Real-time PCR assays that detect genes for botulinum neurotoxin A-G subtypes.

The role of Real-Time PCR assays for surveillance and rapid screening for pathogens is garnering more and more attention because of its versatility and ease of adoption. The goal of this study was to design, test, and evaluate Real-Time TaqMan PCR assays for the detection of botulinum neurotoxin (bont/A-G) genes from currently recognized BoNT subtypes. Assays were computationally designed and then laboratory tested for sensitivity and specificity using DNA preparations containing bont genes from 82 target toxin subtypes, including nine bivalent toxin types; 31 strains representing other clostridial species; and an extensive panel that consisted of DNA from a diverse set of prokaryotic (bacterial) and eukaryotic (fungal, protozoan, plant, and animal) species. In addition to laboratory testing, the assays were computationally evaluated using in silico analysis for their ability to detect bont gene sequences from recently identified toxin subtypes. Seventeen specific assays (two for each of the bont/C, bont/D, bont/E, and bont/G subtypes and three for each of the bont/A, bont/B, and bont/F subtypes) were designed and evaluated for their ability to detect bont genes encoding multiple subtypes from all seven serotypes. These assays could provide an additional tool for the detection of botulinum neurotoxins in clinical, environmental and food samples that can complement other existing methods used in clinical diagnostics, regulatory, public health, and research laboratories.

Botulinum neurotoxin serotype A inhibited ocular angiogenesis through modulating glial activation via SOCS3.

BACKGROUND: Pathological angiogenesis causes significant vision loss in neovascular age-related macular degeneration and other retinopathies with neovascularization (NV). Neuronal/glial-vascular interactions influence the release of angiogenic and neurotrophic factors. We hypothesized that botulinum neurotoxin serotype A (BoNT/A) modulates pathological endothelial cell proliferation through glial cell activation and growth factor release. METHODS: A laser-induced choroidal NV (CNV) was employed to investigate the anti-angiogenic effects of BoNT/A. Fundus fluorescence angiography, immunohistochemistry, and real-time PCR were used to assess BoNT/A efficacy in inhibiting CNV and the molecular mechanisms underlying this inhibition. Neuronal and glial suppressor of cytokine signaling 3 (SOCS3) deficient mice were used to investigate the molecular mechanisms of BoNT/A in inhibiting CNV via SOCS3. FINDINGS: In laser-induced CNV mice with intravitreal BoNT/A treatment, CNV lesions decreased > 30%; vascular leakage and retinal glial activation were suppressed; and Socs3 mRNA expression was induced while vascular endothelial growth factor A (Vegfa) mRNA expression was suppressed. The protective effects of BoNT/A on CNV development were diminished in mice lacking neuronal/glial SOCS3. CONCLUSION: BoNT/A suppressed laser-induced CNV and glial cell activation, in part through SOCS3 induction in neuronal/glial cells. BoNT/A treatment led to a decrease of pro-angiogenic factors, including VEGFA, highlighting the potential of BoNT/A as a therapeutic intervention for pathological angiogenesis in retinopathies.

Prevalence of Haemophilus influenzae in the nasopharynx of children from regions with varying incidence of invasive H. influenzae serotype a disease: Canadian Immunization Research Network (CIRN) study.

Haemophilus influenzae serotype a (Hia) has recently emerged as an important cause of invasive disease in the North American Arctic and Sub-Arctic regions, mainly affecting young Indigenous children. In this study, we addressed the question of whether the prevalence of Hia and all H. influenzae in the nasopharynx differed between paediatric populations from regions with high versus low incidence of invasive Hia disease. Nasopharyngeal specimens from children with acute respiratory tract infections (ARTI) collected for routine diagnostic detection of respiratory viruses were analysed with molecular-genetic methods to identify and serotype H. influenzae. In Nunavut, a region with a high incidence of invasive Hia disease, all H. influenzae and particularly Hia were found in the nasopharynx of 60.6% and 3.0% children. In Southern Ontario (Hamilton region), where Hia invasive disease is rare, the frequencies of all H. influenzae and Hia detection were 38.5% and 0.6%, respectively. In both cohorts, non-typeable H. influenzae was prevalent (57.0% and 37.9%, respectively). Considering that Hia is an important cause of severe invasive disease in Nunavut children, 3% prevalence of Hia among children with ARTI can reflect continuing circulation of the pathogen in the Northern communities that may result in invasive disease outbreaks.

