Evaluation of fecal smear methods for research on Cryptosporidium spp. oocysts in the feces of dairy calves
DOI:
https://doi.org/10.5433/1679-0359.2022v43n2p585Keywords:
Nested PCR, Oocysts, Protozoan, Ziehl-Neeslsen.Abstract
The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified.References
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Cai, M., Guo, Y., Pan, B., Li, N., Wang, X., Tang, C., Xiao L. (2017). Longitudinal monitoring of Cryptosporidium species in pre-weaned dairy calves on five farms in Shanghai, China. Veterinary Parasitology, 241(1), 14-19. doi: 10.1016/j.vetpar.2017.05.005
Chagas, A. C. S. (2015). Diarreia em bezerros leiteiros lactantes: a doença e o manejo em diferentes unidades da EMBRAPA. São Carlos: EMBRAPA Pecuária Sudeste-Documentos (INFOTECA-E). Recuperado de https://www.infoteca.cnptia.embrapa.br/infoteca/bitstream/doc/1035039/1/Documentos120.pdf
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Fayer, R., Santin, M., & Trout, J. M. (2007). Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Veterinary Parasitology, 145(3-4), 260-266. doi: 10.1016/j.vetpar.2006.12.009
Fereig, R. M., Abdelbaky, H. H., Ihara, F., & Nishikawa, Y. (2018). Development and evaluation of the first immunochromatographic test that can detect specific antibodies against Cryptosporidium parvum. Acta Tropica, 185(1), 349-356. doi: 10.1016/j.actatropica.2018.06.019
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Liao, C., Wang, T., Koehler, A. V., Fan, Y., Hu, M., & Gasser, R. B. (2018). Molecular investigation of Cryptosporidium in farmed chickens in Hubei Province, China, identifies ‘zoonotic’ subtypes of C. meleagridis. Parasites & Vectors, 11(1), 484. doi: 10.1186/s13071-018-3056-5
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Matos, L. V. S. de, Silveira, L. da Neto, Oliveira, B. C. M., Makatu, M. Y., Pierucci, J. C., Viol, M. A., Bresciani, K. D. S. (2019). Molecular characterization of Cryptosporidium in calves from rural settlements in the Northwest region of the state of São Paulo, Brazil. Semina: Ciências Agrárias, 40(1), 491-496. doi: 10.5433/1679-0359.2019v40n1p491
Miambo, R. D., Laitela, B., Malatji, M. P., Santana Afonso, S. M. de, Júnior, A. P., Lindh, J., & Mukaratirwa, S. (2019). Prevalence of Giardia and Cryptosporidium in young livestock and dogs in Magude District of Maputo Province, Mozambique. Onderstepoort Journal of Veterinary Research, 86(1), e1-e6. doi: 10.4102/ojvr. v86i1.1709
Ortolani, E. L. (2000). Standardization of the modified Ziehl-Neelsen technique to stain oocysts of Cryptosporidium sp. Brazilian journal of Veterinary Parasitology, 9(1), 29-31.
Osaki, S. C., Soccol, V. T., Costa, A. O., Oliveira-Silva, M. B., Pereira, J. T., & Procopio, A. E. (2013). Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil. Journal of the Brazilian Society of Tropical Medicine, 46(3), 270-276. doi: 10.1590/0037-8682-0053-2013
Ouakli, N., Belkhiri, A., Lucio, A. de, Köster, P. C., Djoudi, M., Dadda, A., Carmena, D. (2018). Cryptosporidium-associated diarrhoea in neonatal calves in Algeria. Veterinary Parasitology Regional Studies and Reports, 12(1), 78-84. doi: 10.1016/j.vprsr.2018.02.005
Rahman, M., Uddin, M., Sultana, R., Moue, A., & Setu, M. (2013). Polymerase chain reaction (PCR): a short review. Anwer Khan Modern Medical College Journal, 4(1), 30-36. doi: 10.3329/akmmcj.v4i1.13682
Razakandrainibe, R., Diawara, E. H. I., Costa, D., Le Goff, L., Lemeteil, D., Ballet, J. J., Favennec, L. (2018). Common occurrence of Cryptosporidium hominis in asymptomatic and symptomatic calves in France. PLoS Neglected Tropical Diseases, 12(3), e0006355. doi: 10.1371/journal.pntd.0006355
