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BackgroundHuman 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).MethodsThe 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.ResultsThe 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.ConclusionThis 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.

Abstract Human brucellosis cases are rare in non-endemic countries, such as Germany, where infections are predominantly caused by Brucella melitensis. The German National Reference Laboratory for Bovine, Porcine, Ovine and Caprine Brucellosis received a suspected Brucella sp. isolate from a patient for identification. Bacteriological tests and PCR-based diagnostics showed the isolate to be B. suis, but did not yield cohesive results regarding the biovar. Whole genome sequencing and subsequent genotyping was employed for a detailed characterization of the isolate and elucidating the reason for failure of the diagnostic PCR to correctly identify the biovar. The isolate was found to be B. suis bv. 5, a rare biovar with limited geographical distribution primarily found in the Northern Caucasus. Due to a deletion in one of the target regions of the diagnostic PCR, the isolate could not be correctly typed. Based on in silico genotyping it could be excluded that the isolate was identical to one of the B. suis bv. 5 reference strains. Here, we report a rare case of a B. suis bv. 5 field isolate. Furthermore, by reporting this finding, we want to make practitioners aware of possible misinterpretation of PCR results, as it cannot be excluded that the detected deletion is common among the B. suis bv. 5 community, as there is currently a lack of field isolates.

Abstract Background Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. Methods Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. Results In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. Conclusion The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

Brucellosis is a bacterial zoonosis worldwide and a potential health risk in Pakistan due to socio-economic reasons and lack of awareness, veterinary health and modern dairy-processing facilities. The aim of this study was to determine the burden of brucellosis in hospitalized patients of selected districts of Punjab, Pakistan. To this end, a total of 280 serum samples were collected from suspected patients in districts Lahore, Okara and Khushab between 2016 and 2018. Anti-Brucella antibodies were detected in 89 (31.8%) sera by Rose Bengal plate test (RBPT), and Brucella abortus DNA was amplified by polymerase chain reaction (PCR) in 42 (15%) samples. In univariable analysis, the seroprevalence varied significantly (p < 0.05) between districts with Lahore indicating 41/101 (40.6%) Odds ratio (OR) = 53.98 (7.22–403.67; 95% CI), Okara 47/99 (47.5%) OR = 71.4 (9.55–533.65; 95% CI) and Khushab 1/80 (1.3%) as reference. Similarly, exposure to aborted materials and a history of blood transfusion did associate significantly (p < 0.05), posing OR = 2.91 (1.58–5.36; 95% CI) and OR = 4.48 (2.56–7.84; 95% CI) risk. However, consumption of raw milk and history of blood transfusion did associate significantly (p < 0.05) in multivariable analysis indicating OR = 1.91 (1.09–3.34; 95% CI) and OR = 4.92 (2.76–8.75; 95% CI), respectively. The association of history of blood transfusion as a brucellosis risk factor underscores the need to create brucellosis awareness programs both at public and institutional levels in the medical and veterinary fields, as well as facilities and capacity building to delve deeper into the epidemiology of brucellosis in the country. Particular attention should be paid to chronically infected patients. Therefore, laboratory modernization, standardization and validation of the diagnostic tests, and staff trainings are required. Finally, pasteurization of the milk is recommended before consumption.

Abstract Background Bacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such as Bacillus anthracis, Brucella species, and Francisella tularensis. Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each of Ba. anthracis, Br. suis and F. tularensis. Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared. Results As previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids of Ba. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades for Ba. anthracis and F. tularensis as well as multilocus sequence types for Br. suis. For F. tularensis, high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. For Ba. anthracis, only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, for Br. suis, high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions. Conclusions In summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible for F. tularensis and Ba. anthracis, but not yet for Br. suis. The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future.

The zoonotic bacteria, Brucella canis, is becoming the leading cause of canine brucellosis in Europe. In dogs, it causes reproductive problems as well as non-specific lameness or discospondilitis. In humans, B. canis can be origin of chronic debilitating conditions characteristic to its genus such as undulant fever, splenomegaly, and lymphadenopathy. Although B. canis shows some pathogenic characteristics similar to B. abortus and B. melitensis, it lacks surface O-polysaccharide, like nonzoonotic B. ovis. This review shows that host–B. canis interactions are still poorly understood, with many knowledge and capability gaps, causing relatively poor sensitivity and specificity of existing diagnostic tools. Currently, there is no vaccine for this rough Brucella species. Besides, antimicrobial therapy does not guarantee bacterial elimination, and infection relapses are frequently reported, increasing the risks of antibiotic resistance development. B. canis has been detected in dogs in almost all European countries which increased human exposure, but currently there is no systematic surveillance. Moreover, B. canis caused brucellosis is not included in Animal Health Law, and therefore there is no legal framework to tackle this emerging infectious disease. To map out the diagnostic strategies, identify risks for human infections and propose management scheme for infected pet and kennel dogs, we present current understanding of canine B. canis caused brucellosis, outline major knowledge gaps and propose future steps. To address and highlight challenges veterinary and public health services encounter in Europe, we developed two B. canis infection scenarios: of a single household pet and of a kennel dog in larger group.

In 2021, a case of canine brucellosis diagnosed in a dog with orchitis was presented to a veterinary practice in Germany. Serological testing excluded Brucella (B.) canis as a causative agent, but molecular analysis revealed the presence of B. suis biovar 1. Since biovar 1 is not endemic in Europe and the dog had no history of travel to endemic areas, a comprehensive epidemiological investigation was conducted using whole genome sequence data to determine the source of infection. We describe the clinical progress of the animal and the potential infection of a veterinary clinic employee. The findings highlight the importance of considering less common Brucella species as possible causes of canine brucellosis. The data also emphasize that it is quite challenging to identify Brucella species in a routine diagnostic laboratory and to conduct epidemiological investigations to unveil possible transmission routes.

