In Vitro and In Vivo Biological Activity of Berberine Chloride against Uropathogenic E. coli Strains Using Galleria mellonella as a Host Model.

Berberine is an alkaloid of the protoberberine type used in traditional oriental medicine. Its biological activities include documented antibacterial properties against a wide variety of microorganisms; nonetheless, its use against Escherichia coli strains isolated from urinary infections has not yet been widely investigated in vivo. The emergence of antimicrobial resistance requires new therapeutic approaches to ensure the continued effectiveness of antibiotics for the treatment and prevention of urinary infections. Moreover, uropathogenic Escherichia coli (UPEC) has developed several virulence factors and resistance to routine antibiotic therapy. To this end, several in vitro and in vivo tests were conducted to assess the activity of berberine on uropathogenic E. coli strains. Galleria mellonella as an infection model was employed to confirm the in vivo translatability of in vitro data on berberine activity and its influence on adhesion and invasion proprieties of E. coli on human bladder cells. In vitro pre-treatment with berberine was able to decrease the adhesive and invasive UPEC ability. In vivo treatment increased the larvae survival infected with UPEC strains and reduced the number of circulating pathogens in larvae hemolymph. These preliminary findings demonstrated the efficacy and reliability of G. mellonella as in vivo model for pre-clinical studies of natural substances.

Effects of different dietary protein sources on the immunological and physiological responses of marron, Cherax cainii (Austin and Ryan, 2002) and its susceptibility to high temperature exposure.

A two phased feeding trial was conducted to evaluate the effects of alternative protein sources on the immunophysiological responses of marron. During the phase I, marron were fed with five alternative protein supplemented diets for 90 days, while in phase II, the same marron were exposed to elevated temperature (30 °C) and their immunophysiological responses were investigated post exposure. Five isoproteic (crude protein 30%) and isoenergetic diets were prepared by containing fishmeal, poultry by-product meal, feather meal, lupin meal, and meat and bone meal as the main protein source. A hundred and fifty juvenile marron (Cherax cainii) of the average weight 9.09 ±â€¯0.21 g were randomly distributed into 15 tanks (three replicates per feeding treatments). In the Phase I, general immune response parameters, such as, total haemocyte count (THC), proportion of hyaline cells, neutral red retention time (NRRT), phagocytic rate (PR), heamolymph bacteraemia, and condition indices of marron were investigated. The highest (P < 0.05) THC among dietary protein sources was obtained in marron fed with PbM at the end of experiment. Marron fed with FeM protein sources resulted in the highest survival rate followed by PbM fed group. Longer microvilli length (3.83 ±â€¯0.18 µm) was demonstrated in marron fed with PbM diet. Diets containing FM and PbM protein sources revealed significantly (P < 0.05) lower number of microvilli/group than diets containing FeM and LM. The results demonstrated that different dietary protein sources in the marron diets did not detect significant (P > 0.05) change of the condition indices throughout the experiment period, however highest Hiw and Hid was recorded in marron fed with PBM at day 45. The PR of marron fed dietary protein from PbM did not change significantly after temperature exposure. Increased NRRT, PR and haemolymph bacteraemia was observed with dietary feeding of FM at the end of the trial. However, results revealed that PbM could be an alternative protein source for culture of marron as reflected in terms of increased THC, longer microvillus length and improved susceptibility to high temperature exposure. Overall, result could serve as useful baseline data in developing cost effective potential diets for marron aquaculture.

Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model.

The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2h post-infection (treatment) or 2h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24h for quantification of bacteria and phages. Survival rates at 24h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI=10; 47% vs. 20%, MOI=1; and 33% vs. 7%, MOI=0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI=10; 53% vs. 13%, MOI=1; 67% vs. 7%, MOI=0.1). By 24h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses.

Use of silkworms to evaluate the pathogenicity of bacteria attached to cedar pollen.

Injection of a Japanese cedar pollen suspension into silkworm hemolymph kills the silkworms. A certain species of bacteria proliferated in the hemolymph of the dead silkworms. A 16S rDNA analysis demonstrated that the proliferating bacteria were Bacillus cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus amyloliquefaciens. Among them, B. cereus, B. thuringiensis, and B. weihenstephanensis exhibited hemolysis against sheep red blood cells and were lethal to mice. A culture filtrate of B. amyloliquefaciens showed enzyme activity toward the pectic membrane of cedar pollen. These results suggest that silkworms as an animal model are useful for evaluating the pathogenicity of bacteria attached to cedar pollen.

