Species In Fecal Samples Research Articles

Presence of Helicobacter and Campylobacter species in faecal samples from zoo mammals

Helicobacter and Campylobacter species (spp.) colonize the gastrointestinal tract of various domesticated animals. Apart from their pathogenic significance in animals, several species are of zoonotic importance as well. For most non-domesticated animal spp., however, little is known on the presence and importance of these agents. Therefore, we investigated the presence of Helicobacter and Campylobacter spp. in marine and terrestrial zoo mammals.Faecal samples of various marine and terrestrial zoo mammals were collected from 6 different zoos in Belgium. These samples were tested for the presence of Helicobacter and Campylobacter spp. by isolation and direct demonstration of DNA using genus-specific PCRs, followed by sequencing of the obtained amplicons.Helicobacter spp. were detected in 12 and Campylobacter spp. in 5 of the 22 faecal samples from marine mammals. In 4 of 5 dolphins, H. cetorum was demonstrated, a well-known pathogen associated with gastritis and gastric ulceration in marine mammals. C. insulaenigrae was isolated from 4 of 6 sea lions and from 1 of 11 seals. To our knowledge, this is the first description of the presence of C. insulaenigrae on the European mainland. Helicobacter spp. were detected in 5 and Campylobacter spp. (mainly C. jejuni subsp. jejuni and C. coli) in 9 of the 26 faecal samples from terrestrial mammals. Potential novel enterohepatic Helicobacter spp. were detected in both marine and terrestrial zoo mammals.For the first time, the presence of several known and unknown Campylobacter and Helicobacter spp. was demonstrated in the gastrointestinal tract of various marine and terrestrial zoo mammals. Further investigation is needed on the pathogenic and zoonotic importance of these species.

Travel-related acquisition of diarrhoeagenic bacteria, enteral viruses and parasites in a prospective cohort of 98 Dutch travellers.

Limited prospective data are available on the acquisition of viral, bacterial and parasitic diarrhoeagenic agents by healthy individuals during travel. To determine the frequency of travel associated acquisition of 19 pathogens in 98 intercontinental travellers, qPCR was used to detect 8 viral pathogens, 6 bacterial enteric pathogens and 5 parasite species in faecal samples collected immediately before and after travel. We found high pre-travel carriage rates of Blastocystis spp. and Dientamoeba fragilis of 32% and 19% respectively. Pre-travel prevalences of all other tested pathogens were below 3%. Blastocystis spp. (10%), Plesiomonas shigelloides (7%), D.fragilis (6%) and Shigella spp. (5%) were the most frequently acquired pathogens and acquisition of enteral viruses and hepatitis E virus in this relatively small group of travellers was rare or non-existent. Our findings suggest that the role of viruses as the cause of persisting traveller's diarrhoea is limited and bacterial pathogens are more likely as a cause of traveller's diarrhoea. The substantial proportion of travellers carrying Blastocystis spp. and D.fragilis before travel warrants cautious interpretation of positive samples in returning travellers with gastrointestinal complaints.

Isolation, identification and differentiation of Campylobacter spp. using multiplex PCR assay from goats in Khartoum State, Sudan

