Melitensis Biovar Research Articles

Analysis of the Brucella melitensis epidemic in Xinjiang: genotyping, polymorphism, antibiotic resistance and tracing

Brucella spp. are facultative intracellular pathogens that cause zoonosis- brucellosis worldwide. There has been a trend of the re-emergence of brucellosis worldwide in recent years. The epidemic situation of brucellosis is serious in Xinjiang. To analyze the epidemic situation of Brucella spp. in Xinjiang among humans and animals, this study identified 144 Brucella isolates from Xinjiang using classical identification and 16 S rRNA sequencing. MLVA, drug resistance testing, and wgSNP detection were also performed. At the same time, analysis was conducted based on the published data of Brucella isolates worldwide. The results showed that the dominant species was B. melitensis biovar 3, which belonged to GT42 (MLVA-8 typing) and the East Mediterranean lineage. The correlation among isolates was high both in humans or animals. The isolates in Xinjiang exhibited higher polymorphism compared to other locations in China, with polymorphism increasing each year since 2010. No amikacin/kanamycin-resistant strains were detected, but six rifampicin-intermediate isolates were identified without rpoB gene variation. The NJ tree of the wgSNP results indicated that there were three main complexes of the B. melitensis epidemic in Xinjiang. Based on the results of this study, the prevention and control of brucellosis in Xinjiang should focus on B. melitensis, particularly strains belonging to B. melitensis bv.3 GT42 (MLVA-8 typing) and East Mediterranean lineage. Additionally, the rifampicin- and trimethoprim-sulfamethoxazole- resistance of isolates in Xinjiang should be closely monitored to avoid compromising the therapeutic efficacy and causing greater losses. These results provide essential data for the prevention and control of brucellosis in Xinjiang and China. Although the isolates from Xinjiang have significant characteristics among Chinese isolates and can reflect the epidemiological situation of brucellosis in China to some extent, this study cannot represent the characteristics of isolates from other regions.

Detection and characterization of Brucella species in rodents: A threat for the persistence of brucellosis in livestock farms

Brucellosis, caused by various Brucella species, poses a significant threat to global public health and livestock industries. This study aims to fill the knowledge gap concerning the presence of Brucella spp. in rodents on livestock farms in Iran. Both bacteriological and molecular surveys were conducted to assess the prevalence of Brucella spp. in these rodent populations. A total of 16 rodents were captured in four seropositive dairy cattle farms (n = 7) and two seropositive sheep farms (n = 9) and were then examined for the presence of the Brucella-infection. Five cow milk samples and 53 bovine lymph node samples from these farms were also tested for Brucella spp. Lymph node samples from dairy cattle farms contained 32 B. abortus biovar 3 isolates and one B. melitensis Rev1 vaccine isolate. The bacterial culture of rodents identified 12.5% of them (Mus musculus and Rattus norvegicus) harboring Brucella strains in dairy cattle farms. The rodents had B. abortus biovar 3 and B. melitensis biovar 1, suggesting a reservoir for these bacteria. A two-step molecular assay, utilizing the Omp28 sequences in tissue samples of rodents, demonstrated that 68.75% (n = 11) of the tested rodents yielded positive results. Bruce-ladder PCR and wboA typing on isolated bacteria revealed a close relationship to field strain of Brucella species. The study reveals that rodents on seropositive livestock farms in Iran harbor Brucella spp., indicating a potential reservoir for these bacteria. This highlights the importance of monitoring rodent populations through the molecular and bacterial methods to manage and control brucellosis in livestock.

