Lentilactobacillus kefiri SGL 13 and Andrographis paniculata alleviate dextran sulfate sodium induced colitis in mice.

Introduction: Inflammatory bowel diseases (IBD) are chronic inflammatory conditions that typically involve diarrhea, abdominal pain, fatigue, and weight loss, with a dramatic impact on patients' quality of life. Standard medications are often associated with adverse side effects. Thus, alternative treatments such as probiotics are of great interest. The purpose of the present study was to evaluate the effects of oral administration of Lentilactobacillus kefiri (basonym: Lactobacillus kefiri) SGL 13 and Andrographis paniculata, namely, Paniculin 13™, on dextran sodium sulfate (DSS)- treated C57BL/6J mice. Methods: Colitis was induced by administering 1.5% DSS in drinking water for 9 days. Forty male mice were divided into four groups, receiving PBS (control), 1.5% DSS, Paniculin 13™ and 1.5% DSS + Paniculin 13™. Results: The results showed that body weight loss and Disease Activity Index (DAI) score were improved by Paniculin 13™. Moreover, Paniculin 13™ ameliorated DSS-induced dysbiosis, by modulating the gut microbiota composition. The gene expression of MPO, TNFα and iNOS in colon tissue was reduced and these data matched with the histological results, supporting the efficacy of Paniculin 13™ in reducing the inflammatory response. No adverse effects were associated to Paniculin 13™ administration. Discussion: In conclusion, Paniculin 13™ could be an effective add-on approach to conventional therapies for IBD.

Development of Immunological Assays Based on Leishmania donovani Antigen for Diagnosis of Canine Visceral Leishmaniasis and Their Multicenter Evaluation in Brazil and Italy.

Canine visceral leishmaniasis (CVL) due to Leishmania infantum infection is a zoonotic disease prevalent in the areas of South America and the Mediterranean. Infected dogs as reservoirs can contribute to disease transmission and can be a scourge to public health. Therefore, early diagnosis of infected dogs may play a pivotal role in circumscribing disease progression. Invasive tissue aspiration and insufficient serological methods impair a single assay for prompt CVL diagnosis. In the present study, we aimed to evaluate the potential of Leishmania donovani isolated membrane protein, LAg, for the diagnosis of CVL through immunological assays. Initially, enzyme-linked immunosorbent assay was done with Brazilian dog sera to evaluate the performance of LAg in diagnosing CVL and found sensitivity and specificity of 92.50% and 95%, respectively. The study further confirmed the diagnostic efficacy of LAg in a dipstick format. The dipstick test of canine sera from three centers in Brazil and one center in Italy collectively showed sensitivity values in the range of 53.33% to 100% in recognizing symptomatic dogs and specificity values between 75% and 100% to rule out healthy dogs. Moreover, a rapid immunochromatographic test was developed and optimized using LAg. This test was able to identify 94.73% of CVL of Brazilian origin with specificity of 97.29%. The current results highlight the reactive potential of the L. donovani antigen, LAg, for L. infantum CVL diagnosis and support our previous findings, which suggest the utility of LAg for the diagnosis of both L. donovani and L. infantum human VL in a variety of endemic regions. LAg as a diagnostic candidate may be employed to identify comprehensive CVL cases in epidemiological areas.

Exploring the wound healing, anti-inflammatory, anti-pathogenic and proteomic effects of lactic acid bacteria on keratinocytes.

