Clinical effectiveness of autohemotherapy as an adjuvant in the control of gastrointestinal nematodes in naturally infected sheep

Luciane Holsback Camile Sanches Silva Petrônio Pinheiro Porto Emília Paiva Porto Ellen Souza Marquez About the authors

Abstract

This study aimed to evaluate the effects of autohemotherapy as an adjuvant in the control of gastrointestinal nematodes in sheep. Four experimental groups were formed: G1, 10 animals receiving autologous venous blood; G2, 10 animals receiving autologous venous blood and vermifuge containing levamisole; G3, 10 animals receiving only vermifuge containing levamisole; and G4, 10 animals as the control group receiving no treatment. We performed fecal egg count (eggs per gram, EPG) of strongyles, larval culture, hemogram, leukogram, and serum protein dosage prior to the start of treatment (D0), and on days 14 (D14) and 42 (D42). There was a significant decrease in the EPG of the groups receiving levamisole (G2 and G3) from D14 to the end of the experimental period. At the end of the evaluations, the mean EPG of G2 and G3 was significantly lower than that of G1 and G4. The most common nematode genus was Haemonchus (88%), and the least common was Trichostrongylus (1%). The Fecal Egg Count Reduction Test (FECRT) of G2 and G3 on D14 were 98.1% and 97.9%, respectively, however, in G1, the FECRT was zero on the two days when evaluation took place. G1 and G2 showed a significant increase in monocyte counts on D14 and D42. There was a significant increase in hematocrit and hemoglobin values in G2 and G3, however, a significant increase in the absolute value of red blood cells was observed only in G2. Two doses of autohemotherapy at 21-day intervals, administered alone or as an adjuvant to levamisole, is ineffective in controlling gastrointestinal nematodes in naturally infected sheep.

Keywords:
autologous whole blood; FECRT; isotherapic; levamisole; small ruminant

Resumo

Este estudo objetivou avaliar os efeitos da auto-hemoterapia como adjuvante no controle de nematódeos gastrointestinais em ovinos. Quatro grupos experimentais foram formados: G1, 10 animais que receberam sangue venoso autólogo; G2, 10 animais que receberam sangue venoso autólogo e vermífugo contendo levamisol; G3, 10 animais que receberam somente vermífugo contendo levamisol; e G4, 10 animais do grupo controle, que não receberam tratamento. Realizamos contagem de ovos nas fezes (ovos por grama, OPG) de estrongilídeos, cultivo de larvas, hemograma, leucograma e dosagem de proteína sérica antes do início do tratamento (D0) e nos dias 14 (D14) e 42 (D42). Houve uma diminuição significativa no OPG dos grupos que receberam levamisole (G2 e G3) do D14 até o final do período experimental. Ao final das avaliações, o OPG médio de G2 e G3 foi significativamente menor do que G1 e G4. O gênero de nematódeo mais comumente encontrado foi Haemonchus (88%) e o menos foi Trichostrongylus (1%). O teste de Redução na Contagem de Ovos nas Fezes (RCOF) de G2 e G3 no D14 foi 98,1% e 97,9%, respectivamente, entretanto, no G1, o RCOF foi zero nos dois dias avaliados. G1 e G2 mostraram aumento significativo na contagem de monócitos em D14 e D42. Houve um aumento significativo nos valores do hematócrito e hemoglobina em G2 e G3, entretanto, um aumento significativo no valor absoluto de hemácias foi observado somente em G2. Duas doses de auto-hemoterapia em intervalos de 21 dias, administradas isoladamente ou como adjuvante do levamisole, não é eficaz no controle de nematóides gastrintestinais em ovinos naturalmente infectados.

Palavras-chave:
sangue autólogo total; RCOF; isoterapia; levamisol; pequeno ruminante

Introduction

The state of Paraná has 588,996 head of sheep and lambs. Approximately 14.9% of sheep and lambs in Brazil are in the south, and 3.0% are in Paraná(11 IBGE. Instituto Brasileiro de Geografia e Estatística; Censo Agropecuário 2019. [cited 2021 jul 20]. Available from: https://www.ibge.gov.br/estatisticas/economicas/agricultura-e-pecuaria/9107-producaoda-pecuaria-municipal.html?=&t=resultados
https://www.ibge.gov.br/estatisticas/eco...
). There has been considerable interest in producing lambs for meat, originating from small and mediumsized creations that have a small number of sheep for maintenance. The sheep industry generates approximately R$ 96 million annually in Paraná(22 DERAL - Departamento de Economia Rural, Divisão de Conjuntura Agropecuária. Boletim semanal,03/2021-22 de janeiro de 2021. cited 2021 aug 2]. Available from: https://www.agricultura.pr.gov.br/Pagina/Conjuntura-Boletim-Semanal-032021
https://www.agricultura.pr.gov.br/Pagina...
).

