Untangling the stranglehold through mathematical modelling of Streptococcus equi subspecies equi transmission.

Strangles, a disease caused by infection with Streptococccus equi subspecies equi (S. equi), is endemic worldwide and one of the most frequently diagnosed infectious diseases of horses. Recent work has improved our knowledge of key parameters of transmission dynamics, but important knowledge gaps remain. Our aim was to apply mathematical modelling of S. equi transmission dynamics to prioritise future research areas, and add precision to estimates of transmission parameters thereby improving understanding of S. equi epidemiology and quantifying the control effort required. A compartmental deterministic model was constructed. Parameter values were estimated from current literature wherever possible. We assessed the sensitivity of estimates for the basic reproduction number on the population scale to varying assumptions for the unknown or uncertain parameters of: (mean) duration of carriership (1∕γC), relative infectiousness of carriers (f), proportion of infections that result in carriership (p), and (mean) duration of immunity after natural infection (1∕γR). Available incidence and (sero-)prevalence data were compared to model outputs to improve point estimates and ranges for these currently unknown or uncertain transmission-related parameters. The required vaccination coverage of an ideal vaccine to prevent major outbreaks under a range of control scenarios was estimated, and compared available data on existing vaccines. The relative infectiousness of carriers (as compared to acutely ill horses) and the duration of carriership were identified as key knowledge gaps. Deterministic compartmental simulations, combined with seroprevalence data, suggest that 0.05

Detection and molecular characterization of Actinomyces denticolens causing lymph node abscessation in horses.

Introduction: Abscessation of equine head lymph nodes can be caused by various bacteria, but Streptococcus equi subsp. equi is mainly involved. At our laboratory, samples of three unrelated horses with submandibular abscesses were found negative for S. equi, and further testing proved the presence of another genus. This raised the question for the exact identity of this pathogen and whether these isolates were epidemiologically related and it warranted further characterization with regards of virulence and resistance factors. Methods: Culture followed by identification using MALDI-TOF MS, MIC testing and whole genome sequencing (WGS) was performed to characterize the bacteria. Results: Bacterial culture and subsequent identification with MALDI-TOF MS resulted in the reliable identification of A. denticolens in two of the three cases. Final confirmation of A. denticolens for all three isolates was achieved by analysis of the WGS data, supported by multilocus sequence typing (MLST). The three isolates showed 95% nucleotide sequence identity. The number of single nucleotide polymorphisms (10,170 to 36,058) indicated that the isolates were not clonal, suggesting that these cases were epidemiologically unrelated. Only four known virulence related genes were detected. The absence of known antibiotic resistance genes was in line with the high susceptibility, as indicated by the susceptibility patterns obtained for two of the three isolates. Conclusion: We conclude that A. denticolens should be included in the differential diagnosis of (submandibular) lymph node abscessation in horses, especially if strangles cannot be confirmed with laboratory diagnostics. Furthermore, we report the first draft genome of A. denticolens isolated from horses.

Ovine and Bovine Congenital Abnormalities Associated With Intrauterine Infection With Schmallenberg Virus.

In December 2011, a previously unknown congenital syndrome of arthrogryposis and hydranencephaly in sheep and cattle appeared in the Netherlands as an emerging epizootic due to Schmallenberg virus (SBV). Gross lesions in 102 lambs and 204 calves included porencephaly, hydranencephaly, cerebellar dysplasia and dysplasia of the brainstem and spinal cord, a flattened skull with brachygnathia inferior, arthrogryposis, and vertebral column malformations. Microscopic lesions in the central nervous system showed rarefaction and cavitation in the white matter, as well as degeneration, necrosis, and loss of neurons in the gray matter. Brain and spinal cord lesions were more severe in lambs than in calves. Ovine and bovine cases examined early in the outbreak showed encephalomyelitis. SBV infection was confirmed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in brain samples in 46 of 102 lambs (45%) and in 32 of 204 calves (16%). Immunohistochemistry, performed on tissue samples from 18 RT-qPCR-positive lambs, confirmed the presence of bunyaviral antigen in neurons of the brain in 16 cases. SBV antibodies were detected by enzyme-linked immunosorbent assay in fetal blood in 56 of 61 sampled ovine cases (92%). In a virus neutralization test, all tested dams of affected newborns, 46 ewes and 190 cows, were seropositive. Compared with other teratogenic viral infections, the pathogenesis and lesions of SBV in sheep and cattle fetuses are similar to those of other ruminant orthobunyaviruses. However, the loss of spinal ventral motor neurons and their tracts, resulting in micromyelia, distinguishes SBV infection from other viral central nervous system lesions in newborn ruminants.

