First Isolation and Multilocus Sequence Typing of Brucella canis from a Subclinically Infected Pet Dog in China

Canine brucellosis, a worldwide zoonotic disease, is mainly caused by Brucella canis. In the present study, we isolated a Brucella strain (CD3) from a subclinically infected pet dog in Sichuan Province, Southwestern China. Classical biotyping methods and molecular biological tests (BCSP31 and BcSS PCR) proved that the strain belonged to B. canis. Furthermore, B. canis CD3 and another two B. canis strains (WJ5 and YA4), which were all isolated from pet dogs in Sichuan, were genotyped using multilocus sequence typing (MLST). Our results showed that the three B. canis strains were identified as the same sequence type (ST21). The present study is the first to report B. canis strain from a subclinically infected pet dog in China, indicating a potential threat to public health posed by subclinical infections in pet dogs. We suggest that screening for B. canis should be incorporated into routine medical examination of pet dogs and other companion animals in areas with a history of animal or human brucellosis.

Occupational Exposure Assessment and Seroprevalence of Brucella Specific Antibodies Among Veterinarians in the Northern Palestine

Background: Brucellosis is one of the most popular zoonosis in the world caused by bacteria belonging to the genus Brucella. The disease is considered an occupational risk to persons dealing with animals and animal products. Brucellosis is endemic in livestock in Palestine. Yet, few studies investigated human brucellosis in Palestine. We aimed to estimate Brucella seropositivity among veterinary healthcare professionals in Northern Palestine, and to assess the associated risk factors.Methods: A cross-sectional study was conducted in four governorates in the Northern West Bank (Jenin, Nablus, Qalqylia, and Tulkarm). A sample of 100 veterinarians was collected. Participants were interviewed using a structured questionnaire to assess risk factors. Blood samples were collected to be screened for the presence of anti-Brucella IgG using the Enzyme-Linked Immunosorbent Assay (ELISA).Results: The seroprevalence of Brucellosis by ELISA was 76%. Risk factors included working in the public sector, dealing with animals' vaccination, longer period of exposure, and advancing in age.Conclusions: Brucellosis is a high-risk occupational disease among veterinarians. Its prevalence rate among veterinary healthcare workers in the Northern West Bank, Palestine was very high compared to neighboring countries and internationally.

Knowledge, attitude and practices of frontline health workers in relation to detection of brucellosis in rural settings of Tanzania: a cross-sectional study

Background Brucellosis an important zoonotic disease worldwide, which frequently presents as an undifferentiated febrile illness with otherwise varied and non-specific clinical manifestations. Despite its importance, there are few reports on its awareness among frontline health workers. This study aimed at assessing the baseline knowledge, attitude and practice (KAP) related to detection and management of brucellosis among frontline health workers (FHWs) namely; healthcare workers (HWs) and community health workers (CHWs). Methods A cross-sectional study was conducted from December 2019 to January 2020 in Kilosa and Chalinze districts of Tanzania. Data on demographic characteristics, knowledge, attitude and practices regarding brucellosis were collected from the study participants using a structured questionnaire. Interviews were conducted with 32 HWs and 32 CHWs who were systematically selected in study districts. Chi square/fisher Exact was used to assess the association between sociodemographic variables and those related to knowledge, attitude and practices. Results Overall, a total of 30 (93.8%) HWs and nine (28.1%) CHWs from the study districts heard about brucellosis, with (34.4%) of HWs having knowledge about the causative organism. Overall, knowledge showed almost half (46.9%) HWs and (28.1%) CHWs were aware of the symptoms, clinical signs, diagnosis and control regarding brucellosis. Knowledge difference was statistically significant with HWs’ age (p = 0.016). Almost half (46.9%) HWs and less than quarter (12.5%) CHWs had good practices regarding brucellosis control. Almost three quarters (71.9%) of HWs and (21.9%) CHWs had positive attitude regarding brucellosis control; overall attitude was statistically significant with CHWs age (p = 0.028) and education level (p = 0.024). Lack of awareness and unavailability of diagnostic tools were the main challenges faced by FHWs in the two districts. Conclusion The majority of participants were not aware of human brucellosis. Moreover, their overall knowledge was inadequate and the common practices were diagnostic tools, and adequate knowledge to manage brucellosis cases. These findings highlight the need to strengthen frontline health workers knowledge, practices and diagnostic capacities related to brucellosis.

