Extensive human presence and regular gentle handling improve growth, survival and immune competence in ostrich chicks.

A total of 416 day-old ostrich chicks were randomly allocated to one of the three different husbandry practices for 3 months after hatch; HP1 (extensive human presence with gentle human voice, visual and gentle physical stimuli), HP2 (similar to HP1 but without physical stimuli) and S (human presence limited to supply of feed and water). Chick weight (kg) was measured at 6 and 12 weeks of age, while mortalities were recorded daily to calculate the survival rate. Finally, chicks' antibody responses to vaccination against Newcastle disease (NCD) was measured using the Hemagglutination-Inhibition (HI) test at 20 weeks of age. While HP1 chicks were heavier and survived better to 6 weeks of age than HP2 and S chicks (p < .05), no difference was observed thereafter (p > .05). Furthermore, HP1 chicks had an improved immune competence, as illustrated by their lower percentage of positive HI titers, compared to HP2 and S chicks (p < .05). Hence, integrating extensive human presence with positive human-chick interactions may assist in alleviating challenges related to chick rearing in the ostrich industry.

DNA Vaccines Against Mycoplasma Elicit Humoral Immune Responses in Ostriches.

In ostriches, the population densities resulting from intensive rearing increases susceptibility to pathogens such as mycoplasmas. In addition to good management practices, vaccination offers an attractive alternative for controlling mycoplasma infections in food animals, instead of using antibiotics, which often leave unacceptable residues. The use of live attenuated vaccines, however, carry the concern of reversion to virulence or genetic recombination with field strains. Currently there are no commercially available vaccines against ostrich-infecting mycoplasmas and this study therefore set out to develop and evaluate the use of a DNA vaccine against mycoplasma infections in ostriches using an OppA protein as antigen. To this end, the oppA gene of "Mycoplasma nasistruthionis sp. nov." str. Ms03 was cloned into two DNA vaccine expression vectors after codon correction by site-directed mutagenesis. Three-months-old ostriches were then vaccinated intramuscularly at different doses followed by a booster vaccination after 6 weeks. The ability of the DNA vaccines to elicit an anti-OppA antibody response was evaluated by ELISA using the recombinant OppA protein of Ms03 as coating antigen. A statistically significant anti-OppA antibody response could be detected after administration of a booster vaccination indicating that the OppA protein was successfully immunogenic. The responses were also both dose and vector dependent. In conclusion, the DNA vaccines were able to elicit an immune response in ostriches and can therefore be viewed as an option for the development of vaccines against mycoplasma infections.

Distribution and developmental changes of ghrelin-immunopositive cells in the pancreas of African ostrich chicks (Struthio camelus).

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is produced by multiple cell types and affects feeding behavior, metabolic regulation, and energy balance. In the mammalian pancreas, the types of endocrine cells that are immunoreactive to ghrelin vary. However, little was known about its distribution and developmental changes in the pancreas of African ostrich chicks (Struthio camelus). In the present study, the distribution, morphological characteristics, and developmental changes of ghrelin-immunopositive (ghrelin-ip) cells in the pancreas of African ostrich chicks were investigated using immunohistochemistry. Ghrelin-ip cells were found in both the pancreatic islets and acinar cell regions. The greatest number of ghrelin-ip cells were found in the pancreatic islets, and were primarily observed at the periphery of the islets; some ghrelin-ip cells were also located in the central portion of the pancreatic islets. Interestingly, from postnatal d 1 to d 90, there was a steady decrease in the number of ghrelin-ip cells in the pancreatic islets and acinar cell regions. These results clearly demonstrated that ghrelin-ip cells exist and decreased with age in the African ostrich pancreas from postnatal d 1 to d90. Thus, these findings indicated that ghrelin may be involved in the development of the pancreas in the African ostrich.

Susceptibility and Status of Avian Influenza in Ostriches.

