Disposable bioprocessing: The future has arrived

Increasing cost pressures are driving the rapid adoption of disposables in bioprocessing. While well ensconced in lab‐scale operations, the lower operating/ validation costs at larger scale and relative ease of use are leading to these systems entering all stages and operations of a typical biopharmaceutical manufacturing process. Here, we focus on progress made in the incorporation of disposable equipment with sensor technology in bioprocessing throughout the development cycle. We note that sensor patch technology is mostly being adapted to disposable cell culture devices, but future adaptation to downstream steps is conceivable. Lastly, regulatory requirements are also briefly assessed in the context of disposables and the Process Analytical Technologies (PAT) and Quality by Design (QbD) initiatives. Biotechnol. Bioeng. 2009;102: 348–356. © 2008 Wiley Periodicals, Inc.     

Bioprocessing of plant cell cultures for mass production of targeted compounds

More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered. This paper is dedicated to Prof. Dr. Mladenka P. Ilieva on the occasion of her 70th birthday.     

Eco-efficiency of disposable and reusable surgical instruments—a scissors case

Purpose

In recent years, the rising costs and infection control lead to an increasing use of disposable surgical instruments in daily hospital practices. Environmental impacts have risen as a result across the life cycle of plastic or stainless steel disposables. Compared with the conventional reusable products, different qualities and quantities of disposable scissors have to be taken into account. An eco-efficiency analysis can shed some light for the potential contribution of those products towards a sustainable development.

Methods

Disposable scissors made of either stainless steel or fibre-reinforced plastic were compared with reusable stainless steel scissors for 4,500 use cycles of surgical scissors used in Germany. A screening life cycle assessment (LCA) and a life cycle costing were performed by following ISO 14040 procedure and total cost of ownership (TCO) from a customer perspective, respectively. Subsequently, their results were used to conduct an eco-efficiency analysis.

Results and discussion

The screening LCA showed a clear ranking regarding the environmental impacts of the three types of scissors. The impacts of the disposable steel product exceeds those of the two others by 80 % (disposable plastic scissors) and 99 % (reusable steel scissors), respectively. Differences in TCO were smaller, however, revealing significant economic advantages of the reusable stainless steel product under the constraints and assumptions of this case study. Accordingly, the reusable stainless steel product was revealed as the most eco-efficient choice. It was followed by the plastic scissors which turned out to be significantly more environmentally sound than the disposable stainless steel scissors but also more cost-intensive.

Conclusions

The overall results of the study prove to be robust against variations of critical parameters for the prescribed case study. The sensitivity analyses were also conducted for LCA and TCO results. LCA results are shown to be reliable throughout all assumptions and data uncertainties. TCO results are more dependent on the choice of case study parameters whereby the price of the disposable products can severely influence the comparison of the stainless steel and the plastic scissors. The costs related to the sterilisation of the reusable product are strongly case-specific and can reduce the economic benefit of the reusable scissors to zero. Differences in environmental and economic break-even analyses underline the comparatively high share of externalised environmental costs in the case of the disposable steel product.     

A computer-aided approach to compare the production economics of fed-batch and perfusion culture under uncertainty

Fed-batch and perfusion culture dominate mammalian cell culture production processes. In this paper, a decision-support tool was employed to evaluate the economic feasibility of both culture modes via a case study based upon the large-scale production of monoclonal antibodies. The trade-offs between the relative simplicity but higher start-up costs of fed-batch processes and the high productivity but higher chances of equipment failure of perfusion processes were analysed. Deterministic analysis showed that whilst there was an insignificant difference (3%) between the cost of goods per gram (COG/g) values, the perfusion option benefited from a 42% reduction in capital investment and a 12% higher projected net present value (NPV). When Monte Carlo simulations were used to account for uncertainties in titre and yield, as well as the risks of contamination and filter fouling, the frequency distributions for the output metrics revealed that neither process route offered the best of both NPV or product output. A product output criterion was formulated and the options that met the criterion were compared based on their reward/risk ratio. The perfusion option was no longer feasible as it failed to meet the product output criterion and the fed-batch option had a 100% higher reward/risk ratio. The tool indicated that in this particular case, the probabilities of contamination and fouling in the perfusion option need to be reduced from 10% to 3% for this option to have the higher reward/risk ratio. The case study highlighted the limitations of relying on deterministic analysis alone.     