Carriage of Haemophilus influenzae serotype A in children: Canadian Immunization Research Network (CIRN) study.

Background: Haemophilus influenzae serotype a (Hia) has recently emerged as an important cause of invasive disease, mainly affecting young Indigenous children. Carriage of H. influenzae is a pre-requisite for invasive disease and reservoir for transmission. To better understand the epidemiology of invasive Hia disease, we initiated a multicentre study of H. influenzae nasopharyngeal carriage among Canadian children. Methods: With prior parental consent, we collected nasotracheal tubes used during general anaesthesia in healthy children following routine dental surgery in a regional hospital of northwestern Ontario and a dental clinic in central Saskatchewan. In northwestern Ontario, all children were Indigenous (median age 48.0 months, 45.8% female); in Saskatchewan, children were from various ethnic groups (62% Indigenous, median age 56.3 months, 43.4% female). Detection of H. influenzae and serotyping were performed using molecular-genetic methods. Results: A total of 438 nasopharyngeal specimens, 286 in northwestern Ontario and 152 in Saskatchewan were analyzed. Hia was identified in 26 (9.1%) and 8 (5.3%) specimens, respectively. In Saskatchewan, seven out of eight children with Hia carriage were Indigenous. Conclusions: The carriage rates of Hia in healthy children in northwestern Ontario and Saskatchewan are comparable to H. influenzae serotype b (Hib) carriage among Alaska Indigenous children in the pre-Hib-vaccine era. To prevent invasive Hia disease, paediatric conjugate Hia vaccines under development have the potential to reduce carriage of Hia, and thus decrease the risk of transmission and disease among susceptible populations. Addressing the social determinants of health may further eliminate conditions favouring Hia transmission in Indigenous communities.

Historique: L'Haemophilus influenzae de sérotype a (Hia) a récemment émergé comme une cause importante de maladie invasive, particulièrement chez les jeunes enfants autochtones. Il faut être porteur de l'H. influenzae pour contracter une maladie invasive et devenir un réservoir de transmission. Pour mieux comprendre l'épidémiologie de l'infection invasive à Hia, les chercheurs ont lancé une étude multicentrique sur le portage nasopharyngé de l'H. influenzae chez les enfants canadiens. Méthodologie: Après avoir obtenu le consentement des parents, les chercheurs ont recueilli les sondes nasotrachéales utilisées pendant l'anesthésie générale chez des enfants en santé après une chirurgie dentaire courante dans un hôpital régional du nord-ouest de l'Ontario et une clinique dentaire du centre de la Saskatchewan. Dans le nord-ouest de l'Ontario, tous les enfants étaient autochtones (âge médian de 48,0 mois, 45,8 % de filles); en Saskatchewan, les enfants provenaient de divers groupes ethniques (62 % d'Autochtones, âge médian de 56,3 mois, 43,4 % de femmes). La détection de l'H. influenzae et le sérotypage ont été effectués au moyen de méthodes de génétique moléculaire. Résultats: Au total, les chercheurs ont analysé 438 échantillons nasopharyngés, soit 286 du nord-ouest de l'Ontario et 152 de la Saskatchewan. L'Hia a été décelé dans 26 (9,1 %) et huit (5,3 %) échantillons, respectivement. En Saskatchewan, sept des huit enfants porteurs de l'Hia étaient autochtones. Conclusions: Le taux de portage de l'Hia chez les enfants en santé du nord-ouest de l'Ontario et de la Saskatchewan était comparable à celui du portage de l'H. influenzae du sérotype b (Hib) chez les enfants autochtones de l'Alaska avant le déploiement des vaccins contre le Hib. Pour éviter l'infection invasive à Hia, les vaccins pédiatriques conjugués contre l'Hia en cours de développement peuvent réduire le portage de l'Hia, et donc le risque de transmission et de maladie dans les populations susceptibles. Le fait d'aborder les déterminants sociaux de la santé pourrait contribuer à éliminer les conditions favorables à la transmission à Hia dans les communautés autochtones.

Evaluation of a Vaccine Candidate Designed for Broad-Spectrum Protection against Type A Foot-and-Mouth Disease in Asia.

