Search

Abstract Background Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA’s roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished. Methods We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar. Results Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3’ end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567. Conclusion B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.

Abstract Background Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. Methods The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. Result The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. Conclusion The study identified thirty chromosome-encoded genes specific to B. anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.

BackgroundAntimicrobial stewardship (AMS) programs are critical in combating antimicrobial resistance (AMR). In Zambia, there is little information regarding the capacity of hospitals to establish and implement AMS programs. The objective of this study was to conduct a baseline assessment of WHO core elements for an AMS program implementation in eight hospitals in Zambia.Materials and methodsWe conducted an exploratory cross-sectional study from September 2023 to December 2023 using a self-scoring Periodic National and Healthcare Facility Assessment Tool from the World Health Organization (WHO) policy guidance on integrated AMS activities in human health. Eight public hospitals were surveyed across the five provinces of Zambia. Data was analyzed using the WHO self-scoring tool and thematic analysis.ResultsOverall, 62.5% (6/8) of the facilities scored low (below 60%) in implementing AMS programs. Most facilities had challenges with reporting AMS feedback within the hospital (average score = 46%), Drugs and Therapeutics Committee (DTC) functionality (average score = 49%), AMS actions (average score = 50%), education and training (average score = 54%), and leadership commitment to AMS activities (average score = 56%). The overall score for all AMS core elements was average (56%). All the hospitals (100%) did not have an allocated budget for AMS programs. Finally, there were neither antibiograms to guide antimicrobial utilization nor AMS-trained staff in more than 50% of the hospitals surveyed.ConclusionThis study found low AMS implementation in these public hospitals, especially where DTCs were non-functional. The identified challenges and gaps require urgent attention for sustainable multidisciplinary AMS programs.

Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site (p < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.

Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.

Antimicrobial resistance (AMR) among Escherichia coli from food animals is a rising problem, and heavy antimicrobial use in poultry is a contributing factor. In Zambia, studies linking poultry-associated AMR and antibiotic use (AMU) are rare. This study aimed to investigate commercial and medium-/small-scale poultry farmers’ usage of antimicrobials based on a questionnaire survey in ten districts of Zambia. In addition, the study characterized extended-spectrum β-lactamase (ESBL)-producing E. coli isolates obtained from poultry in the same districts. Data regarding knowledge and usage of antimicrobials were collected from commercial and medium-/small-scale poultry farmers using a pre-tested structured questionnaire. At the same time, cloacal samples were collected and analyzed. One hundred and fifty E. coli isolates were tested for antimicrobial susceptibility using eight antibiotic classes. The isolates were further screened for ESBL production by streaking them on cefotaxime (CTX)-supplemented MacConkey agar, then subjecting them to sequencing on a NextSeq. The questionnaire survey showed that more medium-/small-scale than commercial poultry farmers used antimicrobials (OR = 7.70, 95% CI = 2.88–20.61) but less prescriptions (OR = 0.02, 95% CI = 0.00–0.08). Susceptibility testing revealed that resistance was highest to ampicillin (128/148, 86.5%) and tetracycline (101/136, 74.3%) and that the prevalence of multidrug resistance (MDR) (28/30, 93.3%) was high. Whole-genome sequencing (WGS) of eight (8/30, 26.7%) isolates with CTX Minimum Inhibitory Concentration (MIC) ≥ 4 µg/mL revealed the presence of ESBL-encoding genes blaCTX-M-14, blaCTX-M-55, and blaTEM. WGS also detected other AMR genes for quinolones, aminoglycosides, phenicols, tetracycline, macrolides, and folate-pathway antagonists. Altogether, the questionnaire survey results showed a higher proportion of AMU and lower prescription usage among medium-/small-scale farmers. In addition, our results emphasize the circulation of ESBL-producing E. coli strains with associated MDR. It is critical to educate farmers about AMR risks and to encourage responsible usage of antimicrobials. Furthermore, there is a need to strengthen regulations limiting access to antimicrobials. Finally, there is a need to establish a one health system to guide public health response.

Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.

Rotavirus is a major cause of diarrhea globally in animals and young children under 5 years old. Here, molecular detection and genetic characterization of porcine rotavirus in smallholder and commercial pig farms in the Lusaka Province of Zambia were conducted. Screening of 148 stool samples by RT-PCR targeting the VP6 gene revealed a prevalence of 22.9% (34/148). Further testing of VP6-positive samples with VP7-specific primers produced 12 positives, which were then Sanger-sequenced. BLASTn of the VP7 positives showed sequence similarity to porcine and human rotavirus strains with identities ranging from 87.5% to 97.1%. By next-generation sequencing, the full-length genetic constellation of the representative strains RVA/pig-wt/ZMB/LSK0137 and RVA/pig-wt/ZMB/LSK0147 were determined. Genotyping of these strains revealed a known Wa-like genetic backbone, and their genetic constellations were G4-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1 and G9-P[13]-I5-R1-C1-M1-A8-N1-T1-E1-H1, respectively. Phylogenetic analysis revealed that these two viruses might have their ancestral origin from pigs, though some of their gene segments were related to human strains. The study shows evidence of reassortment and possible interspecies transmission between pigs and humans in Zambia. Therefore, the “One Health” surveillance approach for rotavirus A in animals and humans is recommended to inform the design of effective control measures.