Culture-independent analysis of bacterial communities in hemolymph of American lobsters with epizootic shell disease.

Epizootic shell disease (ESD) of the American lobster Homarus americanus H. Milne Edwards, 1837 is a disease of the carapace that presents grossly as large, melanized, irregularly shaped lesions, making the lobsters virtually unmarketable because of their grotesque appearance. We analyzed the bacterial communities present in the hemolymph of lobsters with and without ESD using nested-PCR of the 16S rRNA genes followed by denaturing gradient gel electrophoresis. All lobsters tested (n = 42) had bacterial communities in their hemolymph, and the community profiles were highly similar regardless of the sampling location or disease state. A number of bacteria were detected in a high proportion of samples and from numerous locations, including a Sediminibacterium sp. closely related to a symbiont of Tetraponera ants (38/42) and a Ralstonia sp. (27/42). Other bacteria commonly encountered included various Bacteroidetes, Pelomonas aquatica, and a Novosphingobium sp. One bacterium, a different Sediminibacterium sp., was detected in 20% of diseased animals (n = 29), but not in the lobsters without signs of ESD (n = 13). The bacteria in hemolymph were not the same as those known to be present in lesion communities except for the detection of a Thalassobius sp. in 1 individual. This work demonstrates that hemolymph bacteremia and the particular bacterial species present do not correlate with the incidence of ESD, providing further evidence that microbiologically, ESD is a strictly cuticular disease. Furthermore, the high incidence of the same species of bacteria in hemolymph of lobsters may indicate that they have a positive role in lobster fitness, rather than in disease, and further investigation of the role of bacteria in lobster hemolymph is required.

The phenoloxidase activity and antibacterial function of a tyrosinase from scallop Chlamys farreri.

Tyrosinase (TYR), also known as monophenol monooxygenase, is a ubiquitous binuclear copper-containing enzyme which catalyzes the hydroxylation of phenols to catechols and the oxidation of catechols to quinones. In the present study, the cDNA of a tyrosinase (CfTYR) was identified from scallop Chlamys farreri, which encoded a polypeptide of 486 amino acids. The CfTYR mRNA transcripts were expressed in all the tested tissues, including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest level in mantle. The expression level of CfTYR mRNA in haemocytes decreased significantly during 3-6 h after LPS stimulation, and reached the lowest level at 6 h (0.05-fold, P < 0.05). Then, it began to increase at 12 h (0.32-fold, P > 0.05), and reached the highest level at 24 h (2.91-fold, P < 0.05). At 3 h after LPS stimulation, the phenoloxidase activity catalyzing L-dopa and dopamine in haemolymph increased significantly to 53.13 and 40.36 U mg(-1) respectively, but it decreased to 10.82 U mg(-1) and even undetectable level after CfTYR activity was inhibited. Furthermore, the antibacterial activity of haemolymph against Escherichia coli was also increased significantly at 3 h after LPS stimulation, but it decreased significantly when the haemolymph was treated by TYR inhibitor. The recombinant protein of the mature CfTYR peptide expressed in the in vitro Glycoprotein Expression Kit displayed phenoloxidase activity of 64.36 ± 5.51 U mg(-1) in the present of trypsinase and Cu(2+). These results collectively suggested that CfTYR was a homologue of tyrosinase in scallop C. farreri with the copper-dependence phenoloxidase activity, and it could be induced after immune stimulation and mediate immune response for the elimination of invasive pathogens in scallop.

Effects of Artemisia annua L. (Asteracea) on the digestive enzymatic profiles and the cellular immune reactions of the Sunn pest, Eurygaster integriceps (Heteroptera: Scutellaridae), against Beauveria bassiana.

