Sample handling substantially affects Johne's ELISA

Detection methods for Mycobacterium avium subsp. paratuberculosis (MAP) are imperfect, yet crucial for diagnosis of Johne's disease. Our purpose was to test for significant and biologically relevant changes in Johne's ELISA results associated with how field-collected blood samples were transported to the laboratory, prepared and stored prior to testing, while removing potential confounding by test kit and laboratory variables. Blood samples were collected from 21 cows that previously had MAP ELISA scores ranging from negative to highly positive. Samples for immediate laboratory processing were subjected to different transportation temperatures (on ice, 26 °C) and preparation methods (serum separated, hemolyzed and serum separated, clotted whole blood), but were tested using the same ELISA kit in the same laboratory. Samples for laboratory processing after one week of storage were subjected to different storage temperatures (4 °C, −20 °C) and preparation methods (serum separated, hemolyzed and serum separated, clotted whole blood), and again were tested using the same ELISA kit in the same laboratory. Finally, samples were evaluated by time to processing (one day, one week) and storage temperature (4 °C, −20 °C). Data were checked for normality and analyzed with repeated measures ANOVAs. Significantly (P = 0.027) higher MAP ELISA scores were recorded for whole blood and hemolyzed samples transported at 26 °C than serum separated samples. Sample storage for one week at −20 °C resulted in significantly (P < 0.001) lower MAP ELISA scores, regardless of handling method, compared to samples stored at 4 °C for one week. Method of sample preparation, as well as transportation temperature and medium-term storage temperature, affects MAP ELISA results. Such discrepancies will inevitably result in improper classification of MAP-infected cattle, impeding both biosecurity measures on uninfected farms and MAP control programs.     

Candidate gene and genome-wide association studies of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep: a review

Paratuberculosis (Johne's disease), caused by Mycobacterium avium subspecies paratuberculosis, is responsible for significant economic losses in livestock industries worldwide. This organism is also of public health concern due to an unconfirmed link to Crohn's disease. Susceptibility to paratuberculosis has been suggested to have a genetic component. In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohn's disease. These genes include solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1), toll-like receptors, caspase associated recruitment domain 15 (CARD15, formerly NOD2), major histocompatibility complex (MHC) and cytokines (interleukin-10 and interferon-gamma) and their receptors. Genome wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. There are a number of limitations and difficulties in these approaches, some peculiar to paratuberculosis but others generally applicable to identification of genetic associations for complex traits. The technical approaches and available information for paratuberculosis have expanded rapidly, particularly relating to sheep and cattle. Here we review the current published evidence for a genetic association with paratuberculosis susceptibility, technological advances that have progressed the field and potential avenues for future research.     

Cellular Immune Responses to 35 kDa Recombinant Antigen of Mycobacterium avium paratuberculosis

Mycobacterium avium paratuberculosis is the causative agent of Johne disease, a chronic ulcerative intestinal condition in ruminant animals. Owing to the predominance of cellular response in subclinical forms of the infection, identification of M. a. paratuberculosis antigens eliciting host cell-mediated immune (CMI) reaction is crucial for early control of the disease. A 35 kDa protein of M. a. paratuberculosis was studied for its ability to elicit CMI responses using a mouse model. Lymphoproliferation and IFN-γ response were used to measure the CMI response. Recombinant 35 kDa protein (P35) stimulated proliferation of mouse mononuclear splenocytes sensitized with M. a. paratuberculosis. The P35 elicited increased nitrite production from mononuclear splenocytes from M. a. paratuberculosis-sensitized mice. In addition, RT-PCR-based semiquantitative IFN-γ measurement showed that stimulation with P35 is associated with significant expression of IFN-γ mRNA in M. a. paratuberculosis-sensitized mouse splenocytes. The results indicate that the 35 kDa protein of M. a. paratuberculosis is associated with CMI response in the host.     

Paratuberculosis in European wild rabbits from the Iberian Peninsula

Of the non-ruminant wildlife species known to harbor Mycobacterium avium paratuberculosis (MAP), the rabbit (Oryctolagus cuniculus) is thought to pose the greatest risk of transmission to cattle. We analyzed 80 hunter-harvested wild rabbits from a core study area in southern Spain, and sera from 157 wild rabbits sampled opportunistically on seven additional sites. Gross lesions compatible with paratuberculosis were observed in two of 80 necropsied rabbits. Histopathology revealed focal to diffuse multibacillary MAP-compatible lesions in 8 of 10 rabbits examined. Presence of MAP was confirmed in one rabbit with gross lesions by positive amplification curves for both IS900 and ISMAP02. However, no isolate was obtained from 47 samples by culture. We adapted an indirect ELISA for the detection of MAP antibodies. At the established cut-off of 0.5, 6 of 237 wild rabbit sera (2.5%) yielded a positive ELISA result. Antibodies were detected in rabbits from 3 of 8 sampling sites. Considering the increasing relevance of MAP infection for animal health, these results open a challenging field for future research.     

