First Molecular Investigation of lumpy Skin Disease in Buffaloes Using the Qualitative and Quantitative Polymerase Chain Reaction Assays, Iraq

1. Introduction

Lumpy skin disease (LSD) is an infectious bovine disease, which infecting mainly cattle and to less extent buffaloes as a result of exposure to a viral species known as the lumpy skin disease virus (LSDV), belongs to Capripoxvirus genus in the subfamily Chordopoxvirinae and the family Poxviridae ( 1 ). The first describing of clinical syndrome that including high fever, 0.5-5 cm skin nodules in whole skin or subcutaneous tissue with enlargement of the superficial lymph-nodes, and then, development of ulcerative skin lesions, excessive lacrimation and nasal discharge, was restricted to sub-Saharan Africa, especially in Zambia in 1929, and considered initially to be the result of either poisoning or a hypersensitivity to insect bites ( 2 , 3 ). Between 1929 to 1986, more cases and panzootic infections was reported in African countries such as Botswana (1943-1945), Republic of South Africa (1949), Kenya (1957), Sudan (1972), Tanzania, Zimbabwe, Somalia, Cameron (1981-1986) and Egypt (1988) ( 4 ). According to the World Organization from Animal Health (OIE), LSD outbreaks have been suspected or confirmed in the Middle East countries such as Kuwait (1991), Lebanon (1993), Yemen (1995), United Arab Emirates (2000), Bahrain (2003), and Oman (2010) ( 5 ). Incursion of LSD was reported for the first time in Iraq, Turkey and Iran in 2013, indicating that the disease has a potential for further spread to the Asian and European countries ( 6 , 7 ).

The disease can be transmitted from infected animals to susceptible hosts through blood, nasal discharge, lacrimal secretions, semen and saliva as well as through the infected milk to sucking calves resulting in severe economic losses due to chronic debility in affected animals, reduced milk production, poor growth, infertility, abortions, severe damage to hides and mortalities that could be reach 20% ( 8 ). Further, LSDV isolation from flies (Culicoides, Glossina, Tabanidae and Stomoxys) and mosquitoes has been suggested the mechanical transmission of the virus by these vectors ( 9 ).

Worldwide, the tentative diagnosis of disease is usually based on the characteristic clinical signs ( 10 ). However, the application of laboratory molecular assays such as polymerase chain reaction (PCR) have revealed a high reliability, sensitivity and specificity in detection and confirmation of the virus in suspected clinical specimens or biopsies ( 11 , 12 ). In Iraq, LSD outbreaks were reported in cattle among different regions; and consequently, a number of studies have been performed to confirm the LSD in clinically infected animals using PCR assay and histology ( 10 , 13 ). However, no studies or reports were found about the occurrence of disease in buffaloes or even vectors. Hence, the present study was carried out to confirm the LSD in buffaloes existed mainly in southern parts of Iraq in particular Wasit, Dhi-Qar and Maysan province using conventional and real-time PCR assays. Molecular examination of tick samples that collected from the infested buffaloes with estimation the association of positive findings to clinical vital signs and risk factors were aimed, also.

2. Materials and Methods

2.1. Study Animals

Totally, 150 unvaccinated LSD-buffaloes of different ages, both sexes and many regions were selected equally from three Iraqi provinces; Wasit, Dhi-Qar and Maysan. The selection of study animals was based on the information of veterinarians and owners about the recent appearance of clinical LSD cases in cattle and/or buffaloes existed at these areas. Initially, all study buffaloes were subjected to clinical examination to detect of pursued skin lesions and enlargement in lymph nodes, with measurement of vital signs (temperature, pulse and respiratory rates) and collection of tick samples if available. From each study animal, 5 ml of jugular venous blood was collected, aseptically using a disposable syringe into an anticoagulant EDTA tubes and transported to the laboratory.

2.2. Molecular Examination

Following the manufacturer instructions of the two DNA Extraction Kits, iNtRON Biotechnology (Korea) and Genaid (USA), DNAs were extracted from the samples of blood and ticks, respectively. The concentration (ng/L) and purity of extracted DNAs were measured at an absorbance of 260 /280 nm using the Nanodrop system (Thermo-Scientific, UK).

