Evaluating the association between blood genotype or phenotype and haemoplasma infection in UK and Italian cats.

BACKGROUND: In humans, blood groups are associated with varying prevalence of infections. The aim of this study was to determine if associations exist between the feline AB blood group system and haemoplasma infection. METHODS: Data from two studies were combined. In the first study, DNA samples from 131 haemoplasma-infected and 132 haemoplasma-uninfected UK cats underwent pyrosequencing to determine their blood genotype as AA, Ab or bb. In the second study, blood samples from 160 Italian cats of known blood phenotype A, B or AB underwent PCR testing for feline haemoplasma species DNA. RESULTS: Haemoplasma infection was demonstrated in cats of all phenotypes and genotypes. A significantly higher number of Ab genotype cats tested positive for overall haemoplasma infection status (p = 0.04) and for Mycoplasma haemofelis infection (p = 0.03). LIMITATIONS: Haemoplasma-infected Italian cats were few, possibly increasing the chance of type II error, and the presence of purebred cats in the sample population may have had a confounding effect. CONCLUSIONS: Feline haemoplasmas do not appear to preferentially use either blood type A or B antigens as attachment sites for erythrocyte colonisation. Further investigations in a larger number of haemoplasma-infected cats of known blood phenotype are warranted to explain the association between genotype Ab and haemoplasma infection.

Vaccines for Mycoplasma Diseases of Small Ruminants: A Neglected Area of Research.

Mycoplasmas cause some of the most economically important diseases of sheep and goats, including diseases listed by the World Organisation for Animal Health (OIE) such as contagious caprine pleuropneumonia (CCPP) and contagious agalactia (CA). Other important mycoplasma diseases include chronic respiratory and arthritic syndrome (CRAS) and atypical pneumonia, both present on all continents where small ruminants are farmed. Unfortunately, owing to a lack of investment, most commercial vaccines for these diseases are of poor quality, being mostly composed of killed bacteriocins of dubious or unknown efficacy. Several Mediterranean laboratories produce autogenous vaccines, but these can only be used on farms where outbreaks have been officially declared, and consequently have limited impact on disease nationally. Effective live vaccines are available, but their use is often restricted because of safety concerns. With the necessary safeguards in place, we argue for their greater use. This review examines reported vaccines for mycoplasma diseases of small ruminants and attempts to identify new candidate antigens that may enable the development of improved products. Vaccines for CCPP are covered elsewhere.

Detection of Mycoplasma agalactiae in Ticks (Rhipicephalus bursa) Collected by Sheep and Goats in Sicily (South-Italy), Endemic Area for Contagious Agalactia.

The aim of this preliminary study was to investigate the presence of Mycoplasma agalactiae (Ma) or other Contagious Agalactia (CA) causative organisms, in hard ticks infesting milking sheep and goats in endemic areas for CA in Sicily (South-Italy). Although there is accumulating evidence to support the role of ticks in the transmission of blood-borne haemoplasmas, information regarding their role in the transmission of CA, remains scarce. Ticks (n = 152) were collected from 25 lactating sheep and goats from three farms with previous outbreaks of CA. Microbiological and biomolecular, as well as serological analysis were performed on milk, tick, and serum samples, respectively. Rhipicephalus bursa species predominated, comprising 84.8% of the sampled ticks. Mycoplasma-like colonies were isolated from 5/56 (8.9%) tick pools and were identified as Ma by specific PCR and 16S rRNA gene sequencing. Unexpectedly, the organism was isolated from R. bursa ticks recovered only from animals whose milk tested negative for the pathogen. This preliminary demonstration suggests the potential role for ticks to act as a reservoir for the organisms, with potential involvement in the spread and maintenance of CA. Further work is required to determine the location of the organisms within the body of the ticks and to assess transmission potential.

Mycoplasma agalactiae: The Sole Cause of Classical Contagious Agalactia?

