W. J. M. Landman

Coccidiosis in poultry: anticoccidial products, vaccines and other prevention strategies

Coccidiosis in chickens is a parasitic disease with great economic significance, which has been controlled successfully for decades using mainly anticoccidial products. However, large-scale and long-term use of anticoccidial drugs has led to the worldwide development of resistance against all these drugs. In order to minimize the occurrence of resistance, the rotation of various anticoccidial drugs in single and/or shuttle programmes is used. Unfortunately, this has not solved the anticoccidial resistance problem. Recently, live anticoccidial vaccines have been incorporated into rotation programmes, resulting in an increasing incidence of anticoccidial drug-sensitive Eimeria spp. field isolates, which may ameliorate the efficacy of anticoccidial drugs. Nevertheless, possible upcoming bans restricting the use of anticoccidials as feed additives, consumer concerns on residues and increasing regulations have prompted the quest for alternative coccidiosis control strategies. Although management and biosecurity measures could halt the introduction of Eimeria spp. to a farm, in practice they do not suffice to prevent coccidiosis outbreaks. Phytotherapy, aromatherapy and pre- and probiotics either show conflicting, non-consistent or non-convincing results, and have therefore not been applied at a large scale in the field. So far, live attenuated and non-attenuated anticoccidial vaccines have proved to be the most solid and successful coccidiosis prevention and control strategy. Despite the drawbacks associated with their production and use, their popularity is increasing. If with time, the immunogenicity of subunit vaccines can be improved, they could represent the next generation of highly efficient and low-cost anticoccidial strategies.

Induction of eggshell apex abnormalities by Mycoplasma synoviae: field and experimental studies

A novel eggshell pathology, characterized by an altered shell surface, thinning, increased translucency, and cracks and breaks in the eggshell apex, has become increasingly common in layer flocks of various breeds in The Netherlands. Two field studies found an association between the eggshell apex abnormalities (EAA) and infection with Mycoplasma synoviae. M. synoviae was isolated from the oviduct of birds that produced abnormal eggs, but not from birds in control flocks, although both affected and control birds had agglutinating antibodies against M. synoviae. After a single injection with long-acting oxytetracycline, the production of abnormal eggs ceased, but then reoccurred about 12 days later. A causal relationship between EAA and M. synoviae infection was subsequently demonstrated experimentally. EAA occurred after intratracheal inoculation of birds with M. synoviae, and even more frequently in birds infected with infectious bronchitis virus 5 days before inoculation of M. synoviae. EAA also occurred, although less frequently, in birds inoculated intravenously with M. synoviae and infected with infectious bronchitis virus. EAA did not occur in birds only inoculated intravenously with M. synoviae. M. synoviae was only isolated from the oviducts of birds producing abnormal eggs. The mean daily egg production was reduced in all groups infected with M. synoviae. Examination of the eggshells by scanning electron microscopy revealed that the mammillary layer of the calcified zone was absent and that the inner eggshell membranes were thicker. Isolates of M. synoviae from the oviducts of birds from farms producing abnormal eggs were examined by amplified fragment-length polymorphism analysis and did not appear to be clonal.

Ability of Massachusetts-type infectious bronchitis virus to increase colibacillosis susceptibility incommercial broilers: A comparison between vaccine and virulent field virus

The abilities of Massachusetts-type vaccine virus and virulent infectious bronchitis (IB) field virus to increase colibacillosis susceptibility were compared. In four experiments, 29-day-old female commercial broilers housed in isolators, were infected intratracheally and oculonasally with IB vaccine strains (HI20 and H52) or virulent IB field strains (D387 and M41) (4.8 or 6.8 log10 median embryo infective dose, per broiler). Five days later, Escherichia coli 506 strain was given intratracheally (5.6 to 8.8 log10 colony forming units/broiler). The incidence of nasal discharge at 3 and 5 days after IB virus infection was used to assess the clinical effect of the IB infection, while mortality, body weight uniformity and E. coli lesions at 7 days following E. coli inoculation were used as parameters for colibacillosis. Nasal discharge was observed in 61/117 (5%), 26/119 (22%), 35/119 (29%) and 115/120 (96%) of broilers infected with H120, H52, D387 and M41 virus, respectively. Apart from H52 and D387, differences between IBV strains were significant. IB vaccine and virulent IB viruses did not generally differ significantly in their ability to induce colibacillosis susceptibility. Mean colibacillosis lesion scores of H52-infected birds even significantly exceeded those of birds infected with the other IB viruses. The ability of H120 virus to induce colibacillosis susceptibility tended to be the weakest. The practical consequences of these findings are discussed.