The aim of this study was to identify and characterize thermophilic Campylobacter species in faecal samples from goats in Khartoum State, Sudan, by application of multiplex polymerase chain reaction. Campylobacteriosis is a zoonotic disease of global concern, and the organisms can be transmitted to human via food, water and through contact with farm animals and pets. There are five clinically related Campylobacter species: Campylobacter jejuni (C. jejuni). Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus. Conventional cultural methods to diagnose campylobacteriosis are tedious and time consuming. Wide ranges of genes have been reported to be used for PCR-based identification of Campylobacter spp. We used a multiplex PCR assay to simultaneously detect genes from the major five clinically significant Campylobacter spp. The genes selected were hipO (hippuricase) and 23S rRNA from glyA (serine hydroxymethyl transferase) from each of C. jejuni. C. coli, C. lari, and C. upsaliensis; and sapB2 (surface layer protein) from C. fetus subsp. fetus. The assay was used to identify Campylobacter isolates recovered from 336 cultured faecal samples from goats in three localities in Khartoum State. C. coli was the most predominant isolate (234; 69.6%), followed by C. jejuni (19; 5.7%), C. upsaliensis (13; 3.9%), C. fetus subsp. fetus (7; 2.1%) and C. lari (6; 1.8%). Twenty-nine goats showed mixed infection with Campylobacter spp., 21 of which harbored two Campylobacter spp., while eight animals were infected with three species. Ten out of twelve goats that displayed diarrhea harbored C. coli only. C. coli, C. jejuni and C. upsaliensis showed significant variation with localities. The prevalence of C. coli was significantly higher (87; 25.9%) in goats from Omdurman, whereas C. jejuni and C. upsaliensis were significantly higher (11; 3.3%, 9; 2.7%) in goats from Khartoum. The multiplex PCR assay was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing the isolates, even in mixed cultures. The study demonstrated the significance of goats as reservoirs in the dissemination of Campylobacter spp. which could be considered as potential agent of caprine enteritis and abortion as well as contamination of the wider environment posing serious public health concern in Khartoum State.

Prevalence of Campylobacter species in fecal samples of pigs and humans from Zuru Kebbi State, Nigeria

Aim: The study was carried out to survey and determine the prevalence of Campylobacter species among pigs and humans within the pig rearing areas of Zuru Kebbi State, Nigeria. Materials and Methods: A cross-sectional study was conducted among household pigs and humans between September, 2013 and February, 2014. Isolation and characterization of Campylobacter species were performed using standard culture isolation techniques and biochemical characterization. A total of 450 fecal samples comprised of 300 from pigs and 150 from humans was collected and analyzed. Results: Prevalence of Campylobacter were 278 (92.67%) and 94 (62.67%) for pigs and humans respectively. The most encountered Campylobacter species in both cases was Campylobacter coli (276 [74.19]) followed by Campylobacter jejuni (62 [16.66]). The least isolated species in pigs was Campylobacter hyointestinalis 5 (1.8%) while Campylobacter lari 2 (2.13%) was least isolated in humans. C. lari was not found in pigs. No significant association (p>0.05) existed between Campylobacter isolates and the age and sex of both pigs and humans in this study. Conclusion: Both pigs and humans within the pig rearing areas of Zuru have been shown to harbor Campylobacter species and this might be due to extensive system of pig farming with indiscriminate defecation by pigs coupled with unhygienic disposal of human wastes in the environment and poor personal hygiene.

Detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR

The performance of a multiplex real-time PCR for the detection of Blastocystis, Dientamoeba fragilis, Giardia lamblia, Cryptosporidium species and Entamoeba species in faecal samples was evaluated in an observational prospective study. Paediatric patients (0–18 years) presenting with gastrointestinal symptoms and suspected of having enteroparasitic disease were included. A questionnaire on gastrointestinal symptoms and the chosen treatment was completed at the start of the study and after 6 weeks. Of 163 paediatric patients (mean age, 7.8 years), 114 (70%) had a PCR-positive faecal sample. D. fragilis was detected most frequently, in 101 patients, followed by Blastocystis in 49. In faecal samples of 47 patients, more than one protozoan was detected, mainly the combination of D. fragilis and Blastocystis. Reported gastrointestinal symptoms were abdominal pain (78%), nausea (30%), and altered bowel habits (28%). Eighty-nine of the PCR-positive patients were treated with antibiotics. A significant reduction in abdominal pain was observed both in treated and in untreated patients. This study demonstrated that multiplex real-time PCR detects a high percentage of intestinal protozoa in paediatric patients with gastrointestinal symptoms. However, interpretation and determination of the clinical relevance of a positive PCR result in this population are still difficult.

