Jan van den Broek

Wild Birds and Increased Transmission of Highly Pathogenic Avian Influenza (H5N1) among Poultry, Thailand

Wild birds are associated with increased virus spread to poultry.

Investigation of amphibian mortality events in wildlife reveals an on-going ranavirus epidemic in the North of the Netherlands

In the four years following the first detection of ranavirus (genus Ranavirus, family Iridoviridae) infection in Dutch wildlife in 2010, amphibian mortality events were investigated nationwide to detect, characterize and map ranaviruses in amphibians over time, and to establish the affected host species and the clinico-pathological presentation of the disease in these hosts. The ultimate goal was to obtain more insight into ranavirus disease emergence and ecological risk. In total 155 dead amphibians from 52 sites were submitted between 2011 and 2014, and examined using histopathology, immunohistochemistry, virus isolation and molecular genetic characterization. Ranavirus-associated amphibian mortality events occurred at 18 sites (35%), initially only in proximity of the 2010 index site. Specimens belonging to approximately half of the native amphibian species were infected, including the threatened Pelobates fuscus (spadefoot toad). Clustered massive outbreaks involving dead adult specimens and ranavirus genomic identity indicated that one common midwife toad virus (CMTV)-like ranavirus strain is emerging in provinces in the north of the Netherlands. Modelling based on the spatiotemporal pattern of spread showed a high probability that this emerging virus will continue to be detected at new sites (the discrete reproductive power of this outbreak is 0.35). Phylogenetically distinct CMTV-like ranaviruses were found in the south of the Netherlands more recently. In addition to showing that CMTV-like ranaviruses threaten wild amphibian populations not only in Spain but also in the Netherlands, the current spread and risk of establishment reiterate that understanding the underlying causes of CMTV-like ranavirus emergence requires international attention.

Foot and mouth disease vaccine potency testing: The influence of serotype, type of adjuvant, valency, fractionation method, and virus culture on the dose–response curve in cattle

The aim of this study was to determine a relationship between vaccine potency (amount of PD50 per dose) and fraction of clinically protected cattle following homologous challenge with infectious foot-and-mouth disease (FMD) virus, and to determine the effect of method of fractionation, serotype, type of adjuvant, valency and type of virus culture on the dose-response curve. Data from 297 potency tests of FMD vaccines, comprising 4004 vaccinated cattle, performed at the FMD vaccine production facility in the Netherlands, were used for the present study. A generalised linear mixed effect model was used to analyse the results. Our study showed that the relation between FMD vaccine potency and fraction protected was also affected by the serotype and type of adjuvant. No common level of protection could be assigned to all FMD vaccines with the same amount of PD50 per dose, this information is essential when designing a new standard FMD vaccines control.

Silent spread of highly pathogenic Avian Influenza H5N1 virus amongst vaccinated commercial layers

The aim of this study was to determine whether a single vaccination of commercial layer type chickens with an inactivated vaccine containing highly pathogenic avian influenza virus strain H5N1 A/chicken/Legok/2003, carried out on the farm, was sufficient to protect against infection with the homologous virus strain. A transmission experiment was carried out with pairs of chicken of which one was inoculated with H5N1 virus and the other contact-exposed. Results showed that the majority of the vaccinated birds developed haemagglutination inhibition (HI) titres below 4log2. No clinical signs were observed in the vaccinated birds and virus shedding was limited. However, nearly all vaccinated birds showed a four-fold or higher increase of HI titres after challenge or contact-exposure, which is an indication of infection. This implies that virus transmission most likely has occurred. This study showed that a single vaccination applied under field conditions induced clinical protection, but was insufficient to induce protection against virus transmission, suggesting that silent spread of virus in vaccinated commercial flocks may occur.

A prospective study on fitness, workload and reasons for premature training ends and temporary training breaks in two groups of riding horses.