Single-Gene Phylogeny of Brucella Melitensis in Milk Samples of Ewes in Kalar District, Iraqi Kurdistan Region

The present study aimed to investigate the frequency rate of ovine brucellosis in Kalar district, Iraqi Kurdistan Region and to identify Brucella species in the milk of seropositive ewes via DNA sequencing and phylogenetic analyses. Blood samples were collected from 300 ewes and subjected to the Rose Bengal Plate Test (RBPT), and milk samples of the seropositive animals were collected and underwent a PCR assay using genus- and species-specific primers. Eventually, the positive PCR products were processed for nucleotide sequencing and single-gene phylogenetic analyses. The RBPT revealed a high percentage (11.33%) of seropositive ewes (34 out of 300), and the PCR assay revealed 35.3% (12/34) Brucella genus-positive DNA templates. At the species level, 58.3% (7/12) of templates were positive for the melitensis-specific primers and negative for the abortus and ovis-specific primers, whereas the other five were negative for all species-specific primers used in this study (abortus, melitensis and ovis). The nucleotides sequence of Brucella strains detected in this study showed a 99-100% homology with eight GenBank-published B. melitensis strains, and the single gene phylogenetic analysis showed a close relationship between these strains and the B. melitensis biovar 3 strain NI, China. The high percentage of seropositive ewes showed that brucellosis represents a major obstacle to the animals’ production improvement and a major risk to public health in the Iraqi Kurdistan Region and the single gene phylogenetic analysis confirms its transboundary spread and attracts attention for more strict control programs.

Equine brucellosis in Iran: serological, bacteriological and molecular analysis.

Equine brucellosis significantly impacts the health and functionality of horses, leading to complications such as bursitis infection, septic tenosynovitis, septic arthritis, and non-specific lameness resulting from joint infections. In the present study, we used the Rose Bengal plate agglutination test (RBPT), serum agglutination test (SAT), and the 2-mercaptoethanol (2-ME) assays to find equine brucellosis. From June 2018 to September 2022, 876 blood samples were randomly taken from apparently healthy racing horses in certain parts of Iran, such as Kerman, Isfahan, Tehran, Qom, and Kurdistan. DNA extraction was carried out directly on all 63 serum samples identified as seropositive through RBPT. An additional 30 seronegative serum samples were also randomly chosen for study. Bacterial culture was also done on milk, blood, and vaginal swabs taken from seropositive horses.The bacteria that were found in the samples were then put through Bruce-ladder PCR. Our results indicated that 63 (7.1%), 21 (2.3%), and 2 (0.2%) of horses were seropositive using RBPT, SAT, and 2-ME, respectively. Also, none of the 30 DNA-extracted serum samples from seronegative horses tested positive for Brucella DNA, while 44.5% (28/63) of the DNA samples from seropositive horses yielded positive results for Brucella DNA. Out of the seropositive samples, 26 had DNA from Brucella abortus and 2 had DNA from Brucella melitensis. Also, B. melitensis biovar 1 was found in two milk samples from mares in the provinces of Kerman and Isfahan. It was identified using classical biotyping, and molecular assays. It was seen that some of healthy racing horses in some parts of Iran had antibodies against Brucella. The bacteriology and PCR methodologies provide a more comprehensive and reliable means of identifying Brucella spp. infections in horse, especially when the RBPT test came back positive. This underscores the imperative for employing molecular, bacterial, and serological methods in the diagnosis and monitoring of this zoonotic infection. Additionally, this finding suggests that Brucella is being transmitted to equine hosts as a result of its presence in ruminants. The mechanism of transmission may involve interactions between infected ruminants and susceptible equines. This discovery is significant as it underscores the potential cross-species transmission of Brucella and highlights the importance of understanding and managing the spread of the pathogen in both ruminant and equine populations.

Characterization of Brucella spp. circulating in industrial dairy cattle farms in Iran: a field study 2016 - 2023.

Bovine brucellosis, an infectious disease transmitted by Brucella melitensis and Brucella abortus, presents a significant zoonotic risk for agricultural economics and animal health. The primary objective of this study was to present a comprehensive understanding of the prevalence and features of Brucella strains within the industrial dairy farming sector in Iran. Rose Bengal plate test, standard agglutination test, and indirect enzyme linked immunosorbent assay tests were used to confirm all seropositive animals. A total number of 1,311 bovine samples from seropositive animals including were collected from 224 farms in 21 provinces of different regions of Iran and examined. The discovered Brucella isolates were phenotyped and molecularly characterized. The isolates were all B. abortus or B. melitensis. Bacteria analysis revealed that 70.53% of seropositive farms were tested positive for Brucella strains, predominantly B. melitensis biovar 1 (43.42%) and B. abortus biovar 3 (27.11%). Geographical distribution revealed that B. melitensis biovar 1 was the most common in dairy cow farms (16 provinces), followed by B. abortus biovar 3 (six provinces). Also, the prevalence of B. melitensis biovar 2, B. melitensis biovar 3, B. abortus biovar 1, B. abortus biovar 2 and RB51 vaccine were restricted to certain provinces. AMOS (abortus melitensis ovis suis)-polymerase chain reaction and Bruce-ladder PCR confirmed species identification. These results highlighted the complexity of bovine brucellosis in Iran and illustrated that B. melitensis was spread from small ruminants to cattle. This study provided important epidemiological insights for targeting future brucellosis control programs in the Iranian dairy farms.