Foot-and-mouth disease (FMD) vaccines are currently the most powerful protective and preventive measures used to control FMD. In this study, the chimeric vaccine strain containing antigenic epitopes from the FMD virus serotype A, which belongs to the ASIA topotype, was produced and evaluated. The chimeric vaccine strains contain sea-97/G1 (VP4, VP2, VP3) and A22 Iraq (VP1) or G-VII (VP1) for use in FMD vaccines in Asia. The 50% protective dose was determined in mice. Vaccinated mice were challenged with three different type A viruses (Sea-97/G1, Sea-97/G2, G-VII clade) seven days post-vaccination (dpv), and mice that received the vaccine candidates were protected against the three viruses. The protective capability of one of the vaccine candidates was evaluated in pigs. Vaccinated pigs were challenged with three different type A viruses (Sea-97/G1, Sea-97/G2, G-VII clade) at 28 dpv, and pigs that received the vaccine candidate were protected against the three viruses. The results showed that this vaccine candidate, which was designed to provide protection against FMD in Asia, efficiently protected pigs against virus challenge and thus has potential as a broad-spectrum vaccine for various epidemic FMD viruses.

Detection of foot-and-mouth disease viruses from the A/AFRICA/G-I genotype in the Sultanate of Oman.

Rapid identification and characterization of circulating foot-and-mouth disease virus (FMDV) strains is crucial for effective disease control. In Oman, a few serological and molecular studies have been conducted to identify the strains of FMDV responsible for the outbreaks that have been occurring within the country. In this study, 13 oral epithelial tissue samples from cattle were collected from suspected cases of FMD in Ash Sharqiyah North, Al Batinah North, Dhofar and Ad Dhakhyilia governorates of Oman between 2018 and 2021. FMDV RNA was detected in all samples by real-time RT-PCR and viruses were isolated after one- or two-blind passages in the porcine Instituto Biologico-Rim Suino-2 cell line. Antigen capture ELISA characterized all isolates as serotype A and VP1 phylogenetic analysis placed all sequences within a single clade of the G-I genotype within the A/AFRICA topotype. These sequences shared the closest nucleotide identities to viruses circulating in Bahrain in 2021 (93.5% to 99.5%) and Kenya in 2017 (93.4% to 99.1%). To the best of our knowledge, this is the first time that A/AFRICA/G-I viruses have been detected in Oman. Together with the closely related viruses detected recently in Bahrain, these findings reinforce the importance of deploying effective quarantine control measures to minimize the risks of transboundary transmission of FMD associated with the importation of cattle from East Africa.

Novel pathologic findings and viral antigen distribution in cattle and buffalo calves naturally infected with Foot-and-Mouth disease virus.

The Foot-and-Mouth disease is highly contagious acute viral disease of livestock inflicting huge economic loss to the farmers. The limited knowledge regarding the pathological lesions vis-a-vis distribution of the FMDV in lesser explored endocrine glands and important vital organs other than the target organs of infected calves prompted us to take the present investigation to have detailed insight into the pathogenesis. The systematic necropsy of 37 dead calves (cattle-28 and buffalo-9) was conducted, and thin representative tissue pieces from the affected organs were collected in 10% neutral buffered formalin (NBF) for pathological and immunohistochemical investigations. The genomic detection and its serotyping were done by RT-PCR and multiplex-PCR, respectively. Necropsy examination in all cases showed myocardial lesions resembling 'tigroid heart appearance'. Other organ specific lesions include vesiculo-ulcerative stomatitis, edema of the lungs, petechial hemorrhages, edema of the endocrines, and gastroenteritis. Histopathological examination showed varying sizes of vesicles and ulcerations in stratified squamous epithelium of the tongue, acute necrotizing myocarditis, lymphoid depletion in lymphoid tissues, hepatitis, pancreatitis, thymic hyperplasia, thyroiditis, adrenitis, and enteritis. Positive immunolabeling for viral antigens was observed in endocrine glands, lymphoid organs, lungs, liver, kidneys, and intestine, in addition to other typical locations. The thyroid, adrenal glands, and pancreas, in addition to the tongue and heart, are the tissue of choice for sampling in the field during epidemics. Further, the viral genome and serotype A was confirmed in the affected tissues. This study provides insights into novel tissue tropism and pathogenesis in young calves naturally infected with FMDV.