Determination of the discriminant score of intestinal microbiota as a biomarker of disease activity in patients with ulcerative colitis

BackgroundIn recent years, the gut microbiota has been found to provide an important link to the development of inflammatory bowel diseases (IBD) like ulcerative colitis (UC). Accordingly, inter-individual variation in the gut microbial community may be linked to inter-individual variation in the risk of IBD or other diseases. Further, the Terminal Restriction Fragment Length Polymorphism (T-RFLP) is a molecular biology technique for profiling bacterial species in faecal samples. This study was to evaluate a biomarker based on intestinal microbiota.MethodsThe study subjects were 69 patients with UC together with 80 relatives as controls. Twenty-three patients had active UC (group I) and 46 had quiescent UC (group II). The later included 17 patients with mild inflammation in the large intestine (group IIa), 29 without inflammation (group IIb). The patients’ relatives were consanguineous (group III, n = 47), and non-consanguineous (group IV, n = 33). Faecal samples were obtained from all subjects for the investigation of intestinal microbiota by applying the T-RFLP method. The Discriminant analysis of operational-taxonomic-unit (OTU) on T-RFLP fingerprints was performed. The Canonical Discriminant Function Coefficient (Df) for each OTU was calculated. The individual OTUs were multiplied by the Df value, and the sum was termed the Discriminant Score (Ds).ResultsThe Ds decreased thus: group I > group IIa > group IIb > group III > group IV. Significant difference was calculated for group I vs group IV (P < 0.01), group I vs group IIb (P < 0.05), group I vs group III (P < 0.01), group IIa vs IV (P < 0.01), group IIb vs group IV (P < 0.01), group III vs group IV (P < 0.01), indicating a strong association between gut microbial species and the development of UC.ConclusionsIn this study, the Ds related to UC, or otherwise absence of UC in the five groups. Potentially, Ds may become a clinically relevant biomarker of disease activity in UC. To our knowledge, this is the first application of the Ds to the study of microbiota in UC patients, consanguineous and non-consanguineous relatives.Trial registrationClinical trial No: UMIN 000004123.

Investigation of the use of pooled faecal and environmental samples following an enrichment step for the detection of Salmonella enterica by real-time PCR

INFECTION with Salmonella enterica is an important cause of enteric disease and death in horses (Hartnack and others 2012). However, Salmonella can also be associated with subclinical shedding, representing a...

Differential diagnosis of Moniezia benedeni and M. expansa (Anoplocephalidae) by PCR using markers in small ribosomal DNA (18S rDNA)

Moniezia benedeni and M. expansa are common ruminant tapeworms of worldwide distribution, causing gastrointestinal disorders and even death in sheep and goats. In this study, a polymerase chain reaction- (PCR-) based approach for precise species identification was developed and also applied to faecal DNA diagnosis of the tapeworm infection. Since nuclear ribosomal DNA (rDNA) appears to be a useful target for species and/or strain markers, the 18S regions of the rDNA of M. benedeni and M. expansa were amplified and sequenced. The lengths and GC contents of the regions sequenced were 2476-2487 bp and 51.9-52.1% for M. benedeni and 2473 bp and 51.9-52.0% for M. expansa, respectively. Alignment and comparison of the 18S sequences of the two species showed 92.5-93.3% homology. No matches for the 18S regions of M. benedeni and M. expansa were found with other species by BLAST search, which made the 18S sequences appropriate markers for the design of distinctive primers for the two Moniezia species. Our 18S-based PCR could detect as low levels as 0.5 pg genomic DNA or the DNA extracted from 0.2 g faecal sample collected from the rectum of an M. expansa-infected goat. The results indicate that this PCR approach is a reliable alternative for the differential diagnosis of Moniezia species in faecal samples.