The topical application of lactic acid bacteria (LAB) is recognized as a useful approach to improve skin health. This work aims to characterize by a multidisciplinary approach, the wound healing, anti-inflammatory, anti-pathogens and proteomic effects of six LAB lysates, belonging to the genus Lactobacillus. Our results demonstrated that the lysates of tested LAB stimulated the proliferation of keratinocytes, and that L. plantarum SGL 07 and L. salivarius SGL 19 accelerated the re-epithelization by inducing keratinocyte migration. The bacterial lysates also reduced the secretion of specific pro-inflammatory mediators from keratinocytes. Furthermore, viable L. salivarius SGL 19 and L. fermentum SGL 10 had anti-pathogenic effects against S. aureus and S. pyogenes, while L. brevis SGL 12 and L. paracasei SGL 04 inhibited S. aureus and S. pyogenes, respectively. The tested lactobacilli lysates also induced specific proteome modulation of the exposed keratinocytes, involving dysregulation of proteins (such as interleukin enhancer-binding factor 2 and ATP-dependent RNA helicase) and pathways (such as cytokine, NF-kB, Hedgehog, and RUNX signaling) associated with their specific wound healing and anti-inflammatory effects. This study indicates the different potential of selected lactobacilli, suggesting that they may be successfully used in the future together with conventional therapies to bring relief from skin disorders.

Investigating the Proteomic Profile of HT-29 Colon Cancer Cells After Lactobacillus kefiri SGL 13 Exposure Using the SWATH Method.

Despite some studies revealed that kefir acts on different cancers, such as colorectal cancer, the proteomic changes that occur in the colon cancer cells remain to be explored. In this study, the proteomic analysis was combined with determination of kefir characteristics (e.g., adhesion capacity, gastrointestinal and antibiotic resistances), in order to confirm its use as a probiotic. Therefore, a label-free strategy based on SWATH-MS was applied to investigate the proteomic profile of HT-29 cells after exposure for 24 h to a specific strain of Lactobacillus kefiri named SGL 13. We identified a total of 60 differentially expressed proteins in HT-29 cells, among which most are located into the extracellular exosome, playing important/crucial roles in translation and cell adhesion, as indicated by the enrichment analysis. The eIF2 and retinoid X receptor activation pathways appeared to be correlated with the anti-tumoral effect of SGL 13. Immunoblot analysis showed an increase in Bax and a decrease in caspase 3 and mutant p53, and ELISA assay revealed inhibition of IL-8 secretion from HT-29 cells stimulated with LPS upon SGL 13 treatment, suggesting pro-apoptotic and anti-inflammatory properties of kefir. In conclusion, the results of this study, the first of its kind using co-culture of kefir and colon cancer cells, demonstrate that L. kefiri SGL 13 possesses probiotic potency and contribute to elucidate the molecular mechanisms involved in the L. kefiri-colon cancer cell interactions.

Draft Genome Sequence of Lactobacillus kefiri SGL 13, a Potential Probiotic Strain Isolated from Kefir Grains.

In this report, we present the draft genome sequence of a newly discovered potential probiotic strain of Lactobacillus kefiri, SGL 13, isolated from kefir grains. Antibiotic resistance analysis did not reveal evidence of interspecific horizontal gene transfer since the identified bacitracin resistance gene does not have mobile genetic elements.

Draft Genome Sequence of Lactobacillus salivarius SGL 03, a Novel Potential Probiotic Strain.

In this work, we report the draft genome sequence of Lactobacillus salivarius SGL 03, a novel potential probiotic strain isolated from healthy infant stools. Antibiotic resistance analysis revealed the presence of a tetracycline resistance gene without elements potentially responsible for interspecific horizontal gene transfer.

Selection of a monoclonal antibody by ELISA, immunoblotting and Quartz Crystal Microbalance technology for immunohistochemical detection of Mycoplasma mycoides subsp. mycoides.

An immunohistochemical (IHC) technique was optimised using a monoclonal antibody (MAb) to detect Mycoplasma mycoides subsp. mycoides (Mmm), the agent of Contagious Bovine Pleuropneumonia (CBPP), in sections of lung tissue. A panel of MAbs was produced and screened for Mmm speci city and for cross-reactivity against other mycoplasmas belonging and not belonging to the Mycoplasma mycoides cluster, using in parallel indirect ELISA (i-ELISA) and Immunoblotting (IB). Based on i-ELISA and IB characterization data, 1 MAb (clone 3G10E7) was selected and its highest a nity vs Mmm was con rmed by the Quartz Crystal Microbalance (QCM) technology. Afterwards, IHC analyses were conducted to compare MAb 3G10E7 vs rabbit Mmm speci c hyperimmune serum using lung tissue sections of CBPP infected and CBPP negative animals. Results suggest that screening of MAbs using in parallel ELISA, IB, and QCM technology enables to select high a nity target speci c MAbs. Immunohistochemical results demonstrated that MAb 3G10E7 improved IHC performances, showing reduced background staining and no cross-reactivity against Mycoplasma bovis, which is responsible of pneumonia in cattle.