Gated nanoprobe utilizing metal-organic frameworks for identifying and distinguishing between the wild strains and the vaccine strains of brucella.

In the assay for Brucella, the identification and differentiation of wild strains and vaccine strains present a significant challenge. Currently, there aren't any commercially available product to address this issue. In this study, we have developed a novel gated nanoprobe by utilizing Metal-Organic Frameworks (MOFs) as a scaffold and hairpin DNA as a "gating switch". Specifically, Probe 1 with hairpin structure (P1h) targets a gene that is present in both wild strains Y3 (B. melitensis biovar 3) and vaccine strains A19 (Brucella abortus strains A19). We successfully applied this probe to screen positive samples of Brucella without any cross-reactivity with other substances. Additionally, we identified another specific gene exclusively found in wild strains, which serves as Probe 2 with hairpin structure (P2h) to confirm the strain type. Simultaneous detachment of both P1h and P2h from the MOFs leads to the release of Rhodamine 6G (Rho 6G) and Fluorescein (Flu), specifically indicating the presence of wild strains. If only P1h detaches and the Flu signal is detected, it suggests the presence of vaccine strains. Importantly, this method offers high accuracy, with a detection rate of 90% and a recovery rate of 94.71% to 107.65%, while avoiding cross-reactions with MO and TB. This one-step experiment provides reliable identification and differentiation of Y3 and A19, addressing concerns related to long periodicity, interference from individual variations, and the complex design of primers in existing laboratory methods. Furthermore, our approach successfully detects target 1 (T1) and target 2 (T2) at concentrations ranging from 10-6 M to 10-9 M, with a detection limit of 6.7 × 10-10 M and 6.4 × 10-10 M, respectively. Importantly, our strategy is cost-effective (around $1) and offers higher detection efficiency compared to traditional laboratory methods.

Serological, cultural, and molecular analysis of Brucella from Buffalo milk in various regions of Iran.

Brucellosis is a significant infection that causes abortion, decreased milk production, and sterility in livestock, which greatly affects the industry. This study aimed to determine the prevalence of Brucella in buffalo milk samples across various regions of Iran, utilizing serological, molecular, and cultural analyses. A total of 1860 buffalo milk samples were collected from industrial, semi-industrial, and traditional buffalo farms in four major buffalo breeding provinces. The milk ring test agglutination test (MRT) was initially conducted on all milk samples, followed by culture and molecular testing for positive and negative samples in MRT. The study revealed positive results for the presence of Brucella DNA in various provinces of Iran. The MRT had a relatively low sensitivity, with results ranging from 0 to 0.7% in different provinces. However, the AMOS PCR method showed a significantly higher presence of Brucella DNA, ranging from 13 to 46% in these provinces. The highest abundance of Brucella bacterial DNA was found in Ardabil province, while the lowest was in West Azerbaijan province. Brucella abortus was the most commonly detected bacteria, followed by Brucella melitensis. Interestingly, the B. abortus vaccine strain RB51 was detected in 26.3% of positive samples of B. abortus. The culture assay of milk samples further confirmed the presence of B. melitensis biovar 1 in one sample from Khuzestan province. Overall, the study emphasizes that the AMOS PCR method is the most sensitive in detecting Brucella-exposed milk, while the sensitivity of milk sample culture and MRT is relatively lower.

