Histopathological findings,phenotyping of inflammatory cells,and expression of markers of nitritative injury in joint tissue samples from calves after vaccination and intraarticular challenge with Mycoplasma bovis strain 1067

Background

The pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques.

Results

The inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen.

Conclusions

The results suggest that nitritative injury, as in pneumonic lung tissue of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages.     

Variable number tandem repeat analysis of Mycobacterium bovis isolates from Gyeonggi-do, Korea

Bovine tuberculosis (TB) is a major zoonosis that''s caused by Mycobacterium bovis (M. bovis). Being able to detect M. bovis is important to control bovine TB. We applied a molecular technique, the variable number tandem repeat (VNTR) typing method, to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea. From 2003 to 2004, 59 M. bovis clinical strains were isolated from dairy cattle in Gyeonggi-do, Korea, and these cattle had tuberculosis-like lesions. Twenty-four published MIRU-VNTR markers were applied to the M. bovis isolates and ten of them showed allelic diversity. The most discriminatory locus for the M. bovis isolates in Korea was QUB 3336 (h = 0.64). QUB 26 and MIRU 31 also showed high discriminative power (h = 0.35). The allelic diversity by the combination of all VNTR loci was 0.86. Six loci (MIRU 31, ETR-A and QUB-18, -26, -3232, -3336) displayed valuable allelic diversity. Twelve genotypes were identified from the 59 M. bovis isolates that originated from 20 cattle farms that were dispersed throughout the region of Gyenggi-do. Two genotypes [designation index (d.i.) = e, g] showed the highest prevalence (20% of the total farms). For the multiple outbreaks on three farms, two successive outbreaks were caused by the same genotype at two farms. Interestingly, the third outbreak at one farm was caused by both a new genotype and a previous genotype. In conclusion, this study suggests that MIRU-VNTR typing is useful to identify and distinguish the M. bovis isolates from Gyeonggi-do, Korea.     

Mycobacterium bovis infection in the brush-tailed possum (Trichosurus vulpecula): I. Preliminary observations on experimental infection

An Australian cattle strain of Mycobacterium bovis was injected intramuscularly into 10 brush-tailed possums (Trichosurus vulpecula) which were shown to be highly sensitive to experimental infection with this organism. The possums were killed and examined throughout the course of infection. At necropsy, gross and microscopic lesions were recorded and several tissues cultured for recovery of M. bovis. Infection spread rapidly via the lymphatic system from the injection site to the lumbar lymph nodes, then to the spleen. There was a bacteraemia after 2 week and by 6 weeks lesions were present in spleen, lymph nodes, lungs and kidneys; M. bovis proliferated rapidly and host response was minimal. Few organisms were detected in the liver where miliary lesions were found. M. bovis was excreted in large numbers in urine, faeces and discharging sinuses from subcutaneous abscesses. In two possums that died early in the infection, stress had rendered then more susceptible; infection spread more rapidly than in other possums and liver involvement was more severe. Although aerosol transmission was considered to be a possible means of spreading M. bovis, three in-contact possums did not acquire infection.     

Molecular findings and approaches spotlighting Mycobacterium bovis persistence in cattle

Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (M. bovis) are the etiological agents of human and bovine tuberculosis (TB, bTB) respectively, and share genetic identity over 99% at the whole genome level. Progress has been made towards explaining how mycobacteria and their infected hosts remain in balance without producing clinical symptoms of disease, a phenomenon referred to as latency or persistence, which can be mimicked by certain in vitro conditions. Latency/persistence has mainly been studied using Mtb, where the two-component signalling system, dosRS, has been assigned an instrumental role, and even constitutes the current basis for development of new diagnostic methods and treatment addressing this particular stage of TB. M. bovis conserves homolog genes that in Mtb play a role in human latent TB infection and that, by analogy, would allow it to enter a persistent state in infected cattle; nevertheless, little attention has been paid to this stage in bovine hosts. We suggest that many of the advances acquired through the study of Mtb can and should be taken into consideration by research groups and veterinary professionals dealing with bTB. The study of the infection in bovines, paying particular attention to defining the molecular and cellular markers of a M. bovis persistent infection in cattle, presents great opportunities for the development and trial of new diagnostic tests and vaccines, tools that will surely help in promoting eradication of bTB in high-burden settings.     

Single-nucleotide polymorphism-based epidemiological analysis of Korean Mycobacterium bovis isolates

BackgroundBovine tuberculosis (TB) is caused by Mycobacterium bovis, a well-known cause of zoonotic tuberculosis in cattle and deer, and has been investigated in many physiological and molecular studies. However, detailed genome-level studies of M. bovis have not been performed in Korea.ObjectivesTo survey whole genome-wide single-nucleotide polymorphism (SNP) variants in Korean M. bovis field isolates and to define M. bovis groups in Korea by comparing SNP typing with spoligotyping and variable number tandem repeat typing.MethodsA total of 46 M. bovis field isolates, isolated from laryngopharyngeal lymph nodes and lungs of Korean cattle, wild boar, and Korean water deer, were used to identify SNPs by performing whole-genome sequencing. SNP sites were confirmed via polymerase chain reaction using 87 primer pairs.ResultsWe identified 34 SNP sites with different frequencies across M. bovis isolates, and performed SNP typing and epidemiological analysis, which divided the 46 field isolates into 16 subtypes.ConclusionsThrough SNP analysis, detailed differences in samples with identical spoligotypes could be detected. SNP analysis is, therefore, a useful epidemiological tracing tool that could enable better management of bovine TB, thus preventing further outbreaks and reducing the impact of this disease.     

Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells

Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host’s immune defense as well as avoidance of antimicrobial agents.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0194-z) contains supplementary material, which is available to authorized users.     

Induction of immunity in calves to mycoplasma bovis infection of the respiratory tract

Immunity to colonization of the respiratory tract by Mycoplasma bovis (formerly Mycoplasma agalactiae subsp. bovis was induced in calves by inoculation of formalin inactivated organisms. Animals inoculated intramuscularly and then intratracheally with inactivated mycoplasmas had significantly fewer M. bovis in their lungs, compared with non-vaccinated animals, 3 weeks after intratracheal challenge with viable organisms. In contrast, there was no significant difference in the numbers of M. bovis isolated from the lungs of control animals and of calves given two intramuscular inoculations of inactivated organisms. These results indicate that stimulation of the local immune system is important in the development of resistance to M. bovis respiratory infection following vaccination with inactivated organisms.     

Restriction fragment length polymorphism and spacer oligonucleotide typing: A comparative analysis of fingerprinting strategies for Mycobacterium bovis

The combination of conventional investigation and DNA fingerprinting is yielding important insights into the epidemiology of Mycobacterium bovis infections. Various genetic markers used in restriction fragment length polymorphism (RFLP) have recently been exploited for fingerprinting of M. bovis isolates. The newly developed spacer oligonucleotide typing aimed to investigate the polymorphism of M. tuberculosis in the DR locus, has also been applied to the molecular typing of M. bovis isolates. This work compared the performance of the insertion sequence (IS) IS6110, IS1081 and the genetic elements polymorphic G+C-rich repeat (PGRS) and direct repeat (DR) used in RFLP analysis with spoligotyping using a group of 128 Spanish M. bovis isolates. In this study, the most sensitive technique for identifying polymorphism in M. bovis was PGRS–RFLP, closely followed by IS6110–RFLP. We propose several schemes for fingerprinting of these isolates, however, the clear geographical variations found by different authors makes the study of each local situation indispensable. An international consensus in the methods used would be desirable for efficient interlaboratory comparison of strains.     

5-Bromodeoxyuridine (BUdR) and light inactivation of proliferating Mycobacterium bovis and Mycobacterium avium sensitized bovine lymphocytes

Maximum inactivation (83–98%) of proliferating Mycobacterium bovis and/or Mycobacterium avium sensitized bovine lymphocytes was obtained when the lymphocytes were treated with 10^?4 M of 5-bromodeoxyuridine (BUdR) and light. Inactivation was specific though not total whether the cultures were first stimulated with M. bovis PPD or M. avium PPD, though slightly higher inactivation resulted when cultures were first stimulated with M. bovis PPD. There was, however, a statistically significant suppression (P < 0.05) of the counts/min of cultures initially stimulated with either M. bovis PPD or M. avium PPD and later restimulated with either of the antigens after BUdR-light treatment. Thus experiments showed that inactivation occurs only to those cells responding by proliferation to a particular stimulant. There is a potential for the use of this assay for differentiating infection due to either M. bovis or M. avium if total inactivation of proliferating lymphocytes could be achieved.     

The effect of various Mycoplasma bovis isolates on bovine leukocyte responses

Mycoplasma bovis (M. bovis) contributes to a number of clinical syndromes in cattle; in particular, chronic pneumonia that is poorly responsive to therapy has been increasingly recognized as an important cause of morbidity, mortality, and financial loss. M. bovis impairs host immune function, but little is known about whether field isolates vary significantly in their effect on immune function. This research tested the hypothesis that different field isolates vary in their ability to suppress cellular metabolism and cellular production of radical oxygen species (ROS) by bovine leukocytes. Total blood leukocytes from 6 cattle were exposed to six field isolates, two diagnostic lab isolates, and two high passage laboratory isolates of M. bovis, and ROS production was measured by oxidation of dihydrorhodamine 123 (DHR-123). Cellular metabolism was measured by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Significant differences in the response to some field isolates were identified. Three field isolates and both diagnostic lab isolates significantly decreased ROS production by leukocytes from multiple cattle, while the high pass laboratory isolates did not. In contrast, MTT reduction was not significantly impaired by any of the M. bovis strains tested. M. bovis impairs ROS production by bovine leukocytes; the magnitude of the effect appears to be isolate-dependent, and is not related to a general impairment of cellular metabolism. Chronic M. bovis infection in some cattle may be related to impaired ability of leukocytes to produce ROS when exposed to M. bovis.     

Multiple sampling and discriminatory fingerprinting reveals clonally complex and compartmentalized infections by M. bovis in cattle

The combination of new genotyping tools and a more exhaustive sampling policy in the analysis of infection by Mycobacterium tuberculosis has shown that infection by this pathogen is more complex than initially expected. Mixed infections, coexistence of clonal variants from a parental strain, and compartmentalized infections are all different modalities of this clonal complexity. Until recently, genotyping of Mycobacterium bovis in animal populations was based on spoligotyping and analysis of a single isolate per infection; therefore, clonal complexity is probably underdetected. We used multiple sampling combined with highly discriminatory MIRU-VNTR to study compartmentalized infections by M. bovis in a low-tuberculosis prevalence setting. We spoligotyped the M. bovis isolates from two or more anatomic locations sampled from 55 animals on 39 independent farms. Compartmentalized infections, with two different strains infecting independent lymph nodes in the same animal, were found in six cases (10.9%). MIRU-VNTR analysis confirmed that the compartmentalization was strict and that only one strain was present in each infected node. MIRU-VNTR analysis of additional infected animals on one of the farms confirmed that the compartmentalized infection was a consequence of superinfection, since the two strains were independently infecting other animals. This same analysis revealed the emergence of a microevolved clonal variant in one of the lymph nodes of the compartmentalized animal. Clonal complexity must also be taken into consideration in M. bovis infection, even in low-prevalence settings, and analyses must be adapted to detect it and increase the accuracy of molecular epidemiology studies.     

Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals

Mycobacterium (M.) bovis, a bacterium in the M. tuberculosis complex, is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for diagnosing bovine tuberculosis, but this technique is laborious and time-consuming. In the present study, performance of the SD Bioline TB Ag MPT4 Rapid test, an immunochromatographic assay, was evaluated using reference bacterial strains and M. bovis field isolates collected from animals. The SD MPT64 Rapid test produced positive results for 95.5% (63/66) of the M. bovis isolates from cattle and 97.9% (46/47) of the isolates from deer. Additionally, the test had a sensitivity of 96.5% (95% CI, 91.2-99.0), specificity of 100% (95% CI, 96.7-100.0), positive predictive value of 100% (95% CI, 96.7-100.0), and negative predictive value of 92.9% (95% CI, 82.7-98.0) for M. bovis isolates. In conclusion, the SD MPT64 Rapid test is simple to use and may be useful for quickly confirming the presence of M. bovis in animals.     

Seroprevalence of bovine tuberculosis infection in yaks (Bos grunniens) on the Qinghai-Tibetan Plateau of China

Bovine tuberculosis (BTB), a chronic disease caused by Mycobacterium bovis, has been widely reported in bovines in developing countries, but there is little information on this infection in domestic yaks. Seroprevalence of antibodies to M. bovis in yaks from six and three regions of Tibet and Qinghai plateau, China, respectively, was investigated in 2011 by enzyme-linked immunosorbent assay kits. A total of 1,244 (Tibet 938, Qinghai 306) blood samples was collected and the results showed that 24 (2.6 %) of Tibetan samples and 3 (1 %) of Qinghai's samples were positive for BTB. The findings of the present study indicated that M. bovis infection is prevalent in Chinese yaks in Qinghai and Tibet. These observations should raise a serious public health concern considering the extent to which the herdsmen of the study areas are in contact with their animals and the levels at which they use untreated livestock products. This is the first study showing the infection of M. bovis in domestic yaks.     

Extent of Mycobacterium bovis infection in dairy cattle herds subject to partial culling as determined by an interferon-gamma assay

The interferon-gamma (IFN-γ) assay is employed as a complementary diagnostic test for bovine tuberculosis (BTB) in many countries. To simplify this assay, we established a 96-well plate format using the ESAT-6 and CFP-10 antigens and then employed it to determine the extent of Mycobacterium (M.) bovis infection in dairy herds with a history of BTB outbreaks in a country where only selective culling is practiced. The sensitivity and specificity of this IFN-γ assay were 85.9% and 100%, respectively, based on comparison with the conventional single intradermal tuberculin test (SIDT). The IFN-γ assay was also positive in 30.4% and 36.8% of SIDT-negative animals from herds with recent and remote BTB outbreaks, respectively. Of 14 SIDT-negative, IFN-γ positive cattle, five (35.7%) were culture positive and an additional six were positive based on a polymerase chain reaction-based test for M. bovis. Therefore, the IFN-γ assay has the potential to serve as a specific and sensitive test for M. bovis infection in dairy cattle. Further, the results indicated that a substantial portion of SIDT-negative animals in herds with previous BTB outbreaks were actually infected with M. bovis. Accordingly, the present selective-culling strategy may require modifications to include this more sensitive assay.     

Potential use of lymphocyte blastogenic responses in diagnosis of bovine tuberculosis

A comparison of in vitro lymphocyte responses and delayed type tuberculin skin test responses was made in an animal experimentally exposed to a Mycobacterium bovis-infected animal and in cattle naturally infected with M. bovis. Tuberculin skin tests did not suppress in vitro lymphocyte responses to M. bovis PPD and to M. avium PPD tuberculin. The whole blood test used in these studies provided for considerable savings in time as compared to use of purified lymphocytes for evaluating in vitro cellular responses. Variations in the responsiveness of lymphocytes to specific mycobacterial antigens was observed, therefore, it is recommended that profiles be established using three or more tests conducted at 14-day intervals.     

Tuberculosis in domestic animal species

M. bovis and M. caprae, members of the Mycobacterium tuberculosis complex (MTC), are the major causative agents of tuberculosis in domestic animals. Notably, M. bovis exhibits a wide host range; the infection has been reported in many domesticated animals and free or captive wildlife. Despite most of them acting as spill-over hosts in particular epidemiological scenarios, some domesticated species as pigs, camelids and goats may display high rates of infection and possibly play a role in the inter-species transmission of the disease. The aim of this review is to make an updated overview of the susceptibility and the role in the transmission of the disease of the most common domesticated animals species such as small ruminants, pigs, horses, camelids, dogs and cats. An overview of the diagnostic approaches to detect the infection in each of the species included in the review is also presented.     

Immune response of calves following the inoculation of Mycoplasma dispar and Mycoplasma bovis

A small but significant reduction in the number of Mycoplasma dispar colonising the respiratory tract after intratracheal challenge was observed in gnotobiotic-calves previously inoculated subcutaneously three times with formalin-killed organisms and oil adjuvant. Injection of M. dispar by the intramuscular route, with oil adjuvant, and 2 weeks later by the intratracheal route, without adjuvant, failed to induce immunity to subsequent intratracheal challenge.Following the subcutaneous injection of killed M. dispar, the amount of antibody detected by single radial haemolysis (SRH) increased markedly with increasing age in groups of calves with average ages of 16 to 155 days when first injected. Most calves aged less than 40 days failed to produce an antibody response to a singel injection of M. dispar. With M. bovis a smaller difference was observed between antibody levels generated in calves of different ages; also, calves less than 40 days old produced a detectable SRH antibody response following a single injection of killed M. bovis.IgG1 and IgG2 antibody to M. dispar and M. bovis were measured by ELISA. IgG1 appeared before IgG2 antibody and this was particularly pronounced in younger calves. Also, for both mycoplasmas IgG2 antibody levels were lower in younger than older calves. The IgG1 response to M. dispar was compared in three groups of calves with average ages of 16, 55 and 155 days and was greatest in the oldest and least in the youngest animals. In contrast, the IgG1 response to M. bovis varied little in calves of different ages. It therefore appears that the immune response of young calves to M. dispar is impaired or defective.     

Transfection systems for Babesia bovis: A review of methods for the transient and stable expression of exogenous genes

With the recently sequenced Babesia bovis genome, a large pool of genes with unknown function was identified. The ability to complement and knock-out both unknown and previously identified genes would be a valuable tool to better understand gene function in B. bovis parasites. This review describes recent advances in the development of transient and stable transfection systems for B. bovis. Transient transfection constructs were initially generated using the promoter and the 3′ region of the rap-1 genes of B. bovis controlling expression of luciferase as a reporter. Successful expression of luciferase in B. bovis parasites using this plasmid introduced by classic electroporation of B. bovis infected erythrocytes was followed by the identification and characterization of stronger promoters, such as the ef-1α promoter, using transient transfection techniques. Further refinement of the transient transfection technique included development of the ability to transfect free merozoites using nucleofection, an alternative method to electroporation that results in higher transfection yields and improved viability of transfected parasites. Availability of the transient transfection system was critical for the further development of a stable transfection technique using a plasmid designed to target integration of a gfp-bsd gene into the B. bovis ef-1α locus. Several parasite lines resistant to the anti-babesial drug blasticidin (bsd) and constitutively expressing the gfp-bsd gene were generated after transfection. Integration of the gfp-bsd cassette into the genome was demonstrated by Southern blot and sequence analysis. Taken together these experiments demonstrated the feasibility to introduce, integrate and express exogenous genes in B. bovis. The stable transfection protocol was reproducible and used to transfect at least two distinct B. bovis strains. Further development of these transfection systems will facilitate functional analysis of B. bovis genes and will improve our understanding of the biology of and immunological response to this parasite.