Experimental infection of red dwarf honeybee,Apis florea,with Nosema ceranae

This research is the first record of the infection of Apis florea by Nosema ceranae, a newly identified pathogen of honeybee in Thailand which was initially isolated from A. florea workers. Each Nosema free-bee was fed 2 μl of 50% (w/v) sucrose solution containing 0, 10,000 20,000 or 40,000 Nosema spores/bee. The survival rates of treated bees were significantly lower compared to control bees. Infectivity was not statistically different among the three spore concentrations, whereas no infection was found in control bees. Protein content of control bee hypopharyngeal glands 14 days post inoculation (p.i) was significantly higher (21.47 ± 0.17 mg/bee) compared to all treatments. The infection ratio of bees treated with 40,000 spores/bee increased with time after inoculation. These results suggest that N. ceranae has a significant negative effect on honeybee hypopharyngeal gland protein production and contributes to their shortened life span.     

Differential sensitivity of honey bees and bumble bees to a dietary insecticide (imidacloprid)

Currently, there is concern about declining bee populations and the sustainability of pollination services. One potential threat to bees is the unintended impact of systemic insecticides, which are ingested by bees in the nectar and pollen from flowers of treated crops. To establish whether imidacloprid, a systemic neonicotinoid and insect neurotoxin, harms individual bees when ingested at environmentally realistic levels, we exposed adult worker bumble bees, Bombus terrestris L. (Hymenoptera: Apidae), and honey bees, Apis mellifera L. (Hymenoptera: Apidae), to dietary imidacloprid in feeder syrup at dosages between 0.08 and 125 μg l^?1. Honey bees showed no response to dietary imidacloprid on any variable that we measured (feeding, locomotion and longevity). In contrast, bumble bees progressively developed over time a dose-dependent reduction in feeding rate with declines of 10–30% in the environmentally relevant range of up to 10 μg l^?1, but neither their locomotory activity nor longevity varied with diet. To explain their differential sensitivity, we speculate that honey bees are better pre-adapted than bumble bees to feed on nectars containing synthetic alkaloids, such as imidacloprid, by virtue of their ancestral adaptation to tropical nectars in which natural alkaloids are prevalent. We emphasise that our study does not suggest that honey bee colonies are invulnerable to dietary imidacloprid under field conditions, but our findings do raise new concern about the impact of agricultural neonicotinoids on wild bumble bee populations.     

Sperm viability and gene expression in honey bee queens (Apis mellifera) following exposure to the neonicotinoid insecticide imidacloprid and the organophosphate acaricide coumaphos

Honey bee population declines are of global concern. Numerous factors appear to cause these declines including parasites, pathogens, malnutrition and pesticides. Residues of the organophosphate acaricide coumaphos and the neonicotinoid insecticide imidacloprid, widely used to combat Varroa mites and for crop protection in agriculture, respectively, have been detected in wax, pollen and comb samples. Here, we assess the effects of these compounds at different doses on the viability of sperm stored in the honey bee queens’ spermatheca. Our results demonstrate that sub-lethal doses of imidacloprid (0.02 ppm) decreased sperm viability by 50%, 7 days after treatment. Sperm viability was a downward trend (about 33%) in queens treated with high doses of coumaphos (100 ppm), but there was not significant difference. The expression of genes that are involved in development, immune responses and detoxification in honey bee queens and workers exposed to chemicals was measured by qPCR analysis. The data showed that expression levels of specific genes were triggered 1 day after treatment. The expression levels of P450 subfamily genes, CYP306A1, CYP4G11 and CYP6AS14 were decreased in honey bee queens treated with low doses of coumaphos (5 ppm) and imidacloprid (0.02 ppm). Moreover, these two compounds suppressed the expression of genes related to antioxidation, immunity and development in queens at day 1. Up-regulation of antioxidants by these compounds in worker bees was observed at day 1. Coumaphos also caused a repression of CYP306A1 and CYP4G11 in workers. Antioxidants appear to prevent chemical damage to honey bees. We also found that DWV replication increased in workers treated with imidacloprid. This research clearly demonstrates that chemical exposure can affect sperm viability in queen honey bees.     

