Prevention of artificial dental plaque formation in vitro by plant extracts.

AIMS: A number of previous studies have shown that plant extracts can inhibit formation of dental plaque. The ability of extracts of Rosmarinus officianalis L., Salvia officianalis L., unfermented cocoa, red grape seed and green tea to inhibit plaque bacteria, glucosyltransferase activity, glucan and plaque formation in an in vitro model using bovine teeth was examined. METHODS AND RESULTS: The antimicrobial activity of the plant extracts against oral bacteria was determined using a standard susceptibility agar dilution technique. Inhibition of growth and acid production from glucose and sucrose by Streptococcus mutans in liquid culture was investigated. Prevention of plaque formation on bovine teeth initiated by Strep. mutans was studied using an artificial mouth. The plant extracts inhibited the growth of oral bacteria and prevented acid production by Strep. mutans. Extracts inhibited glucosyltransferase activity and glucan production and inhibited adhesion to glass. Extracts of R. officianalis L. and S. officianalis L. at 0·25 mg ml(-1) reduced plaque growth by >80%. Green tea extract completely inhibited plaque formation but resulted in a greenish discolouration of the teeth which could not be removed by scrubbing. CONCLUSIONS: The plant extracts, particularly those from R. officianalis L. and S. officianalis L., inhibited glucosyltranferase activity, glucan production and plaque formation in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the extracts of R. officianalis L. and S. officianalis L. may be useful as antiplaque agents in foods and dental preparations. Bovine teeth can be used as an alternative to hydroxyapatite for studies of plaque formation, but they need to be carefully sterilized before use.

The antibacterial activity of plant extracts containing polyphenols against Streptococcus mutans.

The minimum inhibitory concentrations (MIC) of commercially available and 70% aqueous propanone (P70) extracts from plants chosen for polyphenol content on Streptococcus mutans and other bacteria were determined using a standard susceptibility agar dilution technique to investigate their potential use as anticariogenic agents. The effects on adhesion of S. mutans to glass were also studied. The lowest MICs were for the P70 extracts of red grape skin (0.5 mg ml(-1)) and green tea and sloe berry skin (2 mg ml(-1)). The commercial extracts generally had a lower activity with a minimum MIC of 2 mg ml(-1) for tea extracts, grape seed extracts and Pynogenol (extract of maritime pine). All other extracts had MICs of > or = 4 mg ml(-1). Unfermented cocoa had greater antimicrobial activity than fermented cocoa and the activity of the fractionated extract increased with the extent of epicatechin polymerization. Epicatechin polymer had an MIC of 1 mg ml(-1) and an MBC of 64 mg ml(-1). Selected extracts were tested against other oral bacteria and showed activity against gram-positive organisms. P70 extracts of unfermented cocoa, epicatechin polymer fraction, green tea and red grape seed were bacteriostatic and prevented acid production when added at the MIC to cultures of S. mutans grown in a chemically defined medium supplemented with either glucose or sucrose. There was a reduction in viability which was greater when added to washed cells, but there were some viable cells after 24 h. The extracts also reduced adherence of S. mutans to glass.

The occurrence of the trematode Plagiorchis muris in the wood mouse Apodemus sylvaticus in North Yorkshire, UK.

Wood mice Apodemus sylvaticus were trapped each September over a 13-year period, from 1993 to 2005, in a wooded area adjacent to Malham Tarn, Yorkshire, UK. Plagiorchis muris was found to be the dominant intestinal trematode and occurred in every year of sampling, with an overall prevalence of 16.9%. This appears to be the first record of P. muris in A. sylvaticus within the UK. The mean worm burden was 2.03 and the distribution of the parasite within mice was typically overdispersed. No difference in prevalence relative to host sex was evident although there was a higher prevalence of 21.9% in larger older mice compared with 9.1% in juveniles, which probably relates to a greater foraging activity in older mice. Annual prevalence values of P. muris infections varied significantly over the study period with higher prevalences being associated with years with a high spring/summer rainfall. The second intermediate hosts for P. muris include a variety of aquatic insect larvae and it is likely that the higher rainfall may result in the occurrence of temporary water bodies suitable for the development of insect larvae.

