Within-farm transmission characteristics of bluetongue virus serotype 8 in cattle and sheep in the Netherlands, 2007-2008.

In 2006 and 2007, sheep and cattle farms in the Netherlands were affected by an epidemic of bluetongue virus serotype 8 (BTV-8). In order to obtain insight into the within-farm spread of the virus, five affected cattle and five affected sheep farms were longitudinally monitored between early 2007 and mid or late 2008. The farms were visited between four and seven times to collect blood samples. During each visit, all animals present in the flock or herd were sampled. The samples were analysed for the presence of BTV-8 antibodies (ELISA) and BTV-8 antigen (rRT-PCR). The observed patterns of RT-PCR positives indicate a rapid within-farm virus spread during the vector season. During vector-free periods we observed a complete rRT-PCR positivity decline within a few months. During the vector season a lower bound estimate of the basic reproduction number (R0) ranges from 2.9-6.9 in the cattle herds (one herd not analysed), and from 1.3-3.2 in the sheep flocks in this study.

Exploring contacts facilitating transmission of influenza A(H5N1) virus between poultry farms in West Java, Indonesia: A major role for backyard farms?

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been reported in Asia, including Indonesia since 2003. Although several risk factors related to the HPAIV outbreaks in poultry in Indonesia have been identified, little is known of the contact structure of farms of different poultry production types (backyard chickens, broilers, layers, and ducks). This study aims to quantify the contact rates associated with the movement of people, and movements of live birds and products and equipment that affect the risk of HPAIV H5N1 transmission between poultry farms in Indonesia. On 124 poultry farms in 6 districts in West Java, logbooks were distributed to record the movements of farmers/staff and visitors and their poultry contacts. Most movements in backyard chicken, commercial native chicken, broiler and duck farms were visits to and from other poultry farms, whilst in layer farms visits to and from poultry companies, visits to egg collection houses and visit from other poultry farms were most frequent. Over 75% of persons visiting backyard chicken and duck farms had previously visited other poultry farms on the same day. Visitors of backyard chicken farms had the highest average contact rate, either direct contact with poultry on other farms before the visits (1.35 contact/day) or contact during their visits in the farms (10.03 contact/day). These results suggest that backyard chicken farms are most at risk for transmission of HPAIV compared to farms of the other poultry production types. Since visits of farm-to-farm were high, backyard farms could also a potential source for HPAIV transmission to commercial poultry farms.

Effect of Simultaneous Exposure of Pigs to Streptococcus suis Serotypes 2 and 9 on Their Colonization and Transmission, and on Mortality.

The distribution of Streptococcus suis serotypes isolated from clinically infected pigs differs between geographical areas, and varies over time. In several European countries, predomination of serotype 2 has changed to serotype 9. We hypothesize a relation, with one serotype affecting the other in colonization and invasion. The aim of this study was to evaluate whether simultaneous exposure of pigs to serotypes 2 and 9 affects colonization and transmission of each type, and mortality. Thirty-six caesarean-derived/colostrum-deprived piglets were randomly assigned to three groups, and there housed pair-wise. At six weeks old, one pig per pair was inoculated with either one (serotype 2 or 9; mono-group) or two serotypes simultaneously (dual-group); the other pig was contact-exposed. Tonsillar and nasal samples were collected within three weeks post inoculation. Bacterial loads in samples were quantified using multiplex real-time polymerase chain reaction (PCR). Transmission rates of the serotypes among pigs were estimated using a mathematical Susceptible-Infectious (SI) model. Bacterial loads and transmission rates did not differ significantly between serotypes. Compared to the mono-group, in the dual-group the average serotype 2 load in tonsillar samples from contact pigs was reduced on days 1 to 4 and on day 6. Simultaneous exposure to the serotypes reduced the mortality hazard 6.3 times (95% C.I.: 2.0-19.8) compared to exposure to serotype 2 only, and increased it 6.6 times (95% C.I.: 1.4-30.9) compared to exposure to serotype 9 only. This study indicates that serotype 2 load and mortality were affected in pigs exposed to these two serotypes.

Different cross protection scopes of two avian influenza H5N1 vaccines against infection of layer chickens with a heterologous highly pathogenic virus.