Small ruminants are affected by various sanitary problems, with gastrointestinal nematodes (GIN) being the main barrier to their development. Parasitism caused by GIN species is primarily due to the family Trichostrongylidae(33 Echevarria F, Borba MFS, Pinheiro AC, Waller PJ, Hansen JW. The prevalence of anthelmintic resistance in nematode parasites of the sheep in Southern Latin America: Brazil. Veterinary Parasitology. 1996;62:199-206. English. Available in: https://doi.org/10.1016/0304-4017(95)00906-X
https://doi.org/10.1016/0304-4017(95)009...
,44 Arosemena NAE, Bevilaqua CML, Melo ACF, Girão MD. Seasonal variations of gastrointestinal nematodes in sheep and goats from semi-arid areas in Brazil. Revue de Médecine Vétérinaire. 1999;11: 873-876. English. Available in: https://agris.fao.org/agris-search/search.do?recordID=FR2000000679
https://agris.fao.org/agris-search/searc...
), and results in losses to the national economy(55 Rashid M, Rashid MI, Akbar H, Ahmad L, Hassan MA, Ashraf K, Saeed K, Gharbi MA systematic reviewon modelling approaches for economic losses studies caused by parasites and their associated diseases in cattle. Parasitology. 2019;146:129-141. English. Available in: https://doi.org/10.1017/S0031182018001282
https://doi.org/10.1017/S003118201800128...
).

There are several proposals for alternatives to control GIN, such as the selection of resistant breeds(66 Estrada-Reyes ZM, Tsukahara Y, Amadeu RR, Goetsch A., Gipson TA, Sahlu T, Puchala R., Wang Z, Hart SP, Mateescu RG. Signatures of selection for resistance to Haemonchus contortus in sheep and goats. BMC Genomics, 2019;20:735. English. Available in: http://dx.doi.org/10.1186/s12864-019-6150-y
http://dx.doi.org/10.1186/s12864-019-615...
), phytotherapy and biological therapy(77 Dos Anjos C, Silva BT, Fertonani LHS, Silva CS, Matsumoto LS, da Silva RMG, Mello-Peixoto ECT.Pomegranate extracts on larval inhibition of Haemonchus spp and Cooperia spp obtained from cattle. Bioscience Journal. 2006;32(5):1277-1285. English. Available in: https://doi.org/10.14393/BJv32n5a2016-30217
https://doi.org/10.14393/BJv32n5a2016-30...
,88 Holsback L, Porto PP, Marquez ES, Porto EP. Phyto-biotherapy to control gastrointestinal nematodes ofsheep. Semina: Ciências Agrárias. 2013.34(6):3841-3850. English. Available in: http://www.redalyc.org/articulo.oa?id=445744138013
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,99 Castro LLD, Sprenger LK, Madrid IM, Oliveira FC, Oliveira PA, Castro LM, Berne MEA, Leite FPL. Efeito in vitro e in vivo de extratos de Eugenia uniflora em nematódeos gastrintestinais de ovinos. Ciência Animal Brasileira. 2019;20:1-12. Portuguese. Available in: https://doi.org/10.1590/1089-6891v20e-49037
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), nematophagous fungi(1010 Vilela VLR, Feitosa TF, Braga FR, Santos AD, Bezerra RA, Silva GLL, Athayde ACR, Araujo JV. Use of Duddingtonia flagrans in the control of gastrointestinal nematodes of feedlot goats. Semina: Ciências Agrárias. 2020;41:915. English. Available in: https://doi.org/10.5433/1679-0359.2020v41n3p915
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,1111 Holsback L, Lima HE, Porto PP, Marquez ES, Zacarias FGS, Porto EP. Biological control of nematodes by nematode trapping fungi Duddingtonia flagrans in naturally infected sheep in Southern Brazil. German Journal of Veterinary Research. 2021; eISSN:2703-1322:17-26. English. Available in: https://doi.org/10.51585/gjvr.2021.2.0010
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), vaccines against nematodes(1212 Meeusen E. Rational design of nematode vaccines; natural antigens. International Journal for Parasitology. 1996;26(8-9):813-818. English. Available in: https://doi.org/10.1016/S0020-7519(96)80046-4
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), and tannin-containing fodder(1313 Butter NL, Dawson JM, Wakelin D, Buttery PJ. Effect of dietary tannin and protein concentration onnematode infection (Trichostrongylus colubriformis) in lambs. The Journal Agricultural Science. 2000;134: 89-99. English. Available in: https://doi.org/10.1017/S0021859699007315
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).

Isotherapy was initially used in the 1930s by François Lamson, a French homeopath physician, for the treatment of intestinal parasitoses. This researcher used large dilutions of blood serum to treat amoebic dysentery, oxyuriasis, ascariasis, and teniasis. Autohemotherapy, which consists of removing blood from the animal and injecting it intramuscularly, is considered as an isotherapic treatment by certain researchers. It is suggested that this method stimulates the endothelial reticle system and quadruples the number of macrophages in the organism. In 1924, it was confirmed that this treatment was valuable in numerous dermatoses, particularly in pruritic and furunculosis affections. This is the synthesis of the doctoral thesis conclusion entitled "A auto-hemoterapia nas dermatoses", written by Alberto Carlos David at the University of Porto in 1924. This thesis proves that the technique has been used since the first half of the nineteenth century and presents cases cured through its use(1414 David AC. A auto-hemoterapia nas dermatoses (Tese online). Portugal: Faculdade de Medicina doPorto; 1924. English. Available in: http://repositorio-aberto.up.pt/bitstream/10216/17607/3/210_2_FMP_TD_I_01_P.pdf
http://repositorio-aberto.up.pt/bitstrea...
).

