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Pasteurella multocida is a pathogen that causes bovine respiratory disease, and the development of an effective vaccine is important for improving animal health. Live-attenuated vaccines induce a long-lasting immune response with minimal side effects. The objective of this study was to evaluate potential live vaccine candidates from three P. multocida mutants produced by separately disrupting the genes of filamentous hemagglutinin 2 ( fhaB2 ) , hydrogenase-1 operon ( hyaE ) , and n-acylneuraminate-9-phosphatase ( nanP ) of a serogroup 3 strain (P1062, WT) by clinical testing and transcriptome analysis. Challenge with WT and the three mutants conferred protection against P. multocida , with less lung lesions (4.7–6.2%) compared to 22.4% in the sham group. Transcriptome analysis identified 807 differentially expressed protein-coding transcripts (DETs) in the blood and 6473 DETs in the liver compared to the sham, WT, and each of the mutants. In total, 15 and 64 differentially expressed microRNAs (DEmiRNAs) and 12 and 74 differentially expressed long non-coding RNAs (DElncRNAs) were identified in blood and liver, respectively. The DEmiRNAs were not significantly associated with the DETs within each comparison. DElncRNAs were associated with 12 and 170 DETs in blood and liver respectively. The greatest number of unique DETs were found between hyaE and sham groups in the liver, which agreed with the low colonization rate in the nares and palatine tonsils. For the DETs between sham and WT the under-enriched gene ontology terms in blood were all included in the liver for the DETs identified by WT vs. sham, nanP vs. sham, and hyaE vs. sham, and were related to the signaling pathway, stimulus, and sensory perceptions in biological processes with the molecular function of olfactory receptor activity. The number of identified DETs, decreased percentage of lung lesions, and colonization rates indicate that fhaB2 could be a promising vaccine candidate.

Background Mycoplasma bovis is a prominent pathogen associated with respiratory disease in livestock. Respiratory disease in cattle often involves co-infection, where a primary viral infection can weaken the host immune system and thus enhance subsequent bacterial infection. The objective of this study was to investigate changes in the host (cattle) transcriptome during bacterial-viral co-infection. RNA sequencing was done in whole blood cells (WBC), liver, mesenteric lymph node (MLN), tracheal-bronchial lymph node (TBLN), spleen, and thymus collected from Control animals ( n  = 2), animals infected with M. bovis (MB; n  = 3), and animals infected with M. bovis and bovine viral diarrhea virus (BVDV) (Dual; n  = 3). Results Thymus and spleen had the greatest number of differentially expressed genes (DEGs) out of all tissues analyzed. In spleen, genes involved in maintenance of the extracellular matrix (ECM) including collagen type XV alpha 1 chain ( COL15A1 ), collagen type IV alpha 2 chain ( COL4A2 ), and heparan sulfate proteoglycan 2 ( HSPG2 ) were the most significantly downregulated in Dual compared to Control and MB. In thymus, complement 3 (C3) was a highly significant DEG and upregulated in Dual compared to Control and MB. Interferon alpha inducible protein 6 ( IFI6 ) and interferon-induced transmembrane proteins ( IFITM1 and IFITM3 ), were significantly associated with infection status and upregulated in spleen and thymus of Dual compared to Control and MB. Conclusion Downregulation of ECM components may cause degradation of the ECM and contribute to increased viral spread due to co-infection. Hyperactivation of complement pathway genes may contribute to damage to the thymus and influence severity of co-infection. Co-expression of IFI6 , IFITM1 and IFITM3 across lymphoid tissues may be connected to enhanced pathogenesis in co-infection. These findings suggest co-infection exacerbates disease severity through modulation of ECM components in spleen and complement and coagulation cascades in the thymus. These impacted pathways may underlie thymic atrophy and impaired pathogen clearance due to BVDV and M. bovis co-infection.

Mannheimia haemolytica is the principal agent contributing to bovine respiratory disease and can form biofilms with increased resistance to antibiotic treatment and host immune defenses. To investigate the molecular mechanisms underlying M . haemolytica biofilm formation, transcriptomic analyses were performed with mRNAs sequenced from planktonic and biofilm cultures of pathogenic serotypes 1 (St 1; strain D153) and St 6 (strain D174), and St 2 (strain D35). The three M . haemolytica serotypes were cultured in two different media, Roswell Park Memorial Institute (RPMI) 1640 and brain heart infusion (BHI) to form the biofilms. Transcriptomic analyses revealed that the functions of the differentially expressed genes (DEGs) in biofilm associated cells were not significantly affected by the two media. A total of 476 to 662 DEGs were identified between biofilm associated cells and planktonic cells cultured under BHI medium. Functional analysis of the DEGs indicated that those genes were significantly enriched in translation and many biosynthetic processes. There were 234 DEGs identified in St 1 and 6, but not in St 2. The functions of the DEGs included structural constituents of ribosomes, transmembrane proton transportation, proton channels, and proton-transporting ATP synthase. Potentially, some of the DEGs identified in this study provide insight into the design of new M . haemolytica vaccine candidates.