For conventional PCR, the mastermix tubes were prepared using the Maxime PCR Premix Kit (iNtRON Biotechnology, Korea) at a total volume of 20L through targeting the Envelop Protein 32 (P32) gene [(F: 5´-AGGTTCGCGAAATTTCAGATGT-3´) and (R: 5´-TCCCCCTG TACGAATACA-3´)]. The Thermal Cycler conditions were included 1 cycle initial denaturation (95ºC / 5 minutes); 35 cycles of denaturation (95ºC / 30 seconds), annealing (60ºC / 30 seconds) and extension (72ºC / 1 minute); and 1 cycle final extension (72ºC / 5 minutes). The PCR products were analyzed by electrophoresis of the stained Agarose gel with Ethidium Bromide at 100 volt and 80Am for 1 hour. The positive samples were detected at an amplicon size of ~499bp.

For real-time PCR, GoTaq qPCR Master Mix Kit (iNtRON Biotechnology, Korea) at a final volume of 25L through targeting the Envelop Protein 32 (P32) gene [(F: 5´-AGAGCTAACGA AATGAAAAACGGT-3´) and (R: 5´-TGAGCCATTTTTCCAACTC-3´)]. The qPCR conditions were involved 1 cycle pre-denaturation (95ºC / 5 minutes), 40 cycles denaturation (95ºC / 20 seconds) and annealing/extension with detection (58ºC / 1 minute), and melt cycle (65-90ºC). The qPCR data analysis was carried out in BioRad Real Time PCR Analysis Software by calculation of the threshold cycle number (CT value) the positive amplification and negative control sample.

2.3. Statistical Analysis

The findings of present study were analyzed using the GraphPad Prism version 6.0.1.298 (GraphPad Software Inc., USA) software. Chi-square (x²) was applied to detect significant differences between values of clinical signs of positive and negative animals by molecular assays. Odds ratio was applied to estimate statistical association between age and sex factors and positive results by both CR assays. Values were represented as mean±standard errors (M±SE) or percentage (%), and the statistical differences in study results were considered significant at a level of (P<0.05).

3. Results

The total positive results for conventional PCR examination of 150 buffaloes was 8 (5.33%) using the blood samples. Among totally 13 suspected LSD skin lesions, only 1 (7.69%) sample was found to be positive. Among 29 tick samples collected from the 29 infested buffaloes, the findings revealed no positive samples in these samples (Figures 1 and 2).

Figure 1. Total results for conventional PCR examination of 150 blood samples, 13 skin lesions and 29 tick samples using the qualitative conventional PCR assay. Significance * (P<0.05)

Figure 2. Electrophoresis of PCR products for envelope protein (P32) gene at 499bp using 1.5% Agarose gel stained with Ethidium Bromide at 100 Volt, 80 Am for 1 hour.M: Ladder marker (100-1500bp); Lane (4): Positive Control; Lane (5): Negative Control; Lane (1): Positive skin lesion sample; Lanes (2, 3, 6, 7, 8, 9, 10 and 11): Positive blood samples

The application of real-time PCR assays revealed that the total positive results for testing 150 buffaloes was 23 (15.33%) in the blood samples, and 1 (7.69%) positive sample in a totally 13 suspected LSD skin lesions. However, no positive samples were found through testing of 29 tick samples (Figure 3). The amplification of target DNA was detected using the fluorescent molecules, and the cycle threshold (Cq) number of amplification of positive samples was ranged from 20 to 21 (Figure 4). The melting curve and melting peak for envelope protein (P32) gene in LSDV positive samples was calculated using 2x SYBR Green DNA polymerase mastermix, and the chart was generated by plotting relative fluorescence (RFU) vs. cycle number (Figure 5 and 6).

Figure 3. Total results for real-time PCR examination of 150 blood samples, 13 skin lesions and 29 tick samples using the qualitative conventional PCR assay. Significance * (P<0.05)

Figure 4. Amplification plot for envelope protein (P32) gene in LSDV positive samples using 2x SYBR Green DNA polymerase mastermix. The chart is generated by plotting relative fluorescence (RFU) vs. cycle number

Figure 5. Melting curve for envelope protein (P32) gene in LSDV positive samples using 2x SYBR Green DNA polymerase mastermix. The chart is generated by plotting relative fluorescence (RFU) vs. cycle number

Figure 6. Melting peak for envelope protein (P32) gene in LSDV positive samples using 2X SYBR Green DNA polymerase mastermixIn comparison between both molecular assays, the positive findings of real-time PCR were increased significantly (P<0.029) in blood samples (15.33%) compared to conventional PCR (5.33%), but not in values of skin lesion (P>0.05), (Figure 7).

Figure 7. Total positive results of blood and skin lesion samples by the conventional and real-time PCR assays

The lack of significant differences (P>0.05) was observed between the total findings of temperature, pulse and respiratory rates of LSD positive and negative buffaloes by the conventional and real-time PCR (Table 1).

Statistical correlation between the positive findings of conventional PCR and the groups of risk factors was revealed a significant variation (P<0.05) between their values (Table 2). For age factor, the percentage of positive results was increased significantly (P<0.0381) in the groups of animals aged <1 year (6.94%), 1-4 years (3.7%) and >4-8 years (5.26%) when compared to buffaloes aged > 8 years (0%). However, values of Odds ratio and relative risk of buffaloes aged < 1 year (1.865 and 1.792, respectively) were increased significantly in comparison with the values of other age groups; 1-4 years (0.575 and 0.592, respectively), 4-8 years (0.991 and 0.985, respectively) and 8 years (0 and 0, respectively). Regarding the sex factor, although no significant differences (P<0.515) were found between the positive findings of female (5.93%) and male (3.13%), values of Odds ratio and relative risk of females (1.95 and 1.895, respectively) appeared significantly higher than observed in males (0.513 and 0.528, respectively). Concerning with the findings of region factor, there was significant elevation (P<0.0344) in percentage of positive buffaloes to LSD as well as the Odds ratio in Wasit province (12% and 6.8, respectively) in comparison with those reported in Maysan (4% and 0.65%, respectively) and Dhi-Qar (0% and 0%, respectively) provinces. However, values relative risk were increased significantly (P<0.0395) in provinces of Maysan (6.67) and Wasit ( 6 ) compared to Dhi-Qar (0) province.

In this study, the findings of age factor were showed that the values of positive LSD were increased significantly (P<0.036) in buffaloes aged < 1 year (10.06%), 1-4 years (14.82%) and >4-8 years (10.53%) when compared to buffaloes aged > 8 years (0%). However, values of Odds ratio and relative risk reported a significant increase (P<0.05) in buffaloes aged < 1 year (1.497 and 1.414, respectively) when compared to values of other age groups; 1-4 years (0.941 and 0.947, respectively), >4-8 years (0.618 and 0.658, respectively) and >8 years (0 and 0, respectively). For sex factor, no significant differences (P<0.086) were seen between positive findings of females (16.1%) and males (12.5%); however, there were significant increases (P<0.0201) in values of Odds ratio and relative risk of females (1.343 and 1.288, respectively) than males (0.745 and 776, respectively). Among study regions, the findings of positivity, Odds ratio and relative risk of Wasi province (28%, 3.933 and 3.11, respectively) were increased significantly when compared to values of Dhi-Qar (4%, 0.157 and 0.19, respectively) and Maysan (14%, 0.858 and 0.875, respectively), (Table 3).

4. Discussion

Worldwide, there were a few data available about the occurrence and prevalence of LSD in buffaloes as well as the role of different vectors in transmission of infection between bovine animals; while in Iraq, this study appeared to be the first one carried out in buffaloes and in their infested ticks. In comparison with our findings, variable LSD positive results were reported using different diagnostic assays; 28.2% in South Africa using ELISA ( 14 ), 0% in Egypt by virus isolation and PCR ( 15 ) and 15.2% in other Egyptian study ( 16 ). The lack of positive findings in study tick samples may be caused either by the low numbers of tested tick samples or indicated that this vector is not implicated in transmission of LSDV. Other explanation is that ticks have a low chance to transmit the virus as a result of short period of existence on bodies of infested buffaloes because these animals are covered usually with water in rivers, lakes or even marshes. Although, the application of both PCR assays had targeting the Envelop protein (P32) gene, the findings of real-time PCR assay in blood samples were significantly than reported by conventional PCR assay. Caliendo, Schuurman ( 17 ) demonstrated that the quantitative molecular assay is more sensitive than the qualitative, since the quantitative assay having a lower limit of sensitivity and gave lower viral load values at all concentrations tested. Other studies have mentioned for the ability of real-time PCR to overcome many problems found in conventional PCR such as low sensitivity and resolution, non-automated, poor precision, short dynamic range and size-based discrimination only ( 18 , 19 ).

