Yolk Sac Inoculation Research Articles

Isolation, identification, and pathogenicity of a ALV-K strain from Chinese indigenous chicken breed

Subgroup K avian leukosis virus (ALV-K) is a new subgroup of avian leukosis virus (ALV) first identified in Chinese indigenous chickens in recent years. In this study, an ALV-K strain was isolated from Luhua chicken in Shandong province, China, and designated SD20LH01. The full-length genomic sequence of SD20LH01 was 7491 bp, which had the highest homology with ALV-K reference strains GDFX0601, GDFX0602 and GDFX0603. The nucleotide homology of env gene of SD20LH01 with reference strains of subgroup A, B, C, D, E, and J was ranged from 57.1 to 93.2%, while 94.1 to 99.4% with other ALV-K reference strains. The nucleotide difference of SD20LH01 mainly clustered with gp85 gene and U3 sequence when compared with the reference strain of ALV-K. In order to investigate the pathogenicity of SD20LH01, SPF chicken embryos were infected by yolk sac inoculation, and 1-day-old chickens were infected by intraperitoneal inoculation of SD20LH01. The results showed that yolk sac inoculation of SD20LH01 could induce persistent viremia, growth retardation and reduce the immune response to NDV and AIV-H9 vaccines. However, intraperitoneal inoculation in 1-day-old chickens could only induce a low level of viremia. In addition, no tumors were found in infected chickens during the animal experiments. This study enriched the genomic sequence data of ALV-K isolated in Chinese indigenous chickens, and laid a foundation for further study on the pathogenesis and prevention of ALV-K.

Resistance and Pathogenicity of Salmonella Thompson Isolated from Incubation End of a Poultry Farm.

Simple SummaryNon-typhoid Salmonella is the general term of Salmonella other than typhoid and paratyphoid, which often causes foodborne gastroenteritis in humans, but some serotypes have been proved to be pathogenic to poultry. Salmonella Enterica and Salmonella Typhimurium are the common serotypes pathogenic to poultry and have been systematically studied, but other serotypes have rarely been studied. During Salmonella surveillance in farms, we discovered by chance that Salmonella Thompson, a common non-typhoid Salmonella, is also pathogenic to avian embryos. Therefore, this study aimed to explore antimicrobial resistance and pathogenicity of clinical S. Thompson. Firstly, we found that the core-genome multilocus sequence typing of 14 clinical S. Thompson was consistent with two strains of S. Thompson from humans in China. Secondly, the antimicrobial resistance gene analysis demonstrated that all strains carried the polymyxin resistance gene mcr-9, which had not appeared resistance phenotype. Meanwhile, many essential virulence genes were also found in each S. Thompson isolate. Finally, the bacterial inoculation experiment revealed that clinical S. Thompson was highly pathogenic to newborn chicks after yolk sac inoculation. This study suggests that Salmonella Thompson can circulate between humans and poultry farms and transmit drug resistance genes and demonstrated that Salmonella Thompson is highly pathogenic to chicks and should be guarded against in the hatching stage of poultry farms.Salmonella Thompson, an important foodborne pathogen, is rarely found to be pathogenic to poultry. Accidentally, S. Thompson was found to be pathogenic to embryos of white feather broiler at a poultry farm in China. Therefore, this study aimed to explore antimicrobial resistance and pathogenicity of clinical S. Thompson isolated from dead poultry embryos. The phylogenetic tree based on 16S rRNA and seven housekeeping genes showed that the 14 clinical S. Thompson were closely related. The core-genome multilocus sequence typing of 14 clinical S. Thompson based on whole-genome sequencing was cgST-12774, consistent with the only two strains of S. Thompson from humans in China as reported in the NCBI database. The antimicrobial resistance gene analysis demonstrated that all strains carried aac(6′)-Iaa and the polymyxin resistance gene mcr-9. Antimicrobial sensitivity tests for 18 antibiotics showed that S. Thompson isolates displayed resistance against streptomycin (100%), ampicillin (35.7%), and doxycycline (14.3%), but sensitivity to polymyxin B, proving that the mcr-9 gene had not appeared resistance phenotype. Virulence genes Salmonella pathogenicity island (SPI) SPI1-5, type I fimbriae gene (fimA), flagellar assembly genes (bcfC, flhD, fliA, fliC, fljB, flgK, and lpfC), and other virulence genes (iroN, pagC, and cigR) were found in each S. Thompson isolate. Additionally, the bacterial inoculation experiment with 1-day-old chicks revealed that clinical S. Thompson was highly pathogenic to newborn chicks after yolk sac inoculation. This study highlighted that the S. Thompson isolated from poultry embryos and the S. Thompson causing human foodborne diarrhea in some parts of China belong to the same cgMLST typology (cgST-12774) and showed the pathogenicity of this clinical S. Thompson to chicks.