In recent decades, autohemotherapy has been little known and practiced in the veterinary field by professionals and animal owners. There are few reports on this therapy in humans and animals in the literature. At the start of the last century, autohemotherapy has been indicated as a treatment for various dermatological conditions(1515 Shakman, S.H. Autohemotherapy Reference Manual. Definitive Guide and Historical Review fromBloodletting to Stemcells. Institute of Science www.InstituteOfScience.com; United States of America; 2010. p. 177-179. English.
www.InstituteOfScience.com;...
). Subsequently, autohemotherapy has become a standard treatment for numerous dermatologic disorders, including urticaria and eczema, in Europe, North America, and Japan(1616 Gottheil WS, Satenstein DL: The autoserum treatment in dermatology. JAMA. 1914, English. 63:11901194. 10.1001/jama.1914.02570140044014 English. Available in: https://doi.org/10.1001/jama.1914.02570140044014
https://doi.org/10.1001/jama.1914.025701...
,1717 Mori O, Hashimoto T. Autologous whole blood intramuscular injection as a cure for chronic urticaria: report of a patient in whom intradermal injection of autologous serum continued to cause a weal-andflare response. British Journal of Dermatology. 1999;140(6):1192-1193. English. Available in: https://pubmed.ncbi.nlm.nih.gov/10354108/
https://pubmed.ncbi.nlm.nih.gov/10354108...
,1818 Staubach P, Onnen K, Vonend A, Metz M, Siebenhaar F, Tschentscher I, Opper B, Magerl M, Lüdtke R,Kromminga A, Maurer M. Autologous whole blood injections to patients with chronic urticaria and a positive autologous serum skin test: A placebo-controlled trial. Dermatology. 2006; 212:150-9. English. Available in: https://doi.org/10.1159/000090656
https://doi.org/10.1159/000090656...
). Currently, autohemotherapy has been used to treat dermatologic and other diseases, predominantly by alternative medical providers, particularly in Europe(1919 Schäfer T. Epidemiology of complementary alternative medicine for asthma and allergy in Europe andGermany. Annals of Allergy, Asthma & Immunology. 2004;93:S5-S10. English. Available in: https://doi.org/10.1016/S1081-1206(10)61481-0
https://doi.org/10.1016/S1081-1206(10)61...
) and Latin America(2020 Cortés-Ros OM, Oliveira AS, Figueiredo FNM, Batista MRC, García LP, Olivares AMO. Tratamiento de laPsoriasis vulgar com Autohemoterapia menor. Hospital “Celia Sánches Manduley”. 2016-2018. Multimed Revista Médica. 2019;23(4):758-774. Spanish. Available in: http://scielo.sld.cu/scielo.php? script=sci_arttext&pid=S1028-48182019000400758&lng=es&nrm=iso&tlng=es
http://scielo.sld.cu/scielo.php? script=...
).