Rapid Detection and Isolation of Escherichia coli O104:H4 from Milk Using Monoclonal Antibody-coated Magnetic Beads.

Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 10(3) E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 10(2) E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 10(1) E. coli O104:H4 initial load). The specificity was 100%.

Production of Monoclonal Antibodies in Serum-free Media.

Four hybridoma cell lines (C4B, 10C2G5, 6C5F4C7, 2D10G11) were adapted to grow in serum-free conditions. Cell proliferation, viability, and antibody production in Nutridoma SP and Ex-Cell HSF 610 media were evaluated and results compared with those obtained using DMEM containing 10% fetal bovine serum (control medium). Clone C4B showed the best IgG productivity in control medium, but viability and number of cells per milliliter were similar for the three media. For clone 10C2G5, the highest values of cell viability were obtained with both control medium and Nutridoma SP; no significant differences in IgG yields and number of cells/mL were observed among the three media. Clone 6C5F4C7 provided no significant differences when grown in the two serum-free media and in control medium. Clone 2D10G11 showed the best IgG productivity in control medium and in Ex-Cell HSF 610, even if Ex-Cell HSF 610 provided the lowest number of cells/mL; no significant differences among the three media were obtained for viability. Purified antibodies produced from each hybridoma cell line grown in serum-free media showed a higher degree of purity than those produced by the same cell lines grown in control medium. Purified MAbs were also titrated by ELISA to test MAb-antigen affinity.

Long-term follow-up of dogs with leishmaniosis treated with meglumine antimoniate plus allopurinol versus miltefosine plus allopurinol.

BACKGROUND: Visceral leishmaniosis is a potentially life-threatening illness caused by a protozoan parasite of the genus Leishmania. It is found mainly in areas where both the parasite and its vector are endemic and is one of the most challenging infectious diseases in the world to control. HIV infected patients are vulnerable to Leishmania infections, and the main reservoir hosts of Leishmania infantum parasites are domestic dogs. Here, we evaluated the long-term efficacy of treatment with meglumine antimoniate plus allopurinol (G1) compared to miltefosine plus allopurinol (G2) in dogs naturally infected L. infantum. METHODS: Eighteen dogs with leishmaniosis were divided into the following two groups: G1 (n = 9) was treated subcutaneously with meglumine antimoniate (100 mg/kg/day/30 days) plus allopurinol (10 mg/kg/per day/30 days), while G2 (n = 9) was treated orally with miltefosine (2 mg/Kg/day/30 days) plus allopurinol (10 mg/kg/day/30 days). Thereafter, the same dose of allopurinol was administered to both groups for 6 years. Leishmania DNA in lymph node aspirates from the G1 and G2 dogs was quantified by real-time quantitative PCR at baseline and every 3 months for 24 months, and then at 28, 36, 48, 60 and 72 months. At each assessment, the dogs were examined for signs of disease, and their clinical scores were recorded. RESULTS: Both combination therapies produced significant clinical improvements in the dogs, with a significant reduction in the parasitic load in the lymph nodes of the dogs from both groups after 3 months of treatment. Clinical relapses were observed in four dogs from G2 (miltefosine/allopurinol), and just one dog from G1 (meglumine antimoniate/allopurinol). All dogs that relapsed had increased clinical scores, and increased anti-Leishmania antibody titers and parasitic loads in their lymph nodes. CONCLUSIONS: Long-term, the clinical and laboratory findings of the G1 dogs were more stable than those of the G2 dogs, thus indicating that meglumine antimoniate had better clinical efficacy than miltefosine. The results suggest that treatment with allopurinol as a maintenance therapy is crucial for stabilizing the care of canine leishmaniosis.