Abstract Background Brucellosis, a zoonosis mainly transmitted by consumption of unpasteurized dairy products as well as direct contact with infected animals, is endemic in Kyrgyzstan. However, Brucella species in humans have not been investigated and the origin of the disease remains poorly known in wide parts of Сentral Asia. Thus, molecular characterization of the circulating strains is a critical first step in understanding Brucella diversity in the country. Methods In this study, isolates were collected from patients with suspected brucellosis from different regions in Kyrgyzstan between 2019 and 2020. The detection and identification of Brucella was carried out by Bruce-ladder PCR. Next generation sequencing was used to sequence the 89 Brucella isolates, which were genotyped by cgSNP and cgMLST to identify epidemiological connection between Brucella isolates as well as placing them in the context of the global Brucella phylogeny. Results The Brucella strains isolated from all regions of Kyrgyzstan were identified as B. melitensis. Based on cgSNP analysis, 18 sequence types were differentiated. The highest numbers of different sequence types were found in Batken (n = 8), Osh (n = 8) and Jalal-Abad (n = 6) oblasts. According to cgSNP and cgMLST analyses, different B. melitensis lineages circulate in Kyrgyzstan, all of them belonging to the Eastern Mediterranean group of the global Brucella phylogeny with the highest similarity to strains from Turkmenistan, Iran and Turkey. Conclusion In the present study, B. melitensis was identified as a causative agent of human brucellosis in Kyrgyzstan and different lineages could be identified. Since this study focused on isolates of human origin, the identity of Brucella species and lineages circulating among animal populations remains elusive. Implementing culture techniques and use of most recent molecular, bioinformatic and epidemiological tools are needed to set up a One Health approach to combat brucellosis in Kyrgyzstan. Further, other Сentral Asian countries need to take part in this effort as brucellosis is a transboundary disease in these regions. Graphical Abstract

Abstract Background Brucella suis is a zoonotic pathogen with a serious impact on public health and the pig industry worldwide. Information regarding B. suis in pigs in Egypt is scarce. This study aimed to investigate the prevalence of B. suis in slaughtered domestic pigs at El-Basatin abattoir in Cairo, Egypt. A total of 1,116 domestic pigs slaughtered in 2020 were sampled for Brucella isolation and identification. Identified Brucella isolates were molecularly confirmed at species, and biovar levels using Bruce ladder PCR and Suis ladder multiplex PCR. Additionally, high-risk practices of 16 abattoir workers (4 veterinarians, 10 butchering and evisceration workers, and 2 scalding workers) were investigated using a pre-piloted structured questionnaire. Results Brucella isolates were recovered from 1.3% of examined pigs (n = 14) at consistently low rates (1.1—2.9%) across the year of sampling from February to December 2020. All isolates were confirmed as B. suis biovar (bv) 2. Remarkably, 92.9% (13/14) of isolates showed atypical ability to produce H2S and hence were considered as B. suis bv2 atypical phenotype. The prevalence was higher in males (1.8%) than in females (0.9). However, this difference was not significant (Odds ratio = 1.9; CI 95% 0.7 – 5.7; P = 0.2). No detectable pathological lesions were associated with B. suis bv2 infection in examined pigs. All strains were isolated from cervical lymph nodes, highlighting a potential oral transmission. High-risk practices were recorded among swine abattoir workers in this study: 75% do not wear gloves or disinfect their knives daily, and 18.8% were willing to work with open wound injuries. Conclusions To the best of our knowledge, this is the first isolation of B. suis bv2 in Egypt. Detection of H2S producing B. suis bv2 atypical phenotype is alarming as it may result in misinterpretation of these isolates as highly human pathogenic B. suis bv1 in Egypt and possibly elsewhere. Further epidemiological tracing studies are crucial for the detection of the origin of this biovar. Including pigs in the national surveillance program of brucellosis, and an education program for swine abattoir workers about occupational risk of B. suis is a need in Egypt.

Three species of white-toothed shrews of the order Eulipotyphla are present in central Europe: the bicolored (Crocidura leucodon), greater (Crocidura russula) and lesser (Crocidura suaveolens) white-toothed shrews. Their precise distribution in Germany is ill-defined and little is known about them as reservoirs for zoonotic pathogens (Leptospira spp., Coxiella burnetii, Brucella spp., Anaplasma phagocytophilum, Babesia spp., Neoehrlichia mikurensis and Bartonella spp.). We investigated 372 Crocidura spp. from Germany (n = 341), Austria (n = 18), Luxembourg (n = 2) and Slovakia (n = 11). West European hedgehogs (Erinaceus europaeus) were added to compare the presence of pathogens in co-occurring insectivores. Crocidura russula were distributed mainly in western and C. suaveolens mainly in north-eastern Germany. Crocidura leucodon occurred in overlapping ranges with the other shrews. Leptospira spp. DNA was detected in 28/227 C. russula and 2/78 C. leucodon samples. Further characterization revealed that Leptospira kirschneri had a sequence type (ST) 100. Neoehrlichia mikurensis DNA was detected in spleen tissue from 2/213 C. russula samples. Hedgehogs carried DNA from L. kirschneri (ST 100), L. interrogans (ST 24), A. phagocytophilum and two Bartonella species. This study improves the knowledge of the current distribution of Crocidura shrews and identifies C. russula as carrier of Leptospira kirschneri. However, shrews seem to play little-to-no role in the circulation of the arthropod-borne pathogens investigated.

We collected 10 Burkholderia mallei isolates from equids in 9 districts in India during glanders outbreaks in 2013–2016. Multilocus variable-number tandem-repeat analysis showed 7 outbreak area–related genotypes. The study highlights the utility of this analysis for epidemiologically tracing of specific B. mallei isolates during outbreaks.

