Low Virulent Isolates Research Articles

Calf immunization protocols with low-virulence isolates of Anaplasma marginale: Analysis of post-inoculation effects and protection against natural challenge

Bovine anaplasmosis is endemic and is of fundamental importance worldwide. Therefore, measures for controlling and preventing clinical diseases are warranted to ensure the reduction of associated economic losses. The objective of the present study was to assess the post-inoculation effects and protection conferred by three different protocols of inoculation of low-virulence live strains of Anaplasma marginale (UFMG1 and UFMG3) in field-challenged cattle. Sixty-eight Holstein calves with an average age of 17 days were randomly divided into four groups. The groups received two subcutaneous administrations spaced 40 days apart, at a dosage of 2 × 106 infected erythrocytes of the following A. marginale strains: G1 (UFMG1 + UFMG1); G2 (UFMG3 + UFMG3); G3 (UFMG1 + UFMG3); and G4 (control). Every two days, the animals were evaluated for rectal temperature, Packed Cell Volume (PCV), and blood smears. Blood samples were collected prior to inoculation, before the field challenge, and after the challenge period, nPCR and IFAT techniques were performed. There were no significant differences in rickettsemia levels, reduction in PCV, or antibody detection among the different inoculation strategies. Forty days after the second inoculation, 90 %, 84.6 %, and 90.9 % of the animals in G1, G2, and G3, respectively, tested positive using nPCR. After inoculation, the group G2, which received the UFMG3 inoculum, had a higher frequency of treatment (odds ratio of 6.7; 1.198-38.018 CI; p = 0.03), while groups G1 and G3 demonstrated similar treatment frequencies compared to the control. During the natural challenge phase, 13.3 % of animals in group G1 required treatment (odds ratio of 0.108; 0.018-0.635 CI; p = 0.014) compared to 58.8 % of the control group. Considering the results collectively, the protocol using the UFMG1 strain (G1) stands out for its potential to be safe and induce some degree of immunization against A. marginale, reducing the incidence of clinical disease and the need for treatment during natural challenge.

Histopathologic evaluation system of African swine fever in wild boar infected with high (Arm07) and low virulence (Lv17/WB/Riel) isolates.

To understand the clinicopathological forms of African swine fever (ASF) in wild boar, it is crucial to possess a basic knowledge of the biological characteristics of the currently circulating ASF virus isolates. The aim of this work is to establish an accurate and comprehensive histopathologic grading system to standardize the assessment of the ASF lesions in wild boar. The study evaluated the differences between animals infected with a high virulence genotype II isolate (Arm07) (HVI) through intramuscular (IM) (n = 6) and contact-infected (n = 12) routes, alongside those orally infected with a low virulence isolate (Lv17/WB/Riel) (LVI) (n = 6). The assessment included clinical (CS), macroscopic (MS), and histopathologic (HS) scores, as well as viral loads in blood and tissues by real-time quantitative polymerase chain reaction (qPCR). Tissues examined included skin, lymph nodes, bone marrow, palatine tonsil, lungs, spleen, liver, kidneys, thymus, heart, adrenal glands, pancreas, urinary bladder, brain, and gastrointestinal and reproductive tracts. The HVI group exhibited a 100% mortality rate with elevated CS, MS, and HS values. Animals infected by contact (CS = 12; MS = 58.5; HS = 112) and those intramuscularly infected (CS = 14.8; MS = 47; HS = 104) demonstrated similar values, indicating that the route of infection does not decisively influence the severity of clinical and pathological signs. The LVI group showed a 0% mortality rate, an inconspicuous clinical form, minimal lesions (CS = 0; MS = 12; HS = 29), and a lower viral load. Histopathologic evaluation has proven valuable in advancing our comprehension of ASF pathogenesis in wild boar and paves the groundwork for further research investigating protective mechanisms in vaccinated animals.

Invasion of Chicken Anemia Virus in Specific-Pathogen-Free Chicken Flocks and Its Successful Elimination from the Colony.