Autohemotherapy has been tested as a thrombocytopenia treatment in dogs(2121 dos Anjos DA, Assis MF, Martins RR, Tomacheuski RM, Taffarel MO. Evaluation of hemoacumpuncture in the treatment of dogs with trhombocitopenia. Journal of Veterinary Science and Public Health. 2020;7(2): 112-118. English. Available in: https://doi.org/10.4025/revcivet.v7i2.42405
https://doi.org/10.4025/revcivet.v7i2.42...
), and as an adjuvant in the treatment of mastocytoma(2222 Quessada AM, Carvalho CJS, Oliveira RN, Costa PM, Barbosa SRV, Sousa e Silva SMM. Autohemoterapia como adjuvante no tratamento de mastocitoma em cão: relato de caso. Revista Brasileira de Ciências Veterinárias. 2010;17(3/4):108-110. Portuguese. Available in: http://dx.doi.org/10.4322/rbcv.2014.153
http://dx.doi.org/10.4322/rbcv.2014.153...
), canine parvovirus(2323 Borges OMM, Souza AP, Mendes RS, Araújo KN, Torres LM, Dantas AKFP. Clinical Effectiveness of Autohemotherapy as an Adjuvant Treatment of Canine Parvovirus. Acta Scientiae Veterinariae. 2014;42:1224. English. Available in: http://www.ufrgs.br/actavet/42/PUB%201224.pdf
http://www.ufrgs.br/actavet/42/PUB%20122...
), and oral papillomatosis(2424 Bambo O, Cardoso JMM, Dimande A, Mapatse M, Santos IFC. Auto-hemoterapia no tratamento dapapilomatose oral canina - Relato de caso. MedVep Dermato - Revista de Educação Continuada em Dermatologia e Alergologia Veterinária. 2012;2(2):39-43. Portuguese. Available in: https://pesquisa.bvsalud.org/portal/resource/pt/vti-11395
https://pesquisa.bvsalud.org/portal/reso...
,2525 Borges OMM, Araújo CLM, Ramalho GC, Silva RMN, Tanikawa A, Souza AP. Effects of Autohemotherapy on Hematologic Parameters and Morphology of Canine Oral Papilomatosis. Acta Scientiae Veterinariae. 2017;45(1):211. English. Available in: http://www.ufrgs.br/actavet/45-suple-1/CR_211.pdf
http://www.ufrgs.br/actavet/45-suple-1/C...
). In production animals, the majority of reports are related to papillomatosis treatment in bovines and caprines(2626 Dhule SJ. Auto-hemotherapy for Management of Caprine Papillomatosis. Intas Polivet. 2013; 14(II):425-426. English. Available in: https://www.indianjournals.com/ijor.aspx?target=ijor:ipo&volume=14&issue=2&article=091
https://www.indianjournals.com/ijor.aspx...
,2727 Ranjan R, Ghumman SPS, Bhatt GR, Singh RS. Efficacy of Autogenous Vaccine and Auto-hemotherapyin Bovine Cutaneous Papilomatosis. Intas Polivet. 2013;14(II):411-414. English. Available in: https://www.indianjournals.com/ijor.aspx?target=ijor:ipo&volume=14&issue=2&article=086
https://www.indianjournals.com/ijor.aspx...
,2828 Kavithaa NV, Rajkumar NV, Jiji RS. Papillomatosis in Jersey cows and its different medical treatment.International Journal of Science, Environment. 2014;3(2):692-694. English. Available in: https://www.ijset.net/journal/297.pdf
https://www.ijset.net/journal/297.pdf...
,2929 Nehru PA, Sunandhadevi S, Rama T, Muniyappan N. Efficacy of Auto-Hemotherapy in Bovine TeatPapillomatosis: A Case Report. Advances in Animal and Veterinary Sciences. 2017;5(8):350. English. Available in: http://dx.doi.org/10.17582/journal.aavs/2017/5.8.350.351
http://dx.doi.org/10.17582/journal.aavs/...
). The few reports regarding the use of autohemotherapy in humans have been related to the prevention of postoperative complications(3030 Mettenleiter MW. Autohemotransfusion in preventing postoperative lung complications. AmericanJournal of Surgery. 1936;XXXII(2): 321-323. English. Available in: https://doi.org/10.1016/S0002-9610(36)90155-1
https://doi.org/10.1016/S0002-9610(36)90...
), and as adjuvants in the treatment of herpetic infections(3131 Olwin JH, Ratajcazak HV, House RV. Successful Treatment of Herpetic Infections by Autohemotherapy.The Journal of Alternative and Complementary Medicine. 1997;3(2):155-158. English. Available in: https://doi.org/10.1089/acm.1997.3.155
https://doi.org/10.1089/acm.1997.3.155...
) and allergic diseases(3232 Malka S, Hullet AC, Borges MS, Lozano AP. International Perspectives on Controversial Practices inAllergic Diseases. The South American Experience. Clinical Reviews in Allergy and Immunology. 1996;14:271-287. English. Available in: https://doi.org/10.1007/BF02802219
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,3333 Ziment I. Unconventional Therapy in Asthma. Clinical Reviews in Allergy and Immunology. 1996;14:289-320. English. Available in: https://doi.org/10.1007/BF02802220
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).

There are several reports on the clinical efficacy of autohemotherapy, however, it has been the target of considerable controversies, leading health professionals not to recognize it. It is an alternative therapy, without recognized scientific proof, and therefore, little research has been conducted in this area.

Therefore, this study aimed to evaluate the effects of autohemotherapy as an adjuvant in the control of GIN in sheep, in addition to evaluating the hematological profile of animals during treatment.

Material and methods

This project was approved on December 11, 2013 by the “Comitê de Ética do Uso de Animais” (CEUA) from the Universidade Estadual do Norte do Paraná, Brazil (reference number Secapee 1017/13).

Location, groups, experimental periods, and fecal examination

The study was conducted on a rural property in the municipality of Ribeirão Claro (23°11’38″’’S 49°45’28’’″ O), north of Paraná State, between March and July, 2014. Forty adult ewes with EPG values 150 were selected, which corresponds to the minimum established EPG for resistance and anti-helminth efficiency tests in sheep(3434 Coles GC, Jackson F, Pomroy WE, Prichard RK, von Samson-Himmelstjerna G, Silvestre A, Taylor MA,Vercruysse J. The detection of anthelmintic resistance in nematodes of veterinary importance. Veterinary Parasitology. 2006;136:167-185. English. Available in: https://doi.org/10.1016/j.vetpar.2005.11.019
https://doi.org/10.1016/j.vetpar.2005.11...
).

Four experimental groups were formed after randomization based on the EPG values; G1 group, comprised of 10 healthy, non-pregnant (2-3 years old) ewes, mean EPG 3310, received autologous venous blood; G2 group, comprised of 10 healthy, non-pregnant (2-3 years old) ewes, mean EPG 3240, received autologous venous blood and commercial vermifuge containing oral solution levamisole hydrochloride 5% (5 mg/Kg); G3 group, comprised of 10 healthy, non-pregnant (2-3 years old) ewes, mean EPG 3185, received only commercial vermifuge containing oral solution levamisole hydrochloride 5% (5 mg/Kg); and G4 group, control group, no treatment, comprised of 10 healthy, nonpregnant (2-3 years old) ewes, mean EPG 3580, received 10 mL of oral saline solution. All groups were treated simultaneously. The treatments (autohemotherapy and levamisole) were performed on day 0 (D0) and 21 days later. D0 was defined as the first day of treatment followed by two experimental periods at day 14 (D14) and day 42 (D42) (totaling three experimental periods). During this period, stool harvests were carried out for EPG counting using the technique described by Gordon and Whitlock(3535 Gordon HMCL, Whitlock HV. A new technique for counting nematode eggs in sheep faeces. Journal ofthe Council for Scientific and Industrial Research. 1939;12(1): 50-52. English. Available in: https://publications.csiro.au/rpr/download?pid=procite:21259a33-8a8e-4add-9315-f8338091a3e6&dsid=DS1
https://publications.csiro.au/rpr/downlo...
) at a dilution of 1:25.

To identify the genera of nematodes, larval culture (coproculture) was performed using the technique described by Roberts and Sullivan(3636 Roberts FHS, Sullivan PJ. Methods for egg counts and larval cultures for Strongyles infesting thegastro-intestinal tract of cattle. Australian Journal of Agricultural Research. 1950;1(1): 99-102. English. Available in: https://doi.org/10.1071/AR9500099
https://doi.org/10.1071/AR9500099...
), with the L3 larvae recovered and identified using the criteria presented by Keith(3737 Keith RK. The differentiation of the infective larval of some common nematode parasites of cattle.Australian Journal of Zoology. 1953;1(2):223-230. English. Available in: https://doi.org/10.1071/ZO9530223
https://doi.org/10.1071/ZO9530223...
). The percentage of fecal egg count reduction of nematodes of each identified genus in the culture of the larvae was calculated from these values using the Fecal Egg Count Reduction Test (FECRT) formula(3838 Wood IB, Amaral NK, Bairden K, Duncan JL, Kassai T, Malone JB, Pankavich JA, Slocombe O, Taylor SM,Vercruysse J. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine, ovine, caprine). Veterinary Parasitology. 1995;58:181-213. English. Available in: http://dx.doi.org/10.1016/0304-4017(95)00806-2
http://dx.doi.org/10.1016/0304-4017(95)0...
):

 FECRT =1 - (EPG mean of treated on day x / EPG mean of the control on day x) ×100.

In the present study, FECRT was not used to estimate the resistance of the helminths to treatment, but to verify the percentage of reduction in the parasite load of animals and to identify the GIN species showing a more substantial decrease due to treatments.

Hematologic and Biochemistry analysis

Whole blood samples were collected from the jugular vein using an 18-gauge disposable needle. The first 4 mL of blood was dripped into a tube containing K3EDTA for hematology analysis, and the remaining 6 mL was transferred to a tube with a clot activator for serum chemistry analysis. All analyses were completed within 8 h of blood collection. Blood samples were centrifuged at 2000 × g for 10 min to extract the serum, which was frozen at-20ºC for further analysis.

Blood samples were analyzed using a BC-2800Vet-Mindray Auto Hematology (Mindray Headquartes, Shenzhen, China) system and multispecies software. The following parameters were measured: red blood cell (RBC) count, hemoglobin (Hb), hematocrit (HCT), white blood cell (WBC) count, and percentage and number of neutrophils, lymphocytes, monocytes, and eosinophils. Blood smear slides were prepared in the field immediately after collection prior to transfer to the tubes. The slides were manually stained using Wright’s stain, and the WBC differential count was determined by counting 100 WBCs.

Total plasma protein (TPP) and albumin values were determined using the biuret and bromocresol green methods, respectively. The analyses were performed using the PKL PPC Automatic Chemistry Analyzer (Paramedical, Salerno, Italy) instrument. The globulin values were calculated by subtracting the TPP value from the albumin value.

Autohemotherapy:

Ten milliliters of blood was collected by jugular vein puncture of each animal in groups G1 and G2. After trichotomy and disinfection with 70% ethanol solution, the blood was immediately inoculated (autologous blood application) intramuscularly in the gluteal region. Autohemotherapy was performed on D0 and D21.

Analyzes of data and statistical tests:

Statistical tests were performed using GraphPad Prism 5.0 (GraphPad Software Inc., CA, USA). The Wilcoxon test was used to compare the means of the EPGs of the treated and control groups. The comparison between the means of EPGs under the genera identified after the larvae growth was performed using the Mann-Whitney U test. Differences between the mean values of cell counts obtained by hemograms, leukograms, and plasma protein dosages were analyzed using a paired t-test. Statistical significance was set at p0.05.

Results and discussion

The mean EPG of both levamisole-treated groups treated (G2 and G3) was significantly lower (p<0.005) than that of the control group (G4) and the group treated only with autohemotherapy (G1). Although not significantly different, the mean EPGs of the animals in the autohemotherapy group (G1) were 40% (D14) to 60% (D42) higher than that of the control group (Table 1).

Table 1
Means (± Standard Error of the Mean - SEM) of the EPGs of the animals of the G1 groups (autohemotherapy), G2 (autohemotherapy + levamisole), G3 (levamisole), and G4 (control, no treatment) at the start of treatment (D0), and on days 14 and 42

Animals in the auto-hemotherapy (G1) and control (G4) groups had significant increases (p=0.0078 and 0.027, respectively) in the EPGs 14 days after treatment. These values decreased four weeks later, and returned to the same levels as those at the beginning of the experiment. The two levamisole-treated groups (G2 and G3) showed significant decreases (p=0.0039 and 0.0020, respectively) in their EPGs by the end of the evaluations (Figure 1).

Figure 1
Evolution of means values of the EPGs of the animals in groups G1 (autohemotherapy), G2 (autohemotherapy + levamisole), G3 (levamisole), and G4 (control, no treatment) at the start of treatment (DO), and on days 14 and 42.

Our results demonstrated that, in the experimental model used, autohemotherapy did not contribute to a decrease in gastrointestinal nematode infection levels in infected sheep. The animals were administered two doses of autologous blood via intramuscular (IM) injection at 21-day intervals, however, other researchers who successfully used this treatment administered weekly applications for a longer period than that used in our study(2222 Quessada AM, Carvalho CJS, Oliveira RN, Costa PM, Barbosa SRV, Sousa e Silva SMM. Autohemoterapia como adjuvante no tratamento de mastocitoma em cão: relato de caso. Revista Brasileira de Ciências Veterinárias. 2010;17(3/4):108-110. Portuguese. Available in: http://dx.doi.org/10.4322/rbcv.2014.153
http://dx.doi.org/10.4322/rbcv.2014.153...
,2525 Borges OMM, Araújo CLM, Ramalho GC, Silva RMN, Tanikawa A, Souza AP. Effects of Autohemotherapy on Hematologic Parameters and Morphology of Canine Oral Papilomatosis. Acta Scientiae Veterinariae. 2017;45(1):211. English. Available in: http://www.ufrgs.br/actavet/45-suple-1/CR_211.pdf
http://www.ufrgs.br/actavet/45-suple-1/C...
,2929 Nehru PA, Sunandhadevi S, Rama T, Muniyappan N. Efficacy of Auto-Hemotherapy in Bovine TeatPapillomatosis: A Case Report. Advances in Animal and Veterinary Sciences. 2017;5(8):350. English. Available in: http://dx.doi.org/10.17582/journal.aavs/2017/5.8.350.351
http://dx.doi.org/10.17582/journal.aavs/...
,3939 Junior FC, Bambo OR, Cardoso JMM, Laisse CJM, Gallina MF, Zadra VF, Rahal SC, Santos IFC. Combinação de auto-hemoterapia e sulfato de vincristina no tratamento de tumor venéreo transmissível em cadelas em Moçambique. Acta Scientiae Veterinariae. 2021;49(1): 659. Portuguese. Available in: https://www.seer.ufrgs.br/ActaScientiaeVeterinariae/article/viewFile/111725/pdf
https://www.seer.ufrgs.br/ActaScientiaeV...
). The present study is the first to evaluate the effect of autohemotherapy on the control of GINs in sheep.

The psychobiological effect of placebos that can cause an improvement or worsening of clinical symptoms in humans(4040 Stewart-Williams S, Podd, J. The placebo effect: dissolving the expectancy versus conditioning debate. Psychological Bulletin. 2004;130(2):324-340. English. Available in: https://doi.org/10.1037/0033-2909.130.2.324
https://doi.org/10.1037/0033-2909.130.2....
) is not observed in animals, because they have no discernment of the treatments they are submitted to. If any negative influence of the treatment occurred, it could be attributed to the stress of the physical restraint and the application of autologous blood, because in humans it causes pain(4141 Abdallah MA, Elzamk M, Sallam MA. Autologous serum injection versus autologous blood in thetreatment of chronic autoreactive urticaria. Journal of the Egyptian Womenʼs Dermatologic Society. 201;9:26-31. English. Available in: https://doi.org/10.1097/01.EWX.0000407617.33366.0f
https://doi.org/10.1097/01.EWX.000040761...
). The animals in the control group received saline solution orally, therefore, they were subjected to the same physical restraint as those in the other groups. Although the injection of autologous blood was likely painful, its physiological consequences have not yet been measured in the medium or long term. For this reason, we cannot explain the difference between the mean EPGs of the control group animal and those treated with autohemotherapy. For this investigation, animals in the control group received IM injections of saline solution to imitate the pain caused by autohemotherapy. FECRT was performed to compare the reduction rates of egg counts in the feces of animals in the three treated groups, as well as to compare the reduction rates of trichostrongylides genera identified in the larval culture. We observed a FECRT of 98.1% and 97.9% in animals from G2 and G3, respectively, on D14, and FECRT of 91.4% and 84.8% in animals from G2 and G3, respectively, on D42. In the group treated only with autohemotherapy (G1), the FECRT was zero on the two days evaluated (Table 2).

Table 2
Fecal Egg Count Reduction Test of Haemonchus, Trichostrongylus and Strongyloides (FECRT %) of the animals in G1 (autohemotherapy), G2 (autohemotherapy + levamisole), and G3 (levamisole) on days 14 and 42

The most frequently observed genus in larval cultures was Haemonchus (88%), followed by Cooperia (2%) and Trichostrongylus (1%). The genus Oesophagostomum was not found on any of the experimental days. The genus Strongyloides accounted for 9% of the nematodes identified by egg morphology in the Gordon and Whitlock examination. There were no Cooperia larvae in the control group on D14 and D42; therefore, it was not possible to evaluate the FECRT of Cooperia in the treated groups. Although there was no reduction in egg counts of all nematodes in the animals treated with autohemotherapy (G1), genera Trichostrongylus, and Strongyloides quantified on D0 by coproculture were absent on D42 (Table 3).