Brucellosis is a neglected zoonotic disease of ruminants. It causes severe health problems in humans and significant economic loss. Only a limited number of studies have been conducted in Pakistan to determine the prevalence of human brucellosis and related risk factors. The objectives of the current cross-sectional study were to determine the prevalence of anti-Brucella antibodies in sera collected from symptomatic patients at three hospitals of Abbottabad using a commercial slide agglutination test (SAT) and to determine risk factors for brucellosis for these patients. Five hundred blood samples were collected. A questionnaire was filled in for each patient to obtain information on age, gender, living area, brucellosis associated symptoms, associated risk factors, pregnancy and abortion history. A total of 13.6% (n = 68) patients were found to be SAT positive and in 83.3% (n = 57) of these samples Brucella DNA was detected by genus specific RT-PCR for BCSP-31 gene. Statistical analysis was performed to determine odd ratios, risk ratios, 95% confidence intervals, and p-values. The prevalence of brucellosis by SAT was reported to be higher in women (14.6%, n = 44) than in men (12.1%, n = 24). The age group 25–50 years was found to be at higher risk for brucellosis (14.5%, n = 50) “animal contact” was reported as the main risk factor followed by “consumption of raw animal products.” Out of 131 pregnant women and 21 patients had abortion, the seropositivity of Brucellosis was 9.9% and 23.8%, respectively. The present study reports a striking prevalence of brucellosis among patients including pregnant women at three hospitals of Abbottabad. These findings may foster strategies for controlling human brucellosis at household level, raising of awareness about brucellosis in hospital and family doctors, and finally in setting up an eradication program in the dairy industry.

Brucellosis is a highly contagious and incapacitating disease of humans, livestock and wildlife species globally. Treatment of brucellosis in animals is not recommended, and in humans, combinations of antibiotics recommended by the World Health Organization are used. However, sporadic antimicrobial-resistant (AMR) isolates and relapse cases have been reported from different endemic regions. In the current study, molecular characterization and antibiotic susceptibility testing using the microdilution method for 35 B. abortus and B. melitensis strains isolated from humans, milk and animal were carried out. Additionally, Next-Generation-Sequencing (NGS) technology was applied to confirm Brucella at the species level and investigate AMR and pathogenicity-associated determinants. MALDI-TOF seemed to be a rapid and reliable tool for routine identification of brucellae to the genus level; however, DNA-based identification is indispensable for accurate species identification. Brucella abortus strains were isolated from two human cases and a sheep. Such infections are uncommon in Egypt. Egyptian Brucella strains are still in-vitro susceptible to doxycycline, tetracyclines, gentamicin, ciprofloxacin, levofloxacin, chloramphenicol, streptomycin, trimethoprim/sulfamethoxazole and tigecycline. Probable (no CLSI/EUCAST breakpoints have been defined yet) in-vitro resistance to rifampicin and azithromycin was observed. WGS failed to determine classical AMR genes, and no difference in the distribution of virulence-associated genes in all isolates was found. Isolates of human and non-human origins were still susceptible to the majority of antibiotics used for treatment in humans. The absence of classical AMR genes in genomes of “resistant” Brucella strains may reflect a lack of information in databases, or resistance might not be encoded by single resistance genes. The One Health approach is necessary for tackling brucellosis. Continuous susceptibility testing, updating of breakpoints, assessing mutations that lead to resistance are needed.

Brucellosis is an important bacterial zoonosis of domestic and wildlife species. This disease has a significant public health concern and is characterized by reproductive failure resulting in economic losses in the livestock industry. Among thirteen known species, B. abortus, B. melitensis, B. suis, and B. canis are human pathogens. Brucellosis has been extensively investigated in humans and domestic animals. However, the situation in wildlife is still not completely reported and studied. Therefore, a systematic literature search and screening were done to clarify the situation of brucellosis in wildlife in Europe. Sixty-five articles from a total of 13,424 reports published between 1991 and 2021 were selected, applying defined inclusion criteria. Wild boars and brown hares were the most often studied terrestrial wildlife species, whereas seals and porpoises were the most often investigated marine wildlife. Poland, Croatia, and Belgium showed the highest seroprevalences of wild boars caused by B. suis biovar 2. In marine wildlife, brucellosis was mainly caused by B. ceti and B. pinnipedialis. Most samples were from carcasses. Thus, sera could not be collected. It is worrisome that B.abortus and B. melitensis were reported from both terrestrial and marine wild animals, posing a zoonotic threat to people exposed to wild animals. Currently, there is no approved vaccine available for wild animals. The main challenges are the development of specific diagnostics and their validation for use in wildlife.

Brucellosis is a bacterial zoonotic disease that affects many animal species and can be transmitted to humans via direct contact or via contaminated food. Although brucellosis is a serious health hazard, its public health concern has been neglected in many countries. In some developing countries, such as Pakistan, where brucellosis is endemic, this disease continues to be of importance. A literature search for the past 11 years (2011–2021) provided a comprehensive insight into brucellosis in Pakistan. In this review, particular emphasis was placed on occurrence, diagnostic tests used, and prevention, treatment, and control in the context of the “One Health” approach.

Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller–Hinton without additives for the AST of B. melitensis.

Brucellosis is a globally reemerging and neglected zoonosis causing serious public health problems as well as considerable economic losses due to infection of livestock. Although the epidemiology of brucellosis has been well studied and its various aspects in humans and animals are well understood, it is still one of the most challenging health problems in many developing countries such as Kyrgyzstan. This review describes epidemiological characteristics of brucellosis in humans and animals, its impact on animal production and the role of implemented infection control measures in Kyrgyzstan. Particularly, introduction of mass vaccination in small ruminants evidently contributed to control of brucellosis in Kyrgyzstan, reducing the number of infections in animals as well as humans.

