Dominant Salmonella Serovars in Australian Broiler Breeder Flocks and Hatcheries: a Longitudinal Study.

A longitudinal study was conducted to determine the dominance and prevalence of Salmonella enterica subsp in Australian broiler breeder flocks and hatcheries. Twenty-two flocks (n = 3339 samples) were sampled over 6 time points beginning at placement until week 40. Hatcheries (n = 274 samples) were sampled following removal of chicks hatched from eggs originating from the 22 donor parent flocks. The percent of positive flocks (36%) and frequency of positive samples (15.6%) were highest during rearing at week 7. The frequency of positive samples decreased over the 40 weeks; however, the number of positive flocks remained relatively consistent. Geographical location had a greater influence on Salmonella detection frequency than company sample origin, despite differing management and vaccination protocols within and between companies. Twelve serovars were detected in total. The predominant serovars during rearing were Salmonella Mbandaka (32%), S. Saintpaul (27%), and S. Liverpool (18%). The predominant serovars detected during production were S. Cubana (27%), S. Saintpaul (24%), and S. Havana (13%). Salmonella Typhimurium, S. Ohio, and S. Hessarek were detected in the hatcheries. Of the serovars detected, only S. Typhimurium and S. Ohio were found in both broiler breeder flocks and hatcheries. However, detection did not correspond to the status of the flock eggs feeding into the hatchery. This study provides an up-to-date capture of the current Salmonella serovars circulating in the broiler breeder industry. Continued surveillance within the Australian Chicken Meat industry is imperative to mitigate and reduce the risk of salmonellosis in the community related to chicken meat. IMPORTANCE This study identified prevalent and dominant Salmonella enterica subsp in Australian Broiler Breeder flocks, as well as in hatcheries post chick hatch and removal, from eggs originating from these donor parent flocks. The captured Salmonella data was further compared to the most common Salmonella serovars isolated from broilers, as well as human salmonellosis notification data, which is useful for consideration of the circulating serovars within the chicken meat industry and their significance in public health. As there are multiple entry points for Salmonella during the entire chicken meat production chain that can lead to carcass contamination, it is important to distinguish serovars present between the different stages of vertical integration to implement and enable Salmonella control strategies.

Reduced protein diet and amino acid concentration alter intestinal barrier function and performance of broiler chickens with or without synthetic glucocorticoid.

Little is known about the effect of low-protein (LP) diets on intestinal barrier function and permeability. In the first part of this study, starting on day 9 of age, the growth performance of the birds fed 3 experimental diets in each phase of feeding (G/F: grower/finisher) was investigated. Three experimental diets were as follows: LP (170/150 g/kg CP) fortified with essential amino acids (EAA), standard protein (SP), (202/190 g/kg), and high protein (HP) (220/210 g/kg). LP and SP diets contained a similar level of EAA concentration, while the HP diet contained 10% above the Ross 308 specifications. Each diet was replicated 6 times (10 male birds per replicate). The second part investigated intestinal permeability (IP) and function on additional 72 birds. On days 14, 16, and 20, a total of 36 birds (12 birds per diet) were injected with dexamethasone (DEX) to induce leaky gut. Birds fed LP diets had lower body weight gain (BWG) and higher feed conversion ratio (FCR) compared with SP and HP diets in both grower and finisher phases of feeding. For the challenge part, DEX increased the FCR independent of diets. Diet and DEX interacted for BWG, whereby the effect of diets was only evident in sham-injected birds. Birds fed LP had a higher fluorescein isothiocyanate dextran (FITC-d) concentration indicating a more permeable intestine compared to HP, but similar to SP. DEX increased FITC-d concentration in all dietary treatments. Birds fed LP had less ileal zonula occludens-2 (ZO-2) expression in comparison with SP, but not HP. DEX increased the expression of Claudin3 and ZO-2 and reduced Claudin1 (P < 0.05) and junctional adhesion molecule 2 in the ileum. Expression of ileal Na+-dependent glucose transporter 1 (SGLT1) was upregulated in LP fed group. It is concluded that compared with SP, IP can be maintained in LP by supplementing EAA. However, when compared with HP, feeding birds with LP may lead to a higher IP. DEX had a profound independent effect on intestinal barrier function.

