Pathogenic Field Strain Research Articles

Complete Genome Sequences of Avian Metapneumovirus Subtype B Vaccine Strains from Brazil.

Avian metapneumovirus (aMPV) causes a highly contagious upper respiratory and reproductive disease in chickens, turkeys, and ducks. Here, complete genome sequences of aMPV-B vaccine strains BR/1890/E1/19 (PL21, Nemovac; Boehringer Ingelheim Animal Health, Brazil) and BR/1891/E2/19 (1062; Hipraviar, France) were sequenced and compared with the pathogenic field strain VCO3/60616.

Effects of feed supplementation with 3 different probiotic Bacillus strains and their combination on the performance of broiler chickens challenged with Clostridium perfringens

The application of probiotics in broiler feed, to alleviate performance deficiencies due to mild infections by coccidia and Clostridium perfringens, is of increasing interest for the poultry industry. Therefore, our objective was to evaluate the capacity of 3 Bacillus strains and their combination as probiotics in vitro and in vivo. Thus, protein and carbohydrate degradation and C. perfringens growth inhibition capabilities were assessed by colometry measurement and an agar diffusion bioassay, respectively. A total of 2,250 1-day-old male broiler chicks were assigned to 5 dietary treatments: 1) non–probiotic-supplemented control (control); 2) control + DSM 32324 at 0.8 × 106 cfu/g of feed; 3) control + DSM 32325 at 0.5 × 106 cfu/g of feed; 4) control + DSM 25840 at 0.3 × 106 cfu/g of feed; and 5) control + DSM 32324 + DSM 32325 + DSM 25840 at 1.6 × 106 cfu/g of feed. A pathogenic field strain of C. perfringens was used to induce the necrotic enteritis challenge on day 19, 20, and 21. All birds and remaining feed were weighed on pen basis on day 0, 21, 35, and 42, to calculate BW gain and mortality-adjusted feed conversion. Mortality and mortality due to necrotic enteritis were recorded daily. On day 21, 45 birds per treatment were evaluated for macroscopic intestinal necrotic enteritis lesions. Performance data were statistically analyzed using an ANOVA and subjected to a least significant difference comparison. Necrotic enteritis lesion scores were statistically analyzed using nonparametric Kruskal-Wallis test. Dunn's test was used for treatment comparison. The tested strains showed different abilities of degrading protein and carbohydrates and inhibiting C. perfringens growth in vitro. The birds fed the multi-train combination presented significantly better performance and lower necrotic enteritis lesion score than those in the control group. Dietary supplementation with probiotics resulted in significantly lower necrotic enteritis mortality. The results demonstrate the suitability of the evaluated Bacillus multistrain combination as an effective probiotic in C. perfringens–challenged chickens.

Generation of recombinant avian coronaviruses indicates the S gene is a factor in pathogenicity

The avian coronavirus infectious bronchitis virus (IBV) is the most economically important disease of chickens in the UK, causing significant losses as a result of poor weight gain and reduced egg quality in infected birds. IBV expresses a large spike (S) glycoprotein on the surface of the virion which is responsible for attachment to host cells and is the main antigenic target for neutralising antibodies during infection. Previous work has also demonstrated that the S protein determines cell tropism in vitro. In order to investigate the involvement of the S gene in IBV pathogenesis and explore the potential for vaccine propagation in cell culture, recombinant viruses were generated using vaccinia virus based reverse genetics. Two isolates of the pathogenic M41 strain were mutated to include the S gene from a non-pathogenic lab strain with extended tropism (Beau-R) or a heterologous pathogenic field strain with restricted tropism (4/91), resulting in two recombinant IBVs termed M41K-BeauR(S) and M41K-4/91(S), respectively. These viruses were characterised in vitro and in vivo to determine the involvement of the S gene in IBV replication and pathogenicity. M41K-BeauR(S) was attenuated in vivo but exhibited the extended host tropism of the S donor strain. M41K-4/91(S) remained pathogenic and also adopted the restricted in vitro tropism of 4/91. This indicates that the S gene not only determines the cellular tropism of the virus but also plays a key role during in vivo infections, and that replacing the ectodomain of IBV S can significantly alter the pathogenicity of the resulting virus.

