Is Mycoplasma synoviae outrunning Mycoplasma gallisepticum? A viewpoint from the Netherlands.

Mycoplasma gallisepticum and M. synoviae are the most relevant mycoplasma species for commercial poultry from the clinical and economic point of view. Although the importance of M. gallisepticum was recognized many decades ago, the relevance of M. synoviae has been a matter of debate. Until the turn of the century, only the respiratory and synovitis forms of the disease were reported, while the majority of infections were subclinical. Since the year 2000 M. synoviae strains with oviduct tropism, able to induce eggshell apex abnormalities and egg drops, have been encountered worldwide. A decreasing incidence of M. gallisepticum has been observed, at least in breeding stock, in countries with control and eradication programmes for this Mycoplasma species. In contrast, the sero-prevalence of M. synoviae is much higher, especially in layer flocks, and in most continents exceeds 70%. Given the emergence of virulent M. synoviae strains with oviduct tropism, its ability to also induce joint and respiratory disease, to act synergistically with other pathogens as well as its much higher sero-prevalence, it seems that M. synoviae is outrunning M. gallisepticum, at least in countries with control and eradication programmes for the latter. This stresses the need to update M. synoviae prevention and control strategies. Thus, in January 2013, the Dutch poultry industry implemented a mandatory control and eradication programme for M. synoviae at all levels of poultry farming with the exception of broilers.

Suitability of differently formulated dry powder Newcastle disease vaccines for mass vaccination of poultry.

Dry powders containing a live-attenuated Newcastle disease vaccine (LZ58 strain) and intended for mass vaccination of poultry were prepared by spray drying using mannitol in combination with trehalose or inositol, polyvinylpyrrolidone (PVP) and/or bovine serum albumin (BSA) as stabilizers. These powders were evaluated for vaccine stabilizing capacity during production and storage (at 6 °C and 25 °C), moisture content, hygroscopicity and dry powder dispersibility. A mixture design, varying the ratio of mannitol, inositol and BSA, was used to select the stabilizer combination which resulted in the desired powder properties (i.e. good vaccine stability during production and storage, low moisture content and hygroscopicity and good dry dispersibility). Inositol-containing powders had the same vaccine stabilizing capacity as trehalose powders, but were less hygroscopic. Incorporation of BSA enhanced the vaccine stability in the powders compared to PVP-containing formulations. However, increasing the BSA concentration increased the hygroscopicity and reduced the dry dispersibility of the powder. No valid mathematical model could be calculated for vaccine stability during production or storage, but the individual experiments indicated that a formulation combining mannitol, inositol and BSA in a ratio of 73.3:13.3:13.3 (wt/wt) resulted in the lowest vaccine titre loss during production (1.6-2.0 log(10) 50% egg infectious dose (EID(50)) and storage at 6 °C (max. 0.8 log(10) EID(50) after 6 months) in combination with a low moisture content (1.1-1.4%), low hygroscopicity (1.9-2.1% water uptake at 60% relative humidity) and good dry dispersibility properties.

High seroprevalence of Histomonas meleagridis in Dutch layer chickens.

Histomona meleagridis is a protozoan parasite that may cause outbreaks of histomonosis with high mortality, especially in turkey flocks. Chickens are less susceptible to the disease than are turkeys, but are considered to act as an important reservoir. To determine the seroprevalence of H. meleagridis in Dutch layer chicken flocks, a large scale seroepidemiologic study (3376 samples) was performed by sampling 12 organic flocks, 24 free-ranging flocks, 40 flocks with floor housing, and 40 flocks with cage housing. At the end of the laying period, approximately 30 blood samples per flock were collected for serology. The seroprevalence found was high. In every flock, at least one of the samples tested positive while in 87% of the flocks, at least one of the samples was strongly positive. There were no significant statistical differences in seropositivity between the housing types. To confirm the enzyme-linked immunosorbent assay (ELISA) results, a small-scale seroepidemiologic study (576 samples) was performed in 29 additional layer chicken flocks kept in different housing systems. Subsequently, a subset of five seropositive flocks was selected. Five birds were obtained from each of these flocks in order to detect the parasite using culture and PCR. In all five flocks, H. meleagridis was either isolated from (culture), detected in (PCR), or both, the birds sampled. Together with the previously performed validation studies, the latter results confirm that the positive ELISA serology found is genuine. We conclude that the seroprevalence of H. meleagridis in layers is, as anticipated, high.