Brucellae are Gram-negative, aerobic, non-motile coccobacilli causing brucellosis in man and animals. The disease is one of the most significant yet neglected global zoonoses. Especially in developing countries, brucellosis is causing public health problems and economic losses to private animal owners and national revenues. Composed of oligonucleotides, aptamers are chemical analogues of antibodies that are promising components for developing aptamer-based rapid, sensitive, and specific tests to identify the Brucella group of bacteria. For this purpose, aptamers were generated and selected by an enhanced protocol of cell systematic evolution of ligands by exponential enrichment (cell-SELEX). This enhanced cell-SELEX procedure involved the combination of both conventional and toggle cell-SELEX to boost the specificity and binding affinity to whole Brucella cells. This procedure, combined with high-throughput sequencing of the resulting aptamer pools, comprehensive bioinformatics analysis, and wet lab validation assays, led to the selection of a highly sensitive and specific aptamer for those Brucella species known to circulate in Egypt. The isolated candidate aptamer showed dissociation constant (KD) values of 43.5 ± 11, 61.5 ± 8, and 56 ± 10.8 nM for B. melitensis, B. abortus, and B. suis, respectively. This is the first development of a Brucella-specific aptamer using an enhanced combination of conventional and toggle cell-SELEX to the authors’ best knowledge.

Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks.

Glanders, caused by Burkholderia (B.) mallei is a notifiable zoonotic disease in equidae. For international trade and movement of equids, certificates of negative serological test results for antibodies against B. mallei are required. To date, the complement fixation test (CFT) is the mandatory test to issue these health certificates. The CFT is difficult to standardize and, due to its poor specificity, often leads to false-positive reactions resulting in trade restrictions with considerable financial consequences. In the present study, the new ID Screen Glanders Double Antigen Multispecies ELISA (GLANDA- ELISA) (IDvet, Grabels, France) was evaluated using 400 negative and 370 glanders positive field samples of equidae. The GLANDA-ELISA was significantly more specific (99.8%) than the CFT (97.0%). Considering the comparable sensitivities of CFT (96.5%) and ELISA (98.1%), this new GLANDA-ELISA test appears a suitable confirmatory test and a realistic alternative for serological testing of horses for trade or movement.

Bovine brucellosis is a global zoonosis of public health importance. It is an endemic disease in many developing countries including Pakistan. This study aimed to estimate the seroprevalence and molecular detection of bovine brucellosis and to assess the association of potential risk factors with test results. A total of 176 milk and 402 serum samples were collected from cattle and buffaloes in three districts of upper Punjab, Pakistan. Milk samples were investigated using milk ring test (MRT), while sera were tested by Rose–Bengal plate agglutination test (RBPT) and indirect enzyme-linked immunosorbent assay (i-ELISA). Real-time PCR was used for detection of Brucella DNA in investigated samples. Anti-Brucella antibodies were detected in 37 (21.02%) bovine milk samples using MRT and in 66 (16.4%) and 71 (17.7%) bovine sera using RBPT and i-ELISA, respectively. Real-time PCR detected Brucella DNA in 31 (7.71%) from a total of 402 bovine sera and identified as Brucella abortus. Seroprevalence and molecular identification of bovine brucellosis varied in some regions in Pakistan. With the use of machine learning, the association of test results with risk factors including age, animal species/type, herd size, history of abortion, pregnancy status, lactation status, and geographical location was analyzed. Machine learning confirmed a real observation that lactation status was found to be the highest significant factor, while abortion, age, and pregnancy came second in terms of significance. To the authors' best knowledge, this is the first time to use machine learning to assess brucellosis in Pakistan; this is a model that can be applied for other developing countries in the future. The development of control strategies for bovine brucellosis through the implementation of uninterrupted surveillance and interactive extension programs in Pakistan is highly recommended.

Anthrax is a recurrent zoonosis in the Ukraine with outbreaks occurring repeatedly in certain areas. For determining whether several Bacillus anthracis genotypes are circulating in this region, four strains from various sources isolated from different regions of the Ukraine were investigated. By combining long- and short-read next-generation sequencing techniques, highly accurate genomes were reconstructed, enabling detailed in silico genotyping. Thus, the strains could be assigned to the Tsiankovskii subgroup of the “TransEurAsia” clade, which is commonly found in this region. Their high genetic similarity suggests that the four strains are members of the endemic population whose progenitor was once introduced in the Ukraine and bordering regions. This study provides information on B. anthracis strains from a region where there is little knowledge of the local population, thereby adding to the picture of global B. anthracis genotype distribution. We also emphasize the importance of surveillance and prevention methods regarding anthrax outbreaks, as other studies predicted a higher number of cases in the future due to global warming.

Brucellosis is reportedly endemic in ruminants in Pakistan. Both Brucella abortus and B. melitensis infections have been decumented in domestic animals and humans in the country. This study aimed to identify the burden of anti-Brucella antibodies in small ruminants as well as associated potential risk factors with its occurrence at nine institutional livestock farms in Punjab, Pakistan. The sera collected from equal number of sheep and goats (500 from each species) were screened by indirect-ELISA for anti-smooth-Brucella antibodies followed by a serial detection by real-time PCR. Overall, 5.1% (51/1000) seropositivity was registered corresponding to 5% (25/500) prevalence in goats and 5.2% (26/500) in sheep. Brucella-DNA could not be detected in any of the tested sera by real-time PCR. Multiple logistic regression model indicated that farm location (OR 34.05), >4 years of age (OR 2.88), with history of reproductive disorders (OR 2.69), and with BCS of ≤ 3 (OR 12.37) were more likely to test positive for brucellosis at these farms. A routine screening, stringent biosecurity, and quarantine measures are warranted for monitoring and eradication of the infection. Similarly, isolation and molecular investigation of the etiologic agent(s) are needed to understand the relationship of epidemiology and out-breaks of brucellosis in the country.