Comparative study on the dynamics and performances of Apis mellifera jemenitica and imported hybrid honeybee colonies in southwestern Saudi Arabia

The aims of this study were to assess the seasonal population dynamics and evaluate the performance of Apis mellifera jemenitica (local bee) and introduced hybrid honeybee colonies in the lowlands and highlands of southwestern Saudi Arabia. Data regarding the performance and population dynamics parameters such as brood and adult bee population, amounts of stored pollen and nectar were gathered from the two races (25 colonies of each) for one year (April 2013 through March 2014), and statistically tested. The results indicated that at low lands, local bee colonies maintained relatively high brood and adult bee populations (P < 0.05) than introduced honeybee colonies and produced more (P < 0.05) honey. The local bee colonies were able to hoard three times more (P < 0.05) pollen and built more (P < 0.05) queen cells than introduced bees in both the low and highland areas. The annual survival rate of local bee colonies was almost double (P < 0.05) than that of introduced honeybee colonies. Moreover, local bees had greater (P < 0.05) adult bee and brood populations than imported, throughout the year. The relatively good performance of local colonies could be due to their long year’s adaptation to cope with resource scarcity and unpredictable environmental conditions of the regions. The possible reasons for the dwindling of the imported hybrid colonies could be due to continuing to exhibit adaptive characteristics of their original that might not fit well with the new environment.     

The neonicotinoids thiacloprid,imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.)

A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. We investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24 h oral exposure, 200 μg/l or 2000 μg/l) and imidacloprid (1 μg/l or 10 μg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50–200 μg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity.     

Effects of a neonicotinoid pesticide on thermoregulation of African honey bees (Apis mellifera scutellata)

Thiamethoxam is a widely used neonicotinoid pesticide that, as agonist of the nicotinic acetylcholine receptors, has been shown to elicit a variety of sublethal effects in honey bees. However, information concerning neonicotinoid effects on honey bee thermoregulation is lacking. Thermoregulation is an essential ability for the honey bee that guarantees the success of foraging and many in-hive tasks, especially brood rearing. We tested the effects of acute exposure to thiamethoxam (0.2, 1, 2 ng/bee) on the thorax temperatures of foragers exposed to low (22 °C) and high (33 °C) temperature environments. Thiamethoxam significantly altered honey bee thorax temperature at all doses tested; the effects elicited varied depending on the environmental temperature and pesticide dose to which individuals were exposed. When bees were exposed to the high temperature environment, the high dose of thiamethoxam increased their thorax temperature 1–2 h after exposure. When bees were exposed to the low temperature, the higher doses of the neonicotinoid reduced bee thorax temperatures 60–90 min after treatment. In both experiments, the neonicotinoid decreased the temperature of bees the day following the exposure. After a cold shock (5 min at 4 °C), the two higher doses elicited a decrease of the thorax temperature, while the lower dose caused an increase, compared to the control. These alterations in thermoregulation caused by thiamethoxam may affect bee foraging activity and a variety of in-hive tasks, likely leading to negative consequences at the colony level. Our results shed light on sublethal effect of pesticides which our bees have to deal with.     

Bifidobacterium xylocopae sp. nov. and Bifidobacterium aemilianum sp. nov., from the carpenter bee (Xylocopa violacea) digestive tract

