A time-resolved fluorescence lateral flow immunoassay for rapid and quantitative serodiagnosis of Brucella infection in humans.

Brucella abortus (B. abortus), an important zoonotic pathogen in Brucella spp., is the major causative agent of abortion in cattle (namely bovine brucellosis). Currently, although the isolation and identification of the Brucella abortus were commonly accepted as the gold standard method, it cannot meet to the requirements for early diagnostic strategies. Conventional PCR techniques and immunological tests can realize rapid detection of B. abortus, but the demands for PCR thermal cyclers and/or specific antibodies hinder their application in basic laboratories. Thus, rapid, sensitive and specific diagnostic strategies are essential to prevent and control the spread of the bovine brucellosis. In this work, a novel detection method for the rapid identification of B. abortus, which uses loop-mediated isothermal amplification (LAMP) combined with a label-based polymer nanoparticles lateral flow immunoassay biosensor (LFIA), was established. One set of specific B. abortus-LAMP primers targeting the BruAb2_0168 gene was designed by the online LAMP primer design tool. The B. abortus-LAMP-LFIA assay was optimized and evaluated using various pathogens and whole blood samples. The optimal amplification temperature and time for B. abortus-LAMP-LFIA were determined to be 65°C and 50 min, respectively. The B. abortus-LAMP-LFIA method limit of detection (LoD) was 100 fg per reaction for pure genomic DNA of B. abortus. Meanwhile, the detection specificity was 100%, and there was no cross- reactivity for other Brucella members and non-Brucella strains. Furthermore, the entire procedure, including the DNA preparation for whole blood samples (30 min), isothermal incubation (50 min) and LFIA detection (2-5 min), can be completed in approximately 85 min. Thus, the B. abortus-LAMP-LFIA assay developed was a simple, rapid, sensitive and reliable detection technique, which can be used as a screening and/or diagnostic tool for B. abortus in the field and basic laboratories.

A Label-Based Polymer Nanoparticles Biosensor Combined with Loop-Mediated Isothermal Amplification for Rapid, Sensitive, and Highly Specific Identification of Brucella abortus

Knowledge, Attitudes, and Practices Regarding Brucellosis among Parents in Aseer Region, Southwestern Saudi Arabia.

An outbreak of brucellosis in a mixed dairy farm with 508 animals (370 cows, 120 sheep, and 18 camels) at Fayoum governorate, North Upper Egypt, was investigated. A storm of abortion and several cases of retained placenta were reported among cows and ewes in April 2020. Serodiagnosis of brucellosis was done using Rose Bengal Test (RBT) and Buffered Acidified Plate Antigen Test (BAPAT). The Milk Ring Test (MRT) was applied to the milk of seropositive animals. A total of 89 samples were used for isolation of Brucella and isolates were confirmed using Abortus, Melitensis, Ovis, Suis-PCR (AMOS-PCR). Test and slaughter strategy was applied to eradicate brucellosis from the farm based on RBT every month until three successive negative tests were obtained. Results showed that the seroprevalences of brucellosis based on RBT and BAPAT were 9.5%, 35%, and 50% in cattle, sheep, and camels, respectively. Despite 50% of male camels being seropositive, no clinical signs have been reported. The MRT identified fewer positive cases than BAPAT and RBT, thus, it cannot be used alone to eliminate the infection from the farm. A total of 31 Brucella isolates were recovered from cows and sheep on the farm. All isolates were confirmed as Brucella melitensis bv3 based on bacteriological examination and Brucella AMOS-PCR confirmed all isolates as Brucella melitensis. No positive reactors at the 6th, 7th, 8th, 9th, and 10th examinations were reported after the implementation of the test and slaughter strategy. In conclusion, extensive animal farming and mixed breeding are potential risk factors for interspecies transmission of brucellosis. Additionally, the test and slaughter strategy could be helpful to release the herd out of quarantine, however, application of biosecurity practices and fair compensation policy for owners should be implemented.

Investigation of an outbreak of brucellosis in a mixed dairy farm and evaluation of a test and slaughter strategy to release the herd out of the quarantine

A comparison of logistic regression and classification tree to assess brucellosis associated risk factors in dairy cattle.

Brucellosis is a zoonotic disease of great animal welfare and economic implications worldwide known since ancient times. The emergence of brucellosis in new areas as well as transmission of brucellosis from wild and domestic animals is of great significance in terms of new epidemiological dimensions. Brucellosis poses a major public health threat by the consumption of non-pasteurized milk and milk products produced by unhygienic dairy farms in endemic areas. Regular and meticulous surveillance is essentially required to determine the true picture of brucellosis especially in areas with continuous high prevalence. Additionally, international migration of humans, animals and trade of animal products has created a challenge for disease spread and diagnosis in non-endemic areas. Isolation and identification remain the gold standard test, which requires expertise. The advancement in diagnostic strategies coupled with screening of newly introduced animals is warranted to control the disease. Of note, the diagnostic value of miRNAs for appropriate detection of B. abortus infection has been shown. The most widely used vaccine strains to protect against Brucella infection and related abortions in cattle are strain 19 and RB51. Moreover, it is very important to note that no vaccine, which is highly protective, safe and effective is available either for bovines or human beings. Research results encourage the use of bacteriophage lysates in treatment of bovine brucellosis. One Health approach can aid in control of this disease, both in animals and man.

Baldev Gulati

Papers

Bovine brucellosis - a comprehensive review.