Development and Evaluation of a Combined Contagious Bovine Pleuropneumonia (CBPP) and Lumpy Skin Disease (LSD) Live Vaccine.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP). Lumpy skin disease (LSD) is a viral disease of cattle caused by lumpy skin disease virus (LSDV). LSD and CBPP are both transboundary diseases spreading in the same areas of Africa and Asia. A combination vaccine to control CBPP and LSD offers significant value to small-scale livestock keepers as a single administration. Access to a bivalent vaccine may improve vaccination rates for both pathogens. In the present study, we evaluated the LSDV/CBPP live combined vaccine by testing the generation of virus neutralizing antibodies, immunogenicity, and safety on target species. In-vitro assessment of the Mycoplasma effect on LSDV growth in cell culture was evaluated by infectious virus titration and qPCR during 3 serial passages, whereas in-vivo interference was assessed through the antibody response to vaccination. This combined Mmm/LSDV vaccine could be used to protect cattle against both diseases with a single vaccination in the endemic countries. There were no adverse reactions detected in this study and inoculated cattle produced high levels of specific antibodies starting from day 7 post-vaccination, suggesting that this combination vaccine is both safe and effective.

Beware of Mycoplasma Anti-immunoglobulin Strategies.

Mycoplasmas are small, genome-reduced bacteria. They are obligate parasites that can be found in a wide range of host species, including the majority of livestock animals and humans. Colonization of the host can result in a wide spectrum of outcomes. In many cases, these successful parasites are considered commensal, as they are found in the microbiota of asymptomatic carriers. Conversely, mycoplasmas can also be pathogenic, as they are associated with a range of both acute and chronic inflammatory diseases which are problematic in veterinary and human medicine. The chronicity of mycoplasma infections and the ability of these bacteria to infect even recently vaccinated individuals clearly indicate that they are able to successfully evade their host's humoral immune response. Over the years, multiple strategies of immune evasion have been identified in mycoplasmas, with a number of them aimed at generating important antigenic diversity. More recently, mycoplasma-specific anti-immunoglobulin strategies have also been characterized. Through the expression of the immunoglobulin-binding proteins protein M or mycoplasma immunoglobulin binding (MIB), mycoplasmas have the ability to target the host's antibodies and to prevent them from interacting with their cognate antigens. In this review, we discuss how these discoveries shed new light on the relationship between mycoplasmas and their host's immune system. We also propose that these strategies should be taken into consideration for future studies, as they are key to our understanding of mycoplasma diseases' chronic and inflammatory nature and are probably a contributing factor to reduce vaccine efficacy.

Infection strategies of mycoplasmas: Unraveling the panoply of virulence factors.

Mycoplasmas, the smallest bacteria lacking a cell wall, can cause various diseases in both humans and animals. Mycoplasmas harbor a variety of virulence factors that enable them to overcome numerous barriers of entry into the host; using accessory proteins, mycoplasma adhesins can bind to the receptors or extracellular matrix of the host cell. Although the host immune system can eradicate the invading mycoplasma in most cases, a few sagacious mycoplasmas employ a series of invasion and immune escape strategies to ensure their continued survival within their hosts. For instance, capsular polysaccharides are crucial for anti-phagocytosis and immunomodulation. Invasive enzymes degrade reactive oxygen species, neutrophil extracellular traps, and immunoglobulins. Biofilm formation is important for establishing a persistent infection. During proliferation, successfully surviving mycoplasmas generate numerous metabolites, including hydrogen peroxide, ammonia and hydrogen sulfide; or secrete various exotoxins, such as community-acquired respiratory distress syndrome toxin, and hemolysins; and express various pathogenic enzymes, all of which have potent toxic effects on host cells. Furthermore, some inherent components of mycoplasmas, such as lipids, membrane lipoproteins, and even mycoplasma-generated superantigens, can exert a significant pathogenic impact on the host cells or the immune system. In this review, we describe the proposed virulence factors in the toolkit of notorious mycoplasmas to better understand the pathogenic features of these bacteria, along with their pathogenic mechanisms.

Immune and sex-biased gene expression in the threatened Mojave desert tortoise, Gopherus agassizii.

The immune system of ectotherms, particularly non-avian reptiles, remains poorly characterized regarding the genes involved in immune function, and their function in wild populations. We used RNA-Seq to explore the systemic response of Mojave desert tortoise (Gopherus agassizii) gene expression to three levels of Mycoplasma infection to better understand the host response to this bacterial pathogen. We found over an order of magnitude more genes differentially expressed between male and female tortoises (1,037 genes) than differentially expressed among immune groups (40 genes). There were 8 genes differentially expressed among both variables that can be considered sex-biased immune genes in this tortoise. Among experimental immune groups we find enriched GO biological processes for cysteine catabolism, regulation of type 1 interferon production, and regulation of cytokine production involved in immune response. Sex-biased transcription involves iron ion transport, iron ion homeostasis, and regulation of interferon-beta production to be enriched. More detailed work is needed to assess the seasonal response of the candidate genes found here. How seasonal fluctuation of testosterone and corticosterone modulate the immunosuppression of males and their susceptibility to Mycoplasma infection also warrants further investigation, as well as the importance of iron in the immune function and sex-biased differences of this species. Finally, future transcriptional studies should avoid drawing blood from tortoises via subcarapacial venipuncture as the variable aspiration of lymphatic fluid will confound the differential expression of genes.

Baseline analysis of Mycoplasma mycoides subsp. mycoides antigens as targets for a DIVA assay for use with a subunit vaccine for contagious bovine pleuropneumonia.

BACKGROUND: Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia in cattle. A prototype subunit vaccine is being developed, however, there is currently no diagnostic test that can differentiate between infected cattle and those vaccinated with the prototype subunit vaccine. This study characterized Mmm proteins to identify potential antigens for use in differentiating infected from vaccinated animals. RESULTS: Ten Mmm antigens expressed as recombinant proteins were tested in an indirect ELISA using experimental sera from control groups, infected, and vaccinated animals. Data were imported into R software for analysis and drawing of the box and scatter plots while Cohen's Kappa assessed the level of agreement between the Mmm antigens. Two vaccine antigens (MSC_0499 and MSC_0776) were superior in detecting antibodies in sera of animals vaccinated with the subunit vaccines while two non-vaccine antigens (MSC_0636 and LppB) detected antibodies in sera of infected animals showing all clinical stages of the disease. Sensitivity and specificity of above 87.5% were achieved when the MSC_0499 and MSC_0636 antigens were tested on sera from vaccinated and infected animals. CONCLUSIONS: The MSC_0499 and MSC_0776 antigens were the most promising for detecting vaccinated animals, while MSC_0636 and LppB were the best targets to identify infected animals. Further testing of sera from vaccinated and infected animals collected at different time intervals in the field should help establish how useful a diagnostic test based on a cocktail of these proteins would be.

Impacts of Mycoplasma loads and lung lesions on immune and hematological statuses of pigs in an eight-breed cross heterogeneous population.

Developments of pulmonary diseases, often accompanied by infections of bacteria, severely affect the meat production and welfare of pigs. This study investigated 307 pigs at age of 240 d from an eight-breed cross reared under standardized housing conditions for associations among the extent of lung lesions, bacteria load inferred from 16S rRNA sequencing of bronchoalveolar lavage fluid, as well as 57 immune cells and 25 hematological traits. We showed that the pigs under study suffered substantial and varied lung lesions, and the Mycoplasma is the most associated bacteria genera. At a false discovery rate of 0.05 (FDR < 0.05), the severity of lung lesions were significantly associated with greater CD8+ to CD3+ cell ratio, neutrophil-to-lymphocyte ratio (NLR), and standard deviation of red blood cell volume distribution width (RDW-SD), and lower CD4-CD8-/CD3+, CD3+CD4-CD8-/PBMCs (peripheral blood mononuclear cells) and CD14-CD16-/PBMCs cell ratios, mean corpuscular hemoglobin concentration, lymphocyte count, and lymphocyte count percentage, reflecting an status of inflammation, immune suppression, and hypoxia of the pigs accompanying the progression of the lung lesions. The Mycoplasma abundance showed positive correlations with neutrophil count, neutrophil count percentage, NLR, monocyte count, coefficient of variation in red blood cell volume distribution width , and RDW-SD, and negative correlations with mean corpuscular hemoglobin concentration, lymphocyte count, and lymphocyte count percentage; these correlations are largely consistent with those of lung lesions, supporting the comorbidity of lung lesions and Mycoplasma infection. We also observed nonlinear associations that sharp increases in neutrophil count and neutrophil count percentage occurred only when Mycoplasma abundance raised above the population-average level. The results provide helpful insights into the changes of host immune status in response to Mycoplasma relevant lung diseases in pigs.

Contagious Bovine Pleuropneumonia: A Comprehensive Overview.

Contagious bovine pleuropneumonia (CBPP) is a respiratory disease of cattle that is listed as notifiable by the World Organization for Animal Health. It is endemic in sub-Saharan Africa and causes important productivity losses due to the high mortality and morbidity rates. CBPP is caused by Mycoplasma mycoides subsp. mycoides (Mmm) and is characterized by severe fibrinous bronchopneumonia and pleural effusion during the acute to subacute stages and by pulmonary sequestra in chronic cases. Additional lesions can be detected in the kidneys and in the carpal and tarsal joints of calves. Mmm infection occurs through the inhalation of infected aerosol droplets. After the colonization of bronchioles and alveoli, Mmm invades blood and lymphatic vessels and causes vasculitis. Moreover, Mmm can be occasionally demonstrated in blood and in a variety of other tissues. In the lung, Mmm antigen is commonly detected on bronchiolar and alveolar epithelial cells, in lung phagocytic cells, within the wall of blood and lymphatic vessels, inside necrotic areas, and within tertiary lymphoid follicles. Mmm antigen can also be present in the cytoplasm of macrophages within lymph node sinuses, in the germinal center of lymphoid follicles, in glomerular endothelial cells, and in renal tubules. A complete pathological examination is of great value for a rapid presumptive diagnosis, but laboratory investigations are mandatory for definitive diagnosis. The purpose of this review is to describe the main features of CBPP including the causative agent, history, geographic distribution, epidemiology, clinical course, diagnosis, and control. A special focus is placed on gross and microscopic lesions in order to familiarize veterinarians with the pathology and pathogenesis of CBPP.

Macrophages protect mycoplasma-infected chronic myeloid leukemia cells from natural killer cell killing.

Macrophages (Mϕ) have been reported to downmodulate the cytotoxicity of natural killer (NK) cell against solid tumor cells. However, the collaborative role between NK cells and Mϕ remains underappreciated, especially in hematological cancers, such as chronic myeloid leukemia (CML). We observed a higher ratio of innate immune cells (Mϕ and NK) to adaptive immune cells (T and B cells) in CML bone marrow aspirates, prompting us to investigate the roles of NK and Mϕ in CML. Using coculture models simulating the tumor inflammatory environment, we observed that Mϕ protects CML from NK attack only when CML was itself mycoplasma-infected and under chronic infection-inflammation condition. We found that the Mϕ-protective effect on CML was associated with the maintenance of CD16 level on the NK cell membrane. Although the NK membrane CD16 (mCD16) was actively shed in Mϕ + NK + CML trioculture, the NK mCD16 level was maintained, and this was independent of the modulation of sheddase by tissue inhibitor of metalloproteinase 1 or inhibitory cytokine transforming growth factor beta. Instead, we found that this process of NK mCD16 maintenance was conferred by Mϕ in a contact-dependent manner. We propose a new perspective on anti-CML strategy through abrogating Mϕ-mediated retention of NK surface CD16.

Molecular detection of vector-borne agents in cats in Southern Brazil / Detecção molecular de agentes transmitidos por vetores em felinos domésticos no Sul do Brasil

Abstract This study used serological and molecular methods to investigate the occurrence of vector-borne pathogens (VBP) with zoonotic potential in cats neutered at the University Veterinary Hospital in Canoinhas, Santa Catarina. The combined PCR and serological results revealed that 17 (56.6%) cats were positive for one or more pathogens. The sampled cats had antibodies to Ehrlichia spp. (7/30), Anaplasma phagocytophilum (3/30) and Leishmania infantum (2/30). The PCR assay detected DNA closely related to Ehrlichia canis in 6/30 cats, Mycoplasma haemofelis in 2/30 cats, A. phagocytophilum and Cytauxzoon sp. in one cat each. While Bartonella clarridgeiae and B. henselae were detected in two cats each, and B. koehlerae was detected in one cat.