Evaluation of Chromocult coliform agar for the detection and enumeration of Enterobacteriaceae from faecal samples from healthy subjects.

The purpose of this study was to examine the use of Chromocult agar medium for isolation and enumeration of Enterobacteriaceae from human faecal samples, to compare it to MacConkey agar and to evaluate its usefulness as a possible alternative selective medium in human faecal studies. The medium was shown to be effective in identifying Escherichia coli and coliforms in faeces without the need for extensive accompanying biochemical tests for confirmation of identity. A positive correlation (r=0.86) was found between the recovery of Enterobacteriaceae on the two media, and no significant difference (P>0.05) between overall mean bacterial counts for the whole study group or at different intervals of faecal collection were observed. Chromocult agar is an effective replacement for MacConkey agar in human faecal studies and has the advantage of differentiating E. coli from other coliforms.

The comparative gastrointestinal responses of children and adults following consumption of sweets formulated with sucrose, isomalt and lycasin HBC.

OBJECTIVES: To determine the gastrointestinal responses of children and adults following consumption of sucrose, isomalt and lycasin HBC and to compare these at two different dose levels in adults. DESIGN: Both studies were randomised, double-blind, cross-over designs. SUBJECTS: Fifty-one children aged 6-9 y were recruited from primary schools in the Salford area of Greater Manchester. Forty-eight children completed the study. Fifty healthy adult volunteers aged 18-24 y were recruited from the student population of the University of Salford. All subjects completed the study. INTERVENTIONS: Children consumed either 25 g of sucrose, isomalt or lycasin HBC and adults 25 and 40 g in hard boiled sweets per day for two consecutive test days. Test periods of 2 days were separated by 7 day washout periods. Children consumed sweets throughout test days and adults in no less than 30 min but no more than 90 min. Subjects reported the prevalence and magnitude of flatulence, borborygmi, bloating, colic, bowel movements and watery faeces. RESULTS: Consumption of 25 g isomalt provoked a mild laxative effect in children but not in adults. Consumption of 25 g isomalt significantly increased the prevalence and magnitude of gastrointestinal responses in both children and adults. Consumption of 25 g lycasin HBC significantly increased borborygml in children and adults but no other gastrointestinal responses. Consumption of 40 g lycasin HBC or isomalt by adults significantly increased the mean frequency of bowel movements and the number of subjects passing watery faeces. In adults, 40 g isomalt and lycasin HBC provoked significantly more gastrointestinal responses compared to 25 g of either product. CONCLUSIONS: Consumption of 25 g lycasin HBC does not provoke an unacceptable laxative effect or gastrointestinal response in children or adults compared to 25 g isomalt, which is associated with a mild laxative effect and increase in gastrointestinal responses. In adults gastrointestinal responses following consumption of products were found to be dose dependent.

Comparative gastrointestinal tolerance of sucrose, lactitol, or D-tagatose in chocolate.

D-tagatose is a potential new sugar substitute. Ingested D-tagatose is incompletely absorbed from the small intestine; unabsorbed D-tagatose reaches the colon where it is completely fermented. In a double-blind, controlled crossover study, the gastrointestinal effects were compared following acute consumption of 40 g plain chocolates containing 20 g of sucrose, lactitol, or D-tagatose by 50 healthy adults ages 18 to 24 years. Consumption of D-tagatose was not associated with a significant increase in the frequency of passing feces, or in the number of subjects passing watery feces. However, lactitol consumption was associated with an increase in both of these occurrences. Consumption of chocolate containing D-tagatose and lactitol resulted in significant increases in colic, flatulence, borborygmi, and bloating compared to consumption of the sucrose-containing chocolate, but the majority of symptoms were described as only "slightly more than usual." D-tagatose-containing chocolate did not provoke significantly more of these symptoms than lactitol-containing chocolate. A significant number of subjects reported nausea following consumption of D-tagatose chocolate compared to the sucrose chocolate control, and multiple symptoms occurred in some subjects. Overall, these results demonstrate that a 20-g dose of D-tagatose is tolerated well in comparison to lactitol.

