Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer

BACKGROUND Calculus bovis(CB),used in traditional Chinese medicine,exhibits anti-tumor effects in various cancer models.It also constitutes an integral component of a compound formulation known as Pien Tze Huang,which is indicated for the treatment of liver cancer.However,its impact on the liver cancer tumor microenvironment,particularly on tumor-associated macrophages(TAMs),is not well understood.AIM To elucidate the anti-liver cancer effect of CB by inhibiting M2-TAM polarization via Wnt/β-catenin pathway modulation.METHODS This study identified the active components of CB using UPLC-Q-TOF-MS,evaluated its anti-neoplastic effects in a nude mouse model,and elucidated the underlying mechanisms via network pharmacology,transcriptomics,and molecular docking.In vitro assays were used to investigate the effects of CB-containing serum on HepG2 cells and M2-TAMs,and Wnt pathway modulation was validated by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis.RESULTS This study identified 22 active components in CB,11 of which were detected in the bloodstream.Preclinical investigations have demonstrated the ability of CB to effectively inhibit liver tumor growth.An integrated approach employing network pharmacology,transcriptomics,and molecular docking implicated the Wnt signaling pathway as a target of the antineoplastic activity of CB by suppressing M2-TAM polarization.In vitro and in vivo experiments further confirmed that CB significantly hinders M2-TAM polarization and suppresses Wnt/β-catenin pathway activation.The inhibitory effect of CB on M2-TAMs was reversed when treated with the Wnt agonist SKL2001,confirming its pathway specificity.CONCLUSION This study demonstrated that CB mediates inhibition of M2-TAM polarization through the Wnt/β-catenin pathway,contributing to the suppression of liver cancer growth.

Promise and challenges of traditional Chinese medicine,specifically Calculus bovis,in liver cancer treatment

Liver cancer,one of the most common malignancies worldwide,ranks sixth in incidence and third in mortality.Liver cancer treatment options are diverse,inclu-ding surgical resection,liver transplantation,percutaneous ablation,transarterial chemoembolization,radiotherapy,chemotherapy,targeted therapy,immuno-therapy,and traditional Chinese medicine(TCM).A multidisciplinary team(MDT)is essential to customize treatment plans based on tumor staging,liver function,and performance status(PS),ensuring individualized patient care.Treatment decisions require a MDT to tailor strategies based on tumor staging,liver function,and PS,ensuring personalized care.The approval of new first-line and second-line drugs and the establishment of standard treatments based on immune checkpoint inhibitors have significantly expanded treatment options for advanced liver cancer,improving overall prognosis.However,many patients do not respond effectively to these treatments and ultimately succumb to the disease.Modern oncology treatments,while extending patient survival,often come with severe side effects,resistance,and damage to the body,negatively impacting quality of life.Huang et al's study published at World Journal of Gastroenterology rigorously validates the anticancer properties of Calculus bovis,enhancing our understanding of TCM and contributing to new liver cancer treatment strategies.For over 5000 years,TCM has been used in East Asian countries like China to treat various diseases,including liver conditions.Analysis of real-world clinical data suggests that for patients with advanced-stage tumors lacking effective treatments,integrated TCM therapies could provide significant breakthroughs.

Harnessing the power of Calculus bovis:Anti-cancer properties and Wnt pathway modulation in hepatocellular carcinoma

In this manuscript,we comment on the article,which explores the anti-cancer effects of Calculus bovis(CB)in tumor biology.We highlight its potential,particularly in hepatocellular carcinoma(HCC),where it inhibits the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways and induces apoptosis.CB contains compounds such as oleanolic acid and ursolic acid that target interleukin-6,mitogen-activated protein kinase 8,vascular endothelial growth factor,and caspase-3,offering anti-inflammatory and hepatoprotective benefits.The manuscript also discusses CB sativus(CBS),an artificial substitute,which has shown efficacy in reducing hepatic inflammation and oxidative stress in animal models.We emphasize the need for further research on the effects of CBS on the gut-liver axis and gut microbiota,and on targeting Wnt signaling and M2 tumor-associated macrophage as potential therapeutic strategies against HCC.