Social bees harbor a community of gut mutualistic bacteria, among which bifidobacteria occupy an important niche. Recently, four novel species have been isolated from guts of different bumblebees, thus allowing to suppose that a core bifidobacterial population may be present in wild solitary bees. To date there is sparse information about bifidobacteria in solitary bees such as Xylocopa and Osmia spp., this study is therefore focused on the isolation and characterization of bifidobacterial strains from solitary bees, in particular carpenter bee (Xylocopa violacea), builder bee (Osmia cornuta), and red mason bee (Osmia rufa). Among the isolates from Osmia spp. no new species have been detected whereas among Xylocopa isolates four strains (XV2, XV4, XV10, XV16) belonging to putative new species were found. Isolated strains are Gram-positive, lactate- and acetate-producing and possess the fructose-6-phosphate phosphoketolase enzyme. Full genome sequencing and genome annotation were performed for XV2 and XV10. Phylogenetic relationships were determined using partial and complete 16S rRNA sequences and hsp60 restriction analysis that confirmed the belonging of the new strains to Bifidobacterium genus and the relatedness of the strains XV2 and XV10 with XV16 and XV4, respectively. Phenotypic tests were performed for the proposed type strains, reference strains and their closest neighbor in the phylogenetic tree. The results support the proposal of two novel species Bifidobacterium xylocopae sp. nov. whose type strain is XV2 (=DSM 104955^T = LMG 30142^T), reference strain XV16 and Bifidobacterium aemilianum sp. nov. whose type strain is XV10 (=DSM 104956^T = LMG 30143^T), reference strain XV4.     

Variations of chemical element composition of bee and beekeeping products in different taxons of the biosphere

Geographical variations in element composition of bee products are poorly investigated though a lot of attempts are made to utilize the data in ecological monitoring. So the comparison of chemical element composition of bee and beekeeping products in different taxons of the biosphere may become valuable to test the efficiency of such approach. For this purpose content of 25 elements in bee body, bee bread, propolis and honey from Ribnitsa district of Moldavia (unpolluted area, control), Henty province of Mongolia (selenium deficient area) and Voskresensk district of Moscow region (mineral fertilizers production) were determined by means of the ICP-MS. Among 3 investigated regions Mongolia was characterized by the lowest Se levels and the highest accumulation of Al, Ca, Cd, Cu, Co, K, Mn, Mg, Na, Ni, P, Zn and V in bee bodies. The highest levels of Pb, Cr, Fe, Si, Sr and B, Se, Li, Sn were typical for Voskresensk and Moldavia bees accordingly. The highest correlation coefficients were registered between element concentrations in bee body and bee bread (r = +0.97–0.99, P < 0.0001), less significant – in bee body and propolis (r = +0.5–0.7; P < 0.001) and no correlation was demonstrated between element composition of bee body and honey. Propolis was characterized by significantly higher capacity to accumulate Pb, Cr, Sn and Al than bee body. Compared to bee body honey accumulated the lowest level of Mn and the highest of Si in Se-deficient Mongolia but the opposite phenomenon was demonstrated in Moldavia with moderately increased Se content in the environment. The results suppose that the most promising object for ecological monitoring is bee body. Element composition of propolis seems to reflect prolonged accumulation of elements, especially Pb, Al, Sn and Cr, by plant resin rather than dynamic temporal elements loading. Accumulation levels of elements in bee bread may be used on a par with bee body mineral content only in cases with equal honey content in bee bread. Honey utilization in monitoring of geochemical elements loading should be used with caution due to peculiarities of pollen/nectar elements distribution.     

Using sensillum potential analysis to quantify pheromone sensing of the antennal sensilla of Apis florea Fabricius (1787), foragers and guards

Odor perception via the antennal sensilla in most honeybee species is poorly studied. We measured the antennal sensillum potential in response to Apis florea mandibular gland pheromone and showed that it is robust and reliable in forager and guard bees. Mandibular gland pheromone may be involved in signaling alarm or foraging resource depletion. Changes of antennal sensilla placodea potential of A. florea foragers and guards were measured after exposure to three concentrations of the synthetic pheromones, 2-heptanone and (Z)-11-eicosen-1-ol, using a potentiostat connected to an e-corder (ED401) with microelectrodes. The resting sensillum potential of A. florea foragers and guards were ?55.37 ± 3.44 and ?52.85 ± 5.34 mV, respectively. The sensillum potential of bees exposed to 1.0%, 5.0% and 10.0% (Z)-11-eicosen-1-ol were ?23.35 ± 0.98, ?16.78 ± 1.94 and ?24.24 ± 8.20 mV, respectively, in foragers, and ?21.95 ± 3.21, ?21.42 ± 4.73 and ?13.54 ± 4.16 mV, respectively, for guards. Exposure of bees to 1.0%, 5.0% and 10.0% 2-heptanone induced sensillum potentials of ?10.64 ± 2.44, ?44.88 ± 2.41 and ?48.84 ± 4.40 mV, respectively, in foragers and 15.85 ± 9.38, ?25.48 ± 1.43 and ?15.52 ± 6.61 mV, respectively, in guards. The highest sensillum potential was recorded in foragers exposed to 1.0% 2-heptanone. In general, except for the response to 1.0% 2-heptanone, the sensillum potentials of all bees to (Z)-11-eicosen-1-ol were higher than that of 2-heptanone. These results show that A. florea antennal sensilla in foragers and guard bees exhibit a stronger response to (Z)-11-eicosen-1-ol as compared to 2-heptanone. Our results also provide useful comparative data to explore olfactory perception in non-model honey bee species.     

Transformation of maltose into prebiotic isomaltooligosaccharides by a novel α-glucosidase from Xantophyllomyces dendrorhous

The transglycosylation activity of a novel α-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with α-(1 → 2), α-(1 → 4) and α-(1 → 6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one α-(1 → 6) linkage (panose, 6-O-α-glucosyl-maltotriose and 6-O-α-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the α-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri- and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial α-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides.     

A ‘conovenomic’ analysis of the milked venom from the mollusk-hunting cone snail Conus textile—The pharmacological importance of post-translational modifications

Cone snail venoms provide a largely untapped source of novel peptide drug leads. To enhance the discovery phase, a detailed comparative proteomic analysis was undertaken on milked venom from the mollusk-hunting cone snail, Conus textile, from three different geographic locations (Hawai’i, American Samoa and Australia's Great Barrier Reef). A novel milked venom conopeptide rich in post-translational modifications was discovered, characterized and named α-conotoxin TxIC. We assign this conopeptide to the 4/7 α-conotoxin family based on the peptide's sequence homology and cDNA pre-propeptide alignment. Pharmacologically, α-conotoxin TxIC demonstrates minimal activity on human acetylcholine receptor models (100 μM, <5% inhibition), compared to its high paralytic potency in invertebrates, PD₅₀ = 34.2 nMol kg⁻¹. The non-post-translationally modified form, [Pro]^2,8[Glu]¹⁶α-conotoxin TxIC, demonstrates differential selectivity for the α3β2 isoform of the nicotinic acetylcholine receptor with maximal inhibition of 96% and an observed IC₅₀ of 5.4 ± 0.5 μM. Interestingly its comparative PD₅₀ (3.6 μMol kg⁻¹) in invertebrates was ∼100 fold more than that of the native peptide. Differentiating α-conotoxin TxIC from other α-conotoxins is the high degree of post-translational modification (44% of residues). This includes the incorporation of γ-carboxyglutamic acid, two moieties of 4-trans hydroxyproline, two disulfide bond linkages, and C-terminal amidation. These findings expand upon the known chemical diversity of α-conotoxins and illustrate a potential driver of toxin phyla-selectivity within Conus.     

