Anti-HEV seroprevalence and rate of viremia in a German cohort of dogs, cats, and horses.

Hepatitis E virus (HEV) genotype 3 infections in Germany are mainly transmitted zoonotically through the consumption of swine meat. Furthermore, there is evidence that pets might come into contact with HEV, but the relevance of companion animals as possible sources of HEV transmission in Germany still needs to be defined. A monitoring study was therefore carried out on dogs, cats, and horses from Germany. In total 365 serum samples from pets (124 dogs, 119 cats, and 122 horses) were tested for HEV by PCR and for anti-HEV antibodies by a commercial ELISA. The HEV seroprevalence determined by the sero-assay varied significantly between dogs (10%), cats (6%), and horses (2%). Liver injury-related enzymes, alanine transaminase (ALT), and aspartate transaminase (AST) showed no differences between HEV-positive or negative animals. None of the pet serum samples tested positive for PCR. This serological study suggests that dogs and cats are significantly exposed to HEV in Germany, while horses are of minor relevance.

Ethanol inactivation of orthonairoviruses in ixodid ticks.

Ixodid ticks represent vectors and reservoirs for a broad range of zoonotic pathogens. Collected ticks from field studies are therefore usually stored in ethanol, which in higher concentrations effectively inactivates most of the known tick-borne pathogens. Although commonly practiced as gold standard for inactivation, hardly any scientific data demonstrate that ethanol sufficiently penetrates the comparatively thick cuticula of ticks. Therefore, Amblyomma hebraeum tick pools were stored for 21 days in ethanol (96%). Afterwards, the ethanol was removed and the ticks were homogenized. Quantitative 1H-NMR spectroscopic analysis was applied to determine the residual concentration of ethanol inside the ticks. 1H-NMR spectroscopic analysis revealed that ethanol constituted 28.3-42.6 mg of the total weight of three ticks in the pools (89.9-121.5 mg). In addition, the low-pathogenic Hazara orthonairovirus (HAZV) was used as a cell culture model for this study. The virus was exposed to ethanol concentrations between 0 and 60% and incubated under various temperature conditions for four time periods. Afterwards, the residual virus infectivity was determined by titration. Following ethanol exposure, HAZV did not grow in cells after 9 h of exposure to an ethanol concentration of 25%. These results demonstrate an extremely low ethanol resistance of the virus, which was generally in line with previously reported ethanol inactivation data for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). After prolonged storage and impregnation, comparable ethanol concentrations are achieved in the ticks, indicating the suitability of this inactivation method also for Bunyaviruses in ticks. At the very least, a massive virus inactivation can be assumed. Definitive proof of virus inactivation would require a bioassay of ethanol-treated infected ticks under appropriate biosafety conditions.

Detection of Crimean-Congo hemorrhagic fever virus in blood-fed Hyalomma ticks collected from Mauritanian livestock.

BACKGROUND: Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Orthonairovirus (Nairovididae) and is a (re)emerging tick-borne pathogen. It is endemic in most parts of Africa, Asia and southern Europe, and can cause severe hemorrhagic symptoms in humans, with high fatality rates (5-30%). METHODS: Hyalomma ticks were collected from four different livestock herds (cattle and camels) in Mauritania in 2018. The tick species were determined morphologically and confirmed molecularly by using the cytochrome oxidase 1 gene marker. For the detection of CCHFV, ticks were tested individually by one-step multiplex real-time reverse-transcriptase quantitative polymerase chain reaction. The small segment of all positive samples was sequenced to determine the CCHFV genotype. RESULTS: In total, 39 of the 1523 ticks (2.56%) collected from 63 cattles and 28 camels tested positive for CCHFV. Three Hyalomma species were identified. Hyalomma rufipes had the largest proportion of positivity (5.67%; 16/282), followed by Hyalomma dromedarii (1.89%; 23/1214). No Hyalomma impeltatum tested positive (0%; 0/21). Positive ticks were found in only six out of 91 host animals. Viral sequence analysis revealed the presence of two different CCHFV lineages (Africa I and Africa III). CONCLUSIONS: In this study, 2.56% of Hyalomma ticks collected from camels and cattle in Mauritania tested positive for CCHFV. However, the true prevalence of CCHFV in unfed ticks may be lower, as a considerable number of ticks may have been passively infected during blood-feeding by co-feeding ticks or due to viremia of the host. The results indicate the need to track the actual area of circulation of this virus.

