Characterization of aeromonads and development of vaccine candidate from aeromonas hydrophila isolated from tilapia fish farms in Tanzania

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Life Sciences of Nelson Mandela African Institution of Science and TechnologyTanzania produces less than half of the country annual demand in fish. Therefore, there is an urgent need to produce more fish, particularly through fish farming. However, aeromonads infections cause major lose in aquaculture worldwide and especially in developing countries, including Tanzania, lacking advanced capacity for fish disease control and prevention. Poor fish farming management practices, lack of data on prevalence, emergence of resistances to commonly used drugs, drug residues and limited capacity to control aeromonads bacterial infections emerged as major health problems in fish farming in Tanzania. This study aimed to characterise the aeromonads species circulating in fish farms and then develop a monovalent vaccine candidate from selected prevalent aeromonads specie for supporting tilapia fish farming improvement in Tanzania. A cross sectional study was conducted in Ruvuma, Mbeya, Iringa and Kilimanjaro regions between February 2017 and October 2018. A questionnaire was administered to 32 selected fish farmers to explore their knowledge on pond, fish health and diseases management practices. The results showed that the selected farmers had limited knowledge on pond, fish health and disease management practices. Onfarm training on the same to these farmers would improve their knowledge. A total of 816 whole fish samples were aseptically collected from these 32 fish farms to detect and identify aeromonads using molecular methods in order to establish the prevalence and characterise their virulence properties. The overall prevalence of 24.6% was recorded. Seventy five percent of the isolates had virulence genes of varying combinations and the in-vivo study showed high mortality (98.3%) to isolates with more virulence genes indicating their capacity to establish disease in a favourable environment. The Aeromonas hydrophila strain TZR72018 was selected and attenuated using a novel thermo-continuous sub-culturing method to develop a vaccine candidate. The experimental study was carried out to assess its protective efficacy. The results showed that the vaccine candidate had acceptable protective efficacy of 82.3% and 71.4% when given through intraperitoneal injection (IP) and immersion (IM); respectively. To the best of my knowledge this study reports the development of thermoattenuated and stabilized A. hydrophila vaccine candidate for the first time in Tanzania or elsewhere

Assessing the Awareness, Knowledge, Attitude and Practice of the Community towards Solid Waste Disposal and Identifying the Threats and Extent of Bacteria in the Solid Waste Disposal Sites in Morogoro Municipality in Tanzania

Solid wastes comprise all the wastes arising from human and animal activities that are normally solid, discarded as useless or unwanted materials. Health hazards associated with improper disposal of solid wastes to the community were investigated in Morogoro municipality. The aim of the project was to investigate the solid waste disposal practices and their health hazard implications to the community in Morogoro municipality. The study was conducted by considering several solid waste disposal sites based on three methods; observation, questionnaire survey and microbiological analysis. Based on observation method, several solid waste practices were detected including collection of wastes using trucks, wheel barrow, carriers made from elephant grasses and cement bags. Questionnaire survey pinpointed several diseases caused by solid wastes such as malaria, diarrhea, dysentery, cholera, typhoid and worm diseases from the respondents. From microbiological analysis, several pathogenic bacteria were identified from the solid disposal sites. The bacteria with their frequency of isolation identified were: Salmonella typhimurium (16.7%), Shigella dysenteriae (16.7%), Citrobacter freundii (8.3%), Citrobacter amalonaticus (8.3%), Aerobacter aerogenes (8.3%), Proteus vulgaris (16.7%), Klebsiella oxyotoca (8.3%), Klebsiella (8.3%), E.coli (8.3%). Solid waste generated by the daily activities of the people needs to be properly managed in such a way that it minimizes the risk to the environment and human health. Inadequate collection and disposal of solid waste is a major factor in the spread of disease and environmental degradation. Keywords: Solid wastes, Dumpsites, Awareness, Health risks, Pathogenic bacteria, Waste management, Tanzania

Assessing the Awareness, Knowledge, Attitude and Practice of the Community towards Solid Waste Disposal and Identifying the Threats and Extent of Bacteria in the Solid Waste Disposal Sites in Morogoro Municipality in Tanzania

