Influence of fluorescence of bacteria stained with acridine orange on the enumeration of microorganisms in raw milk

The staining of gram-positive and gram-negative cultures with acridine orange in metabolically active and inactive states was investigated using a Bactoscan, direct epifluorescent filter technique (DEFT), and standard plate count as the reference method. The evaluation of the bacterial cultures in the Bactoscan revealed a linear relationship between Bactoscan counts (pulses) and the quantity of pure culture suspension used. But the proper detection of bacteria with the fluorescence optic methods was dependent on the type of microorganism and the physiological state of the cells. The Bactoscan and DEFT underestimated the bacterial counts of gram-negative cultures as compared with standard plate counting. When stained with acridine orange, metabolically active bacteria showed more orange fluorescence and a lower percentage of green fluorescent cells as compared with inactive bacteria. Bactoscan pulse height analysis (PHA) diagrams, graphs of the detected pulses and their intensity, showed low pulses of inactive bacteria. Many of these weak pulses were eliminated from counting because of their faint fluorescent staining. In contrast, PHA diagrams of metabolically active microorganisms showed bright staining and, therefore, high pulses. A complete count of these bacteria was possible. These investigations point out that discrepancies between the fluorescence optical counting methods and the standard plate count depend strongly on the staining of the cultures with acridine orange and, therefore, on the type of microorganism and the metabolic state of the cells measured.     

Enumeration of clostridia in goat milk using an optimized membrane filtration technique

A membrane filtration technique developed for counting butyric acid bacteria in cow milk was further developed for analysis of goat milk. Reduction of the sample volume, prolongation of incubation time after addition of proteolytic enzyme and detergent, and a novel step of ultrasonic treatment during incubation allowed filtration of goat milk even in the case of somatic cell counts (SCC) exceeding 10⁶/mL. However, filterability was impaired in milk from goats in late lactation. In total, spore counts were assessed in 329 farm bulk goat milk samples. Membrane filtration technique counts were lower than numbers revealed by the classic most probable number technique. Thus, method-specific thresholds for milk to evaluate the risk of late blowing have to be set. As expected, the spore counts of milk samples from suppliers not feeding silage were significantly lower than the spore counts of milk samples from suppliers using silage feeds. Not only were counts different, the clostridial spore population also varied significantly. By using 16S rRNA gene PCR and gene sequencing, 342 strains from 15 clostridial species were identified. The most common Clostridium species were Clostridium tyrobutyricum (40.4%), Clostridium sporogenes (38.3%), Clostridium bifermentans (7.6%), and Clostridium perfringens (5.3%). The 2 most frequently occurring species C. tyrobutyricum and C. sporogenes accounted for 84.7% of the isolates derived from samples of suppliers feeding silage (n = 288). In contrast, in samples from suppliers without silage feeding (n = 55), these species were detected in only 45.5% of the isolates.     

Genetic Variation in Primary Metabolites of Pastinaca sativa; Can Herbivores Act as Selective Agents?

Although insect herbivory has been shown to act as a selective agent on plant secondary metabolism, whether primary metabolites contribute to resistance and can respond to selection by herbivores remains untested. In the wild parsnip (Pastinaca sativa), its principal herbivore, Depressaria pastinacella, acts as a selective agent on furanocoumarin resistance factors. In this study, we determined whether webworms can, by causing differential reductions in fitness, act as selective agents on parsnip primary metabolites. Estimates of narrow-sense heritabilities were significantly different from zero for C18 fatty acids in buds and developing fruits, fructose and sorbitol in buds, fructose, myo-inositol, bergapten, and psoralen in fruits. Wild parsnips protected from webworms by insecticide produced 2.5 times as much seed biomass as unsprayed plants; that webworms accounted for this difference in plant fitness was indicated by a significant negative relationship between reproductive effort and an index of webworm damage. Only a handful of metabolites influenced resistance to webworms; these included osthol, sorbitol, and protein in developing fruits as well as previously documented furanocoumarins. Osthol, a coumarinic compound, enhanced resistance, as did protein content, while sorbitol lowered resistance. Other primary metabolites may affect resistance to webworms, but their effect was context-dependent, that is, their effect depended on concentrations of other metabolites (epistasis). Susceptible plant phenotypes were found to have average chemical compositions. Although there was genetic variation in some of the primary metabolites in parsnips, the epistatic nature of their involvement in resistance and the lack of genetic variation in some suggest that selection on them from webworms will be either inconsistent or ineffective.     