Rift Valley fever virus detection in susceptible hosts with special emphasis in insects.

Rift Valley fever phlebovirus (RVFV, Phenuiviridae) is an emerging arbovirus that can cause potentially fatal disease in many host species including ruminants and humans. Thus, tools to detect this pathogen within tissue samples from routine diagnostic investigations or for research purposes are of major interest. This study compares the immunohistological usefulness of several mono- and polyclonal antibodies against RVFV epitopes in tissue samples derived from natural hosts of epidemiologic importance (sheep), potentially virus transmitting insect species (Culex quinquefasciatus, Aedes aegypti) as well as scientific infection models (mouse, Drosophila melanogaster, C6/36 cell pellet). While the nucleoprotein was the epitope most prominently detected in mammal and mosquito tissue samples, fruit fly tissues showed expression of glycoproteins only. Antibodies against non-structural proteins exhibited single cell reactions in salivary glands of mosquitoes and the C6/36 cell pellet. However, as single antibodies exhibited a cross reactivity of varying degree in non-infected specimens, a careful interpretation of positive reactions and consideration of adequate controls remains of critical importance. The results suggest that primary antibodies directed against viral nucleoproteins and glycoproteins can facilitate RVFV detection in mammals and insects, respectively, and therefore will allow RVFV detection for diagnostic and research purposes.

A possible case of renal oxalate deposit reported in an African fruit bat (Epomops franqueti).

We report a possible spontaneous case of oxalate nephrosis in an African fruit bat (Epomops franqueti), incidentally observed in Ibadan, South-West Nigeria, in an anatomical and serological survey of the species. Wild caught bats underwent sedation, intracardial perfusion, necropsy and histopathology. All 15 wild-caught African fruit bats were apparently healthy. However, light microscopy revealed mild oligofocal tubulonephrosis with intraluminal deposition of polarizing crystals interpreted as subclinical oxalate nephrosis in one case. In summary, we suggest a dietary aetiology, based on seasonal availability of high ascorbic acid or oxalate containing fruits. However, exposure to anthropogenic contaminants cannot be completely ruled out.

Molecular discrimination of Hyalomma tick species serving as reservoirs and vectors for Crimean-Congo hemorrhagic fever virus in sub-Saharan Africa.

The species identification of tick vectors of Crimean-Congo hemorrhagic fever virus (CCHFV), especially Hyalomma (H.) species, is a prerequisite to understand the eco-epidemiology of this disease and to reveal vector and virus reservoir species. However, the morphologic species discrimination can be difficult for damaged or blood-fed ticks and in case of species intercrosses. Therefore, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and restriction fragment length polymorphism (RFLP) analysis to distinguish the most common Hyalomma species from sub-Saharan Africa (H. truncatum, H. rufipes and H. dromedarii). Within the last years, MALDI-TOF MS analysis based on tick leg proteins has been shown to be a reliable method to distinguish several tick species. For this purpose, a reference spectral library of several European, American and African tick species was established. In this study, six different Hyalomma species were tested, all of which were all clearly distinguishable by mass spectrometric analyses. Moreover, MALDI TOF- MS was able to confirm morphologic findings where sequencing provided ambiguous results. In addition, a polymerase chain reaction (PCR) based on the CO1 gene amplification of ticks has been developed for the unequivocal species identification by amplicon sequencing and specific restriction endonuclease cleavage pattern analysis. RFLP proved to be a feasible auxiliary discrimination tool for selected Hyalomma species when access to sequencing methods is not available, as for instance during field studies.

