Brucella melitensis: the evaluation of a putative hemagglutinin gene\u27s effect on virulence in the caprine model

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortions in goats and sheep and Malta fever in humans. The zoonotic disease brucellosis causes severe economic losses in the Mediterranean region and parts of Africa, Asia, and Latin America. With the completion of the genomic sequences of B. abortus 2308 and B. melitensis 16M, no classical virulence factors were found; and the chromosomes were virtually identical. However, in B. melitensis, a putative hemagglutinin gene was identified which is absent in B. abortus. The possibility of the hemagglutinin gene being a potential virulence factor was evaluated via gene replacement/deletion in B. melitensis and expression in trans in B. abortus. The hemagglutinin gene was PCR-amplified, cloned into pBBR1MCS-4, and electroporated into B. abortus 2308 yielding B. abortus 2308-QAE. A kanamycin-Region E-kanamycin disrupted gene fragment (KAN-E-KAN) was also generated and electroporated into B. melitensis 16M. The resulting mutants were characterized biochemically to confirm its Brucella origin and screened by antibiotic selective pressure. A colonization study of non-pregnant goats infected with B. abortus 2308, B. melitensis 16M, B. abortus 2308-QAE, or B. melitensis 16MÄE revealed no attenuation of the 16MÄE mutant when compared to 16M at 4, 7, and 21 days post inoculation. The study also showed that both the variant and the mutant were capable of infecting and disseminating throughout the host. All four strains were introduced into the pregnant goat model and evaluated for pathogenicity. Pregnancy/delivery results revealed 27%, 78%, 67%, and 50% abortion rates in goats infected with 2308, 16M, 2308-QAE, and 16MÄE, respectively. Bacterial culture of tissues from 2308, 16M, 2308-QAE, 16MÄE -exposed goats revealed 45 %, 79%, 75%, and 100% colonization of dam/kid pairs, respectively. The expression of the B. melitensis 16M hemagglutinin gene in trans in 2308-QAE revealed a significant (p\u3c0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking the virulence of B. melitensis 16M in pregnant goats. The B. melitensis 16MÄE disruption mutant colonization and abortion rates demonstrated no attenuation in colonization but did show a 28% reduction in abortions when compared to B. melitensis 16M