Little is known about wastage in riding horses and the factors like fitness and workload that may reduce injuries and maximise welfare. To evaluate fitness, workload and reasons for premature training ends (PTEs) and temporary training breaks (TTBs) during a nine week training period, two groups of riding horses were used: Group A consisting of 58 horses used for student equitation courses (32 with training prior to admission and 26 without) and Group B consisting of 26 horses owned by two riding schools (school-I and school-II). To assess fitness, all horses performed a standardised exercise test (SET) at the start (SET-I) and end of the training period (SET-II) measuring heart rate (HR bpm) and speed (m/s). In addition, all horses were monitored daily during the training period for their health and workload. In Group A, trained horses had significantly lower HRs in SET-I (P=0.05) compared to untrained horses and in SET-II, trained horses tended to have lower HRs than untrained horses, though this was not statistically significant (P=0.057). During the training period all horses received an identical workload. A total of 19.0% of Group A horses ended the training period prematurely for veterinary reasons (PTEV); of those untrained horses had earlier a PTEV in the training period (after 2.8 ± 1.3 weeks) than trained horses (after 4.1 ± 1.5 weeks, P=0.030). In Group B, school-I and school-II horses did not differ significantly in fitness level nor in workload. More school-II horses ended the training period prematurely for veterinary reasons (n=7; 70%) compared to school-I horses (n=4; 25%, P=0.032), although seven (63.6%) of these horses were still continuously used in riding lessons. In both groups (A and B), small injuries (without a temporary training break) were significantly associated with premature training ends for veterinary reasons later on: in Group A small injuries preceded 27.3% of the PTEVs (P=0.005) and in Group B small injuries preceded 54.5% of the PTEVs (P=0.030). In conclusion, as all horses in each subgroup had the same workload, the occurrence of PTEV seemed not associated with the workload. In Group A horses, level of fitness seems to be an important factor for the point in time injuries will occur during the training period. In all horses, injuries were more likely when a temporary training break was not taken following seemingly minor injuries. Since a lot of injured Group B horses were used in riding lessons against veterinary advice, this may indicate that riding school owners have different perception on welfare and if true this may cause serious welfare problems.

Quality improvement intervention to increase adherence to ART prescription policy at HIV treatment clinics in Lusaka, Zambia: A cluster randomized trial

Introduction In urban areas, crowded HIV treatment facilities with long patient wait times can deter patients from attending their clinical appointments and picking up their medications, ultimately disrupting patient care and compromising patient retention and adherence. Methods Formative research at eight facilities in Lusaka revealed that only 46% of stable HIV treatment patients were receiving a three-month refill supply of antiretroviral drugs, despite it being national policy for stable adult patients. We designed a quality improvement intervention to improve the operationalization of this policy. We conducted a cluster-randomized controlled trial in sixteen facilities in Lusaka with the primary objective of examining the intervention’s impact on the proportion of stable patients receiving three-month refills. The secondary objective was examining whether the quality improvement intervention reduced facility congestion measured through two proxy indicators: daily volume of clinic visits and average clinic wait times for services. Results The mean change in the proportion of three-month refills among control facilities from baseline to endline was 10% (from 38% to 48%), compared to a 25% mean change (an increase from 44% to 69%) among intervention facilities. This represents a significant 15% mean difference (95% CI: 2%-29%; P = 0.03) in the change in proportion of patients receiving three-month refills. On average, control facilities had 15 more visits per day in the endline than in the baseline, while intervention facilities had 20 fewer visits per day in endline than in baseline, a mean difference of 35 fewer visits per day (P = 0.1). The change in the mean facility total wait time for intervention facilities dropped 19 minutes between baseline and endline when compared to control facilities (95% CI: -10.2–48.5; P = 0.2). Conclusion A more patient-centred service delivery schedule of three-month prescription refills for stable patients is viable. We encourage the expansion of this sustainable intervention in Zambia’s urban clinics.

Flow cytometric measurement of neutrophil alkaline phosphatase before and during initiation of an induced Escherichia coli mastitis in cattle

In 12 healthy cows, neutrophil alkaline phosphatase (NAP) activity was measured by flow cytometer before and during an experimentally induced Escherichia coli mastitis, to study the role and increase of NAP in Gram-negative bacterial infections. Percentage of neutrophils containing alkaline phosphatase and intensity of NAP activity were measured. Preinfection percentage of neutrophils with enzyme activity varied between 64.0% and 84.4% and the intensity of enzyme activity was low in all cows. After induction of infection, percentage of neutrophils with enzyme activity showed a significant decrease on day 1 followed by an significant increase on day 3. NAP intensity increased significantly on the second and third day after infection. This increase of intensity was significantly, positively correlated with the severity of infection. From this study we may conclude that variation in susceptibility to E. coli mastitis could not be explained by preinfection NAP levels. The post-infection increase of NAP activity, that was found following an induced infection was more a result of increased enzyme intensity per neutrophil, then from an increase of percentage neutrophils with enzyme activity. Furthermore, a strong correlation was found between NAP intensity and severity of inflammation. There was evidence that the more severely diseased animals showed stronger NAP intensity increase.

