Tandem mass tag (TMT)-based proteomic analysis of Cryptosporidium andersoni oocysts before and after excystation

Background Cryptosporidium andersoni initiates infection by releasing sporozoites from oocysts through excystation. However, the proteins involved in excystation are unknown. Determining the proteins that participate in the excystation of C. andersoni oocysts will increase our understanding of the excystation process. Methods Cryptosporidium andersoni oocysts were collected and purified from the feces of naturally infected adult cows. Tandem mass tags (TMT), coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomic analysis, were used to investigate the proteomic expression profiles of C. andersoni oocysts before and after excystation. Results Proteomic analysis identified a total of 1586 proteins, of which 17 were differentially expressed proteins (DEPs) upon excystation. These included 10 upregulated and seven downregulated proteins. The 17 proteins had multiple biological functions associated with control of gene expression at the level of transcription and biosynthetic and metabolic processes. Quantitative real-time RT-PCR of eight selected genes validated the proteomic data. Conclusions This study provides information on the protein composition of C. andersoni oocysts as well as possible excystation factors. The data may be useful in identifying genes for diagnosis, vaccine development, and immunotherapy for Cryptosporidium. Graphical Abstract

TMT-based Proteomic Analysis and Protein Expression During Excystation of Cryptosporidium Anderson

Background: Cryptosporidium andersoni (C. andersoni) initiates infection by the release of sporozoites through excystation. However, the proteins involved in excystation remain unknown. Researching the proteins that participate in the excystation of C. andersoni oocysts will fill the gap in our understanding of the excystation system of this parasitic pathogen. Methods: In this study, C. andersoni oocysts were collected and purified from the feces of naturally infected adult cows. Tandem mass tags (TMT) coupled with liquid chromatograph- tandem mass spectrometry (LC-MS/MS) proteomic analysis was used to investigate the proteomic expression profile of C. andersoni oocysts during excystation. Results: Our proteomic analysis identified a total of 1586 proteins, of which 17 were identified as differentially expressed proteins (DEPs), with 10 upregulated and 7 downregulated proteins. Each of those 17 proteins had multiple biological functions associated with control of gene expression at the level of transcription and biosynthetic and metabolic processes. Quantitative real-time PCR of eight selected genes validated the proteomic data.Conclusions: Our findings provide new information on the protein composition of C. andersoni oocysts as well as possible excystation factors. These data may help us to better understand the pathology of C. andersoni and thus may be useful in diagnosis, vaccine development, and immunotherapy for Cryptosporidium.

First Detection of Cryptosporidium spp. in Migratory Whooper Swans (Cygnus cygnus) in China

Cryptosporidium is an important protozoan parasite that can cause gastrointestinal diseases in humans and that also causes respiratory and gastrointestinal diseases in birds. In this study, we investigated the occurrence of Cryptosporidium species in migratory whooper swans in China. Fecal samples (n = 467) from whooper swans were collected from Sanmenxia Swan Lake National Urban Wetland Park, China. The samples were analyzed for Cryptosporidium species and genotypes with PCR along a sequence analysis of the small subunit rRNA. Cryptosporidium was detected in eight of the 467 (1.7%) samples. The analysis of the small subunit rRNA sequence data revealed two zoonotic species (Cryptosporidium parvum and Cryptosporidium andersoni) and one genotype (Cryptosporidium goose genotype II). These are the first data on the positive rate of Cryptosporidium spp. in whooper swans in China, and they suggest that whooper swans can harbor the zoonotic species C. parvum and C. andersoni in China.

Molecular epidemiology of Cryptosporidium spp. in dairy cattle in Guangdong Province, South China

To determine the prevalence of Cryptosporidium in dairy cattle in Guangdong Province, South China, 1440 fecal samples were collected from 10 farms and screened for Cryptosporidium with PCR. The overall prevalence of Cryptosporidium was 4.38% (63/1440), and the infection rates in preweaned calves, postweaned calves, heifers and adults were 6.4% (19/297), 6.19% (33/533), 1.48% (4/271) and 2.06% (7/339), respectively. Three Cryptosporidium species, Cryptosporidium andersoni (n = 33), Cryptosporidium bovis (n = 22) and Cryptosporidium ryanae (n = 8) were detected by DNA sequence analysis of the 63 positive samples, and C. andersoni was identified as the most common species on the dairy cattle farms. In preweaned calves, C. bovis was the most prevalent species (9/19, 47.4%). In contrast, C. andersoni was the predominant species (19/33, 57.6%) in postweaned calves and the only species found in heifers and adults. The zoonotic species Cryptosporidium parvum was not detected in this study. Twenty-four C. andersoni isolates were successfully classified into three multilocus sequence typing (MLST) subtypes. MLST subtype A4,A4,A4,A1 was the predominant subtype, and MLST subtype A2,A5,A2,A1, previously found in sheep, was detected in cattle for the first time. A linkage disequilibrium analysis showed that the C. andersoni isolates had a clonal genetic population structure. However, further molecular studies are required to better understand the epidemiology of Cryptosporidium in Guangdong.

COMPARAÇÃO DA EFICIÊNCIA DAS COLORAÇÕES DE ZIEHL-NEELSEN MODIFICADO E SAFRANINA MODIFICADA NA DETECÇÃO DE OOCISTOS DE Cryptosporidium spp. (EUCOCCIDIORIDA, CRYPTOSPORIDIIDAE) A PARTIR DE AMOSTRAS FECAIS DE BEZERROS DE 0 A 3 MESES

Resumo A criptosporidiose bovina é causada principalmente por quatro espécies distintas: Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae e Cryptosporidium andersoni. A espécie Cryptosporidium parvum (Ordem: Eucoccidiorida, Família: Cryptosporidiidae) é considerada de alto potencial zoonótico, podendo infectar humanos por intermédio da eliminação de oocistos tanto pelos bovinos quanto pelo próprio humano. O objetivo desta pesquisa foi verificar a ocorrência de oocistos de Cryptosporidium spp. em amostras fecais de bezerros (75 machos e 77 fêmeas), tendo sido coletadas 152 amostras de fezes de animais do nascimento até os três meses de idade. O material foi submetido às técnicas de coloração de Ziehl-Neelsen modificado e Safranina modificada, as lâminas foram observadas em toda sua extensão ao microscópio óptico para a verificação da presença de oocistos desta enteroparasitose. Os resultados demonstraram 17,1% (26/152) de positividade no total das amostras examinadas e a análise estatística revelou não haver diferença entre o sexo e as técnicas de coloração utilizadas neste estudo. Conclui-se que a infecção por Cryptosporidium spp. esta presente nas propriedades avaliadas, porém são necessários mais estudos para que o risco de infecção seja mensurado adequadamente e medidas profiláticas implementadas.