Table 3
Fecal Egg Count (EPG) of Haemonchus, Trichostrongylus, Cooperia and Strongyloides of the animals in G1 groups (autohemotherapy), G2 (autohemotherapy + levamisole), G3 (levamisole), and G4 (control, no treatment) at the start of treatment (D0), and on days 14 and 42

The animals in the groups treated with autohemotherapy did not show significant changes in the leukogram, except for an increase in monocytes on D14 (p = 0.0085) and D42 (p = 0.0011) after treatment. Although the same was observed in the other groups, there was a 400% increase in monocytes in G1, and 97.5, 217, and 256% in G2, G3, and G4, respectively, at the end of the experimental period (D42) (data not shown). Pavão et al.(4747 Symoens J, Schuermans Y. Levamisole. Clinics in Rheumatic Diseases. 1979;5(2): 603-629. English.Available in: https://doi.org/10.1016/S0307-742X(21)00086-2
https://doi.org/10.1016/S0307-742X(21)00...
) also found no changes in neutrophil and lymphocyte counts, however, a slight increase (1-4%) was observed in monocytes of mice infected with Trypanosoma cruzi and subjected to autohemotherapy.

The acquired immune response against gastrointestinal nematode infections in sheep has been associated with the activity of Th2CD4+ lymphocytes, eosinophilia, and an increased number of inflammatory cells in the mucosa, such as eosinophils, mast cells, and leukocytes(4242 Amarante AFT, Amarante MRV. Breeding sheep for resistance to nematode infections. Journal ofAnimal and Veterinary Advances. 2003;2:147-161. English. Available in: https://medwelljournals.com/abstract/?doi=javaa.2003.147.161
https://medwelljournals.com/abstract/?do...
). Neutrophils, eosinophils, and antigen-presenting cells also participate in innate immunity against Strongyloides stercoralis larvae. Moreover, eosinophils have been shown to have the ability to chemotaxis to the parasite microenvironment, kill the parasite, and then present antigens to T cells to induce adaptive immunity to infection(4343 Galioto AM, Hess JA, Nolan TJ, Schad GA, Lee JJ, Abraham D: Role of eosinophils and neutrophils ininnate and adaptive protective immunity to larval Strongyloides stercoralis in mice. Infection and Immunity. 2006;74:5730-5738. English. Available in: https://doi.org/10.1128/IAI.01958-05
https://doi.org/10.1128/IAI.01958-05...
,4444 Stein LH, Redding KM, Lee JJ, Nolan TJ, Schad GA, Lok JB, Abraham D: Eosinophils utilize multiplechemokine receptors for chemotaxis to the parasitic nematode Strongyloides stercoralis. Journal of Innate Immunity. 2009;1:618-630. English. Available in: https://doi.org/10.1159/000233235
https://doi.org/10.1159/000233235...
,4545 Padigel UM, Hess JA, Lee JJ, Lok JB, Nolan TJ, Schad GA, Abraham D: Eosinophils act as antigenpresenting cells to induce immunity to Strongyloides stercoralis in mice. The Journal of Infectious Diseases. 2007;196:1844-1851. English. Available in: https://doi.org/10.1086/522968
https://doi.org/10.1086/522968...
).

Levamisole acts on the immune system in a similar way to thymopoietin, a hormone produced in the thymus, stimulating the action of T cells, the response to antigens, interferon production, increasing the phagocytic activity of macrophages and neutrophils, and stimulating cell-mediated, lymphokine production, and the function of suppressor cells(4747 Symoens J, Schuermans Y. Levamisole. Clinics in Rheumatic Diseases. 1979;5(2): 603-629. English.Available in: https://doi.org/10.1016/S0307-742X(21)00086-2
https://doi.org/10.1016/S0307-742X(21)00...
). It is related to immunomodulatory activity in immunocompromised individuals and increased efficacy in vaccinations(4848 Brunner CJ, Muscoplat CC. Immunomodulatory effects of levamisole. Journal of the American Veterinary Medical Association. 1980;176:1159-1162. English. Available in: https://pubmed.ncbi.nlm.nih.gov/7216893/
https://pubmed.ncbi.nlm.nih.gov/7216893/...
,4949 Mojžišova J, Hromada R, Pauli S, Ondrašovič M, Bajova V. Immune response and immunomodulatoryeffect of levamisol in immunosuppressed dogs vaccinated against parvovirosis. Bulletin of the Veterinary Institute in Pulawy. 2004;48:93-97. English. Available in: https://jvetres.piwet.pulawy.pl/files/archive/20042/20042093098.pdf
https://jvetres.piwet.pulawy.pl/files/ar...
). Qureshi et al.(5050 Qureshi Z.I., Lodhi L.A., Samad H.A., Naz N.A. & Nawaz M. Haematological profile following immunomodulation during late gestation in buffaloes (Bubalis bubalus). Pakistan Veterinary Journal. 2001;21:148-151. English. Available in: http://pvj.com.pk/pdf-files/21_3/148-151.pdf
http://pvj.com.pk/pdf-files/21_3/148-151...
) and Mojzisova et al.(4949 Mojžišova J, Hromada R, Pauli S, Ondrašovič M, Bajova V. Immune response and immunomodulatoryeffect of levamisol in immunosuppressed dogs vaccinated against parvovirosis. Bulletin of the Veterinary Institute in Pulawy. 2004;48:93-97. English. Available in: https://jvetres.piwet.pulawy.pl/files/archive/20042/20042093098.pdf
https://jvetres.piwet.pulawy.pl/files/ar...
) demonstrated increased activity and proliferation of lymphocytes in buffaloes and dogs treated with levamisole. However, in our study, we did not observe any changes in neutrophil and lymphocyte counts in levamisole-treated animals. Holsback et al.(88 Holsback L, Porto PP, Marquez ES, Porto EP. Phyto-biotherapy to control gastrointestinal nematodes ofsheep. Semina: Ciências Agrárias. 2013.34(6):3841-3850. English. Available in: http://www.redalyc.org/articulo.oa?id=445744138013
http://www.redalyc.org/articulo.oa?id=44...
) reported a significant increase in lymphocyte counts of sheep seven days after levamisole treatment at the same dose used in this study, and attributed this increase to the immunomodulatory effect of levamisole.

There was a significant increase in HCT and Hb values only in levamisole-treated animals (Table 4). However, a significant increase (p = 0.012) in the absolute value of RBCs was observed only in G2 animals. Holsback et al.(88 Holsback L, Porto PP, Marquez ES, Porto EP. Phyto-biotherapy to control gastrointestinal nematodes ofsheep. Semina: Ciências Agrárias. 2013.34(6):3841-3850. English. Available in: http://www.redalyc.org/articulo.oa?id=445744138013
http://www.redalyc.org/articulo.oa?id=44...
) reported a return to normal RBC, Hb, HCT, and TPP values in levamisole-treated sheep. All ten ewes in G1 had varying degrees of anemia at the start of the experiment(5151 Kramer JW. Normal haematology of cattle, sheep, and goat. In: Feldman BF, Zinkl JG, Jain NC (Eds). Schalm´s Veterinary Haematology 5.ed. Philadelphia: Lippincott Williams & Wilkins. 2000. p.1075-1084.), however, by the end of the experiment, only four ewes had counts below 8 million RBCs/µL of blood (data not shown), although the mean value was below the minimum normal value for the species (Table 4). Bambo et al.(2424 Bambo O, Cardoso JMM, Dimande A, Mapatse M, Santos IFC. Auto-hemoterapia no tratamento dapapilomatose oral canina - Relato de caso. MedVep Dermato - Revista de Educação Continuada em Dermatologia e Alergologia Veterinária. 2012;2(2):39-43. Portuguese. Available in: https://pesquisa.bvsalud.org/portal/resource/pt/vti-11395
https://pesquisa.bvsalud.org/portal/reso...
) and Faria et al.(5252 Faria BP, Rodrigues PR, Calazans RA, Costa PC. Auto-hemoterapia em cães. Enciclopédia Biosfera.2014;10(19):184-195, 2014. Portuguese. Available in: http://www.conhecer.org.br/enciclop/2014b/AGRARIAS/autohemoterapia.pdf
http://www.conhecer.org.br/enciclop/2014...
) evaluated the effect of autohemotherapy in dogs and observed no significant increase in RBC counts, HCT, and Hb after multiple treatment sessions.

Table 4
Medium ± standard error (σX) of red blood cells (RBC), hemoglobin (Hb), hematocrit (HCT), leukocytes, and total plasma protein (TPP) of the animals in G1 groups (autohemotherapy), G2 (autohemotherapy + levamisole), G3 (levamisole), and G4 (control, no treatment) at the start of treatment (D0), and on days 14 and 42

We observed varying and unrelated hemogram, leucogram, TPP, EPG, and FECRT values, therefore, we cannot infer any benefits or drawbacks of autohemotherapy on GIN control in sheep. We believe that the animals of the two non-treated groups (G1 and G4) were favored with the lower loads of contamination in the pastures probably generated by the treated animals with levamisole, since all animals were housed in the same paddocks during the 42-day duration of this study.

Conclusions

In this study, it was concluded that treatment with two doses of autohemotherapy at 21-day intervals, administered alone or as an adjuvant to levamisole, was not able to control parasitic infections caused by GIN in naturally infected sheep, and did not influence myeloid and lymphoid lineage cells and blood proteins, and therefore produced no beneficial or harmful effects on sheep.

Despite reports of clinical improvement in treated animals, this study did not identify any improvement in animals during the 42-day evaluation period. A longer study period and injections at shorter intervals may clarify the benefits of autohemotherapy in the control of worms in sheep.

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Publication Dates

  • Publication in this collection
    14 Jan 2022
  • Date of issue
    2021

History

  • Received
    12 Aug 2021
  • Accepted
    28 Nov 2021
  • Published
    16 Nov 2021
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