Assessment of the antihistomonal effect of paromomycin and tiamulin.

Histomonosis, a parasitic disease of galliformes and sporadically of other birds caused by Histomonas meleagridis, can result in very high mortality, especially in turkeys. The ban on the last antihistomonal drug prompted an urgent search for alternative prevention and treatment strategies. As both paromomycin and tiamulin have been reported to have antihistomonal activity, these antibiotics were investigated in vitro by adding two-fold serial dilutions ranging from 12.5 to 400 microg/mL to cultures of H. meleagridis. Controls (no antibiotics, or 12.5 microg or 400 microg/mL dimetridazole) were included. Parasites were counted after 3, 20, 28, 44, 51, and 71 hours of incubation. Tiamulin did not have a clear antihistomonal effect, but paromomycin had an inhibitory effect at all concentrations tested. The latter antibiotic was subsequently examined in an in vivo study. Five groups of 20 1-day-old poults, matched by weight and sex, were either not treated (infected and uninfected control groups) or treated with paromomycin (100, 200, or 400 ppm) added to their feed. After 2 weeks all groups, except for the uninfected control group, were intracloacally inoculated with 200,000 histomonads per bird. A clear dose-response effect was found for paromomycin. In the 100-ppm paromomycin group, mortality was similar to that in the untreated control group, whereas about half of the birds died in the 200-ppm paromomycin group; almost complete protection against histomonosis was seen in the 400-ppm paromomycin group. This study shows that paromomycin supplied in feed at 400 ppm is a potentially preventive strategy against H. meleagridis.

Development of a blocking-ELISA for the detection of antibodies against Histomonas meleagridis in chickens and turkeys.

Histomonosis is an infectious disease of mainly galliform birds that can cause high mortality, especially in commercial turkey flocks. Several diagnostic tools were available to detect its causative agent, the flagellated protozoan Histomonas meleagridis. However, serological tools were largely lacking. Recently, an indirect ELISA has been described, but this test was not validated for specificity and might suffer from cross-reactivity with related protozoa. Therefore, a specific blocking-ELISA for the detection of antibodies against H. meleagridis in chicken and turkey sera was developed. For this purpose, monoclonal antibodies were raised against a detergent extracted protein of H. meleagridis. These MAbs bound to morphologically identified histomonads in liver tissue of an infected turkey. The MAbs were conjugated with horseradish peroxidase, after which the most reactive conjugate was used to set-up the blocking-ELISA. Experimentally infected turkeys (n=9) and chickens (n=10) seroconverted in the blocking-ELISA within 2-4 weeks following inoculation with a H. meleagridis field strain. The MAb did not bind to Tetratrichomonas gallinarum antigen and experimentally inoculated T. gallinarum seropositive layer chickens (n=18) showed the same inhibition percentages in the blocking-ELISA as negative control birds did. Therefore, it was concluded that H. meleagridis blocking-ELISA does not cross-react with T. gallinarum, which is a closely related protozoan frequently occurring in poultry. The repeatability and reproducibility of the H. meleagridis blocking-ELISA were high. The new blocking-ELISA is a promising tool for experimental studies; however, further validation of this test with field samples is necessary before it can be used for diagnostic purposes.

Molecular identification and phylogenetic relationships of trichomonad isolates of galliform birds inferred from nuclear small subunit rRNA gene sequences.

Histomonas meleagridis is the etiological agent of histomonosis or blackhead disease. Recently, genotyping, based on polymerase chain reaction and sequencing of internal transcribed spacer-1 sequences was applied to various isolates originating from fowl. Three genotypes were described: types I and II isolates were associated with clinical disease and probably derived from H. meleagridis, whereas, type III isolates were not disease-associated and likely corresponded to Parahistomonas wenrichi according to morphological observations. However, this latter species has never been characterized at the molecular level and its phylogenetic relationships with other parabasalids remained hypothetical. To confirm the identification of these isolates, small subunit rRNA gene sequences were obtained from representatives of types I, II, and III and analyzed in a broad phylogeny including 64 other parabasalid sequences. From our phylogenetic trees, we confirmed that types I and II isolates were closely related, if not identical, to H. meleagridis, while type III isolates represented P. wenrichi. Both species clustered together with high support. This grouping suggested that speciation leading to these two species inhabiting the same hosts and ecological niche occurred recently in birds. In addition, speciation was likely followed by loss of pathogenicity in P. wenrichi.