Calculus bovis in hepatocellular carcinoma:Tumor molecular basis,Wnt/β-catenin pathway role,and protective mechanism

In this editorial,we comment on the recent article by Huang et al.The editorial focuses specifically on the molecular mechanisms of hepatocellular carcinoma(HCC),mechanism of Wnt/β-catenin pathway in HCC,and protective mechanism of Calculus bovis(CB)in HCC.Liver cancer is the fourth most common cause of cancer-related deaths globally.The most prevalent kind of primary liver cancer,HCC,is typically brought on by long-term viral infections(hepatitis B and C),non-alcoholic steatohepatitis,excessive alcohol consumption,and other conditions that can cause the liver to become chronically inflamed and cirrhotic.CB is a wellknown traditional remedy in China and Japan and has been used extensively to treat a variety of diseases,such as high fever,convulsions,and stroke.Disturbances in lipid metabolism,cholesterol metabolism,bile acid metabolism,alcohol metabolism,and xenobiotic detoxification lead to fatty liver disease and liver cirrhosis.Succinate,which is a tricarboxylic acid cycle intermediate,is vital to energy production and mitochondrial metabolism.It is also thought to be a signaling molecule in metabolism and in the development and spread of liver malignancies.The Wnt/β-catenin pathway is made up of a group of proteins that are essential for both adult tissue homeostasis and embryonic development.Cancer is frequently caused by the dysregulation of the Wnt/β-catenin signaling pathway.In HCC liver carcinogenesis,Wnt/β-catenin signaling is activated by the expression of downstream target genes.Communication between the liver and the gut exists via the portal vein,biliary tract,and systemic circulation.This"gutliver axis"controls intestinal physiology.One of the main factors contributing to the development,progression,and treatment resistance of HCC is the abnormal activation of the Wnt/β-Catenin signaling pathway.Therefore,understanding this pathway is essential to treating HCC.Eleven ingredients of CB,particularly oleanolic acid,ergosterol,and ursolic acid,have anti-primary liver cancer properties.Additionally,CB is important in the treatment of primary liver cancer through pathways linked to immune system function and apoptosis.CB also inhibits the proliferation of cancer stem cells and tumor cells and controls the tumor microenvironment.In the future,clinicians may be able to recommend one of many potential new drugs from CB ingredients to treat HCC expression,development,and progress.

From traditional Chinese medicine formulations to effective anticancer agents:Insights from Calculus bovis

This editorial examines the therapeutic potential of traditional Chinese medicine(TCM)for aggressive cancers,particularly liver cancer.It highlights the study by Huang et al,which shows how Calculus bovis,a component of the TCM Pien Tze Huang,suppresses liver cancer by inhibiting M2 macrophage polarization via the Wnt/β-catenin pathway.This research emphasizes the importance of transitioning from effective TCM formulations to isolating active components and understanding their mechanisms.While the study provides valuable insights,it primarily focuses on the Wnt/β-catenin pathway and does not delve deeply into the mechanisms of individual components.Future research should aim to comprehensively study these components,explore their interactions,and validate findings through clinical trials.This approach will integrate traditional wisdom with modern scientific validation,advancing the development of innovative cancer treatments based on TCM formulations.

Anti-tumor efficacy of Calculus bovis: Suppressing liver cancer by targeting tumor-associated macrophages

Despite significant advances in our understanding of the molecular pathogenesis of liver cancer and the availability of novel pharmacotherapies,liver cancer remains the fourth leading cause of cancer-related mortality worldwide.Tumor relapse,resistance to current anti-cancer drugs,metastasis,and organ toxicity are the major challenges that prevent considerable improvements in patient survival and quality of life.Calculus bovis(CB),an ancient Chinese medicinal drug,has been used to treat various pathologies,including stroke,convulsion,epilepsy,pain,and cancer.In this editorial,we discuss the research findings recently published by Huang et al on the therapeutic effects of CB in inhibiting the development of liver cancer.Utilizing the comprehensive transcriptomic analyses,in vitro experiments,and in vivo studies,the authors demonstrated that CB treatment inhibits the tumor-promoting M2 phenotype of tumor-associated macrophages via downregulating Wnt pathway.While multiple studies have been performed to explore the molecular mechanisms regulated by CB,this study uniquely shows its role in modulating the M2 phenotype of macrophages present within the tumor microenvironment.This study opens new avenues of future investigations aimed at investigating this drug’s efficacy in various mouse models including the effects of combination therapy,and against drug-resistant tumors.

Exploring a new chapter in traditional Chinese medicine:The potential of Calculus bovis in liver cancer treatment

In the ongoing quest for new treatments in medicine,traditional Chinese medicine offers unique insights and potential.Recently,studies on the ability of Calculus bovis to inhibit M2-type tumour-associated macrophage polarisation by modulating the Wnt/β-catenin signalling pathway to suppress liver cancer have undoubtedly revealed new benefits and hope for this field of research.The purpose of this article is to comment on this study and explore its strengths and weaknesses,thereby providing ideas for the future treatment of liver cancer.