Su1185 New Test for Profiling of the Gut Microbiota in Non-IBD and IBD Patients

to a labeled end of an amplified gene. T-RFLP is an established technique for analysing bacterial species in faecal samples. Methods: This was a quantitative analysis with a major focus on evaluating a biomarker based on intestinal microbiota. The subjects were 69 UC patients together with an 80 relatives as controls. Twenty-three patients had active UC (group I) and 46 had quiescent UC (group II). The latter included 17 with mild inflammation in the large intestine (group IIa), and 29 without inflammation (group IIb). The patients’ relatives were consanguineous (group III, n = 47), and non-consanguineous (group IV, n = 33). The Discriminant analysis of operationaltaxonomic-unit (OTU) on T-RFLP fingerprints was done. The Canonical Discriminant Function Coefficient (Df) for each OTU was calculated by using group I and group IV, in whom the difference of intestinal flora was thought to be greatest. The individual OTUs were multiplied by the Df value, and the sum was termed the Discriminant Score (Ds). The analysis was by SPSS (IBM Statistics 20.0). Results: The Ds decreased thus: group I > group IIa > group IIb > group III > group IV. As certain strains of bacteria are associated with the development of UC, while others not, this should show up in the OTU values in T-RFLP analysis. The analysis was set to factor this assumption, and the OUTs with 0 value 95% of all cases together with the OTUs of structure matrix value <0.01 according to the Discriminant analysis were excluded. Significant difference was calculated for group I vs. group IV (P< 0.01), group I vs. group IIb (P< 0.05), group I vs. group III (P< 0.01), group IIa vs. group IV (P< 0.01), group IIb vs. group IV (P< 0.01), group III vs. group IV (P< 0.01). Conclusions: In this study, the Ds related to UC activity, or otherwise absence of UC in the five groups. Potentially, Ds may become a clinically relevant biomarker of disease activity in UC. To our knowledge, this is the first application of the Ds to the study of microbiota in UC patients, consanguineous and non-consanguineous relatives.

Waiting for the human intestinal Eukaryotome

Waiting for the human intestinal Eukaryotome

Effects on intestinal microbiota of probiotic fermented milk used for prevention of antibiotic-associated diarrhoea

Probiotic administration could prevent antibiotic-associated diarrhoea due to the restoration of an intestinal microbiota imbalance caused by antibiotics. The effect of the probiotics Lactobacillus acidophilus LA-5, Lactobacillus casei subsp. casei LC-01 and Bifidobacterium animalis subsp. lactis BB-12 on the composition and metabolic activities of the intestinal microbiota in human subjects during antibiotic treatment has been analyzed. Subjects that were placed on antibiotic therapy (amoxiciline/clavulanic or levofloxacin) were randomly assigned to receive a multispecies probiotic fermented milk (n = 122) or a placebo fermented milk (n = 125). Stool samples were collected at different intervals during the intervention study and analysed for faecal microbiota enumeration, probiotic species detection, enzyme activities and short chain fatty acid (SCFA) concentrations. The most predominant microbiota groups were total anaerobes, bacteroides and total aerobes. Probiotic species in fecal samples were analyzed by real-time PCR and results indicated that prevalence of the probiotic species within the probiotic group was below 50 % during the intervention study. Enzyme activities and SCFA concentrations showed a large inter-individual variability over the time in the placebo and probiotic groups, except to β-galactosidase activity, which was the most abundant in both groups over the time. In conclusion, the combination of L. acidophilus LA-5, L. casei subsp. casei LC-01 and B. animalis subsp. lactis Bb-12 taken in daily doses of 109–1010 colony-forming units (CFU) does not significantly influence (P > 0.05) either the fecal microbiota or its metabolic activity in elderly hospitalized patients taking amoxicillin/clavulanate or levofloxacin. In addition, this combination of probiotic strains did not reduce the rate of occurrence of diarrohea in these patients.

