A Novel Bacterial Protease Inhibitor Adjuvant in RBD-Based COVID-19 Vaccine Formulations Containing Alum Increases Neutralizing Antibodies, Specific Germinal Center B Cells and Confers Protection Against SARS-CoV-2 Infection in Mice.

In this work, we evaluated recombinant receptor binding domain (RBD)-based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus alum, AddaS03, AddaVax, or the combination of alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and alum as adjuvants has a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus alum, AddaVax, or AddaS03. Antibodies induced with the formulation containing U-Omp19 and alum not only increased their neutralization capacity against the ancestral virus but also cross-neutralized alpha, lambda, and gamma variants with similar potency. Furthermore, the addition of U-Omp19 to alum vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+alum formulation induced RBD-specific Th1 and CD8+ T-cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge of K18-hACE2 mice.

A Brucella Omp16 Conditional Deletion Strain Is Attenuated in BALB/c Mice.

Brucella spp. are facultative intracellular pathogens that invade, survive and proliferate in numerous phagocytic and non-phagocytic cell types, thereby leading to human and animal brucellosis. Outer membrane proteins (Omps) are major immunogenic and protective antigens that are implicated in Brucella virulence. A strain deleted of the omp16 gene has not been obtained which suggests that the Omp16 protein is vital for Brucella survival. Nevertheless, we previously constructed an omp16 conditional deletion strain of Brucella, ΔOmp16. Here, the virulence and immune response elicted by this strain were assessed in a mouse model of infection. Splenomegaly was significantly reduced at two weeks post-infection in ΔOmp16-infected mice compared to infection with the parental strain. The bacterial load in the spleen also was significantly decreased at this post-infection time point in ΔOmp16-infected mice. Histopathological changes in the spleen were observed via hematoxylineosin staining and microscopic examination which showed that infection with the ΔOmp16 strain alleviated spleen histopathological alterations compared to mice infected with the parental strain. Moreover, the levels of humoral and cellular immunity were similar in both ΔOmp16-infected mice and parental strain-infected mice. The results overall show that the virulence of ΔOmp16 is attenuated markedly, but that the immune responses mediated by the deletion and parental strains in mice are indistinguishable. The data provide important insights that illuminate the pathogenic strategies adopted by Brucella.

Incidence of Brucella infection in various livestock species raised under the pastoral production system in Isiolo County, Kenya.

BACKGROUND: We implemented a longitudinal study to determine the incidence of Brucella infection in cattle, camels, sheep and goats that were being raised in a pastoral area in Isiolo County, Kenya. An initial cross-sectional survey was implemented to identify unexposed animals for follow up; that survey used 141 camels, 216 cattle, 208 sheep and 161 goats. Sera from these animals were screened for Brucella spp. using the Rose Bengal Plate test (RBPT), a modified RBPT, and an indirect multispecies Enzyme Linked Immunosorbent Assay (iELISA). Results of RBPT and iELISA were interpreted in parallel to determine seroprevalence. A total of 30 camels, 31 cattle, 22 sheep and 32 goats that were seronegative by all the above tests were recruited in a subsequent longitudinal study for follow up. These animals were followed for 12 months and tested for anti-Brucella antibodies using iELISA. Seroconversion among these animals was defined by a positive iELISA test following a negative iELISA result in the previous sampling period. All seropositive samples were further tested using real-time PCR-based assays to identify Brucella species. These analyses targeted the alkB and BMEI1162 genes for B. abortus, and B. melitensis, respectively. Data from the longitudinal study were analysed using Cox proportional hazards model that accounted for within-herds clustering of Brucella infections. RESULTS: The overall incidence rate of Brucella infection was 0.024 (95% confidence interval [CI]: 0.014-0.037) cases per animal-months at risk. Brucella infection incidence in camels, cattle, goats and sheep were 0.053 (0.022-0.104), 0.028 (0.010-0.061), 0.013 (0.003-0.036) and 0.006 (0.0002-0.034) cases per animal-month at risk, respectively. The incidence rate of Brucella infection among females and males were 0.020 (0.009-0.036) and 0.016 (0.004-0.091), respectively. Real-time PCR analyses showed that B. abortus was more prevalent than B. melitensis in the area. Results of multivariable Cox regression analysis identified species (camels and cattle) as an important predictor of Brucella spp. exposure in animals. CONCLUSIONS: This study estimated an overall brucellosis incidence of 0.024 cases per animal-months at risk with camels and cattle having higher incidence than sheep and goats. These results will inform surveillance studies in the area.

Documenting the absence of bovine brucellosis in dairy cattle herds in the southern region of Malawi and the associated knowledge, attitudes and practices of farmers.

There is paucity of Brucella prevalence data in Malawi. For this reason, a cross-sectional study was conducted, from 06 January 2020 to 27 February 2020, to estimate the seroprevalence of brucellosis in dairy cattle herds amongst smallholder farmers, government and private dairy farms in the southern region. A total of 529 serum samples were screened for anti-Brucella antibodies using the Rose Bengal test (RBT) and a competitive enzyme-linked immunosorbent assay (cELISA). A pre-tested electronic (Epicollect tool, Wellcome Sanger Institute, United Kingdom) questionnaire was administered to 378 smallholder farmers to assess their knowledge, attitudes and practices towards brucellosis. Descriptive statistics were used to analyse the data in Microsoft Excel® and Statistical Package for Social Sciences (SPSS®) version 21. No animal tested positive for presence of anti-Brucella antibodies, indicating 0% prevalence (individual and herd levels). The majority (94.2%; 95% confidence interval [CI]: 91.8-96.5) of smallholder farmers had never heard about brucellosis. Furthermore, assisting during parturition without protective equipment (41.3%; 95% CI: 36.3-46.2) and using bulls for breeding (75%; 95% CI: 70.2-78.9) were amongst the common risk practices that were identified. We could not detect brucellosis in this study that indicates the disease could be very rare or even absent in the dairy cattle herds of the southern region of Malawi. However, further Brucella studies need to be conducted in cattle, small livestock, wildlife and humans to document the true status of brucellosis in the country. Brucellosis surveillance, monitoring, awareness and preventive measures are required to maintain this favourable situation.

Latent class evaluation of the performance of serological tests for exposure to Brucella spp. in cattle, sheep, and goats in Tanzania.

BACKGROUND: Brucellosis is a neglected zoonosis endemic in many countries, including regions of sub-Saharan Africa. Evaluated diagnostic tools for the detection of exposure to Brucella spp. are important for disease surveillance and guiding prevention and control activities. METHODS AND FINDINGS: Bayesian latent class analysis was used to evaluate performance of the Rose Bengal plate test (RBT) and a competitive ELISA (cELISA) in detecting Brucella spp. exposure at the individual animal-level for cattle, sheep, and goats in Tanzania. Median posterior estimates of RBT sensitivity were: 0.779 (95% Bayesian credibility interval (BCI): 0.570-0.894), 0.893 (0.636-0.989), and 0.807 (0.575-0.966), and for cELISA were: 0.623 (0.443-0.790), 0.409 (0.241-0.644), and 0.561 (0.376-0.713), for cattle, sheep, and goats, respectively. Sensitivity BCIs were wide, with the widest for cELISA in sheep. RBT and cELISA median posterior estimates of specificity were high across species models: RBT ranged between 0.989 (0.980-0.998) and 0.995 (0.985-0.999), and cELISA between 0.984 (0.974-0.995) and 0.996 (0.988-1). Each species model generated seroprevalence estimates for two livestock subpopulations, pastoralist and non-pastoralist. Pastoralist seroprevalence estimates were: 0.063 (0.045-0.090), 0.033 (0.018-0.049), and 0.051 (0.034-0.076), for cattle, sheep, and goats, respectively. Non-pastoralist seroprevalence estimates were below 0.01 for all species models. Series and parallel diagnostic approaches were evaluated. Parallel outperformed a series approach. Median posterior estimates for parallel testing were ≥0.920 (0.760-0.986) for sensitivity and ≥0.973 (0.955-0.992) for specificity, for all species models. CONCLUSIONS: Our findings indicate that Brucella spp. surveillance in Tanzania using RBT and cELISA in parallel at the animal-level would give high test performance. There is a need to evaluate strategies for implementing parallel testing at the herd- and flock-level. Our findings can assist in generating robust Brucella spp. exposure estimates for livestock in Tanzania and wider sub-Saharan Africa. The adoption of locally evaluated robust diagnostic tests in setting-specific surveillance is an important step towards brucellosis prevention and control.

Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein.

BACKGROUND: Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. CONCLUSIONS: B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.

Comparative analysis of the main outer membrane proteins of Brucella in the diagnosis of brucellosis.

Brucellosis has placed a heavy economic burden on numerous countries and has consumed considerable medical resources worldwide. To improve the specificity and sensitivity of serological methods for diagnosing brucellosis, it is important to develop new diagnostic antigens. Brucella outer membrane proteins(omps) possess good immunogenicity, but there is a scarcity of comparative studies of these proteins in the clinical diagnosis of brucellosis. In this study, six recombinant Brucella outer membrane proteins, omp10, omp16, omp19, omp25, omp31 and BP26, were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera of humans, bovines and goats with brucellosis were analyzed by indirect ELISA using these proteins, lipopolysaccharide(LPS) and Rose Bengale Ag, served as positive-control antigens. In diagnosing human and goat serum, BP26 exhibited the highest diagnostic accuracy of 96.45% and 95.00%, respectively, while omp31 exhibited the strongest ability to detect Brucella in bovine serum with an accuracy of 84.03%. Cross-reaction experiments also confirmed that the diagnostic specificities of omp31 and BP26 were higher than those of the LPS and Rose Bengale Ag antigens. The results of this study indicate that omp31 and BP26 are candidate antigens with high potential application value in the clinical diagnosis of brucellosis.

Design of a new multi-epitope vaccine against Brucella based on T and B cell epitopes using bioinformatics methods.

Brucellosis is one of the most serious and widespread zoonotic diseases, which seriously threatens human health and the national economy. This study was based on the T/B dominant epitopes of Brucella outer membrane protein 22 (Omp22), outer membrane protein 19 (Omp19) and outer membrane protein 28 (Omp28), with bioinformatics methods to design a safe and effective multi-epitope vaccine. The amino acid sequences of the proteins were found in the National Center for Biotechnology Information (NCBI) database, and the signal peptides were predicted by the SignaIP-5.0 server. The surface accessibility and hydrophilic regions of proteins were analysed with the ProtScale software and the tertiary structure model of the proteins predicted by I-TASSER software and labelled with the UCSF Chimera software. The software COBEpro, SVMTriP and BepiPred were used to predict B cell epitopes of the proteins. SYFPEITHI, RANKpep and IEDB were employed to predict T cell epitopes of the proteins. The T/B dominant epitopes of three proteins were combined with HEYGAALEREAG and GGGS linkers, and carriers sequences linked to the N- and C-terminus of the vaccine construct with the help of EAAAK linkers. Finally, the tertiary structure and physical and chemical properties of the multi-epitope vaccine construct were analysed. The allergenicity, antigenicity and solubility of the multi-epitope vaccine construct were 7.37-11.30, 0.788 and 0.866, respectively. The Ramachandran diagram of the mock vaccine construct showed 96.0% residues within the favoured and allowed range. Collectively, our results showed that this multi-epitope vaccine construct has a high-quality structure and suitable characteristics, which may provide a theoretical basis for future laboratory experiments.

Brucella Outer Membrane Lipoproteins 19 and 16 Differentially Induce Interleukin-18 Response or Pyroptosis in Human Monocytic Cells.

BACKGROUND: Brucella species are Gram-negative intracellular bacteria that causes severe inflammatory diseases in animals and humans. Two major lipoproteins (L19 and L16) of Brucella outer membrane proteins were studied to explore the association with inflammatory response of human monocytes (THP-1). METHODS: Activated THP-1 cells induced with recombinant L19 and L16 were analyzed in comparison with unlipidated forms (U19 and U16) and lipopolysaccharide (LPS) of Brucella melitensis, respectively. RESULTS: Secretion of inflammatory factors tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß was significantly increased from L19, L16, or both stimulated THP-1 cells. High secretion of IL-18 was detected only from L19-induced cells. Signaling of those cytokine responses was identified mainly through the P38-mitogen-activated protein kinase pathway, and signaling of L19-induced IL-1ß response partly occurred via necrosis factor-κB. While exploring different forms of IL-18, we found that L19-induced production of active IL-18 (18 kD) occurred through upregulating NLRP3 and activating caspase-1, whereas L16-induced production of inactive IL-18 fragments (15 kD and 16 kD) occurred through activating caspase-8/3. We also found that L19 upregulated phosphorylation of XIAP for inhibiting caspase-3 activity to cleave IL-18, whereas L16 activated caspase-3 for producing GSDME-N and leading to pyroptosis of THP-1 cells. CONCLUSIONS: Brucella L19 and L16 differentially induce IL-18 response or pyroptosis in THP-1 cells, respectively.

Serological evidence of single and mixed infections of Rift Valley fever virus, Brucella spp. and Coxiella burnetii in dromedary camels in Kenya.

Camels are increasingly becoming the livestock of choice for pastoralists reeling from effects of climate change in semi-arid and arid parts of Kenya. As the population of camels rises, better understanding of their role in the epidemiology of zoonotic diseases in Kenya is a public health priority. Rift Valley fever (RVF), brucellosis and Q fever are three of the top priority diseases in the country but the involvement of camels in the transmission dynamics of these diseases is poorly understood. We analyzed 120 camel serum samples from northern Kenya to establish seropositivity rates of the three pathogens and to characterize the infecting Brucella species using molecular assays. We found seropositivity of 24.2% (95% confidence interval [CI]: 16.5-31.8%) for Brucella, 20.8% (95% CI: 13.6-28.1%) and 14.2% (95% CI: 7.9-20.4%) for Coxiella burnetii and Rift valley fever virus respectively. We found 27.5% (95% CI: 19.5-35.5%) of the animals were seropositive for at least one pathogen and 13.3% (95% CI: 7.2-19.4%) were seropositive for at least two pathogens. B. melitensis was the only Brucella spp. detected. The high sero-positivity rates are indicative of the endemicity of these pathogens among camel populations and the possible role the species has in the epidemiology of zoonotic diseases. Considering the strong association between human infection and contact with livestock for most zoonotic infections in Kenya, there is immediate need to conduct further research to determine the role of camels in transmission of these zoonoses to other livestock species and humans. This information will be useful for designing more effective surveillance systems and intervention measures.

Epidemiology of brucellosis in cattle and dairy farmers of rural Ludhiana, Punjab.

Brucellosis is a zoonotic disease imposing significant impacts on livestock production and public health worldwide. India is the world's leading milk producer and Punjab is the state which produces the most cattle and buffalo milk per capita. The aim of this study was to investigate the epidemiology of bovine brucellosis to provide evidence for control of the disease in Punjab State, India. A cross-sectional study of dairy farms was conducted in humans and livestock in rural Ludhiana district using a multi-stage sampling strategy. The study suggests that brucellosis is endemic at high levels in cattle and buffalo in the study area with 15.1% of large ruminants testing seropositive and approximately a third of dairy farms having at least one animal test seropositive. In total, 9.7% of those in direct contact with livestock tested seropositive for Brucella spp. Persons that assisted with calving and/or abortion within the last year on a farm with seronegative livestock and people which did not assist with calving/abortion had 0.35 (95% CI: 0.17 to 7.1) and 0.21 (0.09 to 0.46) times the odds of testing seropositive compared to persons assisting with calving/abortion in a seropositive farm, respectively. The study demonstrated that persons in direct contact with cattle and buffalo in the study area have high risk of exposure to Brucella spp. Control of the disease in livestock is likely to result in benefits to both animal and public health sectors.

Sero-epidemiology and associated risk factors of brucellosis among sheep and goat population in the south western Nepal: a comparative study.

