Seroprevalence of Brucella Infection in a Cohort of HIV-Positive Malawian Pregnant Women Living in Urban Areas.

Background: The seroprevalence of Brucella infection in sub-Saharan regions is high, and no recent data are available for Malawi, a country in which >60% of the population is involved in agropastoral activity. Aim: To evaluated the seroprevalence of Brucella in a cohort of HIV-positive pregnant women, living in an urban setting in Malawi. Methods: Sera of 201 pregnant women were tested for Brucella IgG. The Rose Bengal Plate Test and Serum Agglutination Tube test were used to determine antibody titer. Results: Five out of 201 (2.48%) women show positivity to Brucella, consistent with a past exposition to the infection. All five women delivered healthy infants, but two of them reported previous abortion/stillbirths, with a higher rate than those of the rest of the cohort (40% vs. 21.5%). Conclusions: This is one of the first reports of exposure of pregnant women to Brucella infection in Malawi, providing evidence of Brucella occurrence in an urban setting. Control programs should be introduced to reduce its impact on animal and human health.

Production of a Bacillus anthracis Secretome with Suitable Characteristics as Antigen in a Complement Fixation Test.

In this study, we cultured the Bacillus anthracis vaccine strain Sterne 34F2 in a medium containing EDTA, and we assessed the best conditions to inhibit the activity of zinc-dependent metalloproteases to obtain a secretome containing a high concentration of non-degraded PA (PA83), as evaluated by the SDS-PAGE analysis. Then, we used this secretome as the antigen in a Complement Fixation Test (CFT) to monitor the production of antibodies against PA83 in the sera of rabbits vaccinated with Sterne 34F2 and then infected with a B. anthracis virulent strain to evaluate the potency of the vaccine. The PAS-based CFT results were compared with those obtained by using a commercial ELISA kit. The two serological tests gave similar results in terms of specificity and sensitivity, as the kinetics of the antibodies production was very similar. The Sterne 34F2 vaccine induced an antibody response to PA83, whose titer was not inferior to 1:8 in PAS-based CFT and 42 kU/mL in PA83-based ELISA, respectively, in all vaccinated rabbits. Our opinion is that the PAS-based CFT can be successfully employed in humans and in animals for epidemiological retrospective studies or post-vaccination monitoring. We also suggest the use of our method to test the efficacy of veterinary anthrax vaccines.

Brucella melitensis B115-based ELISA to unravel false positive serologic reactions in bovine brucellosis: a field study.

BACKGROUND: Brucellosis is a zoonosis whose incidence is not declining worldwide despite the global effort to control the disease. Accurate and precise diagnosis is a crucial step in any prophylaxis program but single tests to unequivocally detect animals infected with Brucella spp. are currently unavailable. In Italy, serological diagnosis of bovine brucellosis is performed with two official tests: a rapid agglutination test (i.e., Rose Bengal Plate test, RBPT) and a complement fixation test (CFT) that detect antibodies directed mainly to the smooth lipopolysaccharide (S-LPS). Neither of the two tests is able to avoid the detection of false positive serological reactions (FPSRs) caused by bacteria sharing S-LPS components with Brucella spp. and responsible for the single reactors (SR) phenomenon. A B. melitensis R strain-based ELISA showed a good diagnostic performance in unravelling FP animals; however, since a limited number of animals were analyzed in that study, a large field study was conducted here to discriminate between Brucella-infected from FP animals, with the final aim of reducing the unnecessary slaughter of the latter. An ELISA based on a R strain of Brucella, i.e., Brucella melitensis B115, was employed to measure specific IgG responses in a collection of bovine sera (n = 648). Sera were obtained from 180 farms (either officially brucellosis-free or not brucellosis-free) recruited during an extended period of time (2014-2018) and were preliminarily assayed with the official tests by the Italian Reference Centers and then subjected to the ELISA. RESULTS: Negative sera, when subjected to the ELISA, gave O.D. values below the cutoff; SR sera, i.e. RBPT positive and CFT negative, as well as double positive (DP) sera, i.e. RBPT and CFT positive, gave O.D. values that were below the cutoff. All positive sera, i.e. from Brucella-infected animals, were RBPT positive and CFT positive (ICFTU ranging from 20 to 1280) and gave ELISA O.D. values above the cutoff. CONCLUSIONS: The B. melitensis B115-based ELISA systematically unravelled all false positive (FP) sera while confirming the diagnosis in Brucella-infected animals. Thus, the test employed in the present study may complement the official assays to avoid the costly slaughter of FP animals.

