Study area description and clinical observations
The investigation was conducted in seven geographical districts including Mergasur, Khalifan, Choman, Barzan, Shaqlawa, the provincial capital Erbil city and Qushtapa located in Erbil Governorate, in the Kurdistan region, Northern Iraq (Fig. 3A&B). Erbil Governorate (13.165 km2) is adjacent to the international borders of Southern Turkey and Northwestern Iran characterized by a rugged mountain range along the northern border of Iraq where the Mergasur region and Shaqlawa mountains (Barzan) are natural reserves for rearing wild goats (bezoar ibex, Capra aegagrus).
A total of 3529 goats including 3254 domestic goats (local breeds) from ten farms located in five geographical areas (Barzan, Ghoman, Kalfan, Erbil city, Shaqlawa) as well as 275 captive wild goats from seven herds located in five geographical areas (Barzan, Mergasor, Erbil, Shaqlawa, Qushtapa) were studied. A total of seven herds of captive wild goats including four herds reared by free-range grazing in two natural reserves in the mountainous area of Mergasur district and Brazan city, one herd reared in a small animal zoo located in Erbil city, as well as two commercial herds, located in Erbil city and Shaqlawa district were investigated (Table 1). The latter farm transported wild captive goats from mountainous regions of Northern Iran to Northern Iraq for breeding purpose. All farms and herds were visited by local veterinary authorities from August 2015 to September 2016 for clinical examination, data and sample collection. The domestic goat farms were managed and reared in a semi-free range grazing system where the goats spent most of their time (~ 60–70%) grazing on free local rangeland. Of the total ten domestic goat farms and seven captive goat herds, 13 were not immunized against PPR virus while three domestic goat farms were vaccinated with commercial dried live attenuated vaccine strain Nig. 75/1 (Jordan Bio-Industries Center, Amman, Jordan) (Table 1).
Clinical and post-mortem examination and observations
The veterinarians recorded the health status including the form of disease (peracute, acute and subacute) and case history of any symptoms related to PPR infection including pyrexia, ocular and nasal discharge, ulceration and erosion of oral mucosa (stomatitis), emaciation, severity of diarrhea as well as morbidity and mortality status. The infected domestic goats were euthanized using pentobarbital (1 mL per 10 lb. (4.5 kg) body weight) via intravascular injection. The post-mortem investigation was conducted by collecting necropsy samples from euthanized and fresh carcasses at the Directorate of Central Veterinary Laboratory (DCVL), Erbil, Iraq.
c-ELISA
For preparation of serum for c-ELISA, a total of 493 (domestic: n = 396; wild: n = 97) goat blood samples were collected and placed on ice in a cooler and transported to the DCVL, Erbil, Iraq. The serum samples were tested for antibodies against PPR virus using commercial ELISA kit (ID Screen®PPR Competition, Innovative Diagnostics, Grabels, France) and BioTek Absorbance Microplate reader 800 TS (BioTek Instruments GmbH, Bad Friedrichshall, Germany) for detecting anti-PPRV nucleoprotein antibodies in sheep and goat serum. The optical density (OD) was recorded at 450 nm in accordance with the manufacturer’s recommended procedure. The results were recorded as a percent inhibition of the optical density (%OD) reading of the test sample, and the competition percentage (S/N%) for each sample was calculated using following formula: S/N% = ODsample/ODNC × 100. Sample results ≤OD %50 and > OD%60 were considered as positive and negative, respectively. The serological results, based on the proportion of animals that had detectable antibodies in the sample population, were calculated and recorded as a percentage.
Genotypic identification and characterization of PPRV
Sample collection
A total of 486 swabs samples including 270 (n = 220 domestic goats; n = 50 wild goats) swabs from nasal and mouth lesions and 216 (n = 178 domestic goats; n = 38 wild goats) swabs from tissue samples were collected from different organs including lymph nodes, small and large intestine, lung and liver of clinically diseased and suspected healthy animals. All samples were placed on ice in a cooler and delivered to the DCVL, Molecular Department for further investigation.
