<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article  PUBLIC "-//NLM//DTD Journal Publishing DTD v3.0 20080202//EN" "http://dtd.nlm.nih.gov/publishing/3.0/journalpublishing3.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" dtd-version="3.0" xml:lang="en" article-type="research article"><front><journal-meta><journal-id journal-id-type="publisher-id">OJVM</journal-id><journal-title-group><journal-title>Open Journal of Veterinary Medicine</journal-title></journal-title-group><issn pub-type="epub">2165-3356</issn><publisher><publisher-name>Scientific Research Publishing</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.4236/ojvm.2015.56019</article-id><article-id pub-id-type="publisher-id">OJVM-57114</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Medicine&amp;Healthcare</subject></subj-group></article-categories><title-group><article-title>
 
 
  Prevalence of Antibodies to &lt;i&gt;Toxoplasma gondii&lt;/i&gt; and &lt;i&gt;Neospora caninum&lt;/i&gt; in Pigs in Grenada, West Indies
 
</article-title></title-group><contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>avindra</surname><given-names>Nath Sharma</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Keshaw</surname><given-names>Tiwari</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Alfred</surname><given-names>Chikweto</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Claude</surname><given-names>DeAllie</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Muhammad</surname><given-names>Iqbal Bhaiyat</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib></contrib-group><aff id="aff1"><addr-line>Department of Pathobiology, School of Veterinary Medicine, St. George’s University, St. George, Grenada</addr-line></aff><aff id="aff2"><addr-line>Veterinary and Livestock Division, Ministry of Agriculture, Land, Fisheries and Environment, Grenada</addr-line></aff><author-notes><corresp id="cor1">* E-mail:<email>rsharma@sgu.edu(ANS)</email>;</corresp></author-notes><pub-date pub-type="epub"><day>10</day><month>06</month><year>2015</year></pub-date><volume>05</volume><issue>06</issue><fpage>138</fpage><lpage>141</lpage><history><date date-type="received"><day>7</day>	<month>May</month>	<year>2015</year></date><date date-type="rev-recd"><day>accepted</day>	<month>9</month>	<year>June</year>	</date><date date-type="accepted"><day>12</day>	<month>June</month>	<year>2015</year></date></history><permissions><copyright-statement>&#169; Copyright  2014 by authors and Scientific Research Publishing Inc. </copyright-statement><copyright-year>2014</copyright-year><license><license-p>This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/</license-p></license></permissions><abstract><p>
 
 
  The aim of the study was to estimate the prevalence of antibodies to &lt;i&gt;Toxoplasma gondii&lt;/i&gt; (&lt;i&gt;T. gondii&lt;/i&gt;) and &lt;i&gt;Neospora caninum&lt;/i&gt; (&lt;i&gt;N. caninum&lt;/i&gt;) in pigs in Grenada, West Indies. &lt;i&gt;T. gondii&lt;/i&gt; is a serious zoonosis affecting the unborn fetus and immunocompromized individuals. &lt;i&gt;N. caninum&lt;/i&gt; is a similar coccidian parasite, which is not zoonotic, but is the cause of abortion and neonatal mortality in livestock similar to &lt;i&gt;T. gondii&lt;/i&gt;. An earlier study conducted in Grenada and using a modified agglutination test (MAT) revealed seropositivity to &lt;i&gt;T. gondii&lt;/i&gt; in pigs. No information is available on &lt;i&gt;N. caninum&lt;/i&gt; infection of pigs in the Caribbean islands including Grenada. Serum samples from 185 pigs in Grenada, West Indies were tested for antibodies to &lt;i&gt;T. gondii&lt;/i&gt; and &lt;i&gt;N. caninum&lt;/i&gt; using an indirect enzyme linked immunosorbent assay (ELISA). Antibodies to &lt;i&gt;T. gondii&lt;/i&gt; were found in 24.3% of pigs (95% confidence interval (CI): 18.12% to 30.48%) as all the tested pigs were negative for antibodies to &lt;i&gt;N. caninum&lt;/i&gt;. Although, seroprevalence for &lt;i&gt;T. gondii&lt;/i&gt; was higher in females (25.75%) than in males (20.70%), this result was statistically insignificant (p = 0.57). The results were similar to a previous study in Grenada confirming the continuity of infection in pigs. Human Toxoplasmosis is transmitted mainly through ingestion of tissue cysts in contaminated raw or undercooked meat or sporulated oocysts in soil, water or vegetables. Education of farmers and the Grenadian community on epidemiology of these parasites is warranted to prevent infection in pigs and in humans. This is the first report on the seroprevalence of &lt;i&gt;N. caninum&lt;/i&gt; in pigs in the Caribbean region.
