[Japanese encephalitis in Southern Europe]. / Japanse encefalitis in Zuid-Europa?

In 2012, a fragment of the Japanese encephalitis virus (JEV) genome was isolated from a pool of Culex pipiens mosquitoes caught in 2010 and 2011 in Northern Italy. JEV has a broad geographical distribution in South and Southeast Asia and Oceania, and is the most important cause of viral encephalitis in Asia in humans and also causes encephalitis in horses and fertility problems in pigs. However, recently isolated JEV genome fragments in mosquitoes in Italy could be an indication of repeated introduction of JEV, enzootic circulation of JEV or a related virus in Southern Europe. Until more information is available, Japanese encephalitis remains a travel-related infectious disease for travellers to JEV endemic and epidemic areas outside of Europe.

Genetic characterization of an atypical Schmallenberg virus isolated from the brain of a malformed lamb.

A novel orthobunyavirus, named "Schmallenberg virus" (SBV), was first detected in the blood of cattle at the end of the summer in Germany in 2011, and subsequently in late autumn from the brain of a stillborn malformed lamb in The Netherlands. Full genome sequences, including 5' and 3' terminal "panhandle" sequences of the L, M, and S segments of the SBV isolated from lamb brain tissue (named HL1) were determined. In addition, a second SBV strain was isolated from the blood of a dairy cow (named F6) also in The Netherlands. This isolate was passaged on Vero cells, and its genome sequence was determined by next-generation sequencing. Alignments of the two genome sequences revealed 4, 12, and 2 amino acid differences in the open reading frames of the L, M, and S segments, respectively. Eleven of a total of 12 amino acid differences were detected in the M segment encoding the ectodomain of the putative structural glycoprotein Gc. Notably, in the HL1 isolate, positions 737-739 are occupied by isoleucine, arginine, and leucine (IRL), whereas in the majority of other sequenced SBV isolates these positions are occupied by threonine, histidine, and proline, respectively. Moreover, in all sheep, goat, and cattle SBV isolates sequenced and published so far, an IRL sequence was never found. This has brought us to the conclusion that the M segment of the HL1 isolate differed markedly from that of other lamb and cow isolates. Whether this atypical variant resulted from adaptation to the ewe, fetus, or insect vector remains to be investigated.

Estimation of the basic reproduction number for Streptococcus equi spp. equi outbreaks by meta-analysis of strangles outbreak reports.

BACKGROUND: Streptococcus equi spp. equi (S. equi), the cause of strangles in horses, is considered a highly contagious pathogen affecting equines and the equine industry worldwide. Fundamental epidemiological characteristics of outbreaks, such as the basic reproduction number (R0 ), are not well described. OBJECTIVES: Estimate R0 for S. equi in equine populations from outbreak data. STUDY DESIGN: Systematic review and meta-analysis of published and unpublished data. METHODS: A literature search for outbreak reports was carried out. Depending on data available in the reports, the early epidemic growth rate or final attack rate (AR) approach was used to estimate the basic reproduction number for that outbreak. Other recorded outbreak characteristics were the type of housing (group vs. individual). An overall estimate for R0 was computed by meta-analysis. RESULTS: Data from eight outbreaks were extracted from peer-reviewed publications. Data from two additional, non-published outbreaks was also included in the meta-analysis. A conservative estimate for R0 was 2.2 (95% confidence interval [CI] 1.9-2.5). A less conservative estimate, including outbreaks with a 100% AR for which a lower limit R0 was estimated, was 2.7 (95% CI 2.1-3.3). MAIN LIMITATIONS: Few papers describing longitudinal incidence data were found so most estimates were based on the outbreaks' final size. Several outbreaks had a 100% attack rate and could therefore only be included as a lower limit estimate in the meta-analysis. The reported result therefore may be an underestimation. CONCLUSIONS: This estimate for R0 for S. equi informs parameters for future mathematical modelling, quantifies desired preventive vaccine coverage and helps evaluate the effect of prevention strategies through future modelling studies.

