Dairy Cows Experimentally Infected With Bovine Leukemia Virus Showed an Increased Milk Production in Lactation Numbers 3-4: A 4-Year Longitudinal Study.

Bovine leukemia virus (BLV) is widespread in global cattle populations, but the effects of its infection on milk quantity and quality have not been clearly elucidated in animal models. In this study, 30 healthy first-lactation cows were selected from ≈2,988 cows in a BLV-free farm with the same criteria of parity, age, lactation number, as well as milk yield, SCS, and composition (fat, protein, and lactose). Subsequently, these cows were randomly assigned to the intervention (n = 15) or control (n = 15) group, and reared in different cowsheds. Cows in the intervention group were inoculated with 1 × phosphate-buffered solution (PBS) resuspended in peripheral blood mononuclear cells (PBMC) from a BLV-positive cow, while the controls were inoculated with the inactivated PBMC from the same individual. From June 2016 to July 2021, milk weight (kg) was automatically recorded by milk sensors, and milk SCS and composition were originated from monthly performed dairy herd improvement (DHI) testing. Fluorescence resonance energy transfer (FRET)-qPCR and ELISA showed that cows in the intervention group were successfully infected with BLV, while cows in the control group were free of BLV for the entire period. At 45 days post-inoculation (DPI), the numbers of whole blood cells (WBCs) (P = 0.010), lymphocytes (LYMs) (P = 0.002), and monocytes (MNCs) (P = 0.001) and the expression levels of IFN-γ (P = 0.013), IL-10 (P = 0.031), and IL-12p70 (P = 0.008) increased significantly in the BLV infected cows compared to the non-infected. In lactation numbers 2-4, the intervention group had significantly higher overall milk yield (P < 0.001), fat (P = 0.031), and protein (P = 0.050) than the control group, while milk SCS (P = 0.038) and lactose (P = 0.036) decreased significantly. Further analysis indicated that BLV infection was associated with increased milk yield at each lactation stage in lactation numbers 3-4 (P = 0.021 or P < 0.001), but not with SCS and milk composition. Together, this 4-year longitudinal study revealed that artificial inoculation of BLV increased the milk yield in cows in this BLV challenge model.

Development of specific monoclonal antibodies for the detection of natural chicken tumor necrosis factor-alpha.

Tumor necrosis factor alpha (TNF-α) is an important proinflammatory cytokine and the only known cytokine that can directly kill tumor cells. Unlike mammalian counterparts, chicken TNF-α (chTNF-α) gene has not been identified until very recently due to its high GC content (∼70%) and long GC fragments. The biological functions of this newly-identified cytokine and its detection methods remain to be further investigated. In this study, the extracellular domain of chTNF-α was cloned into prokaryotic vector after codon optimization and recombinant chTNF-α protein was expressed. Subsequently, using recombinant chTNF-ɑ as immunogen, rabbit polyclonal antibody (pAb) and eight clones of mouse anti-chTNF-ɑ monoclonal antibodies (mAbs) were produced, respectively. Both the pAb and mAbs specifically recognized recombinant chTNF-ɑ expressed in E.coli and transfected COS-7 cells. Further mapping the antigenic region showed that all the mAbs recognized a region of amino acid residues 195-285 of chTNF-ɑ. Furthermore, an antigen-capture enzyme-linked immunosorbent assay for the detection of chTNF-ɑ was established using one mAb and the pAb. This assay showed no cross-reactivity with irrelevant Trx-fused antigens and could detect natural chTNF-ɑ expressed by mitogen-activated chicken splenocytes in a dose-dependent manner, with a detection limit of 1 ng/mL. Collectively, our results indicated that the mAbs and pAb against chTNF-α are specific and could be used for the study of the biological functions of chTNF-ɑ and the detection of chTNF-ɑ.

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens and Cx. tritaeniorhynchus in China.

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.

Integrative Analysis of miRNA and mRNA Expression Profiles in Mammary Glands of Holstein Cows Artificially Infected with Staphylococcus aureus.