Solid wastes comprise all the wastes arising from human and animal activities that are normally solid, discarded as useless or unwanted materials. Health hazards associated with improper disposal of solid wastes to the community were investigated in Morogoro municipality. The aim of the project was to investigate the solid waste disposal practices and their health hazard implications to the community in Morogoro municipality. The study was conducted by considering several solid waste disposal sites based on three methods; observation, questionnaire survey and microbiological analysis. Based on observation method, several solid waste practices were detected including collection of wastes using trucks, wheel barrow, carriers made from elephant grasses and cement bags. Questionnaire survey pinpointed several diseases caused by solid wastes such as malaria, diarrhea, dysentery, cholera, typhoid and worm diseases from the respondents. From microbiological analysis, several pathogenic bacteria were identified from the solid disposal sites. The bacteria with their frequency of isolation identified were: Salmonella typhimurium (16.7%), Shigella dysenteriae (16.7%), Citrobacter freundii (8.3%), Citrobacter amalonaticus (8.3%), Aerobacter aerogenes (8.3%), Proteus vulgaris (16.7%), Klebsiella oxyotoca (8.3%), Klebsiella (8.3%), E.coli (8.3%). Solid waste generated by the daily activities of the people needs to be properly managed in such a way that it minimizes the risk to the environment and human health. Inadequate collection and disposal of solid waste is a major factor in the spread of disease and environmental degradation. Keywords: Solid wastes, Dumpsites, Awareness, Health risks, Pathogenic bacteria, Waste management, Tanzania

Antimicrobial Profiles of Pathogenic Enteric Bacteria Isolated from Commensal Rodents and Cockroaches from Morogoro Region in Tanzania: An Environmental Vehicle for Resistance Transmission to Human and Animals

The increase in antimicrobial resistance (AMR) is becoming the global health concern and implicated to be a silence pandemic. It reduces the effectiveness of antimicrobials and threatening the health of human and animals. Rodents and cockroaches can play a significant role in the dissemination of resistance bacteria between the environment and humans. This study aimed at determining antimicrobial resistance profile of enteric bacteria isolated from cockroaches and house rats. The study was cross-sectional in design, the target population included house rats (Rattus rattus) and cockroaches (Periplaneta americana). A total of 114 house rats and 57 cockroaches were trapped. Cockroaches were manually trapped with surgical gloves and sealed in sterile plastic containers where by house rats were trapped with wire cages and transported to microbiology laboratory. Bacteria were isolated following standard Microbiological techniques and antimicrobial susceptibility testing was performed using Kirby Bauer diffusion technique. Data analysis was done using Graphpad Prism version 9. A total of 157 isolates of three targeted bacterial species were isolated from the gut of 114 house rats and 57 cockroaches. Among isolated bacteria only 83 were subjected to antimicrobial susceptibility test. Out of the tested antimicrobials; Amoxicillin, Ciprofloxacin and Tetracycline showed the highest resistance. Moreover Multidrug-resistance (MDR) was also observed for 66.3%. Generally, these findings indicate that cockroaches and house rats play a significant role of harboring and disseminating pathogenic resistance bacteria, therefore necessary control measures need to be taken against the infestation of rodents and cockroaches around households so as to minimize transmission of pathogenic and resistant bacteria. Keywords: Antimicrobial resistance (AMR), Enteric bacteria, Rodents and Cockroaches, Multidrug-resistance (MDR) DOI: 10.7176/JHMN/105-06 Publication date: January 31st 202

Evidence of Carriage of Antimicrobial Resistant Salmonella species of Public Health and Veterinary Significance in the Intestines of House Crows \ud (Corvus splendens) in Tanzania

