Anaplasma phagocytophilum is a tick borne bacterium, causing disease in sheep and other mammals, including humans. The bacterium has great economic and animal welfare implications for sheep husbandry in Northern Europe. With the prospect of a warmer and more humid climate, the vector availability will likely increase, resulting in a higher prevalence of A. phagocytophilum. The current preventive measures, as pyrethroids acting on ticks or long acting antibiotics controlling bacterial infection, are suboptimal for prevention of the disease in sheep. Recently, the increased awareness on antibiotic- and pyrethorid resistance, is driving the search for a new prophylactic approach in sheep against A. phagocytophilum. Previous studies have used an attenuated vaccine, which gave insufficient protection from challenge with live bacteria. Other studies have focused on bacterial membrane surface proteins like Asp14 and OmpA. An animal study using homologous proteins to Asp14 and OmpA of A. marginale, showed no protective effect in heifers. In the current study, recombinant proteins of Asp14 (rAsp14) and OmpA (rOmpA) of A. phagocytophilum were produced and prepared as a vaccine for sheep. Ten lambs were vaccinated twice with an adjuvant emulsified with rAsp14 or rOmpA, three weeks apart and challenged with a live strain of A. phagocytophilum (GenBank acc.nr M73220) on day 42. The control group consisted of five lambs injected twice with PBS and adjuvant. Hematology, real time qPCR, immunodiagnostics and flow cytometric analyses of peripheral blood mononuclear cells were performed. Vaccinated lambs responded with clinical signs of A.phagocytophilum infection after challenge and bacterial load in the vaccinated group was not reduced compared to the control group. rAsp14 vaccinated lambs generated an antibody response against the vaccine, but a clear specificity for rAsp14 could not be established. rOmpA-vaccinated lambs developed a strong specific antibody response on days 28 after vaccination and 14 days post-challenge. Immunofluorescent staining and flow cytometric analysis of peripheral blood mononuclear monocytes revealed no difference between the three groups, but the percentage of CD4+, CD8+, γδ TcR+, λ-Light chain+, CD11b+, CD14+ and MHC II+ cells, within the groups changed during the study, most likely due to the adjuvant or challenge with the bacterium. Although an antigen specific antibody response could be detected against rOmpA and possibly rAsp14, the vaccines seemed to be ineffective in reducing clinical signs and bacterial load caused by A. phagocytophilum. This is the first animal study with recombinant Asp14 and OmpA aimed at obtaining clinical protection against A. phagocytophilum in sheep.

中文翻译：

---

评估针对绵羊噬菌体的重组Asp14和OmpA疫苗接种的临床和细菌学结果。

吞噬细胞无浆膜是a传播的细菌，在绵羊和其他哺乳动物，包括人类中引起疾病​​。这种细菌对北欧的绵羊饲养业具有重大的经济和动物福利意义。随着气候变暖和潮湿的前景，载体的可用性可能会增加，从而导致嗜血曲霉的患病率更高。目前的预防措施，如拟除虫菊酯作用于壁虱或控制细菌感染的长效抗生素，在预防绵羊疾病方面是次优的。近来，对抗生素和拟除虫菊酯抗性的认识的提高，正促使人们在绵羊中寻找一种新的预防嗜血曲霉的预防方法。先前的研究已经使用了减毒疫苗，这种疫苗不能有效抵抗活细菌的攻击。其他研究集中在细菌膜表面蛋白，如Asp14和OmpA。一项动物研究使用与A.marginale的Asp14和OmpA同源的蛋白质，对小母牛没有保护作用。在当前的研究中，生产了吞噬曲霉的Asp14（rAsp14）和OmpA（rOmpA）重组蛋白，并将其制备为绵羊疫苗。十只羔羊用佐以rAsp14或rOmpA乳化的佐剂接种两次，间隔三周，并在第42天用噬菌嗜热链球菌（GenBank acc.nr M73220）的活菌株攻击。对照组由五只羔羊组成，两次注射PBS和佐剂。进行了外周血单核细胞的血液学，实时qPCR，免疫诊断和流式细胞仪分析。接种疫苗的羔羊对A的临床症状有反应。与对照组相比，接种组的攻击后吞噬细胞感染和细菌载量没有减少。接种rAsp14的羔羊产生了针对疫苗的抗体反应，但无法确定对rAsp14的明确特异性。接种rOmpA的羔羊在疫苗接种后28天和攻击后14天出现了强烈的特异性抗体反应。免疫荧光染色和外周血单核单核细胞的流式细胞术分析未显示三组之间的差异，但研究期间组中CD4 +，CD8 +，γδTcR +，λ-轻链+，CD11b +，CD14 +和MHC II +细胞的百分比发生了变化，最有可能是由于细菌的佐剂或攻击。尽管可以检测到针对rOmpA以及可能针对rAsp14的抗原特异性抗体反应，该疫苗似乎在减少吞噬曲霉引起的临床体征和细菌负荷方面无效。这是第一个使用重组Asp14和OmpA进行的动物研究，旨在获得针对绵羊中吞噬曲霉的临床保护。