Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA

<p>Abstract</p> <p>Background</p> <p>The epidemiology of <it>E. ruminantium </it>infection in extensively managed young animals is not adequately understood. Thus in this study, we monitored the onset (age at first infection) and kinetics of <it>E. ruminantium </it>infection and antibody response in extensively managed newborn lambs and kids at three sites in The Gambia.</p> <p>Methods</p> <p>We used a nested pCS20 PCR and MAP1-B ELISA in a longitudinal study to monitor the onset (age at first infection) and kinetics of <it>E. ruminantium </it>infection and antibody response respectively, in 77 newborn lambs and kids under a traditional husbandry system at three sites (Kerr Seringe, Keneba, Bansang) in The Gambia where heartwater is known to occur. The animals were monitored for field tick infestation and the comparative performance of the two assays in detecting <it>E. ruminantium </it>infection was also assessed.</p> <p>Results</p> <p>The infection rate detected by pCS20 PCR varied between 8.6% and 54.8% over the 162-day study period. Nineteen per cent of the animals in week 1 post-partum tested positive by pCS20 PCR with half of these infections (7/14) detected in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. The earliest detectable <it>A. variegatum </it>infestation in the animals occurred in week 16 after birth. Antibodies detected by MAP1-B ELISA also varied, between 11.5% and 90%. Although there is considerable evidence that this assay can detect false positives and due to this and other reasons serology is not a reliable predictor of infection at least for heartwater. In contrast to the pCS20 PCR, the serological assay detected the highest proportion of positive animals in week 1 with a gradual decline in seropositivity with increasing age. The pCS20 PCR detected higher <it>E. ruminantium </it>prevalence in the animals with increasing age and both the Spearman's rank test (<it>r</it>_{<it>s </it>}= -0.1512; P = 0.003) and <it>kappa </it>statistic (-0.091 to 0.223) showed a low degree of agreement between the two assays.</p> <p>Conclusion</p> <p>The use of pCS20 PCR supported by transmission studies and clinical data could provide more accurate information on heartwater epidemiology in endemic areas and single-occasion testing of an animal may not reveal its true infection status. The view is supported because both the vector and vertical transmission may play a vital role in the epidemiology of heartwater in young small ruminants; the age range of 4 and 12 weeks corresponds to the period of increased susceptibility to heartwater in traditionally managed small ruminants.</p

A Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Confers Full Protection against Rift Valley Fever Challenge in Sheep

Citation: Faburay, B., Wilson, W. C., Gaudreault, N. N., Davis, A. S., Shivanna, V., Bawa, B., . . . Richt, J. A. (2016). A Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Confers Full Protection against Rift Valley Fever Challenge in Sheep. Scientific Reports, 6, 12. doi:10.1038/srep27719Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease outbreaks in Africa and the Arabian Peninsula. The virus has great potential for transboundary spread due to the presence of competent vectors in non-endemic areas. There is currently no fully licensed vaccine suitable for use in livestock or humans outside endemic areas. Here we report the evaluation of the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins. In a previous study, the vaccine elicited strong virus neutralizing antibody responses in sheep and was DIVA (differentiating naturally infected from vaccinated animals) compatible. In the current efficacy study, a group of sheep (n=5) was vaccinated subcutaneously with the glycoprotein-based subunit vaccine candidate and then subjected to heterologous challenge with the virulent Kenya-128B-15 RVFV strain. The vaccine elicited high virus neutralizing antibody titers and conferred complete protection in all vaccinated sheep, as evidenced by prevention of viremia, fever and absence of RVFV-associated histopathological lesions. We conclude that the subunit vaccine platform represents a promising strategy for the prevention and control of RVFV infections in susceptible hosts

Phylogeny and S1 Gene Variation of Infectious Bronchitis Virus Detected in Broilers and Layers in Turkey

Citation: Yilmaz, H., Altan, E., Cizmecigil, U. Y., Gurel, A., Ozturk, G. Y., Bamac, O. E., . . . Turan, N. (2016). Phylogeny and S1 Gene Variation of Infectious Bronchitis Virus Detected in Broilers and Layers in Turkey. Avian Diseases, 60(3), 596-602. doi:10.1637/11346-120915-Reg.1The avian coronavirus infectious bronchitis virus (AvCoV-IBV) is recognized as an important global pathogen because new variants are a continuous threat to the poultry industry worldwide. This study investigates the genetic origin and diversity of AvCoV-IBV by analysis of the S1 sequence derived from 49 broiler flocks and 14 layer flocks in different regions of Turkey. AvCoV-IBV RNA was detected in 41 (83.6%) broiler flocks and nine (64.2%) of the layer flocks by TaqMan real-time RT-PCR. In addition, AvCoV-IBV RNA was detected in the tracheas 27/30 (90%), lungs 31/49 (62.2%), caecal tonsils 7/22 (31.8%), and kidneys 4/49 (8.1%) of broiler flocks examined. Pathologic lesions, hemorrhages, and mononuclear infiltrations were predominantly observed in tracheas and to a lesser extent in the lungs and a few in kidneys. A phylogenetic tree based on partial S1 sequences of the detected AvCoV-IBVs (including isolates) revealed that 1) viruses detected in five broiler flocks were similar to the IBV vaccines Ma5, H120, M41; 2) viruses detected in 24 broiler flocks were similar to those previously reported from Turkey and to Israel variant-2 strains; 3) viruses detected in seven layer flocks were different from those found in any of the broiler flocks but similar to viruses previously reported from Iran, India, and China (similar to Israel variant-1 and 4/91 serotypes); and 4) that the AVCoV-IBV, Israeli variant-2 strain, found to be circulating in Turkey appears to be undergoing molecular evolution. In conclusion, genetically different AvCoV-IBV strains, including vaccine-like strains, based on their partial S1 sequence, are circulating in broiler and layer chicken flocks in Turkey and the Israeli variant-2 strain is undergoing evolution. © 2016 American Association of Avian Pathologists