The Indian house crow, Corvus splendens (Vieillot) was introduced in Zanzibar, Tanzania by the British and immigrants from India in 1897 to help clean the town. The crow is responsible for polluting the environment, water sources and human surroundings by their droppings and the rubbish they carry. This behavior has led to concern that, the crows may be responsible for the spread of certain pathogens including Salmonella and their persistence in the environment.Given the zoonotic potential of Salmonella, the main aim of this study was to investigate the occurrence of antimicrobial resistant Salmonella infections in Indian house crows and to determine if the isolates were similar to those associated with disease in livestock or humans. Methods: Indian house crows were lured with meat and blood baits to land into the crow live-trap set at the Mabibo compound of the National Institute for Medical Research (NIMR) in Dar es Salaam city in Tanzania. A total of 100 house crows were captured, humanely sacrificed, and their small and large intestines were obtained by using aseptic techniques for microbiological investigations. Culture technique was employed to detect the presence of Salmonella in intestinal contents; and preliminary identification of the isolates was based on colonial characteristics on selective media and microscopic examination of smears following Gram staining. Confirmation of Salmonella species was done by biochemical tests. Antimicrobial susceptibility testing was done by using the disc diffusion method on Mueller Hinton agar. Eight isolates were identified by standard microbiological techniques as Salmonella spp. (6 suggestive of Salmonella gallinarum and 2 suggestive of S. Typhi). All isolates were found to be susceptible to ciprofloxacin but resistant to amoxicillin. Lower levels of susceptibility were noted for chloramphenicol and ceftriaxone. Our results demonstrate the presence of antimicrobial resistant Salmonella spp. in the Indian house crows’ population and provide an indication of potential public and poultry health risks associated with these birds in the coastal area. The occurrence of antibiotic resistant S. Typhi and S. gallinarum among Indian house crows has both veterinary and public health consequences as they may be transmitted to poultry and humans. This therefore provides further rationale for the public action on eradicating the house crows

Phenotypic and molecular detection of Aeromonads infection in farmed Nile tilapia in Southern highland and Northern Tanzania

This research article published by Elsevier Ltd., 201

Current State of Modern Biotechnological-Based Vaccines for Aquaculture: A Systematic Review.

This research article published by Hindawi, 2019This systematic review describes what "the cutting edge vaccines for are". The focus is on types of high tech biotechnological based vaccines, target gene or antigen in developing these vaccines, and challenge model fish species used in vaccines efficacy testing. Vaccines delivery methods, immune response, and their efficacy, adjuvant or carrier systems used, and the overall experimental setup or design of the vaccines under investigation are also described. The search for the original papers published between 2009 and 2018 was conducted in June of 2018, using the PubMed and Google scholar electronic database. Twenty-three (23/4386) studies were included in the final assembly using PRISMA guidelines (Protocol not registered). Recombinant protein vaccines were the highly experimented type of the modern biotechnological based vaccines identified in the selected studies (16/23; 70%). Outer membrane proteins (OMPs) of different -barrels were shown to be a potential antigenic entity for vaccines (57%). Intraperitoneal route with conventional carries or adjuvants was the highly applied delivery system while very few studies used herbal based vaccine adjuvants and nanomaterial as a vaccine carrier. Variation was observed in terms of protection levels in the selected studies. The experimental designs partly contributed to the observed variation. Therefore, recombinant vaccines that use new carrier system technologies and delivered through oral route in feeds would have been of great value for use in the prevention and control of infections in fish. Despite the usefulness as academic tools to identify what is important in pathogenicity of the etiological agent to the host fish, these vaccines are only economically viable in very high-value animals. Therefore, if vaccination is a good option for group, then simple autogenous vaccines based on accurate typing and evidence-based definition of the epidemiological unit for their use would be the most viable approach in terms of both efficacy and economic feasibility especially in low and middle-income countries (LMIC)

Virulence pattern of circulating aeromonads isolated from farmed Nile tilapia in Tanzania and novel antibiotic free attenuation of Aeromonas hydrophila strain TZR7-2018

This research article published by Elsevier B.V., 2020Aeromonads are gram-negative, rod-shaped, facultative anaerobes bacteria known to cause motile aeromonads septicemia diseases (MAS) in warm freshwater farmed fish. Outbreaks are associated with pathogenicity of aeromonads in fish which is partly contributed by virulence characteristics of the etiological agent. The objective of this study was to assess the virulence characteristics of the previously isolated and identified aeromonads, and attenuate potential Aeromonas hydrophila strain TZR7-2018 to serve as local vaccine candidate. Six virulence genes and other virulence characteristics were molecularly and phenotypically assessed both using in-vitro and in-vivo approaches. Attenuation of A. hydrophila parent strain TZR7-2018+ was performed by passaging through thermal continuous sub-culturing 40 times in Tryptic soy agar (TSA). Bacterin was prepared by formalin inactivation from the same parent strain. Humoral responses were assayed using quantitative serological agglutination test (qSAT) while protective efficacy was measured through relative percent survival (RPS). A total 240 Nile tilapia fingerlings with an average weight of 8.1 ± 0.4 g were used in all in-vivo studies. The presence of aerolysin (aer), cytotoxic enterotoxin (act), elastase (ahy), haemolysin (hly), serine (ser) and polar flagella (fla) genes were determined using PCR. Out of 201 isolates, 75.1 % (151/201) of the aeromonads possessed virulence genes (120=A. hydrophila and 31=Aeromonas veronii). The virulence gene pattern of aer/hly/fla was the most prominent with the prevalence of 12.6 %. The attenuated strain TZR7-2018− showed reduced: colon size, multiplication rate, cell size and loss in; haemolysis, motility and capsule. Humoral responses increased gradually and reached maximum at day 28 in both attenuated and bacterin formulation given through intraperitoneal (IP) injection and immersion (IM). A RPS of 82.3 %, 71.4 % and 85.1 %, were recorded to the attenuated vaccine given through IP and IM and bacterin provided through IP respectively. Therefore the attenuated strain TZR7-2018− obtained through thermal continuous subculture technique and the bacterin proved to be efficacious and can serve as vaccine candidate