Reif, J. S., Wimmer, L., Smith, J. A., Dargatz, D. A., & Cheney, J. M. (1989). Human cryptosporidiosis associated with an epizootic in calves. American Journal of Public Health, 79(11), 1528-1530. doi: 10.2105/ajph.79.11.1528
Rigo, C. R., & Franco, R. M. (2002). Comparison between the modified Ziehl-Neelsen and Acid-Fast-Trichrome methods for fecal screening of Cryptosporidium parvum and Isospora belli. Journal of the Brazilian Society of Tropical Medicine, 35(3), 209-214. doi: 10.1590/s0037-86822002000300002
Robertson, L. J., Björkman, C., Axén, C., & Fayer, R. (2014). Cryptosporidiosis in Farmed Animals. In S. M. Cacciò, & G. Widmer (Eds.), Cryptosporidium: Parasite and Disease (pp. 149-235). Springer, Viena. doi: 10.1007/978-3-7091-1562-6_4. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC71 22230/pdf/978-3-7091-1562-6_Chapter_4.pdf
Robertson, L. J., Johansen, Ø. H., Kifleyohannes, T., Efunshile, A. M., & Terefe, G. (2020). Cryptosporidium infections in Africa how important is zoonotic transmission? A review of the evidence. Frontiers in Veterinary Science, 7(1), 724. doi: 10.3389/fvets.2020.575881
Roy, S. L., DeLong, S. M., Stenzel, S. A., Shiferaw, B., Roberts, J. M., Khalakdina, A., Beach, M. J. (2004). Risk factors for sporadic cryptosporidiosis among immunocompetent persons in the United States from 1999 to 2001. Journal of Clinical Microbiology, 42(7), 2944-2951. doi: 10.1128/JCM.42.7.2944-2951. 2004
Ryan, U., Fayer, R., & Xiao, L. (2014). Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology, 141(13), 1667-1685. doi: 10.1017/S0031182014001085
Santín, M., Trout, J. M., & Fayer, R. (2008). A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age. Veterinary Parasitology, 155(1-2), 15-23. doi: 10.1016/j.vetpar.2008.04.018
Santı́n, M., Trout, J. M., Xiao, L., Zhou, L., Greiner, E., & Fayer, R. (2004). Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology, 122(2), 103-117. doi: 10.1016/j.vetpar.2004.03.020
Teixeira, C. S., Almeida, A. J. de, & Oliveira, F. C. R. de. (2008). Oocistos de Cryptosporidium spp. isolados de bezerros bovinos: Pleomorfismo em relação à metodologia de diagnóstico. Journal Brazilian Veterinary Parasitology, 17(1), 230-233.
Thomson, S., Hamilton, C. A., & Hope, J. C. (2017). Bovine cryptosporidiosis: impact, host parasite interaction and control strategies. Veterinary Research, 48(42), 1-16. doi: 10.1186/s13567-017-0447-0
Wegayehu, T., Karim, R., Anberber, M., Adamu, H., Erko, B., & Zhang, L. (2016). Prevalence and genetic characterization of Cryptosporidium species in dairy calves in central Ethiopia. PLoS ONE, 11(5), e0154647. doi: 10.1371/journal.pone.0154647
Xiao, L. (2010). Molecular epidemiology of cryptosporidiosis: an update. Experimental Parasitology, 124(1), 80-89. doi: 10.1016/j.exppara.2009.03.018
Xiao, L., Escalante, L., Yang, C., Sulaiman, I., Escalante, A. A., Montali, R. J., Lal, A. A. (1999). Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Applied an Environmental Microbiology, 65(4), 1578-1583. doi: 10.1128/AEM.65.4.1578-1583.1999
Xiao, L., Sulaiman, I. M., Ryan, U. M., Zhou, L., Atwill, E. R., Tischler, M. L., Lal, A. A. (2002). Host adaptation and host-parasite co-evolution in Cryptosporidium: implications for taxonomy and public health. International Journal for Parasitology, 32(14), 1773-1785. doi: 10.1016/s0020-7519(02)00197-2
Abubakar, I. I., Tillmann, T., & Banerjee, A. (2013). Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. The Lancet, 385(9963), 117-171. doi: 10.1016/s0140-6736(14)61682-2
Björkman, C., Lindström, L., Oweson, C., Ahola, H., Troell, K., & Axén, C. (2018). Cryptosporidium infections in suckler herd beef calves. Parasitology, 142(8), 1108-1114. doi: 10.1017/S0031182015000 426
Bowman, D. D. (2010). Diagnóstico parasitológico. In D. D. Bowman (Ed.), Parasitologia veterinária de Georgis (9a ed., pp. 279-351). São Paulo: Saunders-Elsevier.
