Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Veterinary Clinical Sciences
Non Technical Summary
As the world's population continues to increase, the question of how to supply food to an ever-increasing number of people will become a growing concern. In particular, the increased demand for animal protein has already put a strain on our natural environment. Traditional sources of animal protein, including cattle, swine, and poultry, have led to an increased production of greenhouse gases, deforestation, and the consumption of vast amounts of resources such as water, feed, and energy. Additionally, the demand for fish and other seafood items has led to overfishing and the loss of ocean biodiversity. As both the human population and the demand for animal protein increase, we will continue to see further degradation to our natural environment and increased prices for these types of protein sources. One solution tothis crisis is to adapt human and animal diets to include insects as a source of protein. Insects have been considered a staple food item in some cultures for thousands of years but have only recently started gaining popularity in the Western world. Insects are also being used as a primary protein source in livestock and poultry feeds because of the lower cost associated with producing these invertebrate animals. Insects are easy to raise in captivity, with minimal investment to start up, and require only a small footprint for growth because of our ability to raise them in a vertical space. Additionally, insects require 12 times less feed than cattle to produce the same amount of useable protein. Based on the smaller land and feed requirements, insect farming is considered to be the most economical and sustainable way to raise a source of animal protein for human, livestock, and poultry diets.The cricket industry in the United States currently produces billions of crickets per year, primarily as food for animal consumption; however, there is an interest in the industry of producing these income producing insects as a food for humans, livestock, and poultry. Current revenue from cricket sales for animal feed in the United States is estimated at $100 million USD per year (Fluker Farms, personal communication). Unfortunately, the cricket industry has been plagued by disease outbreaks that have impacted the supply of crickets and thus decreased the total economic revenue generated from this income producing species. Similar to other income producing species of livestock and poultry, high animal densities and management practices put crickets at high risk for infectious diseases, with viral pathogens being the most concerning. Acheta domesticus, the European house cricket, is the predominant species raised in the United States, but has suffered from several viral outbreaks worldwide since the 1970's. The virus most often associated with these outbreaks has been identified as the Acheta domesticus densovirus (AdDV), a virus within the parvoviridae family. The most severe outbreaks in the USA and Europe have resulted in 100% mortality rates and have completely bankrupted some cricket operations. Affected crickets experience a slower growth rate due to a decrease in gastrointestinal motility and inappetence. Affected crickets experience a decreased lifespan, and those reaching adulthood have been shown to produce fewer eggs. Eventually the affected crickets become flaccid, develop hind-limb paralysis, lose their ability to jump, and die. Transmission of the virus is primarily thought to be fecal-oral, but the virus has also been identified in air filters and in other non-cricket insect species grown at the same rearing facilities, suggesting that aerosolization and fomite transmission may also play a role in spread of the virus. To date, no cleaning agents or sanitation protocols have been identified capable of eliminating the virus once it has become established at a cricket rearing facility. If the cricket industry is going to be viable and provide animal protein to growing populations of humans and animals, it is vital that we develop a better understanding of the epidemiology of this virus.A local, large-scale cricket farm has reported increased mortality rates in their A. domesticus breeding colony for the last three years. Losses have approached 50%, which is significantly higher than the historic 10% losses that are expected with cricket rearing. The clinical signs reported in the crickets include reduced food consumption, increased lethargy, flaccid paralysis, and death. Given the high losses, species of cricket, and the clinical signs, AdDV was highly suspected as a potential culprit. A pilot study conducted at the farm confirmed the presence of AdDV on the farm using conventional PCR. Using this farm as a model for other infected farms across the country, we hope to gain further insight into the epidemiology of this virus, focusing on prevalence and two important risk factors, life stage and season (environmental temperature). Anecdotal reports suggest that mortality rates for these viruses are at their highest in later stage animals (>4 weeks old) and during the winter months. The increased mortality in older animals has been attributed to exposure time in the environment, while the higher mortality with temperature is attributed to the lower environmental temperatures associated with the Fall and Winter. Traditionally, most cricket farms attempt to maintain rearing building temperatures between 30-31°C (86-88°F). However, in the winter, it can be a challenge to maintain large warehouse buildings at these temperatures. At the identified farm, the historic mortalities have been low when the air temperatures in the rearing buildings are kept at the traditional rearing temperatures; however, the mortalities increase when the air temperature drops below 29oC (85oF). This suggests that AdDV is sensitive to the effects of high temperatures. The same has been found for several other viruses, including another cricket-specific virus in the dicistroviridae family, the cricket paralysis virus (CrPV). Our research aims to provide preliminary insight into whether life stage or season (temperature) have an impact on the prevalence of this virus in a working A. domesticus farm. This initial work is needed to determine whether certain risk factors are important to the dissemination of the virus and whether, ultimately, management practices can be developed to better control or eliminate the virus.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The primary goal of this study it to characterize the roles of life stage and season on the prevalence of Acheta domesticus densovirus (AdDV) in Acheta domesticus crickets. Accomplishing the goal of this study will provide preliminary insight into the prevalence of an economically important virus in an emerging income producing species at one of the largest cricket farms in the USA. This will represent the first attempt to systematically characterize the prevalence of this virus in the USA. Further, this study will be the first to characterize the role of life stage or season (temperature) in the epidemiology of this virus. Calculating an apparent prevalence for AdDV in crickets is an important benchmark as it can be used to determine true risk, especially if there are life-stage or seasonal differences. Knowledge of the prevalence and potential risk factors associated with the viruses will also enable individuals in the industry to develop biosecurity and management methods to minimize the impact of AdDV in the future.
Project Methods
A cross-sectional study will be done to estimate the prevalence of AdDV in A. domesticus at a single large-scale cricket farm (Fluker Farms, Port Allen, LA) in the USA. Four different life stages of crickets will be sampled: eggs, 2-weeks, 4-weeks, and 6-weeks. Fifteen batches of each life stage will be randomly collected at each sampling period. A random number generator will be used to determine the method of selecting eggs and crickets from the available egg and cricket bins. Twelve eggs and twelve crickets from each life stage will represent one batch; thus, each sampling period will consist of 15 pools of 12 eggs or crickets for a total of 180 individual eggs or crickets. The sample size selected for this study was based on the following a priori data: an alpha=0.05, a power=0.8, and an expected prevalence of >45% in the later stage groups. The 45% a priori prevalence measurement was selected based on a mortality rate of >50% at this farm. The crickets will be sampled at 4 different time periods: March, June, September, and December. Samples will be collected using sterile methods. Eggs will be pipetted from the vermiculite nesting material directly into whirl-pak bags. Crickets will be collected directly into sterile whirl-pak bags, which will be sealed and labeled. The eggs and crickets will be transported on wet ice to the Louisiana State University School of Veterinary Medicine for further processing. Temperature and humidity will be recorded in triplicate in the rearing buildings on the property using a combined digital thermometer and hygrometer. The average temperature and humidity of each building for the two weeks prior to the collection date will be recorded. The average outdoor temperature from the two weeks prior to collection date will also be recorded using data for Port Allen, LA from the National Weather Service website (http://www.srh.noaa.gov).The processing of the crickets and eggs will be similar to that described by Semberg et al.1 Each batch of crickets and eggs will be placed into a mesh bag (Bioreba, Reinach, Switzerland), flash frozen in liquid nitrogen, and ground into a powder using a mortar and pestle.1 Two mL nuclease-free water will be added to the powder and the mixture further homogenized. The cricket homogenate (100 μL) will be further mixed with 180 μL Buffer ATL and 20 μL proteinase K (Qiagen, Hilden, Germany).1 The entire mixture will be incubated at 56 °C for 3 h.1 DNA will be extracted from the mixture using the Qiagen DNA extraction protocol for tissues and Rodent tails (Qiagen, Hilden, Germany).1 The DNA concentration will be estimated using a Qubit 4 Flurometer (Invitrogen, Carlsbad, CA) and stored in −80 °C until final processing. The quantitative real-time PCR assay for AdDV will use primers for amplifying a 305 bp fragment located in the virus capsid protein gene cassette and a 357 bp fragment located in the non-structural region.2 A constant amount of template DNA will be included in each reaction.1 The PCR reactions will be run using the EvaGreen® SYBR Green kit (Bio-Rad, Singapore).1 A 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) will be used to run the samples. The cycling conditions will be as follows: 1) 98 °C for 2 min to activate the enzyme, 2) 40 cycles at 98 °C for 10 s for denaturation, and 3) 58 °C (AdVP-primers) or 62 °C (AdNS-primers) for annealing and extension.1 All assays will be run in duplicate.Statistics: The 95% binomial confidence intervals will be calculated for each proportion. Univariate categorical statistics (chi-square testing) will be used to determine if life stage or sampling month are associated with the presence of each virus. If the p<0.20 for these variables, a logistic regression model will be developed to evaluate both independent variables simultaneously, as well as their interaction term. Hosmer-Lemeshow goodness of fit tests will be used to assess model fit. A Bonferroni correction will be applied; thus, P<0.025 will be used to determine significance. SPSS 24.0 (IBM Statistics, Armonk, NY) will be used to analyze the data.1. Semberg E, De Miranda JR, Low M, Jansson A, Forsgren E, Berggren Å. Diagnostic protocols for the detection of Acheta domesticus densovirus (AdDV) in cricket frass. J Virol Methods. 2018;264:61-64. doi:10.1016/j.jviromet.2018.12.0032. Szelei J, Woodring J, Goettel M, et al. Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics. J Invert Pathol. 2011:106:394-399. doi:10.1016/j.jip.2010.12.009