Staphylococcus aureus- induced mastitis is one of the most intractable problems for the dairy industry, which causes loss of milk yield and early slaughter of cows worldwide. Few studies have used a comprehensive approach based on the integrative analysis of miRNA and mRNA expression profiles to explore molecular mechanism in bovine mastitis caused by S. aureus. In this study, S. aureus (A1, B1 and C1) and sterile phosphate buffered saline (PBS) (A2, B2 and C2) were introduced to different udder quarters of three individual cows, and transcriptome sequencing and microarrays were utilized to detected miRNA and gene expression in mammary glands from the challenged and control groups. A total of 77 differentially expressed microRNAs (DE miRNAs) and 1625 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that multiple DEGs were enriched in significant terms and pathways associated with immunity and inflammation. Integrative analysis between DE miRNAs and DEGs proved that miR-664b, miR-23b-3p, miR-331-5p, miR-19b and miR-2431-3p were potential factors regulating the expression levels of CD14 Molecule (CD14), G protein subunit gamma 2 (GNG2), interleukin 17A (IL17A), collagen type IV alpha 1 chain (COL4A1), microtubule associated protein RP/EB family member 2 (MAPRE2), member of RAS oncogene family (RAP1B), LDOC1 regulator of NFKB signaling (LDOC1), low-density lipoprotein receptor (LDLR) and S100 calcium binding protein A9 (S100A9) in bovine mastitis caused by S. aureus. These findings could enhance the understanding of the underlying immune response in bovine mammary glands against S. aureus infection and provide a useful foundation for future application of the miRNA-mRNA-based genetic regulatory network in the breeding cows resistant to S. aureus.

Isolation and characterization of multidrug-resistant Klebsiella pneumoniae from raw cow milk in Jiangsu and Shandong provinces, China.

Antimicrobials are the most important therapy to bovine mastitis. Bacterial infection and antibiotic treatment of mastitis cycles frequently in dairy farms worldwide, giving rise to concerns about the emergence of multidrug-resistant (MDR) bacteria. In this study, we examined the microbial diversity and antibiotic resistance profiles of bacteria isolated from raw milk from dairy farms in Jiangsu and Shandong provinces, China. Raw milk samples were collected from 857 dairy cattle including 800 apparently healthy individuals and 57 cows with clinical mastitis (CM) and subjected to microbiological culture, antimicrobial susceptibility assay and detection of antibiotic-resistant genes by polymerase chain reaction (PCR) and sequencing. A total of 1,063 isolates belonging to 41 different bacterial genera and 86 species were isolated and identified, of which Pseudomonas spp. (256/1,063, 24.08%), Staphylococcus. spp. (136/1,063, 12.79%), Escherichia coli (116/1,063, 10.91%), Klebsiella spp. (104/1,063, 9.78%) and Bacillus spp. (84/1,063, 7.90%) were most frequently isolated. K. pneumoniae, one of the most prevalent bacteria, was more frequently isolated from the farms in Jiangsu (65/830, 7.83%) than Shandong (1/233, 0.43%) province, and showed a positive association with CM (p < .001). The antimicrobial susceptibility assay revealed that four of the K. pneumoniae isolates (4/66, 6.06%) were MDR bacteria (acquired resistance to ≥three classes of antimicrobials). Furthermore, among 66 isolates of K. pneumoniae, 21.21% (14/66), 13.64% (9/66) and 12.12% (8/66) were resistant to tetracycline, chloramphenicol and aminoglycosides, respectively. However, all K. pneumoniae isolates were sensitive to monobactams and carbapenems. The detection of antibiotic-resistant genes confirmed that the ß-lactamase genes (blaSHV and blaCTX-M ), aminoglycoside modifying enzyme genes [aac(6')-Ib, aph(3')-I and ant(3″)-I], tetracycline efflux pump (tetA) and transposon genetic marker (intI1) were positive in MDR isolates. This study indicated that MDR K. pneumoniae isolates emerged in dairy farms in Jiangsu province and could be a potential threat to food safety and public health.

Molecular characterization of bovine leukemia virus reveals existence of genotype 4 in Chinese dairy cattle.

Bovine leukemia virus (BLV) causes enzootic bovine leucosis and is widely spread worldwide, except several European countries, Australia and New Zealand. Although BLV is highly prevalent in China, information about the genetic diversity and evolutionary dynamics of BLV among Chinese dairy herds is still lacking. To determine the genetic variability of BLV, 219 cows from four cities of Ningxia province of China were screened for BLV infection by fluorescence resonance energy transfer (FRET)-PCR and sequencing, 16 selected positive samples were subjected to molecular characterization. Phylogenetic analysis using the neighbor-joining (NJ) method on complete sequences of envelope (env) gene of BLV obtained from China and those available in GenBank (representing BLV genotypes 1-10) revealed that those Chinese strains belonged to genotypes 4 and 6. Totally, 23 mutations were identified and 16 of them were determined to be unique mutations among Chinese strains. Alignment of the deduced amino acid sequences demonstrated six mutations in glycoprotein 51 (gp51) and three mutations in glycoprotein 30 (gp30) located in the identified neutralizing domain (ND), CD8+ T cell epitope, E-epitope, B-epitope, gp51N12 and cytoplasmic domain of transmembrane protein. This study reported for the first time the BLV genotype 4 in China, and further studies are warranted to compare its immunogenicity and pathogenicity with other BLV genotypes.