A specific-pathogen-free (SPF) chicken colony was maintained with successive groups a month apart in age. The absence of specific pathogens, including chicken anemia virus (CAV), was confirmed through periodic serological tests for each group. However, some groups became CAV seropositive. The procedures of removing seropositive and the adjacent seronegative chickens followed with chemically disinfecting the housing did not halt CAV outbreaks. The full genome sequence of the CAV strain that appeared was closely related to low-virulence isolates in China. The outbreaks of CAV decreased with an increase in the seropositive chicken population, indicating that the progeny is protected from CAV infection by maternal anti-CAV antibodies. The persistence of CAV in erythroid and lymphoid tissues or reproductive tissues from CAV seropositive chickens was examined in chickens of various ages using polymerase chain reaction (PCR). Since a low persistence of CAV was observed in the colony, we isolated eggs from CAV seropositive hens through artificial insemination using semen collected from roosters and confirmed as CAV-free by PCR. Fertilized eggs were transferred to a new SPF facility and used for generating CAV-free progeny. To date, chickens reared in the new facility have been CAV-free for longer than two years. Redirection of eggs from seropositive hens was an effective means of eliminating CAV from chickens.

Tertiary lymphoid organs in wild boar exposed to a low-virulent isolate of African swine fever virus

Despite the great interest in the development of a vaccine against African swine fever (ASF) in wild boar, the immunological mechanisms that induce animal protection are still unknown. For this purpose, tertiary lymphoid organs (TLOs) of wild boar were characterised and compared with mucosa-associated lymphoid tissues (MALTs) by histopathology, histomorphometry and immunohistochemistry (CD3, CD79, PAX5, LYVE1, fibronectin). In addition, real-time polymerase chain reaction (qPCR) and immunohistochemistry (p72) were used to evaluate the presence of ASF virus (ASFV) in blood and tissues samples, respectively. TLOs were observed in animals infected with a low-virulent ASFV isolate (LVI), animals co-infected with low and high-virulent ASFV isolates (LVI-HVI) and animals infected only with the high virulence isolate (HVI). TLOs in LVI and LVI-HVI groups were located adjacent to the mucosa and presented a similar structure to MALT. Immunoexpresion of p72 observed in the inflammatory cells adjacent to TLOs/MALTs confirmed its development and reactivity generated by ASF attenuated isolates. Immunohistochemical evaluation, based on cellular composition (T and B lymphocytes), and histomorphometrical study revealed a more pronounced maturation of TLOs/MALTs in the LVI-HVI group. It is currently unclear whether these formations play a protective role by contributing to local immunity in chronic inflammatory diseases. However, the structural similarities between TLOs and MALTs and the location of TLOs close to the mucosa suggest that they may perform a similar function, facilitating a local protective response. Nevertheless, further investigations are warranted to assess the cellular and humoral dynamics of these lymphoid organs induced by attenuated isolates.

Sequencing and characterization of Helcococcus ovis: a comprehensive comparative genomic analysis of virulence

BackgroundHelcococcus ovis (H. ovis) is an emerging bacterial pathogen that commonly causes opportunistic respiratory, mammary, and uterine infections across mammalian hosts. This study applied long- and short-read whole genome sequencing technologies to identify virulence factors in five H. ovis isolates with low, medium, and high virulence phenotypes.ResultsThe resulting assemblies contained one circular chromosome ranging from 1,744,566 to 1,850,083 bp in length and had a mean GC content of 27.6%. Phylogenetic and nucleotide identity analyses found low virulence strain KG38 to be part of a clade that forms an outgroup apart from the rest of the H. ovis taxon. Assembling the first complete genomes of the species revealed major genomic rearrangements in KG38. One to six prophage regions were identified in each genome. A novel pathogenicity island was found exclusively in the two high virulence strains (KG37 and KG104), along with two hypothetical transmembrane proteins designated as putative VFs. Finally, three zinc ABC transporters and three Type-II/IV secretion systems were identified as possible virulence determinants in this species. The low virulence strain KG38 has fewer intact paralogs of these operons in its genome compared to the higher virulence isolates, which strongly suggests a role in virulence. This strain is also missing four putative virulence factors (VFs) found in other isolates associated with adherence (collagen adhesin precursor), immune evasion (choline-binding protein A and a PspA-like hypothetical protein) and cell wall synthesis (glycerol-3-phosphate cytidylyltransferase).ConclusionsIn this study, we assembled reference-quality complete genomes for five H. ovis strains to identify putative virulence factors. Phylogenetic analyses of H. ovis isolates revealed the presence of a clade representing a potentially novel species within the genus Helcococcus. A novel pathogenicity island and two hypothetical transmembrane proteins were found exclusively in high-virulence strains. The identification of Zinc ABC transporters and Type-II/IV secretion systems as possible virulence determinants, along with the differences in operon content between the low and high virulence isolates, strongly suggests they also play a role in the bacterium’s pathogenicity. Taken together, these findings are a valuable first step toward deciphering the pathogenesis of H. ovis infections.