A Newly Emerging Serotype A Strain in Foot-and-Mouth Disease Virus with Higher Severity and Mortality in Buffalo than in Cattle Calves in North Egypt.

A severe foot-and-mouth disease (FMD) epidemic struck several Egyptian provinces recently, causing significant losses among animals even in vaccinated farms. This study indicated the existence of the newly emerging foot-and-mouth disease virus (FMDV) and first investigated its effect on the Egyptian water buffalo (Bubalus bubalis) and cattle calves in the Beheira province, north Egypt. Twenty tongue epithelial samples from diseased calves in five infected farms were randomly collected, prepared, and propagated using baby hamster kidney-21 (BHK-21) cells. Whole genomic RNA was extracted from the cells of the third passage. A FMDV genome was detected and serotyped using one-step reverse transcription polymerase chain reactions (RT-PCRs). Nucleotide sequencing of the purified serotype-specific PCR bands was performed, and a maximum likelihood phylogenetic tree based on 600 base pairs of VP1 was constructed. The results identified FMDV, serotype A in all infected samples, whereas the serotypes O and SAT2 were negative. The obtained 20 sequences were identical to each other and similar to the newly reported strain in Egypt that belongs to the Europe-South America (Euro-SA) topotype. The epidemiological and clinical parameters associated with such a strain were fully recorded by veterinarians and analyzed in a single infected farm including 70 cattle and buffalo calves. It caused higher peracute mortalities in buffalo (25.7%; 95% CI: 13-43) than in cattle (8.6%; 95% CI: 2-24) calves. Severe clinical signs such as dullness, hypothermia, bradycardia, and cardiac arrhythmia were common to both except in fatal cases, whereas hyperthermia and respiratory signs were prevalent in cattle calves. In conclusion, we first characterized the newly emerging FMDV in the calves of Beheira as more fatal and severe in buffalo than in cattle calves.

Emergence of a novel genetic lineage 'A/ASIA/G-18/2019' of foot and mouth disease virus serotype A in India: A challenge to reckon with.

Foot and mouth disease (FMD) has engendered large scale socioeconomic crises on numerous occasions owing to its extreme contagiousness, transboundary nature, complicated epidemiology, negative impact on productivity, trade embargo, and need for intensive surveillance and expensive control measures. Emerging FMD virus variants have been predicted to have originated and spread from endemic Pool 2, native to South Asia, to other parts of the globe. In this study, 26 Indian serotype A isolates sampled between the year 2015 and 2022 were sequenced for the VP1 region. BLAST and maximum likelihood phylogeny suggest emergence of a novel genetic group within genotype 18, named here as 'A/ASIA/G-18/2019' lineage, that is restricted so far only to India and its eastern neighbour, Bangladesh. The lineage subsequent to its first appearance in 2019 seems to have displaced all other prevalent strains, in support of the phenomenon of 'genotype/lineage turnover'. It has diversified into two distinct sub-clusters, reflecting a phase of active evolution. The rate of evolution of the VP1 region for the Indian serotype A dataset was estimated to be 6.747 × 10-3 substitutions/site/year. India is implementing a vaccination centric FMD control programme. The novel lineage showed good antigenic match with the proposed vaccine candidate A IND 27/2011 when tested in virus neutralization test, while the existing vaccine strain A IND 40/2000 showed homology with only 31% of the isolates. Therefore, in order to combat this challenge of antigenic divergence, A IND 27/2011 could be the preferred strain in the Indian vaccine formulations.

A Pediatric Investigators Collaborative Network on Infections in Children (PICNIC) multi-centre Canadian descriptive analysis of Haemophilus influenzae bacteremia in children: Emerging serotypes.

