We thank Tien-Cheng Li, Yi-Tang Lin, Chia-Jung Tsai, and Ya-Lan Li for their assistance during this study. This study was supported by grant no. 107AS-8.7.1-BQ-B2 (1) from the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan.

1. Safety precautions when handling lyssaviruses
<ol>
	<li>Ensure that all laboratory workers handling bat specimens receive pre-exposure rabies prophylaxis17. Monitor the rabies antibody levels of the workers beforehand and re-examine them every 6 months17. Follow-up rabies vaccination is required for those whose antibody levels are lower than 0.5 IU/mL17.</li>
	<li>Depending on the biosafety regulations of the country where the laboratory is located, ensure that the following procedures are performed in suitable biosafety level laboratories (e.g., BSL-2 laboratories in Taiwan and BSL-3 laboratories in Australia), and that workers are currently qualified and wear proper personal protective equipment18. 
	NOTE: Rabies vaccination provides little to no protection from lyssaviruses belonging to phylogroups II and III18. Workers must be informed that several zoonotic pathogens have been identified in bats19 and should handle samples under suitable biosafety level laboratory conditions with proper personal protective equipment.</li>
</ol>
2. Sample collection
<ol>
	<li>Use found weak or sick bats or carcasses. 
	NOTE: Weak or sick bats are delivered to the Bat Conservation Society of Taipei for veterinary care or research, while carcasses are submitted directly to the Animal Health Research Institute. No healthy bats have been euthanized in this surveillance program.</li>
	<li>Have a bat ecologist identify bat species through morphological characteristics20.</li>
	<li>Perform DNA barcoding of the bat species when lyssavirus positive is diagnosed, using previously published procedures21.</li>
	<li>Submit an information sheet of each bat carcass (collection site, species, clinical signs, etc.).</li>
</ol>
3. Necropsy of bat specimens
<ol>
	<li>Prepare materials.
	<ol>
		<li>Prepare a clean dissection board and place a sterile absorbent pad for necropsy.</li>
		<li>Prepare collection tubes for collecting bat organs.</li>
		<li>Prepare disposable tweezers and scalpels for necropsy. Change tools between each bat&#8217;s necropsy. Prepare cotton balls moistened with 70% ethanol for cleaning tweezers and scalpels during sampling.</li>
		<li>Prepare two microscope slides for the FAT. Collect fresh specimens and fix them in formalin solution for histopathological examination.</li>
	</ol>
	</li>
	<li>Examine all external orifices before necropsy. Photograph the bat&#8217;s external features, especially the head, ears, and wings, for species differentiation.</li>
	<li>Collect an oral swab sample. Place the bat in ventral recumbency on the board and fix the bat&#8217;s head with tweezers. Cut the skin along the midline of the calvaria with a scalpel and pull the skin to the lateral sides. Cut the skull along the midline of the calvaria with a scalpel and open it with tweezers to expose the brain tissue.</li>
	<li>Remove the brain tissue from the skull and place it on a sterile tongue depressor, and make impression smears from the brain tissue (see step 4.2). Collect a small piece of fresh brain tissue and fix it in formalin for histopathological examination. Contain the remaining brain tissue in a tube for nucleic acid extraction.</li>
	<li>Clean tweezers and scalpel with cotton balls moistened with 70% ethanol to remove retained tissues between specimen collection.</li>
	<li>Place the bat on the board in dorsal recumbency and fix it on the board with needles at both sides of the axilla and tail heel.</li>
	<li>Incise the skin along the midline of the body from mandible to anus. Lift and separate the skin and underlying muscle tissues with tweezers. Collect the salivary glands, which are near the mandibular bone.</li>
	<li>Lift the sternum slightly with tweezers, and cut the sternum and abdominal wall along the midline with a scalpel. Cut the clavicles with a scalpel. Fix the left and right rib cages to the board with needles to open the thoracic cavity.</li>
	<li>Record the gross lesions and the degree of post-mortem change.</li>
	<li>Remove the visceral tissues (i.e., heart, lungs, liver, kidneys, intestines) from the carcass using tweezers and a scalpel. Collect the visceral specimens as needed for future research. 
	NOTE: Collection of duplicate samples is recommended. One should be collected for molecular diagnosis, and the other should be frozen at -80 &#176;C with or without viral transport medium for viral culture22.</li>
</ol>
4. Direct fluorescent antibody test (FAT)
<ol>
	<li>Make impression smears of the brain tissue for the FAT. Perform the FAT as previously described18,23 with the following modifications.</li>
	<li>Gently separate the brain tissue from the connected nerve tissue with tweezers and transfer the brain tissue to a sterile tongue depressor. Cut the cross-section of the brain, including the brain stem and cerebellum18,23. Make impression smears of the brain tissue by lightly touching the cut surface of the brain tissue, and press the slide on lens tissue to remove excess tissue.</li>
	<li>Fix the slides with acetone at -20 &#176;C for 30 min. Dry the tested slides and the positive and negative controls before staining with conjugate.</li>
	<li>Using each of the two commercially available FITC-conjugated anti-rabies antibodies for staining lyssavirus antigen is highly recommended23. Determine the working concentration of the commercial conjugate before the first staining. Drop the diluted conjugates through a 0.45 &#956;M syringe filter onto the slides and incubate the slides at 37 &#176;C for 30 min within a wet chamber.</li>
	<li>Drain the excess conjugate from the slides and wash the slides with phosphate-buffered saline (PBS) after incubation.</li>
	<li>Drop a small amount of 10% glycerol on the slides and cover with cover slides.</li>
	<li>Examine the slides with a fluorescent microscope.</li>
</ol>
5. Nucleic acid extraction
<ol>
	<li>Add the proper volume of MEM-10 (minimum essential medium supplemented with 10% fetal bovine serum) to the brain tissue (10% w/v).</li>
	<li>Homogenate the brain tissue with a 5 mm steel bead in a homogenizer instrument and centrifuge at 825 x g for 10 min.</li>
	<li>Extract the nucleic acid, the final volume of which is 50 &#956;L, within 200 &#956;L of the supernatant using the commercially available total nucleic acid extraction kit with instrument.</li>
</ol>
6. RT-PCR and phylogenetic analysis
NOTE: Several primer sets have been published to detect all known lyssaviruses or specific lyssaviruses. The protocol described here is an example that our laboratory uses and may not fit all experimental needs. Select suitable primers according to laboratory needs.
<ol>
	<li>Prepare the one-step RT-PCR reagent as follows: add 5 &#956;L of extracted nucleic acid to the reaction mixture containing 2.5 &#956;L of 10x reaction buffer, 0.5 &#956;L of forward and reverse primers (10 &#956;M of each), 4 &#956;L of 1.25 mM dNTP, 0.3 &#956;L of RNase inhibitor (40 U/&#181;L), 0.3 &#956;L of reverse transcriptase (10 U/&#181;L), 0.4 &#956;L of DNA polymerase (5 U/&#181;L), and 11.5 &#956;L of DEPC-treated water. 
	NOTE: The primer set used in this protocol is JW12 (5&#39;-ATGTAACACCYCTACAATG-3&#39;) and N165-146 (5&#39;-GCAGGGTAYTTRTACTCATA-3&#39;)24. Modify the preparation of reagents and cycling conditions according to the primer sets used.</li>
	<li>Perform the cycling under the following conditions: incubation at 42 &#176;C for 40 min; initial denaturation at 94 &#176;C for 10 min; 35 cycles of 94 &#176;C for 30 s, 55 &#176;C for 30 s, and 72 &#176;C for 30 s; and finally, further extension at 72 &#176;C for 10 min.</li>
	<li>Use another or more primer sets to increase the diagnostic sensitivity.
	<ol>
		<li>Use N113F (5&#39;-GTAGGATGCTATATGGG-3&#39;) and N304R (5&#39;-TTGACGAAGATCTTGCTCAT-3&#39;)25,26 to prepare the one-step RT-PCR reagent as follows: add 5 &#956;L of extracted nucleic acid to a reaction mixture containing 5 &#956;L of 10x buffer, 5 &#956;L of forward and reverse primers (4 &#956;M of each), 5 &#956;L of 1.25 mM dNTP, 0.5 &#956;L of RNase inhibitor (40 U/&#181;L), 0.2 &#956;L of reverse transcriptase (10 U/&#181;L), 1 &#956;L of DNA polymerase (5 U/&#181;L), and 23.3 &#956;L of DEPC-treated water.</li>
		<li>Perform the cycling under the following conditions: incubation at 42 &#176;C for 40 min; initial denaturation at 95 &#176;C for 5 min; 35 cycles of 95 &#176;C for 1 min, 55 &#176;C for 1 min and 20 s, and 72 &#176;C for 1 min; and finally, further extension at 72 &#176;C for 10 min. 
		NOTE: Depending on laboratory needs, choose suitable primers for diagnosis. N113F was originally designed for rabies virus amplification, but it may not work well for other lyssaviruses. The set of N113F and N304R works well for rabies virus (Taiwan ferret badger variant) and Taiwan bat lyssavirus. It will be easier to obtain whole nucleoprotein sequences by using the set of JW12 and N304R primers if the lyssavirus is amplified by both of the above two primer sets.</li>
	</ol>
	</li>
	<li>Analyze the PCR product on 2% agarose gel electrophoresis and visualize by UV light illumination.</li>
	<li>Sequence the PCR product by commercial sequencing service.</li>
	<li>Enter or upload the sequence to the webpage of the Nucleotide Basic Local Alignment Search Tool (BLAST). Select the &#34;others (nr etc.)&#34;&#160;database and enter the organism as Lyssavirus. Select the MegaBlast algorithm and run the BLAST.</li>
</ol>
7. Virus isolation
NOTE: Perform virus isolation when either 1) the FAT or 2) the RT-PCR indicates positivity.
<ol>
	<li>Homogenize the brain specimen in a 10% (w/v) suspension in MEM-10. Centrifuge at 825 x g for 10 min.</li>
	<li>Inoculate 200 &#181;L of supernatant with a suspension of 3 x 106 MNA (mouse neuroblastoma) cells in 1 mL of MEM-10 for 1 h at 37 &#176;C with 1% CO2. Transfer the brain homogenate-cell suspension to a 25 cm2 flask and add 6 mL of MEM-10.</li>
	<li>Cultivate 1 mL of the brain homogenate-cell suspension in a 4 well Teflon-coated glass slide with 6 mm diameter at the same time.</li>
	<li>After 3&#8211;4 days of incubation at 37 &#176;C with 1% CO2, fix the cells on the 4 well slide with 100% acetone (v/v).</li>
	<li>Stain the slides with two FITC-conjugated anti-rabies antibodies following steps 4.4&#8211;4.7. The cells are infected when the intracytoplasmic inclusions are investigated. Record the percentage of infected cells.</li>
	<li>Perform trypsinization and subculture of the inoculated cell culture when the slides are stained as negative:
	<ol>
		<li>Remove medium and rinse the flask with 5 mL of PBS.</li>
		<li>Add 1 mL of trypsin to the flask and firmly strike the bottom of the flask.</li>
		<li>Add 6 mL of MEM-10 and resuspend the cells.</li>
		<li>Put the cell suspension into a new tissue flask (6 mL) and onto a 4 well slide (1 mL).</li>
	</ol>
	</li>
	<li>Repeat steps 7.4&#8211;7.6 until 100% infectivity is reached.</li>
	<li>Collect the supernatant after 24 h of incubation.</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyssavirus+in+Indian+Flying+Foxes,+Sri+Lanka.">Lyssavirus in Indian Flying Foxes, Sri Lanka.</a> Emerging Infectious Diseases. 22 (8), 1456-1459 (2016).</li><li>Hu, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyssavirus+in+Japanese+Pipistrelle,+Taiwan.">Lyssavirus in Japanese Pipistrelle, Taiwan.</a> Emerging Infectious Diseases. 24 (4), 782-785 (2018).</li><li>Nokireki, T., Tammiranta, N., Kokkonen, U. -. 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No conflicts of interest are declared.

This laboratory standard operating procedure (SOP) provides a serial process for testing bat samples for the presence of lyssavirus antigens in Taiwan. The key steps include the employment of FAT and RT-PCR. The selection of suitable samples and successful isolation of the virus are also important. Additionally, some troubleshooting was conducted during the monitoring of bat lyssaviruses. The major difference was the target animals. Initially (2008&#8211;2009), the target animals of bat lyssavirus surveillance were live ...

Viruses within the genus Lyssavirus are zoonotic pathogens. There are at least seven lyssavirus species associated with human cases1. In addition to the 16 species in this genus1,2,3, Taiwan bat lyssavirus (TWBLV)4 and Kotalahti bat lyssavirus5 have been recently identified in bats, but their taxonomic statuses have yet to be determined.
Bats are the natural hosts of most lyssaviruses, with the exception of Mokola lyssavirus and Ikoma lyssavirus, which have yet not been identified in any bats1,2,3,6. The information on lyssaviruses in Asian bats is still limited. Two uncharacterized lyssaviruses in Asian bats (one in India and the other in Thailand)7,8 have been reported. One human rabies case associated with a bat bite in China was reported in 2002, but the diagnosis was made only by clinical observation9. In Central Asia, Aravan lyssavirus was identified in the lesser mouse-eared bat (Myotis blythi) in Kyrgyzstan in 1991, and Khujand lyssavirus was identified in the whiskered bat (Myotis mystacinus) in Tajikistan in 200110. In South Asia, Gannoruwa bat lyssavirus was identified in the Indian flying fox (Pteropus medius) in Sri Lanka in 20153. In Southeast Asia, several serological studies on bats in the Philippines, Thailand, Bangladesh, Cambodia, and Vietnam showed variable seroprevalence11,12,13,14,15. Although Irkut lyssavirus was identified in the greater tube-nosed bat (Murina leucogaster) in Jilin Province, China in 201216, the exact species and locations of lyssaviruses in East Asian bat populations remain unknown.
To assess the presence of lyssavirus in Taiwanese bat populations, a surveillance program employing both direct FAT and RT-PCR was initiated. Taiwan bat lyssavirus was identified in two cases of the Japanese pipistrelle (Pipistrellus abramus)4 in 2016&#8211;2017. In the present article, a laboratory standard operating procedure is introduced for lyssavirus surveillance of the bat population in Taiwan. The flow chart of bat lyssavirus diagnosis in our laboratory is presented in Figure 1.

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		<tr>
			<td>2.5% Trypsin (10x)</td>
			<td>Gibco</td>
			<td>15090046</td>
			<td>Trppsin</td>
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		<tr>
			<td>25 cm2 flask</td>
			<td>Greiner bio-one</td>
			<td>690160</td>
			<td></td>
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		<tr>
			<td>Acetone</td>
			<td>Honeywell</td>
			<td>32201-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose I</td>
			<td>VWR Life Science</td>
			<td>97062-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>NIHON SHIYAKU REAGENT</td>
			<td>NS-32294</td>
			<td></td>
		</tr>
		<tr>
			<td>AMV Reverse Transcriptase</td>
			<td>Promega</td>
			<td>M5101</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic(100X)&#160;</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Braun</td>
			<td>BA215</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemilumineance system</td>
			<td>TOP BIO CO.</td>
			<td>MGIS-21-C2-1M</td>
			<td></td>
		</tr>
		<tr>
			<td>Collection tube</td>
			<td>Qiagen</td>
			<td>990381</td>
			<td></td>
		</tr>
		<tr>
			<td>Collection tube</td>
			<td>SSI</td>
			<td>2341-SO</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>Muto Pure chemical Co., LTD.</td>
			<td>24505</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA analyzer</td>
			<td>Applied Biosystems</td>
			<td>3700XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>FITC Anti-Rabies Monoclonal Globulin</td>
			<td>Fujirebio Diagnostic Inc.</td>
			<td>800-092</td>
			<td>FITC-conjugated anti-rabies antibodies: reagent B</td>
		</tr>
		<tr>
			<td>Four-well Teflon-coating glass slide</td>
			<td>Thermo Fisher Scientific</td>
			<td>30-86H-WHITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Electrophoresis System</td>
			<td>Major Science</td>
			<td>MJ-105-R</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (1x)</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td>Trppsin</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ASTEC</td>
			<td>SCA-165DS</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>LIGHT DIAGNOSTICS Rabies FAT reagent</td>
			<td>EMD Millipore Corporation</td>
			<td>5100</td>
			<td>FITC-conjugated anti-rabies antibodies: reagent A</td>
		</tr>
		<tr>
			<td>MagNA Pure Compact Instrument</td>
			<td>Roche</td>
			<td>03731146001</td>
			<td></td>
		</tr>
		<tr>
			<td>MagNA Pure Compact NA Isolation Kit 1</td>
			<td>Roche</td>
			<td>03730964001</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (10x)</td>
			<td>Gibco</td>
			<td>11430030</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>MEM NEAA (100x)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>MEM vitamin solution</td>
			<td>Gibco</td>
			<td>11120052</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Merck</td>
			<td>1.06329.0500</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Terumo</td>
			<td>NN*2332R9</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Medicago</td>
			<td>09-8912-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer synthesis</td>
			<td>Mission Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin ribonuclease inhibitor</td>
			<td>Promega</td>
			<td>N2111</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing service</td>
			<td>Mission Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slide</td>
			<td>Thermo Scientific</td>
			<td>AA00008032E00MNT10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td>MEM-10</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads</td>
			<td>QIAGEN</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile absorbent pad</td>
			<td>3M</td>
			<td>1604T-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Nalgene</td>
			<td>171-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq polymerase</td>
			<td>JMR Holdings</td>
			<td>JMR-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>2720</td>
			<td></td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>QIAGEN</td>
			<td>85300</td>
			<td></td>
		</tr>
		<tr>
			<td>Tongue depressor</td>
			<td>HONJER CO., LTD.</td>
			<td>122246</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Tennyson medical Instrument developing CO., LTD.</td>
			<td>A0601</td>
			<td></td>
		</tr>
		<tr>
			<td>Tylosin Tartrate</td>
			<td>Sigma</td>
			<td>T6271-10G</td>
			<td>MEM-10</td>
		</tr>
	</tbody>
</table>

standard operating procedure for lyssavirus surveillance of the bat population in taiwan

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are natural reservoirs of most lyssaviruses, a lyssavirus surveillance program of bats has been conducted in Taiwan since 2008 to understand the ecology of these viruses in bats. In this program, non-governmental bat conservation organizations and local animal disease control centers cooperated to collect dead bats or bats dying of weakness or illness. Brain tissues of bats were obtained through necropsy and subjected to direct fluorescent antibody test (FAT) and reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus antigens and nucleic acids. For the FAT, at least two different rabies diagnosis conjugates are recommended. For the RT-PCR, two sets of primers (JW12/N165-146, N113F/N304R) are used to amplify a partial sequence of the lyssavirus nucleoprotein gene. This surveillance program monitors lyssaviruses and other zoonotic agents in bats. Taiwan bat lyssavirus is found in two cases of the Japanese pipistrelle (Pipistrellus abramus) in 2016&#8211;2017. These findings should inform the public, health professionals, and scientists of the potential risks of contacting bats and other wildlife.

From 2014 to May 2017, 332 bat carcasses from 13 species were collected for surveillance. Two tested positive. In the first bat case, the brain impression tested negative using the FAT with one of the commercial FITC-conjugated anti-rabies antibodies (Figure 2), while the RT-PCRs employing each of the two primer sets (JW12/N165-146, N113F/N304R) yielded positive results (Figure 3). A 428 bp sequence of amplicon (amplified with N113F/N304R and containing the part...

Watch this Scientific Journal Video about Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan at JoVE.com

Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan

This protocol introduces a standard laboratory operating procedure for diagnostic testing of lyssavirus antigens in bats in Taiwan.

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are ...

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7.4K Views.  Council of Agriculture, Executive Yuan. This protocol introduces a standard laboratory operating procedure for diagnostic testing of lyssavirus antigens in bats in Taiwan.

Video: Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan

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 ...

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

An Ultra-clean Multilayer Apparatus for Collecting Size Fractionated Marine Plankton and Suspended Particles

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those of suspended/sinking particles. Here, we present an all plastic (Polypropylene and Polycarbonate), multi-layer filtration system for collection of suspended particulate matter (SPM) at sea. This ultra-clean sampling device has been designed and developed specifically for trace element studies. Meticulous selection of all non-metallic materials and utilization of an in-line flow-through procedure minimizes any possible metal contamination during sampling. This system has been successfully tested and tweaked for determining trace metals (e.g., Fe, Al, Mn, Cd, Cu, Ni) on particles of varying size in coastal and open ocean waters. Results from the South China Sea at the South East Asia Time-Series (SEATS) station indicate that diurnal variations and spatial distribution of plankton in the euphotic zone can be easily resolved and recognized. Chemical analysis of size-fractionated particles in surface waters of the Taiwan Strait suggests that the larger particles (&#62;153 &#181;m) were mostly biologically derived, while the smaller particles (10 - 63 &#181;m) were mostly composed of inorganic matter. Apart from Cd, the concentrations of metals (Fe, Al, Mn, Cu, Ni) decreased with increasing size.

Plankton and suspended particles play a major role in the biogeochemical cycles in the ocean. Here, we provide an ultra-clean, low stress method for the collection of various sizes of particles and plankton at sea with the capability of handling large volumes of seawater.

The distributions of many trace elements in the ocean are strongly associated with the growth, death, and re-mineralization of marine plankton and those ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

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 ...

A Video Surveillance System to Monitor Breeding Colonies of Common Terns (Sterna Hirundo)

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread breeding distribution, that has been recently listed as endangered in some habitats of its range. Waterbird monitoring programs exist to track populations through time; however, some of the more intensive approaches require entering colonies and can be disruptive to nesting populations. This paper describes a protocol that utilizes a minimally invasive surveillance system to continuously monitor common tern nesting behavior in typical ground-nesting colonies. The video monitoring system utilizes wireless cameras focused on individual nests as well as over the colony as a whole, and allows for observation without entering the colony. The video system is powered with several 12 V car batteries that are continuously recharged using solar panels. Footage is recorded using a digital video recorder (DVR) connected to a hard drive, which can be replaced when full. The DVR may be placed outside of the colony to reduce disturbance. In this study, 3,624 h of footage recorded over 63 days in weather conditions ranging from 12.8 &#176;C to 35.0 &#176;C produced 3,006 h (83%) of usable behavioral data. The types of data retrieved from the recorded video can vary; we used it to detect external disturbances and measure nesting behavior during incubation. Although the protocol detailed here was designed for ground-nesting waterbirds, the principal system could easily be modified to accommodate alternative scenarios, such as colonial arboreal nesting species, making it widely applicable to a variety of research needs.

A Video Surveillance System to Monitor Breeding Colonies of Common Terns Sterna Hirundo

This paper describes a protocol that uses a remote video monitoring surveillance system to continuously monitor breeding colonies of ground-nesting waterbirds. The system includes five cameras monitoring individual nests and one camera monitoring the colony as a whole, and is powered by car batteries that are recharged via solar panels.

Many waterbird populations have faced declines over the last century, including the common tern (Sterna hirundo), a waterbird species with a widespread ...

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used microscopic and toxin analyses for decades for this purpose. Molecular biological methods, such as high-throughput DNA sequencing and bacterial assemblage-based approaches, are just beginning to be introduced in Chilean HAB monitoring, and the procedures have not yet been standardized. Here, 16S rRNA and 18S rRNA metabarcoding analyses for monitoring Chilean HABs are introduced stepwise. According to a recent hypothesis, algal-bacterial mutualistic association plays a critical synergetic or antagonistic relationship accounting for bloom initiation, maintenance, and regression. Thus, monitoring HAB from algal-bacterial perspectives may provide a broader understanding of HAB mechanisms and the basis for early warning. Metabarcoding analysis is one of the best suited molecular-based tools for this purpose because it can detect massive algal-bacterial taxonomic information in a sample. The visual procedures of sampling to metabarcoding analysis herein provide specific instructions, aiming to reduce errors and collection of reliable data.

This protocol introduces steps of metabarcoding analysis, targeting 16S rRNA and 18S rRNA genes, for monitoring harmful algal blooms and their associated microbiome in seawater samples. It is a powerful molecular-based tool but requires several procedures, which are visually explained here step-by-step.

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used ...