Unexpected human cases of cutaneous anthrax in Latium region, Italy, August 2017: integrated human-animal investigation of epidemiological, clinical, microbiological and ecological factors.

On 31 August, a veterinarian and a farmworker were hospitalised for skin lesions. Both had been exposed to a dead cow on 19 August on a farm near Rome, where eight further cattle died of confirmed anthrax later the same month. At admission, the first case showed a black depressed eschar and another smaller lesion on one hand. The second case presented deep infection of the skin, with involvement of both arms. Anthrax diagnosis was confirmed by detection of B. anthracis DNA in eschar fragments from both patients. T-cell specific immunity was studied by flow cytometry and Elispot assay after stimulation with B. anthracis secretome in blood samples collected from Case 1. Immunoglobulin production was detected by complement fixation assay. In Case 1, specific CD4+ T-cell activation was detected, without antibody production. Specific antibodies were detected only in the second patient with severe cutaneous illness. Both patients recovered. The two human anthrax cases were epidemiologically linked, but anthrax was not suspected at admission in either case. The veterinarian had initially unrecognised professional exposure and the exposed farmworker did initially not report exposure to affected animals. A One Health strategy integrating human and animal investigations was essential to confirm the diagnosis.

Rapid and safe one-step extraction method for the identification of Brucella strains at genus and species level by MALDI-TOF mass spectrometry.

Brucellosis is essentially a disease of domesticated livestock; however, humans can also be infected via the consumption of contaminated meat or dairy products, underlying the need for rapid and accurate identification methods. Procedures for microbiological identification and typing of Brucella spp. are expensive, time-consuming, and must be conducted in biohazard containment facilities to minimize operator risk. The development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based assay has reduced the processing time while maintaining performance standards. In this study, to improve the identification accuracy and suitability of the MALDI-TOF-based assay for routine diagnosis, we developed a new protein extraction protocol and generated a custom reference database containing Brucella strains representative of the most widespread species. The reference library was then challenged with blind-coded field samples isolated from infected animals. The results indicated that the database could be used to correctly identify 99.5% and 97% of Brucella strains at the genus and species level, respectively, indicating that the performance of the assay was not affected by the different culture conditions used for microbial isolation. Moreover, the inactivated samples were stored and shipped to reference laboratories with no ill effect on protein stability, thus confirming the reliability of our method for routine diagnosis. Finally, we evaluated the epidemiological value of the protocol by comparing the clustering analysis results of Brucella melitensis strains obtained via multiple locus variable-number tandem repeat analysis or MALDI-TOF MS. The results showed that the MALDI-TOF assay could not decipher the true phylogenetic tree, suggesting that the protein profile did not correspond with the genetic evolution of Brucella.

Development of a Sterne-Based Complement Fixation Test to Monitor the Humoral Response Induced by Anthrax Vaccines.

Anthrax is a zoonotic disease caused by Bacillus anthracis spore-forming bacterium. Since it is primarily a disease of animals, the control in animals, and humans depend on the prevention in livestock, principally cattle, sheep, and goats. Most veterinary vaccines utilize the toxigenic, uncapsulated (pXO1+/pXO2-) B. anthracis strain 34F2 which affords protection through the production of neutralizing antibodies directed to the toxin components Protective Antigen (PA), Lethal Factor (LF), and Edema Factor (EF). The titration of specific antibodies in sera of vaccinated animals is crucial to evaluate the efficacy of the vaccination and to obtain epidemiological information for an effective anthrax surveillance. In this study, we developed a Sterne-based Complement Fixation Test (CFT) to detect specific antibodies induced in animals vaccinated with Sterne 34F2. We assessed its efficacy in laboratory animals and under field conditions by monitoring the humoral response induced by vaccination in cattle. The results indicated that the Sterne-based CFT is able to correctly identify vaccinated animals. It proved to be a very sensitive and specific test. Moreover, the Sterne-based CFT offers many benefits with regard to costs, standardization and reproducibility of the assay procedure.

Classification and management of animal anthrax outbreaks based on the source of infection.

Anthrax is a non-contagious infectious disease; it primarily affects herbivores, but all mammals, including humans, can be affected. Humans may contract anthrax directly or indirectly from infected animals. Veterinary surveillance systems, providing information about animal and human cases, should increase the efficacy of the animal anthrax management in order to protect population. Any aspect of the disease should be carefully monitored to implement effective prevention and control strategies. In this paper we propose a new, detailed classification of anthrax outbreaks, based on the source of the infection and the risk level for humans. We describe three different types of animal outbreaks and suggest the most effective procedures for their management and prevention.

