Forty wethers (10 per treatment) were used as non-infected controls, others infected with Haemonchus contortus and anti-γδ T-cell antibodies, also some infected with zolodronic acid (a bisphosphonate derivative), all under controlled conditions. We evaluated wethers following the introduction of infections at 7 dpi and 21 dpi. Vitals were examined through weight check, whole blood evaluation through CBC with Differential Count if indicated, and FEC tested. Expression of 7627 genes were identified through different treatment types after 7 dpi. Testing and evaluation were completed through extraction of DNA of the samples of blood utilizing modified protocols of Microbiome DNA Isolation Kit from Norgen Biotek Corp. (Thorold, ON, Canada). Reverse transcribed cDNA libraries were constructed and later sequenced using Swift Biosciences kits. Analysis was evaluated through default Partek Flow software suites (St. Louis, Missouri, USA) for microbiomes using Kraken at 7 dpi (n = 18) and 21 dpi (n = 20). Blood samples datasets were obtained through sequencing using the NGS Illumina RNA-Seq instrument. Quality assurance and quality control analytics were used for 7 dpi (n = 19). In all there were five goat wethers in four treatment groups for the analysis of this research. So, the CBC with Diff Count if indicated and other blood test can distinguish certain markers to notify health and other immune response symptoms.
Animals and treatments
Forty Alpine wethers (114.2 ± 0.92 d of age and 19.4 ± 0.33 kg BW at the start of the experiment) that had been raised in indoor pens at the Langston University Research Farm were used. The wethers were checked for fecal egg counts (FECs) and confirmed to be nematode-parasite free. Whethers were placed in pens by BW and then randomly allocated to treatments within each pen. Thereafter, a small number of treatment assignments were changed to achieve more similar mean and variation in BW. There were no exclusions. All animals were allowed to acclimatize to pens and feeders for daily supplies of 200 g concentrate pellet per animal composed of 500 g of ground grass (50%) and alfalfa (50%) hays. The treatment groups were as designed in Table 2.
On the first (1) day prior to the L3 infection, the AB injection was administered intravenously. ZA were administered intravenously 7 days prior to and 0, 7, and 14 days after the L3 infection. At the beginning of the experiment all kids except Group 1 were given 10,000 H. contortus infective larvae (L3; hatched and isolated from feces being collected from LU goats) by gavage. Five animals from each group were euthanized on 7 dpi for sampling and the other five animals were euthanized 21 dpi.
Blood sample collection and processing
Blood samples were collected from five goats in four treatment groups 7 dpi. Blood samples included red blood cells, white blood cells (total and differential), hemoglobin, platelets, and plasma protein, from the jugular vein were collected in EDTA tubes. Quality assurance/quality control (QA/QC) parameters resulted in blood samples from 19 cDNA libraries that were used from samples collected 7 dpi. The cDNA libraries were sequenced on an Illumina RNA-Seq Next-generation sequencing (NGS) instrument and filtered and normalized using default Partek Flow software suites (St. Louis, Missouri, USA).
Methods of identifying naturally occurring microbial flora in nontreated and treated wethers were identified. Profiles are based on primers spanning the hypervariable regions V1–V9 of the 16S rRNA gene and the internal transcribed spacer (ITS) region 1 amplified with primers ITS1 and ITS2. PCR reactions consisted of 50 ng input DNA per reaction completed to 20 μL reaction mixture. Each sample was amplified with random barcoded primer combinations. Reactions were quantified with a NanoDrop™ 2000 (Thermo Fisher Scientific, Waltham, MA 02451). Analytics for QA/QC for high-throughput barcoded Illumina MiSeq NGS sequencing of 16S rRNA, resulted in 18 samples that were obtained for 7 dpi and 20 samples for 21 dpi.
Total RNA purification
Total RNA was collected from blood samples using a modified TRIzol reagent procedure (Thermo Fisher Scientific, Waltham, MA, USA).
Lysate preparation from blood
The cDNA libraries were constructed by initial first strand synthesis using the Protocol for Non-directional RNA-seq Workflows and NEBNEXT (New England Biolabs, Ipswich, Massachusetts, USA) Ultra II RNA First Strand Synthesis Module (E7771), according to a modified manufacturer’s protocol.
First strand cDNA synthesis reaction
The first strand synthesis reaction was assembled on ice by adding components to the fragmented and primed RNA for a total volume of 20 mL. The reaction was mixed thoroughly by pipetting. The sample was incubated in a preheated thermocycler with the heated lid set at 80 °C as follows: Step 1: 10 min at 25 °C; Step 2: 15 min at 42 °C; Step 3: 15 min at 70 °C; and Step 4: Hold at 4 °C. We then proceeded directly to Swift Biosciences (Ann Arbor, Michigan, USA) ACCEL-NGS 1S PLUS DNA LIBRARY KIT: Single, Dual Combinatorial and Unique Dual Indexing and prepared the DNA Libraries.
Microbiome DNA isolation
Microbiome DNA was collected from blood samples using a modified Microbiome DNA Isolation Kit from NORGEN BIOTEK CORP. (Thorold, ON, Canada).
Sequencing
Quality Assurance/Quality Control of bar-coded sequence prepped samples of cDNA and the cDNA library sequencing with an Illumina RNASeq NGS instrument were completed by the Genomics Core Facility at Oklahoma State University (Stillwater, Oklahoma, USA).
Quality Assurance/Quality Control of bar-coded sequence prepped samples were completed and sequenced for 16S rRNA metagenomics of blood samples by Swift Biosciences (Ann Arbor, Michigan, USA) using an Illumina MiSeq NGS instrument.
Computational analysis
Several methods were utilized to conduct bioinformatic analysis of the obtained sequence data. For gene expression analyses, we used the Partek Flow (St. Louis, Missouri, USA) software suites pipelines that include, but are not limited to the STAR algorithm, Normalization, and the gene set differential analysis method (GSA).
Preliminary analyses included Qiime2 open-source analysis for the metagenomic or microbiome analysis. The results obtained utilized the Kraken pipeline through the Partek Flow (St. Louis, Missouri, USA) software suites. Inflammation comparisons were based on the ratio of F/B ratios that are evident as well as the reduction of microbial diversity.
Mothur (v1.45.3) was also used for microbiome analysis. The MiSeq manual protocol (http://mothur.org/wiki/miseq_sop/) was followed to obtain the 16S rRNA diversity, classify sequences into OTUs and calculate alpha diversity index such as Shannon diversity index and Simpson index. For Shannon and Simpson diversity index values, the student t-test was calculated, and the significance of these indices were based on p-value of < 0.05.
Ethical approval and consent to participate
The treatment of animals is abided by the guidelines of the Langston University Institutional Animal Care and Use Committee (LUACUC) Approval # 2018-14. All experiments were performed in accordance with relevant guidelines and regulations. Furthermore, the reporting in this manuscript is in accordance with ARRIVE guidelines.
Consent for publication
All authors have consented for publication.
