Use Of Enzyme Immunoassay Research Articles

Avian anti-HBV immunoglobulin: New tool to improve hepatitis B diagnosis methods

Immunoglobulin Y (IgY) could be used in serological diagnosis focused on several infectious agents. This study aims to produce IgY anti-hepatitis B virus surface antigen (anti-HBs) and to assess its use in enzyme immunoassays. Antibodies were produced by immunizing chickens with Hepatitis B vaccine associated (group A), or not, with adjuvant CpG-ODN (group B). Eggs were collected for 20 weeks, yolks were purified based on using polyethylene glycol and affinity chromatography. IgY anti-HBs was featured based on SDS-PAGE and Western Blot techniques. Total protein concentration was measured through spectrophotometry. In-house ELISA used to detect HBsAg was developed based on using IgG/HRP conjugate and IgY-anti-HBs sensitized microplates. Thus, IgY anti-HBs were confirmed through molecular pattern based on SDS-PAGE, whereas specificity of anti-HBs was confirmed through Western Blot. Mean total protein reached 3.27 ± 3.00 mg/mL and 3.11 ± 3.12 mg/mL in groups A and B, respectively. In-house ELISA was developed based on using a panel of HBV positive and negative serum samples; it recorded 100 % sensitivity and 78.9 % specificity to detect HBsAg. In conclusion, it was possible producing anti-HBs IgY by immunizing chickens with HBV vaccine; this molecule could be used as capture antibody to help detecting HBsAg in-house ELISA.

Assessing reproduction in wolverines (Gulo gulo): Fecal progesterone levels in zoo and wild females

Wolverines (Gulo gulo) populations are under threat by increased human activities. A better understanding of their reproductive physiology would benefit research and monitoring of this species. The use of enzyme immunoassay (EIA) to measure fecal progesterone metabolites (FPM) is a powerful tool to understand wildlife reproductive physiology, and a non-invasive method for use in both zoo and wild populations. The main objective of this study was to determine reproductive status in female wolverines using FPM levels, first by monitoring of longitudinal changes in FPM in zoo housed females in breeding situations and second to compare those values to FPM measured in feces obtained from wild females observed in the field during reproductively active periods (Dec–Apr). Female reproductive physiology was assessed by measuring FPM in three zoo housed wolverines to determine species-specific levels arising from ovulation encompassing embryonic diapause and ovarian reactivation (resumption of ovarian luteal activity after diapause) during pregnancy or pseudopregnancy, which in turn enabled verification of reproductive status in 15 wild wolverines, monitored over three years. In the zoo females, FPM levels were sustained at basal levels if ovulation and/or breeding did not occur. After ovulation, both pregnancy or pseudopregnancy exhibited a bi-phasic pattern of FPM increases, with a 4.9-fold increase above baseline during embryonic diapause and a further 4.4-fold increase after ovarian reactivation. FPM levels during pregnancy and pseudopregnancy were indistinguishable, thus increases in FPM levels are only a confirmation of ovulation, and not diagnostic for pregnancy. In wild females, assessment of FPM levels in samples collected from December to February was able to predict a potentially pregnant/reproductively active female with levels above 8200 ng/g dried feces, or non-reproductive (nonovulatory) female during that period with levels below 1700 ng/g. Wild wolverine FPM levels indicative of pregnancy aligned with field observations of denning and/or lactation. FPM levels from samples collected from March and April were inconclusive as levels could be attributed to females being either postpartum or non-reproductive at that time. Timing of ovarian reactivation, and subsequent elevation of FPM, varied among individuals suggesting caution is warranted when assessing only one fecal sample per female. Understanding all factors that may impact reproductive success is essential for effective species conservation, particularly for species with low reproductive rates, like the wolverine. This study has demonstrated the potentially useful incorporation of non-invasive fecal hormone analysis for investigation of physiological parameters and reproductive status in wild wolverine females.

Использование иммуноферментного анализа для определения концентрации микотоксинов в органах, тканях и биологических жидкостях: обзор

Представлен обзор опубликованных исследований, касающихся определения различных микотоксинов в органах, тканях и биологических жидкостях сельскохозяйственных животных и человека. Основной упор сделан на подробном описании пробоподготовки гомогенатов органов к иммуноферментному анализу (ИФА), а также приведены данные по сравнению хроматографических методов исследования с иммуноферментным анализом. Учитывая более доступную стоимость ИФА диагностики, по сравнению с хроматографическими методами, а также легковыполнимую подготовку образцов, ИФА более предпочтителен для так называемого скрининга по определению уровня кумуляции микотоксинов на свиноводческих комплексах, птицефабриках и на других крупных сельскохозяйственных производствах. A review of published studies regarding the definition of various mycotoxins in organs, tissues and biological fluids of farm animals and humans is presented. The main emphasis is placed on a detailed description of the sample processing of organ homogenates for enzyme immunoassay (EIA) as well as data compared to chromatographic methods of the research with enzyme immunoassay. Considering the more affordable cost of EIA diagnostics in comparison with chromatographic methods as well as the quite easy preparation of samples, EIA is more preferred for so-called screening to determine the level of mycotoxin cumulation in pig breeding complexes, poultry farms and other large agricultural industries.

Surveillance for avian influenza virus in captive wild birds and indigenous chickens in Nigeria.

Several reports of avian influenza virus (AIV) have been made on commercial chickens and wild birds in sub-Saharan Africa, but there is paucity of information of AIV among captive wild birds and indigenous chickens. Blood samples were obtained randomly from captive wild birds and chickens. AIV nucleoprotein antibody detection involved the use of enzyme immunoassay (ELISA) and subsequent subtyping with H5 and H7 AIV antigens (haemagglutination inhibition). Four hundred birds belonging to nine families and 14 species were sampled, and overall prevalence of 23% (92/400) was obtained (captive wild birds (10.4%, 5/48), indigenous birds (47.3%, 87/184) and exotic commercial birds (0.0%, 0/168)). Twelve ELISA-positive birds (13.04%) were positive to H7 antigen. Univariate analysis indicated statistical significance of AIV prevalence in captive wild birds (p < 0.0001) and exotic birds (p < 0.0001) using indigenous chickens as reference. This study gave an evidence of exposure of captive wild birds and indigenous chickens to AIV in Nigeria. Scavenging activities common among indigenously raised chickens, unrestricted movement of nonflying wild birds within the captive complex and free access by migrating wild birds to captive wild birds and local chickens were likely factors observed to promote AIV transmission. Continuous surveillance can further highlight the roles played by these birds in the epidemiology of AIV.

Immunoassay and discriminant analysis to improve the diagnosis of ovarian cancer in the elderly.

Ovarian cancer is a common pathology in the structure of tumors among elderly women. The aim of the work is to identify the leading indicators of interleukin profile to improve the diagnosis of ovarian cancer in the elderly. Enzyme immunoassay studied serum levels of interleukins in 78 patients with ovarian cancer stage IIB-IIIB according to the classification of FIGO at the age of 65-70 years and 42 women of similar age without ovarian cancer at the time of the study and in history. The differentiation of the compared groups is carried out by means of discriminant method. The leading diagnostic interleukins for ovarian cancer in the elderly are IL-1α, IL-1β, IL-8 and TNF-α. The constructed discriminant models allow to differentiate patients with ovarian cancer and control women with an accuracy of 93.2-93.8%. The use of enzyme immunoassay and discriminant analysis improve the diagnosis of ovarian cancer in the elderly.

Isolation, preparation, and assessment of the immunogenicity effectiveness of a novel inactivated rotavirus vaccine strain: ZTR-68, rotavirus A genotype G1P[8