 
</p></abstract><kwd-group><kwd>&lt;i&gt;Toxoplasma gondii&lt;/i&gt;</kwd><kwd> &lt;i&gt;Neospora caninum&lt;/i&gt;</kwd><kwd> Pigs</kwd><kwd> MAT</kwd><kwd> ELISA</kwd></kwd-group></article-meta></front><body><sec id="s1"><title>1. Introduction</title><p>Toxoplasmosis is a parasitic disease caused by proptozoa Toxoplasma gondii. The parasite infects warm blooded animals including humans. Human infection is mostly through ingestion of tissue cysts in contaminated raw or undercooked meat or sporulated oocysts in soil and water [<xref ref-type="bibr" rid="scirp.57114-ref1">1</xref>] . In addition to the zoonotic potential of T. gondii, it causes mortality in neonatal pigs and reproductive failure in adult pigs. To the author’s knowledge, there is limited data on the prevalence of T. gondii in pigs in the Caribbean countries. The seroprevalence of T. gondii in pigs was first reported in Trinidad [<xref ref-type="bibr" rid="scirp.57114-ref2">2</xref>] indicating seroprevalence of 5.5%. Chikweto et al. [<xref ref-type="bibr" rid="scirp.57114-ref3">3</xref>] using a modified agglutination test (MAT) reported seroprevalence of 23.1% in pigs in Grenada.</p><p>Neosporosis is an infectious disease primarily of cattle and dogs, and it has been reported rarely in other species [<xref ref-type="bibr" rid="scirp.57114-ref4">4</xref>] [<xref ref-type="bibr" rid="scirp.57114-ref5">5</xref>] . N. caninum is not considered to be zoonotic. However, Helmick et al. [<xref ref-type="bibr" rid="scirp.57114-ref6">6</xref>] in UK while working on the prevalence of Neosposis in aborted sheep and swine reported 0.45% seropositivity to N. caninumin aborted sheep and 9.3% in aborted pigs, by ELISA, but all samples were negative when tested by IFAT. Damriyasa et al. [<xref ref-type="bibr" rid="scirp.57114-ref7">7</xref>] using ELISA reported a seropositivity of 3.3% for N. caninum in sows in Hesse Germany. To the authors’ knowledge, there is no data on seroprevalence of N. caninum in pigs in the Caribbean countries.</p><p>The objective of this study is to estimate the seroprevalence of T. gondii and N. caninum in pigs from Grenada, West Indies.</p></sec><sec id="s2"><title>2. Materials and Methods</title><sec id="s2_1"><title>2.1. Ethical Approval</title><p>The research project was approved by Institutional Animal Care and Use Committee (IACUC) of St. George’s University, Grenada.</p></sec><sec id="s2_2"><title>2.2. Collection of Samples</title><p>Grenada is the southernmost island country of the Southeastern Caribbean Sea. Its size is 344 Km<sup>2</sup> and the island is divided in 6 parishes. Jugular vein blood was collected from 185 pigs and after centrifugation at 3000 g for 5 minutes serum was removed and stored at −20˚C until analysis. Blood was collected from 53 boars and 132 sows, ranging in age from 6 months to 2 years.</p></sec><sec id="s2_3"><title>2.3. Methodology</title><p>A Commercial Toxoplasma gondii multispecies ELISA kit and Neospora caninum multispecies ELISA kit from Pourquier Laboratories (IDvet, France) for detection of anti T. gondii and N. caninum antibodies were used according to the manufacturer’s instructions.</p></sec></sec><sec id="s3"><title>3. Results</title><p>Antibodies against T. gondii were found in 45 pigs (24.3%) (95% confidence interval (CI): 18.12% to 30.48%). Out of the positive animals, 11 (20.8%) were males and 34 (25.8%) were females (<xref ref-type="table" rid="table1">Table 1</xref>).</p><p>All serum samples were negative for antibodies against N. caninum.