Orf disease in local goat; clinical and phylogenetic study in Al-Qadisiyah governorate, Iraq

Khalefa Ali Mansour, Muthanna Hadi Hussain, Asaad jassim Abid and Qassim Haleem Kshash

Department of Veterinary Internal and Preventive Medicine, College of Veterinary Medicine, University of Al-Qadisiyah, Al-Qadisiyah, Iraq

khalefa.mansour@qu.edu.iq, 0000-0001-9135-7256

muthanna.hussain@qu.edu.iq, 0000-0003-1605-6116

assad.abid@qu.edu.iq, 0000-0002-3110-2056

qassim.kshash@qu.edu.iq, 0000-0003-4856-625X

Abstract

This study aimed to diagnose orf virus ORFV from infected goats in Al-Qadisiyah Governorate of Iraq, using polymerase chain reaction and construction of phylogenetic tree. Clinical examination was made on 400 goats in different herds and crusty scabs were collected from forty infected goats. All scabs were subjected to (PCR), sequencing, and phylogenetic analysis involving the presence of GIF gene GM-CSF^* inhibitor factor, a characteristic gene of the ORFV. The typical proliferative skin lesion with overall orf prevalence results by PCR was 10% with low mortality rates and high significance variation with age and gender. Two positive PCR amplicons were sent for partial-gene-based sequencing and Phylogenetic analysis. Multiple alignments of sequences presented close identities with two Iraqi global isolates of the virus. This study didn't only report novel sequences from the local orf virus isolates, but it also provides important data about transmission of local sheep orf virus into local goat during closely rearing and grazing and orf outbreak in small ruminant in Iraq might be suspected.

Keywords: Goat, Orf, GIF Gene, Skin, Scabs

مرض الاورف فی الماعز المحلی, دراسة سریریة و تطوریة فی محافظة القادسیة فی العراق

خلیفة علی منصور، مثنى هادی حسین، أسعد جاسم عبد و قاسم حلیم کشاش

فرع الطب الباطنی، کلیة الطب البیطری، جامعة القادسیة، الدیوانیة، العراق

الخلاصة

هدفت هذه الدراسة إلى تشخیص فیروس الأورف من الماعز المصابة فی محافظة القادسیة فی العراق باستخدام تفاعل البلمرة المتسلسل وبناء شجرة النشوء والتطور. تم إجراء الفحص السریری على 400 ماعز من قطعان مختلفة وتم جمع قشور جلدیة من أربعین ماعز مصابة. وتم إخضاع جمیع القشور لفحص تفاعل السلسلة المتبلمرة وتحلیل النشوء والتطور والذی یتضمن وجود جین GIF العامل المثبط  GM-CSFوهو الجین الممیز للفایروس. کانت الآفة الجلدیة التکاثریة النموذجیة للنتائج بواسطة تفاعل البلمرة المتسلسل 10٪ مع معدلات وفیات منخفض وتباین کبیر فی أهمیة الاصابة مع العمر والجنس. تم إرسال اثنین من أمبلیکون الموجبین لفحص التسلسل الجزیئی القائم على الجینات وتحلیل النشوء والتطور. أضهرت للتسلسلات المتعددة قربها مع عزلتین عراقیتین عالمیتین للفیروس. لم تکتف هذه الدراسة بالکشف عن التسلسلات الجدیدة من عزلات الفیروس، ولکنها وفرت أیضا بیانات مهمة حول انتقال فیروس اورف الأغنام المحلی إلى الماعز المحلی أثناء التربیة والرعی وان تفشی المرض فی المجترات الصغیرة فی العراق قد یکون متوقعا.

