The authors give special thanks to the Santa Catarina State University for financial support, to the Pharmacology Laboratory for the animals and space, and to the Hemoparasites and Vectors Biochemistry Laboratory for the cryopreserved blood sample infected with T. evansi used in these experiments.

All experiments were performed using 10-week-old adult female Swiss mice (35-55 g). Animals were housed in polysulfone cages (3-5 animals per cage) in a room with controlled temperature (21 &#177; 1 &#176;C), 12 h light/12 h dark cycle, and standard lab chow and water ad libitum. Ten animals were assigned to each group in every test to demonstrate the consistent effects of drug treatments. The experimental design was submitted and approved by the Ethics Committee of Santa Catarina State University (CEUA) (protocol number: 6019201123). All animal experiments complied with the ARRIVE guidelines (Animal Research: Reporting of in vivo Experiments) and were carried out in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.
1. Trypanosoma evansi preparation and inoculation
CAUTION: Wear a disposable lab coat, gloves, mask, and eye protection when handling infected blood samples, reagents, and other chemical compounds.
<ol>
	<li>Turn on the water bath equipment and adjust it at a constant temperature of 37 &#176;C.</li>
	<li>Take a microcentrifuge tube containing the preserved blood sample out of the ultrafreezer (-80 &#176;C). Place it in the water bath until the blood thaws. 
	NOTE: The same isolate should be used to infect all mice in every group of different experiments to maintain an equal number of blood passages.</li>
	<li>Use phosphate-buffered saline (PBS, 0.1 M) containing 60% D-(+)-glucose (pH 7.4) to perform serial dilution (10:1 v v) until 1 &#215; 104 trypanosomes per 0.1 mL are achieved. Determine the number of parasite cells by manual cell counting using a Neubauer chamber on a microscope with a 100x objective lens2.</li>
	<li>Inoculate 0.1 mL of the diluted blood solution (infected group), or the same volume in 0.9% saline vehicle (control group), intraperitoneally, using a 26 G &#189;&#34; needle coupled to an insulin syringe.</li>
</ol>
2. Electronic von Frey apparatus setup and operation
NOTE: Be careful about how the obtained data are recorded. Some EvF equipment has specific programs to transfer the obtained data from the apparatus to other electronic devices, such as computers, tablets, or smartphones.
<ol>
	<li>Turn the EvF apparatus on. Ensure that the equipment is well charged before starting the experiment, observing the battery level displayed on the equipment screen and charging it if necessary.</li>
	<li>Connect the EvF apparatus to the chosen electronic device via Wi-Fi to transfer and save the obtained data.</li>
	<li>Insert a 10 &#181;L polypropylene tip into the EvF apparatus cone and set the readout to zero (0.00 g) by pressing the button with a paw symbol.</li>
	<li>Observe that once the evaluated tissue is probed; the maximum applied pressure is shown on the EvF apparatus display. After that, transfer it to the chosen electronic device by pressing the button with an antenna symbol for safe recording.</li>
	<li>Reset the readout to zero and get ready to take a new measurement by simply pressing the button with the paw symbol again.</li>
</ol>
3. General considerations for mechanical threshold assessments
<ol>
	<li>Perform all experiments in a quiet room with controlled temperature at 21 &#176;C and a 12 h light/12 h dark cycle, and be sure to respect the mouse circadian cycle by performing all evaluations at the same time of the day2.</li>
	<li>Have the same person perform both baseline and experimental mechanical threshold assessments to reduce bias and ensure measurement consistency; make sure the experiments are blinded.</li>
	<li>Place each mouse gently inside an individual chamber made of polymethyl methacrylate (PMMA) with a perforated top placed on a 5 mm&#178; mesh floor stand2,8.</li>
	<li>Allow the mice to acclimate for 30 min in the PMMA chambers prior to assessing both baseline and experimental mechanical thresholds2.</li>
	<li>Place the mesh floor stand at a comfortable height (upon a steady surface) so the animals are accessible from all sides. Ensure that the abdomen and all four paws of the mice are easily accessible through the mesh floor openings.</li>
	<li>Cover the steady surface used to allocate the chambers with absorbent material to absorb or collect micturition and defecation. Make sure that the arms of the investigator can move freely below the chambers while operating the EvF apparatus.</li>