Susceptibility of Chicken Embryos, Sheep, Cattle, Pigs, and Chickens to Zika Virus Infection.

The susceptibility of sheep, cattle, pigs, chickens and chicken embryos to Zika virus infection was evaluated by experimental inoculation with Zika virus Thailand strain isolated from a Canadian traveler in 2013. The inoculated animals did not develop any clinical signs of disease nor evidence of Zika virus replication in peripheral blood, cerebrospinal fluid and tissues including brain and spinal cord assessed by real-time RT-PCR. Sera were also negative for Zika virus antibodies by Zika virus neutralization assays as well as Zika virus immunoperoxidase staining of Zika infected Vero cells. Chicken embryos were inoculated by different routes including yolk sac (4 day old embryos), chorioallantoic membrane (8 day old embryos), amniotic fluid (8 day old embryos) and intravenous routes (12 day old embryos). Virus replication in chicken embryos was observed in the brain and body tissues following intravenous (IV), yolk sac (YS), chorioallantoic membrane (CAM), and amniotic fluid (AF) inoculation routes. The highest mortality was observed in embryos inoculated via yolk sac. The dead embryos showed diffuse muscular hemorrhages. The yolk sac inoculated chicken embryos showed delayed hatching and displayed neurological signs immediately after hatching. These studies demonstrate that 8 week old sheep, 6 month old cattle, 4 week old pigs, and 4 week old chickens are not susceptible to Zika virus infection when inoculated experimentally and therefore unlikely to pose a risk as Zika virus reservoirs. However, chicken embryos are highly susceptible to Zika virus resulting in clinical disease of chicks after hatching. This study demonstrates that Zika virus has a tropism for embryonic tissue and that chicken embryos can be used as a model to study Zika virus replication and pathogenesis.

Intrabursal immunization as a possible route to overcome immunosuppression due to maternal antibodies

Vaccination is the most effective mean of preventing, controlling, and even eradicating infectious diseases. Poultry are vaccinated through various routes including eye/nose drops, drinking water, vent brush, or injections. The prolonged suppression effect of maternal antibodies on humoral immune response of newly hatched chicks to active immunization has been reported, while the effect of vaccination route on this suppression still unclear. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP or non-specific IgY antibodies were transferred by yolk sac inoculation to newly hatched chicks (chicks of maternal and no-maternal antibodies), and they were immunized with DNP-KLH via intrabursal route at 0.1, 1 and 10 mg/kg body weight (BW) and with 1 mg/kg BW intraperitoneally at 1 and 4 weeks of age. Concentration of anti-DNP antibodies in serum samples of these chicks was measured by using Enzyme- linked immunosorbent assay (ELISA). The immune response to intrabursal immunization was higher in chicks of no-maternal antibodies than that of chicks of maternal antibodies at 5 weeks of age. Intrabursal immunization showed higher response than intraperitoneal one at the same dose. These results confirmed that the immune suppressive effect of maternal antibodies on the immune response of the newly hatched chicks was antigen specific and depends on the ratio of antigen/maternal antibody at the time of immunization. Furthermore, intrabursal vaccination showed promising results than intraperitoneal vaccination at the same dose.

Detection of Avian Reoviruses in Wild Birds in Poland.

IntroductionThe purpose of this study was to determine the occurrence of avian reovirus (ARV) infections in wild birds in Poland and attempt to propagate the selected ARV strains in chicken embryo kidney (CEK) cells or chicken SPF embryos.Material and MethodsThe study included 192 wild birds representing 32 species, collected between 2014 and 2016. A part of the S4 segment encoding the σNS protein of avian reoviruses (ARVs) isolated from different species of wild birds from that period was amplified.ResultsThe presence of ARV was demonstrated in 58 (30.2%) wild birds belonging to nine orders. The isolated strains were propagated in chicken embryos by yolk sac inoculation, and CPE was induced in the infected CEK monolayer. Agar gel precipitation showed that two ARV isolates from rock pigeon and mute swan shared a common group-specific antigen with chicken reovirus S1133. Specific products of predicted size were found in two ARV isolates from the chicken embryo passage and 13 ARVs isolated from CEK cells.ConclusionThe study indicates the high prevalence of ARV among wild birds in Poland and its possible transmission to farmed birds.

Molecular Characterization of Vaccine-Derived Mutants of Avian Meta-Pneumoviruses Isolated from Turkeys in Egypt

Avian metapneumovirus (aMPV) causes acute respiratory infection in turkeys with sudden onset and rapid spread in the infected flocks. Vaccinated flocks frequently showed clinical signs typical like the non-vaccinated flocks in Egypt, this situation suggests a virulent vaccine virus was circulating in the field. Oropharyngeal and nasal sinus swabs were collected from 100 turkeys from 10 commercial farms with mild to moderate respiratory signs. Virus isolation and propagation was done in SPF embryos of chicken eggs via yolk sac inoculation and further passed in Vero cells. RT-PCR for detection of aMPV was done on field samples as well as on virus isolates. Genotyping of viruses was done through RT-PCR targeting Fusion (F) and the surface Glycoprotein (G) genes and partial sequencing of PCR products was used for genetic and phylogenetic analyses. The results were compared with two live attenuated commercial vaccines commonly used in the field. Analysis of nucleotide and amino acid sequences for F and G genes indicated that they were very closely related to each other and belonging to B subtype of aMPV isolated from Europe. The Egyptian viruses were very close to VCO3 vaccine strain, indicating their possible origin. Phylogenetic analysis indicates clustering of the Egyptian field viruses with European strains of B subtype. These results support that they may have a common progenitor from vaccine strains and suggesting evidence of reversion of attenuated vaccine virus to virulence. This is the first report describing the emergence of vaccine-derived mutants from the B subtype aMPV in broiler turkeys in Egypt.

Induction of non-specific suppression in chicks by specific combination of maternal antibody and related antigen.

Specific immune suppression in newly hatched chicks induced by specific maternal antibodies has been reported. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP and non-specific immunoglobulin (Ig) Y antibodies were transferred by yolk sac inoculation to newly hatched chicks, and then, they were immunized with an optimum immunogenic dose of DNP-KLH at 1 and 4 weeks of age. Concentrations of anti-DNP antibodies in serum samples of these chicks were measured by using Enzyme-linked immunosorbent assay (ELISA). Proportions of T-cell subsets in peripheral blood of these chicks were also measured by flow cytometric analysis at 5 weeks of age (one week after the second immunization). Suppression of anti-DNP antibody response and down-regulation of CD3+CD4+ cells were observed in the chicks received high dose of maternal anti-DNP antibodies and immunized with DNP-KLH. On the other hand, normal anti-DNP antibody response and normal proportion of CD3+CD4+ cells were observed in the chicks received high dose of non-specific IgY antibodies and immunized with DNP-KLH. Furthermore, when chicks received high dose of maternal anti-DNP antibodies and immunized with DNP-KLH at 1 and 4 weeks of age and then with rabbit serum albumin (RSA) at 5 and 8 weeks of age, their primary anti-RSA response was also significantly suppressed. We indicate here that specific maternal antibodies can affect both B and T cell responses and induce non-specific suppression against different antigens. However, this non-specific suppression does not continue for a long time.

Pathogenicity testing and antimicrobial susceptibility of Helicobacter pullorum isolates from chicken origin

This work aimed to study the pathogenicity and to investigate the antimicrobial susceptibility and resistance patterns of Helicobacter pullorum (H. pullorum). The minimum inhibitory concentration (MIC) value of ciprofloxacin, ampicillin, gentamycin, erythromycin, colistin sulfate and tetracycline was determined for eight different H. pullorum isolates. H. pullorum resulted into 33.3% mortality of infected chickens with signs of diarrhea, stunted growth and poor conversion rate in survivors. All experimentally infected embryonated chicken eggs showed embryonic mortalities within 48-h post yolk sac inoculation. H. pullorum was re-isolated from cecum, liver, yolk sac and air-sacs of all dead and sacrificed infected chickens. H. pullorum was also re-isolated from dead embryos, embryonic membranes and fluids of infected embryonated chicken eggs. Polymerase Chain Reaction (PCR) assay was used to detect H. pullorum in experimentally infected chickens and embryonated chicken eggs. All tested H. pullorum isolates were resistant to ciprofloxacin, gentamycin and erythromycin, while 7 out of 8 isolates were resistant to tetracycline. All isolates were susceptible to colistin sulfate and ampicillin.

Single and Combination Diagnostic Test Efficiency and Cost Analysis for Detection and Isolation of Avian Influenza Virus from Wild Bird Cloacal Swabs

Effective laboratory methods for identifying avian influenza virus (AIV) in wild bird populations are crucial to understanding the ecology of this pathogen. The standard method has been AIV isolation in chorioallantoic sac (CAS) of specific-pathogen-free embryonating chicken eggs (ECE), but in one study, combined use of yolk-sac (YS) and chorioallantoic membrane inoculation routes increased the number of virus isolations. In addition, cell culture for AIV isolation has been used. Most recently, real-time reverse transcriptase (RRT)-PCR has been used to detect AIV genome in surveillance samples. The purpose of this study was to develop a diagnostic decision tree that would increase AIV isolations from wild bird surveillance samples when using combinations of detection and isolation methods under our laboratory conditions. Attempts to identify AIV for 50 wild bird surveillance samples were accomplished via isolation in ECE using CAS and YS routes of inoculation, and in Madin-Darby canine kidney (MDCK) cells, and by AIV matrix gene detection using RRT-PCR. AIV was isolated from 36% of samples by CAS inoculation and 46% samples by YS inoculation using ECE, isolated from 20% of samples in MDCK cells, and detected in 54% of the samples by RRT-PCR. The AIV was isolated in ECE in 13 samples by both inoculation routes, five additional samples by allantoic, and 10 additional samples by yolk-sac inoculation, increasing the positive isolation of AIV in ECE to 56%. Allantoic inoculation and RRT-PCR detected AIV in 14 samples, with four additional samples by allantoic route alone and 13 additional samples by RRT-PCR. Our data indicate that addition of YS inoculation of ECE will increase isolation of AIV from wild bird surveillance samples. If we exclude the confirmation RT-PCR test, cost analysis for our laboratory indicates that RRT-PCR is an economical choice for screening samples before doing virus isolation in ECE if the AIV frequency is low in the samples. In contrast, isolation in ECE via CAS and YS inoculation routes without prescreening by RRT-PCR was most efficient and cost-effective if the samples had an expected high frequency of AIV.

Induction of Immune Suppression in the Chick by an Optimal Dose of an Immunizing Antigen in the Presence of its Specific Maternal Antibody

Prolonged interference or suppression of maternal antibodies of the humoral immune response of newly hatched chicks to active immunization has been documented; however, the immunological mechanisms responsible for such suppression are still unclear. Laying hens were immunized with dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH). Purified maternal anti-DNP or non-specific IgY antibodies were transferred by yolk sac inoculation to newly hatched chicks, and they were immunized with DNP-KLH or rabbit serum albumen (RSA) at 1 and 4 weeks of age. The concentrations of anti-DNP and anti-RSA antibodies in serum samples of these chicks were measured using an enzyme-linked immunosorbent assay (ELISA). The immune responses of the chicks that received a high dose of maternal anti-DNP antibodies and were immunized with an appropriate dose of DNP-KLH were suppressed. However, those of the chicks that received the same high dose of maternal non-specific IgY antibodies and were immunized with an appropriate dose of DNP-KLH and those of the chicks that received a high dose of maternal anti-DNP antibodies and were immunized with RSA were not suppressed. On the other hand, suppression of anti-DNP antibody production would not be induced if the chicks received a high dose of antigen specific maternal antibodies and were immunized with a high dose of the same antigen. These results revealed that the immune suppressive effect of maternal antibodies on the immune response of the newly hatched chicks was antigen specific and depended mainly on the ratio of antigen/maternal antibody at the time of immunization.

Distribution of Viral Antigens and Development of Lesions in Chicken Embryos Inoculated with Nipah Virus

An isolate of Nipah virus was injected into fertile eggs via the allantoic cavity or yolk sac. Allantoic inoculation resulted in considerable pathological variation and only partial mortality. Dead embryos showed severe necrosis in the brain and congestion in the kidney and the subcutis of limbs. In contrast, yolk sac inoculation led to uniform infection and mortality, the dead embryos exhibiting the same lesions as those described above but without the subcutaneous congestion. Histological lesions in dead embryos inoculated by either route were similar and particularly severe in the central nervous system. Viral antigens were detected mainly in the vasculature and neurons. The results indicated that Nipah virus is highly pathogenic to chicken embryos, and that the route of inoculation is an important determinant of the course of disease. The findings also suggested that yolk sac inoculation can be used for viral titration, and that the chicken embryo represents a useful model for studying the vascular and neuronal tropisms of Nipah virus.

Characterization of Pseudomonas aeruginosa isolates associated with mortality in broiler chicks.

In mid-2000, a broiler chicken company in Alabama experienced high early mortality rates in chicks from two different hatcheries. Five isolates of Pseudomonas aeruginosa, obtained from these contaminated hatcheries and resulting broiler chicks with omphalitis, were selected to determine virulence of the bacteria. One-day-old specific-pathogen-free white leghorn chicks were placed into positive pressure isolation units (10 chicks per unit); feed and water were provided ad libitum. The five isolates of P. aeruginosa (1 x 10(1) or 1 x 10(1) colony-forming units/bird) were used to challenge two replicates of 10 chicks via yolk sac inoculation. Two control groups were injected with 0.1 ml of phosphate-buffered saline, and two groups received no treatment. Mortality was recorded daily, and the chicks that died were necropsied and liver and yolk sacs were cultured. After 14 days, the remaining chickens were euthanatized and necropsied. Bacterial isolates retrieved from liver and yolk sacs were identified by the API 20 NE typing system to confirm that they were the same as the challenge isolate. Virulence varied greatly among the isolates, resulting in mortality rates from 0 to 90%. The challenge isolates produced different and often distinctive postmortem lesion patterns. Antibiotic sensitivity tests showed that all five isolates were resistant to sulfisoxazole, ceftiofur, penicillin, lincomycin, bacitracin, oxytetracycline, erythromycin, naladixic acid, and tetracycline. The isolates varied in sensitivity to other antibiotics, but all isolates were sensitive to gentamicin.

Arthropathic and amyloidogenic Enterococcus faecalis infections in brown layers: A study on infection routes

Intravenous, intra-articular and intraperitoneal inoculation of 6-week-old brown-layer pullets with an arthropathic and amyloidogenic strain of Enterococcus faecalis resulted in amyloid arthropathy, while intramuscular, oral and intratracheal inoculation did not. Oral inoculation of 1-day-old chickens did not cause any pathology. However, intramuscular inoculation with 106 colony forming units resulted in severe growth retardation and arthritis in 60% of the birds, and amyloid arthropathy in approximately 40%. In egg transmission studies, neither egg dipping nor inoculation of the air chamber with E. faecalis reproduced the condition, although a few chicks became septicaemic. Yolk sac inoculation of 6-day-old embryos caused embryonic death within 2 days. In contrast, egg albumen inoculation with E. faecalis led to arthritis in one of six of the progeny, indicating the possibility that vertical transmission of E. faecalis by the oviductal route could lead to arthritis. The presence of antibodies to E. faecalis was confirmed by enzyme-linked immunosorbent assay in 14/15 of experimental birds that had developed arthritis.

Characterization of an Avian Adenovirus Associated with Inclusion Body Hepatitis in Day-Old Turkeys

A group I avian adenovirus isolated from day-old turkeys with inclusion body hepatitis (IBH) was identified as turkey adenovirus serotype 2 (TAV2) based on cross-neutralization assays and DNA restriction endonuclease analyses. Yolk sac inoculation of embryonated turkey eggs resulted in embryo mortality and significantly (P < 0.01) decreased hatchability compared with sham-inoculated controls. Embryo mortality occurred primarily between day 24 of incubation and the time embryos hatched. Focal necrosis was detected in livers of 11/52 virus-inoculated embryos that died postinoculation and 1/27 hatchlings; in three embryos, areas of necrosis contained intranuclear inclusion bodies. These findings identify the IBH isolate as TAV2, incriminate the virus as a potential cause of suboptimal hatchability in turkeys, and provide additional evidence for causal involvement in IBH.

