Seroprevalence and molecular detection of brucellosis among Pakistani women with spontaneous abortion.

Background: Human brucellosis is a neglected disease transmitted to humans from animals such as cattle, goats, dogs, and swine. The causative agents are bacteria of the genus Brucella, intracellular pathogens usually confined to the reproductive organs of their animal hosts causing sterility and abortions. The objective of the study was to determine the seroprevalence of brucellosis among women with spontaneous abortions (SAW) and compare this seroprevalence with that of healthy pregnant women (HPW). Methods: The case-control study was designed to determine the seroprevalence and molecular detection of brucellosis in women who suffered from spontaneous abortion and healthy pregnant women of the Haripur District of Pakistan. A total of 770 blood samples (n = 385 for each group) were collected from 9 public and 11 private hospitals in Haripur District from December 2021-March 2023. Data on demographic features, epidemiological variables, and risk factors were collected from each participant by structured questionnaires. Initial screening for brucellosis was performed by Rose Bengal Plate Test followed by qRT-PCR for molecular detection of the genus-specific BCSP-31 gene of Brucella. Results: The study showed that anti-Brucella antibodies were more found in SAW 23.63% (91/385) than in HPW 1.29% (5/385). Brucella specific DNA was amplified in 89.01% (81/91) seropositive samples of SAW. Demographic features and risk factors such as age, urbanicity, socioeconomic status, education, occupation, and animal contact were found significantly associated with brucellosis (p ≤ 0.05). Consumption of unpasteurized raw milk (OR = 18.28, 95%CI: 8.16-40.94) was found highly concomitant with seroprevalence. Conclusion: This study reports the first evidence of involvement of brucellosis in spontaneous abortions in women of Pakistan. The study can be used to develop strategies for risk management during pregnancy, to raise awareness for brucellosis, and develop control programs.

Neurobrucellosis: laboratory features, clinical characteristics, antibiotic treatment, and clinical outcomes of 21 patients.

BACKGROUND: Neurobrucellosis (NB) is a rare and serious complication of brucellosis. Its clinical manifestations vary, with no obvious specificity. At present, there is no clear clinical diagnosis or treatment for reference. In this study, we retrospectively analyzed the clinical data for 21 patients with NB to provide reference data for its further study. METHODS: We analyzed the epidemiological and clinical manifestations, laboratory tests, imaging examinations, cerebrospinal fluid, and treatment plans of 21 patients diagnosed with NB in the Department of Neurology, Xuanwu Hospital, Capital Medical University Beijing, China. RESULTS: The ages of the patients ranged from 15 to 60 years old (mean age 40.1 ± 13.33 years), the male: female ratio was 4.25:1. Thirteen patients had a history of animal (sheep, cattle) contact, three had no history of animal contact, and the contact status of four was unknown. Brucella can invade various systems of the body and show multi-system symptoms, the main general manifestations were fever (66.67%), fatigue (57.14%) and functional urination or defecation disturbance (42.86%). The main nervous system manifestations were limb weakness (52.38%) and hearing loss (47.62%).The main positive signs of the nervous system included positive pathological signs (71.43%), sensory abnormalities (52.38%), limb paralysis (42.86%). Nervous system lesions mainly included spinal cord damage (66.67%), cranial nerve involvement (61.90%), central demyelination (28.57%) and meningitis (28.57%). In patients with cranial nerve involvement, 69.23% of auditory nerve, 15.38% of optic nerve and 15.38% of oculomotor nerve were involved. The blood of eight patients was cultured for Brucella, and three (37.5%) cultures were positive and five (63.5%) negative. The cerebrospinal fluid (CSF) of eight patients was cultured for Brucella, and two (25.00%) cultures were positive and six (75.00%) negative. Nineteen of the patients underwent a serum agglutination test (SAT), 18 (94.74%) of whom were positive and one (5.26%) of whom were negative. A biochemical analysis of the CSF was performed in 21 patients, and the results were all abnormal. Nineteen patients underwent magnetic resonance imaging (MRI). Twenty-one patients were treated with doxycycline and/or rifampicin, combined with ceftriaxone, quinolone, aminoglycoside, or minocycline. After hospitalization, 15 patients improved (71.43%), two patients did not recover, and the status of four patients was unknown. CONCLUSIONS: The clinical manifestations, CSF parameters, and neurological imaging data for patients with NB show no significant specificity or correlations. When patients with unexplained neurological symptoms accompanied by fever, fatigue, and other systemic manifestations in a brucellosis epidemic area or with a history of contact with cattle, sheep, animals, or raw food are encountered in clinical practice, the possibility of NB should be considered. Treatment is based on the principles of an early, combined, and long course of treatment, and the general prognosis is good.