In this study, values of the vital signs; temperature, pulse and respiratory rates, were showed an insignificant variation between values of positive and negative animals obtained by both conventional and real-time PCR assays. Our findings were in contrast to the results of the only Iraqi study performed on acutely infected buffaloes ( 18 , 19 ). However, insignificant variation in values of vital signs between positive and negative animals could be attributed to that most of these animals were infected sub-acutely. Considering the disease burden, cattle are found to be more sensitive to the illness compared to buffaloes or other ruminants ( 20 ).

Association between positive results of molecular PCR assays and risk factors were reported a significant variation in their values. For conventional PCR, we showed that animals of <1 year age old were more sensitivity to LSD than other age groups, and this in consistent with that reported Ayelet, Haftu ( 21 ). While, the findings of real-time PCR were seen that buffaloes of <1 year, 1-4 and > 4-8 years age old were more sensitive to LSD than those of >8 years. The prevalence of LSD in different age groups might be attributed to the diminished maternal immunity in calves of less than 1 year, or exposing of adult cattle to stress factors of pregnancy, lactation, and fattening. Constable, Hinchcliff ( 1 ) reported that all ages are susceptible to the causative virus, except animals recovered recently from an attack, in which case there is a solid immunity lasting for about 3 months. Also, very young calves, lactating and malnourished bovine animals develop more severe clinical disease during outbreaks. Abd Elmohsen, Selim ( 22 ) demonstrated in cattle that the disease prevalence in older animals (12-50 months) was higher than in young animals (3-12 months) attributing these results to the low frequency of exposure because calves are kept at home away from biting insects. Faris, El-Bayoumi ( 16 ) found that animals aged 1-2 years old and over 2 years have a higher prevalence of LSD than young animals of age less than one year as a result of exposure to stress factors.

In both conventional and real-time PCR assays, no significant variation was showed between the positive females and males suggesting that both genders are susceptible to infection. However, statistical analysis of results revealed that females were at a higher risk of LSD than males, and this might be attributed to that females have increased stress factors than males including lactation and pregnancy ( 10 ). Also, the number of existed males at each farm, or even tested males night be reflected negatively on the total positive results in the current study.

We showed that the prevalence rate and risk of LSD were fluctuated among different localities. The high prevalence rate of positivity and risk observed in Wasit province might be explained by the method of selection of study animals because study buffaloes of this province were subjected selectively for collection of samples. Also, Veterinarian and owners of Wasit province were provided much more helpful during collection of animals’ data and then selection of suspected buffaloes with LSD. In many study areas, recent outbreaks in cattle livestock were targeted to detect study buffaloes since direct contact could play a role in transmission of infection from diseased cattle to sensitive buffaloes. Other explanations include the spread of cultivated areas and swamplands might result in a rapid propagation of insect population, or might be as a result of farmers’ refusal to vaccinate their animals ( 16 , 23 ).

This study provides data indicating infection by LSDV in buffalo population in Iraq; however, the role of these animals in the epidemiology of LSD is still not clear. The application of molecular PCR assays through targeting an Envelop protein (P32) gene shows a high efficacy in detection of disease especially with the real-time PCR assay. Blood represents the sample of choice instead of skin nodules since most infected buffaloes were revealed mild or sub-acute symptoms. The role of tick in transmitting of LSDV to positive LSD buffaloes was not seen in the current study; therefore, furthermore long-term molecular studies are necessary to implicate the role of ticks or other vectors such as mosquitoes and flies in transmission of infection or even in life cycle of LSDV. Additionally, control strategies should be included the consideration of the risk factors identified in this study.

Authors' Contribution

Study concept and design: A. M. A. and O. K. L.

Acquisition of data: A. M. A.

Analysis and interpretation of data: O. K. L.

Drafting of the manuscript: O. K. L.

Critical revision of the manuscript for important intellectual content: A. M. A. and O. K. L.

Statistical analysis: A. M. A.

Administrative, technical, and material support: A. M. A.

Ethics

The current study was approved by and carried out under the license of the Scientific Committee of the Department of Internal and Preventive Veterinary Medicine and the Scientific Committee of the College of Veterinary Medicine, University of Baghdad (Baghdad, Iraq).

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgment

The authors have deeply indebted and grateful to the staff and the Head of Department of Internal and Preventive Medicine (College of Veterinary Medicine, University of Baghdad) for their help and support.

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