Simultaneous saccharification and fermentation and economic evaluation of ultrasonic and jet cooking pretreatment of corn slurry

The potential of ultrasonics to replace hydrocooking in corn-to-ethanol plants was examined in this study. Batch and continuous experiments were conducted on corn slurry with sonication at a frequency of 20 kHz. Batch mode used a catenoidal horn operated at an amplitude of 144 μm peak-to-peak (p–p) for 90 s. Continuous experiments used a donut horn operating at inner radius amplitude of 12 μm p–p. Jet-cooked samples from the same ethanol plant were compared with ultrasonicated samples. The highest starch-to-ethanol conversion was obtained by the jet-cooked samples with a yield of 74% of the theoretical yield. Batch and continuous sonication achieved 71.2% and 68% conversion, respectively, however, statistical analysis showed no significant difference between the jet cooking and ultrasonication. On the basis of the similar performance, an economic analysis was conducted comparing jet cooking and ultrasonic pretreatment. The analysis showed that the capital cost for the ultrasonics system was ~10 times higher compared to the capital cost of a hydrocooker. However,due to the large energy requirements of hydrocookers, the analysis showed lower total overall costs for continuous ultrasonication than that for jet cooking, assuming the current energy prices. Because of the high utility cost calculated for jet cooking, it is concluded that ultrasonication poses as a more economical option than jet cooking. Overall, the study shows that ultrasonics is a technically and economically viable alternative to jet cooking in dry-grind corn ethanol plant.     

Phytoremediation of biosolids from an end-of-life municipal lagoon using cattail (Typha latifolia L.) and switchgrass (Panicum virgatum L.)

Land spreading of biosolids as a disposal option is expensive and can disperse pathogens and contaminants in the environment. This growth room study examined phytoremediation using switchgrass (Panicum virgatum L.) and cattail (Typha latifolia L.) as an alternative to land spreading of biosolids. Seedlings were transplanted into pots containing 3.9 kg of biosolids (dry wt.). Aboveground biomass (AGB) was harvested either once or twice during each 90-day growth period. Switchgrass AGB yield was greater with two harvests than with one harvest during the first 90-day growth period, whereas cattail yield was not affected by harvest frequency. In the second growth period, harvesting frequency did not affect the yield of either plant species. However, repeated harvesting significantly improved nitrogen (N) and phosphorus (P) uptake by both plants in the first period. Phytoextraction of P was significantly greater for switchgrass (3.9% of initial biosolids P content) than for cattail (2.8%), while plant species did not have a significant effect on N phytoextraction. The trace element accumulation in the AGB of both plant species was negligible. Phytoextraction rates attained in this study suggest that phytoremediation can effectively remove P from biosolids and offers a potentially viable alternative to the disposal of biosolids on agricultural land.     

Integrated versus stand-alone second generation ethanol production from sugarcane bagasse and trash

Ethanol production from lignocellulosic materials is often conceived considering independent, stand-alone production plants; in the Brazilian scenario, where part of the potential feedstock (sugarcane bagasse) for second generation ethanol production is already available at conventional first generation production plants, an integrated first and second generation production process seems to be the most obvious option. In this study stand-alone second generation ethanol production from surplus sugarcane bagasse and trash is compared with conventional first generation ethanol production from sugarcane and with integrated first and second generation; simulations were developed to represent the different technological scenarios, which provided data for economic and environmental analysis. Results show that the integrated first and second generation ethanol production process from sugarcane leads to better economic results when compared with the stand-alone plant, especially when advanced hydrolysis technologies and pentoses fermentation are included.     