The epidemiology of brucellosis in the Sultanate of Oman

Brucellosis is a serious disease of cattle in many countries of the world, including Mediterranean and Middle Eastern countries. The study outlined in this thesis was conducted to investigate the epidemiology of brucellosis in the Sultanate of Oman. Thirty of 1267 holdings tested in the Sultanate contained seropositive animals for brucellosis (herd prevalence 2.4%, 95% CI 1.6, 3.4%). The southern governorate (Dhofar) had significantly more seropositive holdings (n = 20, 8.6%, 95% CI 5.3, 13) than did the northern governorates (n = 10, 0.97%, 95% CI 0.5, 1.8) (p < 0.001) highlighting the endemic nature of the disease in Dhofar. Although there were no significant differences between the herd seroprevalence for individual species, the highest herd level seroprevalence was reported in cattle (4.9%) followed by camels (2.3%), goats (1.4%) and sheep (0.6%). The overall individual animal seroprevalence of brucellosis in Oman was generally at a low level (0.4%; 95% CI 0.2, 0.5). The individual seroprevalence level in the different species was also low, being 0.4%, 0.4%, 0.4% and 0.1% in cattle, camels, goats and sheep, respectively. The practice of moving animals without testing between governorates is likely to have allowed the spread of infection throughout Oman. The active importation of live animals from other countries in the Horn of Africa, without prior monitoring of their brucellosis status, inter-species contact, sharing of common pasture, large herd size and the presence of poor biosecurity/unhygienic conditions in herds in the southern governorate may have facilitated the spread of brucellosis in the Dhofar region and from here infection may have been transmitted to other governorates. A logistic-regression analysis was undertaken to identify risk factors for disease. This analysis indicated associations of breed, age, herd size and production system with seropositivity. A higher seroprevalence was found in imported animals (OR 3.71, 95% CI 0.68, 20.43), and the seroprevalence increased with age. The latter finding is possibly because of a higher risk of contracting the disease after puberty through increased contacts with potentially infected animals. Only Brucella melitensis was cultured from different species of animals and biotype 1 was the only type identified in Oman by molecular means and phage typing. Sequencing of DNA revealed that all isolates had a very similar pattern. In the current study although there was no significant difference observed in the seroprevalence detected by different diagnostic assays (cELISA, iELISA and RBPT), the ELISAs were capable of detecting more positive samples than the RBPT and Rapid test. This may reflect the better sensitivity of the ELISAs and it is recommended that these tests be used in the control and eradication of brucellosis in Oman, where vaccination is undertaken. In Oman, human brucellosis was first reported in 1979 in the southern Dhofar governorate. A retrospective analysis of human brucellosis data sourced from the Ministry of Health, Oman from 1995 to 2012 was conducted. Information regarding location, age, gender, nationality of patients and year were included in the analysis. During this period, 2737 human cases of brucellosis were reported, with 96.7% of these in Dhofar. The incidence of disease was highest in young individuals (0-10 years of age), highlighting that these subjects were more at risk of acquiring brucellosis. The incidence of brucellosis was slightly higher in males (56%) than females (44%). Most of the positive patients were Omani nationals, most likely because of more opportunity for contact with infected animals on privately owned farms. The failure of disease control programmes in the southern region (2003 until 2012) could be due to a lack of information, inappropriate planning or administrative issues. With the information gathered from this study, it is considered there is a need to build a strategy to control the disease throughout Oman, rather than restricting control to the Dhofar governorate. However it is recommended that the control program adopted in the southern region (Dhofar), where the seroprevalence is high, be different to that implemented in the northern regions, where the disease prevalence is lower and more manageable. In the southern region, implementing a vaccination programme, along with individual animal identification and disease screening with a plan of intensive involvement and extension in the community, should be considered. In contrast in the northern region a test and slaughter program could be implemented

The Practice of Sheep Veterinary Medicine

This book is intended to be a reference text for veterinarians who provide clinical services to sheep producers. It is directed first and foremost at Australian sheep-raising systems, but the approaches described herein will have wide application in all countries where sheep are raised under extensive grazing conditions. Most of the important conditions of sheep in Australia are relatively straightforward to diagnose, but the establishment of effective and economically sound control strategies is often the most difficult part of health management, particularly for those who are less familiar with sheep production systems. With six initial chapters focusing on providing readers with a basic understanding of the business and science underpinning sheep production, this book focuses its remaining chapters on reproduction and disease conditions, ordered largely on a systems basis. The book provides details about the way disease processes develop and manifest in sheep flocks, with numerous references for those who wish to read further. Australian sheep production is a profitable and fulfilling agricultural pursuit for a large number of farm owners, and this book is intended to assist those who work in the industry to add to the profitability and efficiency of sheep production systems, the quality of sheep products and the welfare of the sheep in those systems

The Design, Construction, and Testing of a Recombinant DNA Vaccine for Brucella abortus and Brucella melitensis

Brucella spp. are a bacterium that cause brucellosis, a zoonotic disease, which is commonly seen in cattle, sheep, goats, swine, and canines. Brucellosis is a problem worldwide, although it is eradicated in some countries (Garin-Bastuji et al. 1998). The reason for designing recombinant DNA (rDNA) vaccines opposed to utilizing the live-attenuated vaccines on the market is that they cannot be given to pregnant animals without potentially causing abortion, while an rDNA vaccine should be safe for pregnant animals since it does not contain viable bacteria. Also, there are no serological tests that can accurately distinguish between an animal vaccinated with the current vaccines available for brucellosis and an animal with an active Brucella infection because the current vaccines are live-attenuated and interfere with diagnostics. Recombinant DNA vaccines eliminate many safety concerns, which is why two rDNA vaccines were constructed in the same way, except for the length of the immunogenic epitopes selected from Brucella abortus. The epitopes from B. abortus that were chosen are present in the other main species of Brucella because the main species of Brucella, such as B. abortus, B. melitensis, and B. suis, are 99% identical at the DNA level. The epitopes range in length from 25 to 70 amino acids. Each vaccine contained a specific insert, with different lengths of epitopes and an antibiotic resistance gene that was incorporated for selection of successfully transfected cells during in vitro testing. After the vaccines were constructed, they were tested in vitro to determine the vaccine’s ability to enter different cell types and show protein expression in a variety of cells. The vaccines were tested in a caprine model to determine the vaccine’s ability to be successfully expressed in mammalian cells in vivo. It was determined that no significant antibody concentration was detected via the testing methods utilized