Characterization of α-phosphoglucomutase isozymes from Toxoplasma gondii

The Toxoplasma gondii genome project has revealed two putative isoforms (TgPGM-I and TgPGM-II) of α-phosphoglucomutase (EC 5.4.2.2). We obtained recombinant proteins of these isoforms from the Beverley strain of T. gondii and characterized their properties, particularly the kinetic properties of these isoforms. The specific activities of TgPGM-I and TgPGM-II for α-d-glucose 1-phosphate were 338 ± 9 and 84 ± 6 μmol/min/mg protein, respectively, at 37 °C under optimal conditions. The Kcat and Km values of TgPGM-I were 398 ± 11/s and 0.19 ± 0.03 mM and those for TgPGM-II were 93 ± 7/s and 3.53 ± 0.91 mM, respectively, for α-d-glucose 1-phosphate. Magnesium ions were the most effective divalent cations for both the enzyme activities. The maximum activities of both the enzymes were obtained in the presence of more than 0.2 mM α-d-glucose 1,6-bisphosphate. Although both enzymes were attached to the α-phosphohexomutase superfamily, amino acid sequence homology between TgPGM-I and TgPGM-II showed very low overall identity (25%). No α-phosphomannomutase (EC 5.4.2.8) activity was detected for either enzyme. The data indicated that TgPGM-I, but not TgPGM-II, may play an important role in α-d-glucose 6-phosphate production.     

Synthesis and characterization of a fluorescent probe for α-tocopherol suitable for fluorescence microscopy

Previously prepared fluorescent derivatives of α-tocopherol have shown tremendous utility in both in vitro exploration of the mechanism of ligand transfer by the α-tocopherol transfer protein (α-TTP) and the intracellular transport of α-tocopherol in cells and tissues. We report here the synthesis of a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) containing α-tocopherol analog having extended conjugation with an alkenyl thiophene group that extends the absorption and emission maxima to longer wavelengths (λ_ex = 571 nm and λ_em = 583 nm). The final fluorophore thienyl-ene-BODIPY-α-tocopherol, 2, binds to recombinant human α-TTP with a K_d = 8.7 ± 1.1 nM and is a suitable probe for monitoring the secretion of α-tocopherol from cultured Mcf7#189 cells.     

Enzymatic properties of a bee venom serine protease from the bumblebee Bombus ignitus

We previously reported that bumblebee (Bombus ignitus) venom serine protease (Bi-VSP) acts as a prophenoloxidase-activating factor in arthropods and a fibrin(ogen)olytic enzyme in mammals. In the present study, we characterized the enzymatic properties of Bi-VSP purified from B. ignitus venom. The 34-kDa active form of Bi-VSP was purified from the venom of B. ignitus worker bees. Glycoprotein staining showed that approximately 20% of the total molecular mass of Bi-VSP is due to carbohydrate moieties. Bi-VSP had an optimal pH and temperature of pH 9.0 and 40 °C, respectively, and was stable at 50 °C for at least 10 min. Bi-VSP activity decreased abruptly below pH 6.0, indicating that Bi-VSP activity is almost completely inhibited at pH 5.4 of B. ignitus venom. The protease activity of Bi-VSP was strongly inhibited by typical serine protease inhibitors such as phenylmethanesulfonyl fluoride, leupeptin, and soybean trypsin inhibitor.     

Characteristics of Korean-Saanen goat milk caseins and somatic cell counts in comparison with Holstein cow milk counterparts

Characteristics of α- and β-casein fractions in the milk of Korean-Saanen goats were compared with those of Holstein cow milk using capillary electrophoresis (CE) analysis. The αs₁-CN content of the Saanen goat milk samples varied from 2.4% to 9.3% of total proteins. Total αs-CN content of the goat milk varied from 10.1% to 17.0%. Total β-CN content containing β₁-CN and the β₂-CN varied from 49.6% to 61.0% of total proteins. Average αs-CN to β-CN ratio of the Saanen goat milk from different farms was 0.24 ± 0.04, ranging from 0.17 to 0.33. The αs-CN (αs₁-CN + αs₀-CN) to β-CN (β_A1-CN + β_A2-CN) ratio of Holstein cow milk was 0.81, which was much higher than that of Korean-Saanen goat milk. The goat milk samples having more than 1.5 million cells/ml somatic cell counts (SCC) contained higher αs-CNs (P < 0.01) and lower β-CNs (P < 0.05) contents than milks with <1.5 million SCC. This resulted in a higher αs-CN to β-CN ratio (P < 0.01) in the milk with >1.5 million SCC.     

Purification and characterization of a novel thermostable α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp.