Epidemiological analysis of an outbreak of foot-and-mouth disease (serotype SAT2) on a large dairy farm in Kenya using regular vaccination.

During August-September 2012, an outbreak of Foot-and-mouth Disease (FMD) due to serotype Southern African Territories-2 (SAT2) occurred on a large, extensively grazed dairy farm in Nakuru County, Kenya. Over 29 days, 400/644 (62.1%) cattle were recorded as displaying clinical signs consistent with FMD. Out of the 18 management groups present, 17 had clinical cases (weighted mean incidence rate 3.5 per 100 cattle-days, 95% CI 2.4, 5.1; range 0.064-10.9). Transmission may have been encouraged when an infected group was moved to a designated isolation paddock. A four to five day minimum incubation period was apparent in five groups for which a point source exposure was evident. Further transmission was associated with the movement of individual animals incubating infection, use of a common dip and milking parlour, and grazing of susceptible groups in paddocks neighbouring to infectious cases. Animals over 18 months old appeared to be at highest risk of disease possibly due to milder clinical signs seen among younger animals resulting in reduced transmission or cases not being recorded. Cows with a breeding pedigree containing a greater proportion of zebu appeared to be at lower risk of disease. The outbreak occurred despite regular vaccination (three times per year) last performed approximately three months before the index case. Incidence risk by the lifetime number of doses received indicated limited or no vaccine effectiveness against clinical disease. Reasons for poor vaccine effectiveness are discussed with antigenic diversity of the SAT2 serotype and poor match between the field and vaccine strain as a likely explanation. Detailed field-derived epidemiological data based on individual animals are rarely presented in the literature for FMD, particularly in East-Africa and with the SAT2 serotype. This study provides a detailed account and therefore provides a greater understanding of FMD outbreaks in this setting. Additionally, this is the first study to provide field-derived evidence of poor vaccine effectiveness using a SAT2 vaccine. Further field-based measures of vaccine effectiveness in line with evaluation of human vaccines are needed to inform FMD control policy which has previously relied heavily upon experimental data and anecdotal experience.

Development and validation of an indirect Enzyme-linked immunosorbent assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants.

To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

Schmallenberg virus outbreak in the Netherlands: routine diagnostics and test results.

At the end of 2011, a new Orthobunyavirus was discovered in Germany and named Schmallenberg virus (SBV). In the Netherlands malformations in new-born ruminants were made notifiable from the 20th of December 2011. After a notification, malformed new-borns were necropsied and brain tissue was sampled for reverse transcription-polymerase chain reaction (RT-PCR). In addition, blood samples from mothers of affected new-borns were tested for antibodies in a virus neutralization test (VNT). The aim of this study was to summarize and evaluate the diagnostic data obtained and to gain insight into the possible regional differences. In total 2166 brains were tested: 800 from lambs, 1301 from calves and 65 from goat kids. Furthermore 1394 blood samples were tested: 458 from ewes, 899 from cows and 37 from goats. Results showed that 29% of the lamb brains, 14% of the calf brains, and 9% of the goat kid brains were RT-PCR positive. The number of malformed and RT-PCR positive lambs decreased over time while the number of malformed and RT-PCR positive calves increased. In the VNT 92% of the ewes, 96% of the cows and 43% of the goats tested positive. Combining RT-PCR and VNT results, 18% of all farms tested positive in both the RT-PCR and VNT. The relative sensitivity and specificity of the RT-PCR are 19% and 97% respectively, and of the VNT 99% and 6%. The results show a widespread exposure to SBV and the regional evaluation seems to indicate an introduction of SBV in the central/eastern part.