Motor polyradiculoneuropathy as an unusual presentation of neurobrucellosis: a case report and literature review.

Brucellosis, a zoonotic disease caused by Brucella species, poses a significant global health concern. Among its diverse clinical manifestations, neurobrucellosis remains an infrequent yet debilitating complication. Here, we present a rare case of neurobrucellosis with unusual presentations in a 45-year-old woman. The patient's clinical course included progressive lower extremity weakness, muscle wasting, and double vision, prompting a comprehensive diagnostic evaluation. Notable findings included polyneuropathy, elevated brucella agglutination titers in both cerebrospinal fluid and blood, abnormal EMG-NCV tests, and resolving symptoms with antibiotic therapy. The clinical presentation, diagnostic challenges, and differentiation from other neurological conditions are discussed. This case underscores the importance of considering neurobrucellosis in regions where brucellosis is prevalent and highlights this rare neurological complication's distinctive clinical and radiological features. Early recognition and appropriate treatment are crucial to mitigate the significant morbidity associated with neurobrucellosis.

Characterization of Brucella spp. and other abortigenic pathogens from aborted tissues of cattle and goats in Rwanda.

BACKGROUND: Abortions cause tremendous economic losses in food-producing animals and may lead to food insecurity. OBJECTIVES: This study aimed to characterize Brucella spp. and other abortigenic pathogens from aborted tissues of cattle. METHODS: For cattle, aborted tissues (n = 19) were cultured, and Brucella spp. were detected using the genus-specific 16S-23S ribosomal DNA interspacer region (ITS) assay and speciated using Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR assays. Brucella negative samples were screened using the eight abortigenic pathogens PCR panel. Samples from an abortion outbreak that occurred within a goat tribe were included in this investigation. Sera of females (n = 8) and males (n = 2) were analyzed using the Rose Bengal Test (RBT) and indirect enzyme-linked immunosorbent assay (i-ELISA), while vaginal swabs (n = 3) and aborted tissues (n = 1) were cultured and characterized. RESULTS: The ITS-PCR detected Brucella DNA in cultures from two aborted tissues of cattle (10.5%, [2/19]), which were identified as B. melitensis (n = 1), and B. abortus (n = 1) using AMOS and Bruce-ladder PCR assays. Campylobacter fetus (n = 7) and Leptospira spp. (n = 4) including co-infections (n = 2) of C. fetus and Leptospira spp. were identified from the Brucella negative samples of cattle. Goats (100.0%, 10/10) were brucellosis seropositive on RBT and i-ELISA. Mixed infections caused by B. melitensis and B. abortus were isolated from the vaginal swabs (n = 3) and aborted tissues (n = 1). DISCUSSION AND CONCLUSIONS: This is the first identification of abortion-associated pathogens in aborted cattle indicating the enormous financial losses and a threat to public health. It is therefore essential to include these identified pathogens in the surveillance scheme of veterinary and human services.

Proteomics-based screening and antibiotic resistance assessment of clinical and sub-clinical Brucella species: An evolution of brucellosis infection control.

Brucellae are intracellular sneaky bacteria and they can elude the host's defensive mechanisms, resulting in therapeutic failure. Therefore, the goal of this investigation was to rapid identification of Brucella species collected from animals and humans in Saudi Arabia, as well as to evaluate their resistance to antibiotics. On selective media, 364 animal samples as well as 70 human blood samples were cultured. Serological and biochemical approaches were initially used to identify a total of 25 probable cultured isolates. The proteomics of Brucella species were identified using the MALDI Biotyper (MBT) system, which was subsequently verified using real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Both Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) were tested for antimicrobial susceptibility using Kirby Bauer method and the E-test. In total, 25 samples were positive for Brucella and included 11 B. melitensis and 14 B. abortus isolates. Twenty-two out of 25 (88%) and 24/25 (96%) of Brucella strains were recognized through the Vitek 2 Compact system. While MBT was magnificently identified 100% of the strains at the species level with a score value more than or equal to 2.00. Trimethoprim-sulfamethoxazole, rifampin, ampicillin-sulbactam, and ampicillin resistance in B. melitensis was 36.36%, 31.82%, 27.27%, and 22.70%, respectively. Rifampin, trimethoprim-sulfamethoxazole, ampicillin, and ampicillin-sulbactam resistance was found in 35.71%, 32.14%, 32.14%, and 28.57% of B. abortus isolates, correspondingly. MBT confirmed by microfluidic electrophoresis is a successful approach for identifying Brucella species at the species level. The resistance of B. melitensis and B. abortus to various antibiotics should be investigated in future studies.