Efficacy of a porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) natural recombinant against a heterologous PRRSV-1 isolate both clustered within the subgroup of BJEU06-1-like isolates

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) has been prevalent in more than 20 provinces of China. However, no PRRSV-1-specific vaccine is commercially available in China. To evaluate the feasibility of using a low virulent PRRSV-1 isolate against potential outbreaks caused by virulent Chinese PRRSV-1 isolates, here we evaluated the efficacy of a low virulent PRRSV-1 HLJB1 strain isolated in 2014 as live vaccine against a virulent PRRSV-1 SD1291 strain isolated in 2022. Genome-based phylogenetic analysis showed that both HLJB1 and SD1291 were grouped within BJEU06–1-like isolates. However, they shared only 85.27% genomic similarity. Piglet inoculation and challenge study showed that HLJB1 inoculation could reduce viremia but did not significantly alleviate clinical signs and tissue lesions. Virus neutralization test indicated that HLJB1 inoculation could induce homologous neutralizing antibodies (NAbs) but no heterologous NAbs at 42 dpi. In addition, flow cytometric analyses showed that no memory T follicular helper (Tfh) cells against SD1291 and SD1291-specific IFN-γ secreting cells were induced by HLJB1 pre-inoculation. These results supported that HLJB1 inoculation only provides partial cross-protection against SD1291 infection even though they are clustered within the same PRRSV-1 subgroup, which is closely related to the failure in conferring cross-protective adaptive immune responses.

Insights into the infection dynamics and interactions between high-virulence and low-virulence isolates of Phytophthora palmivora and durian seedlings

ABSTRACT Durian (Durio zibethinus L.) is susceptible to Phytophthora palmivora, which causes rot disease, leading to significant yield loss. This study aimed to investigate local defense responses of durian to P. palmivora and compare the infection dynamics of high virulence (HV) and low virulence (LV) isolates. Durian leaves on 1-year-old seedlings were inoculated with mycelial plugs. Symptoms were observed within 24 h. The HV-isolate was more aggressive, leading to larger lesions, earlier and denser colonization, and higher sporulation. P. palmivora lifestyle related gene (PpEF1α, PpHmp1, PpCdc14, and PpOPEL) were expressed at a higher level in HV compared to LV. Local defense response showed intense accumulation of H2O2, with a stronger response to the HV. Callose deposition increased initially but declined after inoculation, with different diameters between isolates, but no significantly different intensity patterns. These findings provide insights into the P. palmivora-durian interaction, with potential applications in improving disease management strategies.

Whole-transcriptome analysis reveals virulence-specific pathogen-host interactions at the placenta in bovine neosporosis.

Research on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.

The genetic variability and evolution of red-spotted grouper nervous necrosis virus quasispecies can be associated with its virulence.

Nervous necrosis virus, NNV, is a neurotropic virus that causes viral nervous necrosis disease in a wide range of fish species, including European sea bass (Dicentrarchus labrax). NNV has a bisegmented (+) ssRNA genome consisting of RNA1, which encodes the RNA polymerase, and RNA2, encoding the capsid protein. The most prevalent NNV species in sea bass is red-spotted grouper nervous necrosis virus (RGNNV), causing high mortality in larvae and juveniles. Reverse genetics studies have associated amino acid 270 of the RGNNV capsid protein with RGNNV virulence in sea bass. NNV infection generates quasispecies and reassortants able to adapt to various selective pressures, such as host immune response or switching between host species. To better understand the variability of RGNNV populations and their association with RGNNV virulence, sea bass specimens were infected with two RGNNV recombinant viruses, a wild-type, rDl956, highly virulent to sea bass, and a single-mutant virus, Mut270Dl965, less virulent to this host. Both viral genome segments were quantified in brain by RT-qPCR, and genetic variability of whole-genome quasispecies was studied by Next Generation Sequencing (NGS). Copies of RNA1 and RNA2 in brains of fish infected with the low virulent virus were 1,000-fold lower than those in brains of fish infected with the virulent virus. In addition, differences between the two experimental groups in the Ts/Tv ratio, recombination frequency and genetic heterogeneity of the mutant spectra in the RNA2 segment were found. These results show that the entire quasispecies of a bisegmented RNA virus changes as a consequence of a single point mutation in the consensus sequence of one of its segments. Sea bream (Sparus aurata) is an asymptomatic carrier for RGNNV, thus rDl965 is considered a low-virulence isolate in this species. To assess whether the quasispecies characteristics of rDl965 were conserved in another host showing different susceptibility, juvenile sea bream were infected with rDl965 and analyzed as above described. Interestingly, both viral load and genetic variability of rDl965 in seabream were similar to those of Mut270Dl965 in sea bass. This result suggests that the genetic variability and evolution of RGNNV mutant spectra may be associated with its virulence.

Intrapancreatic accessory spleens in African swine fever infection of wild boar (Sus scrofa).

Intrapancreatic accessory spleen (IPAS) is one of the most frequent congenital splenic anomalies in humans; however, studies in veterinary medicine are scarce. This study aimed to describe the macroscopic, histopathological and immunohistochemical features of 11 suspected cases of IPAS in wild boar piglets of 3-4 months old. Seven of the 11 animals were immunised with a low virulence isolate of African swine fever virus (ASFV) and subsequently challenged with a highly virulent ASFV isolate (LVI-HVI group). The remaining four animals were exclusively infected with a highly virulent isolate of ASFV (HVI group). Grossly, lesions comprised focal or multifocal reddish areas of variable shape, located on the surface of the pancreatic tail or within the parenchyma. Histological and immunohistochemical studies (anti-CD79 and CD3) confirmed the presence of IPAS in eight of the 11 cases. IPAS shared the same histological structure and alterations as those observed in the original spleen. The immunohistochemical study against ASFV revealed the presence of VP72+ cells in both the spleen and IPAS of seven of the eight piglets. The results of this study describe for the first time the presence of IPAS in ASFV infection of wild boar (Sus scrofa) regardless the isolate and suggest that the infection may induce the development of ectopic splenic tissue due to an increased demand for phagocytic cells from the reticuloendothelial system. However, further studies are needed to understand the immunological mechanisms that trigger the formation of these accessory organs.

Transcriptome analysis of sugarcane reveals differential switching of major defense signaling pathways in response to Sporisorium scitamineum isolates with varying virulent attributes.

Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum is one of the most devastating diseases that affect sugarcane production, globally. At present, the most practical and effective management strategy for the disease is the cultivation of resistant cultivars. In this connection, a detailed understanding of the host's defense mechanism in response to smut isolates with varying degrees of virulence at the molecular level would facilitate the development of reliable and durable smut-resistant sugarcane varieties. Hence, in this study, a comparative whole transcriptome analysis was performed employing Illumina RNA-seq in the smut susceptible cultivar Co 97009 inoculated with two distinct S. scitamineum isolates, Ss97009 (high-virulent) and SsV89101 (low-virulent) during the early phases of infection (2 dpi and 5 dpi) and at the phase of sporogenesis (whip emergence) (60 dpi). Though the differential gene expression profiling identified significant transcriptional changes during the early phase of infection in response to both the isolates, the number of differentially expressed genes (DEGs) were more abundant at 60 dpi during interaction with the high virulent isolate Ss97009, as compared to the low virulent isolate SsV89101. Functional analysis of these DEGs revealed that a majority of them were associated with hormone signaling and the synthesis of defense-related metabolites, suggesting a complex network of defense mechanisms is being operated in response to specific isolates of the smut pathogen. For instance, up-regulation of hormone-related genes, transcription factors, and flavonoid biosynthesis pathway genes was observed in response to both the isolates in the early phase of interaction. In comparison to early phases of infection, only a few pathogenesis-related proteins were up-regulated at 60 dpi in response to Ss97009, which might have rendered the host susceptible to infection. Strikingly, few other carbohydrate metabolism-associated genes like invertases were up-regulated in Ss97009 inoculated plants during the whip emergence stage, representing a shift from sucrose storage to smut symptoms. Altogether, this study established the major switching of defense signaling pathways in response to S. scitamineum isolates with different virulence attributes and provided novel insights into the molecular mechanisms of sugarcane-smut interaction.

Cellular and Humoral Immune Responses after Immunisation with Low Virulent African Swine Fever Virus in the Large White Inbred Babraham Line and Outbred Domestic Pigs.

African swine fever virus is currently present in all of the world’s continents apart from Antarctica, and efforts to control the disease are hampered by the lack of a commercially available vaccine. The Babraham large white pig is a highly inbred line that could represent a powerful tool to improve our understanding of the protective immune responses to this complex pathogen; however, previous studies indicated differential vaccine responses after the African swine fever virus challenge of inbred minipigs with different swine leukocyte antigen haplotypes. Lymphocyte numbers and African swine fever virus-specific antibody and T-cell responses were measured in inbred and outbred animals after inoculation with a low virulent African swine fever virus isolate and subsequent challenge with a related virulent virus. Surprisingly, diminished immune responses were observed in the Babraham pigs when compared to the outbred animals, and the inbred pigs were not protected after challenge. Recovery of Babraham pigs after challenge weakly correlated with antibody responses, whereas protective responses in outbred animals more closely correlated with the T-cell response. The Babraham pig may, therefore, represent a useful model for studying the role of antibodies in protection against the African swine fever virus.

Investigation of virulence diversity in Ralstonia solanacearum isolates by a random amplified polymorphic DNA collected from Egyptian potato fields

In this study, a total of ten bacterial isolates obtained from the wilted potato plants in Egypt, identified as Ralstonia solanacearum race 3 (biovar II) were used to study the differentiation among the isolates by RAPD marker analysis. In pathogenicity experiments, all isolates demonstrated typical wilt disease symptoms on potato plants. However, the level of virulence was significantly different as the isolates GK1, GT2, and AM8 were highly virulent one isolate “ES6” was moderate virulent and one isolate “SA9” was virulent.Molecular characterization of virulent, moderate virulent, and low virulent isolates along with five avirulent isolates (originated from the same regions) was performed by RAPD marker analysis using six random ten-mer primers. The results of RAPD marker analysis showed significant variation (9-12) in the number of fragments having molecular weight from 219 to 1120 bp. The results presented here demonstrated that no correlation was observed between the RAPD profile and the geographicaly originated strains.

Genetic grouping and geographic distribution of Piscine orthoreovirus-1 (PRV-1) in farmed Atlantic salmon in Norway

Piscine orthoreovirus-1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). However, it has been shown that PRV-1 variants differ in their ability to induce HSMI. The objective of this work was to identify the PRV-1 variants in Norwegian aquaculture and their geographical distribution. Sequencing and subsequent analysis of the five genomic segments (S1, S4, M2, L1 and L2) putatively linked to virulence, made out the basis of the study. Thirty-seven Norwegian PRV-1 isolates were sequenced, and they grouped into eight genogroups based on combinations of the five analyzed genomic segments. Two groups were defined as high-virulent and two low-virulent, based on comparison with PRV-1 reference isolates with known virulence. The remaining four groups were of unknown virulence. The geographic distribution indicated a higher frequency of the high-virulent isolates in the mid- and northern regions. The present study confirms circulation of both high- and low-virulent isolates of PRV-1 in farmed Atlantic salmon in Norway. To reduce the impact of PRV-1 related disease, detection and differentiation between high- and low-virulent genogroups of PRV-1 could be a targeted approach for reduction of high-virulent variants.

Diverse Host Immune Responses of Different Geographical Populations of the Coconut Rhinoceros Beetle to Oryctes Rhinoceros Nudivirus (OrNV) Infection.

ABSTRACTIncursions of the coconut rhinoceros beetle (CRB), Oryctes rhinoceros, into different islands in the South Pacific have been detected in recent years. It has been suggested that this range expansion is related to an O. rhinoceros haplotype reported to show reduced susceptibility to the well-established classical biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). Our understanding of the genetic characteristics which distinguish the population of O. rhinoceros that has recently established in Solomon Islands from other well-established populations across the region is very limited. Here, we hypothesized that the recently established O. rhinoceros population should have greater innate immune responses when challenged by OrNV than those of well-established and native O. rhinoceros populations. We used the RNA sequencing (RNA-Seq) approach to generate gene expression profiles of midgut tissue from OrNV-infected and noninfected individuals collected in the Solomon Islands (recent incursion), Papua New Guinea and Fiji (previously established), and the Philippines (within the native range). The collections included individuals from each of the three major mitochondrial lineages (CRB-G, CRB-PNG, and CRB-S) known to the region, allowing us to explore the specific responses of each haplotype to infection. Although insects from the Philippines and Solomon Islands that were tested belong to the same mitochondrial lineage (CRB-G), their overall responses to infection were different. The number of differentially expressed genes between OrNV-infected and noninfected wild-caught individuals from the four different locations varied from 148 to 252. Persistent OrNV infection caused a high level of induced antimicrobial activity and immune responses in O. rhinoceros, but the direction and magnitude of the responses were population specific. The insects tested from the Solomon Islands displayed extremely high expression of genes which are known to be involved in immune responses (e.g. coleoptericin, cecropin, and serpin). These variations in the host immune system among insects from different geographical regions might be driven by variations in the virulence of OrNV isolates, and this requires further investigation. Overall, our current findings support the importance of immunity in insect pest incursion and an expansion of the pest’s geographic range.IMPORTANCE Oryctes rhinoceros nudivirus (OrNV) is a double-stranded DNA (dsDNA) virus which has been used as a biocontrol agent to suppress coconut rhinoceros beetle (CRB) in the Pacific Islands. Recently a new wave of CRB incursions in Oceania is thought to be related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles (CRB-G). Our comparative analysis of OrNV-infected and noninfected CRBs revealed that specific sets of genes were induced by viral infection in the beetles. This induction was much stronger in beetles collected from the Solomon Islands, a newly invaded country, than in individuals collected from within the beetle’s native range (the Philippines) or from longer-established populations in its exotic range (Fiji and Papua New Guinea [PNG]). Beetles from the Philippines and the Solomon Islands that were tested in this study all belonged to the CRB-G haplotype, but the country-specific responses of the beetles to OrNV infection were different.

Comparative expression analysis of potential pathogenicity-associated genes of high- and low-virulent Sporisorium scitamineum isolates during interaction with sugarcane.

The online version contains supplementary material available at 10.1007/s13205-021-02893-7.

Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation.

Bovine neosporosis is currently considered one of the main causes of abortion in cattle worldwide and the outcome of the infection is, in part, determined by Neospora caninum isolate virulence. However, the dam and foetal immune responses associated with this factor are largely unknown. We used a model of bovine infection at day 110 of gestation to study the early infection dynamics (10- and 20-days post-infection, dpi) after experimental challenge with high- and low-virulence isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively). In the present work, dam peripheral cellular immune responses were monitored twice a week from -1 to 20 dpi. At different time points, IFN-γ and IL-4 production was investigated in stimulated dam blood and the percentage of monocytes, NK cells, B cells and T cells (CD4+, CD8+ and γδ) in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry. In addition, maternal iliofemoral lymph nodes and foetal spleen and thymus were collected at 10 and 20 dpi for the study of the same cell subpopulations. Peripheral immune response dynamics were similar after the infection with both isolates, with a significant increase in the percentage of CD4+ T cells at 6 and 9 dpi in PBMC, coincident with the higher levels of IFN-γ and IL-4 release. However, the levels of IFN-γ were significantly higher and an increase in CD8+ T cells at 9, 13 and 20 dpi was observed in the dams infected with Nc-Spain7. Nc-Spain1H infection induced higher IL4 levels in stimulated blood and a higher CD4+/CD8+ ratio in PBMC. The analysis of the maternal iliofemoral lymph node showed a significant enhancement in the percentage of NK, CD4+ and CD8+ T cells for the animals infected with the highly virulent isolate and euthanized at 20 dpi. Regarding the foetal responses, the most remarkable result was an increase in the percentage of monocytes at 20 dpi in the spleen of foetuses from both infected groups, which suggests that foetuses were able to respond to N. caninum infection at mid gestation. This work provides insights into how isolate virulence affects the maternal and foetal immune responses generated against N. caninum, which may influence the course of infection.

Comparative analysis of draft genome assemblies developed from whole genome sequences of two Hyaloperonospora brassicae isolate samples differing in field virulence on Brassica napus

Hyaloperonospora brassicae causes downy mildew, a major disease of Brassicaceae species. We sequenced the genomes of two H. brassicae isolates of high (Sample B) and low (Sample C) virulence. Sequencing reads were first assembled de novo with software's SOAPdenovo2, ABySS V2.1 and Velvet V1.1 and later combined to create meta-assemblies with genome sizes of 72.762 and 76.950Mb and predicted gene densities of 1628 and 1644 /Mb, respectively. We could annotate 12.255 and 13,030 genes with high proportions (91-92%) of complete BUSCOs for Sample B and C, respectively. Comparative analysis revealed conserved and varied molecular machinery underlying the physiological specialisation and infection capabilities. BLAST analysis against PHI gene database suggested a relatively higher loss of genes for virulence and pathogenicity in Sample C compared to Sample B, reflecting pathogen evolution through differential rates of mutation and selection. These studies will enable identification and monitoring of H. brassicae virulence factors prevailing in-field.

Identification, Virulence, and Molecular Characterization of a Recombinant Isolate of Grass Carp Reovirus Genotype I.

The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7–98.3% nucleotide identity, a 96.4–100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.

Cerebrospinal fluid (CSF) augments metabolism and virulence expression factors in Acinetobacter baumannii

In a recent report by the Centers for Disease Control and Prevention (CDC), multidrug resistant (MDR) Acinetobacter baumannii is a pathogen described as an “urgent threat.” Infection with this bacterium manifests as different diseases such as community and nosocomial pneumonia, bloodstream infections, endocarditis, infections of the urinary tract, wound infections, burn infections, skin and soft tissue infections, and meningitis. In particular, nosocomial meningitis, an unwelcome complication of neurosurgery caused by extensively-drug resistant (XDR) A. baumannii, is extremely challenging to manage. Therefore, understanding how A. baumannii adapts to different host environments, such as cerebrospinal fluid (CSF) that may trigger changes in expression of virulence factors that are associated with the successful establishment and progress of this infection is necessary. The present in-vitro work describes, the genetic changes that occur during A. baumannii infiltration into CSF and displays A. baumannii’s expansive versatility to persist in a nutrient limited environment while enhancing several virulence factors to survive and persist. While a hypervirulent A. baumannii strain did not show changes in its transcriptome when incubated in the presence of CSF, a low-virulence isolate showed significant differences in gene expression and phenotypic traits. Exposure to 4% CSF caused increased expression of virulence factors such as fimbriae, pilins, and iron chelators, and other virulence determinants that was confirmed in various model systems. Furthermore, although CSF's presence did not enhance bacterial growth, an increase of expression of genes encoding transcription, translation, and the ATP synthesis machinery was observed. This work also explores A. baumannii’s response to an essential component, human serum albumin (HSA), within CSF to trigger the differential expression of genes associated with its pathoadaptibility in this environment.