Helminth parasites of the wild rabbit Oryctolagus cuniculus near Malham Tarn, Yorkshire, UK.

Between 1992 and 1996, 95 rabbits from the immediate locality of Malham Tarn, North Yorkshire, UK were examined for the presence of helminth parasites. All the examinations took place in late September or October. Three species of nematodes, Graphidium strigosum, Passalurus ambiguus and Trichostrongylus retortaeformis and two species of cestodes, Taenia pisiformis and Cittotaenia pectinata were identified. There were no associations between helminth species richness and year of sampling, host weight or sex. A logistic model was fitted to the prevalence data from these helminths as was an over-dispersed Poisson model to the worm burden data. Graphidium strigosum was the most frequently identified species with an average prevalence of 78%. The mean prevalence and intensity of Graphidium infection were significantly effected by sampling year. The lower than normal rainfall recorded at the Tarn during the years 1995 and 1996 may have be one reason for this pattern. The worm burden of G. strigosum was significantly positively associated with rabbit body weight. The intensity of infection with P. ambiguus was significantly higher in female rabbits. There was a significant non-linear relationship between P. ambiguus worm burden and rabbit weight (P = 0.002) with worm burdens being highest in the 1000 g to 1499 g weight cohort. Trichostrongylus retortaeformis was only identified in 1994 and male rabbits harboured significantly higher worm burdens than females (48 vs. 7, P = 0.022). Over the five years, the average Taenia pisiformis prevalence was 31% and there was a significant positive association between worm burden and rabbit weight (P = 0.001). Cittotaenia pectinata had a prevalence of 37% over the whole study period with no interactions between prevalence or intensity and body weight, year of sampling or rabbit sex. All five helminths showed an overdispersed distribution with k values less than 1.

Gastrointestinal tolerance of gamma-cyclodextrin in humans.

The gastrointestinal tolerance of gamma-cyclodextrin (gamma-CD) was examined in 24 healthy human volunteers. In a double-blind, placebo-controlled, randomized cross-over study, single acute doses of 8 g maltodextrin (placebo) or 8 g gamma-CD (test) were consumed as a mid-morning snack after addition to 100 g yogurt. Gastrointestinal symptoms as well as frequency and consistency of stools were recorded before and after lunch at between 3-4 and 7-8 h after intake respectively. The perception of the symptoms was rated on a subjective scale ranging from 1 ('more than normal') to 3 ('exceptionally more than normal'). Following consumption of maltodextrin, five subjects reported a total of 12 symptoms of which seven, two and three were rated as having grade 1, 2 and 3, respectively. Following consumption of gamma-CD, five subjects reported six symptoms all of which were graded as 1. Two subjects reported flatulence, which is a frequent consequence of the consumption of malabsorbed carbohydrates, after placebo and test treatment. The incidence of individual and combined side-effects as well as the number and consistency of faeces passed was not significantly different between placebo and test treatment. It is concluded that single doses of 8 g gamma-CD and maltodextrin are tolerated equally well. This is in keeping with a good digestibility of gamma-CD by salivary and pancreatic amylase.

Tolerance and breath hydrogen excretion following ingestion of maltitol incorporated at two levels into milk chocolate consumed by healthy young adults with and without fasting.