Brucellosis in Herat Province, Afghanistan: Epidemiological Status and Risk Factors

Background: Human brucellosis, also known as Malta fever, is an acute systemic zoonotic disease in several parts of the world. The most pathogenic Brucella specie is Brucella melitensis that occurs in the human population of all age groups and of both sexes. Objectives: The present study aimed to investigate human Brucella infection in Afghanistan. Methods: The participants in this cross-sectional study were 44 patients diagnosed with Brucella infection during eight months and confirmed using Wright test by physicians of Iran Clinic Hospital. For data analysis, a statistical model was used through SPSS. Results: The most affected patients were female housewives (40.9%) and students (18.18%). The most frequent clinical manifestations were recurrent attacks of fever (95.34%), weight loss (81.39%), loss of appetite (79.06%), musculoskeletal pain (69.76%), boredom (67.44%), and lethargy (60.46%). A total of 41 (93.18%) patients mentioned the consumption of unpasteurized milk as the source of infection. Conclusions: The study results revealed that the main route of Brucella transmission in Afghanistan is the consumption of contaminated dairy products. The highest prevalence of brucellosis was observed among the young and middle-aged populations and housewives.

MicroRNA-155 Expression With Brucella Infection in vitro and in vivo and Decreased Serum Levels of MicroRNA-155 in Patients With Brucellosis

Infection by Brucella is characterized by the inhibition of host immune responses. MicroRNA-155 (miR-155) has been implicated in the immune response to many diseases. In this study, miR-155 expression during Brucella 16M infection of macrophages and mice were analyzed. Expression of miR-155 was significantly induced in macrophages at 24 hours post infection. Analysis of infected mice showed that miR-155 was inhibited at 7 and 14 days, but induced at 28 days. Very interestingly, the induction or inhibition trend was reversed at 7 and 14 days in 16M△virB-infected mice. This suggested that decreased expression of miR-155 at an early stage of infection was dependent on intracellular replication. In humans with brucellosis, serum levels of miR-155 were significantly decreased compared to those without brucellosis and healthy volunteers. Significant correlations were observed between serum level of miR-155 and serum anti-Brucella antibody titers and symptom of sweat. The decrease in miR-155 with Brucella infection contrasts with the increase in miR-155 observed in Mycobacterium tuberculosis infection. This contrasting effect suggests that Brucella interferes with miR-155-regulated immune responses through a unique mechanism. Taken together, data from this study indicate that Brucella infection affects miR-155 expression, and that human brucellosis patients show decreased serum levels of miR-155.

Awareness about Human Brucellosis in Healthcare Professional in India

Introduction: Human brucellosis is a widespread zoonosis of serious public health consequences. The infection is transmitted from animal to human through direct contact with infected animals or consumption of infected, unpasteurized animal milk. Being a disease with wide and non-specific clinical manifestations, a case of brucellosis can be detected only if the treating health care professional is aware of the disease and keeps a high rate of suspicion when dealing with suspected cases. We surveyed to find the extent of awareness about Human brucellosis in healthcare professionals in Gujarat, India. Methodology: A cross-sectional study was conducted among healthcare professionals from December 2020 to May 2021 using a self-administrated questionnaire. This study included healthcare professionals including AYUSH practicing in Gujarat. A validated questionnaire consisting of 23 items was administered to assess the knowledge of professionals toward suspecting, diagnosing, preventing, and managing a case of brucellosis. Both physical and Google forms were used to collect data. Data were analyzed using Statistical Package for the Social Sciences (SPSS) program, IBM version 22. Results: Sixty-nine healthcare professionals responded to the questionnaire. The findings of the study showed overall good awareness about brucellosis with a higher rate of knowledge in allopathic healthcare professionals and nurses in comparison to AYUSH doctors. Nearly 50% of respondents did not know the treatment as well as the preventive potential of human brucellosis. Conclusion: The current study suggests a need for creating more awareness in the healthcare professional, particularly AYUSH practitioners about brucellosis for better management and prevention.

