Cloning and Sequence Analysis of the Gene Encoding LipL32 of <Emphasis Type="Italic">Leptospira interrogans</Emphasis> Serovar Sejroe

Leptospira, a member of the order Spirochaetales, is the causative agent of leptospirosis, an important zoonosis encountered worldwide. The Leptospira interrogans serovar Sejroe was grown in EMJH medium and its DNA was isolated using standard techniques. The LipL32 gene was amplified using the reported primer of Kirschneri of LipL32. The amplified product was found to comprise 756 base pairs. This amplified gene fragment of LipL32 lipoprotein was cloned in E. coli (DH5α) cells using pDrive plasmid as a vector. The recombinant cells were selected on LB agar medium containing ampicillin, X-gal and isopropyl-β-d-thiogalactopyranoside. Plasmid was extracted from the recombinant white colonies, and restriction endonuclease (RE) analysis was carried out using PstI and SalI. On partial sequence analysis, the product exhibited 756 base pairs, corresponding to 251 amino acids. The cloned gene could be further used for expression of recombinant protein for serodiagnosis of leptospirosis.     

Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs

An IgG-ELISA used recombinant antigen and a rapid flow-through enzyme immunoassay were developed for rapid screening of leptospiral antibodies in dogs using recombinant LipL41, which is one of the conserved outer membrane proteins in pathogenic leptospires as the coating antigen. Results from this study were compared with the standard microscopic agglutination test and found that the sensitivity and specificity of the enzyme-linked immunosorbent assay were 75.46% and 93.29% and whereas that of flow-through-based dot-immunobinding assay were 87.73% and 89.63%, respectively. Relative merits of these tests were also assessed. The flow-through-based dot-immunobinding assay was thus proved to be a valid screening test for canine leptospirosis.     

雏番鸭细小病毒病诊断技术和试剂的研究

根据番鸭细小病毒单克隆抗体（ＭＰＶ－ＭｃＡｂ）致敏的胶乳可与ＭＰＶ产生特异性凝集反应，而这种特异性凝集反应可被ＭＰＶ抗体所抑制的原理，建立了胶乳凝集试验（ＬＰＡ）和胶乳凝集抑制试验（ＬＰＡＩ）。致敏抗体蛋白浓度为０．５ｇ／Ｌ时，ＬＰＡ可检测出ＭＰＶ最小蛋白量为２μｇ／Ｌ。对ＬＰＡ与ＦＡ、ＬＰＡＩ与ＡＧＰ作了比较，结果：ＬＰＡ阳性率９０．５％，ＦＡ阳性率９２．４％，两者的符合率９２．４％；鸭血清ＬＰＡＩ抗体效价在Ｌｏｇ２５以下时，ＡＧＰ试验均阴性，前者比后者敏感性高３２～６４倍。ＬＰＡＩ抗体效价与保护力相关性试验结果表明，当鸭血清中ＬＰＡＩ抗体效价在Ｌｏｇ２１以上时，雏番鸭均能耐受强毒的攻击，两者呈正相关。致敏胶乳和抗原保存时间在４～８℃时，分别为５个月和６个月；在２２～２８℃时，分别为７ｄ和１５ｄ。     

A multipronged approach for the detection of leptospirosis in captive sloth bears (Melursus ursinus) in Agra and Bannerghatta sloth bear rescue centers in India

Leptospirosis is an exacerbating factor responsible for the drastic decline of sloth bear population in India. In this study, a multipronged approach based on antigen detection using Polymerase Chain Reaction (PCR) employing G1/G2 and LigBF/LigBR primers, antibody detection using Microscopic Agglutination Test (MAT) and recombinant LigBCon1-5 antigen based Latex Agglutination Test (rLigBCon1-5 LAT), serum biochemistry using hepatic (serum glutamate oxalo acetic transaminase (SGOT) and serum glutamate pyruvic transaminase (SGPT) and renal biomarkers (blood urea nitrogen (BUN) and Creatinine) and gross/histopathological evidence in liver and kidneys were employed to investigate leptospirosis in captive sloth bears. A total of 133 serum samples collected from Agra (n=113) and Bannerghatta (n=20) sloth bear rescue centers were screened using MAT and rLigBCon1-5 LAT. A total of 87 and 78 sera tested positive by MAT and LAT respectively. Pyrogenes was the leading serovar obtained using MAT followed by Icterohaemorrhagiae, Javanica, Grippotyphosa, Canicola and Tarassovi. The relative sensitivity, specificity and accuracy of rLigBCon1-5 LAT in comparison to MAT were 89.66%, 100% and 93.23% respectively. PCR performed on hepatic and renal tissues showed amplicon of 285 and 219 base pairs for G1/G2 and LigBF/LigBR primers respectively. Gross evidence (icteric liver, severely engorged hepatic sinusoids, congested kidneys with necrotic white spots on sub capsular surface), histopathology (severe hepatic degeneration and tubulointerstitial nephritis) and elevated hepatic/renal biomarkers were suggestive of leptospirosis. This study suggests that rLigBCon1-5 LAT can be employed as a pen-side test for detecting leptospirosis in sloth bears.     

Evaluation of ELISA for the detection of Toxoplasma antibodies in swine sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from greater than 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving less than 10 EIU were negative in the other tests, and all those with greater than 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10-70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

Prevalence of antibodies of Leptospira serovars in beef cattle in central Queensland.

OBJECTIVE: To obtain up-to-date data on the prevalence of antibodies to Leptospira serovars in central Queensland beef herds preliminary to assessing their role in bovine subfertility and the role of cattle as a zoonotic reservoir. DESIGN: Sera from 2857 female cattle in 68 central Queensland beef herds were tested for antibodies to 14 Leptospira serovars using the microscopic agglutination test. Vaccination use and age of cattle were collected to enable the calculation of crude and age-stratified seroprevalences. RESULTS: The most commonly detected antibodies were to serovars hardjo (15.8% crude seroprevalence), tarassovi (13.9%), pomona (4.0%) and szwajizak (2.2%). Vaccinates were omitted from the hardjo and pomona seroprevalence data. The seroprevalence for hardjo and pomona tended to increase with age of the animals. CONCLUSION: These results are broadly similar to those of previous serological surveys. The data suggest that serovars other than hardjo, pomona and tarassovi, are unlikely to have a significant role in bovine subfertility and that cattle are unlikely to be a source of human infection with them in central Queensland.     

Evaluation of three slide agglutination tests for rapid identification of Staphylococcus aureus.

Three slide agglutination tests for identification of Staphylococcus aureus were compared. The agglutination tests used for evaluation were Staphaurex (Wellcome Diagnostics), Staphyslide-Test (BioMerieux), and ANI S. aureus TEST (Ani Biotech Oy). A total of 347 isolates were analyzed, including 288 strains of S. aureus, 49 of S. epidermis, 11 of S. intermedius, 12 strains of other staphylococci and 14 non-staphylococcal strains. One hundred of the S. aureus strains were isolates from cases of food poisoning, 129 from mastitis and 59 from other clinical cases. The sensitivities of the tests were also compared using diluted suspensions of S. aureus strains and with purified Protein A dilutions. The results showed that the sensitivities of the tests were 98.6%, 97.9% and 99.0% for Staphaurex, Staphyslide-test and ANI S. aureus TEST, respectively. The specificities were 100% for the Staphyslide test and 98.8% for both the ANI S. aureus TEST and the Staphaurex test. The sensitivities measured with diluted S. aureus strain suspensions and Protein A solutions were equal with the Staphaurex and ANI S. aureus TEST. All the agglutination tests studied proved to be practical, easy to use and accurate for the rapid identification of S. aureus strains from culture isolates.     

广西某地区猪伪狂犬病血清学调查

采用乳胶凝集试验对广西某地区的68个养猪场和10个自然村40个散养的1 307头免疫和非免疫猪的血清进行伪狂犬病(PR)抗体检测,结果共有739头份血清抗体阳性,阳性率为56.54%,其中伪狂犬病疫苗免疫猪和非免疫猪的血清抗体阳性率分别为66.73%和18.47%.未免疫的规模猪场和散养户的血清抗体阳性率分别为21.43%和16%.结果表明,部分免疫猪的PRV抗体水平较低,应及时进行强化免疫;部分未免疫猪的血清抗体呈阳性,存在PRV感染现象,而且规模猪场感染程度比散养户严重,应及时采取必要的防控措施.     

Development of a Latex Agglutination Test Using The Major Epitope Domain of Glycoprotein E of Pseudorabies Virus Expressed in <Emphasis Type="Italic">E. coli</Emphasis> to Differentiate Between Immune Responses in Pigs Naturally Infected or Vaccinated with Pseudorabies Virus

A 0.8 kb DNA fragment encoding the major epitope domain of glycoprotein E (gE) of pseudorabies virus (PRV) was inserted downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induction by isopropy1-β-D-thiogalactopyranoside (IPTG), a high level expression of fusion protein was obtained. SDS-PAGE and western immunoblotting analysis showed that the fusion protein was 38 kDa and could bind with antisera against PRV. The protein existed mainly in the form of the inclusion body. After being denatured and renatured, the protein was used to prepare the latex antigen. The concentration of antigen, temperature and time for sensitization were optimized. The latex agglutination test (LAT) was able to differentiate sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs. The diagnostic specificity and sensitivity of the developed gE latex agglutination test (gE-LAT) were also evaluated by using sets of sera. The diagnostic specificity and diagnostic sensitivity of the gE-LAT were 96.77% and 95.76%, respectively. For comparison between gE-LAT and a commercial blocking enzyme-linked immunosorbent assays (ELISA), 260 serum samples were tested. The coincidence frequency of both assays was 96.94% (252/260). No significant difference was found between the two methods (p>0.05). For comparison between the abilities of gE-LAT and gE-ELISA to detect sera with low titres of gE-specific antibody, 66 sera from 22 pigs were tested. The data indicate that the gE-LAT is of similar sensitivity to gE-ELISA. These results indicate that gE-LAT using recombinant gE might be very useful as a routine screening method for the differential diagnosis of PRV infection.     

The Development and Application of the Latex Agglutination Test to Detect Serum Antibodies against Japanese Encephalitis Virus

The attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) was cultured in BHK-21 cells. The viral supernatant was purified and concentrated with PEG (MW 20 000). A suitable concentration of JEV antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity and stability of the antigen were assessed. A latex agglutination test (LAT) was developed for rapidly detecting antibody against JEV infection. The LAT and haemagglutination inhibition (HI) assay were compared by simultaneously testing 35 porcine serum samples from five farms. Ninety per cent (20/23) of the samples were seropositive by both assays. No significant difference was found between the two methods (p > 0.05). Furthermore, when 1613 porcine sera from 120 farms were tested by LAT, the number of positive sera was 652, while that of negative sera was 961, ranging from 20% to 50% positive throughout the year. These results indicate that LAT is an appropriate candidate method for epidemiological surveys for and diagnosis of Japanese encephalitis.     

Evaluation and comparison of native and recombinant LipL21 protein-based ELISAs for diagnosis of bovine leptospirosis

A 21-kDa leptospiral lipoprotein (LipL21) was evaluated for its diagnostic potential to detect bovine leptospirosis by ELISA. Both native LipL21 (nLipL21) and recombinant LipL21 (rLipL21) proteins were tested and compared regarding diagnostic efficiency, and no statistically significant difference was observed. The sensitivity of rLipL21 ELISA for 62 microscopic agglutination test (MAT) positive sera was 100% and the specificity with 378 MAT negative sera was 97.09%. Thus, rLipL21 protein-based ELISA could be used as an alternative to MAT for the diagnosis of bovine leptospirosis.     

乳胶凝集试验检测番鸭呼肠孤病毒方法的建立

用纯化的抗番鸭呼肠孤病毒病抗体致敏乳胶成功地建立了检测番鸭呼肠呼病毒的方法。通过试验，确定了抗体致敏乳胶的最佳浓度1：10(0．4242mg/mL)，最佳致敏温度37℃，最佳致敏时间2个小时。人工攻毒雏番鸭30只，用所建立的方法检测粪便。结果表明，攻毒后的第3天粪便中即可检测到病毒，直至攻毒鸭全部死亡都可从粪便中检测到病毒抗原；同样攻毒1日龄雏番鸭30只，剖检取心、肝、脾按常规方法处理，用所建立的方法的检测。结果表明，首先是脾脏在攻毒后第4天3只中有2只检测结果阳性，第5天2只死亡鸭中有一只肝检测结果阳性，脾脏2只都呈阳性，此后的病死鸭脾和肝均为阳性，而心脏则未见有阳性。这对，临床上诊断与防治番鸭呼肠孤病毒感染具有重要的参考意义。     

牛布鲁菌单克隆抗体乳胶凝集试验方法的建立

建立了用纯化好的牛布鲁菌(B.abortus)单克隆抗体致敏乳胶的检测方法。通过试验确定了抗体致敏乳胶最佳偶联蛋白量为1.23 mg/mL,最佳致敏时间4 h,最佳的乳胶浓度为1%。水样模拟样本的制作,取含1.0×109cfu/mL灭活的B.abortus544A生理盐水菌悬液各1 mL,加入9 mL自来水中,充分混合均匀,各取1 mL,土样、奶样模拟样本制作同上。最低检出率水样为3×104cfu/mL~1.0×105cfu/mL,土样、奶样为5×104cfu/mL~1.0×105cfu/mL。     

A Brief History and Overview of Toxoplasma gondii

Toxoplasma gondii was discovered by scientists working in North Africa and Brazil around 100 years ago. The parasite has since been found to be capable of infecting all warm‐blooded animals including humans making it one of the most successful parasitic organisms worldwide. The pathogenic potential of T. gondii was recognized in the 1920s and 1930s, in congenitally infected children presenting with the classic triad of symptoms, namely hydrocephalus, retinochoroiditis and encephalitis. In addition, around the same time T. gondii parasites were found to be associated with severe intraocular inflammation. In the 1980s, T. gondii emerged as a major cause of death in patients with acquired immunodeficiency syndrome, illustrating the importance of the immune system in controlling T. gondii infection. T. gondii was reported as a major cause of abortion in sheep in New Zealand in the 1950s, which raised questions about potential new transmission routes for the parasite. The discovery of the cat as the definitive host in the 1960s was a very important finding as it helped to complete our understanding of the parasite’s life cycle, and the oocyst stage of T. gondii shed in the faeces of infected cats was found to be an important source of infection for many intermediate hosts and helped to explain infection in herbivorous animals and people with a vegetarian diet. In addition, this stage of the parasite was very robust and could survive in the environment, depending on the climatic conditions, for up to 12–18 months. Knowledge of the parasite’s lifecycle, transmission routes, risk groups and host immune responses has helped in the development of strategies to control the disease, reduce transmission of the parasite and limit environmental contamination.     

免疫学技术在弓形虫检测方面的研究概况

弓形虫病是世界分布最广泛的一种人兽共患病,此病在温暖潮湿的地区感染率极高,所以建立高效敏感的检测方法是非常必要的。目前,国内检测弓形虫病的技术有多种,其中以免疫学技术为主。虽然病原学检测方法的敏感性高,特异性强,但其成本高,对人体会产生严重的副作用,所以并不适宜推广。近年来,通过对弓形虫表面抗原的大量研究,检测弓形虫病的免疫学技术也有了显著提高。从敏感性差、操作复杂到现在的特异性强、快速简便,从单一抗原的检测到现在的多表面抗原的检测,无疑为检测弓形虫带来了新的思路。论文主要对检测弓形虫的ELISA、改良凝集试验以及化学发光免疫分析的最新进展进行了综述。     

北京地区犬猫弓形虫病血清学调查

比较了酶联免疫吸附试验（ELISA）和乳胶凝集试验对犬猫进行龚地弓形虫（Toxoplasma gondii）抗体检测的结果.在66只犬猫的血清中,ELISA方法检测的阳性率为24.0%,乳胶凝集试验阳性率为21.0%,二者的检测结果差异不显著（P＞0.05）.后采用ELISA方法对北京地区家养的159只犬和128只猫进行了弓形虫IGg抗体的检测,共有21只犬（13.21%）和18只猫（14.06%）检测结果为阳性.1岁以下犬（3.70%）、猫（4.35%）低于1至3岁犬（11.29%）和猫（6.98%）的感染率.3～6岁犬猫的感染率分别是27.27%和16.22%,6岁以上犬（30.0%）、猫（32.0%）的感染率最高.不同性别之间感染无明显差异（P＞0.05）.     

Experimental Detection of Canine Haemoglobin (Occult Blood) in Canine Faeces by Reversed Passive Latex Agglutination

A reversed passive latex agglutination test (RPLA) using anti-canine haemoglobin (Hb) antibody was developed for detecting bleeding in the lower digestive organs in dogs, and its applicability as a simple test for faecal occult blood was assessed. In Ouchterlony's gel immunodiffusion test, the anti-canine Hb antibody used to sensitize the latex reacted with canine Hb but not with Hbs, plasmas or meat extracts from pigs, goats, sheep, cattle, horses or chickens, or with fish extracts. Using latex sensitized with 50 µg/mg of anti-canine Hb IgG antibody, the lowest limit of detection for canine Hb was 21 µg/ml, and the latex reacted negatively with all test specimens other than canine Hb. In an in vitro experiment with a mixture of canine faeces and erythrocytes, the antigenicity of the Hb was found to undergo only very slight changes even when the specimens were allowed to stand for 12 h at room temperature. Hb could not be detected by RPLA in any of four successive faecal samples from three experimental dogs after infusion of autologous blood (5, 3 or 1 ml) into the stomach. In 3 other experimental dogs given an infusion of autologous blood (5, 3 or 1 ml) into the ascending colon, the presence of Hb was confirmed by RPLA in all four successive faecal samples obtained from those which received 5 or 3 ml of blood and in all except that obtained following the first defecation from the animal which had received 1 ml of blood.     

Evaluation of 3 Serological Tests for Early Detection Of Leptospira‐specific Antibodies in Experimentally Infected Dogs

Background

Leptospirosis in dogs is a disease of global importance. Early detection and appropriate therapeutic intervention are necessary to resolve infection and prevent zoonotic transmission. However, its diagnosis is hindered by nonspecific clinical signs and lack of rapid diagnostic tests of early infection. Recently, 2 rapid point‐of‐care tests (WITNESS Lepto [WITNESS Lepto, Zoetis LLC, Kalamazoo, MI, USA] and SNAP Lepto [SNAP Lepto, IDEXX Laboratories, Westbrook, ME, USA]) for detection of Leptospira‐specific antibodies in canine sera were developed.

Hypothesis

Immunoglobulin M‐based WITNESS Lepto containing multiple detection antigens can detect Leptospira‐specific antibodies to common leptospiral serovars earlier in the course of infection as compared to microscopic agglutination test (MAT) and SNAP Lepto.

Animals

Four groups of 8 6‐ to 8‐month‐old male Beagle dogs were used.

Methods

Thirty‐two healthy seronegative dogs were inoculated experimentally with serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona (8 dogs/serovar). Acute‐phase sera were collected at regular intervals and monitored for Leptospira‐specific antibodies by WITNESS Lepto, MAT, and SNAP Lepto.

Results

Seroconversion was detected in all dogs by day 10 by WITNESS Lepto and in 30 of 32 dogs by day 14 by MAT. The SNAP Lepto test detected seroconversion in 3 dogs during the 2 weeks postchallenge.

Conclusions

Immunoglobulin M‐based WITNESS Lepto detected immune responses specific to multiple leptospiral serovars early in the course of infection and identified seroconversion in all animals earlier than did the gold standard MAT. The SNAP Lepto test displayed considerably lower and inconsistent performance during the study period. At the point‐of‐care, WITNESS Lepto should be the test of choice for rapid and reliable screening of acutely ill dogs suspected to have leptospirosis.