Progression of lesions in the respiratory tract of broilers after single infection with Escherichia coli compared to superinfection with E. coli after infection with infectious bronchitis virus.

The progression of Escherichia coli lesions was studied in the respiratory tract of 4-week-old commercial broilers. Lesions were induced after a single intratracheal E. coli infection, and after an infection with E. coli preceded 5 days earlier by an oculo-nasal and intratracheal infectious bronchitis virus (IBV) infection of either the virulent M41 strain or the H120 vaccine strain. Trachea, lung and thoracic airsac lesions were examined macroscopically and microscopically. Tissue samples were taken at 3h post-inoculation (hpi), and 1, 2, 4 and 7 days post-inoculation (dpi) with E. coli. The location of both pathogens was assessed by immunohistochemistry. Single E. coli inoculation induced pneumonia and airsacculitis; in case it was preceded by IBV infection, the same macroscopical lesions and also viral tracheitis were found. No clear difference existed between the single and dual infected birds with respect to inflammatory reactions in the lung, which had disappeared within 7 days, except for the presence of more follicles in dual infected birds. IBV antigen was detected in secondary bronchi and airsacs up to 2 dpi and in the trachea up to 4 dpi. E. coli bacteria were found in the tracheal lumen included in purulent material, the parabronchi and airsacs. In lung tissue E. coli antigen was found up to 4 dpi. No clear difference existed between single and dual inoculated birds regarding the presence of E. coli in the lung. In the airsacs, a few bacteria were found from 0.5 hpi up to 4 dpi in E. coli and IBV-E. coli inoculated birds. Although both pathogens were cleared beyond detection at 7 dpi, in IBV-E. coli inoculated birds lesions in the airsac persisted, in contrast to broilers inoculated with E. coli only. In the present study it is shown that 4-week-old broilers are not resistant to intratracheal E. coli inoculation, however, these birds can overcome the induced E. coli infection within a short time span. Moreover, a preceding infection with vaccine or virulent IBV does not seem to impair the clearance of E. coli in the respiratory tract of broilers, but rather induces an exaggerated inflammatory response in the airsacs only, which seems to be the mechanism behind the pattern of airsacculitis in commercial poultry in the field.

Seroprevalence of Mycoplasma synoviae in Dutch commercial poultry farms.

Before the year 2000, Mycoplasma synoviae was associated mainly with subclinical respiratory infections in broilers in the Netherlands and was considered to have low clinical and economic impact. The subsequent occurrence of M. synoviae arthritis and amyloid arthropathy, and of eggshell apex abnormalities, has resulted in an increasing demand for M. synoviae-free poultry. Therefore, a cross-sectional seroprevalence study was carried out over a 12-month period during 2005 and 2006. Ten blood samples per farm were generally used because M. synoviae was expected to spread quickly. However, for grandparent and layer breeder stock, 24 to 60 blood samples per house were available from a voluntary M. synoviae monitoring programme. Sera were tested by means of the rapid plate agglutination test (agglutination at dilution > or =1:8 was considered positive). The numbers of farms sampled out of the national total were: broiler grandparent, 53/53; broiler parent rearing, 34/150; broiler parent, 114/300; broiler, 185/800; layer grandparent, 13/13; layer parent, 40/50; layer, 173/1250; and meat turkey, 50/75. The seroprevalence of M. synoviae in commercial poultry was high, especially in commercial layers where it was 73% (95% confidence interval (CI)=67 to 80); in layer and broiler grandparent stock, the seroprevalence was 0% and 10%, respectively, based on sample sizes equal to the population size. In layer and broiler parent farms, the seroprevalence was 25% (95% CI=19 to 31) and 35% (95% CI=28 to 44); in both broiler parent rearing and broiler farms it was 6% (95% CI=0 to 13 and 95% CI=3 to 9); and in meat turkey, the seroprevalence was 16% (95% CI=10 to 22).

In vitro effect of herbal products against Histomonas meleagridis.

The ban on chemotherapeutics against Histomonas meleagridis in the European Union has left turkey producers without an effective treatment against histomonosis. It has encouraged the development of alternative control methods amongst which are a number of herbal products. In the present study the in vitro effect of four herbal products against H. meleagridis was examined. After suspension of the herbal products in Dwyer's culture medium used for subculturing of a viable culture, parasite growth was monitored by frequently counting the histomonads until 72h of incubation. The solid products Aromabiotictrade mark, Enteroguardtrade mark, and Protophyt SPtrade mark as well as the positive control products dimetridazole and Histostat-50trade mark, were tested in twofold serial dilutions ranging in concentration from 400 to 12.5mug/mL, while the liquid product Protophyt Btrade mark was tested in concentrations ranging from 0.24% to 0.008% (v/v). The herbal products Enteroguardtrade mark and Protophyt Btrade mark as well as dimetridazole and Histostat-50trade mark, two chemotherapeutics with known antihistomonal activity, inhibited the growth of H. meleagridis, while no antihistomonal effect was found for Aromabiotictrade mark and Protophyt SPtrade mark.

In vivo effect of herbal products against Histomonas meleagridis in turkeys.

Histomoniasis is a serious disease in poultry. All chemotherapeutics with known efficacy against its causative agent, Histomonas meleagridis, have been banned from use as prophylactic or therapeutic use in production animals. In a search for possible alternatives, the in vivo effects of the herbal products Enteroguard and Protophyt were examined. Two-week-old turkeys allocated into 13 groups of 18 birds were either sham inoculated (negative control group) or were inoculated with 100, 3162 or 200 000 histomonads per bird. Control groups (no feed additives, dimetridazole, or Histostat-50) were included in the study. No morbidity or mortality was observed in the negative control group or in the groups inoculated with 100 histomonads per bird. Mortality was 100% in the groups inoculated with 200 000 histomonads per bird and either untreated (positive control group) or receiving Protophyt SP, Protophyt SP and Protophyt B, Enteroguard, or Histostat-50. Mortality was 17% in the dimetridazole-treated group. In the groups inoculated with 3162 histomonads per bird, mortality was 100% for the positive control group and the group receiving Enteroguard, and was 94% in the group receiving Protophyt SP. In the present study, Enteroguard or Protophyt was not found to be effective against histomoniasis.

Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both.

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed "B. pulli" also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.

Detection and quantification of classical swine fever virus in air samples originating from infected pigs and experimentally produced aerosols.

During epidemics of classical swine fever (CSF), neighbourhood infections occurred where none of the 'traditional' routes of transmission like direct animal contact, swill feeding, transport contact or transmission by people could be identified. A hypothesized route of virus introduction for these herds was airborne transmission. In order to better understand this possible transmission route, we developed a method to detect and quantify classical swine fever virus (CSFV) in air samples using gelatine filters. The air samples were collected from CSFV-infected pigs after experimental aerosolization of the virus. Furthermore, we studied the viability of the virus with time in aerosolized state. Three strains of CSFV were aerosolized in an empty isolator and air samples were taken at different time intervals. The virus remained infective in aerosolized state for at least 30 min with half-life time values ranging from 4.5 to 15 min. During animal experiments, concentrations of 10(0.3)-10(1.6)TCID(50)/m(3) CSFV were detected in air samples originating from the air of the pig cages and 10(0.4)-10(4.0)TCID(50)/m(3) from the expired air of infected animals. This is the first study describing the isolation and quantification of CSFV from air samples originating from infected pigs and their cages, supporting previous findings that airborne transmission of CSF is feasible.

Improved culture of Histomonas meleagridis in a modification of Dwyer medium.

Dwyer medium is the most frequently employed culture medium for Histomonas meleagridis. Both for subculturing and for resuscitation of H. meleagridis from storage in liquid nitrogen, modified Dwyer medium with an increased rice powder concentration (0.8%) and no chicken embryo extract proved superior to Dwyer standard medium, with threefold (10(6.3) vs. 10(5.8) histomonads/ml) to 10-fold (10(6.7) vs. 10(5.8) histomonads/ml) higher concentrations of parasites after resuscitation or subculturing, respectively.

Detection of egg yolk antibodies reflecting Salmonella enteritidis infections using a surface plasmon resonance biosensor.