Little is known about the gastrointestinal effects of ingesting maltitol in chocolate. This study was designed to determine whether it leads to increased gastrointestinal symptomatology and if that symptomatology is dose related. It was also designed to discover whether breath hydrogen excretion in response to maltitol is dose related. In a double-blind, crossover study, 20 healthy volunteers aged 18-24 y ingested 100 g chocolate containing 40 g sucrose, 10 g sucrose plus 30 g maltitol or 40 g maltitol after fasting (abstinence from food and liquids from 2200 h on the night before chocolate consumption) and not fasting. There was no difference in symptomatology between fasting and nonfasting periods, and consumption order had no effect on symptomatology. Relative to ingestion of sucrose, 30 g maltitol caused no significant difference in symptoms, but 40 g resulted in more mild borborygmi (P < 0.05) and mild flatulence (P < 0.01) but not moderate or severe symptoms. Neither 30 nor 40 g maltitol caused significantly greater laxation than sucrose ingestion (P > 0.05). In a separate study, 10 healthy volunteers aged 18-24 y ate the same test products before breath H2 testing; 40 g maltitol in chocolate caused a greater total breath H2 excretion compared with 30 g maltitol (P < 0.05) or sucrose (P < 0.01). Total breath hydrogen excretion was also greater with 30 g maltitol compared with sucrose (P < 0.05). This dose-related response was consistent with the lower symptomatology after ingestion of 30 vs. 40 g maltitol. We have shown that 30 g maltitol in chocolate causes no significant symptomatology in young adults; however, 40 g caused mild borborygmi and flatus but no increased laxation. An increased breath H2 response indicates colonic fermentation of this polyol.

Ultrastructure of the alimentary tract of third-stage larvae of Litomosoides carinii.

The ultrastructural morphology of the alimentary tract of the third-stage larvae of Litomosoides carinii (Nematoda: Filarioidea) is described. The oesophagus possesses the characteristic tri-radiate lumen and is lined by cuticle. Teh lumen of the oesophagus is tightly occluded anteriorly by particulate material and posteriorly at the oesophagointestional junction. Three glands are present in the glandular region, one dorsal and two subventral. These glands contain secretory granules with similar morphological and staining properties. The lumen of the intestine is ill-defined without microvilli and apparently non-functional. It is likely that the larvae feed via the transcuticular route.

Activities of glycogen phosphorylase, alanine aminotransferase and aspartate aminotransferase in adult worms of Litomosoides carinii recovered from pyridoxine deficient cotton rats (Sigmodon hispidus).

This paper demonstrates that the activities of glycogen phosphorylase (GP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are reduced in adult worms of the filarial nematode Litomosoides carinii recovered from pyridoxine-deficient cotton rats when compared to worms recovered from pyridoxine-sufficient controls. GP, ALT and AST activities were determined in adult worms L. carinii recovered from cotton rat hosts over a 20-week experimental period. Activities of GP, ALT and AST in the parasite showed a direct correlation with the dietary pyridoxine intake of their host. Throughout the experiment, enzyme activities were significantly lower (P < 0.001) in worms from rats fed a pyridoxine-free diet ad libitum that in worms from rats fed either a stock colony diet, a pyridoxine-free diet ad libitum with daily supplementation of 100 micrograms pyridoxine or limited amounts of pyridoxine-free diet with daily supplementation of 100 micrograms pyridoxine. The lower than normal activity of GP, ALT, AST and other enzymes dependent on the biologically active derivative of pyridoxine, the coenzyme pyridoxal-5-phosphate (PLP), interferes with the protein, carbohydrate and lipid metabolism of L. carinii and may in part cause the reduced establishment, development and growth of the parasite in pyridoxine-deficient hosts.

Dose-related gastrointestinal response to the ingestion of either isomalt, lactitol or maltitol in milk chocolate.