BackgroundRotavirus is one of the leading causes of diarrhoea in children in both developed and developing countries, resulting in an estimated 600 000 deaths worldwide each year. In China, roughly 13 400 children younger than 5 years die from rotavirus infection each year. We aimed to isolate and characterise a human virus for use as a candidate human inactivated rotavirus vaccine strain. We studied the preparation of the vaccine and assessed its immunogenicity effectiveness. MethodsFrom September, 2009, to November, 2012, we collected 206 stool specimens from children in Yunnan, China, and tested for the presence of rotaviruses by use of enzyme immunoassay and double-stranded RNA-PAGE. G and P typing of the positive samples were done by one-step RT-PCR and sequencing. A new human rotavirus was isolated from a child aged 3 months with acute gastritis from the First People's Hospital of Zhaotong City, Zhaotong, China, and was designated ZTR-68. We did a full genomic analysis of ZTR-68 and tested for identifiable characteristics of the virus. A scalable inactivated rotavirus vaccine was cultured by rolling bottles, purified using low-velocity centrifuge, microfiltration clarification, ultrafiltration concentration, and size-exclusion chromatography, and inactivated by the addition of formaldehyde. Its immunogenicity was studied in Balb/c mice by intramuscular injection. FindingsStrain ZTR-68 was isolated and plaque purified, and its complete genome was sequenced. ZTR-68 is a rotavirus A of genotype G1P[8] and has a close phylogenetic relation to Wa. We confirmed that our rotavirus vaccine was completely inactivated, and rotavirus antigen can be measured by ELISA. After intramuscular injection of the vaccine into BalB/c mice aged 6 weeks, high titres of rotavirus-specific IgG (>20 000) and neutralising activity (roughly 3000) were recovered in the sera of these mice. InterpretationThis newly isolated rotavirus, ZTR-68, is being used to establish a new strain of inactivated rotavirus vaccine. Our findings show that the inactivated rotavirus vaccine has good immunogenicity and provide evidence for further clinical development as an alternative candidate vaccine. FundingThis research was financially supported by the State Project for Essential Drug Research and Development, the Chinese national “Twelfth Five-Year” plan (project number: 2014ZX09102041004); the National High Technology Research and Development Program (“863” Program) of China (project number: 2012AA02A404); and the Yunnan Research Program of Application Foundation and Advanced Technology, self-raised funds (project number: 2013FZ140).

Usefulness of enzyme immunoassay (EIA) for screening of anti HIV antibodies in urinary specimens: A comparative analysis

BackgroundStandard HIV testing is done using serum or plasma. FDA approved ELISA to screen urine for IgG antibodies to HIV-1 in 1996. It is a simple, noninvasive test and is appropriate for developing countries where health care personnel may not be professionally trained or where clean needles for drawing blood may not always be available. Methods436 individuals with high-risk behavior and strong clinical suspicion of HIV infection were screened for IgG antibodies to HIV-1 in urine by ELISA. Urine HIV testing was performed by enzyme immunoassay, at the ongoing Voluntary Confidential Counseling and Testing Center (VCCTC) at a large tertiary care microbiology lab. The individuals enrolled for the study had high-risk exposure to the virus and majorities were from a state with a high incidence of HIV infection. In all individuals, both serum and urine were tested for IgG antibodies to HIV-1. ResultsOverall, 135 individuals (30.96%) were HIV-positive, of whom 96 (71%) had never previously tested positive; 87% of those who tested positive received their results, and most were referred for medical care. Sensitivity, specificity and predictive values of HIV-1 urine ELISA test kit were determined. Sensitivity was found to be 89.6%; 95% CI [82.9–94.0], specificity 97.3%; 95% CI [94.6–98.8], positive predictive value 93.8%; 95% CI [87.8–97.1] and negative predictive value 95.4%; 95% CI [92.3–97.4]. ConclusionEfficiency, sensitivity, and specificity of the urine-based screening for HIV-1 test kits were excellent as compared to the reference test.

Comparison of Hemagglutination Inhibition Assay and Enzyme Immunoassay for Determination of Mumps and Rubella Immune Status in Health Care Personnel

