An Immunoperoxidase Monolayer Assay (IPMA) for the detection of lumpy skin disease antibodies.

During this study a new Immunoperoxidase Monolayer Assay (IPMA) was developed for the detection of antibodies against lumpy skin disease virus (LSDV) in an easy and low tech setting. Using two dilutions (1:50 and 1:300) in a duplicate format, the test was shown to be highly sensitive, specific and repeatable. In comparison to the VNT and a commercial ELISA, the LSDV-IPMA was able to detect the LSDV antibodies earlier in infected, vaccinated and vaccinated/infected animals. The assay is very flexible as it can be easily adapted for the detection of sheeppox or goatpox antibodies and it can be scaled-up to handle medium size sample sets by preparing the IPMA plates in advance. These plates are safe and can be handled in low biosafety level labs.

Evaluation of different diagnostic methods for diagnosis of Lumpy skin disease in cows.

Viral isolation, polymerase chain reaction (PCR), dot blot hybridization (DBH), and indirect enzyme-linked immunosorbent assay (iELISA) were used for the diagnosis of lumpy skin disease in clinically infected, fevered, and apparently normal dairy cows. Lumpy skin disease virus (LSDV) was isolated from skin biopsies and blood samples collected from clinically infected cows in percentages of 72% and 20%, respectively. The virus recovered from blood samples collected from fevered cows in percentage of 33.3%. Both PCR and DBH detected viral DNA in 100% of skin biopsies collected from clinically infected cows whereas the detection rates in blood samples collected from clinically infected animals were 100% and 84% using PCR and DBH, respectively. Viral DNA was detected in blood samples collected from fevered cows using PCR and DBH in percentages of 77.8% and 66.6%, respectively. Only 19.1% of blood samples collected from in-contact cows was positive for both of PCR and DBH. Detection rates of antibodies against LSDV using iELISA in serum samples collected from clinically infected and fevered cows were 56% and 11.1%, respectively, whereas all in-contact cows had no antibodies against the virus.

Evaluation of indirect fluorescent antibody test (IFAT) for the diagnosis and screening of lumpy skin disease using Bayesian method.

The performance of indirect fluorescence antibody test (IFAT) for serological diagnosis and screening of lumpy skin disease (LSD) was evaluated using methods without gold standard. Virus neutralization test (VNT) was used as the second test and the study sites were selected from two different geographical places in Ethiopia to get different disease prevalence. The analysis of conditional dependent Bayesian model for the accuracy of IFAT showed that sensitivity, specificity, prevalence of the population Pi(1) and the population Pi(2) were 0.92 (0.89-0.95), 0.88 (0.85-0.91), 0.28 (0.25-0.32) and 0.06 (0.048-0.075), respectively. The posterior inferences obtained for VNT sensitivity, specificity and conditional correlation between the tests for sensitivity (rhoD) and specificity (rhoDc) were 0.78 (0.74-0.83), 0.97 (0.95-0.99), 0.052 (-0.03-0.15) and 0.019 (-0.01-0.06), respectively. The interval estimation of conditional correlation for both sensitivity and specificity clusters around zero and thus conditional dependence between the two tests was not significant. Although accuracy measure would not be the only basis for test selection, the result of our study demonstrated that IFAT has a reasonable high accuracy to be used for the diagnosis and sero-surveillance analysis of LSD in the target population.

Elimination of toxicity and enhanced detection of lumpy skin disease virus on cell culture from experimentally infected bovine semen samples.

Lumpy skin disease virus (LSDV), a poxvirus of the genus Capripoxvirus, is shed in the semen of infected bulls. The screening of semen for infectious virus requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the polymerase chain reaction (PCR) are sensitive diagnostic tests which may be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture detects infectious virus and is a sensitive method, there are major difficulties in using this method due to the toxic effect of semen on the cells. The aim of this study was to find a method that decreases the toxic effect of semen and enhances the isolation of LSDV on cell culture. Semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain V248/93, with a titre of 6.5 log TCID50. The semen samples were treated with one of four different methods: centrifugation, serial dilution, filtration and chemical treatment with kaolin. The samples subjected to centrifugation, serial dilution and filtration were supplemented with gentamycin. Semen toxicity on cell cultures was eliminated when supernatants of semen samples centrifuged at 2000 rpm for 1, 3 and 5 min and serially diluted were used to inoculate confluent monolayer bovine dermis cells. The toxicity recorded when the pellet fractions of semen samples centrifuged for 5 min at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. Filtration and kaolin treatment of semen samples did not remove the toxic effect.

The detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques.

Lumpy skin disease (LSD) is a disease of cattle, primarily in Africa and Madagascar and rarely in the Middle East. It is caused by a capripoxvirus that belongs to the family Poxviridae. The disease is of economic importance in endemic areas. Effective control of LSD requires accurate and rapid laboratory techniques to confirm a tentative clinical diagnosis. Comparative studies on different diagnostic tests used at different stages of the disease have not been done. The aim of this study was to compare several of these tests. Six seronegative bulls, between 11 and 20 months of age, were infected intravenously and kept in an insect-free facility. The course of the infection was monitored. During a 3-month period blood samples and skin biopsies were collected for virus isolation and polymerase chain reaction (PCR). Skin biopsies were also examined using transmission electron microscopy (TEM). The incubation period in infected animals varied from 4-5 days. The length of the viraemic period did not correlate with the severity of clinical disease. Viraemia was detected from 1-12 days using virus isolation and from 4-11 days using the PCR, which is longer than has previously been reported. Virus was isolated from skin biopsies until Day 39 post infection (p.i.) and PCR could demonstrate viral DNA until Day 92 p.i. Transmission electron microscopy of negatively stained skin biopsies detected LSD virus only in one of the four bulls that developed skin lesions until Day 33 p.i. The PCR was a fast and sensitive method to demonstrate viral DNA in blood and skin samples. It could detect viral nucleic acid in skin lesions 53 days longer than virus isolation. Virus isolation from blood and skin samples was sensitive and reliable, but as a single test it may be too time-consuming to use although this depends on how rapidly the diagnosis must be confirmed. In conclusion, this study showed the PCR to be superior in detecting LSD virus from blood and skin samples. However, virus isolation is still required when the infectivity of the LSD virus is to be determined.

Use of a polymerase chain reaction assay to detect bovine herpesvirus type 2 DNA in skin lesions from cattle suspected to have pseudo-lumpy skin disease.

Beef cattle from a herd in north Alabama were examined because of an outbreak of nonfatal skin disease characterized by discrete circumscribed areas of inflammation that developed on the skin from the neck to the hips. Areas of inflammation, which tended to be superficial, underwent necrosis and scabbed over. The scabs eventually dropped off leaving discrete, round, whitish, hairless lesions that were 1.2 to 2.5 cm diameter. Because clinical signs were consistent with those expected with pseudo-lumpy skin disease (PLSD) caused by bovine herpesvirus type 2 (BHV-2), samples from 16 representative animals were submitted for BHV-2 testing. All 16 animals were seropositive for BHV-2, but the virus could not be isolated from skin biopsy specimens or buffy coat samples. Results of a polymerase chain reaction assay incorporating primers designed to amplify 2 DNA sequences from BHV-2 were positive for 3 of the 10 cattle, suggesting that skin lesions in these cattle were a result of PLSD. Our findings suggest that PLSD may be more common and widespread in the United States than suggested by the frequency with which BHV-2 has been isolated from cattle with PLSD-like skin lesions.