Identification of Brachyspira hyodysenteriae and Other Pathogenic Brachyspira Species in Chickens from Laying Flocks with Diarrhea or Reduced Production or Both

ABSTRACT 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.

Molecular epidemiology of unilateral amyloid arthropathy in broiler breeders associated with Enterococcus faecalis.

Although symmetrical polyarticular amyloidosis has been described extensively in brown layers, spontaneous unilateral amyloid arthropathy has not been described previously in chickens. Birds from nine flocks of broiler parent stock (PS) had unilateral lameness associated with severe swelling of the left hock joint and the caudal aspect of the metatarsus. Gross pathology was restricted to the left hock joint and the left digital flexor tendons in almost all cases, suggesting an association with administration of Mareks disease vaccine. Amyloid deposits were found in 83% (25/30) of affected joints by histological examination of Congo red stained sections. Systemic amyloidosis, involving mainly the liver and spleen, was found in 59% (10/17) of birds. Enterococcus faecalis was isolated from joints in 77% (23/30) of cases and Staphylococcus aureus was isolated from the joint in one case (1/30). Thirty-five E. faecalis isolates from joints, tendons and blood samples from birds in five affected PS flocks were compared using pulsed-field gel electrophoresis (PFGE) to separate genomic fragments after digestion with Sma I. All but one isolate had identical or closely related restriction endonuclease digestion (RED) patterns that were very similar to a known arthropathic and amyloidogenic E. faecalis isolate. A further 30 E. faecalis isolates from seven grandparent stock (GPS) flocks and two isolates from two unaffected PS flocks of the same genetic background were analysed by PFGE. Among these isolates, 11 originating from four GPS flocks had RED patterns identical to or closely related to the reference amyloid-inducing strain. Moreover, one E. faecalis isolate from amyloidotic joints of brown layers housed in California, USA was included in the analysis and appeared to be identical to the reference strain. This study showed that the E. faecalis isolates involved in these outbreaks of unilateral amyloid arthropathy in broiler breeders belonged to the same clone as that responsible for outbreaks in brown layers.

Enterococcus cecorum infections in broiler breeders and their offspring: molecular epidemiology

Increased mortality and problems with lameness were reported in Dutch broiler flocks from the year 2008 onwards. Therefore, a field inventory, including 10 affected broiler flocks, nine corresponding broiler breeder flocks and five hatcheries, was carried out. The onset of clinical signs (lameness and increased mortality) started at about 2 weeks of age. The flock mortality varied from 3.1 to 8.1% at slaughter. Post-mortem lesions of broiler flocks were characterized by the occurrence of pericarditis/hydropericardium, arthritis and femoral head necrosis. Enterococcus cecorum was isolated from approximately 30% of the lesions. In the broiler breeders, E. cecorum was not isolated from any of the lesions. However, it was isolated from 31 out of 65 (47%) cloacal swabs, from two out of 65 (3%) oviduct samples, from one out 65 (1.5%) bone marrow samples and from two out of 25 (8%) blood samples. E. cecorum was not isolated from the air samples or dead-in-shell originating from the hatcheries involved. In total, 78 isolates were subjected to further typing by means of tRNA intergenic spacer PCR and confirmed as E. cecorum. The genetic relatedness of these cocci was subsequently studied using pulsed-field gel electrophoresis. The banding patterns of approximately 68% of E. cecorum isolates originating from parent stock flocks were clonal to one or more isolates of the same or other parent flocks. In contrast, isolates originating from their diseased offspring showed much greater genetic variation. Therefore, the vertical transmission of E. cecorum could not be demonstrated.