Brucellosis, caused by the bacteria of the genus Brucella, is one of the most neglected common zoonotic diseases globally with a public health significance and a high economic loss among the livestock industry worldwide. Since little is known about the distribution of B. abortus in Egypt, a total of 46 B. abortus isolates recovered between 2012–2020, plus one animal isolate from 2006, were analyzed by examining the whole core genome single nucleotide polymorphism (cgSNP) in comparison to the in silico multilocus variable number of tandem repeat analysis (MLVA). Both cgSNP analysis and MLVA revealed three clusters and one isolate only was distantly related to the others. One cluster identified a rather widely distributed outbreak strain which is repeatedly occurring for at least 16 years with marginal deviations in cgSNP analysis. The other cluster of isolates represents a rather newly introduced outbreak strain. A separate cluster comprised RB51 vaccine related strains, isolated from aborted material. The comparison with MLVA data sets from public databases reveals one near relative from Argentina to the oldest outbreak strain and a related strain from Spain to a newly introduced outbreak strain in Egypt. The distantly related isolate matches with a strain from Portugal in the MLVA profile. Based on cgSNP analysis the oldest outbreak strain clusters with strains from the UK. Compared to the in silico analysis of MLVA, cgSNP analysis using WGS data provides a much higher resolution of genotypes and, when correlated to the associated epidemiological metadata, cgSNP analysis allows the differentiation of outbreaks by defining different outbreak strains. In this respect, MLVA data are error-prone and can lead to incorrect interpretations of outbreak events.

Bovine brucellosis is a contagious zoonotic disease that causes economic losses through abortion and infertility. A cross-sectional study was designed to determine the seroprevalence and associated risk factors of bovine brucellosis in district Gujranwala of Punjab, Pakistan. A total of 220 bovine sera (112 from buffaloes, 108 from cattle) from 46 unvaccinated herds were collected. Parallel testing by the Rose Bengal Plate Test (RBPT) and Indirect Enzyme-linked Immunosorbent Assay (I-ELISA) showed a 58.7% (27/46) herd-level and 22.7% (50/220) animal-level seroprevalence. Seroprevalence was higher (p < 0.001, OR = 7.62) in adult animals (37.2%) compared to younger animals (4.9%). A herd size of >10 animals (p = 0.021, OR = 7.83), less housing space (p = 0.037, OR = 6.39) and history of abortion at the farm (p = 0.023, OR = 5.6) were found as risk factors associated with the seropositivity of brucellosis. There was a substantial agreement between the RBPT and I-ELISA results (Cohen’s kappa coefficient (κ) = 64.16, percent agreement = 89.5%). In conclusion, a relatively higher seroprevalence was found compared to the previous reports from the country. Standardization and validation of the advanced diagnostic tests would be needed. Biosecurity, personal protection, quarantine measures and routine screening of animals at the farm level and disease awareness programs and consumption of pasteurized milk in the human population will be helpful in preventing the transmission/zoonosis of the disease.

Brucellosis is a highly contagious zoonosis that occurs worldwide. Whole-genome sequencing (WGS) has become a widely accepted molecular typing method for outbreak tracing and genomic epidemiology of brucellosis. Twenty-nine Brucella spp. (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were isolated from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats originating from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR. Illumina MiSeq® was used to sequence the 29 Brucella isolates. Using MLST typing, ST11 and ST1 were identified among B. melitensis and B. abortus, respectively. Brucella abortus and B. melitensis isolates were divided into two main clusters (clusters 1 and 2) containing two and nine distinct genotypes by core-genome SNP analysis, respectively. The genotypes were irregularly distributed over time and space in the study area. Both Egyptian B. abortus and B. melitensis isolates proved to be genomically unique upon comparison with publicly available sequencing from strains of neighboring Mediterranean, African, and Asian countries. The antimicrobial resistance mechanism caused by mutations in rpoB, gyrA, and gyrB genes associated with rifampicin and ciprofloxacin resistance were identified. To the best of our knowledge, this is the first study investigating the epidemiology of Brucella isolates from livestock belonging to different localities in Egypt based on whole genome analysis.

Brucellosis is a neglected zoonotic disease, and occupations with a high risk of infection exist. Limited information is available on brucellosis for these employees at high risk in Pakistan. A total of 459 persons with high-risk occupations, i.e., 211 abattoir workers, 63 milkers, 52 dung cake makers, 44 veterinarians, 44 shepherds, and 45 veterinary students of the districts Kasur and Lahore, Pakistan, were tested in this study. Blood samples and information on place of residence, gender, age, urbanicity, type of occupation, socioeconomic status, contact with animals, consumption of raw milk, contact to women who had a miscarriage, contact to aborted animal fetus or abortion material, pregnancy, miscarriage history, or intrauterine fetal death were collected. Serum samples were examined using Rose Bengal tests for anti-Brucella antibodies and seropositive samples were subjected to genus-specific qPCR for the detection of DNA. Data were analyzed using chi-squared and binary regression. Twenty (4.35%) persons were seropositive for anti-Brucella antibodies. Out of these, 18 (90%) were tested positive by Brucella genus-specific qPCR. Positive sera were more often found in Lahore district (8.3%) than in Kasur district (3.1%). Persons older than 36 years were more often seropositive. Persons involved in the handing of milk and in contact with animals were more often seropositive. Contact with women who had had a miscarriage was also a significant factor for seropositivity for brucellosis. Place of residence, gender, age, urbanicity, and contact with animals were identified as potential risk factors in the present study. The present study confirmed that brucellosis is an occupation hazard for abattoir workers, milkers, dung cake makers, and veterinarians in the districts of Lahore and Kasur of Punjab, Pakistan. The study shows the need for strategies for safety at work to minimize the risk of infection. Raising awareness for the prevention and use of proper personal protection equipment during the slaughtering and treatment of animal is highly needed.

