Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China

Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Resumo Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Cross-genera amplification and identification of Colpodella sp. with Cryptosporidium primers in fecal samples of zoo felids from northeast China / Amplificação cruzada de gêneros e identificação de Colpodella sp. com iniciadores de Cryptosporidium em amostras fecais de zoofilia do nordeste da China

Abstract The protozoans include many intracellular human pathogens. Accurate detection of these pathogens is necessary to treat the diseases. In clinical epidemiology, molecular identification of protozoan is considered a more reliable and rapid method for identification than microscopy. Among these protozoans, Cryptosporidium considered being one of the important water-borne zoonotic pathogens and a major cause of a diarrheal disease named cryptosporidiosis in humans, domestic animals, and wild animals. This study was aimed to identify Cryptosporidium in zoo felids (N= 56) belonging to different zoo of China, but accidentlly Colpodella was encountered in the zoo felids sample and phylogenetic data confirmed this unexpected amplification from fecal samples using two-step nested-PCR. Phylogenetic analysis revealed the fact about the specific primers used previously by many researchers and cross-genera amplification. We came to know that genetically sequenced amplicon gives more accurate identification of species. This study suggests more investigation on Colpodella which has been neglected previously but gains the attention of researchers after identified from humans and animals and has been known to correlate with neurological symptoms in patients.

Resumo Os protozoários incluem muitos patógenos humanos intracelulares. A detecção acurada desses patógenos é necessária para tratar as doenças. Na epidemiologia clínica, a identificação molecular de protozoários é considerada o método de identificação mais confiável e rápido do que a microscopia. Entre esses protozoários, o Cryptosporidium é considerado um dos importantes patógenos zoonóticos transmitidos pela água e uma das principais causas de uma doença diarreica denominada criptosporidiose em humanos, animais domésticos e selvagens. Este estudo teve como objetivo identificar Cryptosporidium em zoofelídeos (N = 56) pertencentes a diferentes zoológicos da China, mas acidentalmente Colpodella foi encontrada na amostra de zoofelídeos e os dados filogenéticos confirmaram essa amplificação inesperada de amostras fecais usando nested-PCR em duas etapas. A análise filogenética revelou o fato sobre os primers específicos usados ​​anteriormente por muitos pesquisadores e a amplificação entre gêneros. Ficamos sabendo que o amplicon sequenciado geneticamente fornece uma identificação mais acurada das espécies. Este estudo sugere mais investigação sobre Colpodella, que foi negligenciada anteriormente, mas ganha a atenção dos pesquisadores depois de identificada em humanos e animais e é conhecida por se correlacionar com sintomas neurológicos em pacientes.

Identification of Avian Toll-Like Receptor 3 and 7 and Analysis of Gene Variation Sites

Toll-like receptors 3 and 7 (TLR3 and 7) mediate immune responses through the recognition of viral single-stranded RNA and double-stranded RNA and therefore play important roles in host defense. Differences in TLR3 or 7 may affect host resistance to RNA viral infection. To illuminate these differences, the partial coding sequence (CDS) of TLR3 and 7 genes were cloned and amplified from the Phasianus colchicus and Numida meleagris, total 64 avian species of TLR3 and 7 sequences were later analyzed. Based on the results, 315 non-synonymous mutation sites and 202 synonymous mutation sites were also observed in the avian TLR3, and 227 non-synonymous mutation sites and 174 synonymous mutation sites were observed in the avian TLR7. Among these sites, 44 and 45 sites were observed in functional regions of TLR3 and 7, used common variation of amino acids in most avian species. A number of these different sites appeared to affect the recognition and were also visualized. H59Y, E60K, G64R, E93K, L112S, K117E, N118K, R120H, V123M, L163F, R443Q, R459K, E460D, C485H, and F511L for TLR3, and I432V, M437V, and T732S for TLR7 were considered. It is possible that these sites bind to ligands and play crucial roles in viral recognition. These data indicated that the positive selection has occurred in the avian TLR3 and 7 genes.(AU)

The Effect of Supplementing Tea Polyphenols on Yolk Cholesterol and Production Performance of Laying Hens During the Egg-laying Period

This study aimed to investigate the effect of supplementing 300 mg/kg tea polyphenols (TP) on yolk cholesterol content and production performance of laying hens during the egg-laying period. A total of 600 Roman laying hens aged 24 weeks were randomly divided into two dietary treatment groups. The feeding experiment lasted for 48 weeks. Layers fed basal diet supplemented with 0 (control group) and 300mg/kg TP (TP group) diet, respectively. The yolk cholesterol content, laying performance, and egg quality were determined at 28, 38, 48, 58, and 68 weeks of age. The yolk cholesterol content in the TP group was significantly decreased at 28-68 weeks of age (p<0.01), compared to the control group. There was a significant increase in laying rate in the TP group at 38 weeks of age (p<0.05), compared to the control group, while no significant differences during the other laying periods were obtained (p>0.05). The FCR significantly decreased in the TP group at 38 weeks of age whereas AEW significantly increased in the TP group at 58 weeks of age (p<0.05). Similarly, the eggshell thickness and eggshell strength in the TP group significantly increased (p<0.05), compared with the control group at 38 weeks of age. The albumen height and Haugh unit significantly increased at 28 weeks of age (p<0.05). In conclusion, the results showed that the diet supplemented with 300 mg/kg TP had positive effects on production performance of layers during the egg-laying period, and could lessen yolk cholesterol content significantly at 28-68 weeks of age.(AU)

Prevalence of bovine tuberculosis in the Aksu Region of Xinjiang, China, between 1985 and 2016 / Prevalência de tuberculose bovina na região Aksu de Xinjiang, China, entre 1985 e 2016

Prevalence of bovine tuberculosis infection in cattle in Aksu Prefecture determined by intradermal tuberculin skin test (TST), between 1985 and 2016. Cattle were analyzed according to region, feeding pattern, herds and age. A total of 890,009 cattle were tested, with overall bovine tuberculosis prevalence of 0.13% (1172/890009). Statistically significant difference was found in feeding pattern and herds. Prevalence in cows (0.19%, 615/327022) was higher than that in beeves (P< 0.01, OR= 1.903, 95% CI = 1.696 to 2.134). Significant difference (P< 0.01; OR= 2.238, 95%; CI= 1.937 to 2.585) was evident for rates for bovine tuberculosis in the peasant household (0.12%, 942/802343) and farm groups (0.26%, 230/87666). The overall prevalence of bTB was decreased in the Aksu Prefecture, especially the positive rate was under 0.1% in 2010s. We concluded that the control measures forbovine tuberculosis in the Aksu region cattle herds are effective.(AU)

Prevalência de infecção por tuberculose bovina em gado na prefeitura de Aksu determinada por teste cutâneo tuberculínico (TST) entre 1985 e 2016 foi avaliada. O gado foi analisado de acordo com região, padrão alimentar, rebanho e idade. Um total de 890009 animais foram testados, com prevalência de 0,13% de tuberculose bovina (1172/890009). Diferença estatisticamente significativa foi encontrada em padrão alimentar e rebanhos. Prevalência em vacas (0,19%, 615/327022) foi mais alta que em bois (P< 0,01, OR= 1,903, 95% CI = 1,696 a 2,134). Diferenças significativas (P< 0,01; OR= 2,238, 95%; CI= 1,937 a 2,585) foram evidentes em taxas para tuberculose bovina em casas de camponeses (0,12%, 942/802343) e grupos de fazendeiros (0,26%, 230/87666). A prevalência de bTB caiu na prefeitura Aksu, a taxa positiva se encontrava abaixo de 0.1% a partir de 2010. Conclui-se que as medidas de controle para tuberculose bovina na região de Aksu foram eficazes.(AU)

Prevalence of bovine tuberculosis in the Aksu Region of Xinjiang, China, between 1985 and 2016 / Prevalência de tuberculose bovina na região Aksu de Xinjiang, China, entre 1985 e 2016

Prevalence of bovine tuberculosis infection in cattle in Aksu Prefecture determined by intradermal tuberculin skin test (TST), between 1985 and 2016. Cattle were analyzed according to region, feeding pattern, herds and age. A total of 890,009 cattle were tested, with overall bovine tuberculosis prevalence of 0.13% (1172/890009). Statistically significant difference was found in feeding pattern and herds. Prevalence in cows (0.19%, 615/327022) was higher than that in beeves (P< 0.01, OR= 1.903, 95% CI = 1.696 to 2.134). Significant difference (P< 0.01; OR= 2.238, 95%; CI= 1.937 to 2.585) was evident for rates for bovine tuberculosis in the peasant household (0.12%, 942/802343) and farm groups (0.26%, 230/87666). The overall prevalence of bTB was decreased in the Aksu Prefecture, especially the positive rate was under 0.1% in 2010s. We concluded that the control measures forbovine tuberculosis in the Aksu region cattle herds are effective.(AU)

Prevalência de infecção por tuberculose bovina em gado na prefeitura de Aksu determinada por teste cutâneo tuberculínico (TST) entre 1985 e 2016 foi avaliada. O gado foi analisado de acordo com região, padrão alimentar, rebanho e idade. Um total de 890009 animais foram testados, com prevalência de 0,13% de tuberculose bovina (1172/890009). Diferença estatisticamente significativa foi encontrada em padrão alimentar e rebanhos. Prevalência em vacas (0,19%, 615/327022) foi mais alta que em bois (P< 0,01, OR= 1,903, 95% CI = 1,696 a 2,134). Diferenças significativas (P< 0,01; OR= 2,238, 95%; CI= 1,937 a 2,585) foram evidentes em taxas para tuberculose bovina em casas de camponeses (0,12%, 942/802343) e grupos de fazendeiros (0,26%, 230/87666). A prevalência de bTB caiu na prefeitura Aksu, a taxa positiva se encontrava abaixo de 0.1% a partir de 2010. Conclui-se que as medidas de controle para tuberculose bovina na região de Aksu foram eficazes.(AU)

Salmonella Enteritidis in Layer Farms of Different Sizes Located in Northern China: On-Farm Sampling and Detection by the PCR Method

ABSTRACT Salmonella enterica subspecies enterica serotype Enteritidis (SE) has caused foodborne infections over decades. It is transmitted mainly from contaminated eggs to humans. SE is commonly present in layer houses, and closely interacts with environmental factors. The objective of the present study was to develop a viable PCR method to identify SE in environmental samples collected in layer farms of different sizes, and to evaluate SE contamination status in four main egg-production provinces of northern China. After specificity retrieval using Primer-BLAST against the NCBI database, three SE specific oligonucleotide primers were selected as candidate primers. The primers targeting Prot6e gene were adopted and primers targeting Sdf I were also selected to validate the results, after testing eight different types of pooled poultry environmental samples (overshoe, air, drinking nipple, feed, egg collection belt, eggshell, air inlet, and air outlet) by PCR. A PCR detection limit of 1 CFU/mL was determined using cell lysates from pure cultures. Testing time was less than 48 h. On-farm samples were collected from two layer farm sizes (one housing more than 50,000 layers, and the other, less than 50,000 layers) in each province. The applied PCR method was shown to be simple, inexpensive and effective for screening SE in a large amount of farm samples. The study identified only one SE-positive farm, which a large farm and where nine samples were found to be contaminated with SE: drinking nipples (3), egg collection belt (1), air inlet (1), air (1), overshoe (1) and eggshell (2).(AU)

Salmonella Enteritidis in Layer Farms of Different Sizes Located in Northern China: On-Farm Sampling and Detection by the PCR Method

ABSTRACT Salmonella enterica subspecies enterica serotype Enteritidis (SE) has caused foodborne infections over decades. It is transmitted mainly from contaminated eggs to humans. SE is commonly present in layer houses, and closely interacts with environmental factors. The objective of the present study was to develop a viable PCR method to identify SE in environmental samples collected in layer farms of different sizes, and to evaluate SE contamination status in four main egg-production provinces of northern China. After specificity retrieval using Primer-BLAST against the NCBI database, three SE specific oligonucleotide primers were selected as candidate primers. The primers targeting Prot6e gene were adopted and primers targeting Sdf I were also selected to validate the results, after testing eight different types of pooled poultry environmental samples (overshoe, air, drinking nipple, feed, egg collection belt, eggshell, air inlet, and air outlet) by PCR. A PCR detection limit of 1 CFU/mL was determined using cell lysates from pure cultures. Testing time was less than 48 h. On-farm samples were collected from two layer farm sizes (one housing more than 50,000 layers, and the other, less than 50,000 layers) in each province. The applied PCR method was shown to be simple, inexpensive and effective for screening SE in a large amount of farm samples. The study identified only one SE-positive farm, which a large farm and where nine samples were found to be contaminated with SE: drinking nipples (3), egg collection belt (1), air inlet (1), air (1), overshoe (1) and eggshell (2).

Transcriptome Analysis of Chicken Embryo Fibroblast Cell Infected with Mareks Disease Virus of GX0101 LTR

Mareks disease (MD), a lymphoproliferative disorder of chickens caused by the MD virus (MDV), is economically significant. The resistance/susceptibility to MD is controlled by host genetics. The host response to different virus strains varies. The pathogenicity of REV-LTR deleted GX0101LTR MDV has been previously reported. However, the precise molecular mechanism of the response of chickens to GX0101LTR remains unclear. The current study aimed at identifying the genes and pathways involved in the response to GX0101LTR virus infection in specific pathogen-free chicken embryo fibroblast cells using global transcriptome analysis. A total of 1,633 genes associated with GX0101LTR infection were identified. Functional analysis showed that the cytokine-cytokine receptor interaction plays an important role in the response to GX0101LTR infection.(AU)

Transcriptome Analysis of Chicken Embryo Fibroblast Cell Infected with Mareks Disease Virus of GX0101 LTR

Mareks disease (MD), a lymphoproliferative disorder of chickens caused by the MD virus (MDV), is economically significant. The resistance/susceptibility to MD is controlled by host genetics. The host response to different virus strains varies. The pathogenicity of REV-LTR deleted GX0101LTR MDV has been previously reported. However, the precise molecular mechanism of the response of chickens to GX0101LTR remains unclear. The current study aimed at identifying the genes and pathways involved in the response to GX0101LTR virus infection in specific pathogen-free chicken embryo fibroblast cells using global transcriptome analysis. A total of 1,633 genes associated with GX0101LTR infection were identified. Functional analysis showed that the cytokine-cytokine receptor interaction plays an important role in the response to GX0101LTR infection.