Experimental and field investigations of exposure, replication and transmission of SARS-CoV-2 in pigs in the Netherlands.

In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding-domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40-80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely.

New Insights into the Management of an EHV-1 (Equine Hospital) Outbreak.

In May 2018, Wolvega Equine Hospital (WEH) experienced an EHV-1 outbreak. This outbreak caused significant economic losses and negative publicity for the hospital. How should hospitals prepare themselves for these outbreaks and prevent shedding of the virus on multiple neighboring premises? The hospital transformed most of its activities into mobile practice and the entire infected hospital population was moved to a separate remote location. The hospital was cleaned and disinfected according to the latest recommendations before reopening. Four neighboring professional equine businesses and three privately owned premises were affected by the spread of the virus from the hospital population and initiated quarantine restrictions. Equine hospitals should prepare themselves for EHV-1 outbreaks as the intake of the virus cannot be prevented. A management protocol should include public information protocols, swift client information and quarantine measures that ensure quick containment of the outbreak. Timely reopening of the hospital can be achieved by rehousing the contaminated population. It should also include good regulations with clients and a properly carried out release protocol. Equine sports organizations should establish sufficient vaccination coverage in order to decrease the frequency of EHV-1 outbreaks.

Globetrotting strangles: the unbridled national and international transmission of Streptococcus equi between horses.

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi, establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such 'carrier' animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

Comparison of Two Diagnostic Methods to Detect Insulin Dysregulation in Horses Under Field Conditions.

Straightforward testing procedures to enable the diagnosis of insulin dysregulation (ID) in horses that are suitable for use in daily veterinary practice are needed because of the risk that ID could result in laminitis. In our study (that included 90 horses), we compared the proportion of horses classified as ID-positive, ID-suspect, and ID-not diagnosed according to the basal insulin concentration (BIC) with the proportion of horses classified as ID-positive or ID-negative according to a practical and feasible version of an oral sugar test (OST). Furthermore, BIC, basal glucose concentration, and insulin and glucose concentration after OST were analyzed and compared. In the total study population, the OST detected significantly more ID-positive cases than the BIC, with cutoffs at equivalent specificities. Receiver operating characteristics analysis showed that at a lower cutoff, the sensitivity of the BIC could be increased, but at the cost of a significantly lower specificity. Taking this into account, we found diagnostic performance of the OST to be considerably better than the BIC and therefore considered it more recommendable for use as a screening test for ID in ambulatory practice. Furthermore, we investigated the relationship between body condition score and breed type with glucose and insulin concentration as determined after our version of the OST. For that purpose, the study group was subdivided into lean, moderate, and obese horses and "easy keeper breeds" versus "non-easy keeper breeds". Results supported the general assumption that obese horses and "easy keeper breeds" are more prone to the development of ID.

First detection of foot-and-mouth disease virus O/ME-SA/ Ind2001e sublineage in Jordan.

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease that is caused by the FMD virus (FMDV). This disease affects both wild and domestic cloven-hoofed animals, and the latter of which includes cattle, swine, sheep and goats. FMD is endemic to Jordan and has a severe impact on the productivity of domestic livestock. In January 2017, FMD outbreaks were detected in different animal species across Jordan, resulting in high mortality rates among young lamb and goat populations as well as causing classic FMD symptoms in cattle. In this study, clinical specimens were collected from animals affected by FMD. The results obtained from sequencing the VP1 gene place the studied FMDV isolate within the FMDV O/ME-SA/ Ind2001e sublineage. Phylogenetic analysis of VP1 suggests that the O/JOR/1/2017 isolate is very similar to that of viruses isolated from Saudi Arabia in 2016. The possible introduction of this strain to Jordan might occur through transboundary animal movement or other transmission routes from Saudi Arabia, a neighbouring country.

Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows.

A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.

An international inter-laboratory ring trial to evaluate a real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen.