Detection of Brucella abortus DNA and RNA in different stages of development of the sucking louse Haematopinus tuberculatus.

BACKGROUND: Brucellosis is considered the world's most widespread zoonotic infection. It causes abortion and sterility in livestock leading to serious economic losses and has even more serious medical impact in humans, since it can be a trigger to more than 500,000 infections per year worldwide. The aim of this study was to evaluate the role of Haematopinus tuberculatus, a louse that can parasitize several ruminants, as a new host of brucellosis. Louse specimens were collected from seropositive and seronegative water buffaloes and divided in 3 developmental stages: adults, nymphs and nits. All samples were separately screened for Brucella spp. DNA and RNA detection by Real Time PCR. In particular, primers and probes potentially targeting the 16S rRNA and the Brucella Cell Surface 31 kDalton Protein (bcsp31) genes were used for Real Time PCR and buffalo ß actin was used as a housekeeping gene to quantify host DNA in the sample. A known amount of B. abortus purified DNA was utilized for standard curve preparation and the target DNA amount was divided by the housekeeping gene amount to obtain a normalized target value. A further molecular characterization was performed for Brucella strain typing and genotyping by the Bruce-ladder, AMOS-PCR and MLVA assays. Data were statistically analysed by ANOVA. RESULTS: Brucella abortus DNA and RNA were detected in all developmental stages of the louse, suggesting the presence of viable bacteria. Data obtained by MLVA characterization support this finding, since the strains present in animals and the relative parasites were not always identical, suggesting bacterial replication. Furthermore, the detection of Brucella DNA and RNA in nits samples demonstrate, for the first time, a trans-ovarial transmission of the bacterium into the louse. CONCLUSIONS: These findings identified H. tuberculatus as a new host of brucellosis. Further studies are needed to establish the role of this louse in the epidemiology of the disease, such as vector or reservoir.

Detection of Leishmania parasites in the testis of a dog affected by orchitis: case report.

BACKGROUND: Transmission of canine leishmaniasis (CanL), a severe infection caused by L. infantum, usually occurs through the sand fly bite to the vertebrate host. A venereal route of transmission has also been suggested, but this issue is still controversial. FINDINGS: Here, we report a case of a dog affected by orchitis showing a clinical profile of L. infantum infection. By exploiting a real-time PCR assay, we detected a significantly higher DNA load of the parasite in the lymph node and testis than in blood and urine samples collected from the dog. CONCLUSIONS: Our results suggest that: 1) L. infantum infection can be associated with testicular lesions in naturally infected dogs; 2) genital involvement could result in shedding of the parasites in the semen, favoring venereal transmission of the disease.

Evidence for a relationship between Leishmania load and clinical manifestations.

Visceral leishmaniasis (VL) is a life-threatening disease of medical, social and economic importance in endemic areas. Dogs are the main reservoir of Leishmaniainfantum. In this study, the authors investigated a group of 56 natural infected dogs to establish the relationship between parasite load and various clinical forms of leishmaniasis. The sick dogs were monitored at the beginning from clinical and physiological point of view. Leishmania load was measured by real-time PCR assay on whole blood samples and lymph node aspirates, collected at the time of diagnosis. Our results indicate that a higher quantity of Leishmania DNA was found in the lymph nodes of dogs characterized by maximum clinical score. This interesting finding indicates the presence of a positive relationship between Leishmania load and clinical manifestations in dogs showing a severe clinical form of leishmaniasis.

Study of efficacy of miltefosine and allopurinol in dogs with leishmaniosis.

Visceral leishmaniosis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients, including human immunodeficiency virus-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of miltefosine and allopurinol for the control of human leishmaniosis using the dog as a model. The study included 28 sick dogs treated with miltefosine (2 mg/kg/day PO) administered concurrently with allopurinol (10 mg/kg/day, PO) for 30 days, and then with allopurinol alone, at the same dosage, for 1 year. Eight dogs (four of which relapsed) received a second cycle of miltefosine within 6 months of the first cycle. Efficacy was measured by real-time polymerase chain reaction assay on whole blood samples and lymph node aspirates, collected at baseline and every 3 months for 12 months. Of the total number of animals (28), two showed renal insufficiency and died after the start of therapy with miltefosine. Two other dogs presented some side effects to treatment, such as nausea, vomiting and reduction in white and red blood cell counts, and these animals were excluded from the follow-up. The results showed that the first cycle of therapy with miltefosine and allopurinol induced a drastic and progressive reduction of L. infantum load in lymph node aspirates but the second cycle did not eliminate the parasite.

Canine inflammatory myopathy associated with Leishmania Infantum infection.

Inflammatory myopathy associated with several infectious diseases occurs in dogs including those caused by Toxoplasma gondii, Neospora caninum, Ehrlichia canis and Hepatozoon canis. However, muscle disease due to Leishmania infection has been poorly documented. The aim of this study was to examine the distribution and types of cellular infiltrates and expression of MHC class I and II in muscle biopsies obtained from 15 male beagle dogs from a breeder group with an established diagnosis of leishmaniasis. Myopathic features were characterized by necrosis, regeneration, fibrosis and infiltration of mononuclear inflammatory cells consisting of lymphocytes, plasma cells and histiocytes. The predominant leukocyte populations were CD3+, CD8+ and CD45RA+ with lesser numbers of CD4+ cells. Many muscle fibers had MHC class I and II positivity on the sarcolemma. There was a direct correlation between the severity of pathological changes, clinical signs, and the numbers of Leishmania amastigotes. Our studies provided evidence that: 1) Leishmania should be considered as a cause of IM in dogs; 2) Leishmania is not present within muscle fibers but in macrophages, and that 3) the muscle damage might be related to immunological alterations associated with Leishmania infection. Leishmania spp. should also be considered as a possible cause in the pathogenesis of human myositis.

Interferon-gamma (INF-gamma), IL4 expression levels and Leishmania DNA load as prognostic markers for monitoring response to treatment of leishmaniotic dogs with miltefosine and allopurinol.

In this study, we searched for a connection between Leishmania load and cytokine expression levels in the tissues of Leishmaniainfantum naturally infected dogs and the efficacy of treatment with miltefosine and allopurinol. To this purpose, we exploited a real-time PCR system to detect Leishmania load and the expression levels of IFN-gamma and IL-4 mRNAs at the time of diagnosis and during the follow up post-treatment. In particular, we measured the amount of parasites in blood and lymph node samples, while the expression levels of IFN-gamma and IL-4 cytokines were assessed in the blood of the animals. We employed different targeted real-time PCR assays on 20 naturally infected dogs with clinical signs. Three healthy dogs living in a non-endemic area were selected as negative controls. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels in different kinds of tissue might represent a reliable tool to evaluate the immune response, the efficacy of the therapy and to predict the relapses during the treatment.

Urine sampling for real-time polymerase chain reaction based diagnosis of canine leishmaniasis.

A real-time polymerase chain reaction (PCR) assay was used for quantifying Leishmania infantum DNA in urine samples from naturally infected dogs. Forty-one infected dogs were divided into 3 groups: 22 dogs showing only cutaneous signs (group 1), 12 dogs showing hematuria (group 2), and 7 dogs affected by severe nephropathy (group 3). Groups 2 and 3 dogs showed altered laboratory parameters related to an impairment of renal function. The real-time PCR analysis showed higher levels of Leishmania DNA in the lymph node aspirates from all groups of infected dogs versus those measured in their blood or urine. Interestingly, urine samples from dogs belonging to groups 2 and 3 contained a higher Leishmania DNA load than that detected in their blood. This finding suggests that a real-time PCR analysis of urine from infected dogs could be a useful and noninvasive tool for monitoring the severity of leishmaniasis.