Prevalence of Coxiella burnetii infections in aborted fetuses and stillborn calves.

Coxiella burnetii infections are mostly subclinical in cattle, but can occasionally be associated with abortion. In the present study, 100 aborted fetuses or stillborn calves that were submitted for postmortem examination between September 2007 and March 2008 were examined for infection with C burnetii. Samples of both pooled fetal tissues and placental cotyledon were tested using a real-time PCR assay. In addition, the sections of placental cotyledon were examined using immunohistochemistry (IHC). The IHC of four placentas was positive. The PCR results of the IHC-positive placentas were high positive (HP); the PCR results of the organs of these four fetuses and calves varied from low positive (LP) to HP. The four IHC-positive fetuses had a gestation length of seven to nine months. All four placentas had histological signs of inflammation, but only one of four placentas had gross pathological signs of inflammation possibly due to a concomitant infection with Bacillus licheniformis. Five other IHC-negative placentas had (high) positive PCR results; the PCR results of the organs of these fetuses were LP or negative. The present study indicates that C burnetii infections are detected in a limited percentage of aborted fetuses and stillborn calves by IHC. To assess the importance of placentas with PCR-positive and IHC-negative test results, more research is needed.

Lawsonia intracellularis-associated proliferative enteritis in weanling foals in the Netherlands.

Equine proliferative enteropathy (EPE) is an emerging infectious enteric disease caused by the obligate intracellular gram-negative bacterium Lawsonia intracellularis. EPE was tentatively diagnosed in six weanling foals, aged between 5 and 7 months. Clinical signs included depression, anorexia, ventral oedema, and weight loss. Plasma biochemistry consistently revealed severe hypoproteinaemia. The ante-mortem diagnosis of EPE was based on clinical signs, hypoproteinaemia (6/6), the detection of moderate-to-high titres of L. intracellularis antibody (6/6), and severe thickening of the small intestinal wall on ultrasonography (2/2), or L. intracellularis detected in faeces by PCR (I/2). The first foal died despite treatment and at post-mortem examination the tentative diagnosis was EPE. Three foals from the same farm, which showed similar clinical symptoms were treated with azithromycin and rifampicin; two survived. Post-mortem examination of the foal that died confirmed the tentative clinical diagnosis of EPE on the basis of the lesions found and the detection of L. intracellularis--DNA in the ileum and jejunum. The fifth foal died despite intensive treatment and the post-mortem examination revealed lymphohistiocytic enteritis, typhlitis, and widespread thrombosis in several organs. The sixth foal recovered completely after treatment. This report confirms the presence of clinical L. intracellularis infection in weanling foals in the Netherlands and shows the difficulty in reaching a definitive ante-mortem diagnosis.

[West Nile virus expanding in Europe]. / West-Nijl-virus in opmars in Europa.

The areas of Europe in which West Nile virus (WNV)-transmission to humans is observed have expanded over the last few years, with endemic circulation amongst animals of southern Europe. This situation calls for heightened vigilance to the clinical presentation of WNV infection in humans. The average incubation period lasts 2-6 days. Of those infected, 20% will experience a mild, non-specific disease presentation such as high fever, headache, myalgia, possibly with rash and lymphadenopathy; <1% will develop severe neurological symptoms. Rare complications include: myelitis, optic neuritis, rhombencephalitis, polyradiculitis, myocarditis, pancreatitis and fulminant hepatitis. Clinicians should take WNV infection into consideration when making a differential diagnosis for such symptoms in patients who have returned from areas with potential virus circulation. Given the increase in the spread of WNV within Europe, this now holds true for continental travellers as well as those destined for the Americas, Africa and Asia. It is important to include the patient's travel history, clinical symptoms and any occurrences of vaccination against viruses causing Japanese encephalitis, tick-borne encephalitis and yellow fever into the diagnostic workup, as the antibodies against these diseases show cross-reactivity.

The seroprevalence of Lawsonia intracellularis in horses in The Netherlands.

Equine proliferative enteropathy caused by Lawsonia intracellularis is an emerging disease of weanling foals and affects their growth and development. The prevalence of Lawsonia intracellularis in The Netherlands is not known. The aim of the study was to investigate the seroprevalence of Lawsonia intracellularis in horses in The Netherlands. Blood samples were taken from healthy foals before and after weaning and from healthy yearlings and mature horses on farms throughout The Netherlands. These samples were analysed for the presence of Lawsonia intracellularis-specific antibodies with a blocking ELISA. White blood cell count, packed cell volume, and total protein concentration were also measured in all foals. Information regarding housing, pasture access, and contact with pig manure on the premises was obtained for all animals. The prevalence of Lawsonia intracellularis antibodies in foals increased significantly from 15% before weaning to 23% after weaning (p = 0.019); it was 89% in yearlings and 99% in horses older than 2 years. There was no significant difference in seroprevalence between the pasture-kept and stable-confined adult horses (97% and 100%, respectively), and there was no significant influence of contact with pig manure. None of the sampled animals showed clinical disease. In conclusion, the results suggest that Lawsonia intracellularis is widespread in The Netherlands and that seropositivity is not necessarily associated with clinical problems. The high seroprevalence in adult horses suggests long-term persistence of antibodies against Lawsonia intracellularis or constant exposure to the bacterium.

Prevalence of Coxiella burnetii infection in Dutch dairy herds based on testing bulk tank milk and individual samples by PCR and ELISA.

A study using an ELISA and a real-time PCR assay based on the detection of the repetitive transposon-like gene of Coxiella burnetii revealed that infection with the bacterium was widespread among Dutch dairy herds, with antibodies detected in bulk tank milk (BTM) from 268 of 341 herds (78.6 per cent) and bacterial DNA detected in 193 of 341 herds (56.6 per cent). The BTM samples were taken in November and December 2007. Serological and molecular studies in young and adult cattle selected from 100 herds showed that antibodies were present in the blood of 470 of 2936 (16.0 per cent) lactating cows but only in 19 of 1831 (1.0 per cent) young animals. Bacterial DNA was detected in the milk of 254 of 2925 (8.7 per cent) lactating cows; bacterial DNA was not detected in any of the faecal samples obtained from youngstock. The blood and milk samples were taken from the cattle in the period January to April 2008.

Dairy cows with metritis: Coxiella burnetii test results in uterine, blood and bulk milk samples.

In cattle, Coxiella burnetii infections are generally asymptomatic but can also be associated with reproductive disorders. Metritis is considered as one of the symptoms of C. burnetii infections, but reliable information is lacking. Therefore, information on the presence of C. burnetii in the uterine content of cows with metritis is important to increase our knowledge on this pathogen. In this study, the uterine content of 45 dairy cows with metritis belonging to 12 herds was tested for C. burnetii with a real-time PCR assay. Only one uterine sample tested PCR (highly) positive, all other samples were PCR negative. The PCR positive cow tested also positive for antibodies. Three other cows from other herds tested antibody positive. The bulk milk (BM) samples of these 12 herds were tested by real-time PCR assay and antibody-ELISA. Six BM samples (50%) were positive in PCR and 10 (83%) were positive in ELISA. Culturing the uterus samples by bacteriology, the most frequently cultured bacteria were arcanobacterium (n=24), E. coli (n=16), other streptococci (n=10), Streptococcus uberis (n=8) and Streptococcus dysgalactiae (n=5). This study indicates that C. burnetii is not an important cause for metritis in dairy herds, although apparently C. burnetii was or had been present in most of these herds.