Human gut microbiome viewed across age and geography

Gut microbial communities represent one source of human genetic and metabolic diversity. To examine how gut microbiomes differ between human populations when viewed from the perspective of component microbial lineages, encoded metabolic functions, stage of postnatal development, and environmental exposures, we characterized bacterial species present in fecal samples obtained from 531 individuals representing healthy Amerindians from the Amazonas of Venezuela, residents of rural Malawian communities, and inhabitants of USA metropolitan areas, as well as the gene content of 110 of their microbiomes. This cohort encompassed infants, children, teenagers and adults, parents and offspring, and included mono- and dizygotic twins. Shared features of the functional maturation of the gut microbiome were identified during the first three years of life in all three populations, including age-associated changes in the representation of genes involved in vitamin biosynthesis and metabolism. Pronounced differences in bacterial species assemblages and functional gene repertoires were noted between individuals residing in the USA compared to the other two countries. These distinctive features are evident in early infancy as well as adulthood. In addition, the similarity of fecal microbiomes among family members extends across cultures. These findings underscore the need to consider the microbiome when evaluating human development, nutritional needs, physiological variations, and the impact of Westernization.

Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples

Diarrheal diseases due to notifiable bacterial infections require rapid diagnosis of the causative pathogens. To facilitate detection, a real-time multiplex PCR was developed that identifies common diarrhea-causing bacteria in fecal samples. On the basis of published sequence data, sets of primers and probes were designed that were specific for Campylobacter jejuni, Salmonella, Shigella/enteroinvasive Escherichia coli EIEC, and Yersinia species, suitable for use in a one-tube PCR assay. The assay was assessed using a list of 137 well-defined intestinal bacterial strains or isolates. Furthermore, 393 routine clinical stool samples were analyzed, and the results of real-time multiplex PCR were compared with those obtained by established microbiological methods. The PCR yielded results within 3 h including DNA purification. No false-positive signals or cross-reactions were observed. The analytical sensitivity was 10 3 cfu mL −1 for Campylobacter jejuni, 10 4 cfu mL −1 for Salmonella, and 10 5 cfu mL −1 for Shigella/EIEC and Yersinia, respectively. Compared with culture, PCR detected 79 out of 81 Campylobacter jejuni (97.5%), 71 out of 74 Salmonella (96%), 8 out of 8 Shigella (100%), and 10 out of 10 Yersinia-positive (100%) clinical samples. In culture-negative samples ( n = 192), PCR additionally detected 2 Shigella, 1 Salmonella, and 5 Campylobacter jejuni infections. Thus, the new real-time multiplex PCR provides reliable results within a short time and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of enteropathogenic bacteria.

Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil

We have studied the variability of glutamate dehydrogenase (gdh) and small subunit ribosomal (SSU) rRNA coding genes of Giardia species in fecal samples isolated from wild and exotic animals in Brazil, and compared with homologous sequences of isolates from human and domestic animals characterized in previous studies. Cysts of Giardia duodenalis were obtained from feces of naturally infected monkeys (Alouatta fusca) (n=20), chinchillas (Chinchilla lanigera) (n=3), ostriches (Struthio camelus) (n=2) and jaguar (Panthera onca) (n=1). Assemblage AI was assigned to the unique isolate of jaguar. All the samples from monkeys, chinchillas, and ostriches were assigned to Assemblage B. There was little evolutionary divergence between the referred isolates and isolates described elsewhere. The Assemblage B isolates identified in this study were closely related to Assemblage BIV isolated from humans. The molecular identification of Assemblages A and B of G. duodenalis isolates from exotic and wild animals demonstrates that such hosts may be a potential reservoir for zoonotic transmission of G. duodenalis.

