Mertyn Malkinson

Introduction of West Nile virus in the Middle East by Migrating White Storks

West Nile virus (WNV) was isolated in a flock of 1,200 migrating white storks that landed in Eilat, a town in southern Israel, on August 26, 1998. Strong, hot westerly winds had forced the storks to fly under considerable physical stress before reaching the agricultural land surrounding the town. Most of the flock were fledglings, <1 year old, which had hatched in Europe. Thirteen dead or dying storks were collected 2 days after arrival and submitted to the laboratory for examination. Four WNV isolates were obtained from their brains. Out of 11 storks tested six days after arrival, three had WNV-neutralizing antibodies. Comparative analysis of full-length genomic sequences of a stork isolate and a 1999 flamingo isolate from the USA showed 28 nucleotide (nt) (0.25%) and 10 amino acid (0.3%) changes. Sequence analysis of the envelope gene of the stork isolate showed almost complete identity with isolates from Israeli domestic geese in 1998 and 1999 and from a nonmigrating, white-eyed gull in 1999. Since these storks were migrating southwards for the first time and had not flown over Israel, we assume that they had become infected with WNV at some point along their route of migration in Europe.

Phylogenetic relationships of West Nile viruses isolated from birds and horses in Israel from 1997 to 2001.

In November 1997, an outbreak of a neuroparalytic disease caused by West Nile (WN) virus was diagnosed in young goose flocks. Domestic geese were similarly affected in the late summer and fall of 1998, 1999, 2000 and 2001. WN viruses were also isolated from migratory and wild birds and horses in 1998–2001. A 1278 bp sequence of the envelope gene of 24 Israeli WN virus isolates was compared with those of seven isolates from Africa, Europe and New York. As a result, the Israeli isolates could then be grouped into two clusters. The 15 avian and three equine from 1997–2001 in the first cluster of viruses were shown to be identical to WN-NY99, while the second cluster comprised one goose isolate from 1998 and two goose and two pigeon isolates from 2000. These closely resembled the most recent Old World isolates, and indicate that at least two WN genotypes were co-circulating in the region during this time.

Comparison of serological and virological findings from subgroup J avian leukosis virus-infected neoplastic and non-neoplastic flocks in Israel

Blood samples from nine broiler breeder flocks comprising five flocks clinically affected with myeloid leukosis tumours (ML+) and four tumour-free flocks from the same commercial background (ML−) were compared for avian leukosis virus subgroup J (ALV-J) serum antibodies by enzyme-linked immunosorbent assay (ELISA), for antigenemia (group-specific antigen) by antigen-trapping ELISA and for viremia. Group-specific antigen was detected in the sera of 58.1% of ML+ birds and 46.4% of the ML− birds (P=not significant), while 45.5% of ML+ birds and 24.1% of the ML− birds had ALV-J antibodies (P=0.065). In inoculated cell culture, 64.1% of the ML+ sera were viremic compared with 16.7% of the ML− sera (P=0.001). Similar significant differences were found between the two groups of flocks when ALV-J viremia was detected by immunofluorescence using a monoclonal env antibody (P=0.004), and for proviral DNA by polymerase chain reaction using two different sets of env-gene primers, H5−H7 (P=0.001) and R5–F5 (P=0.001). Using the primer pair R5–F5 the product size was approximately 1 kbp, while some heterogeneity in size among isolates was discernable. Our results indicate that a combination of diagnostic tests should be adopted in routine examination of tumour material in order to rule out false-negative findings.

A Twelve-Month Study of West Nile Virus Antibodies in a Resident and a Migrant Species of Kestrels in Israel

Two species of kestrel, the common and lesser, were caught each month at three geographically defined locations in Israel over a 12-month period, and a total of 306 blood samples were examined for West Nile virus neutralizing antibodies. The prevalences and mean antibody titers were analyzed statistically by the multiple linear regression model and were shown to be significantly affected by two of the independent variables, location and age of the bird. The season had no overall effect on prevalence and titer but a comparison of the mean monthly titers revealed that April was highest and July and August the lowest statistically for the common kestrel which is a resident species. In contrast, the migrating lesser kestrel was caught only in the spring months and principally at the Jerusalem location, where eight out of 29 birds were seropositive. By comparing the serology of the non-migrating, common kestrel with the migrating, lesser kestrel, the effect of seasonality was evaluated in relation to their ecological patterns and yielded evidence for the entry in April of a small number of previously infected common kestrels into Israel. This serological approach based on continuous sampling over an extended period could be used to forecast in the coming years the timing and dispersion of West Nile virus in both Old and New Worlds if surveys are based on a limited number of informative (flag) species.

Kinetics of the appearance of Marek's disease virus DNA and antigens in the feathers of chickens

A comparison was made of the temporal appearance of six isolates of serotype 1 Mareks disease virus (MDV) in the feathers of specific pathogen-free (SPF) infected birds using three assays: agar gel precipitation (AGP), enzyme-linked immunosorbent assay (ELISA) and dot-blot DNA hybridisation. Isolate GA-5 served to standardise the in vivo pathogenicity assay, while the remaining five were recent isolates from Israel. Each isolate was inoculated into susceptible 4-day-old birds housed with an equal number of uninoculated birds. All six caused high mortality (80 to 100%) in the inoculated birds and a wide range of mortality (15 to 80%) in the contact groups. The transmission of infection from the inoculated group to the contact group was demonstrated for all six isolates by AGP and ELISA and for four isolates by dot-blot hybridisation. The other two isolates either showed a concurrent rise in MDV-DNA levels (isolate B) or failed to produce detectable levels of DNA in the inoculated and contact infected groups (isolate Ab). This could be due to the nature of the hybridisation reaction between the probe and the homologous sequence in the genome of isolate Ab. Antigenic activity was detected 11 days post-injection by ELISA, 14 days by AGP in some of the inoculated groups. In the contact infected birds the ELISA and dot-blot assays detected virus about 14 days earlier than did AGP. The time interval between the first detection of virus in the inoculated as compared with the contact infected groups differed for each assay and each isolate, viz; 10 to 14 days by ELISA, 14 to 24 days by AGP and 11 to 18 days by DNA-hybridisation.

Diagnosis of turkey meningoencephalitis virus infection in field cases by RT-PCR compared to virus isolation in embryonated eggs and suckling mice

Turkey meningoencephalitis (TME) is a paralytic epornitic disease of turkeys caused by turkey meningoencephalitis virus (TMEV), an arthopod-borne flavivirus belonging to the Ntaya serogroup VI. A TMEV specific RT-PCR was compared with classical techniques for TMEV diagnosis, which include virus isolation in 8-day-old chicken embryonated eggs and suckling mice, on 17 TME flocks with neurological signs that occurred during the fall of 1997. In 11/17 flocks both the RT-PCR and the virus isolation methods detected virus, in 4/17 flocks a negative diagnosis was obtained by both methods, and two flocks were positive by RT-PCR only. In four flocks RT-PCR only detected virus after inoculation into embryonated eggs or suckling mice. There was a dose response effect in the yield of the RT-PCR product. Direct examination of turkey brains yielded bands of low to medium intensity. Use of RT-PCR after embryo and/or mouse inoculation resulted in products of far greater intensity. Thus, RT-PCR can be successfully used to amplify TMEV RNA in the brains of diseased turkeys but a negative result would require egg and mouse inoculation for enrichment of virus prior to RT-PCR.

Replication of Marek's disease virus in chicken feather tips containing vaccinal turkey herpesvirus DNA.

The presence of herpesvirus of turkeys (HVT) DNA in the feather tips of chickens vaccinated with HVT was assessed by dot blot hybridisation with a probe specific for HVT and lacking homology to MDV DNA. Only small amounts of HVT DNA were detected in the feather tips of chickens that were vaccinated or left in contact with HVT vaccinated chickens. However when chickens were challenged with virulent MDV, HVT DNA was detected in the feather tips of vaccinated chickens and the largest amount was detected 35 days after vaccination. HVT DNA was recovered in significantly higher quantities from some of the MDV-infected chickens than from those infected by contact. This suggests that MDV infection may provide helper functions for HVT. MDV DNA was identified in the feather tips of MDV-challenged chickens from 25 to 45 days after challenge. Thus, immunisation of chickens with HVT did not prevent the replication of MDV in the feather tips but only diminished it.

Langerhans Cells in the Skin of Normal and Lumpy Skin Disease Virus-Infected Cattle

Lumpy skin disease (LSD) is a dermotropic virus infection of cattle caused by a member of the capripox virus family. It presents as dermal thickenings 0.5–5.0 mm in diameter that involve all the skin layers. In addition the regional lymph nodes, mucous membranes and lungs are invariably affected (1). It was reported that during the acute stage of the disease, necrotic areas of the lesions are infiltrated by neutrophils, macrophages and occasionally, eosinophils. As the lesions progress these cells are gradually replaced by round cells (lymphoblasts, lymphocytes, plasma cells and macrophages) and by fibroblasts, while the keratinocytes are swollen and eosinophilic inclusions are present in different cell types (1). The histopathological description is reminiscent of an earlier one concerning sheeppox in which considerable dermal oedema and the relatively early appearance of large numbers of cells of unique appearance, termed “cellules claveleuses” were described (2). These cells show rounded or oval nuclei and cytoplasm of increased basophilia. Their cytoplasmic boundaries are ill-defined but there are often long, irregular processes giving the cell a somewhat stellate appearance (2). From the description of this unique lesion in sheeppox one might conclude that dendritic or even Langerhans cells (LC) were actively involved in its histopathogenesis.