</ol>
4. Baseline mechanical threshold assessment and mouse response to stimuli
<ol>
	<li>For abdomen measurements, divide the abdomen into three virtual parts (cranial, medium, and caudal areas). Choose the cranial area (anatomically containing the liver) as the standard target tissue for visceral allodynia mechanical threshold assessment. For paw measurements, choose the right hind paw for standardization.</li>
	<li>Take a baseline measurement from each mouse 48 h prior to the infection. To do so, place the mice in the containing chambers, prepare the EvF apparatus, and use both hands to raise the probe slowly to stimulate the targeted tissue.</li>
	<li>Gradually increase the pressure on the tissue until the mouse expresses a nociceptive behavior. For right hind paw evaluation, look for paw retraction, paw licking, or four-paw jumping2,8,9. For visceral evaluation, look for a sharp retraction of the abdomen, immediate licking or scratching of the probed site, or four-paw jumping2,11.</li>
	<li>Save the obtained value by transferring the data to the chosen electronic device containing the specific EvF program or writing it in a grade book. Reset the display to zero and repeat the process four times to obtain five measurements8. 
	NOTE: Consider only three similar measurements to estimate the mean value2</li>
	<li>Measure the mechanical threshold of the mouse present in the adjacent chamber until each mouse is probed five times and a mean value is obtained for each animal.</li>
	<li>Repeat the baseline measurements 24 h later and exclude mice with mean values below 3.00 g or with differences between basal measurements higher than 2.00 g2,8. 
	&#8203;NOTE: Only one target tissue must be chosen for each mouse in every experiment so the animals do not get used to being probed, as the number of evaluations would be considerably higher in a short period.</li>
</ol>
5. Experimental right hind paw and visceral allodynia mechanical threshold assessment
<ol>
	<li>Assess the right hind paw or visceral allodynia mechanical threshold 1 h after the infection for day 0 evaluation.</li>
	<li>Repeat the procedure every 24 h over 5 days post infection. 
	NOTE: if there is a treatment to be administered, choose an appropriate time point within the experimental design.</li>
	<li>Return the mice to their original cages to prevent animals from fighting each other, with access to standard lab chow and water ad libitum after measurements are completed.</li>
	<li>Perform a normality test to check the normal distribution of the data and further appropriate statistical analysis of the collected data.</li>
</ol>
6. Mouse grimace scale assessment
<ol>
	<li>Ensure that each mouse is seen easily while inside the containing chamber and examine each mouse prior to the experiment to ensure there are no lesions on the limbs, abdomen, or face and no coat changes.</li>
	<li>Observe the mouse orbital area. Classify open eyes as an absence of pain (score 0) while an obvious pain may be represented when the mouse closes its eyes (score 2).</li>
	<li>Observe the mouse&#39;s nose. Classify a normal nose as the absence of pain while the presence of a bulge on the bridge of the nose represents obvious pain.</li>
	<li>Observe the mouse&#39;s cheeks. Classify normal cheeks as the absence of pain while the presence of a bulge on both cheeks represents obvious pain.</li>
	<li>Observe the position of the mouse&#39;s ears. Classify rounded ears as the absence of pain while ears rotating outwards or backwards, away from the face, in a pointed shape, represent obvious pain. Space between the ears increases with the pain score.</li>
	<li>Observe the mouse&#39;s whiskers. Classify whiskers with their natural downward curve as the absence of pain while whiskers that are either pulled back against the cheek or pulled forward represent obvious pain.</li>
	<li>As innate behaviors, such as self-grooming, may interfere with the facial expression, do not consider these to be pain-related behaviors8. Wait for the mouse to stop this behavior before every action unit evaluation.</li>
	<li>Perform a normality test to check the normal distribution of the data and further appropriate statistical analysis of the collected data. 
	NOTE: At the end of all the experiments, mice were humanely euthanized using ketamine (90 mg/kg) and xylazine (7.5 mg/kg), injected intraperitoneally. After reaching a confirmed deep anesthesia plan, they were submitted to cervical dislocation.</li>
</ol>

Assessing Pain and Allodynia in T. evansi-Infected Animals

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	<li>Desquesnes, M., 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=Trypanosoma+evansi+and+surra:+a+review+and+perspectives+on+origin,+history,+distribution,+taxonomy,+morphology,+hosts,+and+pathogenic+effects.">Trypanosoma evansi and surra: a review and perspectives on origin, history, distribution, taxonomy, morphology, hosts, and pathogenic effects.</a> BioMed Research International. 2013, 194176 (2013).</li><li>Cipriani, D. 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=Experimental+Trypanosoma+evansi+infection+induces+pain+along+with+oxidative+stress,+prevented+by+COX-2+inhibition.">Experimental Trypanosoma evansi infection induces pain along with oxidative stress, prevented by COX-2 inhibition.</a> Experimental Parasitology. 247, 108477 (2023).</li><li>Paim, F. 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=Cytokines+in+rats+experimentally+infected+with+Trypanosoma+evansi.">Cytokines in rats experimentally infected with Trypanosoma evansi.</a> Experimental Parasitology. 128 (4), 365-370 (2011).</li><li>Gillingwater, K., 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=In+vivo+investigations+of+selected+diamidine+compounds+against+Trypanosoma+evansi+using+a+mouse+model.">In vivo investigations of selected diamidine compounds against Trypanosoma evansi using a mouse model.</a> Antimicrobial Agents and Chemotherapy. 53 (12), 5074-5079 (2009).</li><li>Dkhil, M. A., 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=Treatment+of+Trypanosoma+evansi-infected+mice+with+Eucalyptus+camaldulensis+led+to+a+change+in+brain+response+and+spleen+immunomodulation.">Treatment of Trypanosoma evansi-infected mice with Eucalyptus camaldulensis led to a change in brain response and spleen immunomodulation.</a> Frontiers in Microbiology. 13, 833520 (2022).</li><li>Dkhil, M. A., 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=Murine+liver+response+to+Allium+sativum+treatment+during+infection+induced-trypanosomiasis.">Murine liver response to Allium sativum treatment during infection induced-trypanosomiasis.</a> Saudi Journal of Biological Sciences. 28 (6), 3270-3274 (2021).</li><li>Martins de Moraes, 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=Infection+by+Trypanosoma+evansi+in+horses+from+Brazil.">Infection by Trypanosoma evansi in horses from Brazil.</a> Revista Portuguesa de Ci&#234;ncias Veterin&#225;rias. 102 (561-562), 159-163 (2007).</li><li>Martinov, T., 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=Measuring+changes+in+tactile+sensitivity+in+the+hind+paw+of+mice+using+an+electronic+von+Frey+apparatus.">Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus.</a> Journal of Visualized Experiments. 82, e51212 (2013).</li><li>Rodr&#237;guez-Angulo, H., 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=Role+of+TNF+in+sickness+behavior+and+allodynia+during+the+acute+phase+of+Chagas'+disease.">Role of TNF in sickness behavior and allodynia during the acute phase of Chagas' disease.</a> Experimental Parasitology. 134 (4), 422-429 (2013).</li><li>Langford, D. J., 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=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">Coding of facial expressions of pain in the laboratory mouse.</a> Nature Methods. 7, 447-449 (2010).</li><li>Eijkelkamp, N., 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=Increased+visceral+sensitivity+to+capsaicin+after+DSS-induced+colitis+in+mice:+spinal+cord+c-Fos+expression+and+behavior.">Increased visceral sensitivity to capsaicin after DSS-induced colitis in mice: spinal cord c-Fos expression and behavior.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 293 (4), 749-757 (2007).</li><li>Mekata, H., 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=Expression+of+regulatory+dendritic+cell-related+cytokines+in+cattle+experimentally+infected+with+Trypanosoma+evansi.">Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi.</a> Journal of Veterinary Medical Science. 77 (8), 1017-1019 (2015).</li><li>Mukaka, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistics+corner:+a+guide+to+appropriate+use+of+correlation+coefficient+in+medical+research.">Statistics corner: a guide to appropriate use of correlation coefficient in medical research.</a> Malawi Medical Journal. 24 (3), 69-71 (2012).</li><li>Schober, P., 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=Correlation+coefficients:+appropriate+use+and+interpretation.">Correlation coefficients: appropriate use and interpretation.</a> Anesthesia and Analgesia. 126 (5), 1763-1768 (2018).</li><li>Kamidi, C. M., 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=Differential+virulence+of+camel+Trypanosoma+evansi+isolates+in+mice.">Differential virulence of camel Trypanosoma evansi isolates in mice.</a> Parasitology. 145 (9), 1235-1242 (2018).</li><li>Mekata, H., 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=Isolation,+cloning,+and+pathologic+analysis+of+Trypanosoma+evansi+field+isolates.">Isolation, cloning, and pathologic analysis of Trypanosoma evansi field isolates.</a> Parasitology Research. 112, 1513-1521 (2013).</li><li>Silva, A. 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=Trypanosoma+evansi+pathogenicity+strain+in+rats+inoculated+with+parasite+in+fresh+and+cryopreserved+blood.">Trypanosoma evansi pathogenicity strain in rats inoculated with parasite in fresh and cryopreserved blood.</a> Ci&#234;ncia Rural. 39 (6), 1842-1846 (2009).</li><li>Silva, A. 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=Acetylcholinesterase+activity+and+lipid+peroxidation+in+the+brain+and+spinal+cord+of+rats+infected+with+Trypanosoma+evansi.">Acetylcholinesterase activity and lipid peroxidation in the brain and spinal cord of rats infected with Trypanosoma evansi.</a> Veterinary Parasitology. 175, 237-244 (2011).</li><li>Di&#243;genes, A. K. L., 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=Concurrent+validity+of+electronic+von+Frey+as+an+assessment+tool+for+burn+associated+pain.">Concurrent validity of electronic von Frey as an assessment tool for burn associated pain.</a> Burns. 46 (6), 1328-1336 (2020).</li><li>Kalueff, A. V., 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=Neurobiology+of+rodent+self-grooming+and+its+value+for+translational+neuroscience.">Neurobiology of rodent self-grooming and its value for translational neuroscience.</a> Nature Reviews Neuroscience. 17, 45-59 (2016).</li></ol>