Evaluation of the immunogenicity and safety of Brucella melitensis B115 vaccination in pregnant sheep.

In spite of its limitations, Rev.1 is currently recognized as the most suitable vaccine against Brucella melitensis (the causative agent of ovine and caprine brucellosis). However, its use is limited to young animals when test-and-slaughter programs are in place because of the occurrence of false positive-reactions due to Rev.1 vaccination. The B. melitensis B115 rough strain has demonstrated its efficacy against B. melitensis virulent strains in the mouse model, but there is a lack of information regarding its potential use in small ruminants for brucellosis control. Here, the safety and immune response elicited by B115 strain inoculation were evaluated in pregnant ewes vaccinated at their midpregnancy. Vaccinated (n=8) and non-vaccinated (n=3) sheep were periodically sampled and analyzed for the 108 days following inoculations using tests designed for the detection of the response elicited by the B115 strain and routine serological tests for brucellosis [Rose Bengal Test (RBT), Complement Fixation Test (CFT) and blocking ELISA (ELISAb)]. Five out of the 8 vaccinated animals aborted, indicating a significant abortifacient effect of B115 inoculation at midpregnancy. In addition, a smooth strain was recovered from one vaccinated animal, suggesting the occurrence of an in vivo reversion phenomenon. Only one animal was positive in both RBT and CFT simultaneously (91 days after vaccination) confirming the lack of induction of cross-reacting antibody responses interfering with routine brucellosis diagnostic tests in most B115-vaccinated animals.

Suspect vector transmission of human cutaneous anthrax during an animal outbreak in Southern Italy.

During an outbreak of sheep anthrax in Basilicata, southern Italy, the owner of a flock located about 3 km away from the affected farm developed skin lesions attributable to cutaneous anthrax. The DNA extracted from the human scabs confirmed the diagnosis, and a 15-loci multiple locus variable number tandem repeat (VNTR) analysis (MLVA) following single-nucleotide repeat (SNR) analysis yielded the same genotype as that found in the dead sheep. The breeder, who had not had contact with infected or dead animals, reported having been stung by gadflies.

Ground Anthrax Bacillus Refined Isolation (GABRI) method for analyzing environmental samples with low levels of Bacillus anthracis contamination.

BACKGROUND: In this work are reported the results of a qualitative analytical method capable of detecting Bacillus anthracis spores when they are present in very low concentration in the soil. The Ground Anthrax Bacillus Refined Isolation (GABRI) method, assessed in our laboratory, was compared with the classic method. The comparison involved artificially anthrax-contaminated soil samples (500 spores/7.5 grams soil) and naturally contaminated soil samples collected in Bangladesh during a field investigation. RESULTS: The results indicated that, in contrast to the classic method, the GABRI method was able to detect B.anthracis in all contaminated samples. The GABRI method produces a more sensitive measure of anthrax spore presence significantly different from the standard method. In particular, the latter is more sensitive to the presence of normal soil contaminants. CONCLUSION: The main feature of the GABRI method is its ability to strongly reduce the presence of the environmental contaminants, which being much more numerous than B. anthracis tend to inhibit their germination and growth making it extremely difficult to visualize any colonies. The reduction of the microbial environment also allows one to be able to culture and test a larger quantity of potentially contaminated soil and to isolate B. anthracis when the spores are present in very low concentrations in the soil.

Protective role of antibodies induced by Brucella melitensis B115 against B. melitensis and Brucella abortus infections in mice.

It has been demonstrated that antibodies specific for O-PS antigen of Brucella smooth strains are involved in the protective immunity of brucellosis. Since the rough strain Brucella melitensis B115 was able to protect mice against wild Brucella strains brucellosis despite the lack of anti-OPS antibodies, in this study we evaluated the biological significance of antibodies induced by this strain, directed to antigens other than O-PS, passively tranferred to untreated mice prior to infection with Brucella abortus 2308 and B. melitensis 16M virulent strains. The protective ability of specific antisera collected from mice vaccinated with B. melitensis B115, B. abortus RB51 and B. abortus S19 strains was compared. The results indicated that antibodies induced by B115 were able to confer a satisfactory protection, especially against B. abortus 2308, similar to that conferred by the antiserum S19, while the RB51 antiserum was ineffective. These findings suggest that antibodies induced by B115 could act as opsonins as well as antibodies anti-O-PS, thus triggering more efficient internalization and degradation of bacteria within phagocytes. This is the first study assessing the efficacy of antibodies directed to antigens other than O-PS in the course of brucellosis infection.