Microbiological Laboratory Diagnosis of Human Brucellosis: An Overview

Brucella spp. are Gram-negative, non-motile, non-spore-forming, slow-growing, facultative intracellular bacteria causing brucellosis. Brucellosis is an endemic of specific geographic areas and, although underreported, represents the most common zoonotic infection, with an annual global incidence of 500,000 cases among humans. Humans represent an occasional host where the infection is mainly caused by B. melitensis, which is the most virulent; B. abortus; B. suis; and B. canis. A microbiological analysis is crucial to identifying human cases because clinical symptoms of human brucellosis are variable and aspecific. The laboratory diagnosis is based on three different microbiological approaches: (i) direct diagnosis by culture, (ii) indirect diagnosis by serological tests, and (iii) direct rapid diagnosis by molecular PCR-based methods. Despite the established experience with serological tests and highly sensitive nucleic acid amplification tests (NAATs), a culture is still considered the “gold standard” in the laboratory diagnosis of brucellosis due to its clinical and epidemiological relevance. Moreover, the automated BC systems now available have increased the sensitivity of BCs and shortened the time to detection of Brucella species. The main limitations of serological tests are the lack of common interpretative criteria, the suboptimal specificity due to interspecies cross-reactivity, and the low sensitivity during the early stage of disease. Despite that, serological tests remain the main diagnostic tool, especially in endemic areas because they are inexpensive, user friendly, and have high negative predictive value. Promising serological tests based on new synthetic antigens have been recently developed together with novel point-of-care tests without the need for dedicated equipment and expertise. NAATs are rapid tests that can help diagnose brucellosis in a few hours with high sensitivity and specificity. Nevertheless, the interpretation of NAAT-positive results requires attention because it may not necessarily indicate an active infection but rather a low bacterial inoculum, DNA from dead bacteria, or a patient that has recovered. Refined NAATs should be developed, and their performances should be compared with those of commercial and home-made molecular tests before being commercialized for the diagnosis of brucellosis. Here, we review and report the most common and updated microbiological diagnostic methods currently available for the laboratory diagnosis of brucellosis.

Lateral Flow Immunoassay for Visible Detection of Human Brucellosis Based on Blue Silica Nanoparticles

Brucellosis is a highly contagious zoonosis chronic infectious disease with a strong latent capability to endanger human health and economic development via direct or indirect ways. However, the existing methods for brucellosis diagnosis are time-consuming and expensive as they require a tedious experimental procedure and a sophisticated experimental device and performance. To overcome these defects, it is truly necessary to establish a real-time, on-site, and rapid detection method for human brucellosis. Here, a lateral flow immunoassay (LFIA) with a rapid, sensitive, and alternative diagnostic procedure for human brucellosis with a high degree of accuracy was developed based on blue silica nanoparticles (SiNPs), Staphylococcal protein A (SPA), and surface Lipopolysaccharide of Brucella spp. (LPS), which can be applied for rapid and feasible detection of human brucellosis. To our knowledge, this is the first report that uses blue SiNPs as a signal probe of LFIA for the rapid diagnosis of human brucellosis. The precursor of blue SiNPs@SPA such as colorless SiNPs and blue SiNPs was synthesized at first and then coupled with SPA onto the surface of blue SiNPs by covalent bond to prepare blue SiNPs@SPA as a capture signal to catch the antibody in the brucellosis-positive serum. When SPA was combined with the antibodies in the brucellosis-positive serum, it was captured by LPS on the test line, forming an antigen–antibody sandwich structure, resulting in the T line turning blue. Finally, the results showed that it is acceptable to use blue SiNPs as visible labels of LFIA, and standard brucellosis serum (containing Brucella spp. antibody at 1,000 IU/ml) could be detected at a dilution of 10−5 and the detection limit of this method was 0.01 IU/ml. Moreover, it also demonstrated good specificity and accuracy for the detection of real human serum samples. Above all, the blue SiNPs-based LFIA that we developed provides a rapid, highly accurate, and inexpensive on-site diagnosis of human brucellosis, and shows great promise in clinical diagnostics for other diseases.

Spatiotemporal distribution of human brucellosis in Inner Mongolia, China, in 2010–2015, and influencing factors

Human brucellosis is caused by Brucella species and remains a major burden in both human and domesticated animal populations, especially in Inner Mongolia, China. The aims of this study were to analyze the spatiotemporal trends in human brucellosis in Inner Mongolia during 2010 to 2015, to explore the factors affecting the incidence of brucellosis. The results showed that the annual incidence was 29.68–77.67 per 100,000, and peaked from March to June. The majority of human brucellosis was male farmers and herdsmen, aged 40–59 years. The high-risk areas were mainly Xilin Gol League and Hulunbeier City. The incidence of human brucellosis in Inner Mongolia decreased during 2010 to 2015, although the middle and eastern regions were still high-risk areas. The regions with larger number of sheep and cattle, lower GDP per capita, less number of hospital beds, higher wind speed, lower mean temperature more likely to become high-risk areas of human brucellosis.

1026. Following the Hoof Prints: Detecting Coxiella and Brucella infections with A Plasma-based Microbial Cell-Free DNA Next-generation Sequencing Test

Background Coxiella burnetii and Brucella spp. are zoonotic bacterial pathogens responsible for Q fever and Brucellosis, respectively. Both pathogens have a global distribution and Brucellosis is the most common zoonosis in the world. However, the CDC reports only 80-120 cases of human brucellosis and ~150 cases of acute Q fever annually. The diagnosis of these infections can be limited by: (1) their difficulty to culture; (2) the insensitivity and nonspecificity of serology; (3) the clinical overlap with other infections; and (4) the unreliability of epidemiological exposure history for these zoonoses. Unbiased microbial cell free DNA (mcfDNA) next-generation sequencing (NGS) offers a potential solution to overcome these limitations. Methods The Karius TestTM (KT) developed and validated in Karius’s CLIA certified/CAP accredited lab in Redwood City, CA detects mcfDNA in plasma. After mcfDNA is extracted and NGS performed, human reads are removed, and remaining sequences are aligned to a curated database of > 1500 organisms. McfDNA from organisms present above a statistical threshold are reported and quantified in molecules/µL (MPM). KT detections of Coxiella and Brucella were reviewed from August 2017 - present; clinical information was obtained with test requisition or consultation upon result reporting. Results KT detected 8 cases of Coxiella burnetii (1735 MPM +/- 3000) and 5 cases of Brucella melitensis (avg 296 MPM +/- 223) (Table 1), representing approximately 1-2% of all detections in the US during this period. All of the Coxiella detections were in adults (100% male) with 5 cases of fever of unknown origin, 2 cases of culture-negative endocarditis and one case of endovascular graft infection. Brucella detections occurred in 3 adults and 2 children (60% male), 3 with exposure to unpasteurized dairy and included 3 cases of spine infection (2 vertebral osteomyelitis, 1 epidural abscess). Conclusion Open-ended, plasma-based mcfDNA NGS provides a rapid, non-invasive test to diagnose diverse clinical manifestations of zoonotic infections such as Q fever and Brucellosis against competing broad differential diagnoses. Furthermore, these cases highlight the potential of the KT to diagnose infections caused by fastidious/unculturable pathogens with cryptic clinical presentations. Disclosures Nicholas R. Degner, MD, MPH, MS, Karius Inc. (Employee, Shareholder) Ricardo Castillo-Galvan, MD MPH, Karius Inc. (Consultant) Jose Alexander, MD, D(ABMM), FCCM, CIC, SM, MB(ASCP), BCMAS, Karius (Employee) Aparna Arun, MD, Karius (Employee) Ann Macintyre, DO, Karius, Inc. (Employee) Bradley Perkins, MD, Karius, Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee) Matthew Smollin, PharmD, Karius, Inc. (Employee)