</p></sec><sec id="s4"><title>4. Discussion</title><p>A worldwide distribution of T. gondii has been reported based mainly on serological surveys [<xref ref-type="bibr" rid="scirp.57114-ref1">1</xref>] . Seropreva- lence of 23% in market pigs and 43% in breeder pigs, in the USA were detected during a survey in 1983-84. After a decade there was a decline in antibodies in pigs tested from the same areas [<xref ref-type="bibr" rid="scirp.57114-ref8">8</xref>] . Good husbandry practices</p><table-wrap id="table1" ><label><xref ref-type="table" rid="table1">Table 1</xref></label><caption><title> Seroprevalence of T. gondii in pigs in Grenada, West Indies</title></caption><table><tbody><thead><tr><th align="center" valign="middle"  colspan="2"  >Number of pigs tested</th><th align="center" valign="middle" >Number positive</th><th align="center" valign="middle" >Percent positive</th></tr></thead><tr><td align="center" valign="middle" >185</td><td align="center" valign="middle" ></td><td align="center" valign="middle" >45</td><td align="center" valign="middle" >24.3</td></tr><tr><td align="center" valign="middle" >Male</td><td align="center" valign="middle" >53</td><td align="center" valign="middle" >11</td><td align="center" valign="middle" >20.8</td></tr><tr><td align="center" valign="middle" >Female</td><td align="center" valign="middle" >132</td><td align="center" valign="middle" >34</td><td align="center" valign="middle" >25.8</td></tr></tbody></table></table-wrap><p>were attributed to this decline in seroprevalence of T. gondii. Like other food animals, pigs also get infected with T. gondii oocysts from cats. Pigs can also be infected through ingestion of infected rats [<xref ref-type="bibr" rid="scirp.57114-ref6">6</xref>] [<xref ref-type="bibr" rid="scirp.57114-ref9">9</xref>] and through feeding on milk from infected goats [<xref ref-type="bibr" rid="scirp.57114-ref10">10</xref>] [<xref ref-type="bibr" rid="scirp.57114-ref11">11</xref>] .</p><p>Our results showed 24.3% (95% confidence interval [CI]: 18.12% to 30.48%) seropositivity for T. gondii in pigs using an ELISA, This is similar to the first study conducted by Chikweto et al. [<xref ref-type="bibr" rid="scirp.57114-ref3">3</xref>] in Grenada, in which a seroprevalence of 23.1% was reported using a MAT. There is considerable variation in the Seroprevalence of T. gondii from different countries of the world. In Germany it was found to be 19% ([<xref ref-type="bibr" rid="scirp.57114-ref7">7</xref>] ; in the USA, from 5% to 70.83 [<xref ref-type="bibr" rid="scirp.57114-ref1">1</xref>] ; in Romania, 0.4% to 0.5% [<xref ref-type="bibr" rid="scirp.57114-ref12">12</xref>] ; in Poland, 10.5% [<xref ref-type="bibr" rid="scirp.57114-ref13">13</xref>] ; in Italy, 16.3% to 19% [<xref ref-type="bibr" rid="scirp.57114-ref14">14</xref>] ; in the Netherlands, 2.6% [<xref ref-type="bibr" rid="scirp.57114-ref15">15</xref>] ; 0.0% in Malaysia ([<xref ref-type="bibr" rid="scirp.57114-ref16">16</xref>] and in Serbia, 9.3% [<xref ref-type="bibr" rid="scirp.57114-ref17">17</xref>] . These variations are attributed to the type of husbandry, outdoor and indoor farms, access of cats to the farm, age of pigs, and the type of tests used for seroprevalence studies [<xref ref-type="bibr" rid="scirp.57114-ref1">1</xref>] [<xref ref-type="bibr" rid="scirp.57114-ref6">6</xref>] .</p><p>Sera tested in the present study showed higher prevalence of antibodies to T. gondii in females compared to males, but this was statistically insignificant (p = 0.57). Published reports on association of gender and seroprevalence of T. gondii in pigs are scanty. Lonela et al. [<xref ref-type="bibr" rid="scirp.57114-ref9">9</xref>] reported a high seroprevalence of T. gondii in sows (80%) compared to boars (20%). Sergio et al. [<xref ref-type="bibr" rid="scirp.57114-ref18">18</xref>] in Brazil reported no association between gender and seroprevalence of T. gondii in pigs.</p><p>Canids are the final host of N. caninum. Livestock and wild animals get infected by consuming oocysts from infected canids. N. caninum is not zoonotic. Neosporosis is primarily a disease of cattle in which it is a major cause of abortion and reproductive disorders. Neosporosis in sheep and goats is asymptomatic. There have been reports on role of Neosporosis in abortion and neonatal mortality in adult sheep. However, the seroprevalence of N. caninum in sheep and goats varies from 0.6% to as high as 30.8% in various countries of the world [<xref ref-type="bibr" rid="scirp.57114-ref5">5</xref>] .