Santana André M, Silva Daniela G, Thomas Funmilola C, Bernardes Priscila A, Pizauro Lucas J L, Santana Clarissa H, Burchmore Richard J S, Eckersall Peter D, Fagliari José J
Department of Veterinary Clinic and Surgery, School of Agricultural and Veterinary Sciences, São Paulo State University (FCAV/UNESP), Jaboticabal, SP, Brazil.
Department of Veterinary Clinic and Surgery, School of Agricultural and Veterinary Sciences, São Paulo State University (FCAV/UNESP), Jaboticabal, SP, Brazil.
Vet Immunol Immunopathol. 2018 Sep;203:30-39. doi: 10.1016/j.vetimm.2018.07.014. Epub 2018 Aug 4.
The study aimed to evaluate clinical signs, blood serum acute phase proteins (APP) and iron dynamics during the acute phase response (APR) of Salmonella Dublin experimentally infected Murrah buffalo calves. Six buffalo calves constituted the control group (CNT) and six were orally inoculate with 10 CFU of S. Dublin (INF). Clinical evaluation was performed, rectal swabs to detect S. Dublin strains were collected and venous blood was sampled before and throughout seven days after inoculation. The APP fractions β-haptoglobin, α-haptoglobin, ceruloplasmin and transferrin were analyzed by 1-D and 2-D electrophoresis. Proteins were identified using LC/ESI-MS/MS and NCBI database. Plasma fibrinogen, serum iron and serum haptoglobin concentrations were measured. The inoculation of 10 CFU of S. Dublin was effective in inducing clinical signs of Salmonellosis, such as hyperthermia and diarrhea. 1-DE showed that β and α-haptoglobin increased 204% (p = 0.008) and 184% (p = 0.022) 48 h after inoculation (HAI), respectively, with highest concentrations 120 HAI (498% increased, p = 0.012; 431% increased, p = 0.011) and 168 HAI (492% increased, p = 0.019; 523% increased, p = 0.028). 2-DE showed that the expression of two spots, identified as β-haptoglobin, were increased 693% (p = 0.0006) and 580% (p = 0.0003) 168 HAI, respectively, while one spot, identified as α-haptoglobin, increased 714% (p = 0.040). Haptoglobin concentrations increased 1339% (p < 0.0001) 168 HAI. 1-DE showed that ceruloplasmin increased 42% (p = 0.034) 48 HAI, with highest concentration 120 HAI (133% increased, p = 0.022). 2-DE showed that the expression of two spots, identified as ceruloplasmin, were increased 218% (p = 0.0153) and 85% (p = 0.0143) 168 HAI, respectively. Fibrinogen increased 78% (p = 0.012) 96 HAI, with highest concentration 120 HAI (increased 114%, p = 0.002). Iron decreased 33% 24 HAI (p = 0.015) and 37% 72 HAI (p = 0.029), and began to be restored 96 HAI. 1-DE showed that transferrin decreased 23% 120 HAI (p = 0.047), and that values were restored 168 HAI. 2-DE showed that expression patterns of transferrin comparing 0 h and 168 HAI were similar, evidencing that values were restored 168 HAI. In conclusion, the inoculation of 10 CFU was effective in inducing hyperthermia and diahrrea. β and α-haptoglobin, ceruloplasmin and fibrinogen worked as positive APP during the APR to S. Dublin infection and are potential biomarker candidates. Concentrations of iron and transferrin decreased during the infection, highlighting the fact that mechanisms for restricting iron availability are part of the APR triggered against S. Dublin infection in buffalo calves.
本研究旨在评估实验性感染都柏林沙门氏菌的穆拉水牛犊急性期反应（APR）期间的临床症状、血清急性期蛋白（APP）和铁动力学。六头水牛犊组成对照组（CNT），另外六头口服接种10 CFU的都柏林沙门氏菌（INF）。进行临床评估，采集直肠拭子以检测都柏林沙门氏菌菌株，并在接种前及接种后七天内采集静脉血样。通过一维和二维电泳分析APP组分β-触珠蛋白、α-触珠蛋白、铜蓝蛋白和转铁蛋白。使用液相色谱/电喷雾串联质谱法（LC/ESI-MS/MS）和美国国立生物技术信息中心（NCBI）数据库鉴定蛋白质。测定血浆纤维蛋白原、血清铁和血清触珠蛋白浓度。接种10 CFU的都柏林沙门氏菌可有效诱发沙门氏菌病的临床症状，如体温过高和腹泻。一维电泳显示，接种后48小时（HAI）β-触珠蛋白和α-触珠蛋白分别增加204%（p = 0.008）和184%（p = 0.022），在120 HAI时浓度最高（分别增加498%，p = 0.012；增加431%，p = 0.011），在168 HAI时分别增加492%（p = 0.019）和523%（p = 0.028）。二维电泳显示，鉴定为β-触珠蛋白的两个斑点的表达在168 HAI时分别增加693%（p = 0.0006）和580%（p = 0.0003），而鉴定为α-触珠蛋白的一个斑点增加714%（p = 0.040）。触珠蛋白浓度在168 HAI时增加1339%（p < 0.0001）。一维电泳显示，铜蓝蛋白在48 HAI时增加42%（p = 0.034），在120 HAI时浓度最高（增加133%，p = 0.022）。二维电泳显示，鉴定为铜蓝蛋白的两个斑点的表达在168 HAI时分别增加218%（p = 0.0153）和85%（p = 0.0143）。纤维蛋白原在96 HAI时增加78%（p = 0.012），在120 HAI时浓度最高（增加114%，p = 0.002）。铁在24 HAI时降低33%（p = 0.015），在72 HAI时降低37%（p = 0.029），并在96 HAI时开始恢复。一维电泳显示，转铁蛋白在120 HAI时降低23%（p = 0.047），并在168 HAI时恢复到原有水平。二维电泳显示，比较0小时和168 HAI时转铁蛋白的表达模式相似，表明其在168 HAI时恢复到原有水平。总之，接种10 CFU可有效诱发体温过高和腹泻。β-触珠蛋白、α-触珠蛋白、铜蓝蛋白和纤维蛋白原在对都柏林沙门氏菌感染的急性期反应中作为阳性APP起作用，是潜在的生物标志物候选物。感染期间铁和转铁蛋白浓度降低，突出了限制铁可用性的机制是水牛犊针对都柏林沙门氏菌感染触发的急性期反应的一部分这一事实。