Antigenic, immunologic and genetic characterization of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18.

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18. RB51 derived from B. abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B. melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.

B. melitensis rough strain B115 is protective against heterologous Brucella spp. infections.

Brucellosis is one of the most serious zoonoses all over the world, with B. melitensis, B. abortus and B. suis being the most pathogenic species for humans. Vaccination of domesticated livestock still represents the most efficient way to prevent human infection. However, the available Brucella vaccines retain an important residual virulence and induce antibodies interfering with surveillance programs. Moreover, each vaccine shows different protective effects versus different Brucella species and different animal hosts. Nowadays, while B. melitensis and B. suis infections in cattle are emerging as a significant problem, there are no available vaccines to overcome such issue. B. melitensis strain B115, a natural, attenuated rough strain in our previous studies proved to be highly protective against B. melitensis and B. ovis infections in mice, without inducing interfering antibodies. In this study, we tested the efficiency of B115 as vaccine against B. abortus and B. suis. Vaccination of mice with 10(8) CFU/mouse of B. melitensis B115 conferred a satisfactory protection against B. abortus 2308. On the contrary, mice vaccinated once with 10(8) or 10(9) CFU/mouse of B115 were weakly protected against B. suis infection. Conversely, when mice were vaccinated twice with 10(9) CFU B115/mouse, the protective activity significantly increased. Unlike its rough phenotype, B115 showed an adequate persistence in mice accompanied to a solid humoral and cell-mediated immunity. All together, these findings suggest the potential usefulness of B115 to control brucellosis in animal hosts due to heterologous challenges.

Evaluation of the house fly Musca domestica as a mechanical vector for an anthrax.

Anthrax is a disease of human beings and animals caused by the encapsulated, spore-forming, Bacillus anthracis. The potential role of insects in the spread of B. anthracis to humans and domestic animals during an anthrax outbreak has been confirmed by many studies. Among insect vectors, the house fly Musca domestica is considered a potential agent for disease transmission. In this study, laboratory-bred specimens of Musca domestica were infected by feeding on anthrax-infected rabbit carcass or anthrax contaminated blood, and the presence of anthrax spores in their spots (faeces and vomitus) was microbiologically monitored. It was also evaluated if the anthrax spores were able to germinate and replicate in the gut content of insects. These results confirmed the role of insects in spreading anthrax infection. This role, although not major, given the huge size of fly populations often associated with anthrax epidemics in domestic animals, cannot be neglected from an epidemiological point of view and suggest that fly control should be considered as part of anthrax control programs.

Severe anthrax outbreaks in Italy in 2004: considerations on factors involved in the spread of infection.

Anthrax is a disease of humans and animals caused by the encapsulated, spore-forming Bacillus anthracis. In Italy, anthrax is normally a sporadic disease. During the summer 2004, anthrax broke out in the Basilicata, in southern Italy, a region with a low prevalence of anthrax in which vaccination had been suspended since 1998. The disease involved several animals in few weeks and in a large area. Over 41 days, 81 cattle died, as well as 15 sheep, 9 goats, 11 horses and 8 deer. The Multiple-locus Variable-Number Tandem Repeats Analysis (MLVA) showed that all the 53 isolates belonged to the Cluster Ala, genotype 1. The results of the Single Nucleotide Repeats (SNRs) Analysis showed that 48/53 B. anthacis strains belonged to a single clonal lineage, the sub-genotype sgt - eB. Two sporadic mutants, sgt - eB,m1 and sgt - eB,m2, were isolated, only one managing to infect other herds. Factors that could have contributed to the spread of infection, such as the transmission of spores by insect vectors and the favourable weather conditions were evaluated.

SNR analysis: molecular investigation of an anthrax epidemic.