Avian influenza (AI) virus strains vary in antigenicity, and antigenic differences between circulating field virus and vaccine virus will affect the effectiveness of vaccination of poultry. Antigenic relatedness can be assessed by measuring serological cross-reactivity using haemagglutination inhibition (HI) tests. Our study aims to determine the relation between antigenic relatedness expressed by the Archetti-Horsfall ratio, and reduction of virus transmission of highly pathogenic H5N1 AI strains among vaccinated layers. Two vaccines were examined, derived from H5N1 AI virus strains A/Ck/WJava/Sukabumi/006/2008 and A/Ck/CJava/Karanganyar/051/2009. Transmission experiments were carried out in four vaccine and two control groups, with six sets of 16 specified pathogen free (SPF) layer chickens. Birds were vaccinated at 4weeks of age with one strain and challenge-infected with the homologous or heterologous strain at 8weeks of age. No transmission or virus shedding occurred in groups challenged with the homologous strain. In the group vaccinated with the Karanganyar strain, high cross-HI responses were observed, and no transmission of the Sukabumi strain occurred. However, in the group vaccinated with the Sukabumi strain, cross-HI titres were low, virus shedding was not reduced, and multiple transmissions to contact birds were observed. This study showed large differences in cross-protection of two vaccines based on two different highly pathogenic H5N1 virus strains. This implies that extrapolation of in vitro data to clinical protection and reduction of virus transmission might not be straightforward.

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.

Effect of spatial separation of pigs on spread of Streptococcus suis serotype 9.

The spread of an infectious agent in a population can be reduced by interfering in the infectiousness or susceptibility of individuals, and/or in their contact structure. The aim of this study was to quantify the effect of prevention of direct contact between infectious and susceptible pigs on the transmission of Streptococcus suis (S. suis). In three replicate experiments, S. suis-free pigs were housed in boxes either in pairs (25 pairs) or alone (15 pigs). The distance between the boxes was ±1 m. At 7 weeks of age, one pig of each pair was inoculated intranasally with S. suis serotype 9; the other pigs were exposed to S. suis by either direct (pairs) or indirect contact (individually housed pigs). Tonsillar brush and saliva swab samples from all pigs were collected regularly for 4 weeks post inoculation to monitor colonization with S. suis. All inoculated pigs became infected, and their pen mates became colonized within 2 days. Thirteen indirectly exposed pigs became positive within 7-25 days after exposure. The rate of direct transmission ßdir was estimated to be 3.58 per pig per day (95% CI: 2.29-5.60). The rate of indirect transmission increased in time, depending on the cumulative number of days pigs tested positive for the presence of S. suis. The estimate ß'ind was 0.001 (95% CI: 0.0006-0.0017) new infections per pig per day for each day that an infected pig was tested positive for S. suis. We conclude that prevention of direct contact reduces the rate at which susceptible pigs become colonized. Simulation studies using these parameters showed, however, that such intervention measure would not limit S. suis serotype 9 spread in a commercial pig farm to a relevant extent, implying that spatial separation of groups op pigs within a compartment would not be effective on a farm.

Association between transmission rate and disease severity for Actinobacillus pleuropneumoniae infection in pigs.

A better understanding of the variation in infectivity and its relation with clinical signs may help to improve measures to control and prevent (clinical) outbreaks of diseases. Here we investigated the role of disease severity on infectivity and transmission of Actinobacillus pleuropneumoniae, a bacterium causing respiratory problems in pig farms. We carried out transmission experiments with 10 pairs of caesarean-derived, colostrum-deprived pigs. In each pair, one pig was inoculated intranasally with 5×10(6) CFUs of A. pleuropneumoniae strain 1536 and housed together with a contact pig. Clinical signs were scored and the course of infection was observed by bacterial examination and qPCR analysis of tonsillar brush and nasal swab samples. In 6 out of 10 pairs transmission to contact pigs was observed, but disease scores in contact infected pigs were low compared to the score in inoculated pigs. Whereas disease score was positively associated with bacterial load in inoculated pigs and bacterial load with the transmission rate, the disease score had a negative association with transmission. These findings indicate that in pigs with equal bacterial load, those with higher clinical scores transmit A. pleuropneumoniae less efficiently. Finally, the correlation between disease score in inoculated pigs and in positive contact pigs was low. Although translation of experimental work towards farm level has limitations, our results suggest that clinical outbreaks of A. pleuropneumoniae are unlikely to be caused only by spread of the pathogen by clinically diseased pigs, but may rather be the result of development of clinical signs in already infected pigs.

Quantification of dust-borne transmission of highly pathogenic avian influenza virus between chickens.