Evaluation of a Lateral Flow Immunochromatography Assay (LFIA) for Diagnosis and Surveillance of Brucellosis in French Alpine Ibex (Capra ibex).

France has been officially free of bovine brucellosis since 2005. Nevertheless, in 2012, as the source of two human cases, a bovine outbreak due to B. melitensis biovar 3 was confirmed in the French Alpine Bargy massif, due to a spillover from wild, protected Alpine ibex (Capra ibex). In order to reduce high Brucella prevalence in the local ibex population, successive management strategies have been implemented. Lateral flow immunochromatography assay (LFIA) was thus identified as a promising on-site screening test, allowing for a rapid diagnosis far from the laboratory. This study compared a commercial LFIA for brucellosis diagnosis with the WOAH-recommended tests for small ruminants (i.e., Rose Bengal test (RBT), Complement fixation test, (CFT) and Indirect ELISA, (iELISA)). LFIA showed the same analytical sensitivity as iELISA on successive dilutions of the International Standard anti-Brucella melitensis Serum (ISaBmS) and the EU Goat Brucella Standard Serum (EUGBSS). Selectivity was estimated at 100% when vaccinated ibex sera were analyzed. When used on samples from naturally infected ibex, LFIA showed high concordance, as well as relative sensitivity and specificity (>97.25%) in comparison with RBT and CFT. This work shows high reliability and ensures a better standardization of LFIA testing for wild ruminants.

The Control Program of Brucellosis by the Iranian Veterinary Organization in Industrial Dairy Cattle Farms.

Brucellosis is a zoonotic infection in livestock that induces a major public health concern in developing countries, including Iran. Despite the efforts of the Iranian veterinary organization (IVO) to control brucellosis, it is still prevalent in domestic animals. In this regard, the present study aimed to evaluate the efficiency of the control strategy used by the IVO in infected herds on serological, cultural, and molecular methods. For this purpose, blood specimens were sampled from a total of 8750 vaccinated dairy cattle in two Brucella-infected farms. These farms were recognized as positive for Brucella by a screening program. Sera were evaluated by the Rose Bengal Plate Test and Wright test analysis. Positive dairy cattle were slaughtered under IVO supervision. The remaining cattle were evaluated every 3 weeks and positive animals were slaughtered. This procedure continued until the remaining animals revealed three successive negative responses in serological tests. Several lymph nodes and milk samples were collected from 164 seropositive cattle and subjected to bacterial isolation and confirmation by Bruceladder-polymerase chain reaction. Brucella melitensis biovar 1 and RB51 vaccine strains were recovered from milk and lymph node samples, respectively. Shedding of B. melitensis in the milk of vaccinated cows is a serious problem resulting in the further spread of brucellosis. The policy of "test and slaughter" performed on infected dairy cattle farms showed their usefulness for the control of brucellosis outbreaks. For the uncontrolled spread of brucellosis in Iran, effective control of bovine brucellosis required several serological surveillances to identify infected herds, eradication of the reservoirs, and vaccination of young heifers with RB51.

The first report of Brucella melitensis biovar 2 strain isolated from cattle in Turkey

Bovine brucellosis is an infectious zoonotic disease of great impact on animal welfare and has significant economic implications on livestock farm worldwide. The disease is caused primarily by Brucella abortus (B. abortus), while B. melitensis is less common, and B. suis infection is rare. B. melitensis is the most common causative agent of brucellosis in small ruminants and humans. Although the main host of B. melitensis is considered to be small ruminants, this bacterium is also present in large ruminants. Despite brucellosis has been eradicated in many European countries, it is still endemic in Mediterranean countries and Turkey. The most prevalent Brucella species in the Mediterranean basin and Turkey is B. melitensis biovar (bv) 3. Previous studies have reported that B. melitensis bv2 is quite low in Turkey. This is the first study to isolate B. melitensis bv2 from cattle in Turkey. The strains were characterized using classical biotyping methods and then were molecularly confirmed. Multilocus variable number tandem repeat analysis (MLVA-16) typing of the strains revealed a novel genotype (1-5-3-13-3-2-3-2-4-41-8-5-4-3-3-7), which matches the Multilocus sequence typing (MLST) profiles in the database of ST8 (3-2-3-2-1-5-3-2-8). These results indicate that B. melitensis bv2 can easily infect cattle and this has to be considered in the epidemiology and control of bovine brucellosis. Circulating the highly pathogenic B. melitensis bv2 in cattle farms is of public health concern.