During 2014–2015, patients in northeastern Kenya were assessed for brucellosis and characteristics that might help clinicians identify brucellosis. Among 146 confirmed brucellosis patients, 29 (20%) had negative serologic tests. No clinical feature was a good indicator of infection, which was associated with animal contact and drinking raw milk.

Brucella canis is one of many responsible pathogens of discospondylitis in dogs and infections require specific management. Little is known about the epidemiologic situation in Europe. The purpose of the study was to get insights into the occurrence of brucellosis in dogs in Europe. The database of a European veterinary laboratory was screened for Brucella positive samples. Additionally, medical records of a veterinary hospital in Germany were screened for diagnosis of discospondylitis and brucellosis. The laboratory received samples from 20 European countries for Brucella testing in dogs: 3.7% of submitted samples were Brucella spp. PCR-positive (61/1,657), and Brucella canis antibodies were identified in 5.4% of submitted samples (150/2,764). Brucella spp. PCR-positive samples originated from Spain (11.1% of submitted samples), Poland (6.7% of submitted samples) and rarely from Italy and France. Samples with Brucella canis antibodies originated from 13 European countries (Sweden, Belgium, Austria, Switzerland, Italy, Finland, Germany, Denmark, Hungary, Norway, Poland, France, Netherlands). Young dogs (0–24 months) had a 5.4-fold increased risk of PCR positive samples. The supplementary medical records search identified four young female dogs (7–30 months) with Brucella canis discospondylitis in Germany. The four dogs had been imported to Germany from Eastern European countries (Moldavia, Romania, Macedonia). In conclusion, infection with Brucella canis needs to be considered in dogs in Europe and diagnostics for Brucella canis infection appear indicated in young dogs with discospondylitis.

Glanders is a zoonotic contagious disease of equids caused by Burkholderia (B.) mallei. Serodiagnosis of the disease is challenging because of false-positive and false-negative test results. The accuracy of the complement fixation test (CFT) which is prescribed for international trade by the World Organisation for Animal Health (OIE), five ELISAs and a Western blot (WB) were compared for serodiagnosis of glanders using sera from 3,000 glanders-free and 254 glanderous equids. Four ELISA tests are based on recombinant antigens (TssA, TssB, BimA and Hcp1), the IDVet ELISA is based on a semi-purified fraction of B. mallei and WB makes use of a purified LPS-containing B. mallei-antigen. Sensitivity and specificity of tests were estimated using cut-off values recommended by the test developers. The WB and all ELISAs, except BimA, were significantly more specific than the CFT. ELISAs based on TssA, TssB, and BimA antigens had significantly lower sensitivity compared to CFT while the sensitivities of the Hcp1-ELISA, the IDVet-ELISA and the WB did not differ significantly from that of the CFT. Given their comparable sensitivities and specificities, the CFT (98.0%, 96.4%), the WB (96.8%, 99.4%), the Hcp1-ELISA (95.3%, 99.6%) and the IDVet-ELISA (92.5%, 99.5%) should be further developed to meet OIE requirements.

Brucellosis, globally known bacterial zoonosis, is endemic to Pakistan. B. abortus in bovines, B. melitensis in small ruminants and B. canis in dogs mainly cause this disease. A total of 1821 sera (1196 from sheep and 625 from goats) from animal herds near the Pakistan–Afghanistan border were collected. In parallel testing of sera for anti-Brucella antibodies (B. abortus and B. melitensis) was carried out by RBPT and indirect ELISA. The presence of Brucella DNA in sera was tested by real-time PCR. The overall percentage of seropositive samples was 0.99 (18/1821) by both tests. All positive samples originated from Baluchistan territory which translated into 1.76% (18/1021). None of the positive sera had signals for Brucella DNA and none of sera from goats carried detectable antibodies. Both tests showed an almost perfect agreement with Kappa statistics. The flock size was found to be associated with the presence of anti-Brucella antibodies. The samples of Khyber Pakhtunkhwa (KPK) tested negative in both serological tests and hence were not processed for real-time PCR. The present study shows the presence of anti-Brucella antibodies in sheep in the Baluchistan region of Pakistan. Diagnostic services need to be improved and test and slaughter policies might be implemented for eradication of Brucella infection in these areas. Awareness about the infection is needed at the farmer’s level. Isolation and molecular biology of the isolates could help with understanding the prevailing etiology in a better way.

Brucellosis is an important zoonosis worldwide. Equines are susceptible to the infection when in close contact with infected animals. The objective of our study was to update the existing knowledge and detect and differentiate the causative agent of brucellosis in breeding equines in Punjab, Pakistan. A cross-sectional study was designed to evaluate the occurrence and etiology of the infection in the equine population in three districts. A total of 448 equine sera were collected from three prefectures viz. Sahiwal, Khanewal, and Okara of the Punjab Province of Pakistan. Ninety-six (21.4%) samples were found positive by RBPT, 3.56% (16/448) by iELISA, and 4.24% (19/448) by CFT. Real-time PCR demonstrated the presence of Brucella abortus-DNA in sero-positive samples. Age and location were found as risk factors. The study concludes equine brucellosis seroprevalence in the country where Brucella abortus as the main etiology. Fistulous withers and poll evil cases should be treated with care as they could be hazardous and a source of zoonotic transmission. Routine screening at an early age, vaccination in ruminants, and consumption of pasteurized dairy milk in humans is recommended for prevention of the infection. Specific tests need to be standardized and validated.