Background: There has been dramatic reduction in Haemophilus influenzae serotype b (Hib) since introduction of Hib vaccines, but children still experience serious invasive Haemophilus influenzae (Hi) disease caused by various serotype and non-typeable bacteria. The object of this study was to describe the serotype distribution and clinical spectrum of Hi bacteremia in children admitted to Canadian hospitals. Methods: All children with Hi bacteremia admitted 2013 through 2017 to 10 centres across Canada were included. Demographic, clinical, treatment and outcome data were collected. Results: Haemophilus influenzae bacteremia occurred in 118 children of median age 12 months (inter-quartile range: 7-48 months). Forty-three (36%) isolates were non-typeable (NTHi) and 8 were not typed. Of the 67 typeable (THi), Hia (H. influenzae serotype a) (n=36, 54%), Hif (serotype f) (n=19, 26%) and Hib (serotype b) (n=9, 13%) dominated. The THi was more likely than NTHi bacteremia to present as meningitis (p<0.001), particularly serotype a (p=0.04) and less likely to present as pneumonia (p<0.001). Complicated disease (defined as intensive care unit admission, need for surgery, long-term sequelae or death) occurred in 31 (26%) cases and were more likely to have meningitis (p<0.001) than were those with uncomplicated disease. Conclusion: In the era of efficacious conjugate Hib vaccines, NTHi, Hia and Hif have emerged as the leading causes of invasive Hi in Canadian children, with Hia being most likely to result in meningitis and complicated disease. A vaccine for all NTHi and THi would be ideal, but knowledge of the current disease burden from circulating strains will inform prioritization of vaccine targets.

A Thermostable Dissolving Microneedle Vaccine with Recombinant Protein of Botulinum Neurotoxin Serotype A.

BACKGROUND: As a Class A bioterrorism agent, botulinum neurotoxin serotype A (BoNT/A) carries the risk of being used by terrorists to cause mass poisoning. The microneedle (MN) patch has a great potential for application as a novel vaccine delivery method. The aim of this study is to develop a thermally stable, dissolving microneedle patch for the delivery of a recombinant protein vaccine using a recombinant C-terminal heavy chain of BoNT/A (Hc of BoNT/A, AHc) to prevent botulism. METHODS: Fish gelatin, a natural non-toxic and bacteriostatic material, was selected as the microneedle matrix for the preparation of the dissolving microneedle vaccine. Subsequently, the mechanical performance, bacteriostatic properties, vaccination effect, and stability of the microneedle patches were evaluated using instruments such as the displacement-force test station and optical coherence tomography (OCT) scanner. RESULTS: Fish gelatin matrix at high concentrations has good bacteriostatic properties, and excellent mechanical performance and vaccination effect, meeting the necessities of a vaccine. In both in vivo and in vitro neutralization experiments, MN vaccines containing different antigen doses achieved the same protective efficacy as subcutaneous vaccinations, protecting mice against 106 LD50 of BoNT/A injected intraperitoneally. Thermal stability analysis of the MN vaccines revealed that the fish gelatin matrix protected the AHc vaccine from protein denaturation even after 7 days of storage at 37 °C and enabled the vaccine patches to maintain good immunogenicity and protective efficacy even after 6 months of storage at room temperature. CONCLUSION: In this study, we successfully prepared a bacteriostatic MN patch using a fish gelatin matrix that not only has a good vaccination effect, but also obviates the need for a cold chain for the AHc vaccine, providing the possibility of rapid, painless, and large-scale vaccination.

Evolutionary Dynamics of Foot and Mouth Disease Virus Serotype A and Its Endemic Sub-Lineage A/ASIA/Iran-05/SIS-13 in Pakistan.

Foot and mouth disease (FMD) causes severe economic losses to the livestock industry of endemic countries, including Pakistan. Pakistan is part of the endemic pool 3 for foot and mouth disease viruses (FMDV), characterized by co-circulating O, A, and Asia 1 serotypes, as designated by the world reference laboratory for FMD (WRL-FMD). FMDV serotype A lineage ASIA/Iran-05 is widespread in buffalos and cattle populations and was first reported in Pakistan in 2006. This lineage has a high turnover, with as many as 10 sub-lineages reported from Pakistan over the years. In this study, we reconstructed the evolutionary, demographic, and spatial history of serotype A and one of its sub-lineages, A/ASIA/Iran-05/SIS-13, prevalent in Pakistan. We sequenced nearly complete genomes of three isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13. We estimated recombination patterns and natural selection acting on the serotype A genomes. Source and transmission routes in Pakistan were inferred, and the clustering pattern of isolates of the SIS-13 sub-lineage were mapped on a tree. We hereby report nearly complete genome sequences of isolates belonging to sub-lineage A/ASIA/Iran-05/SIS-13, along with purported recombinant genomes, and highlight that complete coding sequences can better elucidate the endemic history and evolutionary pressures acting on long-term co-circulating FMDV strains.

Responses of selected biomarkers, female reproductive hormones and tissue changes in non-pregnant does challenged with Mannheimia haemolytica serotype A2 and its outer membrane protein (OMP) immunogen.

BACKGROUND: Mannheimia haemolytica causative agent of pneumonic mannheimiosis, a common respiratory disease of goat and sheep, which cause huge economic losses to farmers worldwide. Pneumonic mannheimiosis caused by M. haemolytica serotype A2 has been reported among small ruminants in Malaysia. The lipopolysaccharide (LPS) and outer membrane protein (OMP) are major virulence determinants for M. haemolytica serotype A2. Although pneumonic mannheimiosis is known to cause poor reproductive performance in small ruminants under field conditions, there is a dearth of published information on the specific effects of M. haemolytica serotype A2 infection on the female reproductive physiology. In this experiment, we explored the impact of M. haemolytica serotype A2 and its OMP immunogen on selected pro-inflammatory cytokines, acute phase proteins, female reproductive hormones, and cellular changes in visceral and female reproductive organs of non-pregnant does. METHODOLOGY: Twelve healthy, non-pregnant, Boer crossbreds does were divided equally into three groups (n = 4); Group 1 served as the negative control and was challenged with 2 ml of sterile PBS intranasally. Group 2 served as the positive control and was challenged with 2 ml of 109 colonies forming unit (CFU) of M. haemolytica serotype A2 suspension intranasally. Group 3 was challenged with 2 ml of OMP extracted from 109 CFU of M. haemolytica A2 intramuscularly. The experimental does were monitored for clinical signs and responses periodically. Blood samples were collected at 0, 1, 2, 4, 6, 12 and 24 h and 3, 7, 21, 35 and 56 days post treatment for serological analyses. All does were euthanised using the halal slaughter method on day 60 post challenge/treatment. Tissues from the uterus, liver, lung and associated bronchial lymph nodes were collected and fixed in 10% formalin for 14 days for histopathological study. RESULTS: Compared to the control group, the challenged/treated groups showed significant (p < 0.05) increase in the rectal temperature, respiratory rate, heart rate, and rumen motility. Serum analyses revealed that the concentrations of progesterone and estrogen hormones were significantly (p < 0.05) decreased in groups 2 & 3. In contrast, the concentrations of pro-inflammatory cytokines (IL-1ß and IL-6) and acute phase proteins (Hp and SAA) were significantly increased (p < 0.05) in the challenged/treated groups compared to the control group. Histopathological lesion scoring revealed mild to moderate cellular changes characterised by congestion, haemorrhage, degeneration, leucocytic cellular infiltration, and cellular necrosis in the tissues of does from the OMP treatment and bacterial challenge groups compared to the control group. CONCLUSION: The findings from this study suggests that M. haemolytica serotype A2 and its OMP immunogen induced mild to moderate inflammatory and degenerative changes which may potentially interfere with fertilization through hormonal imbalances and cause temporary loss of fertility in infected does.

Outbreaks of Foot-and-Mouth Disease in Burundi, East Africa, in 2016, Caused by Different Serotypes.

Burundi is a small, densely populated country in the African Great Lakes region. In March 2016, several hundreds of cattle were reported with vesicular lesions, suggesting foot-and-mouth disease (FMD). Epithelial samples, saliva, and blood were collected in six of the affected provinces spread over the country. The overall seroprevalence of FMD virus (FMDV) in the affected herds, as determined by antibodies against FMDV non-structural proteins, was estimated at 87%. Antibodies against FMDV serotypes O (52%), A (44%), C (19%), SAT1 (36%), SAT2 (58%), and SAT3 (23%) were detected across the provinces. FMDV genome was detected in samples from five of the six provinces using rRT-PCR. FMDV was isolated from samples from three provinces: in Cibitoke province, serotypes A and SAT2 were isolated, while in Mwaro and Rutana provinces, only serotype SAT2 was isolated. In Bururi and Cankuzo provinces, the serological profile suggested a recent incursion with serotype SAT2, while in Bubanza province, the serological profile suggested past incursions with serotype O and possibly serotype SAT1. The phylogenetic assessments showed the presence of topotypes A/Africa/G-I and SAT2/IV, similarly to previously characterized virus strains from other countries in the region, suggesting a transboundary origin and necessitating a regional approach for vaccination and control of FMD.