Paenibacillus bovis BD3526产胞外多糖的培养条件优化

利用单因素试验和响应面法对Paenibacillus bovis BD3526产胞外多糖发酵条件工艺参数进行优化。结果表明:培养温度、时间、培养基装量与Paenibacillus bovis BD3526胞外多糖产量存在显著的相关性。当麸皮质量浓度为30 g/L时,BD3526产胞外多糖的优选条件为:培养温度29.91℃、培养时间23.96 h、培养基装量59.58m L/250 m L锥形瓶,采取上述优选条件进行培养,理论预测最大胞外多糖产量为11.11 g/L。验证试验实际测得多糖平均产量为10.92 g/L,比理论预测值低1.71%。

Effects of in vitro cultivated Calculus Bovis compound on pulmonary lesions in rabbits with schistosomiasis

AIM:To explore the interventional effects and mechanism of in vitro cultivated Calculus Bovis compound preparation(ICCBco) on pulmonary lesions in portal hypertensive rabbits with schistosomiasis. METHODS:The experimental group included 20 portal hypertensive rabbits with schistosomiasis treated by ICCBco.The control group included 20 portal hypertensive rabbits with schistosomiasis treated by praziquantel. The morphological changes of the pulmonary tissues were observed under light and electron microscopy.The expression of fibronectin(FN) and laminin(LN) in the lung tissues was analyzed by immunohistochemistry. RESULTS:Under light microscope,the alveolar exudation in the lung tissue was more frequently observed in the control group,while the alveolar space was fairly dry in the lung tissue of ICCBco group.Under electron microscope,more alveolar exudation in the lung tissue,and moremacrophages,alveolar angiotelectasis and the blurred three-tier structure of alveolar-capillary barrier could be seen in the control group.In ICCBco group,fibers within the alveolar interspace slightly increased in some lung regions,and the structure of typeⅠepithelium,basement membrane and endodermis was complete,and no obvious exudation from the alveolar space,and novascular congestion could be observed.There was a positive or strong positive expression of FN and LN in the lung tissue of the control group,while there was a negative or weak positive expression of FN and LN in ICCBco group. CONCLUSION:ICCBco can effectively prevent pulmonary complications in portal hypertensive rabbits with schistosomiasis by means of improving lung microcirculation and lowering the content of extracellular matrix.

Association of Streptococcus bovis presence in colonic content with advanced colonic lesion

AIM:To prospectively examine the association between presence of Streptococcus bovis(S.bovis)in colonic suction fluid and the endoscopic findings on colonoscopy.METHODS:From May 2012 to March 2013,203 consecutivepatients who underwent colonoscopy for any reason were enrolled in the study.Exclusion criteria included:antibiotic use in the previous month,age younger than18 years,and inadequate preparation for colonoscopy.The colonoscopy was performed for the total length of the colon or to the occluding tumor.The endoscopic findings were registered.Samples were obtained proximal to the colonoscopic part of the suction tube from each patient and sent to the clinical microbiology laboratory for isolation and identification of S.bovis.Samples were incubated in enrichment media with addition of antibiotic disks for inhibition of growth of Gram-negative rods.The samples were seeded on differential growth media;suspected positive colonies were isolated and identified with Gram staining,catalase,and pyrrolidonyl arylamidase tests,and further identified using a VITEK2 system.Statistical analyses were performed using the Student’s t andχ2 tests.RESULTS:Of the 203 patients recruited,49(24%)patients were found to be S.bovis carriers;of them,the endoscopic findings included:17(34.7%)cases with malignant tumors,11(22.4%)with large polyps,5(10.2%)with medium-sized polyps,6(12.2%)with small polyps,4(8.1%)with colitis,and 6(12.2%)normal colonoscopies.Of 154 patients found negative for S.bovis,the endoscopic findings included:none with malignant tumors,9(5.8%)cases with large polyps,11(7.1%)with medium-sized polyps,26(16.9%)with small polyps,7(4.5%)with colitis,and101(65.6%)normal colonoscopies.S.bovis(Grampositive coccus)is considered part of the normal intestinal flora.There is an association between S.bovis bacteremia and colonic neoplasia.It is not well understood whether the bacterium has a pathogenetic role in the development of neoplasia or constitutes an epiphenomenon of colorectal neoplasms.There was a clear relationship between positivity for S.bovis in colonic suction fluid and findings of malignant tumors and large polyps in the colon.CONCLUSION:There is an association between S.bovis bacteremia and malignant colonic lesions;this should prompt for development of a reliable screening method for advanced colonic lesions.