Brucellosis is one of the most important worldwide zoonoses of many countries including Egypt. Camel brucellosis has not gained much attention in Egypt yet. This study is focused on the three governorates with the highest camel populations and the largest camel markets in the country to determine the disease seroprevalence and identify the Brucella species in local camel holdings. In total, 381 serum samples were collected from male and female camels from Giza, Aswan, and Al-Bahr Al-Ahmar (the Red Sea) governorates. Samples were serologically examined using the Rose–Bengal plate test (RBPT), indirect ELISA (i-ELISA), competitive ELISA (c-ELISA) and complement fixation test (CFT). Brucella antibodies were detected in 59 (15.5%), 87 (22.8%), 77 (20.2%) and 118 (31.0%) of sera by RBPT, i-ELISA, c-ELISA and CFT, respectively. Using real-time PCR, Brucella DNA was amplified in 32 (8.4%) seropositive samples including Brucella abortus (25/32), Brucella suis (5/32) and Brucella melitensis (2/32), defining a complex epidemiological status. To the best of our knowledge, this is the first study reporting Brucella suis DNA in camel serum. The risk-associated factors including age, sex, breed and geographical distribution were statistically analyzed, showing non-significant association with seroprevalence. The results of this study will raise awareness for camel brucellosis and help develop effective control strategies.

Brucellosis is a common zoonotic disease in Egypt. However, there are limited data available on the genetic diversity of brucellae circulating in Egypt and other Mediterranean areas. One hundred and nine Brucella (B.) strains were isolated from different animal species in thirteen Egyptian governorates. Multi-locus variable number tandem repeats (VNTRs) analysis (MLVA-16) was employed to determine the geographical relatedness and the genetic diversity of a panel of selected Egyptian strains (n = 69), with strains originating from Italy (n = 49), Portugal (n = 52), Greece (n = 63), and Tunisia (n = 4). Egyptian B. melitensis strains clustered into two main clusters containing 21 genotypes. Egyptian B. abortus strains clustered into three main clusters containing nine genotypes. The genotypes were irregularly distributed over time and space in the study area. Egyptian strains of B. melitensis showed MLVA-16 patterns closer to that of Italian strains. Egyptian B. abortus strains isolated from cattle share the same genotype with strains from Portugal and similar to strains from Italy with low genetic diversity. Strains with similar MLVA patterns isolated from different governorates highlight the movement of the pathogen among governorates. Hence, it may also reflect the long endemicity of brucellosis in Egypt with earlier dispersal of types and great local genetic diversity. Open markets may contribute to cross-species transmission and dissemination of the new types nationwide. The presence of West Mediterranean lineages of B. melitensis and relatedness of B. abortus strains from the studied countries is a result of the socio-historical connections among the Mediterranean countries. Transnational eradication of brucellosis in the Mediterranean basin is highly demanded.

Bovine anaplasmosis is a tick-borne disease with zoonotic potential, caused by the obligate intracellular bacterium Anaplasma marginale. The disease is distributed worldwide in tropical and subtropical regions. The economic losses from anaplasmosis in animals is of significant importance because it causes severe morbidity and mortality in cattle. Recovered animals may become persistent carriers. Epidemiological information on the actual status of bovine anaplasmosis in Egypt is scarce. Thus, this study aimed to determine anti-Anaplasma antibody and DNA in serum samples using ELISA and PCR, respectively. In total, 758 bovine sera were collected from cattle farms located in 24 Egyptian governorates in 2015 to 2016. Sera were analyzed with the commercially available ‘Anaplasma antibody competitive ELISA v2’ kit and ‘AmpliTest Anaplasma/Ehrlichia spp. real time TaqMan TM PCR. Anaplasma spp. antibodies were detected in 140 (18.5%) (CI: 15.8−21.4%) of the investigated sera by ELISA, and Anaplasma/Ehrlichia-DNA was detected in 40 (5.3%) (CI: 3.8−7.1%) of the positive sera by real time PCR. Co-detection of both Anaplasma spp. and Coxiella burnetii-specific antibodies was proven in 30 (4%) of the investigated sera. The results of this work confirm the significant prevalence of bovine anaplasmosis in Egypt. Raising awareness in decision makers of the public health, veterinarians and animal owners is required to reduce the spread of infection.

Brucellosis is a highly contagious bacterial disease affecting a wide range of animals, as well as humans. The existence of the clinically diagnosed brucellosis in avian species is controversially discussed. In the current study, we set to summarize the current knowledge on the presence of brucellae in avian species. Anti-Brucella antibodies were monitored in different avian species using classical diagnostic tools. Experimental infection of chicken embryos induced the disease and resulted in the development of specific lesions. Few empirical studies have been performed in adult poultry. However, the isolation of brucellae from naturally-infected chickens has not been possible yet.

Brucellosis is a highly contagious zoonosis worldwide with economic and public health impacts. The aim of the present study was to identify Brucella (B.) spp. isolated from animal populations located in different districts of Egypt and to determine their antimicrobial resistance. In total, 34-suspected Brucella isolates were recovered from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR. Antimicrobial susceptibility testing against clinically used antimicrobial agents (chloramphenicol, ciprofloxacin, erythromycin, gentamicin, imipenem, rifampicin, streptomycin, and tetracycline) was performed using E-Test. The antimicrobial resistance-associated genes and mutations in Brucella isolates were confirmed using molecular tools. In total, 29 Brucella isolates (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were identified and typed. The resistance of B. melitensis to ciprofloxacin, erythromycin, imipenem, rifampicin, and streptomycin were 76.2%, 19.0%, 76.2%, 66.7%, and 4.8%, respectively. Whereas, 25.0%, 87.5%, 25.0%, and 37.5% of B. abortus were resistant to ciprofloxacin, erythromycin, imipenem, and rifampicin, respectively. Mutations in the rpoB gene associated with rifampicin resistance were identified in all phenotypically resistant isolates. Mutations in gyrA and gyrB genes associated with ciprofloxacin resistance were identified in four phenotypically resistant isolates of B. melitensis. This is the first study highlighting the antimicrobial resistance in Brucella isolated from different animal species in Egypt. Mutations detected in genes associated with antimicrobial resistance unravel the molecular mechanisms of resistance in Brucella isolates from Egypt. The mutations in the rpoB gene in phenotypically resistant B. abortus isolates in this study were reported for the first time in Egypt.