</p><p>There is paucity of published reports on Neosporosis in pigs. The few available reports indicate a very low seroprevalence of N. caninum in the swine population. Damariyasa et al. [<xref ref-type="bibr" rid="scirp.57114-ref7">7</xref>] reported a seropositivity of 3.3% pigs in Germany. Helmick et al. [<xref ref-type="bibr" rid="scirp.57114-ref6">6</xref>] reported seropositivity of 8.8% in pigs in the UK, but failed to confirm the seropositivity in that pig population by indirect fluorescent antibody test (IFAT) and an inhibition ELISA. A survey conducted in Brazil revealed a seroprevalence of 3.1% [<xref ref-type="bibr" rid="scirp.57114-ref18">18</xref>] . To the authors’ knowledge, there is no report on seroprevalence of N. caninum in pigs in Caribbean countries. This is the first report on the seroprevalence N. caninum in Grenada and the Caribbean region.</p></sec><sec id="s5"><title>5. Conclusion</title><p>Pork is popular meat in Grenada and preferred when cooked through barbeque. The chances of survival of T. gondii oocysts in barbeque cooked meat are increased. Infected animals could thus transmit the disease to humans.</p></sec><sec id="s6"><title>Recommendation</title><p>Pigs in Grenada are moderately exposed to T. gondii. Measures taken to prevent toxoplasmosis in humans should include education of pig farmers and the community at large. Pig farmers need to realize the role of cats and rodents in transmission of infection to pigs. Cats and rodent in free farms with good sanitary practices will aid in the prevention of toxoplasmosis in pigs and humans. Grenadians must be educated about the importance of complete cooking of pork before consumption.</p></sec><sec id="s7"><title>Acknowledgements</title><p>The research was supported by funds from Small Research Grant Initiative (SRGI) at St. George’s University, Grenada.</p></sec><sec id="s8"><title>Competing Interests</title><p>The authors declare that there is no competing interest.</p></sec><sec id="s9"><title>NOTES</title></sec></body><back><ref-list><title>References</title><ref id="scirp.57114-ref1"><label>1</label><mixed-citation publication-type="other" xlink:type="simple">Dubey, J.P. (2009) Toxoplasmosis in Pigs—The Last 20 Years. Veterinary Parasitology, 164, 89-103. http://dx.doi.org/10.1016/j.vetpar.2009.05.018</mixed-citation></ref><ref id="scirp.57114-ref2"><label>2</label><mixed-citation publication-type="other" xlink:type="simple">Adesiyun, A.A. and Kazabon, E.P. (1996) Seroprevalence of Brucellosis, Q Fever and Toxoplasmois in Slaughter Animals in Trinidad. Revue d’Elevage et de Medicine Veterinaire des Pays (in French), 49, 28-30.</mixed-citation></ref><ref id="scirp.57114-ref3"><label>3</label><mixed-citation publication-type="other" xlink:type="simple">Chikweto, A., Kumthekar, S., Tiwari, K., Nyack, B., Deokar, M.S., Stratton, G., Macpherson, C.L.N., Sharma, R.N. and Dubey, J.P. (2011) Seroprevalence of T. gondii in Pigs, Sheep, Goats and Cattle from Grenada and Carriacou. Journal of Parasitology, 97, 950-951. http://dx.doi.org/10.1645/GE-2811.1</mixed-citation></ref><ref id="scirp.57114-ref4"><label>4</label><mixed-citation publication-type="other" xlink:type="simple">Dubey, J.P. (1999) Recent Advances in Neospora and Neosporosis. Veterinary Parasitology, 84, 349-367. http://dx.doi.org/10.1016/S0304-4017(99)00044-8</mixed-citation></ref><ref id="scirp.57114-ref5"><label>5</label><mixed-citation publication-type="other" xlink:type="simple">Dubey, J.P. and Schares, G. (2011) Neosporosis in Animals—The Last Five Years. Veterinary Parasitology, 180, 90- 108.  