BACKGROUND: Understanding the transmission of highly pathogenic avian influenza virus (HPAIv) between poultry flocks is essential to prevent and control epidemics. Dust, produced in infected chicken flocks, has been hypothesized to be an important mechanical vector for between-flock transmission of HPAIv. OBJECTIVES: The aim of our study was to quantify the amount of virus shed by infected birds and its relation to deposition of virus in the environment and the rate of dust-borne transmission between groups of chickens. METHODS: Four replicate experiments were performed, each replicate with two groups of 14 chickens housed in two separate rooms. In one group, chickens were inoculated with HPAIv. Ventilation forced the air from that room to the second (recipient) group through a tube. Deceased birds in the inoculated group were replaced with new susceptible birds up to day 10 p.i. Dust samples were collected daily. Trachea and cloaca swabs were collected daily to determine virus shedding and virus spread to the recipient group. RESULTS: The amount of virus detected in dust samples in the recipient room was, on average, 10(3·7) EID(50) /m(3) . Virus transmission from the inoculated to the recipient group occurred in two experiments. The transmission rate parameter for dust-borne transmission was estimated at 0·08 new infections/infectious chicken/day. CONCLUSIONS: The results of this study are a first step to elucidate the importance of dust-borne transmission of HPAIv between flocks and help interpret environmental samples.

Experimental inoculation of male rats with Coxiella burnetii: successful infection but no transmission to cage mates.

Beginning in 2007, the largest human Q fever outbreak ever described occurred in the Netherlands. Dairy goats from intensive farms were identified as the source, amplifying Coxiella burnetii during gestation and shedding large quantities during abortions. It has been postulated that wild rodents are reservoir hosts from which C. burnetii can be transmitted to domestic animals and humans. However, little is known about the infection dynamics of C. burnetii in wild rodents. The aim of this study was to investigate whether brown rats (Rattus norvegicus) can be experimentally infected with C. burnetii and whether transmission to a cage mates occurs. Fourteen male brown rats (wild type) were intratracheally or intranasally inoculated with a Dutch C. burnetii isolate obtained from a goat. At 3 days postinoculation, a contact rat was placed with each inoculated rat. The pairs were monitored using blood samples and rectal and throat swabs for 8 weeks, and after euthanasia the spleens were collected. Rats became infected by both inoculation routes, and detection of C. burnetii DNA in swabs suggests that excretion occurred. However, based on the negative spleens in PCR and the lack of seroconversion, none of the contact animals was considered infected; thus, no transmission was observed. The reproduction ratio R(0) was estimated to be 0 (95% confidence interval = 0 to 0.6), indicating that it is unlikely that rats act as reservoir host of C. burnetii through sustained transmission between male rats. Future research should focus on other transmission routes, such as vertical transmission or bacterial shedding during parturition.

Oocyst output and transmission rates during successive infections with Eimeria acervulina in experimental broiler flocks.

The infection dynamics of Eimeria species determine the clinical manifestation of the disease coccidiosis in poultry flocks, and a better understanding of the dynamics may contribute to improvement of control measures. Our aim was to study the course of infection and the transmission of Eimeria acervulina in groups of broilers by quantifying the transmission rate parameter and oocyst output. Three transmission experiments were carried out with groups of 20 male SPF broilers. At 2 days of age, one bird in each trial was orally inoculated with five sporulated E. acervulina oocysts (D0 post-inoculation, pi). One day after inoculation (D1 pi), the inoculated bird was housed with 19 non-inoculated contact birds. Individual faecal droppings were examined daily from D3-D32 pi to quantify the number of oocysts per gram faeces. The inoculated bird started shedding oocysts at D5 pi and contact birds between D10 and D17 pi. Contact birds that became infected due to oocyst excretion by the inoculated bird were characterized as first generation contact birds (C1). Contact birds excreting from D15 pi onwards (C2) became infected after the first C1 birds had started shedding and were considered to belong to a successive generation of the flock infection. Oocyst output was significantly lower for C1 compared to C2 birds, but the transmission rate parameter remained constant for both infection generations. These results suggest that although oocyst load increases, the transmission rate of E. acervulina remains constant between successive generations of infection in a flock.

Transmission of a live Eimeria acervulina vaccine strain and response to infection in vaccinated and contact-vaccinated broilers.

Live vaccines for coccidiosis control are infrequently used in broilers, mainly due to variability in efficacy and relatively high costs. More insight in transmission of vaccine and wild-type strains can facilitate optimization of vaccination strategies and might increase its use as an alternative for anticoccidial drugs. The aim of this study was to quantify transmission of a live Eimeria acervulina vaccine strain and to determine the degree of protection against a subsequent infection with a wild-type E. acervulina strain. An experiment was carried out with 4 groups of 22 SPF broilers. At 2 days of age, 11 birds of groups 2 to 4 were vaccinated directly by oral application of E. acervulina oocysts of the Paracox™ vaccine and 11 birds were placed in contact with these birds (contact-vaccinated). Birds in group 1 remained unvaccinated (controls) and were not exposed to vaccinated birds. At day 28 of age, 6 groups of 10 birds were formed, with 2 groups (duplo) for each treatment group, i.e. vaccinated, contact-vaccinated or unvaccinated control birds. Five birds of each group were orally inoculated with wild-type E. acervulina oocysts and five were contact-exposed. Single droppings were examined daily from days 5 to 49 of age for oocyst output and to determine the time of infection. The transmission rate of the vaccine strain was estimated to be 1.6 per day and of the wild-type strain 2.3, 8.7 and 20.8 per day for vaccinated, contact-vaccinated and unvaccinated birds, respectively. Although transmission of wild-type coccidia was not significantly reduced in vaccinated or contact-vaccinated groups, both groups were equally protected against high oocyst output after infection compared to unvaccinated groups. These results suggest that factors influencing transmission of live vaccine strains in flocks may be important targets for improvement of vaccine efficacy and warrant further research.

Wild birds and increased transmission of highly pathogenic avian influenza (H5N1) among poultry, Thailand.

Since the outbreaks of highly pathogenic avian influenza (HPAI) subtype H5N1 virus, wild birds have been suspected of transmitting this virus to poultry. On January 23, 2004, the Ministry of Public Health in Thailand informed the World Health Organization of an avian influenza A (H5N1) outbreak. To determine the epidemiology of this viral infection and its relation to poultry outbreaks in Thailand from 2004 through 2007, we investigated how wild birds play a role in transmission. A total of 24,712 swab samples were collected from migratory and resident wild birds. Reverse transcription PCR showed a 0.7% HPAI (H5N1) prevalence. The highest prevalence was observed during January-February 2004 and March-June 2004, predominantly in central Thailand, which harbors most of the country's poultry flocks. Analysis of the relationship between poultry and wild bird outbreaks was done by using a nonhomogeneous birth and death statistical model. Transmission efficiency among poultry flocks was 1.7 X higher in regions with infected wild birds in the given or preceding month. The joint presence of wild birds and poultry is associated with increased spread among poultry flocks.

A single vaccination of commercial broilers does not reduce transmission of H5N1 highly pathogenic avian influenza.

Vaccination of chickens has become routine practice in Asian countries in which H5N1 highly pathogenic avian influenza (HPAI) is endemically present. This mainly applies to layer and breeder flocks, but broilers are usually left unvaccinated. Here we investigate whether vaccination is able to reduce HPAI H5N1 virus transmission among broiler chickens. Four sets of experiments were carried out, each consisting of 22 replicate trials containing a pair of birds. Experiments 1-3 were carried out with four-week-old birds that were unvaccinated, and vaccinated at day 1 or at day 10 of age. Experiment 4 was carried out with unvaccinated day-old broiler chicks. One chicken in each trial was inoculated with H5N1 HPAI virus. One chicken in each trial was inoculated with virus. The course of the infection chain was monitored by serological analysis, and by virus isolation performed on tracheal and cloacal swabs. The analyses were based on a stochastic SEIR model using a Bayesian inferential framework. When inoculation was carried out at the 28th day of life, transmission was efficient in unvaccinated birds, and in birds vaccinated at first or tenth day of life. In these experiments estimates of the latent period (~1.0 day), infectious period (~3.3 days), and transmission rate parameter (~1.4 per day) were similar, as were estimates of the reproduction number (~4) and generation interval (~1.4 day). Transmission was significantly less efficient in unvaccinated chickens when inoculation was carried out on the first day of life. These results show that vaccination of broiler chickens does not reduce transmission, and suggest that this may be due to the interference of maternal immunity.

Salmonella enteritidis surveillance by egg immunology: impact of the sampling scheme on the release of contaminated table eggs.

Design of surveillance programs to detect infections could benefit from more insight into sampling schemes. We address the effect of sampling schemes for Salmonella Enteritidis surveillance in laying hens. Based on experimental estimates for the transmission rate in flocks, and the characteristics of an egg immunological test, we have simulated outbreaks with various sampling schemes, and with the current boot swab program with a 15-week sampling interval. Declaring a flock infected based on a single positive egg was not possible because test specificity was too low. Thus, a threshold number of positive eggs was defined to declare a flock infected, and, for small sample sizes, eggs from previous samplings had to be included in a cumulative sample to guarantee a minimum flock level specificity. Effectiveness of surveillance was measured by the proportion of outbreaks detected, and by the number of contaminated table eggs brought on the market. The boot swab program detected 90% of the outbreaks, with 75% fewer contaminated eggs compared to no surveillance, whereas the baseline egg program (30 eggs each 15 weeks) detected 86%, with 73% fewer contaminated eggs. We conclude that a larger sample size results in more detected outbreaks, whereas a smaller sampling interval decreases the number of contaminated eggs. Decreasing sample size and interval simultaneously reduces the number of contaminated eggs, but not indefinitely: the advantage of more frequent sampling is counterbalanced by the cumulative sample including less recently laid eggs. Apparently, optimizing surveillance has its limits when test specificity is taken into account.

Reduction of Coxiella burnetii prevalence by vaccination of goats and sheep, The Netherlands.

Recently, the number of human Q fever cases in the Netherlands increased dramatically. In response to this increase, dairy goats and dairy sheep were vaccinated against Coxiella burnetii. All pregnant dairy goats and dairy sheep in herds positive for Q fever were culled. We identified the effect of vaccination on bacterial shedding by small ruminants. On the day of culling, samples of uterine fluid, vaginal mucus, and milk were obtained from 957 pregnant animals in 13 herds. Prevalence and bacterial load were reduced in vaccinated animals compared with unvaccinated animals. These effects were most pronounced in animals during their first pregnancy. Results indicate that vaccination may reduce bacterial load in the environment and human exposure to C. burnetii.

A comparison of transmission characteristics of Salmonella enterica serovar Enteritidis between pair-housed and group-housed laying hens.

Human cases of bacterial gastro-enteritis are often caused by the consumption of eggs contaminated with Salmonella species, mainly Salmonella enterica serovar Enteriditis (Salmonella Enteritidis). To reduce human exposure, in several countries worldwide surveillance programmes are implemented to detect colonized layer flocks. The sampling schemes are based on the within-flock prevalence, and, as this changes over time, knowledge of the within-flock dynamics of Salmonella Enteritidis is required. Transmission of Salmonella Enteritidis has been quantified in pairs of layers, but the question is whether the dynamics in pairs is comparable to transmission in large groups, which are more representative for commercial layer flocks. The aim of this study was to compare results of transmission experiments between pairs and groups of laying hens. Experimental groups of either 2 or 200 hens were housed at similar densities, and 1 or 4 hens were inoculated with Salmonella Enteritidis, respectively. Excretion was monitored by regularly testing of fecal samples for the presence of Salmonella Enteritidis. Using mathematical modeling, the group experiments were simulated with transmission parameter estimates from the pairwise experiments. Transmission of the bacteria did not differ significantly between pairs or groups. This finding suggests that the transmission parameter estimates from small-scale experiments might be extrapolated to the field situation.

Comparison of Campylobacter levels in crops and ceca of broilers at slaughter.

A considerable fraction of the poultry carcasses becomes contaminated with Campylobacter by cross-contamination from the digestive tract of colonized broilers at slaughter. Campylobacter in the crop may serve as a possible source of cross-contamination, because the crop may contain high numbers of Campylobacter and is more likely to rupture during the slaughtering process than intestines. In this study, the correlation between Campylobacter colonization levels in crop and cecum was assessed in 48 broilers of 31 days of age. In addition, the effect of drinking water supplemented with 0.2% volatile fatty acid (VFA) on these Campylobacter colonization levels was studied. No correlation between crop and cecal colonization levels was found (p = 0.09; P = 0.71), indicating that future studies on cross-contamination should include an examination of not only cecal colonization levels but also crop colonization levels. Supplementation of drinking water with VFA did not result in a significant reduction of colonization levels in either the crop (P = 0.50) or the ceca (P = 0.92), indicating that this is not an effective measure to reduce cross-contamination at slaughter.

Use of epidemiologic models in the control of highly pathogenic avian influenza.

In the past decades, mathematical models have become more and more accepted as a tool to develop surveillance programs and to evaluate the efficacy of intervention measures for the control of infectious diseases such as highly pathogenic avian influenza. Predictive models are used to simulate the effect of various control measures on the course of an epidemic; analytical models are used to analyze data from outbreaks or from experiments. A key parameter in both types of models is the reproductive ratio, which indicates whether virus can be transmitted in the population, resulting in an epidemic, or not. Parameters obtained from real data using the analytical models can subsequently be used in predictive models to evaluate control strategies or surveillance programs. Examples of the use of these models are described here.