OBJECTIVES: To determine whether there were differences between different polyols (sugar alcohols) in terms of their ability to stimulate intolerance symptoms when consumed in milk chocolate. Also to discover whether symptomatology can be related to the dose of polyol ingested. DESIGN: The study was of a randomised double-blind cross-over design. SUBJECTS: 59 healthy volunteers aged 18-24 years were recruited from the student population of the University of Salford. All subjects successfully completed the trial. INTERVENTIONS: Subjects ingested 100 g milk chocolate containing 40 g bulk sweetner as either sucrose, isomalt, lactitol or maltitol or a mixture (10:30 w/w) of sucrose and isomalt, sucrose and lactitol or sucrose and maltitol. Each bar was taken as breakfast on one day with following products consumed at 1-week intervals. Subjects reported the incidence and severity of the symptoms of flatulence, borborygms, colic, motion frequency and loose stools. RESULTS: The ingestion of 30 g or 40 g lactitol resulted in a significant increase in the incidence and severity of all symptoms examined compared to reactions after the consumption of standard sucrose-containing chocolate (P <0.01). Similarly, 40 g isomalt led to an increased incidence of all symptoms, including mild laxation (P <0.01), but unlike lactitol none was rated as being severe. A reduction in isomalt to 30 g was marked by increased tolerance with evidence of only mild borborygms (P <0.01), mild flatulence, colic, and laxation (P <0.05), with no increase in motion frequency (P <0.35). Ingestion of 40 g maltitol caused less intolerance than 40 g isomalt, with evidence of only flatulence, borborygms and colic (P <0.01), symptoms being rated as only mild. A reduction to 30 g led to a decrease in all symptoms except mild flatulence. Maltitol did not have any laxative effect when ingested at either 30 g (P = 0.32) or 40 g (P = 0.13) per day. CONCLUSIONS: This work has shown that there are significant differences in the reporting of gastrointestinal symptomatology following the consumption of isomalt, lactitol and maltitol incorporated into milk chocolate. However, with all three polyols the incidence and severity of symptomatology was dose dependent.

The host-parasite relationships in pyridoxine (vitamin B6) deficient cotton rats infected with Litomosoides carinii (Nematoda: Filaroidea).

This paper demonstrates that the establishment and growth of the filarial nematode parasite, Litomosoides carinii, is reduced in pyridoxine-deficient cotton rats. Young cotton rats were assigned to one of three dietary: vitamin B6-deficient cotton rats (B6-AL) were fed a pyridoxine-free diet ad libitum; pair-fed controls (B6 + PF) were fed the same amount of pyridoxine-free diet as animals in the deficient group and given daily oral supplements of 100 micrograms pyridoxine; and pyridoxine-sufficient controls (B6 + AL) were fed the pyridoxine-free diet ad libitum and supplemented daily with 100 micrograms pyridoxine. Half of each group was infected with 50 L3 of L. carinii by subcutaneous injection 8 weeks after the start of the experimental feeding period. B6-deficient cotton rats ate less (P < 0.001) and gained less weight (P < 0.001) than B6-supplemented controls. The levels of microfilaraemia in deficient animals, measured weekly throughout the experiment by taking blood smears, was significantly lower than in supplemented animals (P < 0.001). The deficient rats became latent for L. carinii at 20 weeks post-infection, whereas there was patent microfilaraemia in rats in the other dietary groups until the end of the experiment. Smaller (P < 0.001) and fewer (P < 0.05) adult worms were recovered from the pleural and abdominal cavities of deficient animals than from either pair-fed or sufficient controls at autopsy 28 week post-infection.

Embryogenesis in Litomosoides carinii from pyridoxine deficient cotton rats.

Pyridoxine (vitamin B6) deficiency was induced in cotton rats which were then infected with the filarial parasite Litomosoides carinii. Embryogenesis was assessed microscopically in worms taken from pyridoxine deficient cotton rats and from various categories of control animals. Embryogenesis was retarded in worms from pyridoxine deficient hosts and more abnormal embryos were present in such worms than in those from control animals.

Filariasis: nutritional interactions in human and animal hosts.