An uncommon case of brucellosis with ocular presentation / Um caso incomum de brucelose com apresentação ocular

ABSTRACT We report an unusual case of brucellosis presented with headache, diminished vision, papillitis and multiple peripapillary hemorrhages accompanied by subretinal fluid extending up to macula. Diagnosis of brucellosis was made based on positive polymerase chain reaction of cerebrospinal fluid sample for Brucella species DNA, accompanied by a raised titer of anti-brucella antibodies. Patient showed remarkable improvement on triple drug therapy in form of doxycycline, rifampicin and ceftriaxone.

RESUMO Relatamos um caso incomum de brucelose apresentada com cefaleia, visão diminuída, papilite e múltiplas hemorragias peripapilares acompanhadas por fluido sub-retinal, estendendo-se até a mácula. O diagnóstico de brucelose foi feito com base na reação em cadeia da polimerase positiva de amostra de líquido cefalorraquidiano para DNA de espécies de Brucella, acompanhada por um título elevado de anticorpos antibrucela. O paciente apresentou melhora notável com a terapia tripla com drogas na forma de doxiciclina, rifampicina e ceftriaxona.

Comparison of Different Laboratory Methods for Clinical Detection of Brucella Infection.

The rapidity, accuracy, and detection abilities of different laboratory methods (tube agglutination test (SAT), indirect ELISA, fluorescence polarization test (FPA), and blood culture methods) to detect Brucella in the laboratory. The study included 95 patients with documented and 42 patients with suspected brucellosis and 56 healthy control subjects. For the tests, the positive rates of Brucella infection detection in the confirmed group were significantly higher than in group with suspected infection (p<0.01) and in healthy controls (p<0.01). There was no significant difference between indirect ELISA and FPA in detecting antibodies to Brucella in acute (χ2=0.335), subacute (χ2=0.660), and chronic cases (χ2=5.332). Among the detection methods, indirect ELISA showed the highest sensitivity (98.9%), specificity (100%), and Youden index (0.989). The sensitivity and specificity of FPA were 96.8 and 96.4%, respectively. In order to easily and rapidly diagnose brucellosis in clinical practice, a combination of detection methods is recommended, in which Brucella antibodies are screened by FPA and then confirmed by indirect ELISA.

Differentiating brucella spondylitis from tuberculous spondylitis by the conventional MRI and MR T2 mapping: a prospective study.

BACKGROUND: Brucella spondylitis (BS) and tuberculous spondylitis (TS), caused initially by bacteremia, are the two leading types of granulomatous spinal infections. BS is easy to miss or may be misdiagnosed as TS. Our purpose aims to differentiate BS from TS in conventional MR imaging and MR T2 mapping. METHODS: We performed on 26 BS and 27 TS patients conventional MR imaging and MR T2 mapping. We analyzed the features in conventional MR imaging and measured T2 values of the lesion vertebrae (LV) and unaffected adjacent vertebrae (UAV) in BS and TS patients, respectively. RESULTS: There were no significant differences in sex, age, national between BS and TS. There was significantly lower severity of vertebral destruction, vertebral posterior convex deformity, dead bone, and abscess scope in BS when compared to TS (p < 0.001, p = 0.048, p < 0.001, p < 0.001, respectively). The vertebral hyperplasia was significantly higher in BS when compared to TS (p < 0.001). The T2 value of the LV with BS was markedly higher than that in the UAV with BS and that in the LV and UAV with TS (p < 0.001, p < 0.037, p < 0.001, respectively). The T2 value of the LV with TS was significantly higher than that of the UAV in TS and BS (p < 0.001, p < 0.001, respectively). There were no significant differences in the T2 value of the UAV between BS and TS (p = 0.568). CONCLUSIONS: The qualitative and quantitative evaluation may differentiate BS from TS. The conventional MR imaging helps to distinguish BS from TS by several distinctive features. MR T2 mapping has the additional potential to provide quantitative information between BS and TS.