Introduction

ORF’ is recognized as sore and scabby mouth disease, contagious ecthyma, and contagious pustular dermatitis, considered one of the most important viral skin diseases in goat farms. It mainly affects sheep, goat and some other housetrained and wild ruminants, causing economic loss in the livestock construction. (1). Disease is spread worldwide and widespread in many countries wherever sheep and goats are owned, and the disease also has zoonotic latent affecting the farmers, veterinarians and butchers who are in direct interaction with infected animals especially during shearing, docking, drenching and slaughtering or indirect connection with infected animals (2). The spread within a group is carried out over direct contact between animals during a confrontation or suckling. Morbidity is actually higher in young animals and mortality is usually low, but it may be very great when bacterial or fungal secondary infections occur (3). Orf causes highly economic losses including weight loss, premature culling, treatment and control costs. Many studies indicate the world wide and very high incidence of orf infection in goat herds as in China 60% (4), 14.4% in Malysia (5) in Korea (6), in India (7). In Africa, orf contagion has been stated in sheep and goats in certain countries such as Cameroon, Nigeria, Tanzania and Egypt (8), Gabon (9), in Brazil (10). Clinically, orf develops from erythema to macule, papule, vesicle creation and at that time pustules to dense crusts called scabs. The scabs are often friable and minor trauma makes the lesions bleed simply. These lesions are ordinarily started on muzzle, lips, oral mucosa, ears and round the nostrils. The lesions can also be gotten on feet, eyelids and teats (11). Suspected orf disease can be identified based on clinical signs, tracked by laboratory tests such as serum neutralization test (SNT), electron microscopy, histopathology of the infected tissues and polymerase chain reaction (PCR) (12). Polymerase chain reaction is a consistent method for the diagnosis and can distinguish ORFV from other pox viruses such as sheep pox and goat pox (13). Numerous molecular investigative approaches including (PCR) and quantitative PCR (qPCR) have been established to distinguish orf virus (14).

The present study aimed to detect ORFV in Iraqi goat based on the GIF gene which is responsible for inhibiting the ovine cytokines Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and interleukin-2 (IL-2) as a unique feature and the significant of age and gender on the prevalence rate of Orf (15).

Materials and methods

Ethics statement

Scabs were collected from goat during this study for diagnosis. No animal experimentation was accompanied and sample gathering and procedures were permitted by the cooperative animal ethical committee of College of Veterinary Medicine, Al-Qadisiyah University. All labors were finished to lessen animal pain during the development of sample gathering in orf suspected sickness. Consent was gotten from the animal possessors for the assortment of tissue samples by veterinarian authorities.

Sampling

The study was planned to be done from December, 2018 to January, 2019 in the rural part of AL-Dagharah district positioned in the north east of Al-Qadisiyah province approximately on latitude 31.58°N, longitude 44.500° E. in the middle of Iraq as in figure

(1).

Figure 1: Study location, AL-Dagharah district in Al-Qadisiyah province, Iraq (google maps).

Forty goats had suspected with clinical orf include crusty external skin lesions on the muzzle, nostrils and lips, goats were dull and depressed, they were 28 kids (18 females and 10 males) and 12adults (8 female and 4 male), they reported after examination of three separated private goat flocks. Clinical inspection of the infected goats and the specimen procedure were approved out as the procedure described by (13). Dried scabs from the infected goats were collected and stored in freezer until testing via PCR.

Molecular identification of Orf virus by PCR

Collected scabs from infected goats were homogenized by means of germ-free plasters and pestles within the utility of hygienic sand and physiological buffer saline to yield 20% tissue suspension and had achieved to be used in DNA extraction. DNA extraction was passed out by viral nucleic acid extraction kit (QIAGEN \ Germany) from samples as according to manufacturer’s protocol. The procedure used a specific primer (408 pb) of GIF gene (15), forward primer sequence is 5-GCTCTAGGAAAGATGGCGTG -3 and reverse primer sequence is 5-GTACTCCTGGCTGAAGAGCG -3. PCR procedure was accomplished according to the manufacturer’s instruction. Thermal cycler was automated conferring to the following circumstances; 95oC for 5 mins as a first beginning step, tracked with 30 cycles of 95oC for 30s; 65oC for 30s, 72oC for 45s, and one ending cycle of 72oC for 7 mins. PCR harvests were run in the 2% agars gel and electrophoresed at 100 V for 60 mins, and DNA bands were visualized with ethidium bromide staining.