Cai, M., Guo, Y., Pan, B., Li, N., Wang, X., Tang, C., Xiao L. (2017). Longitudinal monitoring of Cryptosporidium species in pre-weaned dairy calves on five farms in Shanghai, China. Veterinary Parasitology, 241(1), 14-19. doi: 10.1016/j.vetpar.2017.05.005
Chagas, A. C. S. (2015). Diarreia em bezerros leiteiros lactantes: a doença e o manejo em diferentes unidades da EMBRAPA. São Carlos: EMBRAPA Pecuária Sudeste-Documentos (INFOTECA-E). Recuperado de https://www.infoteca.cnptia.embrapa.br/infoteca/bitstream/doc/1035039/1/Documentos120.pdf
Fayer, R. (2010). Taxonomy and species delimitation in Cryptosporidium. Experimental Parasitology, 124(1), 90-97. doi: 10.1016/j.exppara.2009.03.005
Fayer, R., Santin, M., & Trout, J. M. (2007). Prevalence of Cryptosporidium species and genotypes in mature dairy cattle on farms in eastern United States compared with younger cattle from the same locations. Veterinary Parasitology, 145(3-4), 260-266. doi: 10.1016/j.vetpar.2006.12.009
Fereig, R. M., Abdelbaky, H. H., Ihara, F., & Nishikawa, Y. (2018). Development and evaluation of the first immunochromatographic test that can detect specific antibodies against Cryptosporidium parvum. Acta Tropica, 185(1), 349-356. doi: 10.1016/j.actatropica.2018.06.019
Li, N., Wang, R., Cai, M., Jiang, W., Feng, Y., & Xiao, L. (2019). Outbreak of cryptosporidiosis due to Cryptosporidium parvum subtype IIdA19G1 in neonatal calves on a dairy farm in China. International Journal for Parasitology, 49(7), 569-577. doi: 10.1016/j.ijpara.2019.02.006
Liao, C., Wang, T., Koehler, A. V., Fan, Y., Hu, M., & Gasser, R. B. (2018). Molecular investigation of Cryptosporidium in farmed chickens in Hubei Province, China, identifies ‘zoonotic’ subtypes of C. meleagridis. Parasites & Vectors, 11(1), 484. doi: 10.1186/s13071-018-3056-5
Liu, A., Gong, B., Liu, X., Shen, Y., & Wu, Y. (2020). A retrospective epidemiological analysis of human Cryptosporidium infection in China during the past three decades (1987-2018). PLOS Neglected Tropical Diseases, 14(3), e0008146. doi: 10.1371/journal.pntd.0008146
Mammeri, M., Chevillot, A., Chenafi, I., Thomas, M., Julien, C., Vallée, I.,… Adjou, K.T. (2019). Molecular characterization of Cryptosporidium isolates from diarrheal dairy calves in France. Veterinary Parasitology: Regional Studies and Reports, 18(1),100323. doi: 10.1016/j.vprsr.2019.100323
Matos, L. V. S. de, Silveira, L. da Neto, Oliveira, B. C. M., Makatu, M. Y., Pierucci, J. C., Viol, M. A., Bresciani, K. D. S. (2019). Molecular characterization of Cryptosporidium in calves from rural settlements in the Northwest region of the state of São Paulo, Brazil. Semina: Ciências Agrárias, 40(1), 491-496. doi: 10.5433/1679-0359.2019v40n1p491
Miambo, R. D., Laitela, B., Malatji, M. P., Santana Afonso, S. M. de, Júnior, A. P., Lindh, J., & Mukaratirwa, S. (2019). Prevalence of Giardia and Cryptosporidium in young livestock and dogs in Magude District of Maputo Province, Mozambique. Onderstepoort Journal of Veterinary Research, 86(1), e1-e6. doi: 10.4102/ojvr. v86i1.1709
Ortolani, E. L. (2000). Standardization of the modified Ziehl-Neelsen technique to stain oocysts of Cryptosporidium sp. Brazilian journal of Veterinary Parasitology, 9(1), 29-31.
Osaki, S. C., Soccol, V. T., Costa, A. O., Oliveira-Silva, M. B., Pereira, J. T., & Procopio, A. E. (2013). Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil. Journal of the Brazilian Society of Tropical Medicine, 46(3), 270-276. doi: 10.1590/0037-8682-0053-2013
Ouakli, N., Belkhiri, A., Lucio, A. de, Köster, P. C., Djoudi, M., Dadda, A., Carmena, D. (2018). Cryptosporidium-associated diarrhoea in neonatal calves in Algeria. Veterinary Parasitology Regional Studies and Reports, 12(1), 78-84. doi: 10.1016/j.vprsr.2018.02.005
Rahman, M., Uddin, M., Sultana, R., Moue, A., & Setu, M. (2013). Polymerase chain reaction (PCR): a short review. Anwer Khan Modern Medical College Journal, 4(1), 30-36. doi: 10.3329/akmmcj.v4i1.13682
Razakandrainibe, R., Diawara, E. H. I., Costa, D., Le Goff, L., Lemeteil, D., Ballet, J. J., Favennec, L. (2018). Common occurrence of Cryptosporidium hominis in asymptomatic and symptomatic calves in France. PLoS Neglected Tropical Diseases, 12(3), e0006355. doi: 10.1371/journal.pntd.0006355
Reif, J. S., Wimmer, L., Smith, J. A., Dargatz, D. A., & Cheney, J. M. (1989). Human cryptosporidiosis associated with an epizootic in calves. American Journal of Public Health, 79(11), 1528-1530. doi: 10.2105/ajph.79.11.1528
Rigo, C. R., & Franco, R. M. (2002). Comparison between the modified Ziehl-Neelsen and Acid-Fast-Trichrome methods for fecal screening of Cryptosporidium parvum and Isospora belli. Journal of the Brazilian Society of Tropical Medicine, 35(3), 209-214. doi: 10.1590/s0037-86822002000300002
Robertson, L. J., Björkman, C., Axén, C., & Fayer, R. (2014). Cryptosporidiosis in Farmed Animals. In S. M. Cacciò, & G. Widmer (Eds.), Cryptosporidium: Parasite and Disease (pp. 149-235). Springer, Viena. doi: 10.1007/978-3-7091-1562-6_4. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC71 22230/pdf/978-3-7091-1562-6_Chapter_4.pdf
Robertson, L. J., Johansen, Ø. H., Kifleyohannes, T., Efunshile, A. M., & Terefe, G. (2020). Cryptosporidium infections in Africa how important is zoonotic transmission? A review of the evidence. Frontiers in Veterinary Science, 7(1), 724. doi: 10.3389/fvets.2020.575881
Roy, S. L., DeLong, S. M., Stenzel, S. A., Shiferaw, B., Roberts, J. M., Khalakdina, A., Beach, M. J. (2004). Risk factors for sporadic cryptosporidiosis among immunocompetent persons in the United States from 1999 to 2001. Journal of Clinical Microbiology, 42(7), 2944-2951. doi: 10.1128/JCM.42.7.2944-2951. 2004
Ryan, U., Fayer, R., & Xiao, L. (2014). Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology, 141(13), 1667-1685. doi: 10.1017/S0031182014001085
Santín, M., Trout, J. M., & Fayer, R. (2008). A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age. Veterinary Parasitology, 155(1-2), 15-23. doi: 10.1016/j.vetpar.2008.04.018
Santı́n, M., Trout, J. M., Xiao, L., Zhou, L., Greiner, E., & Fayer, R. (2004). Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology, 122(2), 103-117. doi: 10.1016/j.vetpar.2004.03.020
Teixeira, C. S., Almeida, A. J. de, & Oliveira, F. C. R. de. (2008). Oocistos de Cryptosporidium spp. isolados de bezerros bovinos: Pleomorfismo em relação à metodologia de diagnóstico. Journal Brazilian Veterinary Parasitology, 17(1), 230-233.
Thomson, S., Hamilton, C. A., & Hope, J. C. (2017). Bovine cryptosporidiosis: impact, host parasite interaction and control strategies. Veterinary Research, 48(42), 1-16. doi: 10.1186/s13567-017-0447-0
Wegayehu, T., Karim, R., Anberber, M., Adamu, H., Erko, B., & Zhang, L. (2016). Prevalence and genetic characterization of Cryptosporidium species in dairy calves in central Ethiopia. PLoS ONE, 11(5), e0154647. doi: 10.1371/journal.pone.0154647
Xiao, L. (2010). Molecular epidemiology of cryptosporidiosis: an update. Experimental Parasitology, 124(1), 80-89. doi: 10.1016/j.exppara.2009.03.018
Xiao, L., Escalante, L., Yang, C., Sulaiman, I., Escalante, A. A., Montali, R. J., Lal, A. A. (1999). Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Applied an Environmental Microbiology, 65(4), 1578-1583. doi: 10.1128/AEM.65.4.1578-1583.1999
Xiao, L., Sulaiman, I. M., Ryan, U. M., Zhou, L., Atwill, E. R., Tischler, M. L., Lal, A. A. (2002). Host adaptation and host-parasite co-evolution in Cryptosporidium: implications for taxonomy and public health. International Journal for Parasitology, 32(14), 1773-1785. doi: 10.1016/s0020-7519(02)00197-2
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2022-02-25
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Candeias, A. P. M., Huff, G. M., Rosado, A. F., Beninca, A. L. V., Souza, L. Z., Osaki, S. C., & Fernandes, N. L. M. (2022). Evaluation of fecal smear methods for research on Cryptosporidium spp. oocysts in the feces of dairy calves. Semina: Ciências Agrárias, 43(2), 585–598. https://doi.org/10.5433/1679-0359.2022v43n2p585
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