Molecular epidemiology of heartwater (Ehrlichia ruminantium infection) in The Gambia

Heartwater is caused by Ehrlichia ruminantium and transmitted by ticks of the genus Amblyomma. It occurs in sub-Saharan Africa and in the Caribbean and affects domestic ruminants. There is general lack of information on the epidemiology of the disease in The Gambia. Results of a countrywide serological survey (chapter 2) using MAP1-B ELISA showed the infection is widespread throughout the country and showed a gradient of increasing heartwater risk for susceptible livestock from the east to the west of The Gambia. In sheep, the highest seroprevalence was detected in the western part of the country, 88.1% in the Western Division and 62.1% in the Lower River Division. Similarly in goats, the highest seroprevalence was detected in the westerly part of the country, North Bank Division (59%) and Western Division (44.1%). Sheep and goats in the two easterly divisions (Central River and Upper River divisions) showed the lowest seroprevalence rates. In Chapter 3 the nested pCS20 PCR showed the highest detection performance for detection of E. ruminantium in Amblyomma variegatum ticks compared to the nested map1 PCR and reverse line blot hybridization. Application of the pCS20 PCR in a subsequent study of E. ruminantium tick infection rates in The Gambia showed infection rates ranged from 1.6 % to 15.1 % with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank) than sites in the easterly divisions (Central River and Upper River) of the country. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma ovis and Anaplasma marginale infections in The Gambia. Chapter 4 described longitudinal monitoring of E. ruminantium infection in extensively managed newborn lambs and kids using pCS20 PCR and MAP1-B ELISA. Infection rates detected by pCS20 PCR increased with increasing age. Half (7/14) of the newborn PCR-positive animals tested positive in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. In contrast, seropositivity detected by MAP1-B ELISA declined with increasing age suggesting the presence of maternal antibodies. It is concluded that both the vector tick and vertical transmission may play a vital role in the epidemiology of heartwater in young animals; and showed the age range of 4 and 12 weeks corresponds to the period of highest susceptibility to heartwater in traditionally managed small ruminants. The study of genetic diversity of E. ruminantium by restriction fragment profile analysis of map1 gene is described in chapter 5. The results showed the presence of multiple genotypes (at least 11) of the E. ruminantium in the three agroecological zones of The Gambia, Sudano-Guinean (SG), Western Sudano-Sahelian (WSS) and Eastern Sudano-Sahelian (ESS) with the greatest number of diversity detected in the WSS and SG zones. Chapter 6 described the evaluation of an inactivated vaccine, prepared from E. ruminantium (Gardel stock), and a live attenuated vaccine from E. ruminantium (Senegal stock) against heartwater in Sahelian sheep. A local stock of E. ruminantium (Kerr Seringe) was used as challenge material. Inactivated and live attenuated vaccines provided 43 % and 100% protection, respectively, against virulent needle challenge. In a subsequent field trial, the attenuated vaccine protected 75% of sheep against virulent tick challenge, which was fatal for all control sheep

Genetic diversity of Ehrlichia ruminantium in Amblyomma variegatum ticks and small ruminants in The Gambia determined by restriction fragment profile analysis

Understanding genetic diversity of Ehrlichia ruminantium in host and vector populations is an important prerequisite to controlling heartwater by vaccination in traditional livestock systems in sub-Saharan Africa. We carried out a study in two phases: (i) evaluating the usefulness of the PCR-RFLP assay based on the map1 coding sequence of E. ruminantium as a discriminatory tool to characterise genetic diversity, (ii) applying the technique to field samples from Amblyomma variegatum ticks and small ruminants to characterise genotypic diversity of the organism in three main agroecological zones of The Gambia, Sudano-Guinean (SG), Western Sudano-Sahelian (WSS) and Eastern Sudano-Sahelian (ESS). Restriction fragment length polymorphisms were observed among different strains of E. ruminantium supporting the usefulness of the PCR-RFLP technique for studying genetic diversity of the organism. Restriction enzyme map1 profile analysis indicated the presence in The Gambia of multiple genotypes (at least 11) of E. ruminantium with sites in the WSS and SG zones showing comparatively high number of diverse genotypes. Profiles similar to the Kerr Seringe genotype (DQ333230) showed the highest distribution frequency, being present at sites in all three agroecological zones, thereby making the strain a suitable candidate for further characterisation in cross-protection studies. An additional three genotypes showed relatively high distribution frequency and were present in all three zones making them equally important for isolation and subsequent characterisation. The study demonstrated the occurrence of mixed infections with E. ruminantium genotypes in ruminants and ticks

Molecular detection of Ehrlichia ruminantium infection in Amblyomma variegatum ticks in The Gambia

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3-15.1%) than in the easterly divisions (Central River and Upper River; range 1.6-7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes