Distúrbios do sistema nervoso central (SNC) em ruminantes abrangem um importante grupo de enfermidades responsáveis por grandes perdas econômicas em todo o mundo. As principais doenças neurológicas causadas por bactérias em ruminantes e que envolvem processos inflamatórios incluem listeriose, leptomeningites e meningoencefalites supurativas, abscessos cerebrais e medulares, empiema basilar e neurotuberculose. Esta dissertação teve por objetivo a caracterização epidemiológica, patológica, microbiológica e imuno-histoquímica das doenças inflamatórias de origem bacteriana que afetam o SNC de ruminantes no Rio Grande do Sul. Foi realizado um estudo retrospectivo no período compreendido entre janeiro de 1996 a dezembro de 2015, onde um total de 3.274 bovinos, 596 ovinos e 391 caprinos foram avaliados. Destes, 219 bovinos, 21 ovinos e sete caprinos foram diagnosticados com doenças inflamatórias no SNC. As doenças neurológicas inflamatórias de origem bacteriana totalizaram 60 casos divididos em 34 bovinos, 19 ovinos e sete caprinos, os quais foram subdivididas em: meningoencefalite por L. monocytogenes (oito ovinos, cinco caprinos e quatro bovinos), leptomeningite e meningoencefalite supurativa (14 bovinos, dois caprinos e um ovino), abscessos cerebrais (seis bovinos e dois ovinos) e medulares (sete ovinos), empiema basilar (quatro bovinos e um ovino) e neurotuberculose (seis bovinos). O exame imuno-histoquímica foi realizado em todos os casos diagnosticados com listeriose (anticorpo anti-L. monocytogenes), meningite e meningoencefalite supurativa (anticorpo anti-Escherichia coli) e neurotuberculose (anticorpo anti-Mycobacterium tuberculosis). A meningoencefalite por L. monocytogenes representou a principal enfermidade neurológica em ovinos e caprinos, seguido dos abscessos medulares em ovinos. Nos bovinos, a leptomeningite e meningoencefalite supurativa foi a doença neurológica mais prevalente para a espécie, frequentemente relacionada com a falha na transmissão da imunidade passiva. O empiema basilar, frequentemente diagnosticado em bezerros, está diretamente relacionado com o manejo do desmame interrompido através da utilização de tabuletas nasais. A neurotuberculose causada por Mycobacterium spp. é uma importante doença neurológica em bovinos jovens e deve ser considerada como diagnóstico diferencial de doenças neurológicas. Dos dados analisados neste período, as doenças neurológicas inflamatórias bacterianas representaram um total de 24,3% entre as doenças neurológicas inflamatórias diagnosticadas neste período e, com isso, conclui-se que representam importantes causas de mortalidade para os ruminantes domésticos. / Central nervous system (CNS) diseases are worldwide economically important conditions in ruminants. The main neurological bacterial diseases which involve inflammation in ruminants are listeriosis, suppurative leptomeningitis and meningoencephalitis, brain and spinal cord abscesses, basilar empyema and neurotuberculosis. This study aim to describe the epidemiological, pathological, microbiological and immunohistochemical findings of the bacterial inflammatory diseases that affect the CNS of ruminants in Rio Grande do Sul state. A retrospective study was performed from January 1996 to December 2015, during which samples of 3.274 cattle, 596 sheep and 391 goats were evaluated. Of these, 219 cattle, 21 sheep and seven goats were diagnosed with inflammatory diseases affecting the CNS. The neurological inflammatory bacterial diseases accounted for 60 cases, which corresponded to 34 cases in cattle, 19 in sheep and seven in goats. These were further subdivided in: meningoencephalitis by L. monocytogenes (eigth sheep, five goats and four cattle), suppurative leptomeningitis and meningoencephalitis (14 cattle, two goats and one sheep), brain abscesses (six cattle and two sheep) and spinal cord (seven sheep), basilar empyema (four cattle and one sheep) and neurotuberculosis (six cattle). Immunohistochemical exam was performed in all cases diagnosed as listeriosis (antibody anti-L. monocytogenes), as suppurative leptomeningitis and meningoencephalitis (antibody anti-E. coli), and as neurotuberculosis (antibody anti-Mycobacterium tuberculosis). L. monocytogenes meningoencephalitis was the main neurological disease in sheep and goats, followed by spinal cord abscesses in sheep. In cattle, suppurative leptomeningitis and meningoencephalitis was the most frequent neurological disease, and its ocorrunce is related to the failure in passive immunity transmission. Basilar empyema is frequently diagnosed in calves and is directly related to early weaning handling through the use of nose-flaps. Neurotuberculosis caused by Mycobacterium spp. is an important neurological disease in young cattle and should be consired as a differential diagnosis of granulomatous meningoencephalitis. During the described period, neurological inflammatory bacterial diseases accounted for 24.3% of the neurological inflammatory diseases, and, thus, these are important causes of death in domestic ruminants.

The challenges faced by the agricultural sector especially around improving production yields using environmentally friendly solutions have received market attention. Biological intervention can range from application of biological products to enhance the nutritional value of crops or to control plant pathogens. Biostart, a biological product that demonstrated growth enhancement when applied in lettuce crops is currently in the market. The product is comprised of a consortium of bacterial isolates (Bacillus licheniformis, Brevibacillus laterosporus and Bacillus laterosporus) but the contribution of the individual isolates to growth enhancement had not been elucidated. Green house experiments on lettuce seedlings with individual and mixed treatments were commissioned to determine such contribution. There was either no or marginal growth enhancement observed in the experiments. The results showed that the product was effective as a consortium and not as individual isolates. Further isolation and screening for potential Bacilli with antifungal properties was undertaken. An isolate identified as Bacillus subtilis that demonstrated inhibition against a wide spectrum of fungi, and especially the phytopathogenic Verticillium dahliae and Fusarium oxysporum, was successfully identified. The isolate was cryo-preserved and cultivated to significant levels at bench scale. A characterized comparison of different putative products with known systematic fungicide showed potential application even of heat treated products. The product showed control V. dahliae when tested in green houses with potatoes and tomatoes as test crops. This isolate has been targeted for further development as a biological control product.

Waterborne diarrhoeal infections continue to be a major health setback in developing countries, especially in rural areas which lack adequate supply of portable water and sanitation facilities. Globally, waterborne diarrhoeal infections occur with an estimated mortality rate of 10–25 million deaths per year, 95% of which are children under the age of 5 years. The Vibrio species is one of the major groups of enteric pathogens that are responsible for diarrhoeal infections. Many strains of these bacterial species continue to cause epidemics of diarrhoea throughout the world. In this study, the prevalence of Vibrio pathogens in wastewater final effluents was assessed. Wastewater final effluent and discharge point samples were collected monthly between September 2012 and August 2013. All samples were collected aseptically using sterile 1 L Nalgene bottles containing 0.5 ml of sterile sodium thiosulphate solution and transported on ice to the laboratory for analyses within 6 h of collection. The membrane filtration method was used for enumeration of presumptive Vibrio densities on thiosulfate citrate bile salt (TCBS) agar plates. Polymerase chain reaction (PCR) was then used to confirm the identities of the presumptive Vibrio species using the species-specific primers. The confirmed isolates were further subjected to molecular characterization to confirm their respective pathotypes. Presumptive Vibrio densities varied from 0 to 2.11 × 102 cfu/100 ml. Out of 300 confirmed Vibrio isolates; 13.3% (40/300) were Vibrio fluvialis, 22% (66/300) were confirmed to be Vibrio parahaemolyticus, and 24.7% (74/300) proved to be Vibrio vulnificus, and 40% (120/300) were other Vibrio species which were not assessed for in this study. The strains of Vibrio fluvialis were found to exhibit 100% resistance to Polymixin and Tetracycline. However, Gentamicin was active against all the three Vibrio species selected for the purpose of this research. The recovery of Vibrio species in the discharged effluents throughout the sampling period even in adequately disinfected effluents is not acceptable considering the fact that Vibrio is a pathogenic bacterium. The findings of this study underline the need for constant monitoring of the microbiological qualities of discharged effluents and might also be suggestive for a review of the disinfection methods used at the treatment works.

Infection with Helicobacter pylori has been associated with the pathogenesis of numerous stomach and gastroduodenal diseases that pose threats to public health. Eradicaftion of this pathogen is a global challenge due to its alarming rate of multidrug resistance. Consequently, to find an alternative treatment, the search is increasingly focused on new antimicrobial product from natural sources including honey. Honey has been used as medicine in several cultures since ancient time due to its enormous biomedical activities. Its beneficial qualities have been endorsed to its antimicrobial, antioxidant, anti-inflammatory properties added to its phytocomponents. In this study, the anti-H. pylori activity of South African honeys and their solvent extracts as well as the phytochemicals present in the two most active honeys were evaluated. Agar well diffusion test was used to investigate the antimicrobial activity of six honey varieties obtained from different locations in the country. Subsequently, the honeys were extracted with four organic solvents viz n-hexane, diethyl ether, chloroform and ethyl acetate employed in order of increasing polarity. The antibacterial activity of the different solvent extracts of each honey was evaluated by agar well diffusion; broth micro dilution and time kill assays. Different chromatographic techniques (Thin layer & column chromatography) were employed to enumerate the phytochemical constituents in the most active solvent extracts of Pure Honey (PH) and Champagne Royal Train (CRT); and were identified by gas-chromatography linked mass-spectrometry. Linalool pure compound was equally evaluated for anti-H. pylori activity in a bid to trace the antibacterial agent among the variety of compounds identified. Data were analyzed by One-way ANOVA test at 95% confidence interval. Crude honeys and their solvent extracts demonstrated potent anti-H. pylori activity with zone diameter that ranged from [16.0mm (crude) to 22.2mm (extract)] and percentage susceptibilities of test isolates between 73.3% (crude) and 93.3% (extract). The chloroform extracts of PH and CRT were most active with MIC50 in the ranges 0.01- viii 10%v/v and 0.625-10%v/v respectively, not significantly different from amoxicillin (P> 0.05); and efficient bactericidal activity (100% bacterial cells killed) at 1/2MIC and 4xMIC over different time intervals, 36-72hrs and 18-72hrs respectively. The appearance of bands on the thin layer chromatography (TLC) chromatogram spotted with the chloroform extracts of PH and CRT; and developed with hexane: ethyl acetate: acetic acid (HEA) and methanol: acetic acid: water (MAAW) solvent systems indicated the presence of compounds. Purification of the compounds contained in these extracts over silica gel column yielded numerous fractions which were evaluated for antibacterial activity and purity. PHF5 was the most active fraction with a mean MIC50 value of 1.25mg/mL. Volatile compounds belonging to different known chemical families in honey were identified in all the active fractions obtained from PH. Conversely, only four compounds were identified in the active fractions obtained from CRT hence the non volatile constituents could be of prime relevance with respect to antibacterial activity of this honey. Of novelty was the presence of thiophene and N-methyl-D3-azirdine compounds, essential precursors used for the synthesis of natural products and pharmaceuticals with vital biomedical properties. Linalool demonstrated potent inhibitory (MIC95, 0.002- 0.0313mg/mL) and bactericidal activity (0.0039-0.313mg/mL) against the test isolates. On the other hand, a significant difference was recorded (P < 0.05) in comparing the activity of linalool compound to the fractions. PH could serve as a good economic source of bioactive compounds which could be employed as template for the synthesis of novel anti-H. pylori drugs. However, further studies are needed to determine the non volatile active ingredients in PH and CRT as well as toxicological testing

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

Antibiotikaresistens är en av de största globala utmaningarna i sjukvården och om den mängdantibiotika som används idag fortsätter att användas i framtiden riskerar det att kosta 10 miljonermänniskor om livet varje år från år 2050. Antibiotikarester i akvatiska miljöer ökar risken förspridning av antibiotikaresistenta bakterier. Vissa antibiotikum är även toxiska för vattenlevandeorganismer. Dagens vattenreningsverk är inte utformade att rena vatten från antibiotikaresteroch därför följer resterna med vattnet ut i recipienten. För att lösa det här problemet behövervattenreningsverk utvecklas med nya reningsmetoder för att kunna rena antibiotika och vårdenmåste minska användandet i sjukvården. De kopplade globala målen kopplade till detta problemär mål 3 “God hälsa och välbefinnande”, 6 “rent vatten och sanitet”, 11 “hållbara städer ochsamhällen”, 14 “hav och marina resurser” (Globala målen, 2021a; Globala målen, 2021b;Globala målen, 2021c; Globala målen, 2021d). Arbetet undersöker potentiella lösningar förutveckling av vattenreningsverk och alternativa behandlingsmetoder för att minska användandetav antibiotika inom sjukvården. För att göra detta har intervjuer med respondenter på Gävlesjukhus och Duvbackens reningsverk genomförts för att hitta brister i dagens teknik.Förbättringsförslagen är baserade på vetenskapliga artiklar och myndigheters hemsidor. I EUfinns det inga krav gällande att producenterna av läkemedel ska granska den verkligamiljöpåverkan läkemedlet har efter att det har godkänts för försäljning. Antibiotikaläkemedel ärbiologiskt aktiva föreningar vilka, även i små mängder, kan påverka det akvatiska ekosystemetnegativt. Penicillin är det vanligaste använda antibiotikan i Gävles sluten- och primärvård.Slutenvården utgör den största gruppen av antibiotikaanvändare men beaktas bör att det är okänthuruvida dessa användare tidigare kommer från primärvården. Det är också okänt hur stor andelav inköpta läkemedel som faktiskt använts. De reningstekniker bäst lämpade att använda påvattenreningsverk för att rena antibiotikarester varierar och beror bland annat på klimat,resurser och vilken typ av antibiotika som finns i avloppsvattnet. Ozonering är positivt eftersomdet är effektivt mot antibiotikasubstanser med brett spektrum. Det ger heller inte någonpåverkan på slam från vattenreningsverket eftersom ozonet löses upp och blir till syre efterreningen. Ozonering är ofta mest effektiv i kombination med andra reningstekniker vilket kallasför hybridprocesser. Adsorption är effektivt för rening av antibiotikarester och den vanligametoden kolbaserad adsorption är effektiv vid rening av tetracyklinrester. Metoden är effektiveftersom det tar upp mycket av resterna samt att energikostnaden är låg. Sandfilter är en brametod för att rena bort antibiotikaresistenta bakterier och dessutom renar filtrets adsorptionockså rester av tetracyklin. Genom att införa en reningsteknik kombinerad med aktivt kol ochozonering för att rena antibiotikarester från avloppsvattnet samt att använda vakuumassisteradsårbehandling kan användandet av antibiotika minska. / Antibiotic resistance is one of the biggest global challenges in modern day