A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species

A hybridization probe-based real-time multiplex-nested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E. multilocularis, Echinococcus granulosus (G1), Echinococcus ortleppi, Echinococcus canadensis (G6, G7), Taenia crassiceps, Taenia hydatigena, Taenia mustelae, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis, Mesocestoides leptothylacus), carnivores (Vulpes vulpes, Vulpes corsac, Vulpes ferrilata, Canis familiaris, Felis catus, Martes foina), Microtus arvalis and Arvicola terrestris. The analytical sensitivity was 10 fg, evaluated with serially diluted DNA of E. multilocularis to 10 μl total DNA solution from E. multilocularis-negative canid faeces. Based on a comparison of 47 dog samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplex-nested PCR was moderately higher (38% vs. 30%) as when tested with a previously evaluated nested PCR with a sensitivity of 70-100%, depending on the number and gravidity status of worms present in the intestine (Dinkel et al., J Clin Microbiol 36:1871-1876, 1998). To assess the epidemiological applicability of this method, 227 canid faecal samples collected in the field were analysed. This newly developed real-time multiplex-nested PCR system is a specific, sensitive and reliable method for the detection of E. multilocularis and host species in faecal samples for epidemiological purposes.

Strain variation within Campylobacter species in fecal samples from dogs and cats

To investigate the incidence of co-colonization of different strains of Campylobacter species present in canine and feline stool samples, isolates were recovered by culture from 40 samples from dogs ( n = 34) and cats ( n = 6). Animals were of different ages, with diarrhoea or without clinical signs. Three isolation procedures were used: two selective agars and a filtration method. In each stool sample, multiple colonies were identified to the species level by PCR, subsequently genotyped by Amplified Fragment Length Polymorphism (AFLP) and pattern similarities (451 isolates) were calculated to investigate their phylogenetic relationships. Genetic heterogeneity of strains in individual stool samples was detected within the species Campylobacter jejuni, C. upsaliensis and C. helveticus, though to a different degree in dogs and cats. In 3 of the 34 (9%) canine samples, more than one genotype of the same Campylobacter species was present, while strain variation was detected in four of the six feline samples. The results show that preferably, multiple colonies should be analyzed in molecular epidemiological and aetiological studies.

Species composition and size structure of frogs preyed by the Iriomote cat Prionailurus bengalensis iriomotensis

Most species of small felids exclusively prey on groundliving mammals such as rodents (Kruuk 1982). Leopard cats Prionailurus bengalensis are also known as typical mammalian predators (Sunquist and Sunquist 2002), except for the Iriomote cat Prionailurus bengalensis iriomotensis, a subspecies of leopard cats endemic to Iriomote Island (284 km, 24°20'N, 123°49'E) in the southernmost part of the Ryukyu Archipelago, Japan. The cat had been long considered a separate species due to morphological differences with other small felids of Southeast Asia (Imaizumi 1967; Leyhausen and Pfleiderer 1999). Recently, their specific taxonomical distinction has been questioned by molecular methods (Masuda et al. 1994; Johnson et al. 1999) but despite this, the feeding ecology of the Iriomote cat remains unique among the cat family as they do not only prey upon mammals but also a variety of animals, such as birds, reptiles, frogs, and insects (Sakaguchi and Ono 1994; Watanabe et al. 2003). The variety of this cat’s diet may be an illustration of the optimal foraging theory in which animals should sample a variety of prey types and/or sizes to maximize their energy intake (Stephens and Krebs 1986). For instance, the Iriomote cat selectively preys on large-sized skinks during the warm season when skinks are abundant (Sakaguchi and Ono 1994). To get a better understanding of the feeding ecology of the Iriomote cat, its principal prey should be evaluated in detail with regard to species identification and size distribution. Preying on ground-living birds and reptiles has already been examined (Sakaguchi and Nishihira 1988; Sakaguchi and Ono 1994) but examination of the cat preying on frogs has not yet been carried out due probably to the difficulties in identifying the anuran species in fecal samples. Here, we developed a technique to identify species and to estimate body sizes of frogs found in the feces of cats. Following identification, the feeding pattern of Iriomote cats was considered with regard to species and size composition of frogs eaten.

Detection of Campylobacter species in faecal samples by direct Gram stain microscopy

To evaluate the Gram stain with carbol-fuchsin counterstain for the rapid detection of Campylobacter species in faecal samples. In total, 842 consecutive diarrhoeic faecal samples were prospectively examined for Campylobacter species by Gram stain and culture. Campylobacter species were isolated from 84 faecal samples (all Campylobacter jejuni). Compared with culture, Gram stain microscopy had a sensitivity of 89%, specificity of 99.7%, positive predictive value of 97%, and negative predictive value of 99% for detecting Campylobacter species. The direct Gram stain method can provide a presumptive result within 30 minutes of receipt of a faecal sample in the laboratory with relatively high sensitivity, and at low cost. Laboratories in areas where Campylobacter enteritis is common and/or with limited resources for Campylobacter culture should consider adopting this as a routine method.

P0030 PP QUANTITATIVE REAL TIME PCR OF BIFIDOBACTERIAL SPECIES OF INFANTS RECEIVING A PREBIOTIC INFANT FORMULA

Introduction: Breast-fed infants are generally found to have a typical intestinal flora with different bifidobacteria as the predominant species, which is considered to be important for the health of the infants. Recently, it has been described that allergic infants have a different flora compared to healthy infants. The levels of certain bifidobacteria species seem to be different and the levels of for example Bifidobacterium adolescentis are higher in allergic infants (1). Until now, the accurate determination of Bifidobacterium species in feces was impossible, but new molecular methods are now available, allowing the quantification of these species. Methods: For the accurate quantification of Bifidobacterium species in complex ecosystems, like the human gut, duplex 5′ nuclease assays (QRT-PCR) were developed for B. adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis and Bifidobacterium longum. The assays, targeted on rRNA intergenic spacer regions, were tested on specificity, sensitivity, reproducibility and compared to conventional PCR methods described earlier. The validated QRT-PCR assays were subsequently used for determination of the relative amounts of different Bifidobacterium species in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructooligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. Results: The results showed a significant increase in the total amount of fecal bifidobacteria in infants receiving the prebiotic formula (OSF). The bifidobacterial flora of these infants showed a large diversity of Bifidobacterium species similar to breast-fed infants, containing for example high numbers of B. infantis and B. longum. The intestinal flora of infants receiving a standard formula seems to resemble a more adult-like flora and contains relatively more B. catenulatum and B. adolescentis. Furthermore, the prevalence of B. adolescentis decreased significantly during the 6 weeks study period in the breast-fed group as well as in the group of infants receiving the prebiotic formula (OSF). Conclusion: With the use of newly developed QRT-PCR assays, the levels of bifidobacteria species in fecal samples can be determined accurately. From the current study it can be concluded that the prebiotic infant formula tested, stimulates different Bifidobacterium species in the infants’ colon, giving a distribution of the species that is comparable to the distribution in breast-fed infants.

Observations on the Role of Frugivorous Bats as Seed Dispersers in Costa Rican Secondary Humid Forests

The role of frugivorous bats as seed dispersers in humid neotropical tropical forests was evaluated between January and August 1995. Thirty germination trials were set up using seeds defecated by six bat species. Seeds of Piper multiplinervium and Ficus insipida, defecated by Carollia brevicauda and Artibeus jamaicensis, respectively, showed significantly higher germination percentages than seeds not ingested by bats. Of seven seed species regurgitated by bats, only Markea neurantha showed significant increases in percent germination compared to seeds not ingested by bats. Of 19 tents and three active feeding roosts found, 12 tents and three feeding roosts were in primary forest, while six tents were in abandoned plantations. For tent construction, four bat species used six plant species. Fifty-two percent of seeds found under tents and feeding roosts were from secondary forest species. Twenty-two percent of seed species in fecal samples from netted bats in three secondary forest types were primary forest species. Bats in our study dispersed seeds between different habitat types.