Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

Background

Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.

Results

By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).

Conclusions

There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.     

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.     

Tactics of Mycobacterium avium subsp. paratuberculosis for intracellular survival in mononuclear phagocytes

Johne''s disease is a condition that refers to chronic granulomatous enteritis in ruminants. It is believed that survival and replication of Mycobacterium (M.) paratuberculosis in mononuclear phagocytes plays an important role in the pathogenesis of Johne''s disease. However, it is not clear how M. paratuberculosis survives for long time periods in mononuclear phagocytes, nor is it clear which factors trigger multiplication of these bacilli and result in the development of Johne''s disease. Investigating the intracellular fate of M. paratuberculosis is challenging because of its very slow growth (more than two months to form visible colonies on media). Existing animal models also have limitations. Despite those obstacles, there has been progress in understanding the intracellular survival tactics of M. paratuberculosis and the host response against them. In this review, we compare known aspects of the intracellular survival tactics of M. paratuberculosis with those of other mycobacterial species, and consider possible mycobactericidal mechanisms of mononuclear phagocytes.     

Prevalence and distribution of paratuberculosis (Johne's disease) in cattle herds in Ireland

A simple random survey was conducted in Ireland during 2005 to estimate the ELISA-prevalence of paratuberculosis, commonly called Johne's disease (JD), in the cattle population. Serum samples were collected from all 20,322 females/breeding bulls over 12 months-of-age in 639 herds. All samples were tested using a commercially available absorbed ELISA. The overall prevalence of infected herds, based on the presence of at least one ELISA-positive animal, was 21.4% (95% CI 18.4%-24.9%). Herd prevalence levels amongst dairy herds (mean 31.5%; 95% CI: 24.6%, 39.3%) was higher than among beef herds (mean 17.9%; 95% CI: 14.6%-21.8%). However, the animal level prevalence was similar. The true prevalence among all animals tested, was calculated to be 2.86% (95%CI: 2.76, 2.97) and for animals >= 2 yrs, it was 3.30% (95%CI: 3.17, 3.43). For animals in beef herds, true prevalence was 3.09% (95%CI: 2.93, 3.24), and for those in dairy herds, 2.74% (95%CI: 2.59, 2.90). The majority of herds had only one ELISA-positive infected animal. Only 6.4% (95% CI 4.7%-8.7%) of all herds had more than one ELISA-positive infected animal; 13.3% (CI 8.7%-19.7%) of dairy herds ranging from two to eight ELISA-positive infected animals; and, 3.9% beef herds (CI 2.4%-6.2%) ranging from two to five ELISA-positive infected animals. The true prevalence of herds infected and shedding Mycobacterium avium subspecies paratuberculosis is estimated to be 9.5% for all herd types; 20.6% for dairy herds; and 7.6% for beef herds. If ELISA positive animals <2-years-of-age are excluded, the true herd prevalene reduces to: 9.3% for all herd types; 19.6% for dairy herds; and 6.3% for beef herds based on a test specificity (Sp) of 99.8% and test sensitivity (Se) (i.e., ability to detect culture-positive, infected animals shedding at any level) of 27.8-28.9%.     

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。     

Evaluation of ELISA in the serodiagnosis of bovine farcy

Mycobacterium farcinogenes is the causal agent of bovine farcy, a chronic infectious disease of zebu cattle in some parts of tropical Africa. Whole cell homologous antigen of M. farcinogenes was used in the standardization and evaluation of an enzyme linked immunosorbent assay (ELISA) for the detection of circulating antibodies against bovine farcy using sera from confirmed bovine farcy and from bovine farcy-free cattle. The cut-off optical density (OD) value was decided at 1.8 using filter 405nm after one hour of incubation at 37°C. Accordingly, 115 out of 124 (92.7%) serum samples from clinically proven bovine farcy cattle were reported sero-positive. Sera from cattle infected with M. avium and M. paratuberculosis revealed OD value <1.8, indicating the differential diagnostic ability of M. farcinogenes antigen. Our test sensitivity was 92.7% and specificity was 97%, therefore could be routinely employed to support early clinical diagnosis, epidemiological surveys and for screening animals before exportation to farcy-free regions.     

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR

Johne’s disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.     

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

A milk and a serum ELISA for detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) were evaluated against the complement-fixation test (CFT) and culture of faecal samples from 580 cows collected between August 1996 and December 1996. Milk and serum were obtained concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP.

A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80–96% at the cut-off values of 4, 7 and 17 OD%.     

Description of the Infection Status in a Norwegian Cattle Herd Naturally Infected by Mycobacterium avium subsp. paratuberculosis

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-γ (IFN-γ) immunoassay, measurement of antibodies, and pathological and bacteriological examination.In the IFN-γ immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test,10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-γ and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-γ immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously.Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.     