Genetic diversity of Brucella melitensis isolates from sheep and goat milk in Iran.

The genetic diversity of Brucella strains has not been fully understood. To investigate this, the genetic characteristics of 64 isolates of Brucella melitensis from sheep and goats' milk were studied using random fragment length polymorphism (RFLP) and multiple locus variable-number tandem repeat analysis (MLVA-16) methods developed in Orsay, France (MLVA-16Orsay). The RFLP analysis revealed that all 64 isolates were of biovar one. The MLVA-typing showed that one sample was simultaneously infected with two strains of B. melitensis and the genotype of 65 isolate was analyzed. Four genotypes (47, 42, 43, and 63) were identified using MLVA-8 (panel 1), whereas six genotypes (138, 125, 116, 108, and two unknown genotypes) were identified using MLVA11 (panels 1 and 2A). From the review of MLVA-16 (panels 1, 2A, and 2B), panel 2B showed a very high discriminatory power. Two loci of Bruc04 and Bruc30 from this panel had diversity index values higher than 0.71 and the average diversity index was 0.619. So MLVA-16Orsay 34 showed the genotype indicating a low genetic homogeneity among the isolates. The findings of MLVA genotyping of the isolates suggest that strains of B. melitensis isolated from the milk of small ruminants in Iran are most closely related to the isolates from neighboring countries of the Eastern Mediterranean group. To the best of our knowledge, this is the first study to indicate the potential use of MLVA genotyping for simultaneous detection of specimen contamination using two different B. melitensis biovars.

Brucellosis seroprevalence in captive scimitar-horned oryx (Oryx dammah) in the United Arab Emirates and associated risk factors

The scimitar-horned oryx ( Oryx dammah ) (SHO) is a large African antelope that became extinct in the wild just over two decades ago. Conservation of the species is of prime importance, but it might face pathogen stressors. Brucella melitensis biovar 1 was previously confirmed in a high-density captive population of SHO held in Abu-Dhabi emirate. The infection reached 67.0% (95% CI: 64.0 - 70.0) individual seroprevalence (n=959) during testing performed between January 2013 and January 2015. A model based on a multivariable logistic regression analysis showed that the seroprevalence ranged from 51.2 (95% CI: 39.6 - 62.7) to 86.9% (95% CI: 82.4 - 91.4) between six different enclosures, and probability of being seropositive was 1.83 (95% CI: 1.32 - 2.55) higher in females than in males, 3.09 (95% CI: 1.66 - 5.91) and 9.35 (95% CI: 4.66 - 19.44) higher in subadults and adults than in juveniles, respectively. The three serological tests used in this study (Rose Bengal Test, lateral flow assay and in-house i-ELISA) had a perfect or near-perfect agreement (Cohen’s Kappa coefficient >=0.97). Recurrent high seroprevalence in time and congruence of results from three different serological tests point toward a persistent B.melitensis infection in a high-density captive SHO population. Conclusion and significance Testing strategy (Bengal Test, lateral flow assay or in-house i-ELISA) has no effect on the estimation of the brucellosis seroprevalence in SHO permitting the selection of a practical test. We call for an evidence-based control program, and Brucella vaccine efficacy and innocuity studies in this endangered species.

Isolation and characterisation of Brucella melitensis by bacteriological and molecular methods from livestock in North Cyprus