Plant extracts are currently studied more and more because of the possibility of their usage in plant protection. Many of the natural plant compounds which are used in the control of pests are known to affect the digestion and immune functions of insects. In this study, effects of Artemisia annua extract on the digestive enzymatic profiles and the cellular immune reactions of Eurygaster integriceps were investigated to reach a better understanding of its role in the control of this pest as the most destructive one in the production of wheat in the Near and Middle East, eastern and southern Europe and North Africa. Feeding and injection methods were used to study the plant extract effects on digestive enzymes and cellular immunity, respectively. When adult E. integriceps fed on food and water containing plant extracts, activity of the digestive enzymes, including alpha-amylase, alpha- and beta-glucosidases, protease and lipase, in addition to cellular immune reactions (total and differentiate hemocyte numbers, phagocytosis, nodule formation and phenoloxidase activity) against Beauveria bassiana were affected and significantly decreased in comparison with controls, in that the clear dose-response relationships were established with respect to enzyme activities and immune reactions. A. annua extract had a significant effect on kinetic parameters (Vmax and Km) of digestive enzymes and phenoloxidase activity so that the presence of the plant extract decreased the value of Vmax and increased Km, causing the reduction of enzyme affinity to the substrate, overall velocity of the reaction and finally interfering with the rate of breakdown of the enzyme-substrate complex. The understanding of fungal-induced immune responses and identification of factors regarding fungal virulence could be important in accelerating host death in a biological control scenario. Hence, the combination of botanical pesticides and microbes to control insect pest populations would be a safe and possibly rapid method to decrease their damage and environmental risk due to the use of chemical pesticides.

Naturally occurring bacteraemia in American lobsters, Homarus americanus Milne-Edwards, in Long Island Sound.

The health status of the American lobster, Homarus americanus Milne-Edwards, in Long Island Sound (LIS) has been in decline, with seasonal mortality events occurring since 1998. In order to assess the potential effects of environmental conditions on lobster health via haemolymph analysis, lobsters collected from various sites in LIS were examined and sampled while concurrent environmental data (water temperature, salinity and dissolved oxygen) were recorded. The pH of the haemolymph of each lobster was tested, followed by a collection of haemolymph for serum biochemistry analysis and bacterial culture. This report focuses on the results of the bacterial sampling. The majority of bacteria cultured were opportunistic pathogens commonly found in the environment, including some that are associated with sewage and pollution. The prevalence of bacteraemia was correlated with the site of collection, the month in which the lobsters were sampled, and water temperature.

The effect of dietary chitin supplementation on the survival and immune reactivity of the shore crab, Carcinus maenas.

Adult male shore crabs (Carcinus maenas) were maintained on a fish-based diet supplemented with 0, 5 or 10% chitin for 11 weeks. Significantly greater mortality was found during this period in the control group (0% chitin) than those fed 10% chitin. Crabs fed 5 or 10% chitin had lower numbers of cultivatable bacteria in the hepatopancreas than those on the basal diet alone. The addition of chitin had no significant effect on the serum concentrations of protein and glucose, and the levels of glycogen in the hepatopancreas. The total number of circulating hemocytes in the blood was unaffected by the addition of chitin to the diet, however, at week 6 there were significantly more hyaline hemocytes in those crabs fed 10% chitin than the control group. The in vitro phagocytic activity of hemocytes was unaffected by chitin supplementation and crabs challenged with Vibrio alginolyticus showed a similar pattern of susceptibility in the three dietary groups (0, 5 or 10% chitin). Overall although crabs on a chitin-supplemented diet showed greater survival, this was not explained in terms of elevation in the cellular defences of these animals. The enhanced survival of crabs-fed chitin is probably as a result of the removal of potentially pathogenic bacteria from the hepatopancreas. Because chitin appears to 'purge' bacteria from the gut, this may prove to be a useful addition to diets on animals undergoing oral probiotic treatment.

Response of American lobsters Homarus americanus to infection with a field isolate of Aerococcus viridans var. homari (Gaffkemia): survival and haematology.

American lobsters Homarus americanus were inoculated with a field isolate of the Gram-positive bacterium Aerococcus viridans var. homari, causative agent of gaffkemia, at 1 x 10(6), 1 x 10(4) or 1 x 10(2) colony forming units (CFU) kg(-1) or with sterile 3% NaCl and maintained at 10 or 15 degrees C until they died or were euthanised. Progression of disease in individual animals was monitored daily by total haemocyte count (THC) and haemolymph culture. Post-mortem examinations were performed on all lobsters. Effects of both ambient temperature and infective dose on survival time were observed. Marked bacteraemia occurred in all mortalities. Haemocytopenia (THC < 10 x 10(9) cells l(-1)) preceded death in most, but not all, mortalities.