Present status of aquaculture and the challenge of bacterial diseases in freshwater farmed fish in Tanzania; A call for sustainable strategies

This research article published by Elsevier, 2020Aquaculture provides significant contributions to household food security, as the capture of wild fish from lakes, dams, and oceans do not meet the current demand for animal protein in Tanzania. Sustainable aquaculture requires well-established regulatory systems and extension services for good pond management practices and maintaining fish health by fish farmers. Fish farming is practiced widely in Tanzania, from small-to large-scale ponds and these farming systems are moving from extensive normal operations (low input demand) to intensive farming (high input demand). However, the industry is largely still operating at a subsistence level with low production. Bacterial infections have been occurring in these fish farms and will continue to be an issue of concern into the future. This review highlights the current challenges, successes, and prospects towards a sustainable aquaculture industry in Tanzania, including: limited extension services mirroring the limited knowledge by farmers regarding pond management practices; the inadequacy of funds to carry out fish disease research or implement a surveillance system; little expertise in fish disease diagnosis and treatment; and poor management options. To minimize disease outbreaks and optimize production in the future, we suggest a strengthening of extension services, augmented with on-farm knowledge transfer. Emphasis should be on pond management practices and fish disease management; the creation of a well-functioning fish disease surveillance system; and strengthening collaborative research on aquaculture between the government research institutions and academia. Establishing small cooperative fish farmer groups within the Aquaculture Association of Tanzania (AAT) for easy access to information is also recommended

Determination of bacterial load and antibiotic susceptibility testing of bacteria isolated from students’ toilets at Sokoine University of Agriculture, Morogoro, Tanzania

The circulation of infectious diseases in the community settings in urban and rural areas remains to be a hectic problem. One of the sources of microbial diseases is toilets. This study aimed at isolating, identifying and establishing bacterial loads associated with public restrooms in students’ hostels at Sokoine University of Agriculture in Morogoro, Tanzania. Samples were collected from a total of thirty toilets (60 samples) in different surfaces; (i) surfaces associated with toilets (toilet seats and toilet bowls), (ii) surfaces routinely touched with hands (door handles in and out of the restrooms, faucet handles and toilet flush handles) and (iii) the restroom floors. Samples were inoculated in MacConkey and Blood agar and then incubated at 37oC for 24 hours. All isolates were sub cultured and identified based on macro- and micro-morphology and Standard Biochemical Tests. The establishment of total bacteria load was done using Standard Plate Count Method. The sensitivity testing of the isolates were carried out using the Disk Diffusion Method on nutrient agar plate. The following bacteria genera and species were isolated from the students’ toilets; Staphylococcus aureus (25.0%), Escherichia coli (36.7%), Pseudomonas aeruginosa (13.3%), Streptococcus pyogenes (6.7%), Proteus mirabilis (6.7%) and Klebsiella pneumonia (11.6%). The results from total bacterial count indicated that the surfaces routinely touched with hands had highest bacteria load compared to restroom floor and toilet seats. However, the differences of means among the surfaces were not statistically significant (P= 0.6762).  Sensitivity testing of the isolates against commonly used antibiotics in the study area showed that all bacterial isolates tested were resistant and intermediate resistant to at least one antibiotic. Keywords: Pathogenic bacteria, Students’ hostels, bacteria count, antibiotic susceptibility testing