Viral Population Diversity during Co-Infection of Foot-And-Mouth Disease Virus Serotypes SAT1 and SAT2 in African Buffalo in Kenya.

African buffalo are the natural reservoirs of the SAT serotypes of foot-and-mouth disease virus (FMDV) in sub-Saharan Africa. Most buffalo are exposed to multiple FMDV serotypes early in life, and a proportion of them become persistently infected carriers. Understanding the genetic diversity and evolution of FMDV in carrier animals is critical to elucidate how FMDV persists in buffalo populations. In this study, we obtained oropharyngeal (OPF) fluid from naturally infected African buffalo, and characterized the genetic diversity of FMDV. Out of 54 FMDV-positive OPF, 5 were co-infected with SAT1 and SAT2 serotypes. From the five co-infected buffalo, we obtained eighty-nine plaque-purified isolates. Isolates obtained directly from OPF and plaque purification were sequenced using next-generation sequencing (NGS). Phylogenetic analyses of the sequences obtained from recombination-free protein-coding regions revealed a discrepancy in the topology of capsid proteins and non-structural proteins. Despite the high divergence in the capsid phylogeny between SAT1 and SAT2 serotypes, viruses from different serotypes that were collected from the same host had a high genetic similarity in non-structural protein-coding regions P2 and P3, suggesting interserotypic recombination. In two of the SAT1 and SAT2 co-infected buffalo identified at the first passage of viral isolation, the plaque-derived SAT2 genomes were distinctly grouped in two different genotypes. These genotypes were not initially detected with the NGS from the first passage (non-purified) virus isolation sample. In one animal with two SAT2 haplotypes, one plaque-derived chimeric sequence was found. These findings demonstrate within-host evolution through recombination and point mutation contributing to broad viral diversity in the wildlife reservoir. These mechanisms may be critical to FMDV persistence at the individual animal and population levels, and may contribute to the emergence of new viruses that have the ability to spill-over to livestock and other wildlife species.

Phylogenetic Structure and Comparative Genomics of Multi-National Invasive Haemophilus influenzae Serotype a Isolates.

Recent reports have indicated a rise of invasive disease caused by Haemophilus influenzae serotype a (Hia) in North America and some European countries. The whole-genome sequences for a total of 410 invasive Hia isolates were obtained from 12 countries spanning the years of 1998 to 2019 and underwent phylogenetic and comparative genomic analysis in order to characterize the major strains causing disease and the genetic variation present among factors contributing to virulence and antimicrobial resistance. Among 410 isolate sequences received, 408 passed our quality control and underwent genomic analysis. Phylogenetic analysis revealed that the Hia isolates formed four genetically distinct clades: clade 1 (n = 336), clade 2 (n = 13), clade 3 (n = 3) and clade 4 (n = 56). A low diversity subclade 1.1 was found in clade 1 and contained almost exclusively North American isolates. The predominant sequence types in the Hia collection were ST-56 (n = 125), ST-23 (n = 98) and ST-576 (n = 51), which belonged to clade 1, and ST-62 (n = 54), which belonged to clade 4. Clades 1 and 4 contained predominantly North American isolates, and clades 2 and 3 predominantly contained European isolates. Evidence of the presence of capsule duplication was detected in clade 1 and 2 isolates. Seven of the virulence genes involved in endotoxin biosynthesis were absent from all Hia isolates. In general, the presence of known factors contributing to ß-lactam antibiotic resistance was low among Hia isolates. Further tests for virulence and antibiotic susceptibility would be required to determine the impact of these variations among the isolates.

Changes in selected cytokines, acute-phase proteins, gonadal hormones and reproductive organs of non-pregnant does challenged with Mannheimia haemolytica serotype A2 and its LPS endotoxin.

