Comparison of the efficacy of Neethling lumpy skin disease virus and x10RM65 sheep-pox live attenuated vaccines for the prevention of lumpy skin disease - The results of a randomized controlled field study.

Lumpy skin disease (LSD) is a viral disease of cattle and buffalo, caused by a Capripox virus. A field study was performed during an LSD epidemic which occurred in 2012-2013 in Israel, in order to assess the efficacy of two commercial vaccines for protection against LSD. Fifteen dairy herds, vaccinated 2-5 months prior to study onset with a single dose of 10(2.5) TCID50 of RM65 attenuated sheep-pox vaccine, and not affected previously, were enrolled in the study. 4694 cows were randomized to be either vaccinated with a 10(3.5) TCID50/dose of RM65 vaccine (x10RM65) or with a same dose of an attenuated Neethling LSD virus vaccine. A case of LSD was defined as the appearance of at least 5 lesions typical to LSD and a severe case was defined if this sign was accompanied by either fever (>39.5°C) or/and a 20% reduction in milk production. Deep lesion biopsies and blood samples were collected from 64.5% of the cases in an attempt to detect DNA of LSD virus by PCR and to differentiate between the wild strain and the vaccine Neethling strain. Seventy-six cows were affected by LSD in 8 herds with an incidence of 0.3-5.7%. Mantel-Haenszel relative risk (RRMH) for LSD morbidity at least 15 days after vaccination in x10RM65 vs. Neethling was 2.635 (CI95%=1.44-4.82) and 11.2 (2.3-54.7) for severe morbidity. RRMH for laboratory confirmed cases was 4.28 (1.59-11.53). An incidence of 0.38% (9/2356) of Neethling associated disease was observed among Neethling vaccinated cows while no such disease occurred in x10RM65 vaccinated cows. We conclude that the Neethling vaccine is significantly more effective than x10RM65 in preventing LSD morbidity, though it might cause a low incidence of Neethling associated disease. No transmission of the Neethling strain to non-Neethling vaccinated cows was observed in this study.

Appearance of skin lesions in cattle populations vaccinated against lumpy skin disease: statutory challenge.

The ultimate goal of a vaccine is to protect vaccinated animals against re-exposure to the same pathogen and provide sterile immunity. However, a cutaneous clinical manifestation appeared, following re-exposure of cattle that had been vaccinated with the RM65 strain, to LSDV infection during an epidemic in 2006-2007. Four thousand six hundred and seven vaccinated cows entered the study after being re-exposed to LSDV infection. Of them, 513 (11%) presented lumps, and there was a marked difference between the proportions of dairy and feedlot animals that were affected: 146 out of 3517 and 367 out of 1090 (6.6 and 33.7%, respectively). This data suggests that the potency of the vaccine need to be re-assessed for beef cattle.

Sample-based assessment of the microbial etiology of bovine necrotic vulvovaginitis.

A semiquantitative evaluation of potential bacterial pathogens was correlated to the severity of lesions during an outbreak of bovine necrotic vulvovaginitis (BNVV) on an Israeli dairy herd. Bacteriologic examination of 287 vaginal swabs from 104 post-calving heifers showed a highly significant correlation between Porphyromonas levii colony forming unit numbers and the clinical scores of the lesions, when assessed by an ordinal regression statistical model. No such correlation was found for the other bacteria included in the study. Nineteen samples taken for virological examinations resulted negative for bovine herpes viruses 1, 2, 4 and 5. Thus the results of this study substantiate the essential role of P. levii in the etiology of BNVV and indicate that BHV4 is not required as a predisposing factor to the syndrome.

Serological and clinical evidence of a teratogenic Simbu serogroup virus infection of cattle in Israel, 2001-2003.

During the last 35 years, two major outbreaks of Akabane virus (AKAV) infection were recorded in cattle in Israel in 1969/1970 and 2002/2003. Congenital malformations of calves characterised by the appearance of an arthrogryposis and hydranencephaly syndrome first appeared in Israel in 1969. Based on epidemiological, clinical, pathological, histopathological and serological data, this syndrome was strongly correlated with seroreactivity to AKAV, a member of the Bunyaviridae, Simbu serogroup. In February 2002, the first cases of 'blind newborn calves' (BNC) were observed on farms located in the northern valleys of Israel. Microtitre serum neutralisation (SN) tests of serum from malformed calves and their dams were conducted using Akabane and Aino viruses (AINOV). The first SN test was performed at the reference laboratory of the Clinical Virology Section, Kyushu Research Station, National Institute of Animal Health, Kagoshima, Japan. The clear-cut findings of seroreactivity to AKAV by cattle located in the affected zone, in contrast to negative findings in cattle from unaffected farms (87% and 3.7%, respectively) was indicative of AKAV infection. In contrast, seroreactivity to Aino virus was relatively low in both affected and non-affected areas during the 2002 outbreak. In order to establish Israeli laboratory standards for Simbu serogroup diagnosis, 57 serum samples tested by the Japanese laboratory were retested by SN in Israel. An almost complete homology (96.5%) was found between the two SN panels of sera (kappa = 0.92). SN and ELISA kits enabled the surveillance of this arbovirus epidemic in the second consecutive year (2003). Moreover, AKAV was identified in trapped midges by hemi-nested PCR and real-time PCR. With these techniques, the geographical limits of the BNC epidemic that appeared in some areas of Israel was identified for the first time and was recorded in the Arava Rift Valley, 400 km south of the epicentre of the 2002 outbreak. The reintroduction of AKAV into this region, together with some evidence of AINOV activity and epidemics of bluetongue (BT) in the southern parts of Europe and the eastern Mediterranean, and renewed outbreaks of West Nile virus infection in Israel, Italy and southern France, are all evidence of the potential spread of arbovirus activity into southern Europe from the Mediterranean Basin.

Rabies virus detection by RT-PCR in decomposed naturally infected brains.

The warm climate of Israel and mishandling of the cadavers during transit to the laboratory requires an accurate method for diagnosis of rabies in decomposed tissues. By using the reverse transcriptase polymerase chain reaction (RT-PCR) 10 decomposed brain samples that collected between 1998 and 2000 were diagnosed as negative by direct fluorescent antibody test (FAT), were found positive. Three of the 10 decomposed brains were confirmed as positive by isolation of rabies virus in tissue culture and by mouse inoculation (MIT) while the other seven decomposed samples were found positive only by RT-PCR. Direct sequencing and molecular analysis of a 328bp fragment of the N gene of all the rabies sequences confirmed their geographical origin. These results demonstrated the importance of the RT-PCR in the detection of rabies virus in decomposed naturally infected brains, especially in cases when the sample is not suitable for other laboratory assays. Thus, the RT-PCR can provide a positive diagnosis; however, when a negative result is obtained due to the nature of the decomposed tissue that can be caused by technical reasons and a false negative might be the case.

Gene gun-mediate DNA vaccination against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is one of the most dangerous diseases of cloven-hoofed animals and is a constant threat in the Middle-East and other regions throughout the world despite intensive vaccination programs. In this work, we describe the ability of FMDV expression constructs to protect pigs from FMDV challenge when used as a vaccine. The construct consists of encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), the entire P1 and 2A together with 3CD sequences, all in the same reading frame. Another plasmid that was tested, carries the serotype O1 (G) VP1, Asia1 VP1 and O1 (G) 3C. Between each of the genes the 3C cleavage sequences were inserted. All constructs carried the cytomegalo virus (CMV) promoter. Using immunofluorescent and immunoblot techniques, we could show the expression and processing of viral proteins. Following the application of FMDV expression constructs into pigs skin by 'Gene Gun', pigs were partially protected from FMDV challenge.

Functional importance of bovine myometrial and vascular LH receptors and cervical FSH receptors.

Bovine myometrium and cervix contain luteinizing hormone/human chorionic gonadotropin (LH/hCG) binding sites, LH receptor (LH-R) messenger RNA (mRNA), and LH-R protein. Expression of LH-R is dependent on the stage of the cycle. LH-R expression is high during the luteal phase but weak during the follicular phase. In both myometrium and cervix, LH activates both the adenylate cyclase and phospholipase C pathways, and the effect of LH on both pathways at each stage of the cycle is correlated with the amount of LH-R present in the tissue. Because activation of cyclic AMP (cAMP) is associated with myometrial quiescence, we suggest that LH activation of uterine cAMP could serve to keep the uterus quiescent during the luteal phase. On the other hand, in the uterine vein LH-R mRNA and LH-R are maximal during preestrus/estrus as opposed to the luteal phase. In the uterine vein, LH increases the expression of cyclooxygenase and production of both prostaglandin E2 (PGE2) and PGF2 alpha. Because PGF2 alpha is the physiological luteolytic signal in the cow, we suggest that this increase in prostaglandin production by the uterine vein is part of the physiological events leading to luteolysis. In addition to uterine LH-R, the bovine cervix at preestrus/estrus has high levels of follicle-stimulating hormone receptor (FSH-R) and its corresponding mRNA. As with LH-R, activation of FSH-R by FSH is associated with activation of a G protein-coupled receptor family that mediates the cAMP and inositol phosphate signaling pathways. Activation of these signaling pathways is associated with an increase in the expression of cyclooxygenase and production of PGE2. Because expression of the FSH receptor was maximal at the time of the FSH peak in the blood, we suggest a physiological role for FSH in the cervix relaxation and opening at estrus.

Gene integration into bovine sperm genome and its expression in transgenic offspring.

Transgenic bovine sperm were produced by restriction enzyme mediated insertion (REMI). REMI utilizes lipofection of linearized pEGFP and the corresponding restriction enzyme for integration into the sperm genomic DNA. The transgenic sperm were used in IVF to produce morula expressing GFP. When transgenic sperm were used for AI in two cows, the resultant calves expressed the exogenous DNA in their lymphocytes as determined by (a) PCR and RT-PCR; (b) specific emission of green fluorescence by GFP; and (c) Southern blot analysis. Data demonstrate that REMI is an efficient method for the production of transgenic sperm and corresponding offspring by AI or embryos by IVF.

Molecular epidemiology of rabies virus isolates from Israel and other middle- and Near-Eastern countries.

A total of 226 isolates of rabies virus from different areas of Israel, including three human isolates and one sample from South Lebanon were identified between 1993 and 1998 by direct immunofluorescence using monoclonal antibodies to the viral nucleoprotein (N). An epidemiological survey based on nucleotide sequence analysis of 328 bp from the C terminus of the N coding region and the noncoding region between the nucleoprotein and the phosphoprotein (NS gene) was performed. Phylogenetic analysis of the isolates from Israel showed that they were related geographically, but not according to host species. Five variants, related groups distributed among four geographical regions, were identified. In each region, rabies virus was isolated from more than one animal species. A comparison of the sequence analysis of rabies virus samples from the rest of world revealed a 2-nucleotide change that distinguished the Middle East variants from the rest.

Molecular characterization of an unassigned Israeli Newcastle disease virus isolate.

Detection of Newcastle disease virus (NDV) and avian pathotyping of NDV isolates are extremely important because the appearance of virulent virus has significant economic consequences in terms of vaccination, eradication, and the ability to export poultry products. By using nucleotide and amino acid (aa) homology analysis, we could demonstrate that a NDV broiler isolate is a velogenic virus. This analysis was done after mean death time and intracerebral pathogenicity index tests gave inconsistent results. By establishing a nucleotide sequence dendrogram, we found that the disputed Ber-Tuvia was clearly in the same group as the known Herev-Laet, a velogenic isolate. The difference between Ber-Tuvia 92 and the Herev-Laet velogenic isolate was 6% as opposed to > 16% of the meso- and lentogenic isolates. The Ber-Tuvia isolate contains the Arg/Arg and Lys/Arg aa at positions 112, 113 and 115, 116, respectively, in the fusion protein cleavage aa sequence, which is typical for virulent NDV isolates.

Expression of functional luteinizing hormone (LH) receptor and its messenger ribonucleic acid in bovine uterine veins: LH induction of cyclooxygenase and augmentation of prostaglandin production in bovine uterine veins.

We have previously reported that bovine endometrium contains LH/human CG binding receptors and LH induces cyclooxygenase and prostaglandin production in the bovine endometrium. The present study investigated 1) whether bovine uterine vein and artery contain LH receptor messenger RNA (mRNA) and receptor protein and 2) whether LH can regulate the formation of vasoactive eicosanoids by the uterine vein. The uterine vein endothelium, but not the uterine artery, contained LH receptor mRNA transcript essentially identical to that found in the bovine corpus luteum. The uterine vein endothelium also contained a 95-kDa immunoreactive receptor protein that bound to rat anti-LH receptor antibody in Western blots. The LH receptor mRNA and LH receptor were maximally expressed in the uterine vein from cows in proestrus/estrus compared with cows in luteal or postovulatory phases. Incubation of endothelial minces of uterine vein with LH resulted in a 2-fold increase in cyclooxygenase concentration as determined by Western blot using an antibody to ram seminal vesicle cyclooxygenase. The increase in cyclooxygenase was maximal in cows in proestrus/estrus compared with postovulatory and luteal phase cows. Incubation of proestrous/estrous uterine vein or artery minces with LH or mellitin (a phospholipase A2 stimulator) caused increased production of eicosanoids. In the uterine vein, LH caused a significant increase in both PGF2alpha (basal 4.1 +/- 0.4 vs. 5.7 +/- 0.4 ng/100 mg x 6 h, P < 0.01; N = 9 cows) and PGE2 (basal 5.7 +/- 0.3 vs. 7.7 +/- 0.8 ng/100 mg x 6 h, P < 0.01; N = 6 cows) but had no effect on prostaglandin production by the artery. Mellitin increased PGF2alpha production by both uterine vein and artery minces but had no effect on PGE2 production in either tissue. Addition of steroids (progesterone, estradiol) or cytokines (tumor necrosis factor-alpha, IL-6) to the uterine vascular tissues had essentially no effect on prostanoid production. In summary, bovine uterine vein from proestrous/estrous cows expressed the LH receptor and its mRNA. Expression of the receptor may have physiological significance as LH induces cyclooxygenase and increases prostaglandin release in the uterine vein. The maximal stimulation of the receptor and its mRNA at proestrus/ estrus may serve to increase the amounts of prostanoids reaching the regressing corpus luteum either directly by increasing prostanoid production or indirectly by increasing the blood flow to the ovary.

A ribozyme targeted to cleave the polymerase gene sequences of different foot-and-mouth disease virus (FMDV) serotypes.

Vaccinations against foot-and-mouth disease virus (FMDV) has dramatically reduced the number of disease outbreaks. Nevertheless, there are still many outbreaks in different regions around the world. In an effort to find new ways to control the disease, ribozymes able to cleave FMDV were designed and tested. In this work we tested the ability of FRZ4, a ribozyme targeted to the viral polymerase gene, to cleave polymerase sequences of several FMDV. Homology analysis was used to choose target sequences which consist of two conserved GUC which lie 15 bases apart and, their flanking sequences. These were the basis for the FRZ4 ribozyme gene sequence that contains two catalytic domains. We show that polymerase sequences from A, Asia 1, C and two different O1 Israeli isolates could be specifically cleaved by FRZ4. It is suggested that FRZ4 can cleave polymerase gene sequences from any FMDV serotype.

Cleavage of transcripts of foot and mouth disease virus (FMDV), Asia1 serotype, by ribozymes targeted to the VP3 and VP4 genes.

Two ribozyme genes were designed to cut within the VP4 and VP3 sequences of foot and mouth disease virus (FMDV) Asia1 serotype genome. The two genes were synthesized and cloned into pBluescript under the control of the T3 promoter. The ribozyme designed to cut the VP4 gene contained two catalytic sequences targeted to two GUC triplets that are 16 bases apart. The second ribozyme, intended to cut VP3, contained one catalytic sequence. Ribozymes obtained from run-off transcription from both plasmids were able to cleave viral RNA derived from runoff transcripts of plasmids carrying the proper FMDV cDNA inserts. The significance of these findings is discussed.

Detection and subtyping of foot-and-mouth disease virus in infected cattle by polymerase chain reaction and amplified VP1 sequencing.

Fast and accurate detection of foot-and-mouth disease (FMD) outbreaks is needed to limit spread of the disease by proper vaccination. The use of the polymerase chain reaction (PCR) has revolutionized the way in which viral diseases are diagnosed. Sequence analysis of the amplified VP1 sequence can enable the classification of FMD virus detected in the morbid animal. PCR assays were carried out to identify the virus and its serotype in suspect animals from 2 outbreaks of FMD type O virus. Sequence analysis of the amplified VP1 cDNA showed 78% homology with O1K and over 95% homology between the samples. These findings suggest that the 2 outbreaks were due to infection with the same virus serosubtype.

Molecular epidemiology of foot-and-mouth disease (FMD) in Israel in 1994 and in other Middle-Eastern countries in the years 1992-1994.

The reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing were employed in the diagnosis and typing of foot-and-mouth disease virus (FMDV) in samples taken during the 1994 disease outbreak in Israel. Using PCR, virus isolation and serological methods it was shown that the 1994 disease outbreak in Israel and other Middle-Eastern countries was caused by O1 type virus. Direct PCR sequencing of VP1 genes and homology analysis of the virus isolates revealed that there were two distinct outbreaks in Israel. The first originated in Jordan, moved to the West Bank territory and then to the Lower Galilee. The second outbreak, caused by another virus, was responsible for disease outbreaks in South Lebanon, Upper Galilee and the Golan Heights. When viral sequences of isolates from the 1993 outbreaks in Egypt and Lebanon were included in the analysis, they showed a high degree of VP1 sequence homology between themselves, suggesting a common origin.

Foot and mouth disease virus replication in bovine skin Langerhans cells under in vitro conditions detected by RT-PCR.

The replication of foot and mouth disease virus (FMDV) was studied in isolated bovine skin Langerhans cells (LC), in keratinocytes from epidermal cell suspension, and in migrating LC obtained from cultured bovine epidermal sheets in vitro. Viral RNA replication in infected cells was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR) of the negative FMDV RNA strand and by the plaque forming assay of FMDV. It was established that bovine skin LC, keratinocytes, and migratory bovine LC infected with FMDV strain 01 Geshur supported virus replication. This RT-PCR method to detect the negative strand of FMDV RNA in migratory bovine skin LC may be useful for determining FMD virus replication in tissue cells.