Seroprevalence and Associated Risk Factors of Rift Valley Fever in Domestic Small Ruminants in the North Region of Cameroon.

Rift Valley fever (RVF) is a zoonotic vector borne infectious disease of major medical and veterinary importance particularly in sub-Saharan Africa. However, there is dearth of epidemiological knowledge of the disease in Cameroon. We conducted a cross-sectional study (January 2016-January 2017) to investigate the seroprevalence and potential risk factors of Rift Valley fever virus (RVFV) in sheep and goats in the North region of Cameroon. Stratified sampling approach was used to select herds where sera were collected from 680 randomly selected small ruminants (355 goats and 325 sheep) in eight localities (Kismatari, Lagdo, Pitoa, Garoua, Bocklé, Dembo, Poli and Touboro) within three administrative divisions (Bénoué, Mayo-Rey and Faro) in the North region. Anti-RVFV antibodies were detected using a competitive Enzyme-Linked Immunosorbent Assay (ELISA), while a capture ELISA was used for the detection of specific RVFV-Immunoglobulin M (Ig-M) antibodies. We evaluated the associated potential risk factors of RVF in small ruminants based on observations of animal-related intrinsic and extrinsic factors in combination with serological results. The results revealed that 3.4% (95% confidence interval (CI): 2.2-5.1%) of sampled animals and 24.6% (95% CI: 15.1-37.1%) of 65 sampled herds were seropositive for anti-RVFV antibodies and no difference in seropositivity between sheep and goats at individual animal as well as at herd levels was observed. Localities along hydrographic or large water banks such as Kismatari (OR: 14.333, (95% CI: 1.436-145.088)) and Pitoa (OR = 11.467 (95% CI: 1.249-50.306)) were significantly associated to RVFV antibody seroprevalence in a simple logistic regression. In addition, the multiple regression analysis showed that age and access to water points significantly influenced RVFV antibody seroprevalence in small ruminants. This study revealed that anti-RVFV antibodies are present in sheep and goats in the North region of Cameroon. It highlights the likely endemic circulation of RVFV in the considered localities despite the absence of clinical cases reported in animals or humans. Under these conditions, it is necessary to set up an early warning, surveillance and control strategy based on epizootic risk.

Molecular and serological surveillance of Hepatitis E virus in wild and domestic carnivores in Brandenburg, Germany.

Hepatitis E virus (HEV) is a zoonotic virus which circulates in pigs and wild boars as main reservoir species. To reveal the infection rate in carnivores, we have carried out a monitoring study of raccoons, raccoon dogs, dogs and cats sampled in Brandenburg, Germany. In summary, 53.8% (43 of 80) of the raccoons, 34.3% (25 of 73) of the raccoon dogs, 56.6% (47 of 83) of dogs and 32.3% (21 of 65) of cats were tested positive for HEV-specific antibodies. No viral RNA could be detected. This first description of anti-HEV antibodies in raccoons and raccoon dogs worldwide and in dogs and cats in Germany highlights the natural host range expansion of HEV.

Protecting effect of PrP codons M142 and K222 in goats orally challenged with bovine spongiform encephalopathy prions.

Breeding towards genetic resistance to prion disease is effective in eliminating scrapie. In sheep, classical forms of scrapie have been eradicated almost completely in several countries by breeding programs using a prion protein (PrP) gene (PRNP) amino acid polymorphism. For goats, field and experimental studies have provided evidence for several amino acid polymorphisms that are associated with resistance to scrapie, but only limited data are available concerning the susceptibility of caprine PRNP genotypes to BSE. In this study, goat kids representing five PRNP genotypes based on three polymorphisms (M142, Q211 and K222 and the wild type I142, R211 and Q222) were orally challenged with bovine or goat BSE. Wild type goats were killed with clinical signs between 24-28 months post inoculation (mpi) to both challenges, and goats with genotype R/Q211 succumbed between 29-36 mpi. I/M142 goats developed clinical signs at 44-45 mpi and M/M142 goats remained healthy until euthanasia at 48 mpi. None of the Q/K222 goats showed definite clinical signs. Taken together the highest attack ratios were seen in wild type and R/Q211 goats, and the lowest in I/M142, M/M142 and Q/K222. In all genotype groups, one or more goats remained healthy within the incubation period in both challenges and without detectable PrP deposition in the tissues. Our data show that both the K222 and M142 polymorphisms lengthen the incubation period significantly compared to wild type animals, but only K222 was associated with a significant increase in resistance to BSE infection after oral exposure to both BSE sources.

Evidence for enzootic circulation of Rift Valley fever virus among livestock in Cameroon.

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen, causing serious epidemics in Africa and the Arabian Peninsula. In Cameroon serological data indicate the presence of RVFV, but active circulation of RVFV, causing clinical infections has not been proven yet. For this purpose we carried out a serological and molecular study on a total of 1953 randomly selected serum samples of small ruminants and cattle, which were collected in years 2013 and 2014 in Cameroon. In a first step, sera were screened serologically using a variety of assay formats to reveal RVFV specific antibodies. At the second stage, seropositive specimen were assessed for acute RVFV infections via IgM-specific ELISA and quantitative real-time RT-PCR. Our data show a significant difference in the antibody prevalence in cattle (13.5% [95% confidence interval: 11.4-15.7]) and small ruminants (3.4% [95% confidence interval: 2.3-4.7]), with indications for annual fluctuations and significant regional differences of seropositivity. One small ruminant and three bovines were eventually found to be positive in IgM ELISA and indications for viremia were found in one bovine by RVFV genome detection using quantitative real-time RT-PCR. The results of this study therefore corroborate the presence of acute RVFV-infection and its circulation in Cameroon.

Hepatitis E virus in wild rabbits and European brown hares in Germany.

Recently, a change of hepatitis E from being a typical travel-associated disease to an autochthonous zoonosis in Germany was observed. An increasing number of autochthonous infections with the hepatitis E Virus (HEV) have been recognized in developed countries. Venison from wild boar is already known to be a potential source of infection, if not prepared properly by the consumer. In Germany, certain wild animals are known to be a reservoir for HEV. However, current information is missing about European brown hares (Lepus europaeus) and wild rabbits (Oryctolagus cuniculus). Thus, a total of 833 hunting-harvested animals (European brown hares n = 669; wild rabbits n = 164) were tested for the occurrence of HEV RNA and HEV antibodies. For this, liver and blood specimens were taken after hunts in six German federal states. HEV antibodies were found by ELISA in 2.2% (624/14) of European brown hares, but no HEV RNA was detectable by nested real-time RT-PCR. In contrast, a seroprevalence of 37.3% (126/47) was observed for wild rabbits, and 17.1% (164/28) of the samples were HEV RNA positive. Genomic analysis revealed that these partial sequences clustered within the rabbit clade of HEV-3 genotype. In addition, one rabbit sequence segregated into subtype 3g of HEV-3. Highest seroprevalences for hares and rabbits were detected in the federal states of Bavaria and of Schleswig-Holstein, respectively. Comparing urban, rural and insular areas, the highest seroprevalence was shown for wild rabbits in rural areas and for European brown hares on the northern island Fehmarn. This study provides evidence that European brown hares and wild rabbits from Germany can be infected with HEV. The different prevalences indicate that wild rabbits are a potential reservoir for HEV in Germany, whereas European brown hares seem to be only of minor importance for the epidemiology of HEV.

Rift Valley fever virus infections in Egyptian cattle and their prevention.

Rift Valley fever virus (RVFV) causes consistently severe outbreaks with high public health impacts and economic losses in livestock in many African countries and has also been introduced to Saudi Arabia and Yemen. Egypt with its four large outbreaks in the last 40 years represents the northernmost endemic area of RVFV. The purpose of this study was to provide an insight into the current anti-RVFV antibody status in immunized as well as non-immunized dairy cattle from the Nile Delta of Egypt. During 2013-2015, a total of 4,167 dairy cattle from four governorates including Dakahlia, Damietta, Gharbia and Port Said were investigated. All cattle were born after 2007 and therewith after the last reported Egyptian RVFV outbreak in 2003. The samples derived from vaccinated animals from 26 different dairy farms as well as non-immunized cattle from 27 different smallholding flocks. All samples were examined following a three-part analysis including a commercially available competition ELISA, an in-house immunofluorescence assay and a virus neutralization test. Additionally, a subset of samples was analysed for acute infections using IgM ELISA and real-time reverse transcriptase PCR. The results indicated that the RVFV is still circulating in Egypt as about 10% of the non-immunized animals exhibited RVFV-specific antibodies. Surprisingly, the antibody prevalence in immunized animals was not significantly higher than that in non-vaccinated animals which points out the need for further evaluation of the vaccination programme. Due to the substantial role of livestock in the amplification and transmission of RVFV, further recurrent monitoring of the antibody prevalence in susceptible species is highly warranted.

Experimental Evaluation of Faecal Escherichia coli and Hepatitis E Virus as Biological Indicators of Contacts Between Domestic Pigs and Eurasian Wild Boar.

Domestic pigs and Eurasian wild boar (Sus scrofa) share several important viral and bacterial pathogens. Therefore, direct and indirect contacts between domestic pigs and wild boar present a risk of pathogen spillover and can lead to long-term perpetuation of infection. Biological indicators could be a powerful tool to understand and characterize contacts between wild boar and domestic pigs. Here, faecal Escherichia coli and Hepatitis E virus (HEV) were explored as potential biological indicators under experimental conditions. The data gained in our pilot study suggest that faecal E. coli can be used as biological indicator of contact between wild boar and domestic pig. For HEV, faecal transmission was also confirmed. However, molecular studies on full-genome basis did not reveal markers that would allow tracing of transmission direction. Based on these promising results, future field studies will especially target the practicability of E. coli microbiome molecular typing as surrogate of contacts at the wildlife-livestock interface.

Serological and genomic evidence of Rift Valley fever virus during inter-epidemic periods in Mauritania.

Rift Valley fever virus (RVFV) is an emerging pathogen of major concern throughout Africa and the Arabian Peninsula, affecting both livestock and humans. In the past recurrent epidemics were reported in Mauritania and studies focused on the analysis of samples from affected populations during acute outbreaks. To verify characteristics and presence of RVFV during non-epidemic periods we implemented a multi-stage serological and molecular analysis. Serum samples of small ruminants, cattle and camels were obtained from Mauritania during an inter-epidemic period in 2012-2013. This paper presents a comparative analysis of potential variations and shifts of antibody presence and the capability of inter-epidemic infections in Mauritanian livestock. We observed distinct serological differences between tested species (seroprevalence: small ruminants 3·8%, cattle 15·4%, camels 32·0%). In one single bovine from Nouakchott, a recent RVF infection could be identified by the simultaneous detection of IgM antibodies and viral RNA. This study indicates the occurrence of a low-level enzootic RVFV circulation in livestock in Mauritania. Moreover, results indicate that small ruminants can preferably act as sentinels for RVF surveillance.

Vaccination of alpacas against Rift Valley fever virus: Safety, immunogenicity and pathogenicity of MP-12 vaccine.