http://dx.doi.org/10.1016/j.vetpar.2011.05.031</mixed-citation></ref><ref id="scirp.57114-ref6"><label>6</label><mixed-citation publication-type="other" xlink:type="simple">Helmick, B., Otter, A., McGarry, J. and Buxton, D. (2002) Serological Investigation of Aborted Sheep and Pigs for Infection by Neospora caninum. Research in Veterinary Science, 73, 187-189. http://dx.doi.org/10.1016/S0034-5288(02)00093-0</mixed-citation></ref><ref id="scirp.57114-ref7"><label>7</label><mixed-citation publication-type="other" xlink:type="simple">Damariyasa, I.M., Bauer, C., Edelhofer, R., Failing, K., Lind, P., Peterson, E., Schares, G., Tenter, A.M., Volmer, R. and Zahner, H. (2004) Cross Sectional Survey in Pig Breeding Farms in Hesse, Germany: Seroprevalence and Risk Factors of Infections with Toxoplasma gondii, Sarcocystis spp and Neospora caninum in Sows. Veterinary Parasitology, 126, 271-286. http://dx.doi.org/10.1016/j.vetpar.2004.07.016</mixed-citation></ref><ref id="scirp.57114-ref8"><label>8</label><mixed-citation publication-type="other" xlink:type="simple">Dubey, J.P., Weigel, R.M., Seigel, A.M., Thulliez, P., Kitron, U.D., Mitchel, M.A., Mannelli, A., Mateus-Pinilla, N.E., Shen, S.K., Kwok, O.C.H. and Todd, K.S. (1995) Sources and Reservoirs of Toxoplasma gondii Infection on 47 Swine Farms in Illinois. The Journal of Parasitology, 81, 723-729.  