Goat caseins are highly polymorphic proteins that affect milk functional properties. In this study, an in silico approach was employed to analyze the influence of goat casein allelic variants on the quantity and bioactivity potential of peptides released after enzymatic hydrolysis. The reported protein sequences from the most frequent allelic variants in Capra hircus caseins (α-S1, β, α-S2, and κ-casein) were analyzed in the BIOPEP-UWM database to determine the frequency of occurrence of bioactive fragments from each casein. After specific hydrolysis with pepsin, trypsin, and chymotrypsin A, important differences in the peptide profile and bioactivity potential were observed within and between the casein allelic variants. The β-casein A and C alleles, α-S1-casein allele E, and α-S2-casein allele F presented the highest bioactivity potential, and some allele-specific peptides were also released, highlighting the impact of genotype on the predicted bioactivity. The inhibition of angiotensin-converting enzyme (ACE-I) and dipeptidyl peptidase IV (DPP-IV) activities was the most frequent bioactivity of the released peptides, suggesting possible antihypertensive and antidiabetic effects. Once confirmed by experimental studies, the use of goat casein genotyping could direct efforts to enhance the functional quality of goat milk.

The objective was to determine differences in microRNAs (miRNAs) counts in several tissues of calves challenged with Mycoplasma bovis ( M . bovis ) or with M . bovis and bovine viral diarrhea virus (BVDV). Eight calves approximately 2 months of age were randomly assigned to three groups: Control (CT; n = 2), M . bovis (MB; n = 3), and Coinfection (CO; n = 3). On day 0, calves in CO were intranasally challenged with BVDV and calves in MB with M . bovis . On day 6, CO calves were challenged with M . bovis . Calves were euthanized 17 days post-challenge and serum (SER), white blood cells (WBC), liver (LIV), mesenteric (MLN) and tracheal-bronchial (TBLN) lymph nodes, spleen (SPL), and thymus (THY), were collected at necropsy. MiRNAs were extracted from each tissue from each calf. Significant (P< 0.01) differences in miRNAs expression were observed in SER, LIV, MLN, TBLN, SPL, and THY. There were no significant (P> 0.05) miRNAs in WBC. In SER, the CO group had levels of miR-1343-3p significantly higher than the CT and MB groups (P = 0.0071). In LIV and SPL, the CO group had the lowest counts for all significant miRNAs compared to CT and MB. In TBLN, the CT group had the highest counts of miRNAs, compared to MB and CO, in 14 of the 21 significant miRNAs. In THY, the CO group had the highest counts, in 4 of the 6 significant miRNAs compared to CT and MB. BVDV was associated with reduction of miRNAs in LIV, SPL, MLN, and TBLN, and M . bovis reduced counts of miRNAs in only TBLN. Measuring circulating miRNAs to assess disease condition or to develop intervention strategies to minimize respiratory diseases in cattle caused by BVDV or M . bovis will be of limited use unless an alternative approach is developed to use them as indicators of disease.

Background Bovine viral diarrhea virus (BVDV) is a pervasive respiratory pathogen of economic concern for the cattle industry. Transplacental infection results in abortion or the establishment of a tolerant and persistent viral infection. Deletion viral genomes (DelVGs) are naturally occurring products of the viral replication process. These deletion viral genomic transcripts are generated with truncations of various sizes that severely impede or prevent self-replication. DelVGs have been implicated in the establishment of viral persistence. Methods We used a bioinformatic pipeline to discover the presence of BVDV DelVGs. These DelVGs were identified via analysis of Illumina MiSeq reads from 74 BVDV1 field isolates from two closely related subgenotypes and from an  in vitro  passage of a BVDV1a virus at two different multiplicities of infection (MOI). Results After the identification of DelVGs, we assessed their phylogenetic linkage to begin elucidating potential roles in the viral life cycle and persistence. BVDV1a viruses queried generate significantly more DelVGs, with 52% of 5’ and 3’ junctions occurring in the core/capsid (C) region and a major NS2-NS5B deletion species. In contrast, the BVDV1b viruses generated significantly fewer DelVGs, especially a reduction in C region deletions. In vitro  passaging of the BVDV1a Singer virus demonstrated that MOI significantly impacts the generation of DelVGs, with higher MOIs generating more DelVGs and a different deletion profile. Conclusions Here, we report that the BVDV1a and BVDV1b subgenotypes generate diverse species of DelVGs. These DelVGs may play key roles in BVDV evolution and the establishment of persistence during transplacental infection.