Simulation of Maternal Immunity by Inoculation of Immune Yolk Preparations into the Yolk Sac of 1-Day-Old Chickens

Yolk harvested from eggs laid by hens hyperimmunized with killed Newcastle disease virus (NDV) was inoculated into the yolk sac of 1-day-old specific-pathogen-free (SPF) chickens. Serum hemagglutination-inhibition antibody titers reached maximum levels 1 to 4 days after yolk inoculation and declined at a rate similar to that reported for naturally acquired maternal antibody. Expected levels of immune interference were observed when yolk-inoculated chickens were vaccinated with a conventional oil-emulsion NDV vaccine. These results show that yolk-sac inoculation with yolk antibody is a suitable approach for producing maternally immune chickens for laboratory studies.

Abortion in Japanese cows caused by Chlamydia psittaci

An outbreak of abortion in cows occuring in Nigaata Prefecture was shown to be caused by Chlamydia psittaci. Elementary bodies characteristic of Chlamydia were found in the liver of aborted fetuses and C. psittaci antigen was demonstrated by indirect immunofluorescence. Chlamydia was isolated from the liver of aborted fetuses by the yolk sac inoculation of developing chick embryos and by the intraperitoneal inoculation of guinea pigs. Abortion occured mostly in middle or late pregnancy. Aborted fetuses showed subcutaneous edema and gelatinous infiltration, enlarged liver and spleen, and dark red pleural and ascitic fluid. Focal necrosis was shown in the liver, spleen and lymph nodes. Serological findings and isolation of Chlamydia from fecal specimens indicated a wide dissemination of C. psittaci among cows in the area.

Laboratory techniques for the diagnosis of chlamydial infections.

Yolk-sac inoculation of embryonated eggs was superseded 25 years ago by the use of cell cultures (often McCoy) for the isolation of Chlamydia trachomatis. Centrifugation of specimens onto the cell monolayers was shown to increase sensitivity, but little of late has further improved sensitivity which is at least ten-fold greater than that of eggs. However, culture is slow and labour intensive so that non-cultural techniques without these drawbacks have come to dominate. Direct fluorescent antibody (DFA) tests are rapid and have sensitivities that range from 70% to 100% for men and 68% to 100% for women, and specificities that range from 87% to 99% for men and 82% to 100% for women; if the tests are read by competent observers the values are at the top end of the ranges. The detection rate may be enhanced even further by relatively low-speed centrifugation of specimens before staining. Skilled reading is not a feature of enzyme immunoassays (EIAs) which according to the literature have sensitivities that range from 62% to 97% for men and 64% to 100% for women, and specificities that range from 92% to 100% for men and 89% to 100% for women. However, comparison against poor reference tests is responsible for most of the higher values and the clinician should not be misled into believing that EIAs have excellent sensitivity; the lower values in the ranges are closer to reality. Furthermore, EIAs that are being designed for use by general practitioners should be regarded with the greatest caution since lack of sensitivity means that chlamydia-positive patients will go undetected. The polymerase chain reaction (PCR) is not bedevilled by insensitivity but it is no more sensitive than the most sensitive cell culture or DFA tests. PCR is unsuitable for routine diagnosis but has a place as a research tool. For men, examination of "first-catch" urine samples by the best of the non-cultural procedures provides an acceptable non-invasive approach to diagnosis; for women, the value of examining urine may be less, but needs to be thoroughly tested. However, there is little doubt that a Cytobrush used to obtain cervical specimens holds no practical advantage over a swab. Serological tests are reliant on the provision of paired sera for making a diagnosis; high antibody titres in single sera may be suggestive of an aetiological association in deep-seated chlamydial infections (epididymitis, arthritis, salpingitis, etc), but unequivocal interpretation is unusual, particularly in an individual case, since the distinction between a current and past infection is problematical.

Experimental Transmission of Turkey Viral Hepatitis to Day-Old Poults and Identification of Associated Viral Particles Resembling Picornaviruses

Turkey viral hepatitis (TVH) was experimentally reproduced in two experiments in 1-day-old poults. In the first experiment, an infectious inoculum was prepared from filtered yolk materials harvested from dead embryonating chicken eggs (ECE) previously inoculated with suspensions of liver and pancreas tissues collected from TVH-affected birds in commercial turkey flocks. One-day-old poults given a yolk-sac inoculation or oral gavage with this preparation developed lesions in the liver and pancreas characteristic of TVH at 20 days postinoculation (PI) in 60% and 14% of the experimentally infected birds, respectively. With the identical inoculum, embryo mortality occurred at 8 and 10 days PI in embryonating turkey eggs (ETE) inoculated into the yolk sac. In the second experiment, an infectious inoculum was prepared from filtered yolk materials from dead ETE harvested in the first experiment. One-day-old poults given a yolk-sac inoculation with this filtered yolk material developed lesions in the liver and pancreas within 5 days PI. At 20 days PI, 67% of the experimentally infected birds had similar lesions. With the inoculum given to these poults, embryo mortality occurred at 6, 8, and 10 days PI in ETE inoculated into the yolk sac. Virus particles 26-28 nm in diameter with icosahedral morphology typical of picornaviruses were identified by EM in the yolk sacs of ETE that died in both experiments, and inoculated ETE that died following passage of filtered suspensions of pancreatic tissues collected from affected birds in the first experiment.

Dietary Protein and Yolk Sac Inoculation with Escherichia coli in Young Turkeys

An experiment was conducted to examine the effect of pathogenic Escherichia coli inoculated into the yolk sac of day-old turkeys. Escherichia coli was isolated from the yolk sac of stunted poults and inoculated directly into the yolk sac of day-old birds. Poults were administered either .1 ml of uninoculated sterile Todd-Hewitt broth or .1 ml of a 10−3 or 10−2 dilution of a 24-hr E. coli culture containing 3.4 × 108 viable bacteria/ml. In addition, poults were fed either 28 or 22% protein diets from 0 to 21 days of age to form a 3 × 2 factorial arrangement. Body weight gain and feed consumption were measured weekly, and dry matter and protein retention and nitrogen-corrected metabolizable energy were measured from 7 to 10 and 17 to 20 days postinoculation. Intestinal mucosal dipeptidase and maltase activities were determined at 21 days of age.Average mortality by 7 days of age was increased from 1 to 36% from the E. coli inoculation of the yolk sac. Escherichia coli significantly depressed body weight gain and feed consumption 27 and 30, 13 and 16, and 6 and 8%, respectively, during the first, second, and third weeks of the experiment but failed to affect feed efficiency. Feeding a 28% protein diet alleviated the depression in feed consumption and body weight gain to some extent compared with a substantial depression at 22% protein.Nitrogen content and gross energy of the excreta were increased by both dilutions of E. coli for the 7 to 10-day period; this was indicative of a malabsorption of nutrients. Dry matter and nitrogen retention values were variable in the 7 to 10-day period and did not differ during the 17 to 20-day period. Changes in mucosal maltase and dipeptidase activities varied along the length of the intestine at 21 days of age.

In Ovo Pathogenicity of Mycoplasma gallisepticum Strains in the Presence and Absence of Maternal Antibody

Mycoplasma gallisepticum (MG) strains showing marked variation in pathogenicity were examined for virulence in ovo. No correlation was found between in ovo pathogenicity and other in vivo or in vitro methods for pathogenicity evaluation. For certain highly pathogenic strains, there was a clear relationship between the titer of MG inoculated and the embryo mortality and time of death; an LD50 for these strains could be calculated by yolk-sac inoculation. However, not every strain that caused lesions in the respiratory tract in vivo caused embryo mortality. Less pathogenic strains that grow well and colonize the respiratory tract usually caused embryo mortality during the later stages of incubation, and there was no strict correlation between titer of inoculum and embryo mortality. It appeared that embryo death in these cases may have resulted from generalized stress due to mass multiplication of the MG. Embryo mortality due to virulent MG was completely blocked in eggs containing maternal antibody to MG, although the mycoplasma could be reisolated from the yolk-sac membrane of the live embryonated egg after 17 days of incubation. Attempts to mimic the effect of maternal antibody by injecting exogenous MG antiserum were not successful.