Studies on the virulence of Aerococcus viridans (var.) homari, the causative agent of gaffkemia, a fatal disease of homarid lobsters.

Virulent and avirulent strains of Aerococcus viridans (var.) homari were used to extend previous studies to determine and confirm differences between the 2 types. Virulent strains possessed polysaccharide capsules and were not agglutinated by lobster hemolymph serum; avirulent strains did not have capsules, were agglutinated by the lobster hemolymph serum, and most did not grow well in lobster hemolymph serum. Growth of the avirulent strains in sterile lobster hemolymph serum induced the production of capsules (which reached a maximum after 5 to 7 d incubation), eliminated susceptibility of the strains to the lobster serum agglutinin, and restored their virulence against lobsters. The factor(s) in lobster hemolymph serum inducing the long-lasting phenotypic response of virulence was (were) heat labile.

Appendage deformity syndrome--a nutritional disease of Macrobrachium rosenbergii.

Culture of the freshwater prawn Macrobrachium rosenbergii as an alternative to penaeid shrimp has recently increased in coastal areas of southern India in order to avoid numerous problems, particularly with white spot syndrome virus (WSSV). However, M. rosenbergii culture is now threatened by a new disease, appendage deformity syndrome (ADS), that also results in high mortality. Analysis of ADS prawns for viruses such as WSSV, monodon baculovirus (MBV) and infectious hypodermal and hematopoeitic necrosis virus (IHHNV) gave negative results. ADS prawns were also negative for bacterial pathogens and affected animals did not respond to antibiotic therapy. A study of potential nutritional deficiency revealed that carotenoid supplementation in the diet led to a significant decrease in ADS prawns.

Purification, cDNA cloning and modification of a defensin from the coconut rhinoceros beetle, Oryctes rhinoceros.

A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros, immunized with Escherichia coli. A full-size cDNA was cloned by combining reverse-transcription PCR (RT-PCR), and 5'- and 3'-rapid amplification of cDNA ends (RACE). Analysis of the O. rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpighian tubules. O. rhinoceros defensin showed strong antibacterial activity against Staphylococcus aureus. A 9-mer peptide amidated at its C-terminus, AHCLAICRK-NH2 (Ala22-Lys30-NH2), was synthesized based on the deduced amino-acid sequence, assumed to be an active site sequence by analogy with the sequence of a defensin isolated from larvae of the beetle Allomyrina dichotoma. This peptide showed antibacterial activity against S. aureus, methicillin-resistant S. aureus, E. coli and Pseudomonas aeruginosa. We further modified this oligopeptide and synthesized five 9-mer peptides, ALRLAIRKR-NH2, ALLLAIRKR-NH2, AWLLAIRKR-NH2, ALYLAIRKR-NH2 and ALWLAIRKR-NH2. These oligopeptides showed strong antibacterial activity against Gram-negative and Gram-positive bacteria. The antibacterial effect of Ala22-Lys30-NH2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. These Ala22-Lys30-NH2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast cells or macrophages, except for AWLLAIRKR-NH2.

[A preliminary study on alternation of generations of Cordyceps sinensis (Berkey) Sacc].

The insect pathogeny of parasitic hepialus by Cordyceps sinensis is, reported in this paper. The infestation of hepialus larvae by Cordyceps sinensis, growth and reproduction of hypha body in the hemolymph of host larvae, growth of stroma, maturity of hymenium and the abjection and germination of ascospores were observed.

The isolation of Vibrio parahaemolyticus and related vibrios from moribund aquarium lobsters.

Vibrios were isolated in pure culture from the hemolymph of 7 out of 28 dead or dying aquarium lobsters which had been acclimated to 20-22 degrees C. One isolate was identified as Vibrio parahaemolyticus, one as a related marine Vibrio (probably V. marinus), and five as Vibrio alginolyticus. No isolates of halophilic Vibrio species were made from healthy lobsters using thiosulfate citrate bile salts sucrose agar (TCBS).