Escherichia coli (E. coli), a major foodborne disease-causing pathogen found in raw cow milk, has even far more reaching public health ramifications as it encodes for antimicrobial resistance (AMR). This study aimed to identify multidrug-resistant (MDR) E. coli from raw cow’s milk and evaluate their antimicrobial-resistant profiles. In total, 418 pooled raw cow milk samples were collected from milk collection centers and analysed using standard culture methods to isolate E. coli. Antimicrobial Susceptibility Testing (AST) was conducted using the Kirby Bauer disk diffusion method and PCR was used to identify cefotaxime (CTX) resistant genes. Overall isolation of E. coli was 51.2% (214/418) with MDR observed in 21% (45/214) of isolates across different antibiotic combinations. Resistance was observed towards ampicillin (107/214, 50%), tetracycline (86/214, 40.1%), trimethoprim/sulfamethoxazole (61/214, 28.5%), and amoxicillin/clavulanic acid (CTX) (50/214, 23.4%). Notably, 15% (32/214) resistance to CTX was observed, while 12.6% (27/214) exhibited resistance to imipenem. The blaCTX-M and blaTEM genes were detected in CTX-resistant isolates. The findings of MDR E. coli that harbour blaCTX-M and blaTEM genes in raw cow’s milk indicate serious public health risks for consumers.

Bovine tuberculosis (bTB) is an infectious disease with significant socioeconomic, animal, and public health impacts. However, the prevalence of bTB remains largely unclear in Malawi due to a paucity of information. Additionally, the existence of multiple risk factors is postulated to enhance bTB transmission in animals. A cross-sectional survey to estimate the prevalence of bTB, animal characteristics and identify associated risk factors was conducted from slaughtered cattle at three major regional abattoirs (southern, central and northern regions) in Malawi. Out of a total of 1547 cattle examined, 154 (9.95%) had bTB-like lesions in various visceral organs and lymph nodes; one sample per animal was collected, processed, and cultured in the in the BACTEC Mycobacterial growth indicator tube (MGIT) 960 system. From the 154 cattle that showed tuberculous like lesions, only 112 were positive on MGIT and 87 were confirmed to have M. bovis based on multiplex PCR. Cattle from the southern region (odds ratio (OR) = 1.96, 95% CI: 1.03–3.85) and central region (OR = 2.00, 95% CI: 1.16–3.56) were more likely presented with bTB-like lesions at slaughter than from the northern region. The risk of having bTB-like lesions was higher in females (OR = 1.51, CI: 1.00–2.29), older cattle (OR = 2.17, CI: 1.34–3.37), and crossbreeds (OR = 1.67, 95% CI: 1.12–2.47) than in males, younger animals, and Malawi Zebu breed, respectively. The high prevalence of bTB is of critical concern and necessitates active surveillance and strengthening of the current control strategies under a One Health (OH) approach at the animal-human interface.

The emergence of pre-extensively drug-resistant tuberculosis (pre-XDR-TB) is a threat to TB control programs in developing countries such as Zambia. Studies in Zambia have applied molecular techniques to understand drug-resistance-associated mutations, circulating lineages and transmission patterns of multi-drug-resistant (MDR) Mycobacterium tuberculosis. However, none has reported genotypes and mutations associated with pre-XDR TB. This study characterized 63 drug-resistant M. tuberculosis strains from the University Teaching Hospital between 2018 and 2019 using targeted gene sequencing and conveniently selected 50 strains for whole genome sequencing. Sixty strains had resistance mutations associated to MDR, one polyresistant, and two rifampicin resistant. Among MDR strains, seven percent (4/60) had mutations associated with pre-XDR-TB. While four, one and nine strains had mutations associated with ethionamide, para-amino-salicylic acid and streptomycin resistances, respectively. All 50 strains belonged to lineage 4 with the predominant sub-lineage 4.3.4.2.1 (38%). Three of four pre-XDR strains belonged to sub-lineage 4.3.4.2.1. Sub-lineage 4.3.4.2.1 strains were less clustered when compared to sub-lineages L4.9.1 and L4.3.4.1 based on single nucleotide polymorphism differences. The finding that resistances to second-line drugs have emerged among MDR-TB is a threat to TB control. Hence, the study recommends a strengthened routine drug susceptibility testing for second-line TB drugs to stop the progression of pre-XDR to XDR-TB and improve patient treatment outcomes.