The Protective Effects of In Vitro Cultivated Calculus Bovis on the Cerebral and Myocardial Cells in Hypoxic Mice

The protective effects of in vitro cultivated calculus bovis （ICCB） on the cerebral and myocardial cells in hypoxic mice and the mechanism were examined. In one group, mice were intragastrically （i.g.） given ICCB for 15 days and then they were subjected to acute cerebral ischemia by decapitation, and then the panting time was recorded. In the other group, 12 min after exposure to hypoxia, mice was administered the ICCB i.g. for 5 days, and then the blood serum and tissues of brain, heart, liver were harvested and examined for SOD, GSH-px and T-AOC activity and content of MDA. The tissues of brain and heart were observed electron-microscopically for ultrastructural changes. The corpus striatum and hippocampus of brain were collected and examined for content of dopamine （DA） and norepinephrine （NE）. The ultrastrural examination showed that the pathological change in brain and heart in the ICCB group was very slight, while abnormal changes in the control group were obviously more serious. ICCB significantly prolonged the panting time of the hypoxic mice （P〈0.001）, increased the activity of SOD, GSH-px, T-AOC in serum and tissues of brain, liver, heart and elevated the content of DA and NE. ICCB also pronouncedly reduced content of MDA in serum and tissues of brain, heart and liver. Significant differences in these parameters were noted between ICCB group and controls. It is concluded that ICCB can exert protective effect on the cells of brain and myocardium by enhancing the tolerance of the tissues to hypoxia and the body＇s ability to remove free radicals and regulating the neurotransmitters.

Detection of Babesia bovis in blood samples and its effect on the hematological and serum biochemical profile in large ruminants from Southern Punjab

Objective:To determine the presence of Babesia bovis(B.bovis) in large ruminants in southern Punjab and its effect on hematological and serum biochemical profile of host animals.Methods:Blood samples were collected from 144 large ruminants,including 105 cattle and 39 buffaloes,from six districts in southern Punjab including Multan,Layyah,Muzaffar Garh,Bhakar,Bahawalnagar and Vehari.Data on the characteristics of animals and herds were collected through questionnaires.Different blood(hemoglobin,glucose) and serum(ALT,AST,LDH,cholesterol)parameters of calves and cattle were measured and compared between parasite positive and negative samples to demonstrate the effect of B.bovis on the blood and serological profile of infected animals.Results:27 out of 144 animals.from 5 out of 6 sampling districts,produced the541-bp fragment specific for B.bovis.Age of animals(P=0.02).presence of ticks on animals(P=0.04)and presence of ticks on dogs associated with herds(P=0.5) were among the major risk factors involved in the spread of bovine babesiosis in the study area.ALT concentrations were the only serum biochemical values that significantly varied between parasite positive and negative cattle.Conclusions:This study has reported for the first time the presence of B.bovis in large ruminant and the results can lead to the prevention of babesiosis in the region to increase the livestock output.

Disseminated infection due to Mycobacterium bovis after intravesical BCG instillation

Intravesical bacillus Calmette-Guerin(BCG) instillation has been adopted for the treatment of patients with superficial bladder cancer. Severe adverse events due to local instillation of BCG are uncommon, with an overall rate of serious complications of less than 5%. We report the case of an immunocompetent adult patient with multi-system effects, namely pneumonitis, granulomatous hepatitis and meningitis, who responded well to standard treatment for Mycobacterium bovis. This case highlights the importance of a thorough assessment of this type of patient.

Specific inhibition of Streptococcus bovis by endolysin LyJH307 supplementation shifts the rumen microbiota and metabolic pathways related to carbohydrate metabolism

