This work is part of a CICYT research project (AGL 2010-22178) of the Spanish Ministry of Science and Technology. The authors are grateful to Nic Aldam and Kristi Prunty for the English revision of the manuscript.

The protocol described below for inducing CTA towards woody crops follows the animal care guidelines of the &#34;Universitat Aut&#242;noma de Barcelona&#34; (Bellaterra, Spain) and is approved by the Ethical Committee of Animal and Human Experimentation for sheep and goats (CEEAH, references 770 and 998 respectively).
1. Preparation of LiCl Dose
<ol>
	<li>Make a 25% w/v (weight/volume) LiCl solution in distilled water. Alternatively, use clean tap water. The LiCl is extremely hygroscopic therefore it is necessary to take care when handling the powdered chemical.
	<ol>
		<li>Weigh 250 g of LiCl and dissolve it in distilled water (LiCl concentrate solution). As the reaction is exothermic, wait until the solution has returned to room temperature before continuing. Pour the solution in a volumetric flask and add distilled water to bring it to 1,000 ml.</li>
	</ol>
	</li>
	<li>Calculate the volume of solution for each animal (individual dose).
	<ol>
		<li>Weigh the animals to obtain their body weight (BW) and calculate the mL of LiCl needed for each animal following equation 1. The recommended dose for long-term persistence is 225 mg LiCl/kg BW in sheep10&#160;and 200 mg LiCl/kg BW in goats17. However, other doses used for grazing are shown in Table 1. 
		 
		(kg BW x LiCl dose in g/kg BW) /LiCl concentration in g/L = Volume of LiCl (L) [Eq. 1] 
		 
		For example: 
		Sheep = 60 kg BW; dose = 225 mg LiCl/kg BW; concentration of LiCl solution = 250 g/L 
		(60 kg x 0.225 g/kg) /250 g/L = 0.054 L = 54 mL 
		Goat = 35 kg BW; dose = 200 mg LiCl/kg BW; concentration of LiCl solution = 250 g/L 
		(35 kg x 0.200 g/kg) /250 g/L = 0.028 L = 28 mL</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LiCl dose</td>
			<td>Mazorra et al. (2006)19</td>
			<td>Burritt et al. (2013)35</td>
		</tr>
		<tr>
			<td>Low</td>
			<td>100</td>
			<td>125</td>
		</tr>
		<tr>
			<td>Medium</td>
			<td>150</td>
			<td>150</td>
		</tr>
		<tr>
			<td>High</td>
			<td>225</td>
			<td>175</td>
		</tr>
	</tbody>
</table>
Table 1: LiCl dosage range. Range of LiCl doses (mg/kg Body Weight, BW) used by different authors to induce feed aversion.
2. Animal Selection and Facilities
<ol>
	<li>Choose adult, non-pregnant and dry animals, which have never previously eaten the target feed.</li>
	<li>Do not induce the CTA to more than 20 animals at the same time, to better control their behavior. Initially, only induce CTA to 5 animals.</li>
	<li>Allocate the animals in a place where the intake of the target feed can be recorded individually. Feed the animals once-a-day with a basal diet covering all their nutrient requirements and provide free access to water and to a mineral block. Assure an adequate salt intake to prevent LiCl toxic effects, since body cells can use Li instead of Na9.</li>
	<li>If the facilities are new to the animals, give them 1 week of adaptation time to become familiar with the environment and the basal diet.</li>
</ol>
3. Aversion Induction
<ol>
	<li>Remove the basal diet the evening before (day -1) and offer the target feed ad libitum to ensure that animals eat the target feed avidly on the following day12.</li>
	<li>On the following day (day 0), remove the orts in the morning and offer 200 g of target feed to each animal during 30 min. Weigh the orts thereafter.</li>
	<li>If the animals consume more than 20-30 g, administer the corresponding calculated volume of LiCl (ml calculated in 1.2) with a drenching gun as soon as is practically possible, not waiting more than 1 hr after consumption. If animals consume less than 20-30 g, repeat the procedure the following day. Due to their innate neophobic feed behavior, animals could need more than 1 day to begin consuming the target feed10,17,25-27.</li>
	<li>After LiCl administration, wait 2 hr before offering the basal diet.</li>
	<li>From day 1 to 3, do not offer the animals the target feed and check them periodically to detect any possible sign of severe illness, recording daily feed and water intake (by weight or by a check-list), respiratory rate and animal behavior. They could present dropped head and ears, inactivity, diarrhea, increased respiration rate and decreased water and diet intake during the days following the LiCl administration9-11,20,25.</li>
</ol>
4. Aversion Validation
<ol>
	<li>On days 4, 5, 6 and 7 after LiCl administration, repeat step 3.2.</li>
	<li>If the animals consume &#60;10 g (or &#60;4 bites) of the target feed, consider the CTA established.The threshold between zero (no consumption) and 10 g could depend on the circumstance of the target plant (toxic or not).</li>
	<li>If any animal consumes &#62;10 g, administer a new LiCl dose and repeat step 4.1. If the animal persists in consuming the target plant after the second dose, eliminate it from the CTA group. Social facilitation (e.g. mother, siblings and close mates) can modify CTA behavior in livestock. Animals which have been averted to a target plant easily consume it again when they graze with non-averted animals28.</li>
</ol>
5. Pasture Management
Note: The dose of LiCl used to induce CTA is completely excreted in a few days, mainly by urine.
<ol>
	<li>Although Li is widely distributed on the earth&#39;s crust, in order to avoid any possible contamination in the organic crop, wait 9-11 days before moving the animals to the crop (clearance time established with Li&#160;pharmacokinetic)11.</li>
	<li>Ensure that the crop ground cover is abundant and palatable throughout the grazing period to prevent the animals from sampling the target feed. Graze treated animals separately from non-treated.</li>
	<li>Allow the animals to graze a delimitated plot of the entire surface to obtain a uniform control of the grass height. Delimit the plot by using portable electric or metallic fences or using a shepherd to graze the animals between specific crop lines.</li>
	<li>Avoid overgrazing a plot, removing the animals before the ground cover becomes scarce (grass height of 5 cm)29.</li>
</ol>
6. Re-establishing the CTA
<ol>
	<li>Every year, before allowing the animals to return to graze the crop, repeat step 4.</li>
</ol>

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The authors have nothing to disclose.

The CTA is easily established in small ruminants if the target feed is a plant which the animal has never eaten before and does not contain an irreplaceable nutrient. Animals have a positive post-ingestive association with a non-toxic feed unless previous contact makes it difficult to change their perception of that particular feed7,33. The condition taste aversion is produced because LiCl stimulates the emetic system producing malaise or gastrointestinal discomfort34. It has been established that animals consuming LiCl increased rates of respiration, showed head and ear droop, occasionally they would kick themselves in the stomach25, and show decreased milk production20 and food and water intake11. However, these signs were overcome in a few days (2 to 4 days)11,20.
The protocol can be slightly modified according to other CTA studies. Although we indicated that the LiCl should be administered immediately after consumption of the target feed, animals were able to associate intake with sickness within the first 4 hr post-administration24. However, it is important to offer the target feed alone and temporarily separate (at least 1 hr after administration24) from other feeds; such as the basal diet, to prevent cross-CTA. Although we can choose one of the doses indicated in Table 1, we have to consider that those studies did not evaluate long term CTA (&#60;1 year), only the short-term (4 days)35&#160;and mid-term CTA (3 months)19. The use of adult animals is recommended due to young temperament in small ruminants which negatively affect the intensity and persistence of the CTA4.
Some key aspects have to be considered to obtain a successful CTA. Although the drenching gun is routinely used for deworming, the LiCl solution has to be applied with a higher volume than that of anthelmintic drugs (average 40 ml/animal). It is important to choose the correct size of the drenching gun, keep the gun in good condition (clean and lubricated) and carefully administer to the animal. To maintain the long-term aversion it is also important that the green cover is of high quality and palatable for the animals in order to avoid the erratic consumption of the target plant. It is stressed that the aversion becomes weaker every time the animal samples the averted food without suffering negative consequences25,36. CTA might be difficult to establish in a flock of small ruminants if they live in an area where the predominant crop is the target plant (e.g., vineyards, orange trees). One of the reasons for this is that previous contact with the target plant might occur when animals graze or are fed pruning waste in shelter. The solution could be to bring animals from other regions or raise the replacement stock without contact with the target plant.
In the last few years, farmers have been encouraged to implement more sustainable farming techniques due to societal demands. The CTA methodology proposed would change the use of tilling tools or herbicides to a system with small grazing animals. An additional benefit with grazing small ruminants is an increase of pasture surface available since the cultivated lands were forbidden from grazing animals in the past. Mixing grazing animals and crop production enables farmers to obtain a higher value product while improving soil quality.

The use of ground cover between woody crop lines mitigates soil erosion and degradation and increases water, organic carbon and nitrogen retention1-3. Additionally, ground cover maintains and increases biodiversity, supporting the balance between crop pests and their natural predators. Farmers tend to eliminate weeds by applying agrochemical products or using a reaper machine; thus reducing nutrient competition between crops and green cover. A cost effective way to control ground cover would be the use of small ruminant grazing. An additional benefit from animal grazing is the improvement in soil health and fertility. However, farmers are reluctant to implement this practice due to small ruminants damaging the crops by consuming young leaves and sprouts.
To prevent potential crop damage it is useful to induce conditioned taste aversion (CTA) in the sheep or goats within the flock or herd. The CTA is easily established for new feeds, due to the innate feed neophobia behavior4,5 of small ruminants, and because familiar feeds are positively associated with a &#34;learned safety&#34; status which is more difficult to change or manipulate6. Animals learn to reject a specific feed (conditioned stimulus) due to its negative post-ingestive effect (unconditioned stimulus). To induce CTA towards palatable and non-toxic plants, lithium chloride (LiCl; inductor agent) is orally administered after the animal consumes the target plant. While there are others inductor agents (e.g. apomorphine, ciclosphosphamide, thiabendazole), LiCl showed the strongest and most persistent CTA due to its effect on the emetic system by stimulation of the chemoreceptor trigger zone area and gastrointestinal distress7,8&#160;with mild signs of general discomfort. Lithium (Li) is absorbed from the upper gastrointestinal tract and distributed in the total body water space9. The animals can have a recovery period as short as two days7,10,11.
The LiCl can be administered by mixing it with the food12,13, in a gelatin capsule13,14 or in a solution administered orally by a drenching gun15-17. Although LiCl solution is caustic, no injury in mouth or esophagus was described. LiCl is used in the range of 100 to 400 mg LiCl/kg of body weight (BW), with better results (more persistent CTA) using higher doses16,18. Nevertheless, considering the known dosage effects towards different species and breeds, the lethal effect in some cases start at 400 mg LiCl/kg BW. The recommended dosage for an effective long-term CTA begins at 200 mg/kg BW for goats and 225 mg/kg BW for sheep10,17,19. Li used at these dosages is excreted within the first 4 days post-administration, mainly through urine (92 &#177; 4%), followed by feces (6.5 &#177; 1.3%) and milk (2.8 &#177; 0.4%)11. The complete estimated withdrawal period for a single dose of LiCl in plasma is 9 and 11 days for sheep and goats, respectively. Due to the minimal Li excretion in milk, CTA cannot be naturally established in the suckling off-spring11,20.
Long-term CTA persistence in sheep has been reported throughout an entire grazing season (3-4 months) when an alternative forage source was available14,21, being re-established to an almost complete aversion with a single LiCl dose at the next grazing season (9 months later)14. Moreover, CTA persistences of 2 and 3 years have been reported in cows under pasture conditions, without the need for reinforcement doses, when the target feed is a toxic but palatable plant22,23. The option of considering an alternative feed is critical for the animal to maintain the CTA against a non-toxic plant. Each time the animal consumes more than 10 g of the averted plant without resulting in gastrointestinal discomfort, the CTA would be compromised24.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Lithium Chloride PRS</td>
			<td style="width:71px;">Panreac</td>
			<td align="right" style="width:119px;">141392.1209</td>
			<td style="width:192px;">Different amounts of same product can be supplied by the same company.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Labelvage drencher 70 mL</td>
			<td style="width:71px;">Labelvage</td>
			<td align="right" style="width:119px;">240040</td>
			<td style="width:192px;">Similar product can be used (different brand or volume).</td>
		</tr>
	</tbody>
</table>

how to create conditioned taste aversion for grazing ground covers in woody crops with small ruminants

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has been produced. Lithium chloride (LiCl) is the preferred agent used in livestock to induce CTA because it specifically stimulates the vomit center. In addition, LiCl is commercially available, and easy to prepare and administer using a drenching gun. Nevertheless, some factors have to be considered to obtain an effective long-lasting CTA, which allows small ruminants to graze during the cropping season. A key aspect is to use animals with no previous contact with the target plant (the plant chosen to be avoided; new feed). Due to their native neophobic feeding behavior, small ruminants can easily associate the negative feedback effects with the new feed, resulting in a strong and persistent CTA. The recommended doses are 200 and 225 mg LiCl/kg body weight (BW) for goats and sheep, respectively. To induce CTA, 100 g of the target plant should be individually offered for at least 30 min, and LiCl administered thereafter if the intake is greater than 10 g. Each time the animal eats the target plant without negative consequences, the CTA becomes weaker. Consequently, to minimize the risk of target plant consumption, it is essential to have sufficient palatable ground cover available. The presence of an alternative feed (of quality and quantity) prevents the accidental consumption of the target plant. A close monitoring of the flock is recommended to remove and re-dose any animal consuming more than 4 bites or 10 g of the target plant. At the beginning of each grazing season, check the CTA status of each animal before moving them to the crop.

The results described below were obtained by several studies in CTA toward woody crops in small ruminants conducted at the Universitat Aut&#242;noma de Barcelona and they provide evidence to establish the protocol proposed.
Doses of 175 and 200 mg LiCl/kg BW in goats and 200 and 225 mg LiCl/kg BW in sheep were successfully used to induce CTA against woody crops with a single administration, with the exception of two animals which did not completely swallow the administered LiCl dose10,17&#160;(Table 2). Most CTA animals showed dropped heads, inactivity and decreased intake of the basal diet the day after LiCl administration10,11,17; however, animals overcame these signs of gastrointestinal discomfort within 2 days10,11. For all the doses used, the typical CTA behavior observed was that the animals refused to approach the feeding boxes, sniffed the plant material and rejected to eat or consumed &#60;10 g of the target feed10,17. In addition, differences between doses for each species were detected regarding the long-term persistence and the number of animals needing a reinforcing LiCl dose in the short-term. Animals which had received the lower dose (175 and 200 mg LiCl/kg BW for goats and sheep, respectively) showed shorter complete CTA persistence (intake of the target feed &#60;10 g) and more animals needed to be reinforced to strengthen the CTA. Goats receiving the same LiCl dose (200 mg/kg BW) as sheep for inducing CTA against olive tree leaves, showed a longer complete CTA persistence17. On the other hand, CTA persistence differed by breed (Lacaune, Manchega and Ripollesa sheep breeds) using the 200 mg LiCl/kg BW but not when the 225 mg LiCl/kg BW dose was used10.
Although a single LiCl dose induced a complete CTA against a novel feed (i.e., olive tree leaves or grapevine leaves and sprouts), it was not enough to induce CTA against a familiar feed (i.e., concentrate and hay). Sheep which received a LiCl dose to induce CTA against concentrate only showed a slight decrease in the target feed intake compared to the control group on the following day (Figure 1). In addition, the repeated LiCl administration (up to 3 doses on consecutive days) during the learning period was not effective to induce CTA against the familiar feed30.
The CTA induced against grapevine leaves with a 225 mg LiCl/kg BW single dose was complete (intake &#60;10 g) through the first year (Figure 2). Nevertheless, during the following 2 years a re-enforcing dose was necessary to strengthen the CTA when ewes resumed consumption of the target feed. The CTA became weaker when the ground cover availability was scarce during the grazing season. A flock of 6 CTA ewes grazing (11 days; 24 hr/day) on a commercial vineyard plot (8.8 acres) with spontaneous ground cover reduced grass cover by 44 &#177; 4% (dry matter basis), leaving on the ground the more fibrous and less nutritive parts of the plants (Table 3)31.
A herd of 5 goats maintained an effective CTA (intake of the target feed markedly lower than the control group) against olive trees through 14 months after aversion was induced with a 200 mg LiCl/kg BW. A pasture trial of 30 min in a commercial olive grove (a plot of 156 m2 with spontaneous grass cover and 5 olive trees) showed that CTA goats spent 3.1% of the pasture trial time in contact with the olive trees (behavior of sampling, smelling or contact could not be distinguished in the video recorded), whereas control goats spent 50.7%32.
<img alt="Figure 1" src="/files/ftp_upload/53887/53887fig1.jpg" /> 
Figure 1: Familiar vs. Novel feed in CTA. Intake of the target feed after a single LiCl dose (225 mg/kg Body Weight, BW) to induce CTA against a familiar or a novel feed in the averted (CTA) and control (C) ewes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53887/53887fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53887/53887fig2.jpg" /> 
Figure 2: CTA persistence after LiCl administration. Group intake of grapevine leaves during the first year after inducing conditioned taste aversion (CTA). (<img alt="Clear Circle" src="/files/ftp_upload/53887/clearcircle.jpg" />, Control Lacaune; <img alt="Circle" src="/files/ftp_upload/53887/circle.jpg" />, CTA Lacaune; <img alt="Clear Triangle" src="/files/ftp_upload/53887/cleartriangle.jpg" />, Control Manchega; <img alt="Trianlge" src="/files/ftp_upload/53887/triangle.jpg" />, CTA Manchega). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53887/53887fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Species</td>
			<td>N</td>
			<td>LiCl dose</td>
			<td>Target feed</td>
			<td>Success ratio, %1</td>
		</tr>
		<tr>
			<td>Goat</td>
			<td>5</td>
			<td>175 mg/kg BW</td>
			<td>Olive tree</td>
			<td>100 (5/5)</td>
		</tr>
		<tr>
			<td>Goat</td>
			<td>10</td>
			<td>200 mg/kg BW</td>
			<td>Olive tree</td>
			<td>90 (9/10)</td>
		</tr>
		<tr>
			<td>Sheep</td>
			<td>20</td>
			<td>200 mg/kg BW</td>
			<td>Olive tree</td>
			<td>95 (19/20)</td>
		</tr>
		<tr>
			<td>Sheep</td>
			<td>15</td>
			<td>225 mg/kg BW</td>
			<td>Olive tree</td>
			<td>100 (15/15)</td>
		</tr>
		<tr>
			<td>Sheep</td>
			<td>44</td>
			<td>225 mg/kg BW</td>
			<td>Grapevine</td>
			<td>100 (44/44)</td>
		</tr>
		<tr>
			<td colspan="5">1Proportion of CTA induced animals with a single LiCl dose.</td>
		</tr>
	</tbody>
</table>
Table 2: CTA success ratio. Proportion of success for inducing conditioned taste aversion against woody crops with single doses of 175, 200 and 225 mg LiCl/kg Body Weight (BW) for goat and sheep.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Item, %</td>
			<td>Before grazing</td>
			<td>After grazing</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td>Dry matter</td>
			<td>28.6 &#177; 1.4</td>
			<td>45.9 &#177; 5.8</td>
			<td>0.066</td>
		</tr>
		<tr>
			<td>Crude protein</td>
			<td>11.4 &#177; 1.2</td>
			<td>7.8 &#177; 0.5</td>
			<td>0.071</td>
		</tr>
		<tr>
			<td>Crude fiber</td>
			<td>29.5 &#177; 0.9</td>
			<td>35.1 &#177; 1.5</td>
			<td>0.046</td>
		</tr>
		<tr>
			<td>Neutral detergent fiber</td>
			<td>46.2 &#177; 4.4</td>
			<td>57.6 &#177; 3.0</td>
			<td>0.077</td>
		</tr>
		<tr>
			<td>Acid detergent fiber</td>
			<td>28.0 &#177; 2.7</td>
			<td>35.9 &#177; 2.5</td>
			<td>0.041</td>
		</tr>
		<tr>
			<td>Lignin acid detergent</td>
			<td>4.2 &#177; 0.8</td>
			<td>6.2 &#177; 1.1</td>
			<td>0.075</td>
		</tr>
		<tr>
			<td>Ash</td>
			<td>8.9 &#177; 0.3</td>
			<td>9.1 &#177; 0.5</td>
			<td>0.788</td>
		</tr>
	</tbody>
</table>
Table 3: Ground cover chemical composition. Chemical composition (dry matter basis) of spontaneous ground cover of a commercial vineyard before and after grazing by averted ewes.

Watch this Scientific Journal Video about How to Create Conditioned Taste Aversion for Grazing Ground Covers in Woody Crops with Small Ruminants at JoVE.com

How to Create Conditioned Taste Aversion for Grazing Ground Covers in Woody Crops with Small Ruminants

Feeding behavior preferences in livestock can be modified to implement a grazing management plan in woody crops. Here, we present a protocol to show a lithium chloride dose after eating a new plant that induces conditioned taste aversion.

Conditioned taste aversion (CTA) is a learning behavior process where animals are trained to reject certain feed after gastrointestinal discomfort has ...

how-to-create-conditioned-taste-aversion-for-grazing-ground-covers

Nanyang Technological University

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7.2K Views.  Universitat Autònoma de Barcelona. Feeding behavior preferences in livestock can be modified to implement a grazing management plan in woody crops. Here, we present a protocol to show a lithium chloride dose after eating a new plant that induces conditioned taste aversion. 

Video: How to Create Conditioned Taste Aversion for Grazing Ground Covers in Woody Crops with Small Ruminants

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data

Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

Advanced micrometeorological methods have become  increasingly important in soil, crop, and environmental sciences.  For many scientists without formal ...

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

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The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

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A Ground Penetrating Radar (GPR) system based on a ground-coupled, densely populated antenna array was used to collect data during an infiltration experiment conducted at a test site near the Tottori Sand Dune, Japan. The antenna array used in this study consists of 10 transmitting antennas (Tx) and 11 receiving antennas (Rx). For this experiment, the system was configured to use all possible Tx-Rx pairings, resulting in a Multi-Offset Gather (MOG) consisting of 110 Tx-Rx combinations. The array was left stationary at a position directly above the infiltration area and data were collected every 1.5 seconds using a time-based trigger. Common-Offset Gather (COG) and Common Mid-Point (CMP) data cubes were reconstructed from the MOG data during post-processing. There have been few studies that used time-lapse CMP data to estimate changes in velocity of propagation. In this study, electromagnetic (EM) wave velocity was estimated heuristically at 1-minute intervals from the reconstructed CMP data through curve fitting, using the hyperbola equation. We then proceeded to calculate the depth of the wetting front. The evolution of the wetting front over time obtain through this method is consistent with the observations from a soil moisture sensor which was placed at a depth below 20 cm. The results obtained in this study demonstrate the ability of such array GPR system to monitor a subsurface dynamic process like water infiltration accurately and quantitatively.

Here we present a Ground Penetrating Radar (GPR) system based on a ground-coupled, densely populated antenna array for monitoring the dynamic process of subsurface water infiltration. A time-lapse radar image of the infiltration process allowed estimating the depth of the wetting front during the course of the infiltration process.

A Ground Penetrating Radar (GPR) system based on a ground-coupled, densely populated antenna array was used to collect data during an infiltration ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...