Companion planting to attract pollinators increases the yield and quality of strawberry fruit in gardens and allotments

1. Global pollinator declines have led to concern that crop yields might fall as a result of a pollination deficit. Companion planting is a traditional practice thought to increase yield of insect pollinated crops by planting a co-flowering species next to the crop. 2. Using a combination of conventional researcher-led experiments and observational citizen scientist data, we tested the effectiveness of bee-friendly borage (Borago officinalis) as a companion plant to strawberry (Fragaria x ananassa). Insect visitors to the ‘Test’ (strawberry + borage) versus ‘Control’ (strawberry only) plants were observed, and strawberry fruit collected. Strawberries collected during the researcher-led experiment were also subject to fruit measurements and assessments of market quality. 3. Companion plants were found to significantly increase both yield and market quality of strawberries, suggesting an increase in insect pollination per plant. Test strawberries companion planted with borage produced an average of 35% more fruits, and 32% increased yield by weight. Test strawberry plants produced significantly more fruit of higher aesthetic quality when assessed by Marketing Standards for Strawberries. 4. Although there was no significant difference in the overall insect visits, when broken down by broad insect group there were significantly more flies visiting the test strawberries than controls. 5. These results could have implications for both gardeners and commercial growers. As consumers prefer a cosmetically perfect fruit, the production of fruit with increased aesthetics aids food waste reduction.     

Disposable bioreactors for plant liquid cultures at Litre‐scale

Driven by the demands of the market and the manufacturing industry, disposable bioreactors have gained in importance in cell culture‐based processes during the last 10 years. Today they are widely accepted in R&D and also in manufacturing where process simplicity, safety and flexibility have top priority. Although disposable bioreactors are mainly used for cell expansions, glycoprotein secretions and virus generations realised with mammalian and insect cell lines, there are several reports delineating their suitability for the cultivation of plant cell and tissue cultures. This review describes the current disposable bioreactor types suitable for growing plant cell suspensions and organ cultures (hairy roots, meristematic clusters, somatic embryos) at Litre‐scale. Based on a definition of the term “disposable bioreactor”, a categorisation of the prevalent types for plant liquid cultures is presented. We describe the bioreactor regimes, working principles and bioengineering parameters of mechanically and pneumatically agitated bag bioreactors, which have advantages of process scalability and efficiency. Furthermore, results from the literature and data from our own research (obtained during production of undifferentiated bioactive cells, expressions of secondary metabolites and glycoproteins, and micropropagations of plant tissues) are discussed.     

Potential of plant cells in culture for cosmetic application

Plants and plant derived ingredients are common and of major importance in the fields of pharmacy, food and cosmetics. The cosmetic industry is a fast moving market. Products have short life-cycles and the industry has to come up with innovative products constantly. Most cosmetic products and their applications are defined by active ingredients. These active ingredients may derive from either synthetic sources or from plant sources. Beside this, no other origin like human or animal are accepted or allowed in cosmetics nor are genetically modified plant sources. The whole cosmetic research and development society is therefore desperately seeking for new innovative plant ingredients for cosmetic application. Unfortunately, new plant derived ingredients are limited because several plants of cosmetic interest are not to be used due to following facts: the plants contain toxic metabolites, the plants grow too slow and a seasonal harvesting is not possible, the concentration of plant constituents differ from harvest to harvest or the plant is endangered and not allowed to harvest. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plant derived actives. Due to all these findings, we decided to risk the step into plant cell culture derived cosmetic active ingredient production. This article describes the successful establishment of an apple suspension culture producing a high yield of biomass, cultured in disposable, middle-scale bioreactors. The use of a bioactive extract out of these cells for cosmetic application and the efficacy of this extract on mammalian stem cells is also outlined in this article. To obtain a suitable cosmetic product we used the high pressure homogenization technique to decompose the plant cells and release all the beneficial constituents while encapsulating these components at the same time in liquid Nanoparticles. With the plant cell culture technology we bring complete new aspects in the development of novel cosmetic plants derived actives.     

Nucleopolyhedrovirus infection enhances plant defences by increasing plant volatile diversity

Plant–herbivore–entomopathogen tri-trophic interactions and biodiversity are relatively understudied topics in ecology. Particularly, the effects of entomopathogens on herbivore-induced plant volatiles and plant volatile diversity on the defensive function of plants have not been studied in detail. We used soybean (Glycine max), beet armyworm larvae (Spodoptera exigua), and nucleopolyhedrovirus (NPV) as a tri-trophic system to determine whether NPV infection can promote the emission and diversity of volatiles from plants. We also investigated whether NPV infection affects the attraction of Microplitis pallidipes, an important endoparasitoid of larval S. exigua. Uninfested soybean plants released 7 detectable volatile compounds while plants fed upon by healthy and NPV-infected S. exigua larvae released 12 and 15 volatiles, respectively. Female parasitoids were more attracted to the volatiles from plants that were fed upon by NPV-infected larvae than healthy larvae, and more attracted to the volatiles from plants that were fed upon by healthy larvae than no larvae. The selective responses of parasitoids to plant odours increased as plant volatile diversity increased. Our study suggests that the NPV infection facilitates the release of plant volatiles and enhances the defensive function of plants by increasing plant volatile diversity which in turn attracts more parasitoids. Also, this work reveals that plants might accrue two indirect benefits from NPV infection, cessation of herbivore feeding and more parasitisation.     

Comparative life cycle assessment of reused versus disposable dental burs

Purpose

Health care infection control has led to increased utilization of disposable medical devices, which has subsequently led to adverse environmental effects attributed to health care and its supply chain. In dental practice, the dental bur is a commonly used instrument that can be either reused or used once and then disposed. To evaluate the disparities in environmental impacts of disposable and reusable dental burs, a comparative life cycle assessment (LCA) was performed.

Methods

The functional unit was defined as one reusable dental bur, where the maximum instances reused was 30 (or in the case of a disposable, the equivalent functional unit would be 30 disposable dental burs). The system boundary included all cradle-to-grave aspects of both single-use and reused burs, including raw material extraction, manufacturing, packaging, distribution, reuse, and disposal. Primary data included the following: operating parameters for ultrasonic cleaning, manual cleaning, and autoclaving of the burs. The secondary data for raw material extraction and production of dental bur and packaging were obtained directly from life cycle inventory databases. Sensitivity analyses were also performed with respect to ultrasonic and autoclave loading.

Results and discussion

Findings from this research showed that when the ultrasonic and autoclave were loaded optimally, reusable burs had 40 % less of an environmental impact than burs used on a disposable basis. When the autoclave and ultrasonic were loaded to approximately two-third capacity, four environmental impact categories favored reusable burs (i.e., ozone depletion, smog, respiratory effects, exotoxicity), and four impact categories environmentally favored disposables (i.e., acidification, eutrophication, carcinogenics, and non-carcinogenics). When the autoclave and ultrasonic were loaded to approximately one-third capacity, reusable dental burs posed more negative environmental impacts in eight of nine environmental impact categories when compared to disposable burs.

Conclusions

Operational efficiency of ultrasonic and autoclave cleaning equipment should be emphasized to enhance the environmental performance of bur reuse. In fact, improper loading of the ultrasonic and autoclave can lead to greater adverse environmental impacts than if the burs were treated as disposables. The environmental and economic impacts associated with bur reuse are expected to be similar with other dental devices that are designated as disposable but are capable of being reused (e.g., scalpels, forceps).     

Restructuring upstream bioprocessing: technological and economical aspects for production of a generic microbial feedstock from wheat

Restructuring and optimization of the conventional fermentation industry for fuel and chemical production is necessary to replace petrochemical production routes. Guided by this concept, a novel biorefinery process has been developed as an alternative to conventional upstream processing routes, leading to the production of a generic fermentation feedstock from wheat. The robustness of Aspergillus awamori as enzyme producer is exploited in a continuous fungal fermentation on whole wheat flour. Vital gluten is extracted as an added-value byproduct by the conventional Martin process from a fraction of the overall wheat used. Enzymatic hydrolysis of gluten-free flour by the enzyme complex produced by A. awamori during fermentation produces a liquid stream rich in glucose (320 g/L). Autolysis of fungal cells produces a micronutrient-rich solution similar to yeast extract (1.6 g/L nitrogen, 0.5 g/L phosphorus). The case-specific combination of these two liquid streams can provide a nutrient-complete fermentation medium for a spectrum of microbial bioconversions for the production of such chemicals as organic acids, amino acids, bioethanol, glycerol, solvents, and microbial biodegradable plastics. Preliminary economic analysis has shown that the operating cost required to produce the feedstock is dependent on the plant capacity, cereal market price, presence and market value of added-value byproducts, labor costs, and mode of processing (batch or continuous). Integration of this process in an existing fermentation plant could lead to the production of a generic feedstock at an operating cost lower than the market price of glucose syrup (90% to 99% glucose) in the EU, provided that the plant capacity exceeds 410 m(3)/day. Further process improvements are also suggested.     

Strategy for selecting disposable bags for cell culture media applications based on a root‐cause investigation

The use of disposable bags for cell culture media storage has grown significantly in the past decade. Some of the key advantages of using disposable bags relative to non‐disposable containers include increased product throughput, decreased cleaning validation costs, reduced risk of cross contamination and lower facility costs. As the scope of use of disposable bags for cell culture applications increases, problematic bags and scenarios should be identified and addressed to continue improving disposables technologies and meet the biotech industry's needs. In this article, we examine a cell culture application wherein media stored in disposable bags is warmed at 37°C before use for cell culture operations. A problematic bag film was identified through a prospective and retrospective cell culture investigation. The investigation provided information on the scope and variation of the issue with respect to different Chinese hamster ovary (CHO) cell lines, cell culture media, and application‐specific parameters. It also led to the development of application‐specific test methods and enabled a strategy for disposable bag film testing. The strategy was implemented for qualifying an alternative bag film for use in our processes. In this test strategy, multiple lots of 13 bag film types, encompassing eight vendors were evaluated using a three round, cell culture‐based test strategy. The test strategy resulted in the determination of four viable bag film options based on the technical data. The results of this evaluation were used to conclude that a volatile or air‐quenched compound, likely generated by gamma irradiation of the problematic bag film, negatively impacted cell culture performance. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1535–1549, 2013     

Biological control: An effective approach against nematodes using black pepper plants (Piper nigrum L.)

Black pepper (Piper nigrum L.) is one of the oldest spices in the world, additionally, it is highly demanded. Several biotic and abiotic variables pose black pepper production worldwide. Plant-parasitic nematodes play a key role among biotic factors, causing considerable economic losses and affecting the production. Different synthetic nematicides were used for controlling plant nematodes, however the majority of pesticides have been pulled from the market due to substantial non-target effects and environmental risks. As a result, the search for alternative eco-friendly agents for controlling plant-parasitic nematodes populations. Microbial agents are a precious option. In this review the bacterial and fungal agents used as an alternative nematicides, they were studied and confirmed as essential anti-microbial agents against plant nematodes which infected Piper nigrum L. This work examines the most common plant nematodes infected Piper nigrum L., with a focus on root knot and burrowing nematodes, in addition, how to control plant parasitic nematodes using microorganisms.     