Background:Endolysins,the bacteriophage-originated peptidoglycan hydrolases,are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance.The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S.bovis and to explore changes in rumen fermentation and microbiota in an in vitro system.Two treatments were used:1)control,corn grain without LyJH307;and 2)LyJH307,corn grain with LyJH307(4 U/mL).An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers(450±30 kg)and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time.In vitro dry matter digestibility,pH,volatile fatty acids,and lactate concentration were estimated at 12 h,and the gas production was measured at 6,9,and 12 h.The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing.Results:LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S.bovis(approximately 70% compared to control,P=0.0289)and increased ruminal pH(P=0.0335)at the 12 h incubation.The acetate proportion(P=0.0362)was significantly increased after LyJH307 addition,whereas propionate(P=0.0379)was decreased.LyJH307 supplementation increased D-lactate(P=0.0340)without any change in L-lactate concentration(P>0.10).There were no significant differences in Shannon’s index,Simpson’s index,Chao1 estimates,and evenness(P>0.10).Based on Bray-Curtis dissimilarity matrices,the LyJH307 affected the overall shift in microbiota(P=0.097).LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium,WCHB1-41,unclassified genus Selenomonadaceae,Paraprevotella,vadinBE97,Ruminococcus gauvreauii group,Lactobacillus,Anaerorhabdus furcosa group,Victivallaceae,Desulfuromonadaceae,and Sediminispirochaeta.The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism.Conclusions:LyJH307 caused a reduction of S.bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism.This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota.

Niuhuang(Bovis Calculus)-Shexiang(Moschus)combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway

Objective To investigate the effects of Niuhuang(Bovis Calculus,BC)and Shexiang(Moschus)(BC-Moschus)on human hepatocellular carcinoma(HCC)cells SMMC-7721 and a nude mouse model of subcutaneous xenografts,and to explore its anti-HCC mechanism.Methods The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro.SMMC-7721 was divided into the BC-Moschus group and the control group,and different doses(rude drug dosage 0.625,1.25,2.5,and 5 mg/m L)of BC-Moschus extract were used for the intervention.The proliferation ability of HCC cells was detected using the Cell Counting Kit-8(CCK-8)assay,and the migration ability was detected by a wound healing assay.A subcutaneous xenograft model was prepared using nude mice with human HCC.Specific pathogen-free-grade BALB/c nude mice(5-week-old)were randomly divided into the following groups(n=6 per group):control(0.9%physiological saline 0.2 m L/d),BC-Moschus[BC 45.5 mg/(kg·d)+Moschus 13 mg/(kg·d)],and cisplatin(DDP,intraperitoneal injection5 mg/kg per week)groups.All groups were administered for 14 d.The volume and mass of the subcutaneous xenografts in nude mice were observed.The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway,apoptosis-associated factor p70 S6 Kinase(S6K),Bax,Bcl-2,caspase-3,and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR(RT-qPCR)and Western blot.Terminal Deoxynucleotidy Transferase-Mediated d UTP NickEnd Labeling(TUNEL)was used for quantitative analysis of apoptotic cells.Results The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone,and the inhibition effect was dose-and time-dependent(P<0.01).The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration(P<0.01).In the subcutaneous xenograft model of nude mice with human HCC,we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group(P<0.01).The levels of the PI3K/AKT/m TOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased(P<0.01).The expression level of the anti-apoptotic gene Bcl-2 was downregulated(P<0.05),and the expression of the pro-apoptotic gene Baxand apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated(P<0.01).The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC(P<0.01).Conclusion The BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice.The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway,regulation of apoptosis-related protein caspase-3,caspase-9,Bcl-2,and Bax expression,and promotion of apoptosis.

Observation on Culture Characteristics of Mycoplasma bovis with Four Different Antigen Quantification Methods

[Objective] The paper was to explore the antigen harvest time of Mycoplasrna bovis and the antigen quantification alternative method. [Method] M. bovis 08M strain was inoculated in the Thiaueourt＇s medium containing 10% horse serum. Four growth curves were plotted by simultaneously measuring color change units （CCU）, colony forming units （CFU）, protein concentration and nucleic acid levels within 110 h. [ Result] The growth of M. bovis was divided into four phases： the longarithmie phase appeared after being cultured for 10 h; the stationary phase appeared at 30 h with the highest number of viable cells up to 1. 0× 108 CCU/mL and 7.7 × 107 CCU/mL, respectively; and the decline phase started at 75 h. The protein concentration afM. bov/s increased rapidly from 15 to 35 h, reached 72.06 μg/mL at 35 h, then maintained at 53.38 - 70.65 μg/mL. The nucleic acid levels of M. bov/s increased rapidly from 15 h, and the cycle threshold （Ct） values were maintained between 15, 32 and 17.84 after 25 h. [ Conclusion] There was a good correlation between the protein concentration and variable count of M. bov/s at the early stationary phase, which was the best time period to harvest antigen. The protein concentration determination could be an alternative method to quantify antigen content of M. bovis when preparing inactivated M. boviz vaccine. Key words Mycoplasma boyis; Antigen quantification; Color change units; Colony forming units; Protein concentration; Nucleic acid content