The authors have no conflicts of interest to declare.

A critical step in experiments involving infected animals is the control of parasitemia levels. Different strains of T. evansi may behave in divergent ways in mice, leading from acute to chronic infections2,4,5,6. Furthermore, changes in the infection dose may decrease or extend the survival time of mice. Therefore, obtaining a survival curve prior to the experiments is recommended to ...

Trypanosoma evansi is the etiological agent of the disease called &#34;surra&#34; or &#34;mal-das-cadeiras&#34; in South America1,2. It commonly affects equines and cattle, but also wild fauna, being transmitted by hematophagous bats, tabanids, and stomoxes fly bites1,3. Surra is a major disease in domestic animals, which can be fatal in the absence of the correct treatment, exhibiting nonspecific clinical signs such as anemia, loss of appetite and weight, muscle weakness, and abortion, which may vary according to the host and geographic region2,4,5,6.
The expression of allodynia and pain in infected animals during the course of the disease is still a new topic2,7. The urge to understand the pathogenesis behind these signs is an important step to improve and refine the currently used treatment by adding effective analgesic drugs to the classic trypanocidal protocol5,6. In this scenario, the possibility of replicating the disease using a murine model represents an advantage, as mice can be easily kept under controlled environments in the laboratory and therefore, yield results that are more consistent than field experiments using livestock.
A mechanical threshold is commonly obtained using an electronic von Frey (EvF) apparatus for the evaluation of allodynia in a vast number of experiments2,8,9. This device is used to assess tissue sensitivity to mechanical stimulation: once the apparatus touches the paw of the animal, through a 20-200 &#181;L tip attached to the device, the force with which the animal withdraws the paw is recorded.
Rodents are usually used as standard animals in these experiments, and most results are extrapolated to other species, as most researched diseases come from other species in which it would be difficult to conduct a controlled study. Furthermore, allodynia and pain are intimately related. The use of a specific facial scale to evaluate pain in infected mice plays an important role as a confirming adjuvant to affirm the presence of pain during the course of T. evansi infection2,10.
In this protocol, we demonstrate a novel model to evaluate allodynia and pain in mice experimentally infected with T. evansi, showing a high correlation between variables, thus proving to be a strong model. Furthermore, it requires a small number of researchers to carry out the entire procedure, decreasing the chance of human interference during the experiment. It also enables the investigator to replicate the target disease during an acute course and add several different treatment drugs in the experimental design, thereby obtaining consistent results in a short period.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>26 G 1/2&#34; needle coupled to insulin syringe</td>
			<td>TKL</td>
			<td>80288090100</td>
			<td>Used to infund solutions on laboratory animals</td>
		</tr>
		<tr>
			<td>Accessories for von Frey analgesimeter</td>
			<td>INSIGHT</td>
			<td>EFF 303</td>
			<td>Containment box with support for digital analgesimeter assessment</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>SIGMA-ALDRICH</td>
			<td>G7021</td>
			<td>A monosaccharide which is the main source of energy in the form of ATP for living organisms</td>
		</tr>
		<tr>
			<td>Digital analgesimeter</td>
			<td>INSIGHT</td>
			<td>von Frey Wi-Fi</td>
			<td>The von Frey Wi-Fi is a portable device used to assess tissue sensitivity to mechanical stimuli</td>
		</tr>
		<tr>
			<td>Gilson type 10 &#181;L polypropylene tip</td>
			<td>CRALPLAST</td>
			<td>18261</td>
			<td>Polypropylene to be used on eletronic von Frey apparatus, recommended for hind paw allodynia assessment</td>
		</tr>
		<tr>
			<td>Laboratory water bath</td>
			<td>BEING INSTRUMENT</td>
			<td>BW-22P</td>
			<td>Used to heat liquid and semi-solid substances contained in appropriate recipients to specific temperature</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>SIGMA-ALDRICH</td>
			<td>806552</td>
			<td>A balanced salt solution buffer used for a variety of cell culture applications</td>
		</tr>
		<tr>
			<td>Swiss mice (Mus musculus) from both gender</td>
			<td>UFSC</td>
			<td>Swiss Webster</td>
			<td>Laboratory animals used for controlled experiments</td>
		</tr>
		<tr>
			<td>Trypanosoma evansi cryiopreserved sample</td>
			<td>UDESC</td>
			<td>-</td>
			<td>Sample used to infect all mice, ceded by the Hemoparasites and Vectors Biochemistry Laboratory</td>
		</tr>
		<tr>
			<td>Universal type 10 &#181;L polypropylene tip</td>
			<td>CRALPLAST</td>
			<td>18171</td>
			<td>Polypropylene to be used on eletronic von Frey apparatus, recommended for visceral allodynia assessment</td>
		</tr>
	</tbody>
</table>

electronic von frey associated with the mouse grimace scale to assess allodynia and pain in trypanosoma evansi-infected mice

Infectious disease pathogenesis is still a complex field to study. The course of several clinical signs, such as allodynia and pain, may be observed in domestic animals. However, the knowledge of their pathways and correct treatment need controlled experiments, many of them using laboratory animals. Measuring changes in mechanical thresholds of the hind paw and viscera is a useful technique to observe changes in pain perception in rodents. Withdrawal response can be measured first in baseline tests, which creates better control of experimental groups. Subsequent tests can be performed after inducing infection and adding drugs to the protocol. The use of an electronic von Frey apparatus associated with the use of a facial scale to observe pain-like changes allows a simple, precise, and consistent assessment to evaluate allodynia and pain in mice. Thus, experiments using the present methodology for Trypanosoma evansi infection represent a useful method to evaluate allodynia and pain in laboratory-infected animals, which can be applied to the conventional treatment for livestock animals.

Decrease in the mechanical threshold due to T. evansi infection as evaluated by electronic von Frey apparatus on both right hind paw and visceral tissues 
The experiment was conducted over 5 days, according to previous data related to the available T. evansi sample2. Infected mice started to show a significant difference in mechanical threshold on the right hind paw at day 3 post infection and remained significantly lower than the control group during the ne...

Author Spotlight: Assessing Pain and Promoting Animal Welfare in Laboratory Animals Infected with Trypanosoma evansi

Here, we present a protocol to evaluate nociception in mice infected with Trypanosoma evansi, using the electronic von Frey apparatus as a tool, measuring mechanical thresholds of the hind paw and viscera.

Electronic von Frey associated with the Mouse Grimace Scale to Assess Allodynia and Pain in Trypanosoma evansi-Infected Mice

Infectious disease pathogenesis is still a complex field to study. The course of several clinical signs, such as allodynia and pain, may be observed in ...

electronic-von-frey-associated-with-mouse-grimace-scale-to-assess

Research

JoVE Journal

Immunology and Infection

824 Views.  Santa Catarina State University. Here, we present a protocol to evaluate nociception in mice infected with Trypanosoma evansi, using the electronic von Frey apparatus as a tool, measuring mechanical thresholds of the hind paw and viscera.

Video: Author Spotlight: Assessing Pain and Promoting Animal Welfare in Laboratory Animals Infected with Trypanosoma evansi

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.