Previous investigations have revealed that lipopolysaccharide (LPS) endotoxin from certain Gram-negative bacteria could adversely affect the reproductive system of female animals. However, it is unknown whether LPS endotoxin of Mannheimia haemolytica serotype A2, the principal causative bacteria that cause pneumonic mannheimiosis in small ruminants, may also induce similar insidious effects. Therefore, this study aimed to investigate the effects of M. haemolytica serotype A2 and its LPS endotoxin on the responses of female gonadal hormones (progesterone and oestrogen), pro-inflammatory cytokines (interleukin-1ß, interleukin-6), acute-phase proteins (haptoglobin and serum amyloid A) and cellular changes via histopathology study of female reproductive organs of the treatment does. Twelve clinically healthy, non-pregnant, crossbred does were randomly allocated into three equal groups. Group 1 was administered intranasally with 2 ml of sterile phosphate-buffered saline (PBS) and served as a negative control group. Group 2 was challenged intranasally with 2 ml of bacterial inoculum containing 109 colony-forming units (CFU)/ml of M. haemolytica serotype A2, while Group 3 was challenged intravenously with 2 ml of LPS endotoxin extracted from 109 CFU/ml of M. haemolytica serotype A2. Following that, blood samples were collected serially at pre-determined intervals for serological analyses. All does were euthanised 60 days post-challenges, and tissue samples from the ovaries, oviducts, uterine horns, uterine body, cervix and vagina were collected for histopathological study. The serological result revealed a significant increase (p < 0.05) in the mean concentrations of progesterone, oestrogen, interleukin-1ß, interleukin-6, haptoglobin and serum amyloid A for both challenged groups. Histopathologically, all reproductive organs (except the cervix and vagina) from both challenged groups displayed significant cellular alterations (p < 0.05) characterised by haemorrhage and congestion, necrosis and degeneration, inflammatory cell infiltration and oedema. This study provides new information that elucidates the potential role of pneumonic mannheimiosis in the pathogenesis of female infertility amongst small ruminants.

A Vaccine Based on the A/ASIA/G-VII Lineage of Foot-and-Mouth Disease Virus Offers Low Levels of Protection against Circulating Viruses from the A/ASIA/Iran-05 lineage.

The recent emergence and circulation of the A/ASIA/G-VII (A/G-VII) lineage of foot-and-mouth disease virus (FMDV) in the Middle East has resulted in the development of homologous vaccines to ensure susceptible animals are sufficiently protected against clinical disease. However, a second serotype A lineage called A/ASIA/Iran-05 (A/IRN/05) continues to circulate in the region and it is therefore imperative to ensure vaccine strains used will protect against both lineages. In addition, for FMDV vaccine banks that usually hold a limited number of strains, it is necessary to include strains with a broad antigenic coverage. To assess the cross protective ability of an A/G-VII emergency vaccine (formulated at 43 (95% CI 8-230) PD50/dose as determined during homologous challenge), we performed a heterologous potency test according to the European Pharmacopoeia design using a field isolate from the A/IRN/05 lineage as the challenge virus. The estimated heterologous potency in this study was 2.0 (95% CI 0.4-6.0) PD50/dose, which is below the minimum potency recommended by the World Organisation for Animal Health (OIE). Furthermore, the cross-reactive antibody titres against the heterologous challenge virus were poor (≤log10 0.9), even in those cattle that had received the full dose of vaccine. The geometric mean r1-value was 0.2 (95% CI 0.03-0.8), similar to the potency ratio of 0.04 (95% CI 0.004-0.3). Vaccination decreased viraemia and virus excretion compared to the unvaccinated controls. Our results indicate that this A/G-VII vaccine does not provide sufficient protection against viruses belonging to the A/IRN/05 lineage and therefore the A/G-VII vaccine strain cannot replace the A/IRN/05 vaccine strain but could be considered an additional strain for use in vaccines and antigen banks.

Assessment of fitness of foot-and-mouth disease virus A IND 27/2011 as candidate vaccine strain.

Antigenic profiling of recent field outbreak strains of foot-and-mouth disease virus (FMDV) serotype A in India has revealed considerable antigenic drift from the vaccine strain, A IND 40/2000, necessitating the selection of a new strain. The complete genome sequence of A IND 27/2011 was analysed. Vaccine quality attributes of the new candidate strain including potency as an inactivated vaccine in cattle were evaluated. The capsid coding region of A IND 27/2011 showed variation at eight antigenically critical amino acid positions from that of A IND 40/2000. The strain suited well with traits required by a vaccine in terms of its adaptability to adherent and suspension cell line, its immunogenicity, and potency as an inactivated vaccine formulation in cattle. Complete protection was observed upon homologous virus challenge at 4 weeks post-vaccination. Taken together, these data demonstrate the suitability of A IND 27/2011 as an effective vaccine strain of FMDV serotype A.

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1-A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy.