple micro-assay method for estimating blood meal size of the sand fly, Phlebotomus langeroni (Diptera: Psychodidae).

The accurate measurement of blood meal size in Phlebotomus langeroni, the potential vector of infantile visceral leishmaniasis in Egypt, is important to determine the number of parasites taken in fully engorged insects. A simple protein content micro-assay is introduced for that purpose. The accuracy of this method was confirmed by hemoglobin estimation method. Laboratory bred P. langeroni were fed artificially on defibrinated human blood and the fully engorged flies were carefully dissected on ice, within 1-10 min after feeding, since the time of dissection is critical. Serial concentrations of the defibrinated human blood were required as standards. Results show that the full blood meal taken by P. langeroni ranged from 0.76-0.94 mm3 of blood with a mean volume of 0.85 +/- 0.02 mm3 and from 0.71- 0.99 mm3 of blood with a mean volume of 0.83 +/- 0.02 mm3 as measured by protein content and hemoglobin estimation methods respectively. The data showed that there is no significant difference (P=0.27) between the two methods in estimating the blood meal size of P. langeroni. In addition, protein content micro-assay had the advantages of being accurate, rapid, sensitive and reliable.

Induction of some digestive enzymes in the midgut of the sandfly Phlebotomus langeroni after sugar and blood meals.

Changes associated with blood and sugar meal digestion in the sandfly, Phlebotomus langeroni were characterized. Different types of sugars: sucrose, glucose, melibiose, cellobiose, lactose, starch, fig fruits, honey dew and a mixture of sucrose and sugar sources were used for the sandfly feeding. Activities of glycosidases and proteases in the sandfly guts after blood and sugar meals were determined using the endpoint method. The results showed that glycosidases (alpha-glycosidase, beta-glycosidase, alpha-galactosidase, and beta-galactosidase) are present in the sandfly midguts. No activities of the glycosidases (alpha-mannosidase and alpha-amylase) were detected in the sandfly gut. Proteases: trypsin and aminopeptidase showed activities in the sandfly midguts. It is concluded that the midgut glycosidase may play an important role in the vector-parasite interaction. Trypsin and aminopeptidase induction after a blood meal is controlled by a secretogogue mechanism which indirectly influences the outcome of the Leishmania parasite infection.

Development of Leishmania major in the phlebotomine sandflies, Phlebotomus papatasi (Scopoli) and Phlebotomus langeroni (Nitzulescu).

Laboratory bred Phlebolomus papatasi and P. langeroni were examined for their susceptibility to develop Leishmania major promastigotes under laboratory conditions. Promastigotes were demonstrated in the gut of both species when they were given sugar 24 hr before or after an infective blood meal and in flies offered only an infective blood. The overall infection rate was slightly higher in P. langeroni than P. papatasi. Head promastigotes were detected in P. papatasi provided with sugar 24 hr before or after an infective blood meal. No head promastigotes were seen in flies offered only infective blood. In P. langeroni, head promastigotes were only seen in flies fed on sugar before or after an infective blood and maintained at 18 degrees C. Results indicate that sugar plays a major role in the migration of parasites from the gut to the head. Temperature may have a marginal effect on the migration process. Attempts to transmit L. major to hamster by the bite of infected P. papatasi were not successful.

Anopheles sergenti (Theobald) a potential malaria vector in Egypt.

Two immunoassays for malaria sporozoite detection and identification, the immunoradiometric assay (IRMA) and the enzyme-linked immunosorbent assay (ELISA) using the species-specific monoclonal antibodies are routinely performed in our laboratory. We analyzed (573) anopheline mosquitoes of A. sergenti (463), A. pharoensis (81) and A. multicolor (29) collected from Siwa-oases and Faiyum Governorate (two known active malaria foci in Egypt), for detection of P. falciparum and P. vivax sporozoites. P. falciparum sporozoites were detected by both IRMA and ELISA tests in two A. sergenti mosquitoes (one from Siwa 1/389 = (0.26%) and one from Faiyum Governorate 1/74 = (1.35%)). No P. vivax sporozoites were detected. This finding is important in explaining the malaria transmission and provide first incrimination of An. sergenti as the responsible vector of malaria in Siwa-oasis, Egypt.

Vector-host-parasite inter-relationships in leishmaniasis. I. The effect of Leishmania parasites on rate of digestion of blood proteins from various vertebrate hosts by the sand fly Phlebotomus langeroni (Diptera: Psychodidae).

Protein digestion in the gut of Phlebotomus langeroni (Nitzulescu) was studied at four subsequent 24 hour intervals post feeding on human, dog (Canis familiaris), rat (Rattus rattus) and turkey (Melagris gallopava) bloods with and without Leishmania infantum or L. major promastigotes. Most of the proteins of the studied blood meals were digested within 96 hours. The percent of blood proteins digested in the first 48 hours was higher than in the second 48 hours in all cases of the studied blood meals except the normal blood of the turkey in which the ratio of the digested blood proteins in the two periods was 1:1. During the first 48 hours, the percent of the digested blood proteins was lower than normal in the presence of L. infantum in case of human and dog blood meals. The reverse was true in case of the rat and turkey blood meals in the presence of L. infantum and in the blood meals from each of the four vertebrate hosts in the presence of L. major. The significance of these findings in considering L. infantum as a natural parasite of P. langeroni in El Agamy focus was discussed.

Vector-host-parasite inter-relationships in leishmaniasis. II. Influence of blood meal from natural vertebrate hosts with and without Leishmania infantum and L. major on the proteolytic activity in the gut of Phlebotomus langeroni (Diptera: Psychodidae).

Proteolytic activity in the gut of Phlebotomus langeroni (Nitzulescu) was studied at four subsequent 24 hours intervals post feeding on human, dog (Canis familiaris), rat (Rattus rattus) and turkey (Melagris gallopava) bloods with and without Leishmania infantum or L. major promastigotes. The gut proteolytic activity increased gradually after feeding to reach a maximum at 48 hours post feeding on any of the 12 studied blood meals. In all cases, the activity declined after 48 hours and almost terminated by 96 hours. In case of normal bloods, the proteolytic activity, at 48 hours post feeding, was the highest in case of dog followed by human, rat and turkey respectively. At this time interval the activity was relatively lower in case of human and dog blood mixed with L. infantum promastigotes than in their respective normal blood. The reverse was true in all other blood meal combinations. Promastigotes were alive and active in fresh gut smears of P. langeroni fed on human, dog and rat bloods mixed with either L. infantum or L. major, throughout the digestion period (1-4 days). They were arrested in P. langeroni within the first day post feeding on turkey blood mixed with either Leishmania species. The results of the present study indicate that the kind of blood meal and the Leishmania species affect the proteolytic activity of P. langeroni. The decrease or increase of the proteolytic activity of P. langeroni has no effect on the survival of Leishmania parasites present in the gut and the kind of blood meal is responsible for their development.

Vector-host-parasite inter-relationships in leishmaniasis. III. Impact of blood meal from natural vertebrate hosts on the survival and the development of Leishmania infantum and L. major in Phlebotomus langeroni (Diptera: Psychodidae).

Phlebotomus langeroni collected from a leishmaniasis endemic focus at Et Agamy, Alexandria, Egypt, were found to have fed on blood from man, dogs (Canis familiaris) and rats (Rattus rattus). The effect of the kind of blood meal on the development and the life-cycle of L. infantum and L. major in laboratory reared P. langeroni was therefore investigated. A membrane feeding technique was used to infect sand flies. Gut smears of infected females were examined immediately after feeding and daily for 16 days. Nectomonads and short promastigote forms of L. infantum or L. major were detected in females fed on human, dog and rat bloods at all intervals. Paramastigotes (infective stage) were present only in females fed on dog blood containing L. infantum or L. major and in those fed on rat blood containing L. major. It is concluded that among the factors influencing the Leishmania-phlebotomus relationship is the natural medium in which the parasite is present in vivo. The blood of the natural reservoir host(s) is the key factor for the development of the infective parasite form in the sand fly and P. langeroni could be considered a potential vector for transmitting L. infantum from dogs and L. major from rats and dogs but not from man. This investigation offers a new concept for the study of interactions among vector, host and parasites in Leishmania transmission.

Malaria chemoprophylaxis to pregnant women provided by community health workers in Saradidi, Kenya. III. Serologic studies.

Parasitaemia and antimalarial antibodies were examined from May 1983 to March 1984 in monthly samples taken from 930 pregnant women attending antenatal clinics in Saradidi, Kenya, and 317 of their infants; 104 women were taking chloroquine phosphate 300 mg base weekly for chemoprophylaxis. Seropositivity rates in pregnant women were uniformly high, and mean enzyme-linked immunosorbent assay (ELISA) absorbance values were not related to presence of parasitaemia or history of chemoprophylaxis. Parasitaemia was present in 26.5% of 1677 slides from pregnant women and there was little variation by month of sample. Mean ELISA absorbance values varied by month of sample. Seropositivity rates in infants were high as measured in both the indirect fluorescent antibody (IFA) test (81.6% of 938) and ELISA at 1:100 (83.8% of 1025) and 1:1000 (34.8% of 1025) serum dilutions. Seropositivity rates decreased slightly after birth but by four months of age rates were again high. Parasitaemia was present in 26.5% of 1677 slides from pregnant women. Paired comparisons were made on maternal samples collected less than two months before parturition and samples from the infants collected within two months after birth. The paired antibody response by IFA or ELISA was not dependent on the presence of detectable parasitaemia in the mother. Infants from mothers with a history of antimalarial chemoprophylaxis had significantly (P = 0.04) lower IFA titres than other infants. Measuring the absorbance of a 1:100 serum dilution by ELISA appeared to be an excellent method with which to measure longitudinal serologic changes in a population.