Evaluation of the effects of sweet potato (Ipomoea batatas (L.) Lam) in broiler diets.

Cereal grains such as maize and wheat are used extensively in feed formulations for poultry as the primary source of carbohydrates. High cost of these grains in many developing countries necessitates the evaluation of other ingredients that are grown locally. Sweet potato is one such crop. The study was conducted as a proof of concept experiment to test the hypothesis that in the presence and absence of enzyme, sweet potato roots when included in diets of broiler chickens may affect the total metabolisable energy content of the diets which may exert certain influences on dry matter digestibility of these diets as well as impacting on production and certain gut parameters. A total of 120 chicks were raised on a commercial starter feed from day 0 to 19. On day 22, the birds were individually weighed and allocated to 96 single bird metabolism cages to conduct a 7-day classical apparent metabolisable energy (AME) assay. The test diets contained 0% and 25% sweet potato flour (SPF) with and without enzyme supplementation (Rovabio Excel AP T-flex) and replicated 24 times. AME of the control diet with and without enzyme was 14.05 and 13.91 MJ/kg whilst the AME of the SPF diets with and without enzymes were 13.45 and 13.43 MJ/kg respectively. AME of SPF was 12.08 MJ/kg. Birds fed the SPF had significantly reduced end weights (p = .002) and weight gains (p < .001) leading to significantly higher intake (p = .004) and FCRs (p < .001) in birds. These effects in growth parameters highlight the need to balance dietary protein and total amino acids when using SPF in broiler diets and may not be a negative effect of SPF per say as AME and dry matter digestibility of SPF diets were comparable to the control diet. The level of sucrase activity in the jejunum was significantly (p < .001) lower due to enzyme inclusion. Use of SPF in the current study did not negatively influence the activities of the brush border enzymes maltase and sucrase, gut morphology in the jejunum of broilers or the load of Enterobacteriaceae in the caecal of birds. This finding is promising in that the gut parameters associated with digestive capacity and gut health were not compromised with feeding of SPF to broilers.

In vitro invasion capacity of Salmonella Typhimurium DT9 isolates sourced from humans and layer hen environments.

In Australia, Salmonella Typhimurium definitive type 9 is frequently isolated during foodborne outbreaks of salmonellosis. Multiple-locus variable number tandem repeat analysis (MLVA) trace back investigations frequently identify isolate distribution patterns that may be epidemiologically linked to disease outbreaks. In this study, the in vitro virulence potential of S. Typhimurium DT9 isolates possessing different MLVA patterns (03 15 07 11 550, 03 24 11 10 523, 03 15 08 11 550 and 03 14 08 11 550) isolated from either humans or layer hens was assessed using a human colon carcinoma cell line. Four strains per MLVA from each host for a total of 32 isolates were included in these experiments. Bacteria were grown to stationary phase and added to cells at a multiplicity of infection of 100. Across all isolates, mean percent recovery ranged from 7.1 ± 1.1 to 33.3 ± 7.1%. The layer hen isolate, KC900 (MLVA profile 03 15 08 11 550), exhibited the greatest invasion with a mean percent recovery of 33.3 ± 7.1%. Overall, layer hen isolates of S. Typhimurium DT9 had significantly higher invasion into Caco2 cells than human isolates (p = .0021). RAPD and enterobacterial repetitive intergenic consensus genomic fingerprinting was also performed. Irrespective of source, the SalmonellaDT9 isolates included in this study exhibited similar fingerprint patterns.

Oviposition time, flock age, and egg position in clutch in relation to brown eggshell color in laying hens.

