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.

Performance characteristics and costs of serological tests for brucellosis in a pastoralist community of northern Tanzania.

The control of brucellosis across sub-Saharan Africa is hampered by the lack of standardized testing and the use of tests with poor performance. This study evaluated the performance and costs of serological assays for human brucellosis in a pastoralist community in northern Tanzania. Serum collected from 218 febrile hospital patients was used to evaluate the performance of seven index tests, selected based on international recommendation or current use. We evaluated the Rose Bengal test (RBT) using two protocols, four commercial agglutination tests and a competitive enzyme-linked immunosorbent assay (cELISA). The sensitivity, specificity, positive predictive value, negative predictive value, Youden's index, diagnostic accuracy, and per-sample cost of each index test were estimated. The diagnostic accuracy estimates ranged from 95.9 to 97.7% for the RBT, 55.0 to 72.0% for the commercial plate tests, and 89.4% for the cELISA. The per-sample cost range was $0.69-$0.79 for the RBT, $1.03-$1.14 for the commercial plate tests, and $2.51 for the cELISA. The widely used commercial plate tests performed poorly and cost more than the RBT. These findings provide evidence for the public health value of discontinuing the use of commercial agglutination tests for human brucellosis in Tanzania.

Importance of brucellosis control programs of livestock on the improvement of one health.

Brucellosis not only represents an important health restraint on livestock but also causes high economic losses in many developing countries worldwide. Despite considerable efforts made for the control of brucellosis, the disease is still spreading in many regions (such as the Middle East) where it represents one of the most important health hazards impacting both animals and humans. The present review aims to investigate the efficacy of veterinary control programs regarding brucellosis, with a special focus on current prevention, control, and eradication approaches. The reasons for unsuccessful control programs such as the absence of highly effective vaccines and non-certified bulls are also debated, to understand why the prevalence of brucellosis in livestock is not decreasing in many areas despite considerable efforts taken to date. The importance of governmental and regional investment in brucellosis control remains one of the main limiting factors owing to the limited budget allocated to tackle this disease. In this context, one health concept has generated novel comprehensive approaches with multiple economic implications across the livestock industry and public health. However, the implementation of such global preventive strategies appears to be a key issue for many endemic and low-income countries. According to the collected data, epidemiological contexts including management and trade systems along with well-defined agro-ecological zones should be evaluated in brucellosis endemic countries to improve milk production and to enhance the sustainability of the livestock sector at both national and regional levels.

Brucellosis: A rapid risk assessment by a regional outbreak team and its coordinated response with the Directorate-General for Food and Veterinary, North region of Portugal, 2019.

We report a Brucella outbreak with seven cases in the Northern Region of Portugal in 2018-2019, associated with the consumption of fresh cheese. This outbreak has implications for risk assessment in Portuguese migrants related to this area, and it is an example of cooperation between public institutions, in a One Health based approach.

Brucella Infection in Asian Sea Otters (Enhydra lutris lutris) on Bering Island, Russia.

Infection with Brucella spp., long known as a cause of abortion, infertility, and reproductive loss in domestic livestock, has increasingly been documented in marine mammals over the past two decades. We report molecular evidence of Brucella infection in Asian sea otters (Enhydra lutris lutris). Brucella DNA was detected in 3 of 78 (4%) rectal swab samples collected between 2004 and 2006 on Bering Island, Russia. These 78 animals had previously been documented to have a Brucella seroprevalence of 28%, markedly higher than the prevalence documented in sea otters (Enhydra lutris) in North America. All of the DNA sequences amplified were identical to one or more previously isolated Brucella spp. including strains from both terrestrial and marine hosts. Phylogenetic analysis of this sequence suggested that one animal was shedding Brucella spp. DNA with a sequence matching a Brucella abortus strain, whereas two animals yielded a sequence matching a group of strains including isolates classified as Brucella pinnipedialis and Brucella melitensis. Our results highlight the diversity of Brucella spp. within a single sea otter population.

Colony-level assessment of Brucella and Leptospira in the Guadalupe fur seal, Isla Guadalupe, Mexico.

The relatively small population size and restricted distribution of the Guadalupe fur seal Arctocephalus townsendi could make it highly vulnerable to infectious diseases. We performed a colony-level assessment in this species of the prevalence and presence of Brucella spp. and Leptospira spp., pathogenic bacteria that have been reported in several pinniped species worldwide. Forty-six serum samples were collected in 2014 from pups at Isla Guadalupe, the only place where the species effectively reproduces. Samples were tested for Brucella using 3 consecutive serological tests, and for Leptospira using the microscopic agglutination test. For each bacterium, a Bayesian approach was used to estimate prevalence to exposure, and an epidemiological model was used to test the null hypothesis that the bacterium was present in the colony. No serum sample tested positive for Brucella, and the statistical analyses concluded that the colony was bacterium-free with a 96.3% confidence level. However, a Brucella surveillance program would be highly recommendable. Twelve samples were positive (titers 1:50) to 1 or more serovars of Leptospira. The prevalence was calculated at 27.1% (95% credible interval: 15.6-40.3%), and the posterior analyses indicated that the colony was not Leptospira-free with a 100% confidence level. Serovars Icterohaemorrhagiae, Canicola, and Bratislava were detected, but only further research can unveil whether they affect the fur seal population.

Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

BACKGROUND: The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. RESULTS: Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. CONCLUSION: The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

Evaluation of five serological tests for the diagnosis of porcine brucellosis in French Polynesia.

Porcine brucellosis due to Brucella suis biovar 1 raises important issues for pig breeders in French Polynesia. In this region, the disease is enzootic, spreads silently and engenders economic losses in infected farms as well as sporadic human cases. While serological tests are essential in surveillance and control programmes of animal diseases, to date none of the available tests have been shown to be reliable enough to be used as a gold standard in routine individual diagnosis of porcine brucellosis. Few studies about the estimation of the sensitivity and the specificity of porcine brucellosis screening tests have been published, none of them dealing with French Polynesia. The studied population included 1,595 pigs from French Polynesia. Five tests were evaluated: Rose Bengal test, fluorescence polarisation assay, indirect ELISA, and two competitive ELISAs (C-ELISA). The sensitivity and the specificity of each test were estimated. C-ELISA2 was the most sensitive test (Se C-ELISA2=0.954 [0.889; 0.992] 95% credibility interval (CrI)) while both C-ELISA and Rose Bengal test (RBT) were the most specific ones (Sp C-ELISA1=0.856 [0.806; 0.915] 95% CrI; Sp C-ELISA2=0.849 [0.817; 0.879] 95% CrI; Sp RBT=0.853 [0.812; 0.898] 95% CrI).

Cattle producers' economic incentives for preventing bovine brucellosis under uncertainty.

Cattle in the Greater Yellowstone Ecosystem occasionally contract bovine brucellosis from free-ranging elk and bison. Cattle producers use a variety of brucellosis prevention activities to reduce their herds' risk of contracting brucellosis, such as: (1) having state agency personnel haze elk off private land, (2) fencing haystacks, (3) administering adult booster vaccination, (4) spaying heifers, (5) altering the winter-feeding schedule of cattle, (6) hiring riders to prevent cattle-elk commingling, and (7) delaying grazing on high-risk allotments. Their brucellosis prevention decisions are complicated, however, by several sources of uncertainty, including the following: a cattle herd's baseline risk of contracting brucellosis, the inherent randomness of brucellosis outbreaks, the cost of implementing prevention activities, and the activities' effectiveness. This study eliminates one source of uncertainty by estimating the cost of implementing brucellosis prevention activities on a representative cow/calf-long yearling operation in the southern GYE. It then reports the minimum level of effectiveness each prevention activity must achieve to justify investment by a risk-neutral producer. Individual producers face different levels of baseline risk, however, and the US government's brucellosis-response policy is constantly evolving. We therefore estimate breakeven levels of effectiveness for a range of baseline risks and government policies. Producers, animal health experts, and policymakers can use this study's results to determine which brucellosis prevention activities are unlikely to generate sufficient expected benefits to cover their cost of implementation. Results also demonstrate the influence of government policy on producers' incentives to prevent brucellosis. Policies that increase the magnitude of economic loss a producer incurs when their herd contracts brucellosis subsequently decrease prevention activities' breakeven levels of effectiveness, and increase producers' incentives to implement those activities. Producers' incentives to implement prevention activities also increase as activities' costs decrease. Policymakers can easily adapt the results of this analysis to help target cost-share agreements to producers and prevention activities most likely to generate positive expected net benefits. Epidemiologists can also use our results to help prioritize future research on the technical effectiveness of various brucellosis prevention activities.

The human-animal interface of domestic livestock management and production and its relationship to brucellosis in the country of Georgia 2010: a rapid assessment analysis.

CONTEXT: Brucellosis is endemic in the country of Georgia, with the highest incidence of disease in the east of Georgia, in the Kakheti region--which is also home to the majority of sheep and a large portion of the national cattle herd (two species that are natural hosts of zoonotic Brucella spp.). OBJECTIVE: Our purpose was to understand the ruminant livestock management and dairy production as well as the sociological factors in order to relate it to the disease ecology of brucellosis and to understand the framework that contributes too brucellosis transmission in the region. METHODS: In 2010, we examined the aspects of livestock management and production through the use of a semi-structured questionnaire that was administered to 198 villagers and 41 key informants (physicians, veterinarians, dairy production specialists, and laboratory personnel) who were identified by convenience sampling. Results were primarily qualitative, but some were quantified to reveal trends and compared with non-parametric tests. RESULTS: We found that animals are managed at the village level. Male villagers take turns shepherding and herding on both summer pastures (highlands) and winter pastures (lowlands or around the village). Men also do all the sheep-dairy production. Women care for milk cattle as well as make the dairy products from cow milk. Of the households that own livestock, 28% own sheep (50 per flock) and 96% own cattle (3 per herd). The northern-most part of Kakheti (Akhmeta) has the widest distribution of its cheese; the guda cheese from this area is sold all over Kakheti and central Georgia. Typically, cheese is aged in 20% brine for 3d (white cheeses) or 21d (guda cheeses). In addition, raw milk is used for cheese production and heating the milk is believed to decrease the quality of the final product. CONCLUSIONS: Interventions at the animal level will be best carried out in the fall when animals return to winter pastures. Under-employed private veterinarians would be available for extension work and contact with local villagers. Control will be best achieved at the animal level because the local people have a social and cultural resistance to the use of heated or pasteurized milk for cheese production.

Meta-analytical equivalence studies on diagnostic tests for bovine brucellosis allowing assessment of a test against a group of comparative tests.

In the assessment of diagnostic tests the task may arise to show that a candidate test is non-inferior compared to a comparative (standard) test with regard to the diagnostic sensitivity or specificity. This setting is known as "one-sided equivalence" and has been applied to a single comparison between two diagnostic tests (Chen et al., 2003). Recently, the approach has been extended into a meta-analytical framework (EFSA, 2006), allowing for the difference between the sensitivity (or specificity) of two diagnostic tests to be estimated using information gathered through systematic literature review. Using this approach, confounding factors are adjusted by matching of parameter estimates on study population and preferred levels of the confounding factors. However, the power of this approach was found to be limited and therefore Markov chain Monte Carlo logistic regression (MCMCLR) models that allow adjustment for confounding variables have been developed (EFSA, 2006). We report here a refinement of the statistical inference based on the latter approach. The objective was to generate a posterior distribution of the meta-analytical difference statistic for the candidate test and a set of comparative tests. The algorithm for this purpose uses Monte Carlo sampling from the posterior distributions of sensitivity (or specificity) and, for each iteration, (i) identifies the least performant comparative test, (ii) establishes the difference statistics for this test and the candidate test and (iii) compares the difference statistic with a critical threshold value. The proportion of iterations in which the critical threshold was exceeded is then interpreted as the P-value for the one-sided equivalence test for the candidate versus the set of comparative tests. We illustrate and discuss the method using a case study on tests for bovine brucellosis.

[Assessment of polymerase chain reaction (PCR) to diagnose brucellosis in a Brucella infected herd]. / Evaluación de la reacción en cadena de la polimerasa para el diagnóstico de la brucelosis en un rebaño lechero infectado con Brucella spp.

The diagnosis of bovine brucellosis using PCR in blood and milk samples from two dairy herds were compared to in vitro isolation, complement fixation test (CF), competitive ELISA (C-ELISA) in serum, and indirect ELISA (I-ELISA) in milk. Samples were obtained from 99 cows vaccinated with Brucella abortus strain 19, from a naturally infected herd (A), whose cows were also vaccinated with B. abortus strain RB51 as adults, and 100 from brucellosis free herd (B). In herd A, PCR identified 14 B. abortus infected cows: nine infected with wild type, and five with wild type and RB51, B. abortus S 19 was not identified. B. abortus biotype 1 was isolated from one cow. All cows infected with a wild strain of B. abortus were positive in serologic tests. Brucella was not found in herd B using PCR. Serological test showed 100% sensitivity related to PCR. The specificity for CF, C-ELISA and I-ELISA was 100%, 99% and 95% respectively. PCR could be useful to identify Brucella biotypes and to complement serologic tests.

Evaluación de la reacción en cadena de la polimerasa para el diagnóstico de la brucelosis en un rebaño lechero infectado con Brucellaspp / Assessment of polymerase chain reaction (PCR) to diagnose brucellosis in a Brucella infected herd

Para el diagnóstico de la brucelosis bovina en muestras de sangre y/o leche, se comparó la reacción en cadena de la polimerasa (PCR) con el aislamiento in vitro de Brucella abortus, las pruebas serológicas defijación del complemento (FC) e inmunoenzimáticas de competición (ELISA-C) en suero e indirecto (ELISA-I) en leche. Se analizaron muestras de vacas lecheras de un rebaño infectado “A”, vacunadas con B. abortus cepa 19 antes de los 8 meses de edad y revacunadas con B. abortus cepa RB51 como adultas (n= 99) y de otro “B”, libre de brucelosis (n=100), como control. En A, la PCR identificó 14 vacas infectadas con B. abortus: nueve con cepa silvestre y cinco con cepa silvestre y RB51. No se identificó B. abortus cepa 19. El biotipo 1 se aisló en un caso. Las 14 vacas infectadas con la cepa silvestre resultaron positivas en las tres pruebas serológicas. En B, por PCR no se identificó Brucella. Las pruebas serológicas mostraron una sensibilidad del 100% respecto de PCR. La especificidad para FC, ELISA-C y ELISA-I fue del 100%, 99% y 95%, respectivamente. Se concluye que la PCR sería útil como complemento de las pruebas serológicas o cuando no hay un resultado concluyente.

The diagnosis of bovine brucellosis using PCR in blood and milk samples from two dairy herds were compared to in vitro isolation, complement fixation test (CF), competitive ELISA (C-ELISA) in serum, and indirect ELISA (I-ELISA) in milk. Samples were obtained from 99 cows vaccinated with Brucella abortus strain 19, from a naturally infected herd (A), whose cows were also vaccinated with B. abortus strain RB51 as adults, and 100 from brucellosis free herd (B). In herd A, PCR identified 14 B. abortus infected cows: nine infected with wild type, and five with wild type and RB51, B. abortus S 19 was not identified. B. abortus biotype 1 was isolated from one cow. All cows infected with a wild strain of B. abortus were positive in serologic tests. Brucella was not found in herd B using PCR. Serological test showed 100% sensitivity related to PCR. The specificity for CF, C-ELISA and I-ELISA was 100%, 99% and 95% respectively. PCR could be useful to identify Brucella biotypes and to complement serologic tests.

Estudio de un brote de brucelosis y valoración diagnóstica de las pruebas de laboratorio / Study of a brucellosis outbreak and diagnostic evaluation of laboratory

En 1991, en la zona de Villa Corona, Jalisco, se registró un número anormal de casos con fiebre de origen desconocido, con múltiples diagnósticos clínicos. Se revisaron todos los casos estableciéndose la sospecha de un brote de brucelosis. Se consideró caso a cualquier persona entre cinco y 60 años, que desde enero de 1991 presentó fiebre, calosfríos de más de tres días de evolución más cefalea y/o artralgias. Se hizo un muestreo por conglomerado de las viviendas; cada caso se comparó con tres controles, dos convivientes y un vecino del mismo grupo de edad; se realizaron pruebas seriadas y hemocultivo, para comparar la validez y confiabilidad diagnóstica. Se detectaron 31 casos probables y se estudiaron 85 controles (68.5 por ciento de la muestra de controles esperada). Se aisló Brucella melitensis por hemocultivo en un caso. Se definieron 18 casos con brucelosis activa. Se obtuvo una razón de momios (RM) de 12.6 (IC 95 por ciento = 30-56.1); XMH, p<0.000 para la ingesta habitual de lácteos crudos y una RM de 10.0 (IC 95 por ciento=2.5-43.5); Xmh, p<0.000 para la ingesta continua durante seis meses. Este riesgo se ajustó a 3.0 para los pares discordantes que ingerían regularmente leche cruda. Las pruebas seriadas mostraron: Hudlesson, sensibilidad = 50 por ciento y especificidad = 84.6 por ciento; rosa de Bengala, sensibilidad = 94.4 por ciento y especificidad 84.6 por ciento. Los resultados sugieren que la prueba de Hudlesson tiene una baja confiabilidad. La aglutinación en tubo y la prueba de 2 mercaptoetanol tienen resultados serológicos más satisfactorios

[Diagnosis of brucellosis in an endemic area. Evaluation of routine diagnostic tests]. / El diagnóstico de la brucelosis en un área endémica. Valoración de las pruebas diagnósticas habituales.

BACKGROUND: The use of classic methods of diagnosis of brucellosis was analyzed, particularly serologic methods whose use in endemic areas and risk groups has been questioned in the literature. METHODS: Prospective analysis of these methods was performed in a group of 171 patients suspected as having brucellosis proceeding from an endemic area, with progressions of risk and frequent antecedents of brucellosis. The results obtained were compared in 119 patients in whom brucellosis was confirmed (80 with positive cultures and 39 with clinic-serologic diagnosis) and in 52 in whom the diagnosis was excluded. RESULTS: The hemocultures provided a sensitivity of 70% with a mean delay in growth of 13.6 days. The rise of Bengal test showed sensitivity of 95% and specificity of 75%. The most adequate cut-off point for seroagglutination was of 1/160 and for the Coombs test 1/320 with sensitivities of 93 and 92% and specificities of 97 and 100%, respectively. CONCLUSIONS: The routine serologic tests offer good results for the diagnosis of brucellosis in endemic areas upon use of adequate cut-off points and permitting therapeutic decisions to be taken prior to knowledge of the results of the cultures. The rose of Bengal test is valid for initial selection of this type of population.