Resumo Como os felinos podem ser parasitados por diversos patógenos transmitidos por vetores (PTV), alguns com caráter zoonótico, este estudo objetivou detectar por métodos sorológicos e moleculares, patógenos transmitidos por vetores hematófagos, em gatos atendidos em um Hospital Veterinário Universitário em Santa Catarina. Os resultados da PCR e da sorologia combinados, revelaram que 17 (56,6%) gatos foram positivos para um ou mais patógenos. Na sorologia, foram positivos 7/30 gatos para Ehrlichia, 3/30 para Anaplasma phagocytophilum e 2/30 para Leishmania infantum. Na PCR foi detectado DNA filogeneticamente associado a: Ehrlichia canis em 6/30 gatos; Mycoplasma haemofelis, em 2/30 gatos; A. phagocytophilum e Cytauxzoon sp. em 1/30 gatos cada. Enquanto Bartonella clarridgeiae e B. henselae foram detectadas, cada uma, em dois gatos, B. koehlerae foi detectada em um gato.

Molecular detection of vector-borne agents in cats in Southern Brazil.

This study used serological and molecular methods to investigate the occurrence of vector-borne pathogens (VBP) with zoonotic potential in cats neutered at the University Veterinary Hospital in Canoinhas, Santa Catarina. The combined PCR and serological results revealed that 17 (56.6%) cats were positive for one or more pathogens. The sampled cats had antibodies to Ehrlichia spp. (7/30), Anaplasma phagocytophilum (3/30) and Leishmania infantum (2/30). The PCR assay detected DNA closely related to Ehrlichia canis in 6/30 cats, Mycoplasma haemofelis in 2/30 cats, A. phagocytophilum and Cytauxzoon sp. in one cat each. While Bartonella clarridgeiae and B. henselae were detected in two cats each, and B. koehlerae was detected in one cat.

DNA Vaccines Against Mycoplasma Elicit Humoral Immune Responses in Ostriches.

In ostriches, the population densities resulting from intensive rearing increases susceptibility to pathogens such as mycoplasmas. In addition to good management practices, vaccination offers an attractive alternative for controlling mycoplasma infections in food animals, instead of using antibiotics, which often leave unacceptable residues. The use of live attenuated vaccines, however, carry the concern of reversion to virulence or genetic recombination with field strains. Currently there are no commercially available vaccines against ostrich-infecting mycoplasmas and this study therefore set out to develop and evaluate the use of a DNA vaccine against mycoplasma infections in ostriches using an OppA protein as antigen. To this end, the oppA gene of "Mycoplasma nasistruthionis sp. nov." str. Ms03 was cloned into two DNA vaccine expression vectors after codon correction by site-directed mutagenesis. Three-months-old ostriches were then vaccinated intramuscularly at different doses followed by a booster vaccination after 6 weeks. The ability of the DNA vaccines to elicit an anti-OppA antibody response was evaluated by ELISA using the recombinant OppA protein of Ms03 as coating antigen. A statistically significant anti-OppA antibody response could be detected after administration of a booster vaccination indicating that the OppA protein was successfully immunogenic. The responses were also both dose and vector dependent. In conclusion, the DNA vaccines were able to elicit an immune response in ostriches and can therefore be viewed as an option for the development of vaccines against mycoplasma infections.

Upper respiratory tract disease and associated diagnostic tests of mycoplasmosis in Alabama populations of Gopher tortoises, Gopherus polyphemus.