Analysis of the Curative Effect of Posterior Approach on Lumbar Brucellar Spondylitis with Abscess through Magnetic Resonance Imaging under Improved Watershed Algorithm.

To explore the performance of improved watershed algorithm in processing magnetic resonance imaging (MRI) images and the effect of the processed images on the treatment of lumbar brucellar spondylitis (BS) with abscess by the posterior approach, the watershed algorithm was improved by adding constraints such as noise reduction and regional area attribute. 50 patients with abscessed lumbar disc herniation admitted to the hospital from January 2018 to January 2019 were selected, and all of them were examined by MRI. They were rolled into two groups in random. The treatment group (n = 25) accepted surgery with the aid of MRI images processed by the improved watershed algorithm, and the control group (Ctrl group) (n = 25) accepted surgery with the aid of unprocessed MRI images. The improved watershed algorithm can accurately segment the spine, and the segmentation results were relatively excellent. In contrast with the unprocessed MRI image, that processed by the improved watershed algorithm had a positive effect on the operation. In contrast with the Ctrl group, the visual analogue scale pain score (VAS), oxygen desaturation index (ODI), erythrocyte sedimentation rate (ESR), and high sensitivity C-reactive protein (CRP) were obviously lower (p < 0.05). The improved watershed algorithm proposed performs better in MRI image processing and can effectively enhance the resolution of MRI images. At the same time, the posterior approach has a good effect in the treatment of lumbar BS with abscess and is worthy of clinical promotion.

Rift Valley fever and Brucella spp. in ruminants, Somalia.

BACKGROUND: Fourteen-years after the last Rift Valley fever (RVF) virus (RVFV) outbreak, Somalia still suffers from preventable transboundary diseases. The tradition of unheated milk consumption and handling of aborted materials poses a public health risk for zoonotic diseases. Limited data are available on RVF and Brucella spp. in Somali people and their animals. Hence, this study has evaluated the occurrence of RVFV and Brucella spp. antibodies in cattle, goats and sheep sera from Afgoye and Jowhar districts of Somalia. METHODS: Serum samples from 609 ruminants (201 cattle, 203 goats and 205 sheep), were serologically screened for RVF by a commercial cELISA, and Brucella species by modified Rose Bengal Plate Test (mRBPT) and a commercial iELISA. RESULTS: Two out of 609 (0.3 %; 95 %CI: 0.04-1.2 %) ruminants were RVF seropositive, both were female cattle from both districts. Anti-Brucella spp. antibodies were detected in 64/609 (10.5 %; 95 %CI: 8.2-13.2 %) ruminants by mRBPT, which were 39/201 (19.4 %) cattle, 16/203 (7.9 %) goats and 9/205 (4.4 %) sheep. Cattle were 5.2 and 2.8 times more likely to be Brucella-seropositive than sheep (p = 0.000003) and goats (p = 0.001), respectively. When mRBPT-positive samples were tested by iELISA, 29/64 (45.3 %; 95 %CI: 32.8-58.3 %) ruminant sera were positive for Brucella spp. Only 23/39 (58.9 %) cattle sera and 6/16 (37.5 %) goat sera were positive to Brucella spp. by iELISA. CONCLUSIONS: The present study showed the serological evidence of RVF and brucellosis in ruminants from Afgoye and Jowhar districts of Somalia. Considering the negligence of the zoonotic diseases at the human-animal interface in Somali communities, a One Health approach is needed to protect public health.

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.

Herd-level contamination of Neospora caninum, Toxoplasma gondii and Brucella in milk of Iranian dairy farms.

The bulk milk examination is a reliable screening tool for monitoring the quality of milk in the farms. The infection to Neospora caninum, Toxoplasma gondii and Brucella sp. Was evaluated in bulk milk samples of dairy farms in Hamedan province, West part of Iran. All the dairy farms (n = 149) were examined for N. caninum, T. gondii and Brucella infections using milk ring test (MRT), microbiology, serology (Enzyme-linked Immunosorbent Assay), and molecular techniques. Based on molecular methods, Brucella-infection was negative in all farms; while, 55 %, 5.4 % and 2.7 % of samples were positive for N. caninum, T. gondii and mix infection, respectively. The highest Neospora-infection was detected in the farms with history of abortion in fall and winter. There was significant association between Neospora-infection and the presence of dogs and rodents in the farms, herd size, and age of the animals. Also, a significant association was seen between Toxoplasma-infection and the presence of cats and rodents in the farms, as well as age of the animals. Average total bacterial count (TBC) was calculated 1.14 × 106±1.1 × 106. The highest TBC was in the farms from Central locations of studied area (5.7 × 106±2.24 × 106), farms with more than 120 animals (7.9 × 106±2.8 × 106), and farms with ≥50-months age (1.74 × 106±6.3 × 105) in spring and summer (6.9 × 106±3.7 × 106). The number of somatic cells was estimated between 1 × 104 and 2 × 106 (Average = 4.2 × 105±3.39 × 105). The current study was a comprehensive evaluation of Neospora, Toxoplasma and Brucella infections in milk samples of Iranian dairy farms for the first time. Neospora-infection is responsible for economic losses in the region. Health education and milk pasteurization are so helpful for inhibiting the milk borne diseases. To reduce the risk factors, predict and design the appropriate schemes like redundant of heterogeneous animals are recommended.

Clinical Presentations of Brucellosis Over a Four-Year Period at Sultan Qaboos University Hospital and Armed Forces Hospital, Muscat, Oman.

OBJECTIVES: Brucellosis is a highly contagious zoonotic disease which can have serious health implications for affected humans and livestock. This study aimed to evaluate the clinical presentation, geographical distribution and risk factors of brucellosis cases admitted over a four-year period to two hospitals in Muscat, Oman. METHODS: This observational study was conducted from January 2015 to December 2018 at the Sultan Qaboos University Hospital and Armed Forces Hospital in Muscat. All patients with probable or definitive diagnoses of brucellosis according to the diagnostic criteria of the World Health Organization were included. Relevant data were gathered from the patients' medical records, including results from standard agglutination tests, Brucella enzyme-linked immunosorbent assays, bacterial blood or tissue/aspirate cultures and Brucella polymerase chain reaction tests. RESULTS: A total of 64 patients were diagnosed with brucellosis over the study period. The median age was 31.5 years and 73.4% were male. The majority (95.2%) presented with fever, followed by weight loss (51%), transaminitis (48.4%), peripheral arthritis/arthralgia (15.9%) and back pain (spondylodiscitis/sacroiliitis; 23.4%). Overall, 75.5% reported having consumed raw dairy products, while only 25.9% gave a positive history of animal contact. CONCLUSION: Patients with brucellosis presented with a wide range of clinical features, the most predominant of which was fever. The majority of patients were residents of or had recently visited Salalah and had consumed raw dairy products. These findings highlight the need for healthcare practitioners to maintain a high index of suspicion for this diagnosis. Moreover, further regulatory measures are necessary to oversee the sale of raw/unpasteurised dairy products.

Identification of Bacillus anthracis, Brucella spp., and Coxiella burnetii DNA signatures from bushmeat.

Meat from wildlife species (bushmeat) represents a major source of dietary protein in low- and middle-income countries where humans and wildlife live in close proximity. Despite the occurrence of zoonotic pathogens in wildlife, their prevalence in bushmeat remains unknown. To assess the risk of exposure to major pathogens in bushmeat, a total of 3784 samples, both fresh and processed, were collected from three major regions in Tanzania during both rainy and dry seasons, and were screened by real-time PCR for the presence of DNA signatures of Bacillus anthracis (B. anthracis), Brucella spp. (Brucella) and Coxiella burnetii (Coxiella). The analysis identified DNA signatures of B. anthracis (0.48%), Brucella (0.9%), and Coxiella (0.66%) in a total of 77 samples. Highest prevalence rates of B. anthracis, Brucella, and Coxiella were observed in wildebeest (56%), dik-dik (50%), and impala (24%), respectively. Fresh samples, those collected during the rainy season, and samples from Selous or Serengeti had a greater relative risk of being positive. Microbiome characterization identified Firmicutes and Proteobacteria as the most abundant phyla. The results highlight and define potential risks of exposure to endemic wildlife diseases from bushmeat and the need for future investigations to address the public health and emerging infectious disease risks associated with bushmeat harvesting, trade, and consumption.

Surgical management for lumbar brucella spondylitis: Posterior versus anterior approaches.