Process economics of industrial monoclonal antibody manufacture

Pressures for cost-effective manufacture of antibodies are growing given their high doses and increasing market potential that have resulted in significant increases in total site capacities of up to 200,000 L. This paper focuses on the process economic issues associated with manufacturing antibodies and reviews the cost studies published in the literature; many of the issues highlighted are not only specific to antibodies but also apply to recombinant proteins. Data collated at UCL suggest current benchmark investment costs of $660-$1580/ft2 ($7130-$17,000/m2) and $1765-$4220/L for antibody manufacturing facilities with total site capacities in the range of 20,000-200,000 L; the limitations of the data are highlighted. The complications with deriving benchmark cost of goods per gram (COG/g) values are discussed, stressing the importance of stating the annual production rate and either titre or fermentation capacity with the cost so as to allow comparisons. The uses and limitations of the methods for cost analysis and the available software tools for process economics are presented. Specific examples found in the literature of process economic studies related to antibody manufacture for different expression systems are reviewed. The key economic drivers are identified; factors such as fermentation titre and overall yield are critical determinants of economic success. Future trends in antibody manufacture that are driven by economic pressures are discussed, such as the use of alternative expression systems (e.g. transgenics, E. coli and yeast), disposables, and improvements to downstream technology. The hidden costs and the challenges in each case are highlighted.     

微生物种子包衣的应用与研究进展

种子包衣是一种高效、新兴的种子处理技术。该技术将外源性材料与种子紧密结合,从而提高种子性能,最终提高作物产量和品质。植物有益微生物(plant beneficial microorganisms, PBM)是指能够促进植物养分吸收、增强其对生物和非生物胁迫的耐受力,并促进植物生长或减少农业化学投入的微生物。因此,PBM可以作为一种微生物种子包衣剂。微生物种子包衣作为一种能够显著提高作物产量、经济效益和农业系统的可持续性发展的革新性技术,因其生态安全性和社会经济效益被认为是传统农业技术有前途的替代品。本文综述了微生物种子包衣技术及其在作物生产中的应用,并对其局限性和不一致性进行讨论。     

Evaluation of the Single-Use Fixed-Bed Bioreactors in Scalable Virus Production

The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector-manufacturing solutions. The use of disposable upstream technology is gaining traction in clinical manufacturing. Packed-bed or fixed-bed reactors, where column is packed with immobilized biocatalyst particles providing surface to constrain the cells in a particular region of the reactor, have been widely used in bioprocessing applications since mid-1900s. However, the world's first single-use, fully integrated, high cell density, fixed-bed bioreactor was launched only approximately a decade ago. By now, several single-use, fixed-bed technology solutions have been developed in a small scale. Scaling-up the manufacturing can be challenging and for commercial-scale manufacturing, there is practically only one single-use, good manufacturing practice-compliant option available. This study reviews the latest, fully disposable, fixed-bed bioreactors; compares the virus production in the different systems; and discusses important manufacturing cost-related topics. It is predicted that single-use, fixed-bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with the need for higher virus titers and virus yields.     

Consolidated Bioprocessing of Lignocellulosic Feedstocks for Ethanol Fuel Production

Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.     

Comparing the plant virus spread patterns of non-persistently transmitted virus and persistently transmitted virus using an individual-based model

To compare the spread patterns between two types of plant viruses, non-persistent virus (NPV) and persistent virus (PV), we developed a spatially-explicit individual-based model. Our probability-based model is driven by the actions of insect vectors that are affected by interactions with host plants and plant viruses, considering both biological and behavioral components of their relationship. As a model system, we used potato virus y and potato leafroll virus, respectively for NPV and PV, potato for host plant, and Myzus persicae for the insect vector; empirical results from previous studies were acquired and adjusted to be used as our parameter values. Our simulation results showed that initial infection of PV in the field resulted in over 1.3 times greater number of insect vectors while causing approximately 7 times greater number of virus-infected plants compared to NPV by the end of simulation. Furthermore, spatial analysis showed that PV-infected plants showed greater aggregation in the field, forming larger patches compared to NPV-infected plants. Our results demonstrated the importance of host plant and insect vector manipulation by plant viruses as well as biological properties such as infectious period in the insect on the difference in overall spread pattern.