Nucleotide sequencing besides phylogenetic analysis

The pure GIF gene PCR product samples were perpetrated to perform DNA sequencing by (Bioneer. Company, Korea - DNA sequencing system). Sequences were explored and firmed by Clustal W 2 system in the Meg-Align database of Laser-gene ((DNA/STAR Inc. Madison, USA). The sequences were associated with homologous sequences of reference strains from NCBI Gen Bank and phylogenetic diagram for GIF genes of ORFV were assembled using MEGA 6 software. The genetic detachments between sequences were considered and used for building of a neighbor joining tree for local ORFV B2L gene (16).

Data analysis

The data obtained in this study were analyzed by IBM SPSS Statistical software version 22 and the statistical significance was set at p≤ 0.05.

Results

Clinical examination

Orf virus was detected in all crust samples that tested using GIF gene (Figure 2), however, the prevalence of orf in local goat was 10% (40\400). Clinical observation of infected goats were founded dry ulcerative and scabby characteristic lesions as thick wart-like multiple scratches on the oral fissures, lips and nostril part in goat were seen. No visible lesions were found in other locations. On the other hand, adults showed mild fever (40-40.5C^o), respiratory and heart rates were normal in most examined cases. In kids, the acute form was domain which includes fever ranged between (41 to 42C^o), anorexia, depression, serous to mucopurulent nasal discharge, conjunctivitis, abnormal respiratory sounds (wheezing) with difficult inspiration dyspnea. Respiratory rate was increased and heart rate also was higher than normal, and the oral lesions made most infected kids unable to suck and \or graze feed and death were noticed in two cases in kids (mortality rate 0.5% (2\400).

In table (1), there was a scientific important connotation among the orf prevalence and age, 70% (28/40) of kids higher than adult 30% (12\40), there was a higher disease occurrence in females 65% (26\40) than in males 35% (14\40).

Table 1: The effect of gender and age of goats on prevalence rate of orf using PCR

Molecular detection

Detection of the virus using PCR was conducted. The results illustrated the presence of the virus in all samples regarding to the GIF gen as in figure (2).

Figure 2: Agarose gels electrophoresis portrait that demonstrates the PCR product exploration of GIF gene in ORFV positive isolates. M: marker (100-2000bp), lane (1-10) confident ORFV at (408 bp) PCR product.

Phylogenetic analysis

Two amplicons gotten by PCR were sent out for sequencing to be identified and matched up with local two Iraqi and eight global strain references. All sequences were placed in GenBank as shown in (Figure 3).

Figure 3: Multiple sequence arrangement analysis of GIF gene in local ORFV IQ-Goat isolate and NCBI-Genbank in local ORFV IQ-Goat isolate. The multiple alignment analysis was created using ClustalW alignment implement in (MEGA 6.0 version). That show the nucleotide alignment likeness as (*) with substitution mutations in GIF gene.

Built on analysis of the sequence of the GIF gene, the Iraqi goat orf virus strain exhibited 99.05% of character at the nucleotide range with the Iraqi sheep Orf virus (KJ697772.1) and the second Iraqi goat ORFV strain display 99.34% identity at the nucleotide level with the other strain of Iraqi sheep ORFV (KJ653446.1) as in Table (2).

Table 2: BLAST submitted ORFV IQ-Goat isolates

The local isolates presented only a 0.66-0.95 % variance from the Iraqi Orf references virus strains detected in 2015 (Figure 4). Phylogenetics analysis based on the partial GIF genes (Fig. 4) indicated that Iraqi strains were closely related to the Iraqi types, with which it has progressed from a communal progenitor.