Pathobiological and Genomic Characterization of a Cold-Adapted Infectious Bronchitis Virus (BP-caKII).

We established a cold-adapted infectious bronchitis virus (BP-caKII) by passaging a field virus through specific pathogen-free embryonated eggs 20 times at 32 °C. We characterized its growth kinetics and pathogenicity in embryonated eggs, and its tropism and persistence in different tissues from chickens; then, we evaluated pathogenicity by using a new premature reproductive tract pathogenicity model. Furthermore, we determined the complete genomic sequence of BP-caKII to understand the genetic changes related to cold adaptation. According to our results, BP-caKII clustered with the KII genotype viruses K2 and KM91, and showed less pathogenicity than K2, a live attenuated vaccine strain. BP-caKII showed delayed viremia, resulting in its delayed dissemination to the kidneys and cecal tonsils compared to K2 and KM91, the latter of which is a pathogenic field strain. A comparative genomics study revealed similar nucleotide sequences between BP-caKII, K2 and KM91 but clearly showed different mutations among them. BP-caKII shared several mutations with K2 (nsp13, 14, 15 and 16) following embryo adaptation but acquired multiple additional mutations in nonstructural proteins (nsp3, 4 and 12), spike proteins and nucleocapsid proteins following cold adaptation. Thus, the establishment of BP-caKII and the identified mutations in this study may provide insight into the genetic background of embryo and cold adaptations, and the attenuation of coronaviruses.

A new recombined porcine reproductive and respiratory syndrome virus virulent strain in China.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases worldwide. In the present study, a new virulent strain of PRRS virus (PRRSV), GDsg, was isolated in Guangdong province, China, and caused high fever, high morbidity, and high mortality in sows and piglets. The genome of this new strain was 15,413 nucleotides (nt) long, and comparative analysis revealed that GDsg shared 82.4% to 94% identity with type 2 PRRSV strains, but only 61.5% identity with type 1 PRRSV Lelystad virus strain. Phylogenetic analysis indicated that type 2 PRRSV isolates include five subgenotypes (I, II, III, IV, and V), which are represented by NADC30, VR-2332, GM2, CH-1a, and HuN4, respectively. Moreover, GDsg belongs to a newly emerging type 2 PRRSV subgenotype III. More interestingly, the newly isolated GDsg strain has multiple discontinuous nt deletions, 131 (19 + 18 + 94) at position 1404–1540 and a 107 nt insertion in the NSP2 region. Most importantly, the GDsg strain was identified as a virus recombined between low pathogenic field strain QYYZ and vaccine strain JXA1-P80. In conclusion, a new independent subgenotype and recombinant PRRSV strain has emerged in China and could be a new threat to the swine industry of China.

Pathogenicity of a newly emerged recombined porcine reproductive and respiratory syndrome virus strain (subgenotype III) in China

Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe pathogen that causes enormous economic losses to the swine industry worldwide. Since its first report in the 1980s, PRRSV has undergone extensive variation. In the previous study, we demonstrated that the GDsg strain was a new recombined virus between the low pathogenic field strain QYYZ and the vaccine strain JXA1-P80, belonging to the newly emerging type 2 PRRSV subgenotype III. In this study, the pathogenicity of a new recombined strain GDsg for pigs was analyzed. The results of in vivo experiments indicated that GDsg could cause persistently high fever, severe interstitial pneumonia, and high viremia and antibody levels in inoculated piglets. In particular, the brains of inoculated pigs exhibited serious hemorrhage and microscopic lesions. These results suggested that compared with the low pathogenic field strain QYYZ and the vaccine strain JXA1-P80, the new recombined GDsg strain had higher virulence in pigs. This study will help to characterize the relationship between recombination and evolution of PRRSV.

Deep sequencing and variant analysis of an Italian pathogenic field strain of equine infectious anaemia virus.