RNA extraction and complementary DNA synthesis
RNA was extracted from nasal and mouth swabs as well as from organ tissues using RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instruction. Synthesis of cDNA was carried out in a 25 μL reaction where 3.5 μL of the purified RNA was added to a mixture containing 2 μL One Taq One-Step Enzyme, 2.5 μL buffer (New England BioLabs Inc., Ipswich, MA, USA), 2 μL reverse specific forward primer NP3, 4 μL 10 mM dNTP, 1.0 μL MgCl2 and 11 μL Aqua dest using One Taq® One-Step RT-PCR kit (New England BioLabs Inc.). The reaction was carried out at 42 °C for 60 min (one cycle) and 95 °C for 10 min (one cycle) using Eppendorf MasterCycler Gradient PCR system (Eppendorf AG, Hamburg, Germany). The purified complementary DNA (cDNA) was quantified using NanoDrop 2000 (Thermo Fisher Scientific GmbH, Darmstadt, Germany) and stored at − 20 °C for further analysis.
Reverse transcription complementary polymerase chain reaction (RT- cPCR)
The cDNA was investigated for the nucleocapsid protein (N) gene using Goldstar PCR Red Master mix (Eurogenetec, Deutschland GmbH, Köln, Germany), oligonucleotide forward primer NP and reverse primer NP4 (Eurofins Genomics GmbH, Ebersberg, Germany) as previously described [35]. The PCR amplification was performed in a 30 μL reaction mixture with 1 μL (10 pmol μL− 1) of each primer, 15 μL ready-to-use Goldstar PCR Red Master mix and 10 μL Aqua dest. Finally, 3 μL DNA template was added to each reaction tube. The amplification reaction was carried out with the thermocycler program: Initial denaturation at 94 °C for 2 min followed by 35 cycles consisted of 94 °C for 30 s, 55 °C for 60 s, 72 °C for 30 s and final extension 72 °C for 5 min. The amplified PCR products were electrophoresed on 1.5% agarose gel matrix (Biozym Scientific GmbH, Hessisch-Oldendorf, Germany), stained in ethidium bromide (0.5 μg mL− 1) and visualized at 302 nm on a UV transilluminator (Cleaver Scientific Ltd., Warwickshire, UK).
PCR-based sequencing and phylogenetic analysis
The positive PCR amplicons of ten randomly selected samples obtained from domestic and wild goats were further sequenced to gain information about the individual N gene structure. PCR products were purified using QIAquick PCR purification kit (Qiagen, GmbH) according to the manufacturer’s instructions. Quality and concentration of purified PCR products were confirmed as mentioned in the above section. The purified PCR products were sequenced at Seqlab-Sequence Laboratories GmbH (Göttingen, Germany), and the sequences were analyzed using FinchTV (version, 1.4.0). For confirming sequence identity, sequence data was subjected to Nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the global database. Additionally, all sequences were further analyzed and compared by Clustal-V and Clustal-W pairwise multiple sequence alignment using MegAlign (DNASTAR Inc., Madison, WI, USA). The phylogenetic tree was constructed where bootstrapping was performed by creating 1000 trials. Similarly, the nucleotide sequences were converted to amino acid sequences using the translate DNA step of the EditSeq program (DNASTAR Inc.) and the phylogenetic tree was constructed using the MegAlign program.
Reverse transcription quantitative PCR (RT-qPCR)
For the quantitative detection of the specific target gene of the PPR virus, cDNA samples were investigated for the N gene using the VetMAX™ PPRV reagents containing N gene-specific oligonucleotide primers and TaqMan® real-time PCR master mix in accordance to the manufacturer’s instructions (Thermo Fisher Scientific GmbH, Dreieich, Germany). All samples were tested along with external (inactivated bacteria or virus) and internal (exogenous) positive controls (Thermo Fisher Scientific GmbH). The qPCR assay was performed using 7500 real-time PCR (Applied Biosystems GmbH, Darmstadt, Germany) system where each sample was analyzed in duplicate. The samples were considered positive when the cycle threshold (Ct) value was ≤40.0 and ≥ 41 Ct value was considered as negative or indeterminate. The quality and reaction efficiency were calculated based on Ct values whereas correlation coefficient values were obtained from the standard curve.
Data analysis
To compare and assess the geographical difference and rate of morbidity and mortality between domestic goat farms and captive wild goat herds, McNemar Chi-square Contingency and Fisher’s Exact Tests were applied using ELISA and RT q-PCR-based data for identifying significant difference (p < 0.05).
