Gupta Aditya, Duncan Mary, Sweeny Amy R, de Araujo Larissa S, Kwok Oliver C H, Rosenthal Benjamin M, Khan Asis, Grigg Michael E, Dubey Jitender P
United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Beltsville, MD 20705-2350, USA.
Saint Louis Zoo, One Government Drive, St. Louis, MO 63110, USA.
Int J Parasitol. 2023 Dec;53(14):777-785. doi: 10.1016/j.ijpara.2023.08.001. Epub 2023 Aug 29.
Here, we report the first known outbreak of clinical protozoal myeloencephalitis in naturally infected raccoons by the parasite Sarcocystis neurona. The North American opossum (Didelphis virginiana) and the South American opossum (Didelphis albiventris) are its known definitive hosts. Several other animal species are its intermediate or aberrant hosts. The raccoon (Procyon lotor) is considered the most important intermediate host for S. neurona in the USA. More than 50% of raccoons in the USA have sarcocysts in their muscles, however clinical sarcocystosis in raccoons is rare. In 2014, 38 free-living raccoons were found dead or moribund on the grounds of the Saint Louis Zoo, Missouri, USA. Moribund individuals were weak, lethargic, and mildly ataxic; several with oculo-nasal discharge. Seven raccoons were found dead and 31 were humanely euthanized. Postmortem examinations were conducted on nine raccoons. Neural lesions compatible with acute sarcocystosis were detected in eight raccoons. The predominant lesions were meningoencephalitis and perivascular mononuclear cells. Histologic evidence for the Canine Distemper Virus was found in one raccoon. Schizonts and merozoites were present in the encephalitic lesions of four raccoons. Mature sarcocysts were present within myocytes of five raccoons. In six raccoons, S. neurona schizonts and merozoites were confirmed by immunohistochemical staining with S. neurona-specific polyclonal antibodies. Viable S. neurona was isolated from the brains of two raccoons by bioassay in interferon gamma gene knockout mice and in cell cultures seeded directly with raccoon brain homogenate. Molecular characterization was based on raccoon no. 68. Molecular characterization based on multi-locus typing at five surface antigens (SnSAG1-5-6, SnSAG3 and SnSAG4) and the ITS-1 marker within the ssrRNA locus, using DNA isolated from bradyzoites released from sarcocysts in a naturally infected raccoon (no. 68), confirmed the presence of S. neurona antigen type I, the same genotype that caused a mass mortality event in which 40 southern sea otters stranded dead or dying within a 3 week period in April 2004 with S. neurona-associated disease. An expanded set of genotyping markers was next applied. This study reports the following new genotyping markers at 18S rRNA, 28S rRNA, COX1, ITS-1, RON1, RON2, GAPDH1, ROP20, SAG2, SnSRS21 and TUBA1 markers. The identity of Sarcocystis spp. infecting raccoons is discussed.
在此，我们报告首次已知的由肉孢子虫属神经元肉孢子虫自然感染浣熊引发的临床原生动物脑脊髓炎疫情。北美负鼠（弗吉尼亚负鼠）和南美负鼠（白腹负鼠）是其已知的终末宿主。其他几种动物是其中间宿主或异常宿主。浣熊（北美浣熊）被认为是美国神经元肉孢子虫最重要的中间宿主。美国超过50%的浣熊肌肉中有肉孢子虫囊肿，然而浣熊的临床肉孢子虫病却很罕见。2014年，在美国密苏里州圣路易斯动物园的场地内发现38只自由放养的浣熊死亡或濒死。濒死个体虚弱、嗜睡且轻度共济失调；几只伴有眼鼻分泌物。发现7只浣熊死亡，31只被实施安乐死。对9只浣熊进行了尸检。在8只浣熊中检测到与急性肉孢子虫病相符的神经病变。主要病变为脑膜脑炎和血管周围单核细胞。在1只浣熊中发现了犬瘟热病毒的组织学证据。4只浣熊的脑炎性病变中存在裂殖体和裂殖子。5只浣熊的肌细胞内存在成熟的肉孢子虫囊肿。在6只浣熊中，通过用神经元肉孢子虫特异性多克隆抗体进行免疫组织化学染色，证实了神经元肉孢子虫裂殖体和裂殖子的存在。通过在干扰素γ基因敲除小鼠中进行生物测定以及直接接种浣熊脑匀浆的细胞培养，从2只浣熊的脑中分离出了活的神经元肉孢子虫。分子特征分析基于68号浣熊。利用从自然感染浣熊（68号）肉孢子虫囊肿中释放的缓殖子中分离的DNA，基于5个表面抗原（SnSAG1 - 5 - 6、SnSAG3和SnSAG4）以及小亚基核糖体RNA基因座内的ITS - 1标记进行多位点分型的分子特征分析，证实存在神经元肉孢子虫I型抗原，该基因型与2004年4月在3周内导致40只南海獭因神经元肉孢子虫相关疾病而搁浅死亡或濒死的大规模死亡事件的基因型相同。接下来应用了一组扩展的基因分型标记。本研究报告了18S rRNA、28S rRNA,、COX1、ITS - 1、RON1、RON2、GAPDH1、ROP20、SAG2、SnSRS21和TUBA1标记处的以下新基因分型标记。还讨论了感染浣熊的肉孢子虫属的鉴定问题。
