青海省牦牛弓形体病的血清学调查

为摸清青海省牦牛弓形体病的感染情况,对来自12个县（市）的614份牦牛血清采用正向间接血凝试验（IHA）方法进行了血清学检测,结果检出阳性样品47份,阳性率为7.65%,表明青海省牦牛普遍存在弓形体的感染。     

Establishment of Double-antibody Sandwich ELISA to Diagnose Acute Toxoplasmosis

The zoonotic protozoa Toxoplasma gondii is an opportunistic pathogen and distributes worldwide. Acute Toxoplasma infection causes serious pathological damages. Dense granule protein 1(GRA1) secreted by dense granule is an important component of Circulating antigen, which is an indication of acute toxoplasmosis. We aimed to use a monoclonal antibody against TgGRA1 to establish an enzyme-linked immunosorbent assay that targets antigen GRA1 in serum for acute toxoplasmosis diagnosis. First, the spleens of TgGRA1-His immunized mice were fused with SP2/0 cells,then we screened hybridomas that can constantly secret monoclonal antibody to the supernatant and injected them into mice to produce a large amount of mAbs. After the identification and purification of ascites, we choose one mAb as a capture antibody, HRP conjugated mouse anti-TgGRA1 polyclonal antibody as a detection antibody to develop sandwich ELISA. This method was used to detect samples from swine and mice artificially infected with Toxoplasma gondii. Besides, the results were compared with that of nPCR and two commercial kits to evaluate the efficiency of sandwich ELISA. We successfully got 4 mAbs with ascitic titers of 10⁶-10⁷, their subtypes are IgG1. Indirect fluorescent assay and Western blot showed that all of them can react specifically with TgGRA1.1G2 mAb and HRP conjugated mouse anti-TgGRA1 polyclonal antibody were used subsequently to establish sandwich ELISA for diagnosing acute infection. After optimization, sandwich ELISA can specifically detect 1.563 ng·mL^-1 GRA1 or 100 ng·mL^-1 ESA in serum. When detecting experimental animal samples, the sandwich ELISA exhibited the high consistency with the results of nPCR and showed higher efficiency than the commercial kits. In summary, we established a sandwich ELISA for acute toxoplasmosis diagnosis that captures one certain toxoplasma antigen GRA1, samples of artificially infected animals can be detected by this method, which makes acute toxoplasmosis diagnosis more reliable. It has guiding significance for clinical treatment of acute toxoplasmosis.     

A case of fatal systemic toxoplasmosis in a cat being treated with cyclosporin A for feline atopy

Acute systemic toxoplasmosis was diagnosed in a 4-5-year-old, male, Domestic Short Hair cat, which had been on cyclosporine A immunomodulatory therapy for feline atopy, over an 8-month period. Cyclosporin A (CsA) has shown promising results as a immunosuppressive agent in the cat for the treatment of eosinophilic plaque and granulomas, allergic cervico-facial pruritus, feline atopy and other immune-mediated dermatoses. However, inhibition of T-lymphocyte function by CsA is believed to have predisposed this cat to the development of a newly acquired, acute Toxoplasma gondii infection, as characterized by severe hepatic and pancreatic pathology in conjunction with the heavy parasite load demonstrated on immunohistochemical (IHC) stains for T. gondii. Cats on CsA therapy appear to be at risk of developing fatal systemic toxoplasmosis.     

绒山羊布氏杆菌病、衣原体和弓形虫病的血清学检测

采用布氏杆菌虎红平板凝集试验、弓形虫和衣原体间接血凝试验对360份绒山羊血清进行布氏杆菌病、弓形虫病和衣原体病的血清学调查。结果在绒山羊的血清样品中检出布氏杆菌病阳性血清8份,布氏杆菌病的血清阳性率为2．22％;检出弓形虫15份,弓形虫病血清阳性率为4．17％;检出衣原体23份,衣原体病血清阳性率为6．39％。     

Isolated lepromatous conjunctivo-corneal granuloma in a cat from Italy

Objective  To describe a case of a conjunctivo-corneal mass in a cat associated with acid-fast bacilli.
Methods  A 2-year-old female black European Short-Hair cat, living outdoors in a suburban environment in Italy, was referred for evaluation of a nodular, vascularized mass of 2 weeks duration. The mass involved the dorsal bulbar conjunctiva at the temporal canthus of OS and invaded the sclera and cornea. Routine ophthalmic and systemic examination, serologic testing, cytology and histology of the mass were performed. Mycobacterium specific polymerase chain reaction (PCR) of variable regions 1, 2 and 3 of the 16S ribosomal RNA (rRNA) gene was also performed.
Results  Neutrophils, lymphocytes, macrophages and giant cells with intracytoplasmic acid-fast bacilli were seen on cytological examination. The histological examination confirmed the presence of a granulomatous lesion with acid-fast bacilli within macrophages. Bacteriological culture of the material from the lesion was negative for Mycobacterium spp. Mycobacterium 16S rRNA gene specific PCR was positive.
A diagnosis of feline leprosy was made. The owners refused any treatment, and 1 year later the lesion was still present.
Conclusions  Veterinary ophthalmologists should be aware of conjunctivo-corneal leproma as an unusual symptom of leprosy.     

1种检测弓形虫急性感染的夹心ELISA方法

弓形虫（Toxoplasma gondii）是一种人畜共患机会性致病原虫,其急性感染可导致宿主产生明显的临床症状和严重的病理损伤。弓形虫致密颗粒蛋白1（dense granuleprotein 1,GRA1）是一种良好的诊断抗原,也是弓形虫急性感染的标志物循环抗原（circulating antigen,CAg）的重要组分。本研究利用TgGRA1单克隆抗体建立双抗体夹心ELISA方法,为急性弓形虫感染的检测提供依据。将免疫GRA1-His的小鼠脾细胞与SP2/0进行融合,筛选出能稳定分泌抗体的杂交瘤细胞。选择其中一种单抗与HRP标记后的鼠源GRA1多抗配对,建立1种双抗体夹心ELISA方法,检测人工感染弓形虫的猪和小鼠血清样品,并将检测效果与巢式PCR（nest PCR,nPCR）和商品化试剂盒进行比较。结果筛选到4株杂交瘤细胞,腹水效价为10⁶~10⁷,亚型均为IgG1;IFA和Western blot结果显示,4株单抗均具有良好的反应性和特异性。选择1G2单抗和HRP标记多抗配对,建立了循环抗原双抗体夹心ELISA方法,最低能够检测到血清中1.563 ng·mL^-1 GRA1抗原,或者100 ng·mL^-1 ESA。该方法与nPCR相比具有较高的一致性,较市售商品化试剂盒更为准确可靠。本研究第1次将GRA1抗原作为急性弓形虫感染的诊断指标,建立相应的检测方法,成功地在人工感染样品中检测到弓形虫急性感染,可为弓形虫急性感染的诊断提供参考,对临床上急性弓形虫病的治疗有指导意义。