Nadeeka K. Wawegama

Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

ABSTRACT Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

Membrane proteins of Mycoplasma bovis and their role in pathogenesis

Mycoplasma membrane proteins influence cell shape, cell division, motility and adhesion to host cells, and are thought to be integrally involved in the pathogenesis of mycoplasmoses. Many of the membrane proteins predicted from mycoplasma genome sequences remain hypothetical, as their presence in cellular protein preparations is yet to be established experimentally. Recent genome sequences of several strains of Mycoplasma bovis have provided further insight into the potential role of the membrane proteins of this pathogen in colonisation and infection. This review highlights recent advances in knowledge about the influence of M. bovis membrane proteins on the pathogenesis of infection with this species and identifies future research directions for enhancing our understanding of the role of these proteins.

Evaluation of an IgG Enzyme-Linked Immunosorbent Assay as a Serological Assay for Detection of Mycoplasma bovis Infection in Feedlot Cattle

ABSTRACT Mycoplasma bovis is a pathogen of emerging significance in cattle throughout the world that is causing a range of diseases, including mastitis, arthritis, and pneumonia. The limited availability and efficacy of current diagnostic and prophylactic tools for its control and its increasing antimicrobial resistance are contributing to its increasing importance in beef and dairy cattle. We have developed an indirect IgG enzyme-linked immunosorbent assay (ELISA) based on a recombinant fragment of the MilA protein and have shown its potential as an effective diagnostic tool. To more comprehensively estimate the diagnostic sensitivity and specificity of this IgG ELISA for detection of infection with M. bovis in cattle and to define a suitable cutoff for use in the field, we further assessed its performance in experimentally infected calves in a closed beef herd and by applying Bayesian latent class modeling to laboratory testing results from 7,448 cattle entering Australian feedlots. The most effective cutoff points were estimated to be 68.6 antibody units (AU) for experimentally infected calves and to be 58.7 AU for a closed adult herd. Under field conditions, in feedlot cattle the globally optimal cutoff was estimated to be 105 AU. At this cutoff, the diagnostic sensitivity was 94.3% (95% probability interval [PI], 89.9% to 99.6%) with a diagnostic specificity of 94.4% (95% PI, 90.3% to 99.6%). Applying this 105 AU cutoff, 13.1% of cattle were seropositive for infection with M. bovis on entry into feedlots, and 73.5% were seropositive when followed up approximately 6 weeks later suggesting a high risk of infection shortly after entry into feedlots.

Histochemical and morphometric characterization of broncho- pneumonia in calves caused by infection with Mycoplasma bovis

The aim of this study was to identify morphometric histological features of pneumonia caused by Mycoplasma bovis in calves. Eight three-month-old calves were infected with M. bovis and samples of their lung tissue, three weeks after exposure, compared to samples from four uninfected calves. In the uninfected animals the goblet cells were clustered in the crypt area of the epithelial folds, while in the infected calves they had migrated towards the tips of the folds and were distributed evenly throughout the folds. In infected lung tissue there was goblet cell hyperplasia and metaplasia in the bronchioles and an increased epithelial height. Goblet cell mucin in uninfected calves was acidic, but in infected calves most goblet cells contained neutral mucins. These morphometric and histochemical bronco-epithelial changes may be able to be used as markers of the severity of bovine respiratory mycoplasmosis.

The performance of three immune assays to assess the serological status of cattle experimentally exposed to mycoplasma bovis

Mycoplasma bovis is associated with several clinical syndromes of cattle. Currently, limited information is available on the sensitivity (Se) and specificity (Sp) of serological assays used for the detection of M. bovis-specific antibodies. Consequently, it is difficult to critically evaluate the outcomes of studies that use these assays. Therefore, the current study used bovine sera sourced from M. bovis exposure studies from three countries to estimate the Se and Sp of two commercial M. bovis enzyme-linked immunosorbent assays (ELISA), BIO K302 and BIO K260, and Western blotting. Western blotting had the highest Se estimate of 74% (95% confidence interval (CI): 16–98%), compared to the BIO K302: 47% (95% CI: 10–87%) and BIO K260: 28% (95% CI: 1–92%). However, for Sp, the BIO K302: 96% (95% CI: 87–99%) and the BIO K260: 100% (95% CI: 93–100%) out-performed Western blotting: 88% (95% CI: 56–98%). Western blotting was the best assay for detecting seroconversion, correctly identifying 61% (95% CI: 29–86%) of exposed animals compared to 35% for BIO K302 (95% CI: 21–54%) and 8% for BIO K260 (95% CI: 0–87%). While none of the methods assessed had high Se and Sp, the availability of these estimates will aid in the interpretation of studies that use these assays. The results of this study highlight the difficulties encountered when using serology to detect exposure to M. bovis in cattle.

Reproduction of respiratory mycoplasmosis in calves by exposure to an aerosolised culture of Mycoplasma bovis

Mycoplasma bovis is an important pathogen of cattle, causing pneumonia, arthritis and otitis media in young calves, and mastitis in lactating cows, resulting in increased morbidity and, in some instances, mortality. The objective of this study was to evaluate the survival of a M. bovis isolate following nebulisation and to establish whether respiratory disease similar to that seen in the field could be induced in calves by exposing them to an aerosolised culture of M. bovis. A group of eight M. bovis-free calves 14-28days old were exposed to an aerosolised culture of a field isolate of M. bovis that had originally been recovered from a joint lesion in a calf. Three weeks after aerosol exposure necropsies were conducted on all calves. Lung lesions were seen in 7 of 8 calves exposed to the aerosol of M. bovis, whilst calves exposed to the culture medium alone did not develop lesions. Two calves in the infected group had detectable concentrations of serum antibody against M. bovis on day 7 post infection and 4 calves had detectable concentrations of serum antibody against M. bovis on day 21 post infection when tested by MilA IgG ELISA. M. bovis was reisolated from the upper trachea of 6 of the 8 infected calves. The infection method described here appeared to induce lung lesions typical of naturally occurring disease associated with infection with M. bovis and should be applicable to testing the safety and efficacy of attenuated vaccine candidates to control disease caused by this pathogen.

Immune responses to vaccination and infection with Mycoplasma gallisepticum in turkeys

ABSTRACT Infection with Mycoplasma gallisepticum induces severe lymphoproliferative lesions in multiple sites along the respiratory tract in chickens and turkeys. These immunopathological responses have been well-characterized in chickens, but have not been studied closely in turkeys. The aim of the study described here was to examine the immune responses of turkeys after live vaccination and infection with M. gallisepticum. In a strain comparison study, the mean log10 antibody titre of birds exposed to an aerosol culture of M. gallisepticum strain Ap3AS was found to be significantly higher at day 14 than that of birds exposed to strain 100809/31. In a dose–response study, there was a significant difference in the mean log10 antibody titre between birds exposed to mycoplasma broth and birds exposed to the highest dose of strain Ap3AS at day 7 after exposure. Immunohistochemical analysis of the tracheal mucosa and the air sacs revealed similar patterns of distribution of CD4+ and CD8+ lymphocytes to those seen in the tracheal mucosa of chickens, implicating these cell types in the pathogenesis of respiratory mycoplasmosis in turkeys. Turkeys that had been vaccinated with M. gallisepticum GapA+ ts-11 had significantly higher antibody titres than unvaccinated birds at both 7 and 14 days after challenge with strain Ap3AS. Vaccination with GapA+ ts-11 protected against the lymphoproliferative response to infection with virulent M. gallisepticum in both the tracheal mucosa and the air sacs, suggesting that this strain may be a useful vaccine candidate for use in turkeys.

Improvements in diagnosis of disease caused by Mycoplasma bovis in cattle

The major disease problems associated with Mycoplasma bovis are contagious mastitis and respiratory disease in cattle. Its importance has increased worldwide due to its increasing resistance to antimicrobial agents and the lack of an effective vaccine. Chronically infected silent carriers introduce infection to naive herds and M. bovis diseases can cause significant economic losses to the affected herds. Availability of a suitable diagnostic tool for extensive screening will improve the ability to determine the appropriate treatment and the full impact of M. bovis in both beef and dairy cattle industries. The present review focuses on the past and present improvements in the diagnosis of disease caused by M. bovis in cattle.

Evaluation of a conserved HA274–288 epitope to detect antibodies to highly pathogenic avian influenza virus H5N1 in Indonesian commercial poultry

ABSTRACT A peptide enzyme linked immunosorbent assay (ELISA) based on an epitope in the haemagglutinin (HA) of avian influenza virus H5N1, amino acid positions 274–288 (HA274–288) was evaluated for detection of H5N1-specific antibodies. An optimized ELISA based on the tetrameric form of the HA274–288 epitope designated MP15 gave low background with non-immune chicken sera and detected vaccinated and infected birds. The HA274–288 epitope was highly conserved in Indonesian H5N1 strains and antibody responses were detected in the majority of the vaccinated chickens regardless of the H5N1 strain used for vaccination. The HA274–288 epitope was also conserved in the majority of H5N1 strains from the neighbouring Asian region, and other H5 subtypes potentially allowing for a wider use of the MP15 ELISA in H5N1 vaccinated and infected flocks. The MP15 ELISA results correlated significantly with haemagglutination inhibition (HI) test results and test sensitivity and specificity were 87% and 92%, respectively. The MP15 ELISA titres were significantly higher than the HI titres in all immune sera allowing for sera to be tested at a single dilution of 1:400 which is of advantage in routine surveillance. The study indicated that the MP15 ELISA is potentially useful for serological detection of H5N1 vaccinated or infected poultry and to have some advantages over the standard HI test for routine monitoring of flocks’ immunity after vaccination.

The C-terminal end of the capsid protein of Avian Nephritis Virus is antigenic and induces broadly cross-reactive antibodies

Avian nephritis virus (ANV) has been isolated frequently from commercial broilers in many countries. The prevalence and economic impact of ANV however has been difficult to ascertain due to the lack of convenient serological techniques. In this study the full-length and fragments of the ANV capsid protein were expressed in Baculovirus and affinity purified recombinant proteins used for the detection of ANV antibodies in ELISA. The crystal structure of Human Astrovirus (HAstV) was used as a model to determine potential homologous C-terminal antigenic regions in ANV. The rp37 fragment from three ANV strains NSW_3, ANV-1 and ANV-2, and a shorter NSW_3 fragment (rp33) were compared for their ability to detect ANV antibodies in seven reference chicken sera. The ANV-1 rp37 antigen was the most strain specific whereas the NSW_3 rp37 and rp33 antigens detected antibodies in all heterologous sera, including ANV-1 serum. Irrespective of the strain used, the two NSW_3 protein fragments rp37 and rp33 were found to be superior as antigens for ELISA when compared to the full-length capsid protein rp75. An ELISA designed using the NSW_3 rp33 could reliably differentiate between uninfected and infected commercial broiler flocks, as demonstrated by statistically significant differences between the OD values. This study identified an ANV immunogenic region and successfully used recombinant protein expression of this region to detect cross-reactive ANV antibodies. The results of this study facilitate future studies into the epidemiology and importance of ANV infections in commercial poultry.