The purification and characterization of an extracellular α-l-arabinofuranosidase (α-l-AFase) from Chaetomium sp. was investigated in this report. The α-l-AFase was purified to homogeneity with a purification fold of 1030. The purified α-l-AFase had a specific activity of 20.6 U mg^?1. The molecular mass of the enzyme was estimated to be 52.9 kDa and 51.6 kDa by SDS–PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.0 and 70 °C, respectively. The enzyme was stable over a broad pH range of 4.0–10.0 and also exhibited excellent thermostability, i.e., the residual activities reached 75% after treatment at 60 °C for 1 h. The enzyme showed strict substrate specificity for the α-l-arabinofuranosyl linkage. The K_m and V_max values for p-nitrophenyl (pNP)-α-l-arabinofuranoside were calculated to be 1.43 mM and 68.3 μmol min^?1 mg^?1 protein, respectively. Furthermore, the gene encoding α-l-AFase was cloned and sequenced and found to contain a catalytic domain belonging to the glycoside hydrolase (GH) family 43 α-l-AFase. The deduced amino acid sequence of the gene showed the highest identity (67%) to the putative α-l-AFase from Neurospora crassa. This is the first report on the purification, characterization and gene sequence of an α-l-AFase from Chaetomium sp.     

Glucomannan and branched (1 → 3)(1 → 6) β-glucan from the aposymbiotically grown Physcia kalbii mycobiont

Cultures of the mycobiont Physcia kalbii were obtained from germinated ascospores and cultivated on Sabouraud-Sucrose-agar medium. Alkaline extraction of freeze-dried mycelia provided a branched (1 → 3),(1 → 6)-β-glucan and a glucomannan, whose chemical structure was determined by monosaccharide composition, methylation, controlled Smith degradation and NMR spectroscopic analysis. The β-glucan had a (1 → 3)-linked β-glucopyranosyl backbone, partially substituted (approx. 50% of the units) at O-6. The side chains were formed by 6-O- (∼82%) and 2,6-O-linked-β-Glcp units, while the non-reducing ends were formed by β-glucopyranosyl residues. The glucomannan had (1 → 6)-linked α-Manp units in the main chain, almost all being substituted at O-2 by α-Manp and α-Glcp units. This glucomannan could be a typical polysaccharide of lichens from the family Physciaceae.     

Pseudomonas contamination of a fungus-based biopesticide: Implications for honey bee (Hymenoptera: Apidae) health and Varroa mite (Acari: Varroidae) control

The ectoparasitic mite Varroa destructor is a major honey bee pest, and its control using pathogen-based biopesticides would resolve many of the problems, such as contamination and pesticide resistance, experienced with chemical control. A biopesticide, formulated with commercially-prepared conidia of a strain of Beauveria bassiana isolated from V. destructor was tested against the mites in bee colonies in southern France. The impact of treatment on hive survivorship, weight and mite infestation levels were very different from those of previous experiments using laboratory-prepared conidia: bee hives treated with the biopesticide died at a higher rate, lost more weight, and had higher mite densities at the end of the study than control hives. The biopesticide was subsequently found to be contaminated with bacteria. Two strains of bacteria were identified, by biotyping and sequencing data of the 16S rRNA and rpoB regions, and while the strains were distinct both were Pseudomonas sp. belonging to the P. fluorescens group. In dual cultures B. bassiana growth was slowed or suppressed when bacterial cfu density was about equal or greater than that of B. bassiana. Experiments using caged adult bees showed that bees ingesting diet and sugar solution treated with B. bassiana and kept at 30 °C had significantly lower survival times than those treated with one of the bacterial strains, but the opposite was true at 33 °C. Because one arthropod (honey bees) was treated for infestation by another (V. destructor), the impact of bacterial contamination was likely more noticeable than in most uses of biopesticides, such as treating plants against phytophagous insects. To reduce such risk in biopesticide development, a systematic screening for bacterial contamination prior to field application is recommended.