Rift valley fever (RVF) is a mosquito-borne disease of animals and humans. Although RVF outbreaks are usually reported at 5-15-year intervals in sub-Saharan Africa, Zambia has experienced an unusually long inter-epizootic/-epidemic period of more than three decades. However, serological evidence of RVF virus (RVFV) infection in domestic ruminants during this period underscores the need for comprehensive investigation of the mechanisms of virus perpetuation and disease emergence. Mosquitoes (n = 16,778) captured from eight of the ten provinces of Zambia between April 2014 and May 2019 were pooled (n = 961) and screened for RVFV genome by a pan-phlebo RT-PCR assay. Aedes mosquito pools (n = 85) were further screened by nested RT-PCR assay. Sera from sheep (n = 13), goats (n = 259) and wild ungulates (n = 285) were screened for RVFV antibodies by ELISA while genome detection in pooled sera (n = 276) from domestic (n = 248) and wild ungulates (n = 37) was performed by real-time RT-PCR assay. To examine the association between the long inter-epizootic period and climatic variables, we examined El Niño-Southern Oscillation indices, precipitation anomalies, and normalized difference vegetation index. We then derived RVF risk maps by exploring climatic variables that would favor emergence of primary RVFV vectors. While no RVFV genome could be detected in pooled mosquito and serum samples, seroprevalence was significantly high (OR = 8.13, 95% CI [4.63-14.25]) in wild ungulates (33.7%; 96/285) compared to domestic ruminants (5.6%; 16/272). Retrospective analysis of RVF epizootics in Zambia showed a positive correlation between anomalous precipitation (La Niña) and disease emergence. On risk mapping, whilst northern and eastern parts of the country were at high risk, domestic ruminant population density was low (< 21 animals/km2) in these areas compared to low risk areas (>21 animals/km2). Besides evidence of silent circulation of RVFV and the risk of disease emergence in some areas, wildlife may play a role in the maintenance of RVFV in Zambia.

Abstract Background The epidemiology of extended-spectrum β-lactamases (ESBLs) has undergone dramatic changes, with CTX-M-type enzymes prevailing over other types. bla CTX-M genes, encoding CTX-M-type ESBLs, are usually found on plasmids, but chromosomal location is becoming common. Given that bla CTX-M-harboring strains often exhibit multidrug resistance (MDR), it is important to investigate the association between chromosomally integrated bla CTX-M and the presence of additional antimicrobial resistance (AMR) genes, and to identify other relevant genetic elements. Methods A total of 46 clinical isolates of cefotaxime-resistant Enterobacteriaceae (1 Enterobacter cloacae, 9 Klebsiella pneumoniae, and 36 Escherichia coli) from Zambia were subjected to whole-genome sequencing (WGS) using MiSeq and MinION. By reconstructing nearly complete genomes, bla CTX-M genes were categorized as either chromosomal or plasmid-borne. Results WGS-based genotyping identified 58 AMR genes, including four bla CTX-M alleles (i.e., bla CTX-M-14, bla CTX-M-15, bla CTX-M-27, and bla CTX-M-55). Hierarchical clustering using selected phenotypic and genotypic characteristics suggested clonal dissemination of bla CTX-M genes. Out of 45 bla CTX-M gene-carrying strains, 7 harbored the gene in their chromosome. In one E. cloacae and three E. coli strains, chromosomal bla CTX-M-15 was located on insertions longer than 10 kb. These insertions were bounded by ISEcp1 at one end, exhibited a high degree of nucleotide sequence homology with previously reported plasmids, and carried multiple AMR genes that corresponded with phenotypic AMR profiles. Conclusion Our study revealed the co-occurrence of ISEcp1-bla CTX-M-15 and multiple AMR genes on chromosomal insertions in E. cloacae and E. coli, suggesting that ISEcp1 may be responsible for the transposition of diverse AMR genes from plasmids to chromosomes. Stable retention of such insertions in chromosomes may facilitate the successful propagation of MDR clones among these Enterobacteriaceae species.

While evidence suggests presence of HEV infection in humans in Zambia, currently, there is no information on its occurrence in domestic pigs. Here, we investigated the presence of HEV antibodies and genome in domestic pigs in Zambia. Sera (n = 484) from domestic pigs were screened for antibodies against HEV by ELISA while genome detection in fecal (n = 25) and liver (n = 100) samples from slaughter pigs was conducted using nested RT–PCR assay. Overall, seroprevalence was 47.7% (231/484) while zoonotic genotype 3 HEV RNA was detected in 16.0% (20/125) of slaughtered pigs. This is the first report to highlight occurrence of HEV infection in domestic pigs in Zambia. This finding suggests possible contamination of the pork supply chain. Moreover, there is a potential risk of zoonotic transmission of HEV to abattoir workers, pig farmers and handlers.