Young Friesian horses show familial aggregation in fitness response to a 7-week performance test

The aim of this study was to monitor the fitness level of young Friesian horses and to assess whether fitness data are predictive for final performance score and whether familial aggregation of response to training could be detected. Sixty-six young Friesian horses, the offspring of six different stallions (A, B, C, D, E and F), underwent a 7-week performance test. The horses were given a performance score for their ability for dressage (in weeks 5 and 7; 0-110 points) and were evaluated for fitness using standardised exercise tests (SETs) at the beginning (week 2, SET-I) and the end (week 6, SET-II) of the period. Heart rate (HR, beats/min) was measured in both SETs, and plasma lactate concentration (LA, mmol/L) was measured only in SET-II. Fitness of the horses improved moderately but significantly between SET-I and SET-II (P=0.015). There was a large heterogeneity in responsiveness to training; some horses were high responders, whereas others were non- or low responders. There was a familial aggregation of HR canter-1 response to training (P=0.039), while the HR of stallion Cs offspring decreased significantly more than those of stallions A (P=0.09), D (P=0.013) and F (P=0.009). Horses that were reluctant to exercise did not differ in HR or LA concentrations compared to those that completed the SET, which may have been a sign of overreaching. HR had no predictive value for the performance score, but horses that did not reach the anaerobic threshold in SET-II scored significantly better (73.8 ± 5.6 points) than horses that did not (69.9 ± 5.9 points, P=0.025). The findings demonstrate for the first time in the horse a familial aggregation of HR response to training, as has been reported previously in humans. Familial aggregation suggests a genetic influence on the effect of training on fitness in horses. HR could not predict final performance score, but LA concentrations during SET-II were predictive.

Monitoring ranavirus-associated mortality in a Dutch heathland in the aftermath of a ranavirus disease outbreak

Abstract The ranaviruses are an emerging group of viruses that infect amphibians, fish, and reptiles. Although ranaviruses have not been linked to extinctions, emergence in amphibian communities has resulted in population declines for some species. We present the results of ranavirus-associated mortality in a Dutch national park in the aftermath of an outbreak associated with a common midwife toad virus (CMTV)–like ranavirus. We monitored five bodies of water across Dwingelderveld National Park, the Netherlands, in 2011–13. Dead and live amphibians were counted weekly July–September and every 2 wk in June and October. Dead amphibians were collected and tested for ranavirus infection. In addition, we measured biologic, chemical, and physical site characteristics to test for a correlation with ranavirus-associated mortality. Ranavirus infection was widespread in our study area and we observed nearly continuous presence of dead, ranavirus-infected amphibians in the presence of asymptomatic, live amphibians throughout our study. Fatalities occurred in larval, subadult, and adult amphibians. Ranavirus infection prevalence (based on fatal cases) was significantly associated with increasing fractions of adults and subadults compared to juveniles and larvae in the population, but was unrelated to any other measured site characteristics. Our findings showed that a CMTV-like ranavirus can persist long term in an ecosystem, affecting a diversity of amphibian species and life stages for a prolonged period. This study illustrates the importance of monitoring the modes of spread for ranaviruses and their impact on amphibian populations.

Ranavirus genotypes in the Netherlands and their potential association with virulence in water frogs (Pelophylax spp.)

Ranaviruses are pathogenic viruses for poikilothermic vertebrates worldwide. The identification of a common midwife toad virus (CMTV) associated with massive die-offs in water frogs (Pelophylax spp.) in the Netherlands has increased awareness for emerging viruses in amphibians in the country. Complete genome sequencing of 13 ranavirus isolates collected from ten different sites in the period 2011–2016 revealed three CMTV groups present in distinct geographical areas in the Netherlands. Phylogenetic analysis showed that emerging viruses from the northern part of the Netherlands belonged to CMTV-NL group I. Group II and III viruses were derived from the animals located in the center-east and south of the country, and shared a more recent common ancestor to CMTV-amphibian associated ranaviruses reported in China, Italy, Denmark, and Switzerland. Field monitoring revealed differences in water frog host abundance at sites where distinct ranavirus groups occur; with ranavirus-associated deaths, host counts decreasing progressively, and few juveniles found in the north where CMTV-NL group I occurs but not in the south with CMTV-NL group III. Investigation of tandem repeats of coding genes gave no conclusive information about phylo-geographical clustering, while genetic analysis of the genomes revealed truncations in 17 genes across CMTV-NL groups II and III compared to group I. Further studies are needed to elucidate the contribution of these genes as well as environmental variables to explain the observed differences in host abundance.