Diagnosis and Treatment Report of the First Case of Mycoplasma bovis in Shandong Province

[Objective] The paper was to introduce the first case of Mycoplasma bovis in Shandong Province.[Method] Samples were collected from sick cattle,and identified through M.bovis isolation,biochemical identification,PCR,16S RNA sequence analysis and clinical treatment and other research work.[Result] A strain of M.bovis was successfully isolated,and typical colonies in fried egg shape were grown.It was further confirmed as M.bovis via molecular biological detection.The use of vaccine and sensitive drugs in clinical could quickly control the epidemic situation of cat-tle herds.[Conclusion] This is the first reported case of M.bovis in Shandong Province,and its diagnosis and treatment process is conducive to further prevention and control of the disease.

Differential IL10 mRNA Profiles Associated to <i>Babesia bovis</i>and <i>B. bigemina</i>Infection Levels in Persistently Infected Animals

This work aimed to find quantitative phenotypic traits that can be used to discriminate the levels of resistance/susceptibility to B. bovis and B. bigemina in two groups of cattle presenting the highest (H) or lowest (L) infection levels and Rhipicephalus microplus ticks count. The animals were selected from a previous study of 50 Canchim (5/8 Charolais/zebu) heifers raised in an endemic area for these parasites. These animals were evaluated regarding their TNFα, IL10, IFN-γ, IL12 and iNOS mRNA levels. No differences were found between these groups regarding TNFα, IFN-γ, IL12β or iNOS transcripts. However, the IL10 transcripts were significantly higher in the H group compared to the L group. Moreover, significant correlation coefficients were observed between B. bovis loads and both IL10 and IFN-γ transcripts, while no correlations were found for B. bigemina loads and all tested immune-related transcripts, suggesting that differential IL10 mRNA profiles were closely associated to B. bovis loads. Our results have contributed to a better understanding of the immune responses against Babesia infection, as we demonstrated that the IL10 cytokine levels might also influence or be influenced by parasitemia levels in persistently infected animals.

Seroprevalence of <i>Mycoplasma bovis</i>Infection in Dairy Cows in Ho Chi Minh, Vietnam

A cross-sectional study was carried out to investigate the seroprevalence of M. bovis. A total of 606 serum samples were randomly collected from dairy cows in Ho Chi Minh City, Vietnam. Commercial ELISA kit was used for detection of antibody to M. bovis. The results indicated that overall seroprevalence was 80.2% (486/606). There were no significant differences between regions or age categories (P > 0.05). Degree of infection to M. bovis commonly distributed at positive degree 1 (68.5%) and 2 (24.1%). Seroprevalence at highest positive degree 4 were found in District 9, District 12 and Thu Duc (6.7%, 6.3% and 1.6%, respectively). This study is the first report of seroprevalence of M. bovis in Vietnam. The results suggested that M. bovis was spreading among dairy cow populations although degree of positivity was low. It should be considered as a high risk pathogen to dairy cows in Vietnam.

Repeated inoculations of Mycobacterium bovis Bacille Calmette-Guérin (BCG) are needed to induce a strong humoral immune response against antigens expressed by the bacteria

The cellular immune response elicited by Mycobacterium bovis Bacille Calmette-Guérin (BCG) has been carefully investigated, but the humoral immune response has been partially neglected. BALB/c mice were immunized with BCG strain used to immunize humans. Anti-BCG antibodies, as assayed by ELISA, began to appear in the sera after the third week of immunization and plateaued three weeks after the 8th immunization. The total immunoglobulins (Igs) were purified by caprylic acid method from pooled serum collected after the 8th immunization. Anti-BCG antigen antibodies were detected in the total Igs preparation as well as in IgG, IgM, IgA, IgG1, IgG2a, and IgG2b, but not in the IgG3. Distinct BCG proteins were recognized the IgGs in Western blot analysis. Opsonization of BCG bacilli by the purified Igs potentiated internalization of the bacteria by murine Raw 264.7 macrophages. The intracellular BCG elimination coincided with the induction of NO production, which was more pronounced in cells infected with opsonized BCG compared to those infected with the non-opsonized bacteria. Coincidently, the production of NO was also higher in macrophages infected with opsonized BCG (maximal NO production at 48 h of incubation). The obtained results demonstrate that repeated inoculations of BCG effectively activate the humoral immune response, justifying the use of BCG as a live recombinant vaccine vector to insert genes encoding virulence factors controlled by antibodies.