BACKGROUND: Brucellosis is a zoonotic disease caused by Brucella spp. In Nepal, the presence of brucellosis in small ruminants, namely sheep and goats, has impacted farmers' livelihood and the food safety of consumers. A cross-sectional study was conducted in Rupandehi district of Nepal during January to March 2020 to investigate the seroepidemiology and associated risk factors of brucellosis in the sheep and goat population. Altogether, 19 sheep and 60 goat farms in the district were visited. Owners were interviewed to get information on animals, including their management and movement patterns. Three hundred fifty-seven samples (80 sheep and 277 goat samples) were collected proportionately based on farm sizes. Each serum sample was tested with Rose Bengal Test and ELISA to estimate the seropositivity of brucellosis. Logistic regression was carried out to calculate corresponding odds ratios of each variable associated with detection of brucellosis. RESULTS: At the farm level, 31.6% (6/19; 95% CI: 12, 54%) of sheep farms and 3.3% (2/60, 95% CI: 0.9, 11.4%) of goat farms were seropositive to brucellosis. Out of 80 sheep serum samples, 12 (15%; 95% CI: 8.79-24.41%) and out of 277 goat serum samples, three (1.1%; 95% CI: 0.37-3.14%) were seropositive to brucellosis. Age greater than 1.5 years (OR = 5.56, 95% CI: 1.39, 29.38; p = 0.02) and herd size of greater than 100 (OR = 4.74, 95% CI: 1.23, 20.32, p = 0.03) were identified as significant risk factors for seropositivity of brucellosis in the sheep population. While in the goat population, none of the variables was identified as a significant risk factor. CONCLUSION: The study provides evidence that the older sheep and the sheep from the large herds were at higher risk of brucellosis. A control program should be put in place immediately in the sheep population because they may transmit infections to other livestock as they were regularly moved for grazing and selling purposes. Also, strict biosecurity measures should be implemented among pastoralists to prevent brucellosis transmission in them. We suggest further one health-based study to reveal the transmission dynamics of brucellosis between animals and humans.

A serological survey of brucellosis in wildlife in four major National Parks of Uganda.

BACKGROUND: Brucellosis is a contagious zoonotic disease of great public health and economic significance especially in developing countries. The disease affects humans and several species of livestock and wildlife. Studies on Brucellosis in wildlife in Uganda have been limited to single populations particularly in Queen Elizabeth National Park. This study aimed at estimating the percentage of positive samples of Brucella spp. in wildlife in four major national parks of Uganda. This was a retrospective survey which utilized archived samples collected from wildlife during the annual disease surveillance activities between 2013 and 2017. RESULTS: A total of 241 samples from seven species namely African buffalo (Syncerus caffer, n = 109), African elephant (Loxodonta africana, n = 22), giraffe (Giraffa camelopardalis rothschildi, n = 41), Uganda kob (Kobus kob thomasi, n = 36), lion (Panthera leo, n = 6), plain zebra (Equus quagga, n = 25), and bushbuck (Tragelaphus scriptus, n = 2), were tested for antibodies using the Rose Bengal Plate Test. The overall percentage of positive samples in the four national parks was 31.1% (75/241; 95% CI: 25.6-37.2). Kidepo Valley National Park had a significantly higher percentage of positive samples of 55.9% (19/34; 95% CI: 39.5-71.1) compared to other sampled national parks (p < 0.05). Lions had significantly higher percentage of positive samples at 66.7% (4/6) than African buffalo at 48.6% (53/109, p < 0.0001). There were no antibodies for Brucella spp. detected in African elephant and bushbuck. CONCLUSION: This study shows variations in percentage of positive samples with Brucella spp. between species and across national parks and notably a high percentage with Brucella spp. in wildlife in Uganda than that recorded elsewhere in sub-Saharan region of Africa. Potential for transmission to other wildlife and spill over to livestock is high especially in national parks with high livestock-wildlife interaction.

Development and evaluation of a gold nanoparticle based Lateral Flow assay (LFA) strip test for detection of Brucella spp.

The widely used serodiagnostic test (RBPT, CFT, I-ELISA and FPA) for diagnosis of brucellosis cannot detect vertically infected or carrier animals that are seronegative, a persistent source of infection to other susceptible animals in the herd. For reducing transmission of disease within the herd, these animals must be detected using a rapid, sensitive, user friendly penside diagnostic test. In the present study, Lateral Flow immunoassay (LFA) strip test was developed for detection of Brucellaspp. from clinical samples (bovine aborted foetal stomach contents). The LFA strip was fabricated by printing anti-Brucella polyclonal antibodies (PAb) and anti-bovine antibodies IgG on test and control line, respectively. For conjugation, colloidal gold nanoparticles (30 nm GNP, Sigma, USA) were conjugated with anti-brucella PAb. The LFA strip test was able to detect 107 cfu/ml B.abortus S99 inactivated organism in PBS and it did not exhibit any cross reactivity with selected non Brucella pathogens. To further validate, 115 clinical specimens were tested using LFA strip test. The relative sensitivity (DSn) and relative specificity (DSp) of LFA strip test was determined by ROC curve analysis using PCR and culture method as reference standard. DSn and DSp of LFA strip test was observed as 78.57% (95%CI: 49.2-95.3); 93.07% (95%CI: 86.2-97.2) and 80.0% (95%CI:51.9-95.7); 94.0% (95%CI:0.795-0.925) using culture and PCR as reference diagnostic tests, respectively. It may be concluded that, the LFA strip test can be used as a rapid penside diagnostic test for screening of brucellosis. To the best of our knowledge, this is the first report on development of GNP based LFA strip test for detection of Brucella spp. from bovine aborted fetal content samples.

ANTIBODY PREVALENCE TO AFRICAN SWINE FEVER VIRUS, MYCOBACTERIUM BOVIS, FOOT-AND-MOUTH DISEASE VIRUS, RIFT VALLEY FEVER VIRUS, INFLUENZA A VIRUS, AND BRUCELLA AND LEPTOSPIRA SPP. IN FREE-RANGING WARTHOG (PHACOCHOERUS AFRICANUS) POPULATIONS IN SOUTH AFRICA.

The warthog (Phacochoerus africanus) can be used as a model for investigating disease transmission at the human, wildlife, and livestock interface. An omnivore and scavenger, a warthog moves freely between natural ecotypes, farmland, and human communities and is susceptible to diseases of zoonotic, agricultural, and conservation concern. A retrospective study using 100 individual serum samples collected from May 1999 to August 2016 was performed to determine antibody prevalence to seven pathogens in warthogs from five locations in northeastern South Africa. Higher prevalence of antibodies to African swine fever virus and Mycobacterium bovis were detected in warthogs from the Greater Kruger National Park ecosystem in comparison to lower prevalence of antibodies to M. bovis and no antibodies to African swine fever virus in warthogs from uMhkuze Game Reserve. Low prevalence of antibodies to foot-and-mouth disease virus, Rift Valley fever virus, and influenza A virus was detected in all locations, and no antibodies against Brucella and Leptospira spp. were detected. No statistically significant difference in antibody prevalence was found between sexes for any disease. At the univariate analysis, M. bovis seropositivity was significantly different among age categories, with 49% (35/71) of adults found positive versus 29% (4/14) of juveniles and 9% (1/11) of sub-adults (Fisher's exact test, P=0.020), and between the sampling locations (Fisher's exact test, P=0.001). The multivariate model results indicated that juvenile warthogs had lower odds of testing positive to M. bovis antibodies than adults (juveniles' odds ratio [OR]=0.17, 95% confidence interval [CI]: 0.02-1.0), although this result was not statistically significant at the 5% level (P=0.052). For warthogs sampled at Satara Buffalo Camp, the odds (OR=0.22, 95% CI: 0.035-0.96) of being M. bovis antibody positive were significantly lower (P=0.043) than for warthogs sampled at Skukuza. Of particular interest in this study was the detection of warthogs seropositive for influenza A virus.

Uncovering the Hidden Credentials of Brucella Virulence.

Bacteria in the genus Brucella are important human and veterinary pathogens. The abortion and infertility they cause in food animals produce economic hardships in areas where the disease has not been controlled, and human brucellosis is one of the world's most common zoonoses. Brucella strains have also been isolated from wildlife, but we know much less about the pathobiology and epidemiology of these infections than we do about brucellosis in domestic animals. The brucellae maintain predominantly an intracellular lifestyle in their mammalian hosts, and their ability to subvert the host immune response and survive and replicate in macrophages and placental trophoblasts underlies their success as pathogens. We are just beginning to understand how these bacteria evolved from a progenitor alphaproteobacterium with an environmental niche and diverged to become highly host-adapted and host-specific pathogens. Two important virulence determinants played critical roles in this evolution: (i) a type IV secretion system that secretes effector molecules into the host cell cytoplasm that direct the intracellular trafficking of the brucellae and modulate host immune responses and (ii) a lipopolysaccharide moiety which poorly stimulates host inflammatory responses. This review highlights what we presently know about how these and other virulence determinants contribute to Brucella pathogenesis. Gaining a better understanding of how the brucellae produce disease will provide us with information that can be used to design better strategies for preventing brucellosis in animals and for preventing and treating this disease in humans.

High incidence of human brucellosis in a rural Pastoralist community in Kenya, 2015.

BACKGROUND: Brucellosis occurs globally with highly variable incidence in humans from very low in North America and Western Europe to high in the Middle East and Asia. There are few data in Sub-Saharan Africa. This study estimated the incidence of human brucellosis in a pastoralist community in Kenya. METHODS: Between February 2015 and January 2016, we enrolled persons living in randomly selected households in Kajiado County. Free health care was offered at three facilities in the study area. Those who met the study clinical case definition completed a standardized questionnaire on demographics, clinical history and presentation. A blood sample was collected and tested by Rose Bengal test (RBT), then later tested at the Kenya Medical Research Institute laboratory for Brucella IgG and IgM by ELISA. Those who tested positive by both RBT and ELISA (IgG or IgM antibodies) were classified as confirmed while those that only tested positive for IgG or IgM antibodies were classified as probable. Further, sera were tested by polymerase chain reaction using a TaqMan Array Card (TAC) for a panel of pathogens causing AFI including Brucella spp. Annual incidence of brucellosis was calculated as the number of confirmed cases in one year/total number in the study population. RESULTS: We enrolled a cohort of 4746 persons in 804 households. Over half (52.3%) were males and the median age was 18 years (Interquartile range (IQR) 9 months- 32 years). A total of 236 patients were enrolled at three health facilities; 64% were females and the median age was 40.5 years (IQR 28-53 years). Thirty-nine (16.5%) were positive for Brucella antibodies by IgG ELISA, 5/236 (2.1%) by IgM ELISA and 4/236 (1.7%) by RBT. Ten percent (22/217) were positive by TAC. We confirmed four (1.7%) brucellosis cases giving an annual incidence of 84/100,000 persons/year (95% CI 82, 87). The incidence did not significantly vary by gender, age and location of residence. CONCLUSION: We report a high incidence of brucellosis in humans among members of this pastoralist community. Brucellosis was the most common cause of febrile illness in this community.

Brucellosis screening and follow-up of seropositive asymptomatic subjects among household members of shepherds in China.

To measure the seroprevalence of high-exposure populations in brucellosis endemic areas and report the outcome and duration of seropositive asymptomatic subjects, we screened 595 family members of shepherds in Jilin Province, China and then followed up 15 seropositive asymptomatic subjects for 18 months. We found that the seropositive rate of 15.5%. Nearly half of seropositive asymptomatic subjects (7/15) developed into brucellosis in the short term; others were still seropositive asymptomatic or had decreased SAT titer in a longer time.

Study on immunogenicity and antigenicity of a novel brucella multiepitope recombined protein.

Currently, brucellosis is a reemerged zoonotic infectious disease with an increased incidence in recent years. A simple, rapid and sensitive method for diagnosing brucellosis can help to reduce medical burden and economic loss. Previously, a multiple epitope recombinant protein was constructed based on linear B-cell epitope prediction tools. In this study, the recombinant protein was used as an antigen to study the immune response produced by immunized mice, and goat serum was used to verify its diagnostic accuracy. The production of antibodies was successfully induced in the vaccinated mice. Flow cytometric analysis revealed that the percentage of CD4+, CD8+ and the CD4+/CD8+ ratios were increased by T cell subsets in mouse splenocytes, indicating that the recombinant protein induced a strong immune response had strong immunoreactivity. Using indirect ELISA, the recombinant protein correctly diagnosed positive and negative brucellosis samples. Compared with the whole bacterial antigen, the recombinant protein had a weaker sensitivity but a stronger specificity. Animal experiments showed that the recombinant protein had good antigenicity, and indirect ELISA indicates that it can be used as an antigen to diagnose brucellosis. Therefore, the recombinant protein is a potential candidate antigen for brucellosis vaccine development and serological diagnosis.