ABSTRACT: There has been no ideal surgical approach for lumbar brucella spondylitis (LBS). This study aims to compare clinical efficacy and safety of posterior versus anterior approaches for the treatment of LBS.From April 2005 to January 2015, a total of 27 adult patients with lumbar brucella spondylitis were recruited in this study. The patients were divided into 2 groups according to surgical approaches. Thirteen cases in group A underwent 1-stage anterior debridement, fusion, and fixation, and 14 cases in group B underwent posterior debridement, bone graft, and fixation. The clinical and surgical outcomes were compared in terms of operative time, intraoperative blood loss, hospitalizations, bony fusion time, complications, visual analog scale score, recovery of neurological function, deformity correction.Lumbar brucella spondylitis was cured, and the grafted bones were fused within 11 months in all cases. It was obviously that the operative time and intraoperative blood loss of group A were more than those of group B (P = .045, P = .009, respectively). Kyphotic deformity was signifcantly corrected in both groups after surgery; however, the correction rate was higher in group B than in group A (P = .043). There were no significant differences between the two groups in hospitalizations, bony fusion time, and visual analog scale score in the last follow-up (P = .055, P = .364, P = .125, respectively).Our results suggested that both anterior and posterior approaches can effectively cure lumbar brucella spondylitis. Nevertheless, posterior approach gives better kyphotic deformity correction, less surgical invasiveness, and less complications.

Direct identification and molecular characterization of zoonotic hazards in raw milk by metagenomics using Brucella as a model pathogen.

Metagenomics is a valuable diagnostic tool for enhancing microbial food safety because (i) it enables the untargeted detection of pathogens, (ii) it is fast since primary isolation of micro-organisms is not required, and (iii) it has high discriminatory power allowing for a detailed molecular characterization of pathogens. For shotgun metagenomics, total nucleic acids (NAs) are isolated from complex samples such as foodstuff. Along with microbial NAs, high amounts of matrix NAs are extracted that might outcompete microbial NAs during next-generation sequencing and compromise sensitivity for the detection of low abundance micro-organisms. Sensitive laboratory methods are indispensable for detecting highly pathogenic foodborne bacteria like Brucella spp., because a low infectious dose is sufficient to cause human disease through the consumption of contaminated dairy or meat products. In our study, we applied shotgun metagenomic sequencing for the identification and characterization of Brucella spp. in artificially and naturally contaminated raw milk from various ruminant species. With the depletion of eukaryotic cells prior to DNA extraction, Brucella was detectable at 10 bacterial cells ml-1, while at the same time microbiological culture and isolation of the fastidious bacteria commonly failed. Moreover, we were able to retrieve the genotype of a Brucella isolate from a metagenomic dataset, indicating the potential of metagenomics for outbreak investigations using SNPs and core-genome multilocus sequence typing (cgMLST). To improve diagnostic applications, we developed a new bioinformatics approach for strain prediction based on SNPs to identify the correct species and define a certain strain with only low numbers of genus-specific reads per sample. This pipeline turned out to be more sensitive and specific than Mash Screen. In raw milk samples, we simultaneously detected numerous other zoonotic pathogens, antimicrobial resistance genes and virulence factors. Our study showed that metagenomics is a highly sensitive tool for biological risk assessment of foodstuffs, particularly when pathogen isolation is hazardous or challenging.

Morphological and molecular diagnosis of diseases of free-ranging crab-eating foxes (Cerdocyon thous) / [Diagnóstico morfológico e molecular de doenças de cachorro-do-mato (Cerdocyon thous) de vida livre]

The aim of this study was to investigate the occurrence of diseases in free-ranging wild canids that were roadkill on highways in the State of Espírito Santo, Brazil. PCR tests were performed for the detection of Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. in the spleen. Morphological evaluation and identification of parasites was performed in the liver and lung. Twenty specimens of C. thous were necropsied at the Animal Pathology Sector of the Veterinary Hospital of the Universidade Federal do Espírito Santo. Tissue samples were processed for histopathological examination and polymerase chain reaction (PCR) analysis. There was no PCR amplification of genomic DNA sequences of Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. using DNA extracted from the spleen as template. Histologically, lesions associated with parasitism by Platynosomum sp. and Angiostrongylus sp. were observed in the liver and lung, respectively. This is the first report of Platynosomum sp. and Angiostrongylus sp. parasitism in C. thous in the state of Espírito Santo, Brazil. Therefore, this study demonstrated parasitism of crab-eating foxes by Platynosomum sp. and Angiostrongylus sp. Importantly, no evidence of infection with Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. was obtained by PCR analysis.(AU)