Figure 4: Phylogenetic tree analysis based on GIF gene partial sequence in local ORFV IQ-Goat isolate.1 isolates that was run for genetic relationship documentation. The phylogenetic map was made by unweight Pair Group way with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version). The native ORFV IQ-Goat isolate No. 1- No.2 were indicated closed related to NCBI-BLAST ORFV isolate No5.DSANA GM-CSF/IL-2 and ORFV isolate NO10.DMUHN GM-CSF (KJ697772.1) and (KJ653446.1) respectively at total genetic changes (0.0005-0.0015%).

Discussion

The Overall prevalence rates of orf founded through this research were very much lesser likened to that of 60% in India, 60% in Ethiopia, 34.89% in China and closely to infection rates reported in Malaysia 14.4%, 13% in India (17). However, orf in local goat in Iraq was determined by PCR as Orf sickness have been described in numerous countries, such as China, Croatia, Italy and Taiwan. Clinically; propagative lesions were seen on the lips, muzzle, eye & everywhere the nostrils with sever clinical signs in kids than in adult, these answers were in treaty with those of (18) and matching with alike clinical symbols were stated by additional researches on spreadable ecthyma in goats and sheep.

The incidence of orf illness can be prejudiced by age and gender which was higher in kids and this finding is in agreement with (10) documented a higher orf infection in kids (70%) and Similarly, by (4), and the younger animals can be extremely infected (3). Controversially, (14) specified that in adult goats the clinical marks might be extra severe, while, (5) presented orf is independent age disease. Meanwhile, female gender had the highest orf incidence; this harmonizes with a preceding study by (5,12). However, unfit sample size by which bulk of the topics studied was female may donate to unreliable statement. In the meantime, earlier annotations exposed that orf contagion have not intolerant predispositions between gender (2,18). In adults the lesion is milder; it may be related to the previous infection when they were kids.

The mortality rate was 0.5%, that closely to reports the small mortality rate of orf, extending from 0.8% to 24.7% (18). Meanwhile, (17) observed no mortality in orf unless the sick animal develops secondary bacterial or mycotic infection or being neglected without nursing.

Orf is a debilitating disease not fatal, but it can be fetal if kids are prohibited from suckling or exposure to minor bacterial or fungal infections. Increasing amount of ORFV contagion in goats may be owed to lack of vaccination policy, mixing rearing system is including sheep, goat and cattle together, continuous moving infected animals between different feeding area which spread ORFV, this observation is in agreement with (19). The two isolates of Iraqi infection were clustered in a single bunch, indicating great level of genetic homology between the isolates. BLAST searches in Gen-Bank revealed these sequences of two Iraqi orf isolates to be 99.05%-99.34% matching to Iraqi ORFV strains that previously submitted in Gen-Bank at 2015, and they also were acquiesced to the NCBI Gen Bank database and allocated the accession numbers KP869116-KP869120. Homology investigates displayed that the five strains bunch together, with related nucleotide variation. This phylogenetic characterization specifies that the GIF gene is an extremely well-maintained section in ORFV. The phylogenetic analysis displayed that these two orf goat viruses are strictly related antigenetic. Furthermore, homology based phylogenetic inquiry presented that the goat ORFV GIF gene is closely similar to that of Iraqi orf sheep, this indicates that ORFV contagion is endemic in small ruminants in Iraq, with incessant hazard of transmission and outbreak occurrence.

This result; like in (20); indicates that ORFV of dairy goats might transmitted from infected sheep because in rural areas of Iraq small ruminants are farmed together with frequent contacts between them without quarantine, the infected animals are aggregate at night all in narrow closed pens especially in winter months. Like other diseases detected by (21-24) it is highly regarded that orf is an important disease because of its effect on the animal’s health and the economic loses made during the disease onset and the treatment.

Conclusions

We concluded that the Iraqi ORFV infecting the local goat was closely related phylogenetically to Iraqi ORFV strains infecting sheep. The age and gender have a significant effect on orf occurrence.

Acknowledgements

We would like to thank the labors of owners and all the work of molecular diagnostic lab for their support and taking part in case and facts assortment of this research.

Conflict of interests

The writers don't have any fight of interest concerning the contented of this research.