Fluorescence-based detection of Leishmania infantum DNA in phlebotomus vectors.

The geographical diffusion of leishmaniasis can depend on the factors limiting the distribution of sandfly vectors. In Sicily, as in all Mediterranean areas, sandflies are present almost all year round because the climate permits perpetuation of this vector's life cycle. Transmission can occurs in rural and domestic habitats through the bite of infected female phlebotomine sandflies. In Italy, the visceral form of leishmaniasis is commonly found, which is due exclusively to L. infantum. Two species of sandfly are considered the main vectors: Phlebotomus perniciosus and P. perfiliewi. Fluorescence resonance energy transfer technology was used to determine the parasite load in phlebotomine vectors, and the test was targeted on a 117-bp fragment of kinetoplast DNA minicircles. The assay was evaluated, focusing on analytical sensitivity, discriminatory power, and reliability of quantification. During 2005, a total of 1686 sandflies were trapped in various Sicilian provinces and in farms randomly selected using black light traps. We found 20, 30, and 16 sandflies positive for Leishmania for each kind of analyzed phlebotomine sandfly, respectively, corresponding to 6.5% for the gravid, 2.7% for the fed, and 6.3% for other groups. Previously the insects were identified on the basis of morphology and the most prevalent sandfly species were P. Sergentomyia minuta, P. perfiliewi, and P. perniciosus.

Leishmania DNA quantification by real-time PCR in naturally infected dogs treated with miltefosine.

A new drug that has just become available in India for treatment of human visceral leishmaniasis (VL) is miltefosine, an alkyphospholipid that was originally developed as an oral antineoplastic agent. Miltefosine is not only directly toxic for Leishmania parasite, but it also enhances both T cell and macrophage activation and production of microbicidal reactive nitrogen and oxygen intermediates. It is highly effective in the treatment of Leishmania infection in mice and human beings. However, adverse effects in dogs treated with miltefosine have been reported, but there are no data on the efficacy of this drug for the treatment of canine visceral leishmaniasis (CVL). The aim of this study was to use a real-time PCR assay to monitor the Leishmania load in the blood samples and lymph node aspirates of 18 naturally infected dogs before and after treatment with miltefosine (2 mg/kg for 30 days). The results of our study showed that the therapy with miltefosine shows a drastic and progressive reduction of parasite load in lymph node aspirates, but does not suppress the parasite in lymph nodes. In all dogs the real-time PCR assay demonstrated an irregular presence of parasites in blood. Therefore, blood does not seem a suitable substrate for the purpose of quantifying Leishmania DNA.

Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol.

A real-time PCR assay was exploited for monitoring the Leishmania DNA load in different tissues from 18 naturally-infected dogs before and after treatment with a combination of meglumine antimoniate (100mg/kg/day, subcutaneously) and allopurinol (10mg/kg/day, orally) for 30 days. After the combined therapy, allopurinol was continued at the same dose until the end of the observation period. Whole blood samples, lymph node aspirates, and skin biopsies were collected at the time of diagnosis, 1 month after starting therapy, and every 3 months for 2 years. In six dogs parasite load assessments continued every 6 months for a further 3 years. At each assessment, the dogs were examined for signs of disease and a clinical score was recorded. At diagnosis, the highest Leishmania DNA load was detected in lymph node aspirates. From 1-6 months post-therapy a general improvement in clinical conditions was recorded in all dogs, which correlated with a decrease in the parasite DNA load in all tested tissues, even though it was less pronounced in lymph node aspirates. In the period from 9-24 months post-therapy, a re-increase in parasite load was observed in the tissues of some dogs, concomitant with a disease relapse. The results show that the combined therapy with meglumine antimoniate and allopurinol promoted a clinical improvement which was accompanied by a reduction in the parasitic load in the blood, skin and lymph nodes but, even after long period of allopurinol administration alone, Leishmania may persist in dog tissues.