BACKGROUND: In Italy, anthrax is endemic but occurs sporadically. During the summer of 2004, in the Pollino National Park, Basilicata, Southern Italy, an anthrax epidemic consisting of 41 outbreaks occurred; it claimed the lives of 124 animals belonging to different mammal species. This study is a retrospective molecular epidemiological investigation carried out on 53 isolates collected during the epidemic. A 25-loci Multiple Locus VNTR Analysis (MLVA) MLVA was initially performed to define genetic relationships, followed by an investigation of genetic diversity between epidemic strains through Single Nucleotide Repeat (SNR) analysis. RESULTS: 53 Bacillus anthracis strains were isolated. The 25-loci MLVA analysis identified all of them as belonging to a single genotype, while the SNR analysis was able to detect the existence of five subgenotypes (SGTs), allowing a detailed epidemic investigation. SGT-1 was the most frequent (46/53); SGTs 2 (4/53), 3 (1/53) 4 (1/53) and 5 (1/53) were detected in the remaining seven isolates. CONCLUSIONS: The analysis revealed the prevalent spread, during this epidemic, of a single anthrax clone. SGT-1--widely distributed across the epidemic area and present throughout the period in question - may, thus, be the ancestral form. SGTs 2, 3 and 4 differed from SGT-1 at only one locus, suggesting that they could have evolved directly from the latter during the course of this epidemic. SGT-5 differed from the other SGTs at 2-3 loci. This isolate, thus, appears to be more distantly related to SGT-1 and may not be a direct descendant of the lineage responsible for the majority of cases in this epidemic. These data confirm the importance of molecular typing and subtyping methods for in-depth epidemiological analyses of anthrax epidemics.

Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping.

The rpoB gene encoding the beta subunit of the DNA-dependent RNA polymerase was molecularly characterized by PCR amplification and DNA sequencing in 26 Brucella reference strains by using primers selected according to the B. melitensis 16 M rpoB published sequence. Comparison of the rpoB nucleotide sequence of all Brucella strains analysed revealed specific nucleotide variations associated with different Brucella species and biovars. 17 rpoB alleles were recognized and new Brucella typing is proposed. Our results suggest that the rpoB gene polymorphism can be used to identify all Brucella species and most of the biovars, offering an improvement over conventional typing methods.

Molecular study of genes involved in virulence regulatory pathways in Bacillus anthracis vaccine strain "Carbosap".

This study investigated the genetic bases of attenuation in the Bacillus anthracis vaccine strain "Carbosap" used in Italy against anthrax in cattle and sheep. Twelve genes involved in virulence regulatory pathways underwent sequence analysis in comparison with a B. anthracis virulent strain.

In vitro stimulation of murine peritoneal monocytes induced by alginates.

In this trial we assessed the effect of soluble alginates on murine cells. Mouse peritoneal monocytes were stimulated in vitro with a solution of alginate. The production of TNF-alpha and nitric oxide (NO), the expression of surface molecules CD80 and CD86, and the ability of monocytes to phagocyte bacteria were assessed, in order to evaluate the effect of alginate on cell functionality. We showed that mouse peritoneal monocytes stimulated with alginate produce NO and TNF-alpha. In addition, alginate is able also to increase their phagocytic activity and to a lesser extent also to increase the expression of CD80. Even with different degrees, it implies that alginates per se act directly on immune response, being able to effectively stimulate proinflammatory activity. These findings corroborate the idea that alginates can represent interesting adjuvants to use to increase the efficacy of antigenic stimulation.

Genetic bases of the rifampin resistance phenotype in Brucella spp.

Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens. Its bactericidal activity is due to its ability to bind to the beta subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene. Mutations of the rpoB gene have been characterized in rifampin-resistant (Rif(r)) strains of Escherichia coli and Mycobacterium tuberculosis. The genetic bases of Rif(r) in Brucella spp. are still unknown. In the present study, the nucleotide sequences of the rpoB gene of the Rif(r) vaccine strain Brucella abortus RB51 and of 20 Rif(r) clones derived in our laboratory from two Brucella melitensis isolates were determined. These sequences were then compared to those of the respective rifampin-susceptible (Rif(s)) parental strains and to the published B. melitensis strain 16M. All Rif(r) strains carried one or more missense mutations mapping in two regions of the rpoB gene. These two "hot" regions were investigated in eight additional Rif(r) Brucella laboratory mutants and in 20 reference Rif(s) Brucella strains. rpoB mutations were found in all Rif(r) mutants. In contrast, no missense mutations were found in any analyzed Rif(s) strains. Our results represent the first from a study of the molecular characterization of rpoB mutations in resistant Brucella strains and provide an additional proof of the association of specific rpoB mutations with the development of the Rif(r) phenotype in prokaryotes. In addition, because of the relationship between Rif(r) and the attenuation of virulence in Brucella spp., studies of virulence in these mutants may provide useful information about the genetic basis of pathogenesis in Brucella.