Effects of furanocoumarins on feeding behavior of parsnip webworms Depressaria pastinacella

The parsnip webworm, Depressaria pastinacella, exhibits limited physiological resistance to furanocoumarin toxins in its principal host, the wild parsnip, Pastinaca sativa. These insects are typically found attacking individual plants low in furanocoumarins, relative to others within populations. They also feed preferentially on parthenocarpic fruits, which are lower in furanocoumarin content than are normal fruits. However, in a previous study with artificial diets, they did not appear to discriminate between high and low concentrations of furanocoumarins. In this study, the ability of webworms to distinguish between diets differing in furanocoumarin content was examined with an artificial diet containing wild parsnip and in green parsnip fruits with and without supplemental furanocoumarins. Larvae showed no preference for high or low furanocoumarin diets containing equal amounts of freeze-dried parsnip fruit powder. When given a choice between otherwise similar wild parsnip fruits, webworms strongly preferred fruits that were not augmented with furanocoumarins in one plant but showed no preference or only a weak preference for nonaugmented fruits in four other plants. In order to identify chemical constituents other than furanocoumarins that might determine feeding preferences, we compared the chemical profile of parthenocarpic fruits (which are preferred) to that of normal fruit. Octyl butyrate, a known deterrent to webworms, is highly correlated with furanocoumarin content, occurs in all plants, and differs significantly among normal and parthenocarpic fruit, suggesting that webworms may be able to avoid furanocoumarins by virtue of their behavioral response to octyl butyrate.     

Characterization of furanocoumarin metabolites in parsnip webworm,Depressaria pastinacella

Although metabolites of furanocoumarins have been characterized in a wide range of organisms, to date they have been identified in only a single insect species, Papilio polyxenes. Depressaria pastinacella, the parsnip webworm, like P. polyxenes a specialist on Apiaceae, routinely consumes plant tissues higher in furanocoumarin content than does P. polyxenes and is capable of faster cytochrome P-450-mediated detoxification of these compounds. In this study, we characterized metabolites of xanthotoxin, a linear furanocoumarin, and sphondin, an angular furanocoumarin, in midguts and frass of parsnip webworms. Two metabolites were isolated and identified from webworms fed artificial diet containing xanthotoxin. LC-ESI-MS analysis resulted in the determination of a MW of 266 for the compound in the frass and one of the compounds in the midgut; ¹H NMR confirmed its structure as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA). The second compound from the midgut had a MW of 252 and was identified by ¹H NMR and ¹³C NMR analysis as 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol) (HMCH). Whereas HCHA has been found in frass of Papilio polyxenes fed xanthotoxin, HMCH has not been reported previously in insects. Although the first step of metabolism of xanthotoxin in webworms as well as P. polyxenes is likely the formation of an epoxide on the furan ring, angular furanocoumarin metabolism in webworms appears to differ. The principal metabolite of sphondin was identified as demethylated sphondin (6-hydroxy-2H-furo[2,3-h]-1-benzopyran-2-one) by LC-ESI-MS and confirmed by ¹H NMR and ¹³C NMR analyses. That webworms produce metabolites of xanthotoxin in common not only with other Lepidoptera (e.g., HCHA) but with other vertebrates (e.g., HMCH) suggests a remarkable conservatism in the metabolic capabilities of cytochrome P-450s and raises the possibility that insects may share other detoxification reactions with vertebrates with respect to toxins in foodplants.     

Survival of Listeria monocytogenes after cleaning and sanitation of wooden shelves used for cheese ripening

The effect of cleaning and heat disinfection processes of wooden shelves used for cheese ripening on the survival of Listeria monocytogenes was examined. The cut boards were inoculated with a suspension containing 5.5?×?10⁷ colony forming units (CFU)/ml of L.??monocytogenes. Survival of L.??monocytogenes was investigated in the wood shavings. During the 24-hour incubation period in a humidity chamber, Listeria counts increased by 0.8 log units on average (n?=?9), compared to results 1?h after incubation, indicating the absence of antimicrobial properties of the wood in use. Additionally, after incubation for 24?h at room temperature, the boards were cleaned by soaking them for 15?min in a solution of hot alkaline detergent followed by brushing and rinsing with warm water. Some of the cleaned boards were subsequently heat treated at 80°C for 5?min and at 65°C for 15?min, respectively. The cleaning procedure alone was not sufficient to render L.??monocytogenes from the upper 2?mm wood layer inactive. In the case of both temperature-time combinations for heat disinfection, however, L.??monocytogenes was not detectable. The present study shows that the use of wooden shelves does not affect the hygienic safety of cheeses if such shelves are in good repair and are thoroughly cleaned and sanitized by heat treatment. Therefore, there is no reason to replace wood employed in cheese ripening processes with other materials.     

Impact of Elevated Levels of Atmospheric CO<Subscript>2</Subscript> and Herbivory on Flavonoids of Soybean (<Emphasis Type="Italic">Glycine max</Emphasis> Linnaeus)

Atmospheric levels of carbon dioxide (CO₂) have been increasing steadily over the last century. Plants grown under elevated CO₂ conditions experience physiological changes, particularly in phytochemical content, that can influence their suitability as food for insects. Flavonoids are important plant defense compounds and antioxidants that can have a large effect on leaf palatability and herbivore longevity. In this study, flavonoid content was examined in foliage of soybean (Glycine max Linnaeus) grown under ambient and elevated levels of CO₂ and subjected to damage by herbivores in three feeding guilds: leaf skeletonizer (Popillia japonica Newman), leaf chewer (Vanessa cardui Linnaeus), and phloem feeder (Aphis glycines Matsumura). Flavonoid content also was examined in foliage of soybean grown under ambient and elevated levels of O₃ and subjected to damage by the leaf skeletonizer P. japonica. The presence of the isoflavones genistein and daidzein and the flavonols quercetin and kaempferol was confirmed in all plants examined, as were their glycosides. All compounds significantly increased in concentration as the growing season progressed. Concentrations of quercetin glycosides were higher in plants grown under elevated levels of CO₂. The majority of compounds in foliage were induced in response to leaf skeletonization damage but remained unchanged in response to non-skeletonizing feeding or phloem-feeding. Most compounds increased in concentration in plants grown under elevated levels of O₃. Insects feeding on G. max foliage growing under elevated levels of CO₂ may derive additional antioxidant benefits from their host plants as a consequence of the change in ratios of flavonoid classes. This nutritional benefit could lead to increased herbivore longevity and increased damage to soybean (and perhaps other crop plants) in the future.     

Effects of Phytic Acid and Xanthotoxin on Growth and Detoxification in Caterpillars

Phytic acid is abundant in the fruits and seeds of many plants and is found in foliage to a lesser extent. Among its several properties, phytic acid is a potent chelator of essential minerals and proteins; thus, the possibility exists that heme-based enzymes such as cytochrome P450 monooxygenases in herbivores are detrimentally affected by phytic acid via chelation of dietary iron. Mortality, growth performance, and P450-mediated metabolism of xanthotoxin, a plant allelochemical, were examined in the presence of phytic acid in three lepidopteran species: a polyphagous seed-feeding species (Heliothis virescens), a polyphagous foliage-feeding species (Trichoplusia ni), and a species oligophagous on immature reproductive structures of two genera of Apiaceae (Depressaria pastinacella). While first instar H. virescens experienced no increase in mortality after 120 hours on a diet containing 1% phytic acid compared to a control diet, both T. ni and D. pastinacella experienced virtually complete mortality over the same time period. Ultimate instars of all three species experienced reductions in relative growth rates (RGR) and relative consumption rates (RCR) in the presence of phytic acid, although the only species to experience reduced digestive efficiency (ECI) was H. virescens. Cytochrome P450-mediated metabolism of xanthotoxin was reduced 60% in the presence of phytic acid in D. pastinacella, although metabolism remained unaffected in the two noctuids. These studies suggest a defensive function of phytic acid in addition to its primary functions of phosphorus storage, energy storage, and cell wall precursor source.     

Tritrophic Effects of Xanthotoxin on the Polyembryonic Parasitoid <Emphasis Type="Italic">Copidosoma sosares</Emphasis> (Hymenoptera: Encyrtidae)

Plant chemistry can have deleterious effects on insect parasitoids, which include the reduction in body size, increased development time, and increased mortality. We examined the effects of xanthotoxin, a linear furanocoumarin, on the polyembryonic encyrtid wasp Copidosoma sosares, a specialist parasitoid that attacks the parsnip webworm, Depressaria pastinacella, itself a specialist on furanocoumarin-producing plants. Furanocoumarins, allelochemicals abundant in the Apiaceae and Rutaceae, are toxic to a wide range of herbivores. In this study, we reared parasitized webworms on artificial diets containing no xanthotoxin (control) or low or high concentrations of xanthotoxin. Clutch sizes of both male and female C. sosares broods were more than 20% smaller when they developed in hosts fed the diet containing high concentrations of xanthotoxin. Xanthotoxin concentration in the artificial diet had no effect on the development time of C. sosares, nor did it have an effect on the body size (length of hind tibia) of individual adult male and female C. sosares in single-sex broods. Webworms fed artificial diets containing low or high concentrations of xanthotoxin were not significantly smaller, and their development time was similar to that of webworms fed a xanthotoxin-free diet. Mortality of webworms was not affected by xanthotoxin in their artificial diet. Therefore, dietary xanthotoxin did not appear to affect C. sosares via impairment of host health. However, unmetabolized xanthotoxin was found in D. pastinacella hemolymph where C. sosares embryos develop. Hemolymph concentrations were fourfold greater in webworms fed the high-xanthotoxin-containing diet than in webworms fed the low-xanthotoxin-containing diet. We failed to detect any xanthotoxin metabolism by either C. sosares embryos or precocious larvae. Therefore, the observed tritrophic effects of xanthotoxin are likely to be due to the effects of xanthotoxin after direct contact in the hemolymph rather than to the effects of compromised host quality.