The use of polymerase chain reaction for rapid diagnosis and differentiation of para- and ortho-pox virus infections in camels

Camels are important livestock resource uniquely adapted to hot and arid environment. Two camel diseases are of economic importance; Camel Pox (CP) caused by an orthopoxvirus and Camel Contagious Ecthyma (CCE) caused by a Parapoxvirus. Clinically CCE is indistinguishable from CP, especially when both diseases co-exist in the same locality. The current traditional methods for laboratory diagnosis of both diseases are not reliable, time consuming or not available in countries where these diseases are endemic. Nucleic acid hybridization techniques based on the polymerase chain reaction (PCR) are now used for detection and characterization of many viruses including Poxvirus. The aim of this investigation is to develop a rapid and reliable technique to separate CCE from CP and to overcome costly and time- consuming traditional techniques. A number of 22 CCE-suspected and 4 CP-suspected skin scabs collected from camels in the Sudan as well as 3 skin scabs collected from sheep affected with Contagious Pustular Dermatitis (Orf) were used. As a reference, MR I strain (Parapoxvirus) and Vaccinia Elstree strain (Orthopoxvirus) were included. Skin scabs were cut into small pieces and homogenized. Scab homogenates were either used directly in the PCR or purified through a 36% sucrose cushion. Viral DNA was extracted from skin scabs using a commercial DNA Isolation kit (Gentra Systems). Accordingly, 3 PCR methods were followed; 1) using DNA prepared directly from skin scabs (PCR 1); 2) scab homogenates were added to the PCR minus the polymerase and then the reaction heated at 100 deg. C as described by Ireland and Binepal (PCR 2); 3) purification of the poxvirus in scab homogenate and virus pellets added to the PCR reaction and then treated as in 2 (PCR 3). In order to compare the PCR methods with traditional techniques for diagnosis, a small volume of each sample was examined by negative contrast electron microscopy (EM). Portion of the same samples were also inoculated onto bovine embryonic oesophageal cells (KOP) and Vero cells for virus isolation. For the amplification of parapoxvirus-specific DNA by PCR, the primers and reaction conditions were used as described by Sullivan et al. For the amplification of orthopoxvirus-specific DNA were used as described by Meyer et al. Results are summarized. The size of the amplification products exactly correspond to those obtained with the reference parapoxvirus (MR I strain) and the reference orthopoxvrus (Vaccinia strain). A total of 8 skin scab samples were negative by all diagnostic techniques used. Method 1 in which viral DNA was extracted directly from scab samples followed by PCR proved to be superior and more sensitive than the other two methods. The results showed that the diagnostic sensitivity of this PCR method is greater than traditional diagnosis techniques of electron microscopy and virus isolation in cell culture. Well-equipped laboratories with cell culture and electron microscopy facilities are not needed and results can be obtained within 24 h of sample receipt in small laboratory. The PCR test described in this report is a valuable addition to the current methods for diagnosis and differentiation of para- and orthopoxvirs infection in camels