http://dx.doi.org/10.2307/3283961</mixed-citation></ref><ref id="scirp.57114-ref9"><label>9</label><mixed-citation publication-type="other" xlink:type="simple">Ionela, G.H., Darabus, M.S., Ilie, K., Imre, A., Balint, D., Indre, M.I. Denisa, S., Roberta, C., Luminita, C. and Corina, P. (2010) Seroprevalence of Toxoplasma gondii Infection in Pigs Reared in Intensive System from TIMIS County. Lucrari Stiintfice Medicina Veterinara, XLIII, 68-72.</mixed-citation></ref><ref id="scirp.57114-ref10"><label>10</label><mixed-citation publication-type="other" xlink:type="simple">Jittapalapong, S., Sangavaranond, A., Pinyopanuwat, N., Chimnoi, W., Khachaeram, W., Koizumi, S. and Maruyama, S. (2005) Seroprevalence of Toxoplasma gondii Infection in Domestic Goats in Satun Province, Thailand. Veterinary Parasitology, 127, 17-22. http://dx.doi.org/10.1016/j.vetpar.2004.08.019</mixed-citation></ref><ref id="scirp.57114-ref11"><label>11</label><mixed-citation publication-type="other" xlink:type="simple">Meerburg, B.C., van Riel, J.W., Cornelissen, J.B., Kijlstra, A. M.F. and Mul, A.M.F. (2006) Cats and Goat Whey Associated with Toxoplasma gondii Infection in Pigs. Vector-Borne and Zoonotic Diseases, 6, 266-274. http://dx.doi.org/10.1089/vbz.2006.6.266</mixed-citation></ref><ref id="scirp.57114-ref12"><label>12</label><mixed-citation publication-type="other" xlink:type="simple">Anamaria, B., Anamaria, I.P., Adriana, G., Viorica, M. and Vasile, C. (2012) The Dynamics of Anti Toxoplasma gondii antibodies (IgG) in Small Ruminants and Pigs from Cluj County, Romania. Scientia Parasitologica, 13, 163-168.</mixed-citation></ref><ref id="scirp.57114-ref13"><label>13</label><mixed-citation publication-type="other" xlink:type="simple">Jacek, S., Jacek, Z. and Jacek, D. (2007) Seroprevalence of Toxoplasma gondii in Farm and Wild Animals from the Area of Lublin Province. Bulletin of the Veterinary Institute in Pulawy, 51, 535-540.</mixed-citation></ref><ref id="scirp.57114-ref14"><label>14</label><mixed-citation publication-type="other" xlink:type="simple">Villari, S., Vesco, G., Peterson, E., Crispo, A. and Boffolano, W. (2009) Risk Factors for Toxoplasmosis in Pigs Bred in Silicy, Southern Italy. Veterinary Parasitology, 161, 1-6. http://dx.doi.org/10.1016/j.vetpar.2009.01.019</mixed-citation></ref><ref id="scirp.57114-ref15"><label>15</label><mixed-citation publication-type="other" xlink:type="simple">Knapen, F., van Kremers, A.F., J.H. Franchimont, J.H. and Narucka, U. (1995) Prevalence of Antibodies to Toxoplasma gondii in Cattle, and Swine in Netherlands: Towards an Integrated Control of Livestock Production. Veterinary Quarterly, 17, 87-91. http://dx.doi.org/10.1080/01652176.1995.9694539</mixed-citation></ref><ref id="scirp.57114-ref16"><label>16</label><mixed-citation publication-type="other" xlink:type="simple">Chandrawadhani, P., Nurulaini, R., Zanin, C.M., Premaalatha, B., Adnan, M., Jamnah, O., Khor, S.K., Khadijah, S., Lai, S.Z., Shaik, M.A.B., Seath, T.C. and Zatil, S.A. (2008) Seroprevalence of Toxoplasma gondii Antibodies in Pigs, Goats, Cattle, Dogs and Cats in Peninsular Malaysia. Tropical Biomedicine, 25, 257-258.</mixed-citation></ref><ref id="scirp.57114-ref17"><label>17</label><mixed-citation publication-type="other" xlink:type="simple">Klun, I., Vujanic, M., Yera, H., Nikolic, A., Ivovic, V., Bobic, B., Bradonjic, S., Dupouy-Camet, J. and Djurkovic- Djakovic, O. (2011) Toxoplasma gondii Infection in Slaughter Pigs in Serbia: Seroprevalence and Demonstration of Parasites in Blood. Veterinary Research, 42, 17-22. http://dx.doi.org/10.1186/1297-9716-42-17</mixed-citation></ref><ref id="scirp.57114-ref18"><label>18</label><mixed-citation publication-type="other" xlink:type="simple">Sergio, S., Hilda, F., Clebert, J.A., Antonio, A., Roberio, M.O., Pavlo, M., Gereon, S. and Solange, M.G. (2010) Prevalence of Anti-Toxoplasma gondii and Anti-Neospora caninum Antibodies in Swine from Northeastern Brazil. Brazilian Journal of Veterinary Parasitology, 19, 80-84.  
http://dx.doi.org/10.4322/rbpv.01902002</mixed-citation></ref></ref-list></back></article>