Rift Valley fever (RVF) is an emerging zoonosis of major public health concern in Africa and Arabia. Previous outbreaks attributed camelids a significant role in the epidemiology of Rift Valley fever virus (RVFV), making them an important target species for vaccination. Using three alpacas as model-organisms for dromedary camels, the safety, immunogenicity and pathogenicity of the MP-12 vaccine were evaluated in this study. To compare both acute and subacute effects, animals were euthanized at 3 and 31days post infection (dpi). Clinical monitoring, analysis of liver enzymes and hematological parameters demonstrated the tolerability of the vaccine, as no significant adverse effects were observed. Comprehensive analysis of serological parameters illustrated the immunogenicity of the vaccine, eliciting high neutralizing antibody titers and antibodies targeting different viral antigens. RVFV was detected in serum and liver of the alpaca euthanized 3dpi, whereas no virus was detectable at 31dpi. Viral replication was confirmed by detection of various RVFV-antigens in hepatocytes by immunohistochemistry and the presence of mild multifocal necrotizing hepatitis. In conclusion, results indicate that MP-12 is a promising vaccine candidate but still has a residual pathogenicity, which requires further investigation.

West Nile viral infection of equids.

West Nile virus (WNV) is a flavivirus transmitted between certain species of birds and mosquito vectors. Tangential infections of equids and subsequent equine epizootics have occurred historically. Although the attack rate has been estimated to be below 10%, mortality rates can approach 50% in horses that present clinical disease. Symptoms are most commonly presenting in the form of encephalitis with ataxia as well as limb weakness, recumbency and muscle fasciculation. The most effective strategy for prevention of equine disease is proper vaccination with one of the numerous commercially available vaccines available in North America or the European Union. Recently, WNV has been increasingly associated with equine epizootics resulting from novel non-lineage-1a viruses in expanding geographic areas. However, specific experimental data on the virulence of these novel virus strains is lacking and questions remain as to the etiology of the expanded epizootics: whether it be a function of inherent virulence or ecological and/or climactic factors that could precipitate the altered epidemiological patterns observed.

Analysis of prion protein aggregates in blood and brain from pre-clinical and clinical BSE cases.

Prion diseases are infectious neurodegenerative diseases affecting humans and animals. The food-borne bovine spongiform encephalopathy (BSE) had serious impact on both economy and public health, respectively. To follow the pathogenesis of BSE, oral challenge studies were previously conducted, among others on the Isle of Riems, Germany (Balkema-Buschmann et al., 2011b). In the present work brain and plasma samples from this pathogenesis study were subjected to surface fluorescence distribution analysis (sFIDA). sFIDA is a diagnostic tool that exploits the aggregated state of the disease-related prion protein (PrP) as a biomarker for prion disorders. With the exception of one animal, all tested brain samples from clinical cattle exhibited a high titer of PrP particles. Moreover we could detect PrP aggregates already 16 and 24 months after infection. In contrast to our previous demonstration of PrP particles in blood plasma from scrapie sheep, however, no aggregates could be identified in plasma from pre-clinical and clinical cattle. This is in accordance with other studies suggesting a restriction of the BSE infection to the central nervous system.

A novel indirect ELISA based on glycoprotein Gn for the detection of IgG antibodies against Rift Valley fever virus in small ruminants.

Rift Valley fever virus (RVFV) is an emerging zoonotic pathogen that causes high morbidity and mortality in humans and livestock. In this paper, we describe the cloning, expression and purification of RVFV glycoprotein Gn and its application as a diagnostic antigen in an indirect ELISA for the specific detection of RVF IgG antibodies in sheep and goats. The performance of this Gn based ELISA is validated using a panel of almost 2000 field samples from sheep and goats from Mozambique, Senegal, Uganda and Yemen. All serum samples were also tested by virus neutralization test (VNT), the gold standard method for RVFV serological testing. Compared to the VNT results the Gn based ELISA proved to have an excellent sensitivity (94.56%) and specificity (95.57%). Apart from establishing this new diagnostic assay, these results also demonstrate a close correlation between the presence of RVFV Gn and neutralizing antibodies.