Is the gamma interferon assay in cattle influenced by multiple tuberculin injections?

Along with other developed countries, Canada is interested in adopting the gamma interferon (IFN-γ) assay to test for bovine tuberculosis (TB). This study compared results of using the IFN-γ assay in a large number of field-tested cattle in Manitoba, some previously tested with a caudal fold test (CFT) only, and others injected with tuberculins for both a CFT and a comparative cervical test (CCT). Parallel testing further compared the IFN-γ assay and CCT results with the confirmed TB status of the animal (culture, histopathologic examination, polymerase chain reaction). Results from IFN-γ assays did not differ following the CFT versus CFT and CCT injections. Parallel testing demonstrated an apparent higher prevalence of tuberculosis for the IFN-γ assay versus CCT, which will assist in earlier removal of exposed animals and, ultimately, prevent populations from becoming infected.     

Evaluation of the interferon-gamma (IFN-γ) assay to diagnose Mycobacterium bovis infection in pigs

Mycobacterium bovis recognizes as hosts a wide spectrum of animal species. In particular epidemiological situations, high prevalence of infection is found also in pigs. In the present study, we evaluated the capability of the interferon-gamma (IFN-γ) assay to identify pigs infected with M. bovis. The results of the immune-diagnosis were correlated to the findings of the post mortem inspection and the bacterial culture of lymph nodes. Blood samples of 146 pigs, belonging to a local breed of Sicily reared in free or semi-free roaming conditions, were collected to assess the specificity and the sensibility of the IFN-γ assay. Thirty-one pigs, from M. bovis free herds, did not react to the IFN-γ assay, yielding a specificity of 100%. The IFN-γ assay identified 15 out of 19 animals positive to the bacterial culture and 22 out of 26 animals with tuberculous lesions, with a sensibility of 78.9-84.6%, respectively. Out of 26 reactors to the test, 15 pigs (57.7%) confirmed to be infected after the bacterial culture and 22 (84.6%) had tuberculous lesions. The IFN-γ assay was able to reveal 4 animals with no visible lesions (NVL). Together, these findings support the feasible use of the IFN-γ assay as an intra vitam tool for the surveillance and management of M. bovis infection in swine populations.     

Evaluation of a fluorescence-polarization assay for the diagnosis of bovine brucellosis in México.

A homogeneous fluorescence-polarization assay (FPA) was used for the serological diagnosis of bovine brucellosis in México. The assay uses O-polysaccharide prepared from Brucella abortus lipoplysaccharide (20-30 kDa) conjugated with fluorescein isothiocyanate as a tracer. To measure the fluorescence polarization, a FPM-1 fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. (1996b). A cut-off value of 90 millipolarization (mP) units was used for testing 560 bovine sera from different areas of México. (305 positive sera and 255 negative sera according to the complement fixation test; CFT.) Some were tested with the Rose Bengal plate (RB) test (n = 490) and some with the rivanol-agglutination (RIV) test (n = 190). Sensitivities were 98.3%, 99.3% and 99.0%, and specificities were 68.8%, 55.4% and 96.9%, respectively, for RB, RIV and FPA. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.96, while RB and RIV (relative to the CFT) gave coefficients of 0.70 and 0.61, respectively. Finally, ROC analysis suggested a cut-off value which agreed with the one recommended in the test procedure. We concluded that FPA is a suitable test to be used instead of the CFT in Mexican conditions.     

Factors influencing the performance of an interferon-γ assay for the diagnosis of tuberculosis in goats

The interferon-gamma (IFN-γ) assay is an effective tool for the diagnosis of tuberculosis (Tb) in goats. The objectives of this study were to evaluate factors that might affect assay performance: (1) the phenol concentration of the purified protein derivative (PPD, tuberculin) used; (2) dialysis of PPD; and (3) delaying antigenic stimulation of blood samples for 8, 16 and 24h after collection. The assay was performed in duplicate with two cut-off points. Dialysis of PPD reduced test sensitivity, whereas the concentration of phenol did not significantly affect test outcome. Delaying antigenic stimulation of samples >8h resulted in a reduction in test sensitivity, compromising the capacity of the assay to detect infected animals. Performing the assay in duplicate was unnecessary, which has implications for reducing assay costs. These findings will facilitate the effective application of the IFN-γ assay as an ancillary test in Tb eradication programmes in goats.     

Immune protection assay of Newcastle disease live vaccine （LaSota Strain） against a genotype VII NDV isolate

从山东省发病鸡群分离鉴定了一株新城疫病毒（NDV）,命名为SDLY01。经蚀斑纯化后进行毒力测定和序列分析表明分离株SDLY01属于基因VII型NDV强毒。20只7日龄SPF鸡免疫新城疫活疫苗LaSota后14d分别用NDV标准强毒F48E8和分离株SDLY01攻毒,同时设同日龄SPF鸡为对照组,未免疫任何疫苗。攻毒后观察10d,免疫组在攻毒后食欲、精神均正常;对照组在攻毒后2～4d发病死亡,并表现ND典型的临床症状和病理变化。攻毒后第3、5、7、9d对免疫组试验鸡取喉头、泄殖腔棉拭进行病毒分离,F48E8攻毒组病毒分离均为NDV阴性,SDLY01攻毒组第5d病毒分离NDV阳性,第3、7和9d病毒分离阴性。本研究结果表明LaSota活疫苗对F48E8和SDLY01均能提供100％免疫保护,但不能完全抑制基因VIINDV分离株在体内的复制和排毒。     

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.     

Development of an Heal amino acid digestibility assay for the growing chicken—effects of time after feeding and site of sampling

1. The study aimed to establish the optimum time after ingestion and optimum sampling site for the development of an ileal amino acid digestibility assay for broiler chickens.

2. To establish the optimal sampling time, 4‐week‐old broiler chickens were given one of 6 protein sources (meat‐and‐bone, soyabean, cottonseed, fish, maize and wheat meals) as the sole source of protein in a test diet. The diets contained chromic oxide as an indigestible marker. The birds were starved for 24 h, fed and subsequently killed for sampling of ileal digesta (terminal 15 cm) at 2, 3, 4, 5 and 6 h after the start of feeding.

3. For the soyabean, fish, wheat and maize meal diets, sampling time had no significant effect on apparent ileal nitrogen digestibility, whereas for the meat‐and‐bone and cottonseed meal diets there was a significant quadratic effect of sampling time. The amount of digesta collected was maximised and the mean apparent ileal nitrogen digestibility had the lowest variation around the 4 h sampling time.

4. To establish the optimum sampling site, 4‐week‐old chickens were given either a meat‐and‐bone, a soyabean or a wheat bran meal‐based diet. The birds were killed 4 h after the start of feeding, and digesta were sampled from 0.10, 0.15, 0.20 or 0.25 cm of terminal ileum.

5. There was no significant effect of sampling site on the apparent ileal digestibility of dietary nitrogen. The terminal 15 cm of ileum was considered a preferred site for sampling ileal digesta from broiler chickens.     

Development and application of a real-time TaqMan(®) qPCR assay for detection and quantification of 'Candidatus Mycoplasma haemolamae' in South American camelids

Two alpacas from a herd in southwest Switzerland died for unknown reasons. Necropsy revealed chronic weight loss and pale mucous membranes. Infection with hemotropic mycoplasmas was suspected and subsequently confirmed by molecular methods. In order to investigate the epidemiological situation in this herd, a real-time TaqMan(?) qPCR assay for the specific detection and quantification of hemoplasma infection in South American camelids was developed. This assay was based on the 16S rRNA gene and amplified 'Candidatus Mycoplasma haemolamae' DNA, but not DNA from other hemoplasmas or non-hemotropic mycoplasma species. The lower detection limit was one copy/PCR, and the amplification efficiency was 97.4%. In 11 out of 24 clinically healthy herd mates of the two infected alpacas, 'Candidatus M. haemolamae' infection was confirmed. No correlation was found between bacterial load and clinical signs or anemia. The assay described herein enables to detect and quantify 'Candidatus M. haemolamae' and may be used in future studies to investigate the prevalence, pathogenesis and treatment follow-up of hemoplasma infections in South American camelids.     

Ileal amino acid digestibility assay for the growing meat chicken—effect of the imposition of a fasting period and the nature of the test diet

1. The objective of this study was to investigate the effects of cold‐pelleting, the length of the fasting period before feeding of the test diet and the nature of die test diet on apparent ileal nitrogen (N) digestibility in the broiler chicken.

2. Four‐week‐old broiler chickens were given a pelleted or non‐pelleted maize/soyabean meal (basal) diet. The birds were starved for 24 h, given a single test meal (25 g) by intubation and killed 4 h after the start of feeding by administration of a barbiturate, to allow sampling of ileal digesta (terminal 15 cm). Cold‐pelleting did not affect apparent ileal N digestibility.

3. Four‐week‐old broiler chickens were fasted for 12 or 24 h and then received a test meal (1 h free access) of either a pelleted soyabean meal or a pelleted meat‐and‐bone meal diet or were continuously fed on one of the two diets. The imposition of a fast did not affect apparent ileal N digestibility. However, a 24 h fasting procedure was preferred, as the between animal variation for apparent ileal N digestibility was lower than for the 12 h fast or for continuous feeding.

4. Four‐week‐old broiler chickens were given either semi‐synthetic starch‐based diets containing maize, wheat bran, meat‐and‐bone meal or fish meal as the sole sources of protein or each of these diets in combination with the basal diet (50:50 on a dry matter basis). With the exception of the maize diet, the apparent ileal N digestibility values calculated by correcting for the digestibility of the basal dietary component were significantly lower than when digestibility was determined directly using a diet in which the respective proteins were the sole protein source. This implies that interactions between the dietary ingredients influence estimates of apparent ileal N digestibility.     

Serological detection of chicken flocks naturally infected with salmonella enteritidis,using an enzyme‐linked immunosorbent assay based on monoclonal antibodies against the flagellar antigen

Summary

The Dutch Salmonella enteritidis monitoring and eradication programme for poultry prescribes a periodic examination of all breeding flocks for the presence of S. enteritidis. For the first years of the programme this was done by bacteriological examination of 50 faecal samples per visit per flock.

In this study we compare the results of bacteriological examination of faecal samples taken at 1580 visits from 545 flocks with those of a S. enteritidis enzyme‐linked immunosorbent assay (ELISA) applied on 24 serum samples per visit per flock. Two flocks were found positive for S. enteritidis by bacteriological examination; both flocks were also detected by ELISA. Ten flocks, bacteriologically negative for S. enteritidis were found positive by ELISA. S. enteritidis was isolated from three of these flocks by repeated and extensive bacteriological examination for verification. Verification was not possible in the fourth EL1SA positive flock. S. enteritidis infections were likely in three other flocks because of the farm histories.

On the basis of the results of this study it was decided to use this ELISA, starting from April 1992, as screening technique in the Dutch S. enteritidis programme instead of bacteriological examination of faecal samples. The ELISA is regarded as a flock test; an extensive, confirmatory bacteriological investigation for S. enteritidis is carried out in ELISA positive flocks to decide whether the flocks are truly infected.     

Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in vitro assay for a direct read-out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evaluate for less severe or immunocompromising infections. Here we investigated the performance of the assay when recombinant co-stimulatory cytokines IL-12 and/or IL-18 were added along with Ag or PBS to cultures of whole-blood from pigs infected with Lawsonia intracellularis. In pigs recovering from a natural infection, addition of rIL-12 or rIL-18 alone increased the Ag-specific IFN-γ release while addition of both cytokines resulted in increased IFN-γ release also in PBS cultures. In analyses after experimental infections with L. intracellularis, significant increased levels of Ag-specific IFN-γ production were measured in Ag+rIL-18 cultures from infected pigs compared to the background response in PBS+rIL-18 control samples (p<0.01) or to Ag+rIL-18 cultures from non-inoculated control pigs (p<0.05). Flow cytometry identified two lymphocyte subsets as the Ag-specific IFN-γ producers. The highest IFN-γ production was by CD4(+)CD8(+) cells while a more numerous population of CD4(-)CD8(+) cells produced lower amounts of IFN-γ in response to rIL-18 and L. intracellularis Ag.     

Effects of clopidogrel therapy on whole blood platelet aggregation,the Plateletworks® assay and coagulation parameters in cats with asymptomatic hypertrophic cardiomyopathy: a pilot study

Background: Although scientific evidence is limited, clopidogrel is frequently used as prophylaxis for arterial thromboembolism in cats with hypertrophic cardiomyopathy (HCM).

Objectives: Evaluating effects of clopidogrel therapy in asymptomatic cats with HCM on (1) conventional whole blood aggregation (WBA), (2) alternative platelet aggregation assessed with tubes of the Plateletworks® assay and (3) standard coagulation parameters.

Animals and methods: Prospective, randomized, double-blind, placebo-controlled pilot study. Fourteen asymptomatic HCM cats were randomly allocated to receive placebo (n = 5) or clopidogrel (18.75 mg/cat q24h, n = 9) as part of a larger study. Aggregation responses (to 20 µM adenosine diphosphate (ADP) and 10 µg/ml collagen) in WBA and the Plateletworks® assay and standard coagulation parameters were evaluated at baseline and after seven days of therapy.

Results: Clopidogrel therapy significantly reduced aggregation responses to ADP and collagen in the Plateletworks® agonists tubes (ADP and collagen: P < 0.001), but did not significantly reduce aggregation responses to ADP and collagen in the WBA technique (ADP: P = 0.07, collagen: P = 0.30). Clopidogrel therapy did not show a significant effect on prothrombin time, activated partial thromboplastin time, antithrombin, D-dimers and fibrinogen concentrations.

Conclusion and clinical importance: Clopidogrel therapy at a dose of 18.75 mg/cat q24h for seven days causes a significant decrease in in vitro platelet aggregation evaluated with the Plateletworks® assay, without affecting standard coagulation parameters in cats with asymptomatic HCM.     

Evaluation of two cocktails containing ESAT-6, CFP-10 and Rv-3615c in the intradermal test and the interferon-γ assay for diagnosis of bovine tuberculosis

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n=23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p>0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p>0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis.     

Determination of decisional cut-off values for the optimal diagnosis of bovine tuberculosis with a modified IFNγ assay (Bovigam®) in a low prevalence area in France

The Bovigam(?) gamma interferon (IFNγ) assay was used to complement official skin-test screening in a low bovine tuberculosis (bTB) prevalence region in France. The aim of our work was to determine decisional cut-off values for protein purified derivatives (PPD) and ESAT6-CFP10 antigens (R) in order to optimize the efficacy of the modified Bovigam(?) test, in this low-prevalence area, for optimal classification of infected or non-infected herds following positive skin tests. The sensitivity of the IFNγ assay relative to post-mortem bTB-positive animals (Se(r)) was studied in 60 cattle from 20 bTB-infected herds. Its absolute specificity (Sp) was studied in 492 cattle from 25 bTB-free herds from a bTB-free zone. Its operational specificity (relative to the positive skin test) (Sp(r)) was also studied in 547 skin-test positive cattle from 172 bTB-free herds from an infected zone. Using normalized interpretations for individual (PPD or R) results, the cut-off values at 0.02 for PPD and 0.01 for R were obtained with a view to employ them in low prevalence areas with no previously observed non-specific reactions to SITT. Concerning its use after positive skin tests, cut-off values were set at 0.05 for PPD and at 0.03 for R. The choice of an interpretation method considering positive results with PPD and/or R (PPDUR), justified in a high risk context, provided a test Se(r) of 93% [84-98] and Sp(r) of 71.8% [67.9-75.6]. Analysis of positive results with PPD and R (PPDUR), ideal for low-risk contexts, provided a test Sp(r) of 94.3% [92.0-96.1] and Se(r) of 77% [64-87]. Thus, adapting the criteria to the region's infection status and to the conditions for its application is essential for the appropriate use of the IFNγ assay.