Aspects of the epidemiology, research, and control of lentiviral infections of small ruminants and their relevance to Dutch sheep and goat farming.

In 1862, the veterinarian Loman reported the first sheep in The Netherlands with symptoms associated with lentiviral infection, although at the time the symptoms were ascribed to ovine progressive pneumonia. In the following century, similar cases were reported by South African, French, American, and Icelandic researchers. Extensive research into the pathology, aetiology, and epidemiology of this slowly progressive and ultimately fatal disease was initiated in several countries, including the Netherlands. Studies of the causative agents--maedi visna virus (MVV) in sheep and caprine arthritis encephalitis virus (CAEV) in goats, comprising the heterogeneous group of the small ruminant lentiviruses (SRLV)--prompted the development of diagnostic methods and the initiation of disease control programmes in many European countries including the Netherlands, as a pioneer in 1982, and in the U.S.A. and Canada.

Evaluation of an indirect ELISA for detection of antibodies in bulk milk against bluetongue virus infections in the Netherlands.

After the introduction of bluetongue virus serotype 8 (BTV-8) in western Europe in 2006, an indirect ELISA for detection of serogroup-specific antibodies against BTV in serum samples was validated for individual milk samples by the Central Veterinary Institute and the Animal Health Service in the Netherlands (Kramps et al., 2008). In order to develop a cost-effective monitoring tool, we now have evaluated this ELISA also for use in bulk milk. Therefore, bulk milk samples and individual milk samples were collected from 92 herds in the affected southern region in the Netherlands in 2007, before the start of the vaccination campaign. In addition, bulk milk samples collected from 88 herds before the bluetongue introduction in 2006 ("historically negative" samples) have been tested. With these results ROC analyses were performed and herd specificity and herd sensitivity of the bulk milk ELISA were estimated. All "historically negative" bulk milk samples were negative in the ELISA, with a mean S/P ratio of 10 ± 0.8%. The herd sensitivity and herd specificity of the ELISA in bulk milk samples depend on the cut-off that is chosen. In order to detect a within-herd-prevalence of 1%, the optimal cut-off S/P ratio 13% was found. A few herds with one or two milk-positive animals would then be missed. The specificity will be 100%. A within-herd-prevalence of 10% can be detected with 100% sensitivity at a cut-off S/P ratio of 96%. In conclusion, the indirect ELISA in bulk milk samples is a very specific and sensitive test which can be implemented in monitoring and surveillance systems in unvaccinated populations.

Experimental infection with neuropathogenic equid herpesvirus type 1 (EHV-1) in adult horses.

Equid herpesvirus type 1 (EHV-1)-associated myeloencephalopathy (EHM) may follow an infection with the virus in horses. This study tested three hypotheses: (1) a large inhaled dose of a neuropathogenic EHV-1 strain would induce a cell-associated viraemia in all infected horses; (2) neurological disease will only occur in viraemic horses, and (3) the cerebrospinal fluid (CSF) composition following EHV-1 viraemia will be an indicator for EHM. Four EHV-1 seronegative horses were inoculated with EHV-1 by inhalation. Three developed clinical signs of neurological disease, which were mild in two horses and lacking typical EHM histopathological findings, but moderately severe in the third horse. This latter animal was the only one found to be viraemic, with xanthochromic CSF and spinal cord histopathology findings characteristic of EHM. This study showed that cell-associated viraemia was not guaranteed, despite a large-dose inoculation with EHV-1, yet viraemia was probably a pre-requisite for subsequent development of EHM. The histopathological changes used to confirm EHM may be predicted from CSF analysis.

Diagnostic performance of ELISA and PCR in identifying SRLV-infected sheep and goats using serum, plasma and milk samples and in early detection of infection in dairy flocks through bulk milk testing.