Contagious agalactia (CA) is suspected when small ruminants show all or several of the following clinical signs: mastitis, arthritis, keratoconjunctivitis and occasionally abortion. It is confirmed following mycoplasma isolation or detection. The historical and major cause is Mycoplasma agalactiae which was first isolated from sheep in 1923. Over the last thirty years, three other mycoplasmas (Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens) have been added to the etiology of CA because they can occasionally cause clinically similar outcomes though nearly always in goats. However, only M. agalactiae is subject to animal disease regulations nationally and internationally. Consequently, it makes little sense to list mycoplasmas other than M. agalactiae as causes of the OIE-listed CA when they are not officially reported by the veterinary authorities and unlikely to be so in the future. Indeed, encouraging countries just to report M. agalactiae may bring about a better understanding of the importance of CA. In conclusion, we recommend that CA should only be diagnosed and confirmed when M. agalactiae is detected either by isolation or molecular methods, and that the other three mycoplasmas be removed from the OIE Manual of Diagnostic Tests and Vaccines in Terrestrial Animals and associated sources.

Mycoplasmas: Brain invaders?

Mycoplasmas of humans and animals are usually associated with respiratory, autoimmune, genital and joint diseases. Human mycoplasmas have also been known to affect the brain. Severe central nervous system (CNS) diseases, such as encephalitis, have been linked to Mycoplasma pneumoniae and ureaplasma infections. Less well known is the sheep and goat pathogen, Mycoplasma agalactiae, which has been found in large quantities in the brain where it may be responsible for non-purulent encephalitis as well as ataxia in young animals. Experimental intra-mammary infections of sheep with this mycoplasma have resulted in histopathological changes in the CNS. The cattle pathogen, M. bovis, has been reported occasionally in the brains of calves and adult cattle showing a range of histopathological lesions including abscesses and fibrinous meningitis. Two avian pathogens, M. gallisepticum and M. synoviae have been isolated from the brains of poultry showing meningeal vasculitis and encephalitis. There have been no reported detections of two other avian pathogens, M. meleagridis or M. iowae in the CNS. Over the last few decades, mycoplasmas have been isolated from the brains of sea mammals dying in large numbers in the North Sea although it was concluded that their role may be secondary to underlying viral disease. Finally, evidence has been advanced that certain Spiroplasma species may have a role in the development of the transmissible spongiform encephalopathies (TSE). Invasion of the brain by mycoplasmas may be as a result of direct entry following damage to the inner ear as seen with M. bovis or across the blood brain barrier by mechanisms as yet uncertain.

Spray dried hyaluronic acid microparticles for adhesion controlled aggregation and potential stimulation of stem cells.

Spray-dried microparticles of a derivative of hyaluronic acid (HA) have been engineered to obtain a controlled aggregation with Human Mesenchymal Stem Cells (hMSCs) into 3D constructs. We demonstrated the utility of chemical functionalization of a native constituent of the extracellular matrix to improve processing performances and to control on stem cell adhesion and differentiation. Native hyaluronic acid (HA), cell adhesive peptides (RGD), transforming growth factor ß3, dexamethasone are biological agents potentially suitable for chondrogenic stimulation of hMSCS. However unmodified HA suffers of drawbacks in terms of stability and versatility of processing. Functionalization strategies are needed to overcome these drawbacks. In this paper microparticles were produced by spray-drying of an aliphatic and amino functionalized HA derivative. Hydrophobic derivatization of HA allowed the production of microparticles stabilized by physical crosslinking and to load and to control dexamethasone release. The presence of pendant amino groups was exploited to tether cyclic RGD and transforming growth factor ß3via maleimide chemistry; cyRGDC functionalization controlled hMSCs/microparticles aggregation. Chondrogenic potential was preliminary assayed by qualitative immunohistological detection.

A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells.

Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32-44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated ß-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression.