Vector-borne nematodes of the Order Filarioidea produce chronic, debilitating human infections which are usually nonfatal but are associated with a high degree of severe morbidity. Weight loss often accompanies infection and is probably a consequence of the increased energy cost associated with filarial fever, lymphangitis and lymphadenitis. In onchocerciasis, weight loss is associated with heavy worm loads as assessed by abundant nodules and large numbers of skin microfilariae. Experimental infections using rodent filariae have confirmed these observations and have also shown that low protein diets render hosts more susceptible to infection; nevertheless, parasite growth and embryogenesis is retarded in stunted female worms from protein deficient animals. In the absence of appropriate evidence, studies of experimental filariasis suggest that human protein-energy malnutrition may delay the development of stage-specific acquired immunity with a corresponding prolongation of patency. Epidemiological and experimental evidence shows that filarial nematodes acquire certain nutrients directly from their hosts. Of major importance in this respect is vitamin A which is taken up preferentially by human and rodent filariae; in humans, symptoms of hypovitaminosis A often accompany infection and could be an aggravating factor in onchocerciasis. Filariae also appear to require other specific nutrients such as iodine, thiamine and pyridoxine; dietary levels of these nutrients affect the host-parasite relationships in filariasis and pyridoxine seems to be of particular importance in this respect. Filarial parasites obviously compete with their hosts for available nutrients and, in the real world, human filariasis is often associated with a deterioration in the plane of nutrition of infected individuals.

Treatment of Nippostrongylus brasiliensis in normal and SPF rats using tetramisole loaded into zeolite.

Administration to rats of tetramisole loaded into zeolite was more successful in killing adults of Nippostrongylus brasiliensis than the administration of tetramisole alone. The most successful treatment occurred in SPF (Specific Pathogen Free) rats dosed with tetramisole loaded into zeolite and no worms were present in this group at autopsy eight days post-infection. It is concluded that the slow release of drug from the zeolite matrix improved its efficacy, especially in removing worms from low-grade infections.

Observations on third-stage larvae and adults of Litomosoides carinii (Nematoda: Filarioidea) by scanning and transmission electron microscopy.

The morphology of the body wall of the infective third-stage larvae and adults of Litomosoides carinii was studied by scanning and transmission electron microscopy. The mouth of both stages is surrounded by four inner papillae and two outer papillae; a pair of amphidial glands open laterally between the inner papillae. Female larvae and adults have two lateral caudal papillae; male larvae have a single ventral one, and adult males have two ventro-lateral lines of caudal papillae. The larval cuticle is two-layered, with fibres in the inner layer, whereas the adult cuticle has four layers apparently traversed by channels. Except for the chordal regions, larvae and adults both have an exceptionally thin hypodermis which is separated from both the cuticle and the underlying muscle cells by highly-convoluted membranes. The dorsal and ventral hypodermal chords contain nervous tissue, whereas the lateral hypodermis contains mainly mitochondria, golgi and micro-organisms. The muscle cells of larvae and adults contain thick and thin myofilaments arranged parallel to the long axis of the worms. In larvae each section has nine to 12 muscle cells, each of which has an inner non-sarcoplasmic zone containing the nucleus. The muscle cells of adult worms, although fewer in number, are more complex, and those of male worms in particular have lamellae which are deeply invaginated to form dense rectangular bands. Glycogen and lipid droplets are absent from larval muscle cells, whereas they are present in those of adult worms.

An examination of the mobilisation of elements from the skin and bone of the bog body Lindow II and a comparison with Lindow III.

The elemental content of samples of tissues derived from a 2,000-year old bog body, Lindow II, are described and the interchange of elements between the body and the surrounding or encompassing anaerobic peat medium is examined. A comparison is made of the chemical 'fingerprint' of Lindow II as compared with another bog body referred to as Lindow III. The application of body paint is noted. The bone has retained much of its structure and the Haversian Canal system is recognisable. The presence of a fungus mycelium on the skin samples is noted.