Six laboratories participated in a ring trial to evaluate the reliability of a real-time PCR assay for the detection of bovine herpesvirus 1 (BoHV-1) from extended bovine semen. Sets of coded samples were prepared and distributed to each of the laboratories. The sample panel contained semen from naturally and artificially infected bulls, serial dilutions of positive semen with negative semen, semen from uninfected seronegative bulls, negative semen spiked with virus, as well as serial dilutions of reference virus. The samples were tested using a previously validated real-time PCR assay for the detection of BoHV-1 in each participating laboratory. The PCR tests were conducted with four different real-time PCR amplification platforms, including RotorGene 3000, Stratagene MX 3000/4000, ABI 7900, and Roche LightCycler 2.0. Virus isolation using one set of samples was performed in one laboratory. The results of the laboratories were compared with one another, and with those of virus isolation. It was found that the sensitivity and specificity of the real-time PCR test was greater than those of virus isolation (82.7% versus 53.6% and 93.6% versus 84.6%, respectively). A high level of agreement on PCR testing results between the laboratories was achieved (kappa value 0.59-0.95). The results of this study indicate that the real-time PCR assay is suitable for the detection of BoHV-1 in extended semen, and would be a good substitute for the slow and laborious virus isolation, for the screening testing at artificial insemination centres and for international trade.

Validation of a real-time PCR assay for the detection of bovine herpesvirus 1 in bovine semen.

A real-time polymerase chain reaction (PCR) assay was developed for detection of the presence of bovine herpesvirus type 1 (BoHV-1) in extended bovine semen. The assay detects a region encoding a highly conserved glycoprotein B gene. The real-time PCR assay was validated for specificity, sensitivity and repeatability using spiked semen and semen from naturally infected animals. The real-time PCR was very rapid, highly repeatable and more sensitive (lower detection limits) than conventional virus isolation method for the detection of BoHV-1 in extended semen. The specificity of the assay is as expected. The assay had an analytical sensitivity of 0.38 TCID(50) virus spiked into negative semen. The second real-time PCR system for the detection of the bovine growth hormone (bGH) gene was applied as an internal control for the DNA extraction and PCR. The bGH PCR can be performed separately to BoHV-1 PCR, or in a duplex format. The real-time PCR assay is intended for use in international trade. The complete validation dossier based on this study and an international inter-laboratory ring trial has been accredited by the Office International des Epizooties (OIE) and has been recommended to be adopted as a prescribed test for international trade.

Diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in individual milk and bulk milk samples of dairy herds.

The objective of the study was to determine the diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (Map) in individual milk samples and in bulk milk samples. For individual milk samples the specificity of the Pourquier ELISA was estimated by testing a panel of individual milk samples from certified Map-free herds. The relative sensitivity of the assay in individual milk samples and agreement of the results with those of serum samples was estimated by testing panels of paired serum-milk samples from seropositive cattle, whole-herd investigations, and moderate or heavy shedders. The specificity of the ELISA for individual milk samples was still 99.8% at a cut-off of 20% sample to positive (S/P) value, clearly lower than the cut-off defined by the manufacturer (30% S/P). The relative sensitivity for individual milk samples as compared with positive serum samples was 87% for a cut-off of 20% S/P, and 80% for a cut-off of 30% S/P. The sensitivity of this ELISA for detection of high shedders was >90% both for individual milk and serum samples, also agreement was very good (kappa=0.91 for all paired samples). The specificity of the Pourquier ELISA in bulk milk samples was investigated by testing bulk milk samples from certified Map-free herds. Feasibility of bulk milk testing was investigated by titrating ELISA positive individual milk samples in negative milk. In addition, 383 bulk milk samples from herds with a known within-herd seroprevalence were tested. The specificity of the ELISA for bulk milk samples was 100% at a cut-off of 12.5% S/P. At the cut-off recommended by the manufacturer (30% S/P) performance of the bulk milk ELISA related to herd status (> or =2 seropositive cows) was rather poor, corresponding with a sensitivity of 24% and a specificity of 99% relative to serology. However, at the revised cut-off for bulk milk of 12.5% S/P and a within-herd seroprevalence of > or =3%, sensitivity and specificity relative to serology were 85% and 96%, respectively. Given the current herd-level seroprevalence in The Netherlands, these test characteristics corresponded with positive and negative predictive values for bulk milk of 67% and 94%, respectively. In conclusion, the diagnostic performance of the Pourquier ELISA for individual milk samples creates opportunities for a cheaper and more feasible testing scheme, while the diagnostic performance for bulk milk samples warrants further consideration.