The extensive nature of ostrich farming production systems bears the continual risk of point introductions of avian influenza virus (AIV) from wild birds, but immune status, management, population density, and other causes of stress in ostriches are the ultimate determinants of the severity of the disease in this species. From January 2012 to December 2014, more than 70 incidents of AIV in ostriches were reported in South Africa. These included H5N2 and H7N1 low pathogenicity avian influenza (LPAI) in 2012, H7N7 LPAI in 2013, and H5N2 LPAI in 2014. To resolve the molecular epidemiology in South Africa, the entire South African viral repository from ostriches and wild birds from 1991 to 2013 (n = 42) was resequenced by next-generation sequencing technology to obtain complete genomes for comparison. The phylogenetic results were supplemented with serological data for ostriches from 2012 to 2014, and AIV-detection data from surveillance of 17 762 wild birds sampled over the same period. Phylogenetic evidence pointed to wild birds, e.g., African sacred ibis (Threskiornis aethiopicus), in the dissemination of H7N1 LPAI to ostriches in the Eastern and Western Cape provinces during 2012, in separate incidents that could not be epidemiologically linked. In contrast, the H7N7 LPAI outbreaks in 2013 that were restricted to the Western Cape Province appear to have originated from a single-point introduction from wild birds. Two H5N2 viruses detected in ostriches in 2012 were determined to be LPAI strains that were new introductions, epidemiologically unrelated to the 2011 highly pathogenic avian influenza (HPAI) outbreaks. Seventeen of 27 (63%) ostrich viruses contained the polymerase basic 2 (PB2) E627K marker, and 2 of the ostrich isolates that lacked E627K contained the compensatory Q591K mutation, whereas a third virus had a D701N mutation. Ostriches maintain a low upper- to midtracheal temperature as part of their adaptive physiology for desert survival, which may explain the selection in ratites for E627K or its compensatory mutations-markers that facilitate AIV replication at lower temperatures. An AIV prevalence of 5.6% in wild birds was recorded between 2012 and 2014, considerably higher than AIV prevalence for the southern African region of 2.5%-3.6% reported in the period 2007-2009. Serological prevalence of AI in ostriches was 3.7%, 3.6%, and 6.1% for 2012, 2013, and 2014, respectively. An annual seasonal dip in incidence was evident around March/April (late summer/autumn), with peaks around July/August (mid to late winter). H5, H6, H7, and unidentified serotypes were present at varying levels over the 3-yr period.

Multiple IgH Isotypes Including IgD, Subclasses of IgM, and IgY Are Expressed in the Common Ancestors of Modern Birds.

Although evolutionarily just as ancient as IgM, it has been thought for many years that IgD is not present in birds. Based on the recently sequenced genomes of 48 bird species as well as high-throughput transcriptome sequencing of immune-related tissues, we demonstrate in this work that the ostrich (Struthio camelus) possesses a functional δ gene that encodes a membrane-bound IgD H chain with seven CH domains. Furthermore, δ sequences were clearly identified in many other bird species, demonstrating that the δ gene is widely distributed among birds and is only absent in certain bird species. We also show that the ostrich possesses two µ genes (µ1, µ2) and two υ genes (υ1, υ2), in addition to the δ and α genes. Phylogenetic analyses suggest that subclass diversification of both the µ and υ genes occurred during the early stages of bird evolution, after their divergence from nonavian reptiles. Although the positions of the two υ genes are unknown, physical mapping showed that the remaining genes are organized in the order µ1-δ-α-µ2, with the α gene being inverted relative to the others. Together with previous studies, our data suggest that birds and nonavian reptile species most likely shared a common ancestral IgH gene locus containing a δ gene and an inverted α gene. The δ gene was then evolutionarily lost in selected birds, whereas the α gene lost in selected nonavian reptiles. The data obtained in this study provide significant insights into the understanding of IgH gene evolution in tetrapods.

Molecular cloning, expression and bioactivity of B cell activating factor (BAFF) in African ostrich.

B cell activating factor (BAFF), which belongs to the tumor necrosis factor (TNF) family, is testified to play a critical role in B cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, the cDNA of open reading frame (ORF) in African ostrich (Struthio camelus) BAFF (designated OsBAFF) was cloned by reverse transcription-PCR (RT-PCR). The OsBAFF gene encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like BAFFs from chicken (cBAFF), quail (qBAFF), duck (dBAFF), goose (gBAFF) and dove (doBAFF). RT-PCR analysis showed that the OsBAFF gene is strongly expressed in the bursa of Fabricius, thymus, spleen, and bone marrow. The soluble OsBAFF had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in Escherichia coli Rosset (DE3). In vitro, purified OsBAFF was not only able to promote the survival of African ostrich bursal lymphocytes, but also able to co-stimulate proliferation of mouse splenic B cells. The expression of OsBAFF in lymphocyte cells was higher than the control after LPS stimulation. These findings indicated that OsBAFF plays an important role in survival and proliferation of African ostrich bursal lymphocytes, which may provide valuable information for research into the immune system of African ostrich and OsBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and any other birds.