Brucellosis in Ethiopia: epidemiology and public health significance.

ABSTRACT Brucellosis is a common bacterial zoonotic disease that has important veterinary and public health concerns and economic impact in Sub-Saharan Africa including Ethiopia. A cross-sectional study was conducted in Addis Ababa Dairy farms and four selected districts of Borena Pastoral setting in Southern Ethiopia from November 2016 to February 2018 to estimate the prevalence of brucellosis and associated risk factors in cattle, sheep and goats and occupationally associated humans. A total of 2300 cattle, 882 small ruminants and 341 humans were serum sampled and serially tested for antibody against Brucella infection using Rose Bengal Test for screening and further confirmed by Competitive-Enzyme Linked Immunosorbent Assay. The overall individual animal level seroprevalence was 0.06 % (95% CI: 0.002 -0.4) in commercial dairy cattle in Addis Ababa, 2.4 % (CI: 1.4-3.7) in cattle, and 3.2 % (CI: 2.1-4.6) in small ruminants; both in Borena pastoral region, Southern Ethiopia. The seroprevalence recorded in humans occupationally linked to livestock production systems in Borena was 2.6 % (CI: 1.2-5). Herd size, parity, and history of abortion were risk factors associated with Brucella seropositivity (p<0.05) in cattle whereas in small ruminants the results show that district, age group, flock size, and history of abortion were found to be significantly associated risk factors with Brucella infection (P<0.05). The risk factors for Brucella infection in humans were assisting in calving and presence of seropositive animals at household (p<0.05). Existence of more than one seroreactor animal species in some villages in Borena indicates more credence to the possibility of cross-species transmission of Brucella infections. Human seropositivity in the households with seropositive animals provides evidence of a public health hazard. In addition, an outbreak investigation of abortion at Adami Tullu Agricultural Research centre, central Ethiopia, was made to isolate and characterise the causative agents using cultural and molecular tools. Fifteen Brucella abortus were isolated from various tissue samples and vaginal swabs collected during postmortem examination of seropositive animals in the centre. The B. abortus isolates and two B. melitensis previously isolated from vaginal swab of goats with history of abortion were whole genome sequenced (WGS). Subsequent phylogenetic analysis involved whole genome SNPs,core-genome SNPs, and in silico analysis of Multilocus Sequence Typing and Multiple Loci Variable Number of Tandem Repeats to characterise and determine global clustering of Ethiopian isolates. The result indicated that the B. abortus isolates from the outbreak were distinct and there were eight genotypes comprising single, double and triple locus variants circulating in the centre. Panel 1 and Panel 2A markers in MLVA-16 typing displayed no diversity among the neotypes suggesting that these are an early branching genotypes of the same strain. The core-genome phylogeny revealed that Ethiopian B. abortus form a distinct African clade branching basally with isolates previously described from Mozambique and Kenya. This clade was previously represented by only a small number of isolates, and thus the placement of Ethiopian B. abortus from the current study within this group substantially increases the representation of these basal B. abortus strains in genomic databases. On the other hand, B. melitensis isolates fall into a distinct African cluster with most isolates from Eastern African countries such as Somalia, Kenya, Eritrea, and Tanzania. The first isolation and molecular characterisation of circulating Brucella species in Ethiopia is a crucial step in planning and designing intervention strategies in the country. A coordinated One Health approach involving active involvement of human and animal health efforts to enhance public health and improve livestock productivity is recommended

Sub-Unit Vaccines for <i>Brucella</i>

Brucella species are the causative agents of brucellosis, which is regarded as the world's most prevalent zoonotic disease. Brucellosis is endemic to the Middle East, Mexico, Asia, South America and the Mediterranean and causes significant economic losses in livestock in these areas of the world, as well as being a reservoir for human brucellosis. Although there are live attenuated vaccines available for brucellosis, they have many drawbacks when used in animals, and are still infectious to humans. It is clear from this that there is a need for a new effective vaccine for brucellosis for both animal and human use. There are increasing instances of ATP-binding cassette (ABC) systems being identified as virulence factors or potential vaccine candidates. In this study inventories of all the ABC systems encoded by five sequenced Brucella strains were compiled. These inventories were compared and differences have been found that could aid in the identification of virulence factors of Brucella. This study also explores the potential of ATP-binding cassette transporters as sub-unit vaccines against Brucella melitensis 16M. Eight ABC transporter proteins (PotD, PotF, Cgt, CydD, LolE, FbpA, OppA and ZnuA) have been produced using recombinant protein technologies, and their protective efficacy was evaluated in a number of studies using the murine model of B. melitensis infection. Of the eight vaccine candidates selected two have shown potential as novel vaccines against brucellosis. PotD and PotF are putative polyamine binding proteins of Brucella and show protection against experimental challenge of approximately 1x104 CFU of B. melitensis 16M when expressed by DNA vaccines or used in a PotD/PotF combination vaccine. Through this work a duel adjuvant system (ISCOMs and CpG) has been identified as a more effective adjuvant for Brucella vaccines than others previously used

Small ruminant research and development in Africa: proceedings of the Third Biennial Conference of the African Small Ruminant Research Network : UICC, Kampala, Uganda, 5-9 December 1994

This volume contains papers and abstracts of the Third Biennial Conference of the African Small Ruminant Research Network. In addition to the keynote address, there are nine papers on Genetic Resources Enhancement and Utilisation, seven papers on Production Systems, Policy and Economics, six papers on Management and Health, 12 papers and Feeding Systems and 10 papers on Performance and Reproduction. Six poster abstracts covering the above topics add to the volume

Veterinary Microbiology & Parasitology

Numerous pathogens affect animal health and wellbeing and production efficiency. These pathogens also have a considerable impact on social economics, food safety and security, and human health. Infectious diseases that originate from both domesticated animals and wildlife represent one of the greatest threats to human health. Recent studies show that domesticated species harbor approximately 84 times more zoonotic viruses than wild species. Eight of the top 10 mammalian species with the highest number of zoonotic viruses are domestic, such as pigs, cattle, and horses. Many animal parasites are also zoonotic, constituting an additional burden on human health. Furthermore, the rapid emergence and spread of drug-resistant pathogen strains pose new threats to animal and human health. Climate changes will undoubtedly alter the interactions between animals and between animals and humans, which will have a huge impact on the transmission rate of existing pathogens and the emergence of new pathogens or the reemergence of old pathogens. In this special collection, interactions of all major pathogen types, including viruses, bacteria, mites and flies, protozoans, and helminths, and their hosts, such as wild and companion animals and livestock species, are discussed. Further, anthelmintic activities of natural products are evaluated. The relevance and utility of cutting-edge tools, such as immunology, genomics and genetics, microbiome studies and metabolomics, and molecular epidemiology, in dissecting host-pathogen interactions are also discussed. This special collection provides a broad knowledge base that encourages dialogue across a wide distribution of the research community in veterinary microbiology and parasitology

Bovine Science

Bovine Science - Challenges and Advances presents up-to-date knowledge of bovine health, covering both introductory topics and more advanced concepts. Chapters cover such topics as new techniques in bovine science and development, health and risk factors and diagnosis of disease in bovines, and production and reproductive technologies and advancements