A surface plasmon resonance (SPR) biosensor assay was developed on the basis of a lipopolysaccharide antigen of Salmonella enterica serovar enteritidis (S. enterica serovar enteritidis) to detect egg yolk antibodies against S. enterica serovar enteritidis. This biosensor assay was compared to two commercial ELISA kits based on LPS antigen and flagellar antigen. A number of 163 egg yolk and combined egg white and yolk samples from chickens experimentally infected with S. enterica serovar enteritidis and 90 egg yolk and combined egg white and yolk samples from uninfected chickens were analyzed. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 82% and a diagnostic specificity of 100%. The within-day coefficient of variation of a positive internal-control egg yolk was 1%. The SPR biosensor assay was able to detect antibodies in a significantly higher percentage of known positive samples than the commercial ELISA's. The anticipated use of the SPR biosensor assay is to determine the S. enterica serovar enteritidis serostatus of non-vaccinated layer hens.

Genotyping of Histomonas meleagridis isolates based on Internal Transcribed Spacer-1 sequences.

C-profiling is a novel genotyping method for protozoan pathogens, based on polymerase chain reaction and sequencing of AT-rich Internal Transcribed Spacer-1 sequences. It was applied to various Histomonas meleagridis isolates originating from outbreaks of histomoniasis in six Dutch turkey and chicken flocks. Three different H. meleagridis genotypes were identified. Type I and type II were associated with clinical disease. In two flocks, both having recovered from an outbreak of histomoniasis, a type III strain was found that was also morphologically slightly different from the type I and type II isolates. C-profiling is a promising technique to differentiate between H. meleagridis subtypes, making it useful for epidemiological studies.

High yield of parasites and prolonged in vitro culture of Histomonas meleagridis.

Dwyer's medium is a frequently employed culture medium for Histomonas meleagridis with rice powder as an essential ingredient. The effect of adding larger quantities of rice powder to the culture medium and the influence of the size of the rice particles on the growth of H. meleagridis was examined. Increasing the amount of rice powder from the standard amount of 10 to 12 mg to 50 to 100 mg per 12.5 ml medium resulted in approximately a 10-fold increase of parasites. Larger quantities of rice powder did not give better yields. The particle size of the rice powder proved relatively unimportant, although the addition of only large rice powder particles (>250 microm) resulted in a somewhat lesser yield. H. meleagridis cultures could be prolonged from approximately 4 days to at least 2 weeks without subculturing by supplementing the culture medium with rice powder only.

Activated vitronectin as a target for anticancer therapy with human antibodies.

The formation of a provisional extracellular matrix represents an important step during tumor growth and angiogenesis. Proteins that participate in this process become activated and undergo conformational changes that expose biologically active cryptic sites. Activated matrix proteins express epitopes not found on their native counterparts. We hypothesized that these epitopes may have a restricted tissue distribution, rendering them suitable targets for therapeutic human monoclonal antibodies (huMabs). In this study, we exploited phage antibody display technology and subtractive phage selection to generate human monoclonal antibody fragments that discriminate between the activated and native conformation of the extracellular matrix protein vitronectin. One of the selected antibody fragments, scFv VN18, was used to construct a fully human IgG/kappa monoclonal antibody with an affinity of 9.3 nM. In immunohistochemical analysis, scFv and huMab VN18 recognized activated vitronectin in tumor tissues, whereas hardly any activated vitronectin was detectable in normal tissues. Iodine 123-radiolabeled huMabVN18 was shown to target to Rous sarcoma virus-induced tumors in chickens, an animal model in which the epitope for huMab VN18 is exposed during tumor development. Our results establish activated vitronectin as a potential target for tumor therapy in humans.

Immunological basis of differences in disease resistance in the chicken.

Genetic resistance to diseases is a multigenic trait governed mainly by the immune system and its interactions with many physiologic and environmental factors. In the adaptive immunity, T cell and B cell responses, the specific recognition of antigens and interactions between antigen presenting cells, T cells and B cells are crucial. It occurs through a network of mediator proteins such as the molecules of the major histocompatibility complex (MHC), T cell receptors, immunoglobulins and secreted proteins such as the cytokines and antibodies. The diversity of these proteins that mainly is due to an intrinsic polymorphism of the genes causes phenotypic variation in disease resistance. The well-known linkage of MHC polymorphism and Marek's disease resistance difference represents a classic model revealing immunological factors in resistance differences and diversity of mediator molecules. The molecular bases in any resistance variation to infectious pathogens are vaguely understood. This paper presents a review of the major immune mediators involved in resistance and susceptibility to infectious diseases and their functional mechanisms in the chicken. The genetic interaction of disease resistance with production traits and the environment is mentioned.