Factors associated with variation in Neospora caninum bulk-milk S/P ratios in initially bulk-milk negative testing Dutch dairy herds.

We conducted a study on 81 initially bulk-milk ELISA negative dairy herds taken from a random sample of Dutch dairy herds to evaluate variation in bulk-milk S/P ratios and to study reasons for bulk-milk conversion. These herds were repeatedly (3-month intervals) tested between April 2004 and August 2005 and serostatus of all animals had previously been established as negative (N), low-positive (LP) or high-positive (HP). Of these herds, herd- and test-related factors associated with variation in sample over positive (S/P) ratios were analysed using a multivariable linear-mixed model with 'herd' as random effect. In addition, changes of animal serostatus in converting herds were described. S/P ratios were calculated as the optical density of the bulk-milk sample minus the optical density of the negative serum control divided by the difference in optical density between the positive and negative serum control. Sixteen bulk-milk conversions in 12 dairy herds were seen with few indications of serological conversion in lactating cattle except for one herd in which recrudescence of infection appeared likely in nine cows. The effect of HP serostatus on bulk-milk S/P ratio was 2-3 times stronger compared with LP serostatus. In addition, bulk-milk S/P-ratio increased when the proportion of HP animals between 1 and 60 days in milk increased and decreased when the average milk-production level of the herd increased. Besides these herd-related factors, the use of different ELISA-testkits between test rounds had a significant effect on the S/P-ratio in bulk-milk samples.

Seroprevalence of Foot-and-Mouth Disease in Susceptible Wildlife in Israel.

Foot-and-mouth disease (FMD) epidemics recur in Israel almost every year. Wild even-toed ungulates are seldom affected during these epidemics. The seroprevalence of FMD in wild ungulates during 2000 and 2005-2013 was estimated using anti-non-structural proteins ELISA. Overall, 209 samples were tested, comprising sera of 120 wild boar (Sus scrofa lybicus), 64 mountain gazelles (Gazella gazella gazella), 6 water buffaloes (Bubalus bubalis), and 19 Persian fallow deer (Dama dama mesopotamica). None of the tested animals presented clinical signs of FMD during blood collection. Sixteen samples [7.7% (95% confidence interval (CI95%) = 4.4-12.1%)] were found to be seropositive. Fifteen out of 120 samples (12.5%) from wild boar were seropositive, compared with only 1 out of 89 samples (1.1%) from all other species combined (Fisher's exact test: p = 0.003). Most of the positive samples obtained from wild boar [13/15 (86.7%)] were collected during 2007, and analysis was restricted to that year and species only. The seroprevalence of FMD in this species during 2007 was estimated at 54.2% (CI95% = 32.8-74.5%; n = 24). A significant infection cluster, comprising nine seropositive samples collected in three different locations, was identified in the north-eastern part of Israel. These findings indicate that wild boar was affected during the 2007 FMD epidemic, even though wild boar presenting FMD typical clinical signs were not observed during that year. The actual role of wild boar in the spread of FMD virus in this epidemic, however, could not be determined. The negligible seroprevalence of FMD found for all other surveillance years indicates that ongoing circulation of FMD among wildlife in Israel is unlikely. It is concluded that while the role of wildlife species in the dynamics of FMD in Israel is usually limited, there might be occasions, in which wildlife plays a part in the spread of the virus.

Prevalence and risk factors for foot and mouth disease infection in small ruminants in Israel.