O objetivo deste estudo foi investigar a ocorrência de doenças em canídeos silvestres de vida livre que foram atropelados em rodovias no estado do Espírito Santo, Brasil. Testes de PCR foram realizados para a detecção de Brucella sp., Babesia sp., Rangelia sp. e Hepatozoon sp. no baço. A avaliação morfológica e a identificação de parasitas foram realizadaa no fígado e no pulmão. Vinte espécimes de C. thous foram necropsiados no Setor de Patologia Animal do Hospital Veterinário da Universidade Federal do Espírito Santo. Amostras de tecido foram processadas para exame histopatológico e análise de reação em cadeia da polimerase (PCR). Não houve amplificação por PCR das sequências de DNA genômico de Brucella sp., Babesia sp., Rangelia sp. e Hepatozoon sp. usando-se DNA extraído do baço como modelo. Histologicamente, lesões associadas ao parasitismo por Platynosomum sp. e Angiostrongylus sp. foram observadas no fígado e no pulmão, respectivamente. Este é o primeiro relato de Platynosomum sp. e Angiostrongylus sp. parasitismo em C. thous no estado do Espírito Santo, Brasil. Portanto, este estudo demonstrou parasitismo de cachorro-do-mato por Platynosomum sp. e Angiostrongylus sp. É importante detacar que não há evidências de infecção por Brucella sp., Babesia sp., Rangelia sp. e Hepatozoon sp. por análise de PCR.(AU)

Multicenter evaluation of the VITEK MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry system for identification of bacteria, including Brucella, and yeasts.

The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has proven to be rapid and accurate for the majority of clinical isolates. Some gaps remain concerning rare, emerging, or highly pathogenic species, showing the need to continuously expand the databases. In this multicenter study, we evaluated the accuracy of the VITEK MS v3.2 database in identifying 1172 unique isolates compared to identification by DNA sequence analysis. A total of 93.6% of the isolates were identified to species or group/complex level. A remaining 5.2% of the isolates were identified to the genus level. Forty tests gave a result of no identification (0.9%) and 12 tests (0.3%) gave a discordant identification compared to the reference identification. VITEK MS is also the first MALDI-TOF MS system that is able to delineate the four members of the Acinetobacter baumannii complex at species level without any specific protocol or special analysis method. These findings demonstrate that the VITEK MS v3.2 database is highly accurate for the identification of bacteria and fungi encountered in the clinical laboratory as well as emerging species like Candida auris and the highly pathogenic Brucella species.

Prevalence of Brucella spp. in raw milk and artisanal cheese tested via real-time qPCR and culture assay.

Brucellosis is one of the most prevalent zoonotic diseases with worldwide distribution. Although the incidence of brucellosis varies widely in different regions, it is a major concern for public health around the world. This study aimed to determine the prevalence and quantity of Brucella spp. in sheep and goat raw milk, as well as artisanal cheeses produced in the North-west of Iran. To evaluate the intrinsic parameters that may affect the survival of Brucella spp., some of the cheese properties (e.g., pH, salt, moisture, and storage time before selling) were also assessed. A total of 572 samples consisting in 214 sheep raw milk, 92 goat raw milk, and 266 local artisanal cheese samples were collected. The artisanal cheeses were manufactured from a mixture of raw sheep and goat milk. According to the results, using quantitative real-time PCR (qPCR), 17.29%, 15.22%, and 22.93% of the sheep raw milk, goat raw milk, and artisanal cheese samples were found positive for Brucella spp., respectively. In comparison with culture assay, qPCR was 3.5 to 5 times (p < 0.05) more sensitive in the detection of Brucella spp. The results also revealed that the mean values of Brucella spp. load in sheep and goat raw milk and artisanal cheeses were 1.22, 1.55, and 1.43 log cell/ml or g, respectively. A positive correlation was found between Brucella load and successful detection of Brucella spp. by culture assay. Data also suggested a correlation (p < 0.01) between the load of Brucella spp. estimated by qPCR and pH value, salt content, and storage period of the cheese samples. However, Brucella spp. load did not correlate significantly with the moisture content. Based on the results, in any of the cheese samples with a pH value less than 4.5 and a storage period more than five months, no contamination with Brucella spp. was detected.