Faecal shedding detected earlier than immune responses in goats naturally infected with Mycobacterium avium subsp. paratuberculosis

Paratuberculosis was diagnosed in a goat herd that participated in a sanitation program against Mycobacterium avium subsp. paratuberculosis. The aim of this study was to characterise the development of gamma interferon (IFN-γ) and antibody responses as well as the occurrence of faecal shedding. Faecal culture appeared surprisingly sensitive as about 18% and 40% of the goats were positive at 9 and 15-17 months of age, respectively, and shedding was often seen prior to peripheral immune responses. Peripheral IFN-γ responses were not related to protection as clinical and high shedding goats often had high responses. An IFN-γ response usually preceded a humoral response. However, positive antibody titers could sometimes be seen simultaneously with, and even prior to, IFN-γ responses. In conclusion, faecal culture appeared as sensitive as IFN-γ testing. Furthermore, the antibody ELISA and the IFN-γ assay may perform equally well in an infected herd if surveillance is conducted annually.     

Flock-level factors associated with the risk of Mycobacterium avium subsp. paratuberculosis (MAP) infection in Greek dairy goat flocks

In this cross-sectional study we identified flock-level risk factors for Mycobacterium avium subsp. paratuberculosis (MAP) infection, in Greek dairy goat flocks. We collected 1599 milk samples from does that were at the last stage of lactation in 58 randomly selected dairy goat flocks, during May to September 2012. The collected samples were tested with a commercial milk ELISA (IdexxPourquier, Montpellier, France) and the results were interpreted at a cut-off that optimized the accuracy of the diagnostic process. For the analysis of the data we used Bayesian models that adjusted for the imperfect Se and Sp of the milk-ELISA. Flock was included as a random effect. Does in flocks that used common water troughs and communal grazing grounds had 4.6 [95% credible interval (CI): 1.5; 17.4] times higher odds of being MAP-infected compared to does in flocks that had no contact with other flocks. Does of flocks supplied with surface water from either streams or shallow wells had 3.7 (1.4; 10.4) times higher odds of being infected compared to those in flocks watered by underground and piped water sources. When kids were spending equal to or more than 10 h per day with their dams they had 2.6 (1.1; 6.4) times higher odds of being MAP infected compared to kids that were separated from their dams for less than 10 h per day. Finally, does in flocks that continuously used the same anti-parasitic compound had 2.2 (1.0; 4.6) times higher odds of MAP infection compared to those in flocks alternating anti-parasitic compounds. These results should be considered in the development of a nationwide future control program fοr caprine paratuberculosis in Greece.     

Quantitative real-time PCR and culture examination of Mycobacterium avium subsp. paratuberculosis at farm level

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants and may contribute to Crohn's disease in humans. The aim of this study was to determine the occurrence and quantity of MAP in cattle feces and milk in the Iranian context. In addition, we evaluated the effect of cattle age as well as farming system as risk factors contributing to MAP load. For this, a total sample of 373 consisting of 150 cattle feces (CF), 150 individual cow's milk (ICM), as well as 73 bulk-tank milk (BTM) was collected randomly and regardless of the cattle health status. The samples were assayed using F57 quantitative real-time PCR (qPCR) and culture method. According to the results of qPCR which was found ∼10 times more sensitive than culture assay, MAP was detected in 68.66% (103/150) of the CF, 12% (18/150) of the ICM and 52.05% (38/73) of the BTM samples. In contrast to the previous reports, the quantity of MAP in the BTM (2.03–5.97 log cfu/50 ml) was statistically (p < 0.01) higher than the ICM (0.90–1.97 log cfu/50 ml). Data suggested a direct relation (p < 0.01) between the cattle age and the quantity of MAP in the CF samples, while the relation was not statistically significant (p > 0.05) for the ICM. In addition, MAP load in the BTM samples obtained from traditional farms was significantly (p < 0.01) higher than that of the industrial ones, while the differences in CF and ICM was not significant (p > 0.05).     

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded “echA12_2” in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2–7 months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.     

Tissue localisation of Mycobacterium avium subspecies paratuberculosis following artificially induced intracellular and naked bacteraemia

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants. While Johne's disease is primarily expressed in the gastrointestinal tract, isolation of MAP from extra-intestinal tissues indicates that microbial dissemination via the haematogenous route may occur during the infection. This study examined the movement of peripheral blood mononuclear cells (PBMCs) infected with MAP and the dissemination of MAP following mycobacteraemia induced by IV inoculation over a time frame of 3 days.     

Assessing the within-herd prevalence of Coxiella burnetii milk-shedder cows using a real-time PCR applied to bulk tank milk

Thirty-seven bulk tank milk (BTM) and individual milk samples of all contributing cows were tested for Coxiella burnetii detection by a real-time PCR assay and used to assess the relationship between the BTM PCR-response and (i) the within-herd prevalence of milk-shedder cows and (ii) the proportion of heavy milk-shedder cows. The within-herd prevalence of milk-shedder cows (i) was found to be significantly higher in herds with a positive BTM and (ii) increased significantly with the estimated titre in Coxiella burnetii obtained in positive BTM. The proportion of heavy milk-shedder cows among the milk-shedder cows increased significantly with an increased estimated titre in Coxiella burnetii in positive BTM. Therefore, a real-time PCR assay applied to BTM samples collected repeatedly over time appears to be a valuable tool to assess on a larger scale the status of herds towards Coxiella shedding, and to evaluate the efficiency of control actions aimed at controlling and/or preventing Coxiella shedding in dairy herds.     

Evaluation of the specificity of three enzyme-linked immunosorbent assays for detection of antibodies against Salmonella in bovine bulk milk

Background

The Swedish Salmonella control program has been running for decades and has resulted in a low prevalence of Salmonella in Swedish food producing animals. Routine bacteriology is used to detect Salmonella, however, bacteriology is time consuming, costly and has a low sensitivity. Different enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of antibodies against Salmonella Dublin and S. Typhimurium in bovine bulk milk, individual milk samples as well as in sera. Screening bulk milk for antibodies against Salmonella spp. could improve the cost-effectiveness of the surveillance in Swedish dairy cattle, but as characteristics of tests may vary in different populations, tests should always be evaluated in the specific population where they will be used. Hence, the aim of this study was to evaluate the specificities of three bovine ELISAs when used to analyse bulk milk samples from Swedish dairy cattle. A second aim was to compare the performance of the two Dublin ELISAs tested.

Methods

Bulk milk samples for analysis were randomly selected from samples collected within the Swedish bulk milk sampling scheme and analyzed with the three ELISAs; a Danish in-house Dublin ELISA, PrioCHECK® Salmonella Ab bovine Dublin ELISA and PrioCHECK® Salmonella Ab bovine ELISA (hereafter named mixed ELISA). The specificities of the ELISAs were calculated assuming a disease-free status in Sweden i.e. that all test positive samples were assumed to be false positive results. This assumption can be used when a disease is known to be infrequent.

Results

The calculated specificities of the two Dublin ELISAs and the mixed ELISA, when using the producer’s recommended cut-off value of the corrected optic-density percent (ODC%) were 99.4% (95% Confidence Interval (CI): 98.8% -99.8%), 99.4% (95% CI: 98.8% -99.8%) and 97.9% (95% CI: 96.8% -98.7%), respectively. The correlation between the ODC% values of the two Dublin ELISAs was 0.83.

Conclusions

We conclude that the evaluated ELISAs have sufficiently high specificities to be used as supplement to bacteriological examinations in the Swedish Salmonella control program in cattle as well as a primary screening test in routine surveillance for S. Dublin.     

Bulk milk ELISA and the diagnosis of parasite infections in dairy herds: a review

The bulk milk enzyme-linked immune sorbent assay (ELISA) is a rapid and inexpensive method of assessing herd exposure to pathogens that is increasingly being used for the diagnosis of parasite infections in dairy herds. In this paper, with the dairy herd health veterinarian in mind, we review the principles of the assay and the recent literature on the potential role of bulk milk ELISA for the diagnosis of ostertagiosis, fasciolosis, parasitic bronchitis due to cattle lung worm and neosporosis. It is generally accepted that assay results reflect exposure to the parasite rather than the presence of active infection. Bulk milk ELISA can be a useful tool for the veterinary practitioner as a component of a herd health monitoring programme or in the context of a herd health investigation. It can also play a role in regional or national surveillance programmes. However, the results need to be interpreted within the context of the herd-specific health management, the milk production pattern and the parasite life cycle.     

Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle

OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.     

Validation of a bulk tank milk antibody ELISA to detect dairy herds likely infected with bovine viral diarrhoea virus in New Zealand

AIMS: To assess the sensitivity and specificity of a bulk tank milk (BTM) antibody enzyme-linked immunosorbent assay (ELISA) to detect likely infection of a dairy herd with bovine vi- ral diarrhoea virus (BVDV). The ELISA was subsequently used to estimate the prevalence of likely infected herds in parts of the North Island of New Zealand.

METHODS: BTM samples from 724 randomly selected dairy herds in the Waikato, Bay of Plenty and Northland regions of New Zealand were tested for BVDV antibodies. From this group, 20 herds were again randomly selected from each of the quartiles of the ELISA percentage inhibition (%INH) result. From each participant herd, serum from 15 randomly selected calves aged 6–18 months and 15 cows was collected and tested using an indirect blocking ELISA for BVDV antibodies.

RESULTS: Among serum results from calves from 50 herds available for analysis, 34 (68%) herds were classified as likely non-infected (0-3 seropositive among 15 calves) and 16 (32%) as likely infected (5–15 seropositive among 15 calves). Receiver- operator characteristic (ROC) analysis identified an optimal cut-off for BTM of 80%INH associated with 81% sensitivity and 91% specificity for likely herd infection. The prevalence of BVDV antibodies in cows within herds and %INH for BVDV in bulk milk were positively correlated (p<0.01). The association between bulk milk %INH and the prevalence of BVDV antibodies in calves was stronger than the same association in cows. Based on the threshold of 80%INH, the 95% confidence interval (CI) for prevalence of likely infection in the 724 herds in the Waikato, Bay of Plenty and Northland regions of New Zealand was 12–17%. Vaccination against BVDV was not significantly associated with the likely infection status of the herd based on prevalence of BVDV antibodies among calves.

CONCLUSION: An ELISA test result for BVDV antibodies in BTM ≥80%INH can be used as a threshold to indicate the presence of likely infection with BVDV in dairy herds in New Zealand, with 81% sensitivity and 91% specificity.     

猪轮状病毒重组VP7蛋白抗原间接ELISA诊断方法的建立

本研究以纯化的原核表达的猪轮状病毒VP7抗原表位区域为抗原,建立了检测猪轮状病毒抗体的间接ELISA诊断方法。特异性试验表明,该抗原与其他7种常见猪病病毒（TGEV、PEDV、CSFV、PCV2、PRRSV、PPV、PrV）的阳性血清不发生交叉反应,批内和批间重复性试验的变异系数均小于10％;对来自不同猪场的血清的检测结果表明,该ELISA方法与中和试验检测结果符合率达94．8％。本试验建立的ELISA诊断方法具有良好的重复性、敏感性和特异性,为PRV的快速诊断、免疫猪群抗体监测和轮状病毒流行病学调查提供了一种快速、简便的血清学诊断方法。     

Association between antibodies to Coxiella burnetii in bulk tank milk and perinatal mortality of Danish dairy calves

Background

Coxiella burnetii is a well-known cause of placentitis and subsequent abortion in ruminants, but there are no reports on the relationship with perinatal mortality. The study was performed to determine the influence of level and change of bulk tank milk (BTM) antibodies to C. burnetii on two outcomes associated with parturition in cattle: a) stillbirth; and b) stillbirth and neonatal mortality combined (perinatal death).

Methods

Twenty-four Danish dairy herds were tested repeatedly for antibodies to C. burnetii in BTM using a commercial ELISA. Samples were collected monthly from July 2008 to July 2009. Information on the 2,362 calvings occurring in the study period was obtained from the Danish Cattle Database. Two multilevel logistic regression models were created for the two outcomes stillbirth and perinatal mortality. One model included the level of BTM antibodies in a specified period before or after the outcome had occurred. The other model included the change in antibodies over time. These predictors were included both at herd and animal level. Furthermore, all models included parity and breed.

Results

The individual monthly BTM antibody levels were highly correlated within herds. Consequently, changes in BTM antibody levels were not found to be associated with neither risk of stillbirth nor the risk of perinatal mortality. However, the risk of stillborn calves and perinatal death was higher with high level of BTM antibodies 8 to 9 months after the incident, but not outside this period.

Conclusion

We conclude that the level of antibodies to C. burnetii in BTM may be associated with perinatal mortality, but the association was not persistent and should be investigated further.     

Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was evaluated in comparison with the indirect fluorescent antibody test (IFAT) in Australia and Zimbabwe. Positive and negative threshold values for the ELISA were set using sera from cattle of known infection status. Sensitivity and specificity estimates for the ELISA based on 158 positive sera from cattle experimentally infected with Australian isolates of B. bovis and 318 negative sera collected from B. bovis-free herds in Australia were 100% and 99.4%, respectively. The specificity of the assay in Africa, based on 328 sera from B. bovis-free herds in Kenya and South Africa, was 99.7%. The ELISA was compared with the IFAT using sequential sera from 16 calves experiencing primary B. bovis infections, and a total of 777 field sera collected from B. bovis-endemic herds in Australia and Zimbabwe. In primary infections, the ELISA and IFAT detected antibodies at or about the same time. With sera from endemic herds, the performance of the ELISA was at least comparable with that of the IFAT. Two hundred and fourteen of 221 sera that were negative by IFAT, were negative by ELISA, and 428 of 439 sera that were clearly positive by IFAT were positive by ELISA. Of 117 sera that gave equivocal (suspect or weak positive) results in the IFAT, 20 were positive by ELISA, 7 were suspect and 90 were negative. We conclude that the ELISA will be useful for epidemiological studies on B. bovis in Australia and Zimbabwe, and probably elsewhere.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

重组N蛋白间接ELISA检测猪繁殖与呼吸综合征病毒抗体

用纯化的猪繁殖与呼吸综合征病毒重组N蛋白作为包被抗原，建立了检测猪繁殖与呼吸综合征病毒抗体的间接ELISA方法，并确定了ELISA最佳工作条件：抗原包被浓度为0．27μg/ml，37C1h加4C过夜，血清(1：40)和酶标免抗猪IgG(1：400)分别在37℃温育30min，底物溶液37℃显色15min。经重复性试验、交叉试验、阻断试验等试验结果表明该方法重复性好、特异性强、灵敏度高；与美国IDEXX试剂盒相比较，特异性和敏感性分别为97．6％和92．1％，无显著性差异。用已建立的方法检测临床血清样本187份，总阳性率为30．5％。     

猪繁殖与呼吸综合征病毒重组N蛋白间接ELISA方法的建立

用纯化的猪繁殖与呼吸综合征病毒(PRRSV)重组核衣壳(N)蛋白作为包被抗原,建立了PRRSV抗体检测的间接ELISA方法,并优化确定了ELISA最佳工作条件:重组抗原包被浓度为13.3 μg/mL,37℃1h或4℃过夜;5％脱脂乳37℃封闭2h;血清1:40稀释,37℃作用90 min;酶标二抗1∶4000稀释,37...     

流行性乙型脑炎病毒E蛋白的截短表达与间接ELISA诊断方法的建立

为建立一种快速检测流行性乙型脑炎病毒抗体的方法,本研究参照已发表的JEV基因组序列,应用RT-PCR扩增了长约1000bp的E基因片段,连接pET30a表达载体中,经诱导后获得了以包涵体形式表达的重组E蛋白。重组蛋白纯化后,经免疫印迹检测证明其具有良好的抗原性和特异性。以该蛋白作为诊断抗原,建立了检测流行性乙型脑炎病毒抗体的E-ELISA诊断方法。该诊断方法具有良好的敏感性、特异性和重复性,为JEV的快速诊断、免疫猪群抗体监测和JEV流行病学调查提供了一种快速、简便的血清学诊断方法。     

Comparison and evaluation of three serological tests for detection of antibodies to infectious bovine rhinotracheitis

Abstract

AIMS: To retrospectively describe clinical features of dogs that were presented to a small animal clinic between 2003–10 with macroscopic haematuria, and investigate whether signalment of the dog and severity and duration of the haematuria at admission were associated with specific aetiologies.

METHODS: Medical records were evaluated of 162 dogs with macroscopic haematuria admitted to a University-based small animal clinic in Thessaloniki, Greece, from January 2003 to December 2010. The inclusion criteria were discolouration of the urine sediment combined with abnormal numbers of erythrocytes, when examined microscopically. Data collected from the medical records included signalment, severity, frequency and duration of haematuria, and diagnosis.

RESULTS: Between January 2007 and December 2010, 8,893 dogs were admitted to the clinic; of these 99 (1.1%) were admitted with haematuria. Of the 162 dogs with records of haematuria, 80 (49.4%) were aged between 5.1–10 years, presented with acute (96/162; 59.3%), constant (99/162; 61.1%) and mild/moderate (150/162; 92.6%) haematuria. Of 147 dogs with a recorded diagnosis, the commonest diagnoses were urinary tract infection (UTI, 42/147; 28.6%), urolithiasis (38/147; 25.9%), prostatic disease (25/147; 17.0%) and urinary tumours (13/147; 8.8%). The prevalence of UTI was higher in female (22/56; 39%) than male (20/91; 22%) dogs, and in medium sized (22/52; 42%) than small (6/40; 15%) dogs. Urolithiasis was most prevalent in small (21/40; 52.5%) dogs, and all dogs with urolithiasis presented with mild/moderate haematuria. The prevalence of prostatic disease was highest in large (11/46; 24%) and giant (3/9; 33%) sized dogs and in dogs aged >10 years (8/30; 27%).

CONCLUSIONS AND CLINICAL RELEVANCE: In this retrospective study from one small animal clinic, UTI, urolithiasis, prostatic disease and urinary tumours predominated among the causes of canine haematuria. The consideration of sex, age, and size of the dog and characteristics of haematuria were found to be useful parameters when forming the list of differential diagnoses.     

A radial growth precipitation test and its comparison with certain other serological tests for the detection of antibody in bovine sera to Mycoplasma agalactiae subsp. bovis

A radial growth precipitation test is described for measuring antibody to Mycoplasma agalactiae subsp. bovis. The test is quantitative and appears to depend on the production of soluble antigen by growing organisms. When compared with indirect haemagglutination, complement fixation and inhibition of film production, for measuring antibody to M. agalactiae subsp. bovis in bovine sera, it was found to have a sensitivity comparable to that of the complement fixation test.