During the last decade, 27% of the foot and mouth disease (FMD) outbreaks in Israel affected small ruminant (SR) farms. FMD outbreaks reoccur in Israel despite vaccination of all livestock and application of control measures. We performed a cross-sectional serological study, aimed at estimating the prevalence of FMD infection in SR in Israel and the possible risk factors for infection. Overall, 2305 samples of adult sheep (n=1948) and goats (n=357) were collected during 2011-14 in two separate surveys. One survey was based on random sampling of intensive management system farms and the other was originally aimed at the detection of Brucella melitensis at extensive and semi-intensive management system farms. Sera were tested by NS blocking ELISA (PrioCHECK(®)). The serological prevalence of antibodies against non structural proteins (NSP) of FMD virus was estimated at 3.7% (95% confidence interval (CI95%)=3.0% -4.5%). Additionally, a significantly lower infection prevalence (p value=0.049) of 1.0% (CI95%=0.1%-3.6%) was found in a small sample (197 sera) of young SR, collected during 2012. The positive samples from adult SR were scattered all over Israel, though two significant infection clusters were found by the spatial scan statistic. Occurrence of an outbreak on a non-SR farm within 5km distance was associated with a fifteen times increase in the risk of FMD infection of SR in the univariable analysis. Yet, this variable was not included in the multivariable analysis due to collinearities with the other independent variables. Multivariable logistic regression modeling found significantly negative associations (P value<0.05) of grazing and being in a herd larger than 500 animals with risk of infection. Grazing herds and herds larger than 500 animals, both represent farms that are intensively or semi-intensively managed. Higher maintenance of bio-safety, fewer introductions of new animals and higher vaccination compliance in these farms may explain their lower risk of infection by FMD virus. We conclude that despite the wide distribution of infection among SR farms, low farm level prevalence indicates that in Israel SR pose only limited role in the transmission and dissemination of FMD. This conclusion may be applicable for other endemic countries in which, similar to Israel, all livestock are vaccinated against FMD.

Prevalence and risk factors for foot and mouth disease infection in cattle in Israel.

Foot and mouth disease (FMD) is a highly contagious viral disease with major economic consequences. In Israel, FMD epidemics recur almost every year and mostly affect cattle. The highest number of outbreaks occurs among beef cattle farms, followed by feedlot farms and dairy farms. We performed several cross-sectional serological studies in Israel during 2006-2014, aimed to reveal if the virus is endemic among cattle and to determine the sero-prevalence of antibodies directed against non-structural proteins (NSP) of FMD virus. Additionally we aimed to determine the risk factors for such sero-positivity. A risk based sampling was performed and the presence of anti-NSP antibodies was estimated using the PrioCHECK(®) ELISA kit. Beef cattle showed the highest sero-prevalence (13.2%, CI95%=10.8-15.8%). Higher FMD sero-prevalence in beef cattle sampled in 2014 was associated with previous FMD outbreaks in the farm and with age (adult cows versus calves (p<0.05)). Sero-prevalence in feedlot calves was significantly lower with only one sero-positive calf out of 256 (0.4%, CI95%=0-2.2%). Sero-prevalence among dairy cattle was 2.7% (CI95%=2-3.6%) with location of up to 3km from FMD outbreaks in multiple farms and location of up to 5km from the nearest border standing out as significant (p<0.05) risk factors for sero-positivity. The extremely low sero-prevalence of FMD in feedlot cattle and the significant association of infection in beef cattle with previous outbreaks suggest absence of virus circulation between these two populations during the study period, although previous data show that during outbreaks such transmission can occur. Low sero-prevalence in dairy cattle located in areas adjacent to previous FMD outbreaks may be attributed to intense routine vaccination and stringent control measures that were applied during outbreaks such as emergency vaccination and strict quarantine. Early detection of FMD outbreaks among grazing beef herds as well as the implementation of control measures among these farms are therefore the methods of choice to prevent future outbreaks in Israel.

Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study.

BACKGROUND: A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. METHODS: We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. FINDINGS: 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies. INTERPRETATION: MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection. FUNDING: European Union, European Centre For Disease Prevention and Control, Deutsche Forschungsgemeinschaft.