Equine infectious anaemia virus (EIAV) is a lentivirus with an almost worldwide distribution that causes persistent infections in equids. Technical limitations have restricted genetic analysis of EIAV field isolates predominantly to gag sequences resulting in very little published information concerning the extent of inter-strain variation in pol, env and the three ancillary open reading frames (ORFs). Here, we describe the use of long-range PCR in conjunction with next-generation sequencing (NGS) for rapid molecular characterization of all viral ORFs and known transcription factor binding motifs within the long terminal repeat of two EIAV isolates from the 2006 Italian outbreak. These isolates were from foals believed to have been exposed to the same source material but with different clinical histories: one died 53days post-infection (SA) while the other (DE) survived 5months despite experiencing multiple febrile episodes. Nucleotide sequence identity between the isolates was 99.358% confirming infection with the same EIAV strain with most differences comprising single nucleotide polymorphisms in env and the second exon of rev. Although the synonymous:non-synonymous nucleotide substitution ratio was approximately 2:1 in gag and pol, the situation is reversed in env and ORF3 suggesting these sequences are subjected to host-mediated selective pressure. EIAV proviral quasispecies complexity invivo has not been extensively investigated; however, analysis suggests it was relatively low in SA at the time of death. These results highlight advantages of NGS for molecular characterization of EIAV namely it avoids potential artefacts generated by traditional composite sequencing strategies and can provide information about viral quasispecies complexity.

Recombinant viral-vectored vaccines for the control of avian influenza in poultry

Vaccination is a commonly used tool for the control of both low pathogenic and highly pathogenic avian influenza (AI) viruses. Traditionally, inactivated adjuvanted vaccines made from a low pathogenic field strain have been used for vaccination, but advances in molecular biology have allowed a number of different viral vectored vaccines, expressing the AI virus hemagglutinin (HA) gene, to be developed and licensed for use for control of AI. This review summarizes the licensed vector vaccines available for use in poultry. As a group, these vaccines can stimulate both a cellular and humoral immune response and, when antigenically well matched to the target AI strain, are effective at preventing clinical disease and reducing virus shedding if vaccinated birds do become infected. The vaccines can often be given to one-day old chicks in the hatchery, which can provide early protection and is a cost effective route of administration of the vaccine. All the licensed vectored vaccines, because they only express the HA gene, can potentially be used to differentiate vaccinated from vaccinated and infected birds, which is often referred to as a DIVA strategy. Although a potentially valuable tool for the surveillance of the virus in countries that vaccinate, the DIVA principle has currently not been applied. Concern remains that maternal antibody or pre-existing immunity to the vector or to the AI HA insert can suppress the immune response to the vaccine. The viral vectored vaccines appear to work well with a prime boost strategy where the vectored vaccine is given first and a different type of vaccine, often a killed adjuvanted vaccine is given two or three weeks later.

Characterization of a pathogenic full-length cDNA clone of a virulent porcine epidemic diarrhea virus strain AH2012/12 in China

Since 2010, outbreaks of variant porcine epidemic diarrhea virus (PEDV) have swept across the world causing substantial economic losses. The development of new, more effective vaccines has been hampered by difficulties in isolating strains and viral genome manipulation. In the present study, we successfully isolated a highly pathogenic field strain AH2012/12, from a pig farm reporting severe diarrhea in China. Phylogenetic analysis revealed that the new isolate belongs to group G2, which represents epidemic and pandemic field strains. Furthermore, we constructed an infectious cDNA clone of the newly isolated strain, rAH2012/12, and the rescued virus displayed phenotypic properties identical to the parental virus in vitro. In vivo experiments demonstrated that the rescued virus displayed similar pathogenicity to the parental isolate, causing high mortality rates in suckling pigs. This study provided a strong basis for the development of live attenuated vaccines and for research into the pathogenic mechanisms of this virus.

Proteomic Characterisation of Two Strains of Mycoplasma mycoides Subsp. Mycoides of Differing Pathogenicity

Mycoplasma mycoides subsp mycoides (Mmm) is a major pathogen of cattle, causing contagious bovine pleuropneumonia (CBPP), a severe–frequently fatal-disease that is widespread in sub-tropical countries. Despite recognition as a pathogen in the 19th century and advances made through genome sequencing and synthetic biology, many of the molecular characteristics of Mmm remain poorly characterized. Using a proteomics approach, this study aimed to define Mmm major protein determinants, including surface membrane proteins and those involved in host colonization and pathogenicity. For this purpose, both whole cells and a Triton X-114 enriched membrane fraction of a highly pathogenic African field strain (N6) and a vaccine strain (KH3J) were analysed and compared using gelbased methodologies i.e. 2-DGE and SDS-PAGE in combination with MALDI-MS/MS and LC-ESI-MS/MS. A total of 318 proteins, (around 31% of the predicted proteome) were identified, comprising lipoproteins, prolipoproteins and hypothetical proteins with predicted membrane locations and possible roles in pathogenicity. One hundred and forty-five of these proteins, with different predicted sub-cellular locations and functions but also including uncharacterized proteins, have not previously been reported as expressed by Mmm. Many more proteins were detected by LC-ESI-MS/MS (n=315) than by 2-DGE and MALDI-MS (n=52), however 3 proteins were detected only by the latter approach. Most proteins were observed to be common to both strains although a substantial number of proteins were detected exclusively in only one strain with possible implications for fitness and pathogenicity. To our knowledge, this study represents the most extensive proteomics characterization of Mmm to date and has identified characteristics on which to base future studies of the physiology and pathogenicity of this prokaryote.

Genetic characterization by composite sequence analysis of a new pathogenic field strain of equine infectious anemia virus from the 2006 outbreak in Ireland

Equine infectious anemia virus (EIAV), the causative agent of equine infectious anaemia (EIA), possesses the least-complex genomic organization of any known extant lentivirus. Despite this relative genetic simplicity, all of the complete genomic sequences published to date are derived from just two viruses, namely the North American EIAV(WYOMING) (EIAV(WY)) and Chinese EIAV(LIAONING) (EIAV(LIA)) strains. In 2006, an outbreak of EIA occurred in Ireland, apparently as a result of the importation of contaminated horse plasma from Italy and subsequent iatrogenic transmission to foals. This EIA outbreak was characterized by cases of severe, sometimes fatal, disease. To begin to understand the molecular mechanisms underlying this pathogenic phenotype, complete proviral genomic sequences in the form of 12 overlapping PCR-generated fragments were obtained from four of the EIAV-infected animals, including two of the index cases. Sequence analysis of multiple molecular clones produced from each fragment demonstrated the extent of diversity within individual viral genes and permitted construction of consensus whole-genome sequences for each of the four viral isolates. In addition, complete env gene sequences were obtained from 11 animals with differing clinical profiles, despite exposure to a common EIAV source. Although the overall genomic organization of the Irish EIAV isolates was typical of that seen in all other strains, the European viruses possessed ≤80 % nucleotide sequence identity with either EIAV(WY) or EIAV(LIA). Furthermore, phylogenetic analysis suggested that the Irish EIAV isolates developed independently of the North American and Chinese viruses and that they constitute a separate monophyletic group.

Assessing the effectiveness of intubation as a challenge model in contagious bovine pleuropneumonia vaccine experiments

A study was carried out to assess the effectiveness of a bronchoscope in administering a pathogenic field strain of Mycoplasma mycoides subsp. mycoides (MmmSC) in cattle challenge experiments. Out of 16 animals inoculated using the bronchoscope, 10 (62.2%) showed clinical disease as evidenced by fever and 15 (93.8%) displayed typical lesions of CBPP from which MmmSC was isolated. Serum samples collected weekly were tested by Complement Fixation Test (CFT) and competitive enzyme-linked immunosorbent assay (c-ELISA). Antibodies to MmmSC were detected in 10 out of the 16 animals by the CFT and 11 out of the 16 animals by c-ELISA. The onset of clinical disease was as early as 2 days post-inoculation, and most of the animals developed clinical disease 2 to 3 weeks post-infection. These results clearly demonstrate that nasotracheal inoculation of pathogenic strain of MmmSC with the aid of a bronchoscope can lead to early onset of clinical disease; similar to previous studies but with higher numbers of animals showing clinical disease. This is in contrast with previous studies where early clinical disease was observed in as little as 15% of inoculated animals. This nasotracheal inoculation method using a bronchoscope can, therefore, be adopted for use in experimental challenge infections of cattle. This method is found to be a better replacement to the contact transmission method whose drawback includes extra cost of donor animals and unpredictable rate and timing of transmission from intubated to challenge animals.