BackgroundScreening tests are available to determine immunity to vaccine‐preventable diseases, such as mumps and rubella. We aimed to define better assay for detecting immune status of health care personnel to vaccine‐preventable diseases.MethodsMumps and rubella antibodies of health care personnel at Shimane University Hospital were examined by hemagglutination inhibition assay (HI), comparing with those by enzyme immunoassay (EIA).ResultsA total of 910 sera from health care personnel were tested. There was poor correlation between HI and EIA in detecting mumps antibodies with correlation coefficient values (r) = 0.190 (P < 0.001), but in rubella antibodies HI and EIA were relatively well correlated (r = 0.930, P < 0.001). Seropositivity rate of HI versus EIA was found to be 65.7 versus 93.2, and 89.5 versus 86.5% for mumps and rubella, respectively. As compared with EIA, HI identified sixfold larger seronegative subjects in mumps. Moreover, in mumps, 88.8% of seronegative subjects detected by HI were seropositive by EIA, while 3.7% of seropositive subjects detected by HI were seronegative by EIA. In rubella, 2.1% of seronegative subjects detected by HI were seropositive by EIA, and 1.7% of seropositive by HI was seronegative by EIA.ConclusionConsiderable difference between HI and EIA in determining immune status of health care personnel to mumps and rubella suggests beneficial use of EIA for the identification of accurate susceptible personnel who subsequently undergo an effective vaccination programs. Seroprevalence survey of health care personnel by using appropriate assay is essential for prevention and infection control strategies in health care settings.

Observations on human parvovirus B19 infection diagnosed in 2011

The incidence of human parvovirus B19 infection is unknown. A retrospective analysis of clinical and laboratory findings was carried out in patients diagnosed with human parvovirus B19 infection in 2011 in a virologic laboratory of a single centre in Hungary. Clinical and laboratory data of patients with proven human parvovirus B19 infection were analysed using in- and out-patient files. In 2011, 72 patients proved to have human parvovirus B19 infection with the use of enzyme immunoassay. The clinical diagnoses of these patients were as follows: human parvovirus B19 infection (30.6%), transient aplastic crisis (16.7%), arthritis (8.3%) and acute hepatitis (4.1%). Symptoms of each of the four phases of the infection occurred in various combinations with the exception of the monophase of cheek exanthema. This occurred without the presence of other symptoms in some cases. Leading symptoms and signs were exanthema (in 74.6% of cases), haematological disorders (in 69% of cases), fever (in 54.9% of cases) and arthritis (in 33.8% of cases). Several atypical dermatological symptoms were also observed. Acute arthritis without exanthema was noted in 8 patients. Of the 72 patients with proven human parvovirus B19 infection there were 7 pregnant women, and one of them had hydrops foetalis resulting spontaneous abortion. In 16 patients (22.5%) human parvovirus B19 IgG was undetectable despite an optimal time for testing. The observations of this study may contribute to a better recognition of clinical symptoms of human parvovirus B19 infection.

Clinical Specificity of the Enzyme Immunoassay Test for Coccidioidomycosis Varies According to the Reason for Its Performance

The diagnosis of coccidioidomycosis relies heavily on serologic test results in addition to clinical history, physical examination, and radiographic findings. Use of the enzyme immunoassay (EIA) has increased because it is rapidly performed and does not require referral to a reference laboratory, as do complement fixation and immunodiffusion tests. However, interpretation of immunoglobulin M (IgM) reactivity by EIA in the absence of immunoglobulin G (IgG) reactivity has been problematic. We conducted a retrospective medical record review of all patients with such IgM reactivity at our institution to identify situations where the finding was more likely to be clinically specific for coccidioidal infection. From 1 January 2004 through 31 December 2008, a total of 1,117 patients had positive EIA coccidioidal serology or EIA IgM-only reactivity; of these, 102 patients (9%) had EIA IgM-only reactivity. Among the 102 patients with EIA IgM-only reactivity, 60 were tested to evaluate symptomatic illness, 13 for follow-up of previously abnormal serology, and 29 for screening purposes. Of the 102 patients, 80 (78%) had positive serologic findings by other methods or had positive culture or histology. Fifty-four (90%) of the 60 patients whose serology was performed to evaluate symptomatic illness had coccidioidal infection, whereas 13 (45%) of 29 patients whose serology was performed for screening purposes had coccidioidal infection. Of the 102 patients with isolated IgM reactivity by EIA, 12 later seroconverted to IgG and IgM reactivity. The use of EIA for screening in 29 asymptomatic persons was associated with unconfirmable results in 13 (45%). Although the majority of patients in our study with isolated IgM reactivity by EIA had probable or confirmed coccidioidomycosis, this result must be interpreted with caution for asymptomatic patients.

Epidemiology of Rotavirus Gastroenteritis among Children &lt;5 Years of Age in Morocco during 1 Year of Sentinel Hospital Surveillance, June 2006–May 2007

In anticipation of vaccine introduction, we assessed the epidemiology, burden, and genotype of infecting strains of rotavirus disease among Moroccan children hospitalized for acute gastroenteritis. From June 2006 through May 2007, 345 children <5 years of age who had acute gastroenteritis and were admitted to 4 sentinel hospitals in different regions of Morocco were enrolled in this surveillance study, and stool specimens were tested for the presence of rotavirus with use of enzyme immunoassay. RNA from positive samples was genotyped by reverse-transcriptase polymerase chain reaction. Overall, 314 children had complete data available, and among these, 138 (44%) tested positive for rotavirus. Rotavirus infection was most common among children <24 months of age (95% of all hospitalizations for rotavirus infection). Rotavirus infection was detected year-round at all 4 sites but was most prevalent from September through January. Genotype analysis demonstrated that 30.6% of samples were G1[P8], 26% were G9[P8], 7.5% were G2[P6], 3.7% were G1[P6], and 0.7% were G2[P8]. Nucleotide sequencing analysis of G- or P-untypeable strains showed that 4.5% were G9[P8], 2.2% were G1[8], 2.2% were G2[P6], and 1.5% were G2[P4]. A high frequency of mixed infection (21%) was found, of which G1G2[P8] accounted for the majority (16.4%). Rotavirus was responsible for 44% of all hospitalizations for diarrhea among young children at these 4 separate sites in Morocco. These data will help inform a decision on the introduction of rotavirus vaccine in Morocco. Continued and extended surveillance in Morocco will be important to monitor changes in the epidemiology of rotavirus disease and the impact of vaccination after introduction.

Population‐Based Incidence of Intussusception and a Case‐Control Study to Examine the Association of Intussusception with Natural Rotavirus Infection among Indian Children

A rotavirus vaccine previously licensed in the United States was withdrawn because it caused intussusception. Data on background intussusception rates in developing countries are required to plan pre- and postlicensure safety studies for new rotavirus vaccines. Also, it is unclear whether natural rotavirus infection is associated with intussusception. Passive surveillance for intussusception in a large, well-defined, poor, urban population in Delhi, India, was conducted in 2 phases. Intussusception was confirmed by ultrasonography or surgery. Fecal samples obtained from patients with intussusception at study hospitals (irrespective of their residence in study areas) and healthy control subjects were tested for rotavirus with use of enzyme immunoassay. If available, resected intestinal tissue samples were tested for rotavirus with use of immunohistochemistical analysis and reverse-transcription polymerase chain reaction. The incidence of intussusception requiring hospitalization was 17.7 cases per 100,000 infant-years of follow-up (95% confidence interval, 5.9-41.4 cases per 100,000 infant-years). Detection rates of rotavirus in stool samples did not differ significantly between case patients and control subjects (4 of 42 case patients vs 6 of 92 control subjects), and no evidence of rotavirus was detected in any of the 22 patients with intussusception for whom intestinal tissue samples were available. The incidence of intussusception among Indian infants appears to be lower than that reported in other middle- and high-income countries. Natural rotavirus infection does not appear to be a major cause of intussusception in Indian infants.

In vitro effects of lactoferrin on lipopolysaccharide-induced proliferation, gene expression, and prostanoid production by bovine peripheral blood mononuclear cells

To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro. PBMCs isolated from 15 Holstein bull calves. Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 microg/mL) in a checkerboard design. Incorporation of 3H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E2 production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays. Lactoferrin supplementation significantly reduced LPS-induced incorporation of 3H-thymidine and production of prostaglandin E2 by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA. Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E2 production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.

Antibodies to Pneumococcal Surface Protein A Families 1 and 2 in Serum and Saliva of Children and the Risk of Pneumococcal Acute Otitis Media

Pneumococcal surface protein A (PspA) is a highly variable yet cross-reactive protein that exists as 2 major families. We assessed the development of human serum and salivary antibodies against the PspA families 1 (PspA1) and 2 (PspA2) in early childhood and their role in the prevention of pneumococcal acute otitis media (AOM). Serum levels of IgG and salivary levels of IgA antibodies to PspA1 and PspA2 were measured by use of enzyme immunoassay from the samples from the Finnish Otitis Media Cohort Study obtained at the ages of 12 months (287 and 160 samples, respectively) and 18 months (258 and 131 samples, respectively). The Cox proportional hazard model was used to evaluate the relative risk (RR) of pneumococcal AOM during the 6 months after sampling relative to concentration of serum or presence of salivary anti-PspA in the samples. Anti-PspA1 and anti-PspA2 concentrations at 12 and 18 months were related to prior culture-confirmed pneumococcal exposure. The concentrations of serum anti-PspA were not significantly associated with the risk of pneumococcal AOM. At 18 months, the presence of salivary anti-PspA was significantly associated with a lower risk of pneumococcal AOM during the 6 months after sampling (RR, 0.27 [95% confidence interval, 0.11-0.69]). The lowered risk of pneumococcal AOM associated with the presence of salivary anti-PspA at 18 months suggests that mucosal anti-PspA antibodies have a role in the prevention of pneumococcal AOM.

Surveillance for Shiga Toxin–producingEscherichia coli,Michigan, 2001–2005

A surveillance system used different detection methods to estimate prevalence of Shiga toxin–producing Escherichia coli during 2003–2005 and 2001–2002. More non-O157 serotypes were detected by enzyme immunoassay than by evaluation of non-sorbitol–fermenting E. coli isolates. We therefore recommend use of enzyme immunoassay and culture-based methods.

Chlamydia pneumoniae antibody levels before coronary events in the Helsinki Heart Study as measured by different methods

The lack of specific tests for the diagnosis of chronic Chlamydia pneumoniae infection has led to the use of enzyme immunoassay (EIA) instead of the gold standard, that is, microimmunofluorescence (MIF), in the measurement of C. pneumoniae antibodies. We assessed the predictive values of C. pneumoniae antibody levels and seroconversions measured by MIF and EIA for coronary events in the prospective Helsinki Heart Study. Sera from 239 cases with coronary events and 239 controls were available at the baseline and data from 210 cases and 211 controls before and after the event. The agreement between MIF and EIA antibody levels was best in high antibody titers. In conditional logistic regression analysis, only high IgA MIF titers (≥40) at the baseline predicted future coronary events, and the participants with MIF seroconversion between consecutive sera had a higher (nonsignificant) risk for coronary events than the controls. The difference in the kinetics of EIA and MIF antibodies demonstrated that MIF should remain the gold standard.

Virulence markers of LCR plasmid in Indian isolates of Yersinia pestis

Presence of 10 important yop genes in Yersinia pestis isolates (18 in number) of Indian origin from 1994 plague outbreak regions of Maharashtra (6 Rattus rattus & Tetera indica rodents) and Gujarat (11 from human patients, 1 from R. rattus) and from plague endemic regions of the Deccan plateau (8 from T. indica) was located by PCR and specific enzyme immunoassay. PCRs were standardized for six effector yops (YopE, YopH, YopJ, YopM, YopO and YopT), three translocator yops (YopB, YopD and YopK) and a regulator LcrV gene. Amplification of all the 10 yop genes was observed in isolates recovered from pneumonic patients and in 5 of 7 rodents from outbreak regions. Among these, amplification of the yopD gene was absent in all eight isolates, and that of yopM in all except one (10R). One of the isolates from rodents of the Deccan plateau (24H) was consistently negative for all the yops. Cloning and expression of truncated yopM (780 bp), yopB (700 bp) and lcrV (796 bp) genes in pQE vectors with SG13009 host cells yielded recombinant proteins for generation of monoclonal antibodies for further use in enzyme immunoassay. Ten stable reactive clones for YopB, nine for YopM and six for LcrV were obtained, all of them exhibiting specific reactions only to Y. pestis. Testing of 26 Y. pestis isolates by monoclonal antibody dot-ELISA and Western blotting provided results identical to PCR, suggesting that the isolates that failed to show PCR amplification also had no expression of their respective proteins. The Y. pestis isolates of outbreak regions had their virulence factors intact in the LCR plasmid. Yersinia pestis isolates recovered from rodents of the Deccan plateau were relatively heterogeneous. It appears that a long residency of Y. pestis of nearly 100 years in the enzootic plague foci has resulted in shedding of virulence genes in the LCR plasmid region in a fairly large proportion of the organisms, possibly due to natural recombination.

Faecal corticosteroid levels as an indicator of well-being in the tammar wallaby, Macropus eugenii

Faecal corticosteroid levels were measured in five female tammar wallabies, Macropus eugenii, at Macquarie University, NSW, Australia, to assess their reliability as indicators of well-being in this species. Animals were challenged with a change in conditions over the course of approximately 1 week, comprising movement from group yards to isolation in individual yards, in order to impose a disturbance to homeostasis (“stress”). Faecal samples were collected in 24-h intervals during the study period and analysed for corticosteroid concentration. The use of enzyme immunoassay for the measurement of corticosteroids in marsupial faecal pellets was validated. We observed a significant increase in faecal corticosteroids upon isolation and movement. Faecal corticosteroids remained above initial levels in all five animals throughout the study period, suggesting that faecal corticosteroid concentrations may be a useful indicator of a change in animal well-being. Faecal corticosteroid levels did not correlate with serum cortisol levels, implying that the use of noninvasive methods in a representative marsupial, the tammar wallaby, has the potential to provide information that is not readily apparent using blood-based protocols. Faecal corticosteroid analysis therefore has the potential for application in monitoring the well-being of captive and managed marsupial populations, as part of an integrated system of measures of animal health and well-being.

Are serum cortisol levels a reliable indicator of wellbeing in the tammar wallaby, Macropus eugenii?

Serum corticosteroid levels were measured in six female tammar wallabies ( Macropus eugenii) at Macquarie University, NSW, Australia, to assess their reliability as indicators of wellbeing in this species. Animals were challenged with a change in conditions over the course of approximately 3 weeks, comprising (i) isolation and movement; (ii) altered feeding routine, in order to impose a disturbance to homeostasis (“stress”). Blood samples were collected five times during the study period, and analysed for corticosteroid concentration and a number of haematological and biochemical measures. The use of enzyme immunoassay for the measurement of corticosteroids in marsupial serum was validated. We found that there was no significant change in serum corticosteroid concentrations in response to either of the changed conditions suggesting that serum corticosteroid concentrations are not reflective of a change in wellbeing. Cortisol was the dominant serum corticosteroid, with concentrations up to 50 times higher than corticosterone. Significant differences were observed in aspartate amino-transferase, alanine amino-transferase, haemoglobin, total erythrocyte count and mean corpuscular haemoglobin concentration over the period of study. However, no significant correlations emerged to justify any of these measures, or serum corticosteroid levels, as reliable indicators of compromised wellbeing in the tammar wallaby.

Isolation of antigens recognized by coeliac disease autoantibodies and their use in enzyme immunoassay of endomysium and reticulin antibody-positive human sera.

Components were isolated from rat liver, sheep lung, rhesus and orang-utan intestine. In enzyme immunoassay, these components detected 57%, 72%, 84% and 88% of human sera containing endomysium and reticulin antibodies (EmA and ARA). At most, 7% of EmA/ARA-negative sera reacted with the antigens. The spectrum of EmA/ARA-positive sera reactive with the various components was different but overlapping. When the antigens of sheep lung and orang-utan intestine were used as a cocktail, 98% of EmA/ARA-positive sera (45/46), but only 2% of control sera (1/46) were detected. The isolated antigens from sheep lung and monkey intestines were able to absorb EmA and ARA from human sera and thus should be suspected to contain the epitopes recognized by EmA and ARA, whereas the rat liver component did not bind. Therefore, the spectrum of autoantibodies in coeliac disease might comprise more than that covered by the terms EmA and ARA. The N-terminal amino acid sequence of some of the antigens was homologous with casein, gliadin, fibrinogen, and collagen.