healthcare and ifcontinued use of antibiotics in the same amount as today, it will kill 10 million people every yeararound 2050. Antibiotic residues in aquatic environments increase the risk of spreading ofspreading antibiotic resistant bacteria. Some antibiotics are also toxic to aquatic organisms.Because the wastewater plants are not designed to purify the water from antibiotics the residuescontinue with the water to the recipient. To solve the problem the wastewater plants needs toupgrade with new technology and the healthcare industry reduces the amount prescribedantibiotics. The UN global goals connected to this issue is goal 3 “Good health and well-being”,6 “clean water and sanitary”, 11 “sustainable cities and communities” and 14 “life below water”(Global goals). This study explores potential developments in the wastewater plants andalternatives for antibiotics in healthcare by interviewing respondents at the hospital in Gävle andthe wastewater plant Duvbacken to find the deficiencies of today. Included suggestions are basedon scientific articles and Swedish agencies. There is no requirement for the medicalmanufacturers within the EU to examine the environmental impact of medicines after they havebeen approved for sale. Antibiotic drugs are biologically active compounds which, even in smallamounts, can adversely affect the aquatic ecosystem. Penicillin is the most widely used antibioticin Gävle's inpatient and outpatient care. Outpatient care constitutes the largest group ofantibiotic users, but it should be taken into account that people from this group may havepreviously belonged to inpatient care. It is also unknown how much of each purchased drug thatis used. The treatment techniques that are best suited to use at water treatment plants to purifyantibiotic residues vary and depend on the climate, resources and the type of antibiotics found inthe wastewater. Ozonation is positive as it is effective for broad spectrum antibiotics. It also hasno effect on sludge from the water treatment plant because the ozone dissolves and becomesoxygen after the treatment. Ozonation is often most effective when combined with otherpurification techniques which is called hybrid processes. Adsorption is effective for thepurification of antibiotic residues and the usual method of carbon-based adsorption is effective inthe purification of tetracycline residues. The method is effective because it absorbs much of theresidues at a low energy cost. Sand filter is an effective method to clean away antibiotic-resistantbacteria and in addition, the adsorption of the filter also cleans residues of tetracycline. Byintroducing a purification technique combined with activated carbon and ozonation to purify thewastewater in wastewater plants and to use vacuum assisted wound treatment, the use ofantibiotics can be reduced.

Piscine francisellosis in an infectious emerging bacterial disease that affects several marine and fresh water fish species worldwide, including farmed salmon, wild and farmed cod, farmed tilapia and several ornamental species, for which no commercial treatment or vaccine exists. During 2011 and the first semester of 2012, chronic episodes of moderate to high levels of mortality with nonspecific clinical signs, and widespread multifocal white nodules as the most consistent gross pathological lesion were experienced by farmed tilapia fingerlings at two different locations in Northern Europe. In this study such outbreaks of granulomatous disease were diagnosed as francisellosis with a genus-specific PCR, and 10 new isolates of the bacterium including the one named STIR-GUS-F2f7, were recovered on a new selective “cysteine blood-tilapia” agar and cysteine heart agar with bovine haemoglobin. Ultrastructural observations of the pathogen in Nile tilapia (O. niloticus) tissues suggested the secretion of outer membrane vesicles (OMVs) by the bacterial cells during infection in these fish. This represented the first documented report of isolation of pathogenic Francisella strains from tilapia in Europe. The phenotypic characterisation indicated that isolates recovered were able to metabolise dextrin, N-acetyl-D glucosamine, D-fructose, α-D-glucose, D-mannose, methyl pyruvate, acetic acid, α-keto butyric acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-glutamic acid, L-proline, L-serine, L-threonine, inosine, uridine, glycerol, D L-α-glycerol phosphate, glucose-1-phosphate and glucose-6-phosphate. The predominant structural fatty acids of the isolates were 24:1 (20.3%), 18:1n-9 (16.9%), 24:0 (13.1%) 14:0 (10.9%), 22:0 (7.8%), 16:0 (7.6%) and 18:0 (5.5%). Anti-microbial resistance analyses indicated that STIR-GUS-F2f7 was susceptible to neomycin, novobiocin, amikacin, ciprofloxacin, imipenem, gatifloxacin, meropenem, tobramycin, nitrofurantoin, and levofloxacin using the quantitative broth micro-dilution method, while the qualitative disc diffusion method indicated susceptibility to enrofloxacin, kanamycin, gentamicin, tetracycline, oxytetracycline, florfenicol, oxolinic acid and streptomycin. The use of the following housekeeping genes: mdh, dnaA, mutS, 16SrRNA-ITS-23SrRNA, prfB putA rpoA, rpoB and tpiA indicated 100% similarity with other isolates belonging to the subspecies F. noatunensis orientalis (Fno). Koch’s postulates were successfully fulfilled by establishing an intraperitoneal injection (IP) challenge model with STIR-GUS-F2f7 in Nile tilapia. Moreover, the challenge model was used to investigate the susceptibility of 3 genetic groups of tilapia to STIR-GUS-F2f7. The lowest amount of bacteria required to cause mortality was 12 CFU/ml and this was seen as early as only 24 hours post