Brucellosis is considered as endemic disease of animals and humans since thousands of years in Egypt. However, brucellosis in pigs has never been reported in Egypt. Thus, serological and molecular assays were applied to detect anti-Brucella antibodies and DNA in serum samples collected from pigs. In total 331 blood samples collected from male and female pigs at slaughterhouses of Cairo and Giza governorates were investigated using Brucella c- and i-ELISA and Brucella real-time PCR. Anti-Brucella antibodies were detected in 16 (4.83%) and 36 (10.8%) sera by i-ELISA and c-ELISA, respectively. Brucella DNA was detected in 10 (3.02%) seropositive samples and identified as Brucella melitensis (7/10) and Brucella suis (3/10). A higher prevelance was found in boars. This is the first study investigating pig brucellosis in Egypt. The results of this study will raise awareness for brucellosis in these farm animals and will help to develop effective control strategies.

Trueperella pyogenes was isolated from a dog and a cat with a mixed infection with Brucella abortus. Both lived on a dairy cattle farm with a history of regular cases of abortion and mastitis. Identification of the bacteria was done by means of MALDI-TOF MS, loop-mediated isothermal amplification (LAMP) based on cpn60, partial 16S rRNA sequencing, and growth on Loeffler Serum Medium. Isolation of Trueperella pyogenes on the dairy farm highlights its neglected role in reproduction failure and draws attention to its effects in the dairy industry in Egypt. Diagnosis and control of abortion in Egypt should include Trueperella pyogenes as one of possible causes of abortion.

Here, we provide the dataset associated with our research article on comprehensive screening of Brucella immunoreactive proteins using sera of naturally infected hosts published in Biochemical and Biophysical Research Communications Wareth et al., 2015 [1]. Whole-cell protein extracts were prepared from Brucella abortus and Brucella melitensis, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently western blotting was carried out using sera from bovines (cows and buffaloes) and small ruminants (goats and sheep). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [2] with the dataset identifiers PXD001270 and DOI:10.6019/PXD001270.

Brucellosis is an infectious disease caused by bacteria of the genus Brucella (B.), affecting both animals and humans, causing severe economic loses and severe illness, respectively. The objective of the present study was to determine the seroprevalence and the risk factors associated with caprine, ovine, and bovine brucellosis in selected districts of Punjab, Pakistan. A total of 1083 blood samples were randomly collected from animals (goats = 440, sheep = 203, cows = 206, and buffaloes = 234). Questionnaires were used to collect data on risk factors associated with brucellosis on the sampling day. All samples were initially screened for anti-Brucella antibodies using the rose bengal plate test (RBPT). The seropositive serum samples were confirmed by a quantitative real-time polymerase chain reaction (PCR) assay for the detection of the Brucella genus- and Brucella species-specific DNA (B. abortus and B. melitensis). Univariant and binary logistic regression were used to identify important risk factors of brucellosis. Anti-Brucella antibodies and DNA were detected in 35 (3.23%) serum samples. Thirty-four (97.1%) DNA samples were confirmed as B. melitensis by qRT-PCR. Abortion history and natural mating were found to be potential risk factors. Brucella melitensis was identified as the causative agent of caprine, ovine, and bovine brucellosis in the selected districts of Punjab, Pakistan. Diseased animals may act as a source of infection for other animals. The elimination of positive seroreactors, development of control strategies for brucellosis, and education programs regarding the control of zoonotic disease are highly needed in developing countries like Pakistan.

Brucellosis is a zoonosis occurring worldwide, with economic and public health impacts. Its diagnosis remains a challenge in endemic countries and basically relies on serology. The present study was carried out on two dairy cattle farms allegedly free from brucellosis, but with sporadic cases of abortion. The aim of this study was to investigate the presence of Brucella (B.) spp. in uterine discharge of seronegative cows after abortion. In farm I, B. melitensis biovar (bv) 3 was cultured from two of five cows after abortion, while in farm II, B. abortus bv 1 was cultured from three of eleven cows after abortion. These cows had been intrauterinely infected but remained seronegative until abortion and seroconverted only thereafter. Shedding of brucellae in uterine discharge of culture positive/seronegative aborting cows is a serious problem resulting in maintenance and further spread of infection. Thus, serosurveys in endemic countries have to be accompanied by molecular detection and/or culture of aborted material to close the diagnostic window and to hinder uncontrolled spread.

Brucellosis is a worldwide distributed zoonosis that causes important economic losses to animal production. In Brazil, information on the distribution of biovars and genotypes of Brucella spp. is scarce or unavailable. This study aimed (i) to biotype and genotype 137 Brazilian cattle isolates (from 1977 to 2008) of B. abortus and (ii) to analyze their distribution. B. abortus biovars 1, 2 and 3 (subgroup 3b) were confirmed and biovars 4 and 6 were first described in Brazil. Genotyping by the panel 1 revealed two groups, one clustering around genotype 40 and another around genotype 28. Panels 2A and 2B disclosed a high diversity among Brazilian B. abortus strains. Eighty-nine genotypes were found by MLVA16. MLVA16 panel 1 and 2 showed geographic clustering of some genotypes. Biotyping and MLVA16 genotyping of Brazilian B. abortus isolates were useful to better understand the epidemiology of bovine brucellosis in the region.

Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.