Limited phenotypic and functional maturation of bovine monocyte-derived dendritic cells following Mycobacterium avium subspecies paratuberculosis infection in vitro

After encountering antigen, dendritic cells (DC) must differentiate into a fully mature phenotype to induce a protective, lasting T cell immunity. Paratuberculosis is a disease caused by the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and is characterized by a transient cell mediated immune response, that when dissipates correlates to the onset of clinical disease. In order to study the mechanism of early cellular immunity associated with M. paratuberculosis infection, we tested the hypothesis that M. paratuberculosis infected bovine DC have impaired activation and maturation thus are defective in the initiation of a sustainable and protective Th1 immune response locally. Our results demonstrate that M. paratuberculosis infected DC showed decreased endocytosis of ovalbumin, indicating some functional maturation. Co-stimulatory molecules CD40 and CD80 mRNA expression from M. paratuberculosis infected DC was increased over untreated immature DC. M. paratuberculosis infection induced chemokine receptor CCR7 increase in DC, yet CCR5 remained high. MHC II surface expression remained low on M. paratuberculosis infected DC. M. paratuberculosis infection inhibited pro-inflammatory cytokine IL-12 production and promoted IL-10 secretion by bovine DC. Together, our findings showed evidence of phenotypic and functional maturation of DC. However, we did not see the expected antigen presentation via MHC II and cytokine responses as a fully mature DC. This may suggest semi-mature DC phenotype induced by M. paratuberculosis infection.     

The contribution of molecular biology to Mycobacteriumavium subspecies Paratuberculosis research

Molecular biology has contributed to our knowledge and understanding of the structure of Mycobacteriumavium subspecies paratuberculosis and has been particularly useful in determining those components that elicit immune responses in the host or discriminate M. avium paratuberculosis from other closely related environmental mycobacteria. As such, it has made a significant impact in the field of diagnosis, and has been instrumental in the development of specific and sensitive diagnostic tests. The next decade will see exciting new developments in paratuberculosis research as a consequence of substantial advances made in the construction of gene transfer systems in mycobacteria. These will provide opportunities for applying new strategies to determine the genetic basis for pathogenesis and the mechanisms of drug resistance and will offer new prospects for the rational design of efficient vaccines.     

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.     

Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture

In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 microl of 10-fold dilutions containing from 5 x 10 to 5 x 10(3)Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential "false negative" result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.     

Molecular epidemiology of Mycobacterium avium subspecies paratuberculosis: IS900 PCR identification and IS1311 polymorphism analysis from ruminants in the Punjab region of India

Johne's disease is chronic granulomatous infectious enteritis of animals caused by Mycobacterium avium subspecies paratuberculosis. A total of 153 animals from 19 dairy farms, 2 gaushalas (unproductive-animal rehabilitation centers), 2 goat and 2 sheep farms from different districts of the Punjab region were selected on the basis of clinical signs of disease. All samples from cattle (n = 86), buffalo (n = 34), goat (n = 25) and sheep (n = 26) were subjected to Ziehl-Neelsen staining and DNA extraction by a freeze and thaw method. Ziehl-Neelsen staining detected 71% samples positive for acid-fast bacilli whereas IS900 PCR detected 55% positive for Map DNA. IS1311 PCR-REA analysis of IS900 positive samples revealed ‘Bison’ type as the most prevalent (82%) genotype of Map, infecting all domestic ruminants. ‘Cattle’ type was present in a minority of cases (15%) from cattle, buffaloes and goats. This is the first report of ‘Cattle’ type Map from buffalo and goat species in India.     

副结核分枝杆菌MAP0862、MAP1345基因的串联表达及其在间接ELISA中的初步应用

为探索副结核特异性蛋白MAP0862与MAP1345在牛副结核病血清学诊断中的作用,将通过串联表达获得的融合蛋白MAP0862-1345纯化定量后包被酶标板,经过对反应条件的优化,初步建立了基于融合蛋白MAP0862-1345的间接ELISA诊断方法。使用建立的ELISA方法对牛副结核病阳性血清、牛结核病阳性血清、牛布病阳性血清、卡介苗免疫牛血清、健康牛血清检测的结果表明其具有较好的特异性;使用该方法与IDEXX副结核病抗体检测试剂盒共同对300份临床血清样本检测,总符合率为92.7%。     

High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA

We conducted a seroepidemiological survey to determine the prevalence of caseous lymphadenitis (CLA) in goat herds in Minas Gerais state, Brazil. Serum samples were collected from goats (n = 676) from 108 rural properties in 2001, covering most of the sub-regions of this ca. 586,500 square kilometer state. Antibodies against Corynebacterium pseudotuberculosis secreted proteins were detected by an indirect enzyme-linked immunosorbent assay (ELISA). Most of the animals (78.9%) tested positive for CLA; 98% of flocks presented at least one seropositive animal. Goats managed under an extensive production system had a significantly higher seroprevalence of CLA than those in intensive and semi-intensive operations. The age distribution of the animals in the flocks affected the prevalence of this disease; however, goat breed did not. We found seropositivity against C. pseudotuberculosis to be highly prevalent in these Brazilian goat herds; consequently, appropriate management practices for the control of CLA should be implemented.     

spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea

Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I νSaβ. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently.     

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.