In this study, the isolation, biotyping and molecular characterisation of Brucella melitensis from cattle, sheep and goats in North Cyprus are reported on. A total of 319 raw milk samples obtained from seropositive dairy livestock (190 cattle, 74 sheep and 55 goats) and tissue samples including the liver, spleen and abomasal contents obtained from 32 aborted foetal samples (5 cattle, 18 sheep and 9 goats) were analysed for the presence and characterisation of the agent. B. melitensis was isolated and identified from 90 out of 319 (28.2%) milk and 19 out of 32 (59.4%) foetal samples by conventional bacteriological methods. Identification of all 109 isolates was confirmed by using real-time PCR with genus and species-specific primers. Following the preliminary identification, 27 selected isolates representing various counties and herds were further analysed by conventional methods. Twenty (74.1%) isolates were identified as B. melitensis biovar 1 and seven (25.9%) were identified as B. melitensis biovar 3. The Bruce-ladder multiplex PCR assay revealed that all the isolates were field strains. The results of the present study confirmed the presence of B. melitensis in livestock including the cattle population in North Cyprus. Even though the majority of the samples came from seropositive cattle, Brucella abortus was not isolated in the study. The results also revealed the potential public health risk of brucellosis in livestock emphasising the need of implementing strict control and eradication strategies against the disease in animal populations in order to protect human health.

Using a Relative Quantitative Proteomic Method to Identify Differentially Abundant Proteins in Brucella melitensis Biovar 3 and Brucella melitensis M5-90.

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.

The prevalence of camel brucellosis and associated risk factors

Camel brucellosis is a widespread and critical zoonotic infection caused by Brucella abortus and Brucella melitensis that negatively impact dairy and meat products. The current meta-analysis covers studies published over a course of 40 years, between 1 January 1980 and 1 April 2021, aiming to investigate the global prevalence of Brucella spp. in camels and related risk factors in different camel-keeping areas. Chi-squared test (I2 index) was applied to evaluate the heterogeneity of studies. A random-effect model (REM) estimated the pooled prevalence among subgroups. The literature search on camel brucellosis was performed among the available databases, including CABI, PubMed, Embase, Web of Science, Cochrane, Scielo, Scopus, and Science Direct. A total of 56 publications, comprising 205 data reports, were evaluated. Our results showed that the overall prevalence of camel brucellosis in the world was 9.23%. The lowest and highest prevalence rates of Brucella spp. infections among camels were reported in Oman (0.34%, 95%CI: 0.18–0.55) and Sudan (37.41%, 95%CI: 25.27–50.31), respectively. According to different risk factors, subgroup analysis showed that the location, sex, herd size, age, and mixed rearing with other animals could be considered the main risk factors impacting the occurrence of brucellosis in camels. The reported Brucella spp. in dromedary camel was Brucella melitensis (Biovar 1, 2, and 3), Brucella abortus (Biovar 3, 5, and 6), and Brucella suis. Our study represented remarkable differences between the prevalence of Brucella spp. in male (6.83% in 41 studies) and female (9.64% among 62 studies) camels. However, the present meta-epidemiological study would help improve control and surveillance approaches to prevent the spread of camel brucellosis among herds.

Evaluation of the genetic profiles of Brucella melitensis strain from Turkey using multilocus variable number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) techniques

Brucellosis is caused by Brucella, and Brucella melitensis is highly prevalent in small ruminants in Turkey. Our aim was to genotype 50 B. melitensis strains isolated from sheep, goat, and cattle abortion samples from different farms in seven geographical regions of Turkey between 2009 and 2017. Forty-six different genotypes were detected in 50 isolates studied according to the MLVA-16. Thirty out of 50 isolate profiles matched profiles from the database exactly, and the remaining 20 were absent. Of these 30 isolates, 93.3% were identical to human isolates previously present in the database. All B. melitensis strains belonged to the eastern Mediterranean group. Genotype 43 was the most common isolate profile, and sequence typing (ST8) was dominant and detected in 39 strains. MLST analysis revealed a novel profile in 11 strains. On comparing the sequences of ST8 and the novel ST, a glucokinase gene variation was detected. In the MLST and MLVA analyses, no distinction was made between B. melitensis biovars. Moreover, there was no significant difference between the strains based on host, region, and year. Consequently, the discrimination power of MLVA was higher than that of MLST in this study. Contrastingly, MLST was useful in distinguishing strains according to geographic origins, as determined by performer studies. Profiles determined by MLVA were the same as those in humans. This raises concerns in regard to One Health and transition between hosts, as it is clear that protecting animal health is very important for human health.