In this paper, we derive some sufficient second order optimality conditions for control problems of partial differential equations (PDEs) when the cost functional does not involve the usual quadratic term for the control or higher nonlinearities for it. Though not always, in this situation the optimal control is typically bang-bang. Two different control problems are studied. The second differs from the first in the presence of the $L^1$ norm of the control. This term leads to optimal controls that are sparse and usually take only three different values (we call them bang-bang-bang controls). Though the proofs are detailed in the case of a semilinear elliptic state equation, the approach can be extended to parabolic control problems. Some hints are provided in the last section to extend the results. [PUBLICATION ABSTRACT]

Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasures to control MDR Salmonella outbreaks. In this study, antimicrobial activity of cationic α-helical bovine NK-lysin-derived antimicrobial peptides was evaluated against MDR Salmonella outbreak isolates. NK2A and NK2B strongly inhibited MDR Salmonella growth while NK1 and NK2C showed minimum-to-no growth inhibition. Scrambled-NK2A, which is devoid of α-helicity but has the same net positive charge as NK2A, also failed to inhibit bacterial growth. Incubation of negatively charged MDR Salmonella with NK2A showed increased Zeta potential, indicating bacterial-peptide electrostatic attraction. Confocal and transmission electron microscopy studies revealed NK2A-mediated damage to MDR Salmonella membranes. LPS inhibited NK2A-mediated growth suppression in a dose-dependent response, suggesting irreversible NK2A-LPS binding. LPS-NK2A binding and bacterial membrane disruption was also confirmed via electron microscopy using gold nanoparticle-NK2A conjugates. Finally, NK2A-loaded polyanhydride nanoparticles showed sustained peptide delivery and anti-bacterial activity. Together, these findings indicate that NK2A α-helicity and positive charge are prerequisites for antimicrobial activity and that MDR Salmonella killing is mediated by direct interaction of NK2A with LPS and the inner membrane, leading to bacterial membrane permeabilization. With further optimization using nano-carriers, NK2A has the potential to become a potent anti-MDR Salmonella agent.

Bovine leukemia virus (BLV) infection in cattle is omnipresent, which causes significantly economical losses worldwide. The objective of this study was to determine microRNA (miRNA) and transcript profiles and to establish their relationship in response to exposure to the virus. Small noncoding and messenger RNA were extracted and sequenced from serum and white blood cells (WBCs) derived from seven BLV seropositive and seven seronegative cows. Transcriptomic profiles were generated by sequencing RNA libraries from WBC. Bta-miR-206 and bta-miR-133a-3p were differentially expressed in serum ( P < 0.05). In WBC, bta-miR-335-3p, bta-miR-375, and bta-novel-miR76-3p were differentially expressed ( P < 0.03). There were 64 differentially expressed transcripts (DETs). Gene ontology (GO) analysis of the DETs overexpressed in the seropositive group with GOs of response to stimulus and immune system process predicted that the DETs could potentially negatively regulate viral life cycle and viral entry or release from host cells. In addition, the DETs depleted in the seropositive group could play a role in the downregulation of antigen processing and presentation of endogenous peptide antigen via MHC class I. The differentially expressed miRNAs targeted 17 DETs, among which the expressions of bta-miR-133a-3p and bta-miR-335-3p were significantly negatively correlated with the expressions of ENSBTAT00000079143 and ENSBTAT00000066733, respectively. Under high prediction criteria, 90 targets of the differentially expressed miRNAs were all non-DETs. The most enriched biological process GO term of the targets was the RNA-dependent DNA biosynthetic process, which could be associated with virus replication. These results suggested that the differentially expressed miRNAs fine-tune most of the target genes in responding to BLV exposure. In addition, Bta-miR-206 interacted with BLV regulatory genes rex and tax by targeting their coding regions. A further study of the miRNAs and the genes may reveal the molecular mechanisms of BLV infection and uncover possible ways to prevent the infection.

Background Pasteurella multocida is a Gram-negative coccobacillus and is the causative agent of fowl cholera in avian species. P. multocida expresses two large filamentous hemagglutinin (FhaB) proteins encoded by fhaB 1 and fhaB 2 genes. Previously, it was demonstrated that P. multocida FhaB2 is an important virulence factor in the development of fowl cholera disease. In the current study, we examined the potential role of FhaB1 in fowl cholera disease development. An fhaB1 deletion mutant, devoid of foreign DNA, was constructed using a temperature sensitive plasmid in a well-characterized P. multocida avian strain P-1059 (A:3). Results Real-time PCR assay confirmed the expression of full-length fhaB1 mRNA in the wild-type parent strain and truncated fhaB1 mRNA in the Δ fhaB1 mutant strain. Both parent and the mutant strain produced biofilm; however, the Δ fhaB1 mutant produced significantly lower amounts of biofilm. Turkey poults were challenged intranasally and intramuscularly to assess the virulence of the fhaB1 mutant and the wild-type parent strains. Contrary to our expectation, inactivation of fhaB1 did not reduce virulence by either challenge route. Conclusions These findings indicate that this large and highly conserved FhaB1 protein is not necessary for the development of acute fowl cholera disease in turkeys.