Cytotoxic activity of the novel small molecule AKT inhibitor SC66 in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is characterized by limited response to current drug therapies. Here, we report that SC66, a novel AKT inhibitor, reduced cell viability in a dose- and time-dependent manner, inhibited colony formation and induced apoptosis in HCC cells. SC66 treatment led to a reduction in total and phospho-AKT levels. This was associated with alterations in cytoskeleton organization, a reduction in expression levels of E-cadherin, ß-catenin and phospho-FAK, together with up-regulation of Snail protein levels. All these alterations were coupled with anoikis cell death induction. In addition, SC66 induced the production of reactive oxygen species (ROS) and DNA damage. Pre-treatment with the ROS scavenger N-Acetyl-cysteine (NAC) prevented SC66-induced cell growth inhibition and anoikis. SC66 significantly potentiated the effects of both conventional chemotherapeutic and targeted agents, doxorubicin and everolimus, respectively. In vivo, SC66 inhibited tumor growth of Hep3B cells in xenograft models, with a similar mechanism observed in the in vitro model. Taken together, these data indicate that the AKT inhibitor SC66 had antitumor effects on HCC cells. This was mediated by ROS production, induction of anoikis-mediated cell death and inhibition of the AKT cell survival pathway. Our results provide a rational basis for the use of SC66 in HCC treatment.

Timing of activation of CD4+ memory cells as a possible marker to establish the efficacy of vaccines against contagious agalactia in sheep.

Mycoplasma agalactiae is a major pathogen of sheep and goats in many areas of the world and particularly in Mediterranean countries. It causes contagious agalactia, an infectious disease primarily affecting mammary glands. Many vaccines against the pathogen are currently under development. The aim of the study was to investigate the involvement of T cell-mediated immunity during vaccination and challenge experiments against Mycoplasma agalactiae. A comparison of the antigen-specific expansion of interferon gamma positive T cell memory and naïve subsets was performed between vaccinated and non-vaccinated sheep to identify cellular subsets whose activation was different between protected and non-protected sheep. Data reported in this manuscript demonstrated that two out of the three vaccines used in this study protected sheep from the disease. In the protected groups CD4(+) memory interferon-γ(+) T cells underwent an early expansion (p<0.05 when compared to unprotected groups), whilst memory CD8(+) Interferon-γ(+) T cells increased in non-protected animals 7 days after infection (p<0.05). γδ(+) Interferon-γ(+) T cells reached peaks of expansion in infected and in two vaccinated groups thus indicating that these cells are not preferentially involved in protection or pathogenesis (p<0.05). Hereby we propose that the early activation of CD4(+) memory Interferon-γ(+) T cells could be considered as a marker of protection from the disease as well as a tool to establish vaccine efficacy.

Biochemical characterisation of some non fermenting, non arginine hydrolysing mycoplasmas of ruminants.

The pattern and kinetics of substrate oxidation by type and recent field strains of Mycoplasma agalactiae, Mycoplasma bovis, Mycoplasma bovigenitalium and Mycoplasma ovine/caprine serogroup 11 were investigated by measurement of oxygen uptake. Metabolism of a range of organic acids, sugars and alcohols was detected. All the test strains were unable to oxidise sugars, glycerol and the organic acids, fumarate, malate and alpha-ketoglutarate (1 mM). All strains oxidised organic acid l-lactate, 2-oxobutyrate and pyruvate and demonstrated the ability to oxidise alcohols, particularly isopropanol, which was oxidised at a high rate and high affinity (0.5 mol/mol isopropanol). Its oxidation was consistent with acetone formation, which may be of important in relation to pathogenicity. All strains oxidised similar substrates, however differences were observed between strains in terms of the relative rates and kinetic values for some substrates.

Pan-Mediterranean comparison for the molecular detection of Theileria annulata.

Seven laboratories decided to compare their molecular diagnostic techniques to identify Mediterranean theileriosis caused by Theileria annulata. Each laboratory used either PCR or PCR and reverse line blot hybridization (RLB) to identify T. annulata. Five laboratories sent their own samples to laboratory 4 to be recoded and passed on to at least two other laboratories. A total of 120 blood samples were analyzed during this study, generating 540 results. Laboratory 1 sent only T. annulata-infected samples (positive control batch), and all the laboratories testing this batch found 100% infection. Laboratory 2 sent only negative samples from a Mediterranean area where T. annulata was unknown, and two laboratories out of three found a few positive samples in these negative samples. For the remaining samples, detection performance was variable. Agreement between laboratories ranged from 21.4 to 91.3%. The overall mismatch between laboratories was around 30% by whatever technique used. This paper describes the methodological parameters that could explain the variation of results.