The objective of the study was to evaluate the diagnostic performances of the ELITEST-MVV ELISA for detection of antibodies against small ruminant lentiviruses and of two recently published PCRs for the detection of proviral DNA of SRLV in blood and corresponding individual milk samples. In addition, the feasibility of bulk milk testing was investigated by titrating ELISA positive pooled milk samples in negative milk, and by investigating bulk milk samples by ELISA and PCR in relation to the SRLV-status of the flocks. The results show that plasma and milk are suitable replacements for serum. For sheep, both PCRs showed a better diagnostic performance than for goats. ELISA results for bulk milk samples were promising with a putative detection limit of <3% within-herd prevalence using 1/10 pre-diluted samples and even <1% within-herd prevalence when samples were tested undiluted. In a panel of 249 bulk milk samples, all samples from SRLV free flocks (n=138) tested negative in the ELISA, while 50% of the samples from flocks with an unknown SRLV-status (n=111) were positive. For a subset of 59 bulk milk samples, agreement between ELISA results and leader-gag PCR results was almost 100%. These results demonstrate the potential of bulk milk testing as a cost effective tool for early detection of infection in dairy flocks, which is essential for SRLV-monitoring programs.

Use of serology and polymerase chain reaction for the rapid eradication of small ruminant lentivirus infections from a sheep flock: a case report.

A SRLV-free sheep flock incurred infection which led to an SRLV infection rate of over 50% of the ewes (34/64) within a 30 months period, indicating that environmental conditions were favourable to transmission. An intensive regimen of sampling at short intervals and testing for SRLV antibodies and proviral DNA combined with strict management was implemented for the entire flock, lambs and yearlings included. This resulted in eradication of the infection within two testing and culling rounds with a 3 months interval. The additional value of the proviral DNA detection by PCR in identifying infected animals was clear in that nine infected animals were found that would have been missed if tested by serology alone. PCR also saved two lambs from being culled; they were sero-positive probably due to maternal antibodies, but not infected.

Evaluation of Ziehl-Neelsen stained faecal smear and ELISA as tools for surveillance of clinical paratuberculosis in cattle in the Netherlands.

Testing cattle suspected of clinical paratuberculosis is an important element of surveillance of paratuberculosis. The aim of this study was to evaluate the diagnostic-test characteristics of microscopic examination of Ziehl-Neelsen stained faecal smears for acid-fast Mycobacteria (ZN-test) and serum-ELISA in cattle suspected of clinical paratuberculosis in the Netherlands. Results of all samples submitted for ZN-test and serum-ELISA between April 2003 and April 2006 to our laboratory were retrieved. Results from cattle for which both tests were performed were analysed using two Bayesian latent-class models for evaluation of diagnostic tests in two populations without a gold standard, assuming (a) conditional independence of tests, or (b) conditional dependence of tests in both infected and non-infected cattle. Sampled cattle were divided into two populations in different ways using four known risk factors for clinical paratuberculosis: region, soil type, clinical signs, and age. For 892 cattle suspected of clinical paratuberculosis, both ZN-test and serum-ELISA results were retrieved: 250 ZN-positive and ELISA-positive, 12 ZN-positive and ELISA-negative, 260 ZN-negative and ELISA-positive, and 370 ZN-negative and ELISA-negative cattle. With priors based on the available literature, the posterior estimates of sensitivity, specificity, and positive and negative predictive values of the ELISA were always higher than those of the ZN-test. Furthermore, lower limits of the 95% credibility intervals of the posterior positive predictive values of the ELISA were >or=99.7%, and of the negative predictive values of the ELISA >or=56.4%. We conclude that the ELISA is preferred to the ZN-test to confirm the presumptive diagnosis of clinical paratuberculosis in the Netherlands. Little diagnostic information can be gained by performing the ZN-test in addition to the ELISA.