Upper respiratory tract disease (URTD) in North American tortoises (Gopherus) has been the focus of numerous laboratory and field investigations, yet the prevalence and importance of this disease remains unclear across many tortoise populations. Furthermore, much research has been focused on understanding diagnostic biomarkers of two known agents of URTD, Mycoplasma agassizii and Mycoplasma testudineum, yet the reliability and importance of these diagnostic biomarkers across populations is unclear. Gopher Tortoises (Gopherus polyphemus) have experienced significant declines and are currently protected range wide. Geographically, Alabama represents an important connection for Gopher Tortoise populations between the core and periphery of this species' distribution. Herein, we systematically sampled 197 Gopher Tortoises for URTD across seven sites in south-central and south-eastern Alabama. Plasma samples were assayed for antibodies to M. agassizii and M. testudineum; nasal lavage samples were assayed for the presence of viable pathogens as well as pathogen DNA. Lastly, animals were scored for the presence of external symptoms and nasal scarring consistent with URTD. External symptoms of URTD were present in G. polyphemus in all sites sampled in Alabama. There was no relationship between active symptoms of URTD and Mycoplasma antibodies, however the presence of URTD nasal scarring was positively related to M. agassizii antibodies (P = 0.032). For a single site that was sampled in three sequential years, seroprevalence to M. agassizii significantly varied among years (P < 0.0001). Mycoplasma agassizii DNA was isolated from four of the seven sites using quantitative PCR, yet none of the samples were culture positive for either of the pathogens. An analysis of disease status and condition indicated that there was a significant, positive relationship between the severity of URTD symptoms and relative body mass (P < 0.05). This study highlights the need for continued monitoring of disease in wild populations. Specifically, focus must be placed on identifying other likely pathogens and relevant biomarkers that may be important drivers of URTD in North American tortoises. Special consideration should be given to environmental contexts that may render wild populations more susceptible to disease.

Vector-borne pathogens affecting shelter dogs in eastern Crete, Greece.

Canine pathogens transmitted by blood-sucking arthropods are of significant importance for veterinary and, in some cases, human health. However, they are still underestimated and rarely investigated in many parts of the Mediterranean region, mostly due to financial reasons. Therefore, in the present paper, we investigated the occurrence of blood-associated pathogens affecting dogs in Crete, Greece. For this purpose, blood samples from 103 shelter dogs were screened for the pathogens by PCR and serological tests. Overall, samples from 43 dogs scored positive for at least one pathogen (41.8%). In particular, antibodies to Leishmania infantum were detected in 26 dogs (25.2%), and 15 and 11 animals were positive for Hepatozoon canis (14.6%) and Mycoplasma haemocanis (10.7%) by PCR, respectively. Co-infections were recorded in nine animals. Clinical signs indicative of infection (alterations of skin or coat or reduced body condition) were detected in 10 animals, four of which were infected with one pathogen, three with two pathogens. Based on the results obtained, dogs from Crete appear to be frequently exposed to several blood-borne pathogens, including agents of zoonotic concern. Given that some of the pathogens were reported for the first time in this area, results presented in our study should improve the awareness of the local veterinarians and of dog rescue organisations in order to reduce disease burden on stray and owned dogs and to control the spread of canine vector-borne diseases from Greece to non-endemic areas by travelling or exported infected dogs.

Effects of mycoplasma infection on the host organism response via p53/NF-κB signaling.

Mycoplasmas are bacteria lacking the cell wall, which is the major characteristic of this taxonomic class (Mollicutes). Among bacteria, mycoplasmas possess the smallest genome known for free-living organisms. This feature limits the autonomy of bacteria and makes them increasingly susceptible to changes in the host organism. Many mycoplasmas themselves cause pathological changes in the host organism, often complicated by immune disorders. Infection with certain strains of mycoplasma results in the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells, which is the major mediator of the inflammatory response. Furthermore, mycoplasmas can inhibit p53-mediated checkpoint control of cell cycle and apoptosis. Collectively, these properties indicate that mycoplasmas might act as cancer-promoting factors. In this review, we summarize the information known to date on the role of mycoplasmas in the regulation of the host immune response and their functional interactions with p53.

Livestock abundance predicts vampire bat demography, immune profiles and bacterial infection risk.

Human activities create novel food resources that can alter wildlife-pathogen interactions. If resources amplify or dampen, pathogen transmission probably depends on both host ecology and pathogen biology, but studies that measure responses to provisioning across both scales are rare. We tested these relationships with a 4-year study of 369 common vampire bats across 10 sites in Peru and Belize that differ in the abundance of livestock, an important anthropogenic food source. We quantified innate and adaptive immunity from bats and assessed infection with two common bacteria. We predicted that abundant livestock could reduce starvation and foraging effort, allowing for greater investments in immunity. Bats from high-livestock sites had higher microbicidal activity and proportions of neutrophils but lower immunoglobulin G and proportions of lymphocytes, suggesting more investment in innate relative to adaptive immunity and either greater chronic stress or pathogen exposure. This relationship was most pronounced in reproductive bats, which were also more common in high-livestock sites, suggesting feedbacks between demographic correlates of provisioning and immunity. Infection with both Bartonella and haemoplasmas were correlated with similar immune profiles, and both pathogens tended to be less prevalent in high-livestock sites, although effects were weaker for haemoplasmas. These differing responses to provisioning might therefore reflect distinct transmission processes. Predicting how provisioning alters host-pathogen interactions requires considering how both within-host processes and transmission modes respond to resource shifts.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'.

Clinical evaluation of outdoor cats exposed to ectoparasites and associated risk for vector-borne infections in southern Italy.

BACKGROUND: Cats can be carriers of infected arthropods and be infected with several vector-borne pathogens (VBP) but there is limited knowledge about their pathogenic role in cats. RESULTS: A cross-sectional controlled study investigated the clinical status and antibody (Bartonella henselae, Rickettsia conorii, Ehrlichia canis, Anaplasma phagocytophilum, Babesia microti and Leishmania infantum) and/or blood PCR (Mycoplasma spp., Bartonella spp., Rickettsia spp., Ehrlichia/Anaplasma spp., piroplasmids, L. infantum, Hepatozoon felis) prevalence in 197 cats. Outdoor cats lacking ectoparasiticide treatment or hosting ectoparasites (study group [SG], n = 134) and indoor cats treated against ectoparasites (control group [CG], n = 63) were enrolled. Clinical data and retroviral co-infections were compared between the two groups. Multivariable analysis tested associations between variables and VBP exposure. Lymphadenia, stomatitis, and various haematological abnormalities were statistically more frequent in SG. Antibodies against R. conorii, B. henselae, A. phagocytophylum, B. microti, E. canis and L. infantum were detected. Bartonella henselae, Bartonella clarridgeiae, Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum" and "Candidatus Mycoplasma turicensis" DNA were identified. Very high antibody (87.8%) and PCR (40.1%) positivity to at least one pathogen were detected and were significantly higher in SG. Co-infections were confirmed in about one-third of the cats and were more frequent in SG cats. Molecular and overall (antibody and PCR) positivity to Bartonella and antibody positivity to R. conorii were higher in SG. Multivariable analysis found significant associations of Bartonella spp. infection with Feline Immunodeficiency Virus (FIV) infection and increased globulins, and of Mycoplasma spp. infection with adult age, FIV infection, anaemia, and increased creatinine. CONCLUSIONS: A very high prevalence of exposure to zoonotic VBP was found in cats, with Rickettsia and Bartonella infections being most prevalent. Some risk factors were documented namely for Mycoplasma spp. and Bartonella spp. The lifestyle of cats is clinically relevant and requires specific preventative measures to protect their health.

Acute phase proteins response in cats naturally infected by hemotropic mycoplasmas.

Information about the acute phase proteins (APP) response in cats naturally infected with hemoplasmas and in cats co-infected with different species of hemoplasmas is lacking. This study evaluated serum amyloid A (SAA), haptoglobin (Hp) and albumin in 48 cats naturally infected with hemoplasmas, including 25 with Candidatus Mycoplasma haemominutum and 23 co-infected with different hemoplasmas agents; and in 10 healthy control cats. Infected cats had significantly higher Hp and lower albumin than controls. Symptomatic cats had significantly higher SAA and Hp, and lower albumin than asymptomatic animals, and also than controls. Asymptomatic cats had significantly higher Hp than controls. Concentrations of APP were not significantly different between single infected and co-infected cats. According with these results, hemoplasmosis should be considered when alterations in APP are detected in diseased cats with compatible clinical signs. Furthermore, a subclinical infection should be considered in apparently healthy cats from endemic areas with increased Hp.

Development of an antigen specific colloidal gold immunochromatographic assay for detection of antibody to M. wenyonii in bovine sera.

The goal of this research was to develop a colloidal gold immunochromatographic strip test for detection of antibody to Mycoplasma wenyonii (M. wenyonii) in bovine using specific antigen. M. wenyonii was isolated from blood samples from the spontaneously infected cattle in Hebei province, China. Suspensions of the M. wenyonii antigenic proteins were prepared by freeze-thaw cycles and ultrasonication. Candidate antigens were screened with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The specific bands of the most antigenic proteins were excised from the gel and were purified by using a gel extraction kit. A colloidal gold immunochromatographic assay using the purified specific proteins as the coating antigen (sp-GICA) was developed for detection of antibody to M. wenyonii. Blood samples from cows in the field were tested for antibody to M. wenyonii by the sp-GICA strip and enzyme-linked immunosorbent assay (ELISA) simultaneously to compare the specificity, sensitivity and accuracy. The results showed that the specific proteins bands with sufficient immunoreactivity have been identified. The apparent molecular weights of the proteins were 115 kDa and 60 kDa, respectively. The stability and reproducibility were quite excellent after the storage of the strip at room temperature for 5 months. This sp-GICA showed 95.48% (148/155), 92.86% (39/42) and 94.92% (187/197) in terms of specificity, sensitivity and accuracy compared to ELISA. The sp-GICA described here shows excellent agreement with ELISA and it is shown to be a simple, convenient, specific and highly sensitive assay for detection of serum antibodies to M. wenyonii.

Mapping of a Mycoplasma-Neutralizing Epitope on the Mycoplasmal p37 Protein.

Many studies have shown that the mycoplasmal membrane protein p37 enhances cancer cell migration, invasion, and metastasis. Previously, we generated 6 monoclonal antibodies (MAbs) against the mycoplasmal protein p37 and showed the presence of mycoplasma-infected circulating tumor cells in the blood of hepatocellular carcinoma patients by using CA27, one of the six MAbs. When mycoplasmas were incubated with cancer cells in the presence of CA27, mycoplasma infection was completely inhibited, suggesting that CA27 is a neutralizing antibody inhibiting mycoplasma infection. To examine the neutralizing epitope of CA27, we generated a series of glutathione S-transferase (GST)-fused p37 deletion mutant proteins in which p37 was partly deleted. To express p37-coding sequences in E.coli, mycoplasmal TGA codons were substituted with TGG in the p37 deletion mutant genes. GST-fused p37 deletion mutant proteins were then screened to identify the epitope targeted by CA27. Western blots showed that CA27 bound to the residues 216-246 on the middle part of the p37 protein while it did not bind to the residues 183-219 and 216-240. Fine mapping showed that CA27 was able to bind to the residues 226-246, but its binding activity was relatively weakened as compared to that to the residues 216-246, suggesting that the residues 226-246 is essential for optimal binding activity of CA27. Interestingly, the treatment of the purified GST-tagged epitopes with urea showed that CA27 binding to the epitope was sodium dodecyl sulfate-resistant but urea-sensitive. The same 226-246 residues were also recognized by two other anti-p37 MAbs, suggesting that the epitope is immunodominant. The identification of the novel neutralizing epitope may provide new insight into the interaction between the p37 protein and host receptors.

Alice in Wonderland syndrome associated with mycoplasma infection.

Alice in Wonderland syndrome (AIWS) is a rare condition in which patients report distorted size perception of objects and their own bodies. Although specific causes and pathology have not been elucidated, an association between AIWS and infection has been suggested. To our knowledge, mycoplasma-induced AIWS has not been examined. A girl aged 7 years 11 months presented with fever (temperature, 40°C) and cough. Although the fever disappeared after approximately 10 days, she complained that her mother's face suddenly appeared smaller to her. Subsequently, she complained that objects intermittently appeared smaller than normal. Particle agglutination test indicated elevated serum antibodies against Mycoplasma pneumoniae. The patient was therefore diagnosed the patient with AIWS secondary to mycoplasma infection. Although mycoplasma infection is known to cause various central nervous system symptoms, this is the first report involving AIWS, suggesting that mycoplasma could affect visual function in children.