Intragenomic Variation in the Internal Transcribed Spacer 1 Region of Dientamoeba fragilis as a Molecular Epidemiological Marker

ABSTRACT Dientamoeba fragilis is a parasite that has been recognized to be a causative agent of gastrointestinal symptoms. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. The search for genetic variation in D. fragilis was based on the small-subunit rRNA gene, which was not found to be useful for molecular epidemiology. In this report, we describe the isolation and characterization of additional rRNA gene cluster sequences, the internal transcribed spacer 1 (ITS-1)-5.8S rRNA gene-ITS-2 region. For comparative purposes, we also isolated the ITS-1-5.8S rRNA gene-ITS-2 region of Histomonas meleagridis, a protozoan parasite of birds and a close relative of D. fragilis. This region was found to be highly variable, and 11 different alleles of the ITS-1 sequence could be identified. Variation in the ITS-1 region was found to be intragenomic, with up to four different alleles in a single isolate. So-called C profiles were produced from the ITS-1 repertoire of single isolates,. Analysis of the C profiles of isolates from nonrelated patients identified several clearly distinguishable strains of D. fragilis. Within families, it was shown that members can be infected with the same or different strains of D. fragilis. In conclusion, the ITS-1 region can serve as a molecular epidemiological tool for the subtyping of D. fragilis directly from feces. This may serve as a means of studying the transmission, geographical distribution, and relationships between strains and the pathogenicity of this parasite.

The role of various agents in chicken amyloid arthropathy

The results of an inventory of field cases of amyloid arthropathy in chickens and of routine post-mortem recordings over a two years period are described. Studies were also performed to evaluate the amyloidogenic potential of arthrotropic bacterial species (Staphylococcus aureus, Escherichia coli and Salmonella enteritidis) isolated from chickens as well as several Enterococcus faecalis isolates compared to the amyloidogenic E. faecalis isolate (previously isolated from amyloidotic joints). As chicken anemia virus was also isolated from amyloidotic joints of field cases, it was also screened for its amyloidogenic potential. In another experiment, Mycoplasma synoviae, inactivated E. faecalis isolate 6085.94, Freunds adjuvant and an arthrotropic reovirus field isolate were also screened for amyloidogenicity by intra-articular injection. These studies showed that the ability to elicit extensive amyloid arthropathy is reserved primarily to E. faecalis, but that this property is not common to every E. faecalis isolate. Intra-articular application of complete Freunds adjuvant led to the formation of extensive joint amyloid deposits. Of the other micro-organisms studied, S. aureus, S. enteritidis and E. coli were also able to cause joint amyloidosis, but in very small amounts. Inactivated E. faecalis, chicken anemia virus and reovirus did not cause amyloid arthropathy after intra-articular inoculation. This study is consistent with results of the analyses of previous field cases and of the induction of amyloid arthropathy in chickens, suggesting a considerable role for E. faecalis in this clinical-pathological entity. Finally, strain typing by analysis of chromosomal DNA restriction endonuclease digests by pulsed-field gel electrophoresis (PFGE) of amyloidogenic, non-amyloidogenic, amyloid-associated and other E. faecalis isolates from various origins showed that all amyloidogenic and amyloid-associated E. faecalis isolates had similar restriction digests, suggesting clonal spread.

Induction of amyloid arthropathy in chickens

The present report describes a novel chicken model in which reactive (AA) joint amyloidosis arises ajier a single injection of Enterococcus faecalis isolated from jield outbreaks of reactive amyloid arthropathy in domestic fowl. All six week old brown layer pullets injected intravenously with 109 or 109 colony forming units developed reactive amyloid arthropathy. Amyloid masses were also present in internal organs. The first articular amyloid deposits were observed 5 days after injection. in internal organs, deposits were encountered fiom day 13 onwards. on intra-articular injection, amyloid arthropathy developed particularly in Ihe target joint and in the internal organs.This model ofAA amyloid arthropathy may contribute in future studies to unravel the pathogenesis of the tissue predilection of amyloid deposition and 05 amyloid arthropathy in general.

Chicken joint amyloid protein is of the AA-type. I. Characterization of the amyloid protein.

Amyloid fibrils were extracted from deposits in joint tissue of heavy breed layers with spontaneous amyloid arthropathy and characterized as being of the AA‐type. Amino acid sequencing revealed a pattern quite similar to duck AA. Acute phase sera of chicken experimentally injected with Enterococcus faecalis showed a SAA‐protein like band cross reacting with anti‐chicken AA in immunoblot.