Isolation of Brucella melitensis from Lumbar Vertebrae in Patient with Initial Misdiagnosis: A Rare Case with Spinal Epidural Abscess, Spinal Stenosis, and Brain Lesion

Introduction: Brucellar spondylodiscitis is among the most typical forms of osteoarticular involvement that still challenges clinicians and scientists for early diagnosis. Case Presentation: We describe the isolation of Brucella melitensis from vertebrae in a man with spondylodiscitis who had osteoarthritis as an underlying condition. The patient showed negative results on blood samples' serological, molecular, and culture tests and had low-back pain, restricted lumbar movements, headache, poor appetite, and fatigue for the past nine months. He had a history of regular ingestion of raw cow milk and milk products for a long time. First, he was misdiagnosed as lumbar disc herniation and then underwent nonsteroidal anti-inflammatory drugs and myorelaxants treatment. The lack of fast diagnosis and appropriate treatment led to severe complications of the disease. Three months after the first magnetic resonance imaging (MRI), the findings of the second MRI without intravenous contrast showed right lateral recess and canal stenosis at L4 - L5 with narrowing the thecal sac at the disc space. Abnormal enhancement of the endplates at L4 - L5 with relating epidural space-enhancing tissue in the setting of spondylodiscitis and the associated epidural abscess was seen behind L4. Moreover, extensive high signal abnormalities in paraspinal tissues at L3, L4, L5, S1, S2, and S3 were notable. The diagnosis was approved by isolating B. melitensis biovar 1 from the culture of the vertebrate body. The Brucella isolate was characterized by Bruce-ladder PCR, AMOS PCR, and classical biotyping. The patient was treated successfully with surgical intervention and triple-antibiotic, including oral doxycycline 100 mg/12 h plus oral rifampin 300 mg/12 h for three months and intramuscular streptomycin 1 g daily for the first two weeks. The patient’s general condition and low-back pain were remarkably improved in the follow-up. Conclusions: Patient histories and a series of different diagnostic procedures such as MRI, serology, molecular, and cultural tests are essential to make a rapid and accurate diagnosis of brucellar spondylodiscitis, thereby reducing the delay for brucellar spondylodiscitis treatment.

Seroprevalence of brucellosis and chlamydiosis in sheep and goats with history of abortion in Iran

• Brucellosis and chlamydiosis are common causes of abortion in livestock. • The prevalence of brucellosis was significantly higher in goats compared to sheep. • Inversely, goats showed a lower prevalence of chlamydiosis compared to sheep. • Brucella melitensis biovars 1 was identified in sheep and goats aborted fetuses. Abortion in small ruminants is a critical issue in many developing countries including Iran and causes devastating economic losses in small ruminant industry. The aim of this study was to evaluate the seroprevalence of brucellosis and chlamydiosis in sheep and goats with a history of abortion during lambing seasons in Iran. Samples were collected during 2019 from sheep and goat flocks located in Fars province (Southern Iran). A total of 124 sera (101 sheep and 23 goats) from aborted females as well as 29 aborted fetuses were sampled and analyzed for Chlamydia abortus and Brucella spp. using indirect enzyme-linked immunosorbent assay (i-ELISA) for chlamydiosis and a set of serological (RBT, SAT and 2-ME and i-ELISA) and bacterial tests for brucellosis. Seropositivity to C. abortus was also detected in 21.7 % and 4.3 % of sheep and goats, respectively. Brucella specific antibodies were found in 29.1 %, 22.6 %, 20.9 % and 23.4 % of tested sheep and goats using RBT, SAT,2-ME and i-ELISA, respectively. Bacterial isolation and biotyping led to the characterization of B. melitensis biovars 1 in sheep and goats aborted fetuses of Fars province. High prevalence of infection associated with brucellosis and chlamydiosis in both sheep and goat with a history of abortion highlights risks of human zoonotic infections by these pathogens. Therefore, special attention is required for further study and control program to counteract these infections in animals and humans.

Trans-species transmission of Brucellae among ruminants hampering brucellosis control efforts in Egypt.