The epidemiology of Rift Valley fever (RVF) is poorly understood in Malawi. Here, a cross-sectional study was conducted (March–June 2020) to investigate the seroprevalence and potential risk factors of RVF virus (RVFV) in cattle, goats, and sheep in three ecological zones of Malawi. A total of 1523 serum samples were tested for anti-RVFV IgG and IgM antibodies by ELISA. Additionally, a questionnaire survey was used to assess potential RVF risk factors. The overall seroprevalence was 17.14% (261/1523; 95% CI = 15.33–19.11) for individual livestock and 33.24% (120/361; 95% CI = 28.18–38.11) for the livestock herd. Seroprevalence was significantly high in sheep (25.68%, 95% CI = 19.31–33.26) compared with cattle (21.35%, 95% CI = 18.74–24.22) and goats (7.72%, 95% CI = 5.72–10.34), (p = 0.047). At the individual livestock level, the risk was elevated in female livestock (OR: 1.74, 95% CI = 1.08–12.82) (p = 0.016), while at the herd level, areas receiving approximately 1001–1500 mm of rainfall (OR: 2.47, 95% CI = 1.14–5.37) (p = 0.022), areas of rainfall amount greater than approximately 1600 mm (OR: 2.239, 95% CI = 1.07–8.82) (p = 0.023), and mixed species herds (OR: 10.410, 95% CI = 3.04–35.59) (p = 0.001), were significant risk factors. The detection of IgM antibodies confirmed active circulation of RVFV in Malawi. Therefore, monitoring of RVF in animals, humans, and vectors using a “One Health” approach, along with community sensitization among the high-risk populations, could help mitigate the threat posed by this zoonotic disease in Malawi.

Anthrax is a worldwide zoonotic disease. Anthrax has long been a public health and socio-economic issue in Mongolia. Presently, there is no spatial information on carcass burial sites as a potential hazard of future anthrax outbreaks and possible risk factors associated with anthrax occurrences in Mongolia. Here, we analyze retrospective data (1986–2015) on the disposal sites of livestock carcasses to describe historical spatio-temporal patterns of livestock anthrax in Khuvsgul Province, which showed the highest anthrax incidence rate in Mongolia. From the results of spatial mean and standard deviational ellipse analyses, we found that the anthrax spatial distribution in livestock did not change over the study period, indicating a localized source of exposure. The multi-distance spatial cluster analysis showed that carcass sites distributed in the study area are clustered. Using kernel density estimation analysis on carcass sites, we identified two anthrax hotspots in low-lying areas around the south and north regions. Notably, this study disclosed a new hotspot in the northern part that emerged in the last decade of the 30-year study period. The highest proportion of cases was recorded in cattle, whose prevalence per area was highest in six districts (i.e., Murun, Chandmani-Undur, Khatgal, Ikh-Uul, Tosontsengel, and Tsagaan-Uul), suggesting that vaccination should prioritize cattle in these districts. Furthermore, size of outbreaks was influenced by the annual summer mean air temperature of Khuvsgul Province, probably by affecting the permafrost freeze-thawing activity.

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.

Globally, tuberculosis (TB) is a major cause of death due to antimicrobial resistance. Mycobacterium tuberculosis CAS1-Kili strains that belong to lineage 3 (Central Asian Strain, CAS) were previously implicated in the spread of multidrug-resistant (MDR)-TB in Lusaka, Zambia. Thus, we investigated recent transmission of those strains by whole-genome sequencing (WGS) with Illumina MiSeq platform. Twelve MDR CAS1-Kili isolates clustered by traditional methods (MIRU-VNTR and spoligotyping) were used. A total of 92% (11/12) of isolates belonged to a cluster (≤12 SNPs) while 50% (6/12) were involved in recent transmission events, as they differed by ≤5 SNPs. All the isolates had KatG Ser315Thr (isoniazid resistance), EmbB Met306 substitutions (ethambutol resistance) and several kinds of rpoB mutations (rifampicin resistance). WGS also revealed compensatory mutations including a novel deletion in embA regulatory region (−35A > del). Several strains shared the same combinations of drug-resistance-associated mutations indicating transmission of MDR strains. Zambian strains belonged to the same clade as Tanzanian, Malawian and European strains, although most of those were pan-drug-susceptible. Hence, complimentary use of WGS to traditional epidemiological methods provides an in-depth insight on transmission and drug resistance patterns which can guide targeted control measures to stop the spread of MDR-TB.