Research Article, J Vet Sci Med Diagn Vol: 7 Issue: 2
Preliminary Findings of Lipoprotein B in Detecting Cattle Chronically Infected with Contagious Bovine Pleuropneumonia
*Corresponding Author : Harrison Osundwa Lutta
Kenya Agricultural and Livestock Research Organization, Biotechnology Research Institute, P.O. Box 14733-00800 Nairobi, Kenya
Tel: +254-722-29 6494
Received: April 26, 2018 Accepted: May 07, 2018 Published: May 12, 2018
Citation: Lutta HO, Mather A, Maina TW, Odongo D, Ndiwa NN, et al. (2018) Preliminary Findings of Lipoprotein B in Detecting Cattle Chronically Infected with Contagious Bovine Pleuropneumonia. J Vet Sci Med Diagn 7:2. doi: 10.4172/2325-9590.1000254
Contagious Bovine Pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides is an important disease of cattle affecting international trade. Complement Fixation Test (CFT) and competitive Enzyme Linked Immunosorbent Assay (c-ELISA) are the only serological tests recognized for diagnosis of CBPP. The performance of CFT depends on the quality of the antigen and the combination of reagents used and only detects CBPP during the acute phase of the disease. There is a need to develop a more sensitive and specific test. Therefore, one mycoplasma protein previously identified as being a potential diagnostic antigen, Lipoprotein B (LppB), was tested for its usefulness to detect infected cattle in an indirect ELISA (i-ELISA). Samples obtained from pre-challenged and challenged cattle were compared using CFT and an i-ELISA based on lipoprotein B. The i-ELISA developed with the new antigen, detected more positive samples than the CFT, which is considered a gold standard. Therefore, the LppB recombinant protein could be explored further as target for screening cattle infected chronically with CBPP.
Keywords: CBPP; Mycoplasma mycoides; CFT; LppB; Sensitivity; Specificity
Mycoplasma mycoides subsp. mycoides (Mmm) is the causal agent of contagious bovine pleuropneumonia (CBPP) that causes serious economic losses in cattle in Africa . It is a disease that mainly affects domestic cattle and symptoms range from acute, to sub-acute and chronic forms . Up to date, no single test is able to detect all clinical stages.
Serological tests recommended by the Office of International Epizootics (OIE) for serological diagnosis of CBPP employs either complement fixation test (CFT) and/or competitive enzyme linked immunosorbent assay (c-ELISA). These tests have drawbacks necessitating the need to develop more robust tests. CFT has a high specificity but takes long to perform and requires more elaborate training of staff. Additionally, it is less effective at diagnosing animals with chronic lesions . The c-ELISA test on the other hand, is simpler to carry out. However, validation studies are still insufficient to make good conclusions about its use in surveillance studies where vaccinations are undertaken in enzootic areas of infected zones. In addition, the test is sensitive but its specificity is uncertain . There is a need for development of another test as sensitive as c-ELISA and as specific as CFT.
Preliminary studies have been carried out to introduce surface proteins of the mycoplasma Mmm strain to develop sensitive and specific diagnostics and efficacious vaccines [5-7]. In this study, a few serum samples that had been tested and confirmed positive by CFT were re-tested against LppB recombinant protein to determine if there are differences in antibody responses, sensitivity and specificity between the two tests.
Materials and Methods
All cattle experiments from which the samples were derived were carried out in accordance with the Kenya Agricultural and Livestock Research Organization (KALRO), Veterinary Science Research Institute (VSRI)-Institutional Animal Care and Use Committee (IACUC), numbers: KALRO/VSRI/IACUC/1/29092009 and KALRO/ VSRI/IACUC/2/00122010.
The cattle used in the study were purchased from Kakamega in western Kenya, a CBPP-free zone. The samples were obtained following challenge by contact transmission using a field isolate of Mmm referred to as strain B237 from Thika, Kenya .
Expression and purification of lipoprotein B
Inoculated 5 ml Luria-Broth (LB) medium containing 30 μg/mL kanamycin with a single colony of BL21DE3 STAR cells containing a pETite expression construct of LppB. Shaken overnight at 220 rpm and 370 C in shaker incubator. The following morning, 0.5% glucose was added before addition of 5 ml to 250 ml LB media plus kanamycin. The cultures were grown in a shaker incubator until the optical density (OD at 600 nm) was between 0.5-0.7, before inducing with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h. Cells were centrifuged at 14,000 x g for 30 min at 150 C, then purified under denaturing conditions as described by Miltiadou et al. .