This study aimed to identify the genotypic fingerprinting of Brucella melitensis biovar 3 isolates from ruminants in Kafr El-Sheikh, Egypt, to compare with other peers globally and to highlight the epidemiology and potential causes of brucellosis control failure. A multilocus variable-number tandem-repeat analysis (MLVA 16) was carried out on 41 B.melitensis bv3 isolates, 31 from the preferential hosts (28 sheep and three goats) and 10 from atypical hosts (nine cattle and one buffalo), identified by bacteriological and molecular techniques. MLVA-16 analysis revealed 19 genotypes with nine as singletons. The most prevalent genotypes were M3_K.E (3,5,3,13,1,1,3,3,7,43,8,7,6,7,5,3), M13_K.E (3,5,3,13,1,1,3,3,7,43,8,5,8,7,7,3) and M5_K.E (3,5,3,13,1,1,3,3,7,43,8,4,8,7,11,3) circulating between different animal species. The B.melitensis isolation from aborted cows in farms that had never reared small ruminants indicates the likelihood of cow to cow B.melitensis transmission. Different genotypes of B.melitensis could be isolated from the same animal. The local geographic distribution of genotypes showed a very close genetic relatedness with genotypes reported outside the study area. Worldwide, our genotypes were mostly related to the Western Mediterranean lineage and less likely to the America's clonal lineage. There is a high genetic similarity of B.melitensis bv3 genotypes among different ruminant species, and the same animal could be infected with different genotypes. There is a high probability of spreading of B.melitensis among atypical hosts in the absence of the original hosts. The genetic relatedness of B.melitensis bv3 genotypes in the study area with other different geographic areas highlighted the national and international ruminants movement role as a potential factor for maintaining B.melitensis infection. Further investigations are required to understand the impact of the presence of more than one genotype of B.melitensis in the same animal on the efficacy of brucellosis control strategies.

Risk factors and Molecular genotyping of Brucella melitensis strains recovered from humans and their owned cattle in Upper Egypt

Brucellosis is a zoonosis that has a devastating impact on the economy and public health, particularly in the Middle East, including Egypt. This study aimed to define risk factors associated with brucellosis in humans and in their cattle in Fayoum governorate - Upper Egypt. Also, molecular genotyping of recovered Brucella isolates from human cases and their cattle to assess the potential cross-species transmission in the study region. Data were obtained via double matched case–control studies for brucellosis in humans (106 cases and 160 controls) and in their cattle (78 cattle cases and 105 cattle controls). The results of multivariate regression analysis revealed that predictors of human brucellosis were animal-related occupations (OR 2.1, P 0.02), previous infection in other household members (OR 3.2, P 0.007), eating home-made soft cheese (OR 2.3, P 0.03), and exposure to cattle abortions (OR 6.9, P < 0.001). For cattle, predictors of brucellosis were maturity ≥2 years of age (OR 2.9, P 0.01), ≥2 animals reared by the same household (OR 3.7–6.9, P ≤ 0.001), and recent abortion (OR 15.2, P 0.01). Twelve Brucella isolates were recovered from eight human cases (7.5%, 8/106) and four cattle cases (6.2%, 4/65). All isolates were B. melitensis biovar 3. Analysis of the IS711 gene sequence revealed complete homology (100%) between isolates. Six virulence genes were utilized for virutyping: virB (100%), omp25 (100%), amiC (100%), ure (91.7%), wbkA (91.7%), and bvfA (75%). Virutyping revealed four virutypes: V1 (lack bvfA, 16.7%), V2 (harbored all genes, 66.7%), V3 (lack wbkA, 8.3%), and V4 (lack wbkA and ure, 8.3%). Repetitive extragenic palindromic PCR (REP-PCR) typing revealed two REP types. Combined REP-PCR/virulence genotyping revealed five different genotypes (G1–G5) for the detected isolates and a unique genotype for the reference strain (G6, B. melitensis bv3 Ether). Human and cattle isolates from the same household had matched genotypes. In conclusion, there were widespread risk factors among the cases studied. Health education for high-risk groups is essential for disease prevention, and combined REP-PCR/virulence genotyping is a quick tool for traceability, particularly in developing countries endemic with brucellosis as Egypt.