In Australia and other parts of the world, table eggs with uniform brown eggshell color are well regarded by consumers. Brown eggshell color has been positively correlated with certain egg characteristics such as shell strength and egg specific gravity, along with specific antibacterial functions. In the current study, the effect of hen oviposition time, flock age, and egg position in-clutch on the intensity of brown eggshell color was studied in commercial laying hens. The collected eggs were processed to measure egg weight, shell reflectivity, shell color (L*a*b*), quantification of protoporphyrin IX (PP IX), and shell thickness. Hen oviposition time had a statistically significant effect (P < 0.05) on egg weight, L*a*b*, amount of PP IX, and shell thickness. L* increased from 59.72 in the first half hour after lights on to 61.67 6 hours later, and PP IX per gram of eggshell decreased from 1.32×10(-7) mM to 1.26×10(-7) mM. Flock age had a significant effect on egg weight, L*a*b*, shell reflectivity, PP IX, and shell thickness. The mean egg weight increased from 55.4 g at 25 wk of flock age to 63.3 g at 75 wk of flock age. PP IX per gram of eggshell was 1.45×10(-7) mM at 25 wk and declined to 1.31×10(-7) mM at 75 wk of flock age. Individual hen clutch length was highly variable, ranging from 22 to 123 eggs in a single clutch. Egg position in a clutch had a significant effect on all egg quality variables measured; however, the R(2) values for each variable measured were low. The eggshell color declined to a greater extent with increasing position in a clutch for long clutches compared with short and medium clutches. In conclusion, hen oviposition time affected brown eggshell color with darker brown eggs laid early in the d and lighter colored brown eggs laid later in the morning. The intensity of brown color decreased with flock age, and egg position in-clutch had relatively little effect on brown eggshell color.

Eggshell color in brown-egg laying hens - a review.

The major pigment in eggshells of brown-egg laying hens is protoporphyrin IX, but traces of biliverdin and its zinc chelates are also present. The pigment appears to be synthesized in the shell gland. The protoporphyrin IX synthetic pathway is well defined, but precisely where and how it is synthesized in the shell gland of the brown-egg laying hen is still ambiguous. The pigment is deposited onto all shell layers including the shell membranes, but most of it is concentrated in the outermost layer of the calcareous shell and in the cuticle. Recently, the genes that are involved in pigment synthesis have been identified, but the genetic control of synthesis and deposition of brown pigment in the commercial laying hen is not fully understood. The brown coloration of the shell is an important shell quality parameter and has a positive influence on consumer preference. The extent of pigment deposition is influenced by the housing system, hen age, hen strain, diet, stressors, and certain diseases such as infectious bronchitis. In this article, the physiological and biochemical characteristics of the brown pigment in commercial brown-egg layers are reviewed in relation to its various functions in the poultry industry.

Pathogenicity of Salmonella strains isolated from egg shells and the layer farm environment in australia.

In Australia, the egg industry is periodically implicated during outbreaks of Salmonella food poisoning. Salmonella enterica serovar Typhimurium and other nontyphoidal Salmonella spp., in particular, are a major concern for Australian public health. Several definitive types of Salmonella Typhimurium strains, but primarily Salmonella Typhimurium definitive type 9 (DT9), have been frequently reported during egg-related food poisoning outbreaks in Australia. The aim of the present study was to generate a pathogenicity profile of nontyphoidal Salmonella isolates obtained from Australian egg farms. To achieve this, we assessed the capacity of Salmonella isolates to cause gastrointestinal disease using both in vitro and in vivo model systems. Data from in vitro experiments demonstrated that the invasion capacity of Salmonella serovars cultured to stationary phase (liquid phase) in LB medium was between 90- and 300-fold higher than bacterial suspensions in normal saline (cultured in solid phase). During the in vivo infection trial, clinical signs of infection and mortality were observed only for mice infected with either 10(3) or 10(5) CFU of S. Typhimurium DT9. No mortality was observed for mice infected with Salmonella serovars with medium or low invasive capacity in Caco-2 cells. Pathogenicity gene profiles were also generated for all serovars included in this study. The majority of serovars tested were positive for selected virulence genes. No relationship between the presence or absence of virulence genes by PCR and either in vitro invasive capacity or in vivo pathogenicity was detected. Our data expand the knowledge of strain-to-strain variation in the pathogenicity of Australian egg industry-related Salmonella spp.

Eggshell penetration by Salmonella Typhimurium in table eggs: Examination of underlying eggshell structures by micro-computed tomography and scanning electron microscopy.

Horizontal infection of table eggs by food borne, human infection causative agents such as Salmonella is a serious concern for consumers and industry. In this study, we investigated the relationship between eggshell translucency, mammillary layer abnormalities and pore structure using Computed Tomography (CT) and scanning electron microscopy. The effects of eggshell pore structure, size and number on Salmonella Typhimurium penetration was also investigated. The eggs were infected with S. Typhimurium and were incubated at 37°C for 3 or 6days. Micro CT results comparing shell features to shell translucency found that there was a significantly increased incidence of externally branching pores found in the high translucency score eggshell group, and more straight pores found in the low translucency score group. Different pore structures, the total number of pores and the shell thickness do not appear to play a role in the horizontal infection of eggs by the S. Typhimurium strain used in this study. While it is likely that the presence of shell pores is responsible for shell penetration, other unknown shell factors must also play a role, and eggshells with a higher incidence of shell pores are not penetrated at a higher rate.

Effects of egg shell quality and washing on Salmonella Infantis penetration.

The vast majority of eggs in Australia are washed prior to packing to remove dirt and fecal material and to reduce the microbial contamination of the egg shell. The egg contents can be an ideal growth medium for microorganisms which can result in human illness if eggs are stored improperly and eaten raw or undercooked, and it is estimated that egg-related salmonellosis is costing Australia $44 million per year. Egg shell characteristics such as shell thickness, amount of cuticle present, and thickness of individual egg shell layers can affect the ease with which bacteria can penetrate the egg shell and washing could partially or completely remove the cuticle layer. The current study was conducted to investigate the effects of egg washing on cuticle cover and effects of egg shell quality and cuticle cover on Salmonella Infantis penetration of the egg shell. A higher incidence of unfavorable ultrastructural variables of the mammillary layer such as late fusion, type B bodies, type A bodies, poor cap quality, alignment, depression, erosion and cubics were recorded in Salmonella penetrated areas of egg shells. The influence of egg washing on the ability of Salmonella Infantis on the egg shell surface to enter the egg internal contents was also investigated using culture-based agar egg penetration and real-time qPCR based experiments. The results from the current study indicate that washing affected cuticle cover. There were no significant differences in Salmonella Infantis penetration of washed or unwashed eggs. Egg shell translucency may have effects on Salmonella Infantis penetration of the egg shell. The qPCR assay was more sensitive for detection of Salmonella Infantis from egg shell wash and internal contents than traditional microbiological methods. The agar egg and whole egg inoculation experiments indicated that Salmonella Infantis penetrated the egg shells. Egg washing not only can be highly effective at removing Salmonella Infantis from the egg shell surface, but also allows subsequent trans-shell and trans-membrane penetration into the egg. Consequently, it is important to prevent recontamination of the egg after washing.

Survey of Enterobacteriaceae contamination of table eggs collected from layer flocks in Australia.

In the present study, eggs from commercial caged layer flocks at different stages of lay in Australia were collected. Enterobacteriaceae populations from eggshell surface and eggshell pore were enumerated and these populations characterized using API® Rapid 20E strips. The eggshell surface, eggshell pore and egg internal content samples were also processed for the isolation of Salmonella and these isolates were tested for the presence or absence of several virulence genes (prgH, sopB, spiC, orfL, invA, sifA, sitC, misL). Results indicated that there was no significant difference in total Enterobacteriaceae count on the eggs of the flock from early, mid or late lay flocks. Enterobacteriaceae isolates were of 11 different genera which included: Cedecea, Citrobacter, Enterobacter, Escherichia, Klebsiella, Kluyvera, Leclercia, Pantoea, Salmonella, Serratia and Yersinia. Out of all 153 identified Enterobacteriaceae isolates, the Escherichia genus was reported most frequently (60.78%). Results also indicated that overall there were 4.51% (14/310) Salmonella positive pooled samples. In this study, 14 Salmonella strains were isolated, serotyping confirmed that 12 out of them were Salmonella Infantis and the 2 others were Salmonella enterica subsp. enterica serovar 4,12:d: Polymerase chain reaction results indicated that all Salmonella Infantis isolates harboured invA, misL, orfL, prgH, sifA, sitC, sopB and spiC genes which suggests that Salmonella Infantis strains isolated from eggshell surface may have the capacity to invade and survive in macrophages.

Presence of Ureaplasma diversum in the Australian cattle population.

In cattle, Ureaplasma diversum has been associated with decreased fertility, granular vulvovaginitis, endometritis, salpingitis and spontaneous abortion in cows and seminal vesiculitis, balanoposthitis and changes in bull sperm. The presence of U. diversum within the Australian cattle population has not been established. One of the aims of this study was to determine if U. diversum was present in Australian cattle, using culture and polymerase chain reaction (PCR), both of which are considered to be gold standards for bacterial identification. Of 100 samples collected from 66 male and 34 female cattle, 15 were positive for U. diversum. Therefore, Australia can no longer be considered free of U. diversum. Further studies should be conducted to ascertain the effects of U. diversum within Australian cattle herds and, if warranted, to investigate prevention, treatment and eradication protocols.

Ureaplasma diversum in bull semen in Australia: its detection and potential effects.

BACKGROUND: The primary objective of this study was to confirm the infection status of Ureaplasma diversum in Australian bulls and to identify morphological changes of sperm from U. diversum-positive bulls. METHODS: Fresh semen samples were taken from 29 sexually active beef bulls from suspect herds in the Riverina/Upper Murray region. U. diversum was identified using PCR analyses and culture of the organism. RESULTS: Nine of the bulls were PCR-positive for U. diversum but none of these had genital lesions. Sperm from infected bulls showed increased incidence of abnormal tails (bent and coiled), as well as surface abnormalities (i.e. small protuberances or lumps). CONCLUSIONS: The results suggest impairment of sperm function and possibly fertility. Further investigations into the potential role of U. diversum as a pathogen for Australian cattle are warranted.

Recovery of Salmonella from eggshell wash, eggshell crush, and egg internal contents of unwashed commercial shell eggs in Australia.

The experiment was conducted to study the prevalence of Salmonella spp. on the eggshell surface, eggshell membranes or pores, and in egg internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Eggshell rinsate, shell crush, and egg internal contents (yolk and albumen) of eggs were processed for Salmonella spp. Salmonella Infantis and Salmonella subspecies 1, serotype 4,12:d were isolated from the eggshell surface. Salmonella spp. were not isolated from any eggshell crush or egg internal contents. It would appear that the occurrence of Salmonella in the Australian egg industry is low.

Polymerase chain reaction for detection of Ureaplasma diversum from urogenital swabs in cattle in Australia.

BACKGROUND: Ureaplasma diversum has been associated with various reproductive problems in cattle, including granular vulvovaginitis, endometritis, salpingitis, early embryonic death, weak calves, decreased conception rates, balanoprosthitis, impaired spermatozoids and seminal vesiculitis in bulls. METHODS: This study briefly outlines the use of polymerase chain reaction (PCR) for the rapid detection of U. diversum directly from urogenital swabs collected from Australian beef cattle. RESULTS: The 16S ribosomal RNA gene sequences obtained from the PCR products of the clinical samples were closely related to U. diversum strain A417. CONCLUSION: The present test enabled detection of the organism directly from clinical swabs collected from animals with or without lesions.

Prevalence of antibodies to Mycoplasma synoviae in laying hens and possible effects on egg shell quality.

Mycoplasma synoviae (M. synoviae) can cause respiratory disease, synovitis, or result in a silent infection in chickens and turkeys. The importance of M. synoviae is well established in broilers but only a few studies have been conducted in layers. In the present study, the prevalence of M. synoviae in commercial layer flocks was estimated using ELISA. For this study, 19 commercial layer flocks were selected randomly from New South Wales and Queensland region of Australia from producers who were willing to participate in the survey. Sixty eggs per flocks were randomly collected, out of these 30 eggs were used for ELISA and remaining 30 eggs were used to estimate various egg shell quality parameters. Subsequently, association between the serological status of eggs for M. synoviae and egg shell quality was studied. In the flocks under study, seroprevalence of M. synoviae was found to be high at 69% (95% confidence interval (CI)=41.3-89.0). Statistical analysis showed an association between serological status for M. synoviae and egg quality parameters such as translucency, shell breaking strength, % shell reflectivity and shell deformation. On the other hand, there was no significant association between serological status for M. synoviae and other egg quality parameters such egg weight, egg shell weight, % egg shell or shell thickness.

Detection of infectious bronchitis virus strain N1/88 from the oviduct and feces of experimentally infected vaccinated and unvaccinated hens.

Hens were vaccinated during the rearing phase with infectious bronchitis virus (IBV) vaccines commercially available in Australia (Vic S and A3) or left unvaccinated and then challenged with the N1/88 strain of IBV at 30 wk of age. Oviduct and fecal samples were collected at regular intervals after N1/88 challenge. A locked nucleic acid probe-based reverse transcription real-time PCR test was designed and used to detect the IBV strain N1/88 from the oviduct and feces of unvaccinated and vaccinated laying hens. Using a recombinant plasmid standard, the detection limit of the reaction was found to be 100 copies and independent assay runs showed reproducible threshold cycle values. Viral RNA was detected in the oviduct of 12 unvaccinated then challenged hens and viral RNA increased sharply on d 10 and 12 postinfection (p.i.). By contrast, among the hens in the vaccinated group, N1/88 was detectable only in the oviduct of 2 hens at 8 and 12 d p.i. N1/88 challenge. Viral RNA was detected in feces of 2 unvaccinated hens up to 4 wk p.i. and in 1 vaccinated hen up to 3 wk p.i. This shows that rearing phase vaccination lowers the total viral RNA of the strain N1/88, even though this strain shows considerable antigenic and genetic variation from the vaccine strain. This new test will be useful for the rapid identification of the N1/88 strain of IBV from oviduct and fecal samples.

Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs.

This experiment was conducted to study the prevalence of Salmonella and Escherichia coli (E. coli). from the surface of egg shells, egg shell membranes or pores, and internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Egg shell swabs, shell crush and egg internal contents (yolk and albumen) of an individual egg were processed for bacteriological examination. Salmonella spp. were not detected from any of the egg shell surfaces, egg shell crush or egg internal contents. Thirty five E. coli isolates were isolated from the egg shell surface. Ten E. coli strains were also isolated from shell crush. However, the internal contents of eggs appeared to be sterile. Polymerase chain reaction was performed on forty-five E. coli isolates using primers for heat stable enterotoxin genes A and B (STa and STb) and also for colicin V gene (cvaC). STa gene was detected in four E. coli isolates isolated from egg shell surfaces. All the E. coli isolates were negative for STb and cvaC genes. These data provide useful information regarding the prevalence of virulent E. coli and Salmonella spp. on and in unwashed eggs collected from layer farms. These data also suggest that unwashed eggs collected from caged layer farms are unlikely to be sources of Salmonella outbreaks. Egg shell translucency could be due to changes in the mammillary layer and mamillary cores during the early phases of egg shell formation and has the potential to increase the incidence of microcracks in egg shells, and hence, may be responsible for bacterial penetration. There was a significant correlation between egg shell translucency and egg shell penetration by Salmonella Infantis and E coli. Both strains of bacteria were able to penetrate the translucent egg shells even at very low doses. The penetration, however, was hindered in both translucent and non translucent eggs at 4 degrees C, as compared with room temperature which highlights the importance of storage of eggs at refrigerated temperatures.

Effects of infectious bronchitis virus vaccine on the oviduct of hens.

In Australia, currently, all pullets reared for egg production are vaccinated with live attenuated strains of infectious bronchitis virus. Various vaccines and protocols to control this viral disease have been developed, although the severity of the disease varies from place to place and flock to flock. In the present trial, the effects of vaccine strains A3 and Vic S on the oviduct of laying hens were assessed by histopathology, electron microscopy, serology and also by determining the presence and persistence of viral RNA in the oviduct by real time PCR following the experimental infection. Birds were either unvaccinated or vaccinated with both A3 and Vic S and then mock-infected, challenged with the same attenuated strains, either A3 or Vic S. Some respiratory signs were observed, but were mild and short-lived. There was no drop in egg production in any of the groups. However, there was visual loss of shell colour in the unvaccinated hens challenged with the Vic S strain. Mild histopathology was recorded only in terms of lymphocyte infiltration and occasional submucosal oedema in the infundibulum and very mild gland dilatation in the magnum. Microscopic lesions were not recorded in the isthmus, tubular shell gland or shell gland pouch. Cilia loss was not observed in any region of the oviduct using scanning electron microscopy. Both the A3 and Vic S vaccine strains were detected in the oviduct of vaccinated and unvaccinated hens, mainly on the 12th day p.i. These results indicate that the A3 and Vic S strains replicate at a low level in the oviduct without causing significant damage and hence are safe for the oviduct.

LNA probe-based real-time RT-PCR for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens.

In the present study, LNA-probe based real-time PCR was designed for the detection and absolute quantification of infectious bronchitis virus (IBV) from the oviduct of unvaccinated and vaccinated hens after IBV challenge. Using a recombinant plasmid standard, the detection limit of the reaction was found to be 10 copies and independent assay runs showed reproducible Ct values. Amongst the unvaccinated hens, the virus could be detected between 6 and 20 days post-infection (p.i.), with a peak of viral load between 10 and 14 days p.i. The virus was also detectable in the oviduct of vaccinated, challenged hens although the viral load was much lower compared to the viral load in the oviduct of unvaccinated, challenged hens. This indicates that rearing phase vaccination can offer significant protection of the fully functional oviduct against a pathogenic strain of IBV. The present test will be useful for the rapid identification of IBV directly from clinical samples. Most vaccination trials investigating the efficacy of vaccines for layer and breeder hens have been conducted based on the respiratory tract response. Evaluation of viral load from the oviduct of vaccinated and unvaccinated hens is an efficient method for assessing oviduct protection in commercial laying hens.

Ultrastructural changes in the oviduct of the laying hen during the laying cycle.

The ultrastructural changes occurring in the fully functional oviduct of Isa Brown laying hens were studied during various stages of the laying cycle. Hens were killed at different positions of the egg in the oviduct. The oviduct was lined by ciliated and non-ciliated cells (also referred to as granular cells). The granular cells in the infundibulum contributed to secretion during egg formation, whereas ciliated cells showed little evidence of secretion. Ultrastructural changes were recorded in the granular and glandular cells of the distal infundibulum. In the magnum, the surface ultrastructure revealed glandular openings associated with the ciliated and granular cells. Cyclic changes were recorded in the glandular cells of the magnum. With respect to the three observed types of glands, the structure of gland type A and C cells varied at different egg positions in the oviduct, whereas type B cells represented a different type of gland cell containing amorphous secretory granules. The surface epithelium of the isthmus was also lined by mitochondrial cells. Two types of glandular cell (types 1 and 2) were recorded in the isthmus during the laying cycle. Intracisternal granules were found in type 2 cells of the isthmus. A predominance of glycogen particles occurred in the tubular shell gland. The granular cells in the shell gland contain many vacuoles. During egg formation, these vacuoles regressed following the formation of extensive rough endoplasmic reticulum; the reverse also occurred. The disintegrated material found in the vacuoles may have been derived from the disintegrating granules.