Antibody responses as determined by CFT and LppB
Serum samples were selected on the basis of CFT results and the presence or absence of lesions. They were tested using an indirect ELISA (i-ELISA) based on the LppB antigen. An analysis was then carried out to compare antibody responses, sensitivities and specificities of the two tests. The CFT was carried out according to Campbell et al. .
The LppB assay was performed as follows: Wells of polysorp 96-micro-well plates (Nalge International, Roskilde Denmark) were pre-coated with 150 μl of 1.2 μg/μl LppB recombinant antigen in phosphate buffered saline (PBS) pH 7.4 for 2 h at 370 C. The wells were blocked with 300 μl of blocking buffer (0.2% casein in PBS) for 20 min at 370 C and washed 3 times in washing buffer (PBS + 0.1% Tween 20). Serum test samples were diluted 1:400 in 1% skimmed milk in PBS, 150 μl added in duplicate on the plate and incubated at 370 C for 40 min before washing 5 times in washing buffer. One hundred and fifty microlitres of horse reddish peroxidase (HRP)-conjugated antibovine IgG (Svanova, Sweden) were added at a dilution of 1:10, 000 in PBS and the plate incubated at 370 C for 30 min. The diammonium salt 2, 2’-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) was used as chromogen and H2O2 as substrate for peroxidase to detect bovine anti-LppB antibodies. The plate was incubated in the dark with gentle shaking for 30 min before OD readings were obtained using an Immunoskan ELISA plate reader at 405 nm.
Determination of sensitivities and specificities between tests
Determination of sensitivities and specificities between tests were done using a 2 × 2 contingency table as shown in Table 1 . All cattle were assumed to be positive after challenge and negative before challenge.
|Type of test||Tested (+)||Tested (-)|
|Sensitivity (Se)= Specificity (Sp)=|
Table 1: Calculation of Sensitivities and Specificities.
Data was entered into Microsoft excel 2010 and SPSS 22.0 where Cohen’s Kappa test was used to determine if there was an agreement between CFT and LppB serological tests in detecting positive and negative serum samples.
Antibody responses as determined by CFT and LppB
Table 2 shows results of pre and post-challenge sera, tested with the two assays. Post-challenge sera were further split into sera from animals without lesions, with sequestrae or from early acute cases. These post-challenge sera were selected from a total of 36 cattle, where 20/36 had no lesions, 9/36 had sequestrae and 7/36 had acute infection. The eight pre-challenge samples were samples collected 21 days before challenge. Acute sera were collected within 1 month after challenge while sera from animals with sequestrae and no visible lesions were collected between 3 to 12 months after challenge. The negative and positive sera in various disease stages were collected at same time points. LppB-ELISA was very efficient in detecting serum samples from infected animals; in particular, from animals with sequestrae (100%), and just over half of sera from animals without lesions. However, it did not identify any sample from acutely infected cattle. It also detected one of the pre-vaccination serum samples.
|Test||Positive serum samples of animals|
|Pre-challenge (n = 8)||Post-challenge (n = 36)|
|Without lesions||With sequestrae||With acute infection|
|CFT (KALRO)||0/8 (0%)||2/20 (10%)||3/9 (33.3%)||1/7 (14.3%)|
|LppB (i-ELISA)||1/8 (12.5%)||14/20 (70%)||9/9 (100%)||0/7 (0%)|
Table 2: Number of Serum Samples Testing Positive Pre-Challenge and at Various Disease Stages Post-Challenge.
Determination of sensitivities and specificities between different assays
Table 2 shows CFT and LppB results on post-challenge serum samples for animals without lesions, with sequestrae and early acute cases. Mmm SC was isolated from lungs of all challenged cattle. Upon challenge, fever was first recorded on the 40th day post-contact. Eight out of nine animals with sequestrae presented with fever. The longest period with fever recorded was 14 days in one animal while the shortest fever period recorded lasted 2 days in two animals. All 7 animals with early acute lesions presented with fever. The highest fever recorded lasted 13 days in one animal while the least fever recorded lasted one day in one cattle.
Calculation of sensitivities and specificities
Sensitivities of the assays was calculated as follows: it was assumed that all infected animals were positive, including those that had no lesions since Mmm SC was isolated from lung tissues of all cattle. Specificity was calculated using pre-challenge samples only since these were assumed to be true negatives. In Table 3 below, sensitivity of LppB was found to be higher than that of CFT while specificity of CFT was higher.
|Type of test||CFT||LppB|
Table 3: Results of Sensitivities and Specificities of Two Tests.
Determination of agreement between CFT and LppB
Table 4 below presents the frequency counts for positive and negative serum samples as assessed using CFT and the indirect ELISAs based on LppB recombinant antigen. Although there was no agreement between CFT and LppB in detection of antibodies in no lesions and sequestrae cases respectively, there was significant agreement between CFT and LppB in detection of antibodies in acute cases (p = 0.659).
|Acute Lesions (N=7)||CFT & LppB||-0.167||0.659|
|Sequestrae (N=9)||CFT & LppB||0.000a||0.000|
|No Lesion (N=20)||CFT & LppB||-0.212||0.023|
Table 4: A Comparison of Similarities and Differences between Different Tests.
The observed differences between the sensitivities obtained in our study and previous studies [10,11], are probably due to the disease status of the animals after challenge and the time point at which sera were collected. Animals in our study were infected by in-contact challenge, and there is always uncertainty on the time point at which a particular animal gets infected . In our study, CFT had a significantly lower sensitivity, 17%. In our study the test was performed on serum samples at the end of 3rd week for acute cases, end of 4th month for cases with sequestrae and end of also 4th month for cases with no lesion. In addition, the immune status of the individual animal seems to play a role in the time course and level of specific antibody production . During the acute phases of the disease, CFT detects both IgM and IgG1 that bind complement but not IgG2 . Antibodies detected by CFT decrease early and the number of positive results decreases dramatically when a blood sampling has occurred 3 months later in an outbreak. This possibly explains the low sensitivity of the CFT assay, which depends on complement binding antibodies, mainly IgM . In contrast, c-ELISA strongly detects IgG2 and therefore will identify positive animals in later stages of disease compared to CFT [14,16].
The sensitivities of the i-ELISA with LppB, detecting mainly specific IgG, was 64%. This figure is comparable to the sensitivity of an assay based on another recombinant protein, LppQ , at 69% but with a specificity of 100%. It is known that LppQ induces a very high antibody response , and is also specific for Mmm. However, our i-ELISA assays detected the highest number (100%) of positive serum samples in cases with sequestrae, while 70% of animals without lesions (Table 2). Progress of CBPP in animals with the acute form of disease is rapid and it is possible that such animals have not had time to produce high enough IgG antibody titres to the proteins. High levels of mycoplasma antigens in the acute form will mop up any specific antibodies generated early in the infection, resulting in negative diagnosis, as suggested before . In contrast, animals with sequestrae have been infected for a longer time and therefore IgG antibody titres may have had time to build up in the blood. In animals without lesions, antigen is relatively low compared to animals with sequestrae, so the number of positive samples detected is intermediate.
The two serological tests showed different results. The LppBbased test detected more positive serum samples than CFT. Since LppB detected all animals with sequestrae, it holds the possibility that a diagnostic test could be developed to identify animals with carrier status.
All authors contributed to conception and design of the study; HOL, JN and HOW wrote the paper.
Acknowledgements for Funding
HOL, JN and HOW were supported by the Canadian International Food Security Research Fund (CIFSRF) grant 106929 of the International Development Research Centre (IDRC) and VACNADA grant DCI-FOOD/2009/226-469. HOL was also supported by the BecA-ILRI Hub through the African Biosciences Challenge Fund (ABCF) Program. The ABCF Progarm is funded by the Australian Department for Foreign Affairs and Trade (DFAT) through the BecA-CSIRO partnership; the Syngenta Foundation for Sustainable Agriculture (SFSA); the Bill & Melinda Gates Foundation (BMGF); the UK Department for International Development (DFID) and the Swedish International Development Cooperation Agency (Sida).
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