Chicken B Lymphoma DT40 Cells as a Useful Tool for in vitro Analysis of Pathogenic Infectious Bursal Disease Virus

Susceptibility of DT40 cells to pathogenic field strains of infectious bursal disease virus (IBDV) including very virulent and classical virulent strains were studied. After the first and second passage of the virus in DT40 cells, IBDV-specific antigen was readily detected in DT40 cells inoculated with the pathogenic field strain infected bursal homogenates. Nucleotide sequence analysis in the VP2 hypervariable domain, which is critical for the virulence of IBDV, revealed no common amino acid substitutions among the pathogenic IBDVs in accordance with the propagation in DT40 cells. These results indicate that DT40 cells are a useful tool for rapid isolation of pathogenic field strains and successive in vitro analysis of IBDV.

Comparative efficacy of an indigenous ‘inactivated vaccine’ using highly pathogenic field strain of Mycobacterium avium subspecies paratuberculosis ‘Bison type’ with a commercial vaccine for the control of Capri-paratuberculosis in India

Johne's disease (JD) is endemic in goatherds located at Central Institute for Research on Goats, Makhdoom, since 1979 and lately it has been reported from farmer's herds in equal frequencies. Despite using test and slaughter method for the control of JD for more than 25 years in these herds, incidence of JD has not been reduced. Efficacy of ‘indigenous vaccine’ containing native ‘Bison type’ genotype of Mycobacterium avium subspecies paratuberculosis (MAP) was compared with commercial vaccine using challenge studies with homologous strain of MAP. Goat kids (85) were randomly divided in to three groups. Kids were vaccinated with 1 ml of vaccine subcutaneously and Sham-immunized with 1 ml of sterile PBS. All kids except 3 in each group were challenged twice at 75- and 275-day post-vaccination (DPV). Four goats each from three groups were sacrificed at 200-day post-challenge to evaluate carcass and histopathologically for vaccine and challenge response in kids of different groups. Samples (blood, serum and fecal) were screened for LTT, ELISA and shedding of bacilli and data on live animal traits, mortality and experimental sacrifice were compared. Average body weights gained by goats in three groups at different stages of trials (0, 1–75, 76–275, 276–425 DPV) showed marked improvements in performance of vaccinated groups over ‘Sham-immunized’ group. Effect of vaccines against challenge became visible in terms of body weights gained at 276–425 DPV (‘Bison’ group gained significantly higher body weights than ‘Sham-immunized’). Mortality was significantly less in two vaccinated as compared to ‘Sham-immunized’. Vaccinated groups also had significant stimulation and sero-conversion for cell mediated and humoral immune response, respectively as compared to ‘Sham-immunized’. Results of post-challenged fecal culture showed significant reduction in shedding of MAP in both vaccinated groups than in ‘Sham-immunized’. There was significant improvement in external and internal body traits and histological lesions in case of vaccinated than ‘Sham-immunized’ group.

Response of Chickens Carrying Germline Insert ALVA11 to Challenge with a Field Strain of Subgroup A Avian Leukosis Virus

The ALVA11 germline insert in chickens is a defective subgroup A avian leukosis virus (ALV) proviral insert that expresses a low-to-moderate level of subgroup A ALV envelope glycoprotein. Chicks carrying or lacking ALVA11 were evaluated for response to challenge by RPL-42, a pathogenic field strain of subgroup A ALV, by either exposure to chicks shedding RPL-42 or direct injection with various doses of RPL-42. Chicks carrying ALVA11 were significantly more resistant, as measured by infectious virus and viral antibody status, to horizontal infection and direct injection of RPL-42 than chicks lacking ALVA11.