Case Study: Supplementation of cow-calf pairs grazing smooth bromegrass pastures with ethanol by-products and low-quality forages.

Multiparous, lactating, crossbred (Simmental × Angus) beef cows with spring-born calves at side (n = 16 per year; 4 per pasture) were used each of 3 yr to evaluate supplementing modified distillers grains plus solubles mixed with low-quality forage on cow and calf performance while grazing. Cow-calf pairs were assigned randomly to treatment with 2 replications (pasture) per year for 3 yr. Treatments were (1) recommended stocking rate of 9.46 animal-unit month/ha with no supplementation (CON) or (2) double the recommended stocking rate (18.9 animal-unit month/ha) and supplemented with a 30:70 modified distillers grains plus solubles:cornstalks (DM) mixture (SUPP). To replace 50% of grazed forage DMI, SUPP pairs were fed an average of 1.13% of BW (DM) over the grazing season. Pairs grazed adjacent smooth bromegrass pastures for 130 d during the summer. Gain was not different (P = 0.19) between SUPP and CON cows (0.28 vs. 0.19 kg/d, respectively). Ending cow BW was not affected (P = 0.46) by treatment. Similarly, calf gain was not affected (P = 0.31) by supplementation. In studies where confined cow-calf pairs were fed average-quality (IVDMD = 52.9%) forage, DMI was 2.58% of pair BW. Based on these data, CON and SUPP pairs consumed 18.6 and 19.1 kg of DM, respectively, of total feed per pair daily. The SUPP pairs consumed 7.1 kg of DM/pair daily of the supplement, replacing approximately 35% of grazed forage intake. These data suggest mixtures of ethanol co-products and low-quality forages can be supplemented to replace grazed forage intake of cattle, allowing for increased stocking rate without affecting animal performance.

Nitrogen fixation associated with sago (Metroxylon sagu) and some implications.

AIMS: To determine the presence and contribution of diazotrophic bacteria to nitrogen concentrations in edible starch derived from the sago palm (Metroxylon sagu). METHODS AND RESULTS: Isolation of diazotrophic bacteria and analysis of nitrogen fixation were conducted on pith, root and sago starch samples. Acetylene reduction showed that five of ten starch samples were fixing nitrogen. Two presumptive nitrogen-fixing bacteria from starch fixed nitrogen in pure culture and five isolates were positive for the nif H gene. Nitrogen concentrations in 51 starch samples were low (37 samples <0·2 g kg(-1); 14 ranging from 0·2 to 2·0 g kg(-1)). CONCLUSIONS: Nitrogen fixation occurs in sago starch, which undoubtedly plays a role in fermentation ecology. Nitrogen levels are considered too low to be of nutritional benefit and to protect against nutritional-associated illnesses. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch does not add significantly to the protein calorie intake and may be associated with susceptibility to nutritional-associated illness.

Spontaneous fermentation of traditional sago starch in Papua New Guinea.

Sago starch is an important dietary carbohydrate in lowland Papua New Guinea (PNG). An investigation was conducted to determine whether microbes play a role in its preservation using traditional methods. In 12 stored sago samples collected from PNG villages, lactic acid bacteria (LAB) were present (> or = 3.6 x 10(4)cfu/g) and pH ranged from 6.8 to 4.2. Acetic and propionic acids were detected in all samples, while butyric, lactic and valeric acids were present in six or more. In freshly prepared sago, held in sealed containers in the laboratory at 30 degrees C, spontaneous fermentation by endogenous microflora of sago starch was observed. This was evident by increasing concentrations of acetic, butyric and lactic acids over 4 weeks, and pH reducing from 4.9 to 3.1: both LAB and yeasts were involved. Survival of potential bacterial pathogens was monitored by seeding sago starch with approximately 10(4)/g of selected organisms. Numbers of Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus fell to < 30/g within 7 days. Salmonella sp. was present only in low numbers after 7 days (< 36/g), but Escherichia coli was still detectable after three weeks (> 10(2)/g). Fermentation appeared to increase the storability and safety of the product.

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea.

AIMS: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. METHODS AND RESULTS: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. CONCLUSIONS: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.

The epidemiology of melioidosis in the Balimo region of Papua New Guinea.

The distribution of Burkholderia pseudomallei was determined in soil collected from a rural district in Papua New Guinea (PNG) where melioidosis had recently been described, predominately affecting children. In 274 samples, 2.6% tested culture-positive for B. pseudomallei. Pulsed-field gel electrophoresis using SpeI digests and rapid polymorphic DNA PCR with five primers demonstrated a single clone amongst clinical isolates and isolates cultured from the environment that was commonly used by children from whom the clinical isolates were derived. We concluded that individuals in this region most probably acquired the organism through close contact with the environment at these sites. Burkholderia thailandensis, a closely related Burkholderia sp. was isolated from 5.5% of samples tested, an observation similar to that of melioidosis-endemic areas in Thailand. This is the first report of an environmental reservoir for melioidosis in PNG and confirms the Balimo district in PNG as melioidosis endemic.

Bacterial contamination of sago starch in Papua New Guinea.

Sago starch is an important food in lowland Papua New Guinea. Extraction of the starch from the palm and storage were performed by way of traditional methods that have been used for thousands of years. Currently, very little is known about the microbiology of sago starch. Sago samples were collected from areas of high starch utilization and analyzed for the presence of bacterial pathogens and indicator organisms. Storage methods and duration were recorded at the time of collection, and pH and water activity on arrival at the laboratory. Sago starch was found to harbor high levels of fecal contamination, as well as various food pathogens including Salmonella, Bacillus cereus, and coagulase-positive staphylococci. Clostridium perfringens was only present infrequently in samples and in very low numbers, while Listeria monocytogenes was not isolated from sago starch. The presence of high levels of fecal contamination in sago starch is of particular concern, and may contribute to diarrheal disease in rural Papua New Guinea.

Fungal colonization of sago starch in Papua New Guinea.

Sago starch is an important source of dietary carbohydrates in lowland Papua New Guinea. Over the past 30 years there have been sporadic reports of severe illness following consumption of sago starch. A common assumption is that fungal metabolites might be associated with the illness, leading to the need for a more thorough investigation of the mycoflora of sago starch. Sago starch was collected from areas of high sago consumption in Papua New Guinea for fungal analysis (69 samples). Storage methods and duration were recorded at the time of collection and pH on arrival at the laboratory. Yeasts were isolated from all samples except two, ranging from 1.2 x 10(3) to 8.3 x 10(7) cfu/g. Moulds were isolated from 65 of the 69 samples, ranging from 1.0 x 10(2) to 3.0 x 10(6) cfu/g. Of 44 samples tested for ergosterol content, 42 samples showed the presence of fungal biomass. Statistical analyses indicated that sago starch stored for greater than five weeks yielded significantly higher ergosterol content and higher numbers of moulds than sago stored for less than five weeks. The method of storage was also shown to influence mould numbers with storage in natural woven fibre containers returning significantly greater numbers than present in other storage methods tested. Potentially mycotoxigenic genera of moulds including Aspergillus and Penicillium were commonly isolated from sago starch, and as such storage factors that influence the growth of these and other filamentous fungi might contribute to the safety of traditional sago starch in PNG.

Melioidosis in a rural community of Western Province, Papua New Guinea.

A prospective study was conducted to determine the significance of melioidosis in the Balimo district of Western Province, Papua New Guinea. During 1998, after the establishment of laboratory procedures and increasing local clinical awareness, the disease was found in 1.8% (95% CI 0.37-5.1%) of individuals presenting with fever refractory to standard treatment. The clinical incidence was 20.0 per 100,000 population (95% CI 12.2-30.9). The median age of culture-confirmed cases was 9.5 years (interquartile range 8.3-14.8 years). The seroprevalence of 747 community children in the region tested was 8.2% (95% CI 6.2-10.4%). Most individuals presented during the rainy season with a febrile disease refractory to standard treatment, sometimes mimicking tuberculosis. Some family clustering was apparent. All patients with bacteraemic melioidosis died, but treatment with the available conventional therapies of chloramphenicol, co-trimoxazole or doxycycline resulted in survival and cure in six patients with subacute/localised melioidosis. Further studies are needed to ascertain the local epidemiology and why children appear particularly at risk, as well as to establish the true extent of melioidosis in Papua New Guinea.

Randomly amplified polymorphic DNA analysis of clinical and environmental isolates of Vibrio vulnificus and other vibrio species.

Vibrio vulnificus is an estuarine bacterium that is capable of causing a rapidly fatal infection in humans. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed for use in detecting V. vulnificus, as well as other members of the genus Vibrio. The resulting RAPD profiles were analyzed by using RFLPScan software. This RAPD method clearly differentiated between members of the genus Vibrio and between isolates of V. vulnificus. Each V. vulnificus strain produced a unique band pattern, indicating that the members of this species are genetically quite heterogeneous. All of the vibrios were found to have amplification products whose sizes were within four common molecular weight ranges, while the V. vulnificus strains had an additional two molecular weight range bands in common. All of the V. vulnificus strains isolated from clinical specimens produced an additional band that was only occasionally found in environmental strains; this suggests that, as is the case with the Kanagawa hemolysin of Vibrio parahaemolyticus, the presence of this band may be correlated with the ability of a strain to produce an infection in humans. In addition, band pattern differences were observed between encapsulated and nonencapsulated isogenic morphotypes of the same strain of V. vulnificus.

Randomly amplified polymorphic DNA analysis of starved and viable but nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is an estuarine bacterium capable of causing a rapidly fatal infection in humans. Because of the low nutrient levels and temperature fluctuations found in the organism's natural habitat, the starvation state and viable but nonculturable (VBNC) state are of particular interest. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed previously for the detection of V. vulnificus strains grown in rich media and has been applied to starved and VBNC cells of V. vulnificus in the present study. As cells were subjected to starvation in artificial seawater, changes in the RAPD profile were detected as early as 15 min into the starvation period. Most noticeable was a uniform loss of RAPD amplification products. By 4 h of starvation, the cells were undetectable by the RAPD method. Cells that had been starved for up to 1 year again became detectable by the RAPD method when nutrients were added to the starvation microcosm. The same loss of signal, but at a lower rate, was also seen as cells entered the VBNC state. VBNC cells were resuscitated by a temperature upshift and were once again detectable by the RAPD method. The addition of chloramphenicol prevented the RAPD signal from being lost in both the starvation and VBNC states. This suggests that DNA binding proteins produced during starvation and entrance into the VBNC state may be responsible for the inability of the RAPD method to amplify V. vulnificus DNA in these states.

Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus.

A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer. The RAPD-PCR results were scanned, and the images were analyzed with a computer program. Ribotype membranes were evaluated visually. Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous. Ribotyping enabled us to differentiate U.S. and Danish strains and V. vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V. vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes. Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V. vulnificus biotype 2 is an opportunistic pathogen for humans. One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains. This is, to our knowledge, the first time the isolation of V. vulnificus biotype 2 from coastal waters has been described.