infection in the red Nile tilapia and in the wild type after 26 days, no mortalities were seen in the species O. mossambicus with this dose. The mortality in red O. niloticus was significantly higher than that of the other two tilapia groups when 12 and 120 CFU/fish were injected. It was also observed that when a dose of 1200 CFU/ml was used, the mortality in O. niloticus wild type was significantly lower than that of the other two tilapia groups and no differences were seen among the 3 groups when the highest dose (1.2 x105 CFU/fish) was used. The median lethal dose (LD50) of O. niloticus wild type was the most stable during the experiment (values around 104 CFU/ml) and the highest of the three groups after day 25 post infection. At the end of the experiment (day 45) the LD50 was 30 CFU/ml in the red Nile tilapia, 2.3x104 CFU/ml for the wild type and 3.3x102 CFU/ml for O. mossambicus. This pattern, where the LD50 of the red tilapia was lower than that of the other two groups, was observed during the whole experiment. The outcomes of these experiments suggested that the red Nile tilapia family appeared to be the most susceptible while the wild type Nile tilapia family the most resistant. The complete genome of STIR-GUS-F2f7 was sequenced using next generation sequencing (NGS) Illumina Hi-Seq platform™, and the annotation of the assembled genome predicted 1970 protein coding sequences and 63 non-coding rRNA sequences distributed in 328 sub-systems. The taxonomy of the species Francisella noatunensis was revised using genomic-derived parameters form STIR-GUS-F2f7 and other strains in combination with a polyphasic approach that included ecologic, chemotaxonomic and phenotypic analyses. The results indicated that STIR-GUS-F2f7 and all the other strains from warm water fish represent a new bacterial species for which the name Francisella orientalis was assigned. Moreover the description of F. noatunensis was emended and the creation of a new subspecies within this taxon i.e. Francisella noatunensis subsp. chilense was proposed. The results of this study led to the development of a highly efficacious vaccine to protect tilapia against francisellosis.

Nile tilapia, Oreochromis niloticus, is one of the most important farmed fish globally. One of the most serious bacterial diseases constraining global tilapia production is Francisellosis caused by Francisella noatunensis subsp. orientalis (Fno). Although outbreaks of Fno are increasing worldwide, there are no licenced commercial vaccines to prevent the disease for use on tilapia farms. Thus, the current treatment of choice is the use of antibiotics combined with increasing water temperature up to 30°C. Studies investigating the diversity of circulating Fno isolates and the immune response of tilapia elicited by vaccination against piscine francisellosis are lacking. In addition, the current conventional and molecular tools used for detection of Fno have many drawbacks, making detection of Fno a challenging process. In this study, five clinical isolates of Fno from diverse geographical locations (UK, Costa Rica, Mexico, Japan and Austria), previously characterised by morphology, genotype, antimicrobial susceptibility and virulence, were used in a proteomic study. The whole proteomic cell profile of the five isolates were homogenous by one-dimension sodium dodecyl polyacrylamide gel electrophoresis (1D-SDS-PAGE), while minor differences in the intensity of 15 proteins between the strains were observed by two-dimension SDS-PAGE (2DE), including some important virulence related proteins. The UK isolate was the most significantly different isolate when compared to the other Fno isolates in the current study. The Fno UK isolate had significantly higher abundance of 10/15 of the significantly expressed proteins including four of the essential pathogenicity and virulence related proteins (IglC, GroEL, DnaK, ClpB) compared to the other used Fno isolates. The antigenic profiles of the five Fno isolates were studied by 1D western blotting using tilapia hyper immune sera which recognised an immunodominant band of a molecular weight of ~ 17-28 kDa in all tested Fno isolates. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS/MS) identified 47 proteins in this antigenic band. Some of the identified proteins are associated with Fno pathogenicity. 2D western blot analysis of the vaccine isolate (Fno UK) revealed differential antigen recognition between sera from vaccinated and non-vaccinated fish following experimental challenge (26 antigenic spots recognised by sera from vaccinated fish; 31 antigenic spots recognised by sera from vaccinated and challenged fish and 30 antigenic spots recognised by non-vaccinated and challenged fish). The identity of these proteins was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of them are known Francisella virulence related proteins. Bioinformatics analyses revealed diverse categories of proteins with high biological functions, however the vast majority of these proteins are involved in energy production and metabolic pathways of the bacteria. This detailed analysis will facilitate the development of cross-strain protective subunit Fno vaccines and antigen-targeted Fno diagnostics. The outer membrane proteins (OMPs) of the same five Fno isolates were extracted using the ionic detergent sarkosyl. The OMP fraction of the different isolates were separated via 1D-SDS PAGE and the digested peptides of the UK isolate were analysed by LC/ESI/MS/MS. High degree of similarity was observed in the OMP profile of the five Fno isolates with an abundant protein band at 17-28 kDa, which was found to be antigenic by 1D western blot using convalescent tilapia sera. LC/ESI/MS/MS analysis of the OMPs of the Fno UK isolate identified 239 proteins, including 44 proteins in the antigenic band (17-28 kDa). Comparison between the proteins identified in the immunogenic band of whole cell lysate and OMP fraction of the Fno UK isolate showed 30 common proteins between the two preparations, 17 proteins were identified only in the whole cell extract and 14 were identified only in OMP fraction. Outer membrane proteins (e.g. Omp-A), virulence related proteins such (e.g. IglC) and other stress related proteins (e.g. AhpC/TSA family peroxiredoxin) were more abundant in the OMP fraction than the whole cell lysate. In silico analysis enabled prediction of the function and location of the OMPs identified by Mass-spectrometry. The findings of this study provide preliminary data on bacterial surface proteins that exist in direct contact with the host immune defence during infection and offering an insight into their potential role as novel targets for Fno diagnostics and vaccine development. The efficacy of an injectable whole cell oil-adjuvanted vaccine was evaluated against challenge with heterologous Fno isolates in Nile tilapia, Oreochromis niloticus. Three duplicate groups of 130 healthy Nile tilapia (~15 g) were intraperitoneally (i.p.) injected with the vaccine, adjuvant-alone or PBS followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection to all immunised tilapia groups with a significantly higher relative percent survival (RPS) of 82.3% against homologous challenge, compared to 69.8% and 65.9% after heterologous challenge. Protection correlated with significantly elevated specific antibody responses and western blot analysis demonstrated cross-isolate antigenicity with sera from fish post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by quantitative real-time polymerase chain reaction (qPCR) in conjunction with significantly greater expression of IgM, IL-1β, TNF-a and MHCII 72 hours post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to those of adjuvant-alone and control fish. The latter results suggested stimulation of protective immune responses following vaccination. In addition, a whole cell formalin killed autogenous immersion vaccine against Fno was developed using the same isolate used for the injectable vaccine. Duplicate tanks of 35 tilapia fry were immersed in the vaccine or in sterile Modified Muller Hinton broth (MMHB) diluted in tank water (1:10 dilution) for 30 s and at 30 days post-vaccination (dpv), all fish groups were immersion challenged with the homologous Fno isolate and monitored for 21 days. A moderate RPS of 43.7% was provided by the vaccine. Serum IgM levels were below the threshold in 30 % of the vaccinated fry 30 dpv. Also, the IgM levels of the vaccinated fry were not significantly different from control fry 21 days-post challenge. A recombinase polymerase amplification (RPA) assay was developed and validated for rapid detection of Fno. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in fish farms, but also by screening 78 Nile tilapia and 5 water samples collected from UK and Thailand. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other species of bacteria tested. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The Fno-RPA was rapid, giving results in approximately 6 min in contrast to the qPCR that required approximately 90 min to reach the same detection limits. Moreover, the RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to control the infection through prompt on-site detection of Fno. The overall results of this study indicated that Fno isolates from different origins share a high degree of homology in their proteomic and antigenic profile. Proteomic characterisation data of Fno isolates has contributed to understanding the diversity of Fno isolates and assisted in identifying suitable candidates for developing an effective Fno vaccine. / Moreover, this study has proven the efficacy of a cross protective Fno injection vaccine in tilapia fingerlings, with further optimisation needed for immersion vaccination of fry, and given insights into the immune response of tilapia to vaccination against francisellosis. In addition, it provided a rapid, sensitive, specific and robust molecular tool for detection of Fno that can assist surveillance and control of piscine francisellosis on tilapia farms.

The genus Mycobacterium represents more than 120 species of bacteria including the pathogenic M. tuberculosis, the etiological agent of tuberculosis. The host mounts a robust inflammatory and cell-mediated response to contain the spread of pathogenic mycobacteria. While macrophages, dendritic cells (DCs) and neutrophils are known to facilitate early responses, the effector functions of CD4+ and CD8+ T cells are critical for containment of the mycobacteria. The type I T helper (Th1) subset of CD4+ T cell population orchestrates the protective immunity through cytokines like interferon (IFN)-γ, interleukin (IL)-12, IL-23 and tumor necrosis factor (TNF)-α However, it is known that despite such responses, host can only contain but not eradicate the infection. Additionally, infection of over one-third of the world’s population with pathogenic mycobacteria is a testimony of its success as a pathogen. Much of its success is attributed to the multiple evasion strategies employed such as inhibition of phagosome-lysosome fusion, secretion of reactive oxygen intermediates antagonistic proteins like superoxide dismutase and catalase, downregulation of antigen presentation to T cells, downregulation of the pro-inflammatory cytokines, skewing the immune balance toward the less effective Th2 responses, inhibition of autophagy, induction of regulatory T cells (Tregs) and immunosuppressive cytokines etc. Thus, an effective check on the infection would be possible if we understand the mechanisms underlying such evasion and survival strategies. In this perspective, evaluation of the host-pathogen interactions in terms of integration of key signaling centers, particularly that during mycobacteria-macrophage or mycobacteria-DC interactions, would underscore as a critical requisite to detail the immune responses and its regulation. This study addresses three such immune evasion and survival strategies employed by the mycobacteria; downregulation of IFN-γ-induced autophagy in macrophages, expansion of Tregs by modulating DC phenotype and finally epigenetic regulation of genes involved in foamy macrophage generation. Autophagy is one of the major immune mechanisms engaged to clear intracellular infectious agents. It contributes to both innate and adaptive immune responses to infections and plays an essential role in restricting intracellular pathogens and delivering pathogen-derived antigens for major histocompatibility complex class II presentation. Nonetheless, several pathogens, especially viruses such as herpes simplex virus, human immunodeficiency virus, influenza; and bacteria like Mycobacteria, Shigella and Listeria exhibit multiple mechanisms to evade autophagy. However, the identities and contributions of host signaling molecules and mechanisms by which pathogens modulate autophagy have not been explored in depth. Here, we demonstrate that M. bovis BCG, Shigella flexneri and Listeria monocytogenes but not Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli inhibit IFN-γ-induced autophagy in macrophages by evoking selective and robust activation of WNT and sonic hedgehog (SHH) pathways via mechanistic target of rapamycin (mTOR). Utilization of macrophages derived from mir155-null mice or by conventional siRNA or miRNA mimics emphasized the role for mTOR-responsive epigenetic modifications in the induction of microRNAs, miR-155 and miR-31 to fine-tune autophagy. Importantly, cellular levels of PP2A, a phosphatase, were regulated by miR-155 and miR-31. Diminished expression of PP2A led to inhibition of glycogen synthase kinase (GSK)-3β, a negative regulator and a nodal link that regulate WNT and SHH pathways. This facilitated the prolonged activation of WNT and SHH signaling pathways. Further, sustained WNT and SHH signaling effectuated the expression of anti-inflammatory lipoxygenases (ALOX5 and ALOX15), which in tandem inhibited IFN-γ-induced janus kinase (JAK)- signal transducer of activated (STAT) signaling and contributed to evasion of autophagy. Together, we have identified novel molecular mechanisms and host factors that are crucial to control autophagy and help the bacterial pathogens like mycobacteria to evade the host immune responses. Much of the protective immunity against mycobacterial infection is mediated by Th1 CD4+ T cells. However, suppressive T cell populations such as CD4+CD25+FoxP3+ Tregs or a less effective Th2 cells are exploited by mycobacteria to subvert the protective host immune response. In this perspective, the molecular mechanisms underlying mycobacteria-induced Treg expansion are unclear. Utilizing cues from the previous reports from others’ and our laboratory, we explored the role for host signaling pathways such as SHH, WNT and NOTCH1 signaling during mycobacteria-mediated DC maturation and Treg generation/expansion. We demonstrate that while inhibition of SHH signaling markedly reduced the ability of the infected DCs to expand Tregs, NOTCH1 signaling functioned to suppress M. bovis BCG-induced Treg expansion. Though SHH and NOTCH1 signaling did not regulate the DC maturation during infection in terms of the maturation markers CD1a, HLA-DR, CD40, CD83, CD80 and CD86, pro-inflammatory cytokines such as TNF-α, IL-2, IL-1β and IL-6 were moderately NOTCH1-responsive and suppressed by SHH signaling. Further, M. bovis BCG-induced SHH signaling and Treg expansion was mediated by the classical phosphoinositide 3-kinase (PI3K)-mTOR-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) cascade. Recent studies have attributed the role for programmed death ligand (PD-L)1 and cyclooxygenase (COX)-2-catalyzed prostaglandin (PG)E2 during expansion of Tregs. Experiments utilizing pharmacological inhibitors and conventional siRNAs indicated that both PD-L1 and COX-2/PGE2 were induced upon M. bovis BCG and M. tuberculosis infection in DCs and were regulated by SHH signaling. While SHH-responsive transcription factor, GLI1 arbitrated COX-2 expression, mycobacteria-stimulated SHH signaling was found to suppress miR-324 and miR-338, bonafide miRNAs that target PD-L1, to aid increased expression of PD-L1 and Treg expansion. This highlights the bi-functional role of SHH signaling during mycobacterial infection of DCs. Further, we found interesting cross-regulation of NOTCH and SHH pathway functions during M. bovis BCG infection of DCs. Inhibition of NOTCH1 signaling resulted in elevated expression of infection-induced PD-L1 whereas inhibition of SHH signaling showed increased transcripts of JAGGED2 (JAG2), a NOTCH1 ligand, and NOTCH intracellular domain (NICD), a marker for NOTCH activation. Thus, our results demonstrate that Mycobacterium directs a fine-balance of host signaling pathways and molecular regulators in DCs to determine the functional outcome of the immune responses including Tregs expansion that favours its survival. Foamy macrophages (FMs) are integral components of granulomas during mycobacterial pathogenesis. FMs are one of the morphotypes differentiated from macrophages characterized by the presence of lipid bodies (LBs)/droplets. The lipids provide nutrients to mycobacteria, leading to an enhanced ability to survive and replicate in host FMs. LBs are also known to regulate lipid metabolism, membrane trafficking, intracellular signaling and inflammatory mediator production. Interestingly, LBs are stores for various immune mediators including arachidonic acid, COX-2, ALOX5, ALOX15 and leukotrienes, underscoring the significance of FMs in the current study. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs in the course of mycobacterial infection are not clear. Here, we analyzed the role for one of the histone modifications widely implicated in shaping the immune responses, Histone H3 lysine 27 trimethylation (H3K27me3), a known marker for gene silencing. While the trimethylation of H3K27 is catalyzed by EZH2, a component of Polycomb-repressive complex (PRC)2, Jumonji C (JmjC) domain protein (JMJD3) is a well-established H3K27me3 demethylase. Unlike M. smegmatis, infection of macrophages with M. tuberculosis or M. bovis BCG displayed JMJD3-dependent LB formation. Supporting this observation, the genes involved in lipid biosynthesis (Ascl1, Adrp, Psap) and uptake (Fat (CD36) and Msr1) were significantly upregulated with M. tuberculosis or M. bovis BCG infection of macrophages in a JMJD3- and TLR2-dependent manner. Abca1 and Abcg1, genes assisting in lipid export were downregulated or remained unchanged with M. tuberculosis or M. bovis BCG infection. Chromatin immunoprecipitation analysis revealed a reduced H3K27me3 mark on the promoters of the selected genes that were upregulated on mycobacterial infections. Corresponding, elevated recruitment of JMJD3 to these promoters was observed. Interestingly, NOTCH1 signaling-responsive MUSASHI (MSI), an evolutionarily conserved RNA-binding protein that inhibits translation of the mRNA, was found to positively regulate infection-induced JMJD3 expression. MSI targeted a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein (MINT/ SPEN), in the infected macrophages to aid in FM formation. Immunohistochemistry and immunofluorescence experiments utilizing in vivo murine granuloma model using M. bovis BCG substantiated these observations. Thus, our study has unveiled novel roles for JMJD3 and its regulators in epigenetic regulation of LB generation in FMs. Altogether, we have established significant roles for several new host factors and inhibitory, survival mechanisms employed by pathogenic mycobacteria. Emphasis on functions of miRNAs and epigenetic regulation in the study has underscored the importance of fine-tuning immune responses during mycobacterial pathogenesis to determine the cell-fate and shape the course of infection. Further understanding and evaluation of these molecular regulators bears potential importance in disease control by aiding the search for effective drugs and therapeutics.

Water is an essential need that stimulates health and well being. Increase in population size and urbanization negatively affect water resources due to high demands of effluent outputs. Wastewater is an important reservoir for Escherichia coli and can present significant acute toxicity if released into receiving water sources without being adequately treated. E. coli is used as indicator organism for the detection of faecal contamination. These strains have been considered to be one of the primary causes of diarrhoeal infections worldwide. The present study was conducted between September 2012 and June 2013 to assess the prevalence of pathogenic E. coli strains in the final effluents of four wastewater treatment plants in Chris Hani and Buffalo City Municipalities in the Eastern Cape Province of South Africa. Standard membrane filtration technique was used for bacteriological analysis and molecular based technique was used for identification of E. coli pathotypes. The results were recorded in colony forming units/100 ml. Faecal coliforms ranged between 0-9.6×10³ CFU/100 ml for the wwtp-Q and E. coli densities ranged between 0-8.4×10³ CFU/100ml. Faecal coliforms ranged between 4×10²-9.7×10³ CFU/100 ml for wwtp-M and E. coli densities ranged between 1.2×10¹-8.4×10³ CFU/100 ml. The wwtp-E showed to have bacterial counts of faecal coliforms ranging between 4.0×10³-8.2×10³ CFU/100 ml and E. coli densities ranging between 3.5×10¹-7.1×10³ CFU/100 ml. The WWTP-K in this study was only assessed for the presence of E. coli. Faecal coliforms were assessed by the other members of the group. This plant showed to have E. coli densities ranging between 0-7.5×10²CFU/100 ml. A total of 200 presumptive E. coli isolates were subjected to screening by conventional PCR in which (29%) of the wwtp-M isolates were positively identified as E. coli, (16%) of the wwtp-K, (22%) of the wwtp-Q and (34%) of the wwtp-E isolates were positively confirmed as E. coli. A total of 100 randomly selected E. coli isolates were characterised into different pathotypes. (16%) of positive isolates were detected as EPEC and 11% were detected as UPEC strains. There was no detection for the ETEC strains. Antibiotic susceptibility patterns of E. coli strains showed high levels of resistance to Penicillin G, Erythromycin, Tetracycline and Sulfamethoxazole. High levels of Susceptibility were observed in antibiotics such as Chloramphenicol, Amoxicillin and Tetracycline. The results of this study reveal that the plants were above the recommended Standard limit of zero CFU/100 ml for effluents meant to be discharge into receiving water sources. This study reveals inadequacy of the plants studied to produce effluents of acceptable quality.