Heterologous Expression of a Gene Encoding a 35 kDa Protein of Mycobacterium avium paratuberculosis in Escherichia coli

The full-length open reading frame coding for a potentially immunogenic 35 kDa protein of Mycobacterium avium paratuberculosis was generated using polymerase chain reaction technology. The gene was inserted in-frame into Escherichia coli expression plasmid pQE32. The resulting recombinant plasmid pPMP35 was transformed into E. coli M15. Analysis of the E. coli induced with isopropyl-beta-D-thiogalactopyranoside revealed that the protein accumulated into the cytoplasm as insoluble inclusion bodies. The level of expression of the recombinant 35 kDa protein (P35) was more than 30% of the total protein of E. coli cells. Expression of the recombinant protein was confirmed by immunoblotting. The P35 reacted with a rabbit antiserum raised against a sonicate of M. a. paratuberculosis. The protein was also recognized by serum from a goat with clinical paratuberculosis. Further, a polyclonal antiserum against P35 recognized a 35 kDa band in a membrane fraction of M. a. paratuberculosis. Also, the protein provoked a significant skin reaction in outbred guinea pigs sensitized with M. a. paratuberculosis, as well as in those sensitized with Mycobacterium avium. The results indicate that the 35 kDa protein of M. a. paratuberculosis is a membrane protein, having a role in the cellular immune response.     

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated ‘A’, ‘B’, ‘C’ and ‘E’) were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern ‘A’, was found in 46 isolates (75%). The second, pattern ‘E’, included 8 isolates (13%), while the third, pattern ‘B’, included 6 isolates (10%). Pattern ‘C’ was found for only one isolate. All of the deer isolates were classified as pattern ‘A’, while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern ‘A’, was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns ‘B’, ‘C’ and ‘E’, were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.     

Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants

Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46 kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.     

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.     

Ovine Johne's disease in Australia--the first 20 years

OBJECTIVE: To review the history of ovine Johne's disease in Australia. PROCEDURE: Relevant publications and reports were identified and reviewed to document the spread of ovine Johne's disease (OJD) from 1980 until the end of 2000, as well as the response of industry and government to the spread of this disease. RESULTS: OJD was first diagnosed in the central tablelands region of New South Wales in 1980. Since then it has spread, either from the initial focus or through separate introductions so that by December 2000 a total of 823 infected flocks had been identified. Cases have been confirmed in New South Wales, Victoria, the Australian Capital Territory, on Flinders Island in Tasmania, on Kangaroo Island in South Australia and in Western Australia. In early 1999, agreement was reached to fund and implement a 6-year, $40 million National OJD Control and Evaluation Program (NOJDP). This program is jointly funded by the sheep industries (national and state), and Commonwealth and State governments, and is managed by Animal Health Australia. CONCLUSION: A national program is now in place to support the control of OJD and research to determine the feasibility and cost-effectiveness of eradication. The development of new diagnostic techniques, such as abattoir surveillance and pooled faecal culture, provide opportunities to refine surveillance strategies and to define better the distribution and prevalence of this disease, as required by the national program. Effective control measures, combined with quality surveillance data, will enable informed decision making for the future national management of OJD.     

牛副结核分枝杆菌实时荧光定量 PCR 检测方法的建立

根据GenBank上公布的牛副结核分枝杆菌C-2染色体的ISMav2基因保守区域序列设计合成1对特异性引物,建立了一套SYBR GreenⅠ荧光定量PCR检测牛副结核分枝杆菌(Mycobacterium paratuberculosis)的方法。以实验室构建的牛副结核分枝杆菌pMD-ISMav2阳性重组质粒为标准品,通过优化反应条件,建立了标准曲线,其相关系数为0.999。以构建的标准品为模板,进行了特异性和敏感性试验。结果显示,该方法检测布氏杆菌、大肠杆菌、沙门氏菌、链球菌DNA均为阴性;最低可检测到相当于每微升1.96×10¹拷贝数的标准品阳性质粒。本研究建立的实时荧光定量PCR具有特异、敏感和快速等优点,可用于牛副结核杆菌病的监测。     

Risk factors for <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">paratuberculosis</Emphasis> in Fars province (Southern Iran) dairy herds

A cross-sectional study was conducted from March to August 2006 in dairy herds in Fars province, southern Iran to determine the herd-level risk factors for infection with Mycobacterium avium subspecies paratuberculosis (MAP). Statistical analysis using multivariable logistic regression showed that contamination of udders of periparturient cows with manure (OR = 6.4, P = 0.02) and history of having suspected cases of Johne's disease in the herd (OR = 6.7, P = 0.04) were significantly associated with the herd infection status. No relationship between breed, herd size and other management practices with the infection status of the herd were found in this study. Implementing high sanitary measures in the farm, particularly with respect to manure handling and cleaning could be considered as one of the important aspects in controlling disease in the region as well as in the future educational effort.