High resolution fluorescence microscopy evidence on the transport of immunoglobulins. Differences between mammalian IgG, F(ab')2 and avian IgY.

We describe the subcellular localization of horse F(ab')(2) and IgG, and ostrich IgY labeled with fluorescein isothiocyanate (FITC) administered IV to mice. We used wide field high sensitivity fluorescence microscopy deblurred by 3-dimensional blind deconvolution of kidney, liver, lungs and brain sections. Sections were obtained from mice sacrificed 15 min, 1 or 5 h after receiving FITC-immunoproteins, counter-stained with DAPI (4',6'-diamidino-2-phenylindole) and Evans blue. FITC-IgG and its fractions are rapidly taken up and extravasated by vascular endothelium. FITC-IgG and FITC-F(ab')(2) appear to be quickly secreted by glomeruli endothelium and to be reabsorbed along all nephron segments. FITC-IgG and FITC-F(ab')(2) appeared 15 min after IV injection within bronchial, alveolar and bile duct epithelium. Hepatocytes were loaded with fluorescence after 15 min of administration. Fluorescence was absent from brain slices, except for the endothelium of some vessels in brain ventricles which appeared intensely fluorescent. Fluorescence appeared in intracellular vesicles which conferred the tissues a glowing foamy aspect for up to 5 h after inoculation. Arterial elastic layers were intensely green after horse FITC-Ig inoculation. Ostrich FITC-IgY behaved completely differently to horse Ig's; only 1 h after injection it was possible to observe small brightly green scarce vesicles in vascular endothelium of arteries, interstitial kidney capillaries between nephron tubules and were also scarce in glomeruli endothelium; FITC-IgY appeared only in hepatic sinusoids in the liver. No IgY was seen in bronchial and alveolar endothelium, in bile ducts or in hepatocytes.

Evaluation of different serological tests for the detection of antibodies against highly pathogenic avian influenza in experimentally infected ostriches (Struthio camelus).

In the present study we collected 177 serum samples from ostriches (Struthio camelus) infected experimentally with A/ostrich/South Africa/Middleton/2004 (H5N2) highly pathogenic avian influenza virus. We tested these samples using the haemagglutination inhibition (HI) test, the agar gel immunodiffusion test and three enzyme-linked immunosorbent assay kits. We considered the HI test, with homologous antigen and including pre-treatment of sera with 10% chicken red blood cells, as the gold standard. Detectable specific antibodies appeared on day 7 post-infection and persisted until the termination of the experiment. The relative sensitivity and specificity of the tests under evaluation and Cohen's K value were calculated. The results reported herein could be of assistance to decision-makers in drafting guidelines for the definition of the health status of ostriches and for trade purposes.

Assessment of the stresses imposed on adult ostriches (Struthio camelus) during handling, loading, transportation and unloading.

The stresses imposed during the handling, loading and unloading of 250 adult ostriches (Struthio camelus) transported by road were evaluated, weighted, scored and later compared with some objective physiological indices of stress measured after the journey. During handling, the numbers of slips and falls, incidents of aggressive behaviour, the calculated behavioural points, the number of injuries recorded per ostrich and the time spent were significantly (P<0.01) greater than the values recorded during loading and unloading. During handling and loading, 45 per cent of the ostriches had a good score (1.1 to 2 points), 15.5 per cent had a fair score (2.1 to 3 points) and 39.4 per cent had a poor or bad score (more than 3 points). The behavioural scores were significantly and positively correlated with the heterophil:lymphocyte ratio, the rectal temperature and the number of injuries sustained by the ostriches. The results showed that the poorer the behavioural score, the higher the level of stress suffered by the ostriches during handling and loading.

Antibody responses in ostriches (Struthio camelus) vaccinated with commercial live and killed Newcastle disease vaccines.

Three ostriches (Struthio camelus) were immunized with commercially available live and killed Newcastle disease (ND) vaccines for chickens and the antibody responses to the ND vaccines were evaluated by a virus-neutralization (VN) test. Primary vaccination with the live vaccine, B1, by eye drop was followed with two shots of alum-precipitated killed vaccine via subcutaneous injection in the neck. As a final booster, another live vaccine, Clone 30, was used by eye drop. A VN antibody titer, more than 1:10 was observed for 6 months. This is the first report on the use of a live vaccine by eye drop as a booster in ostriches as well as evaluating responses to ND vaccines using the VN test in this avian species.

Detection and quantification of antibodies to Newcastle disease virus in ostrich and rhea sera using a liquid phase blocking enzyme-linked immunosorbent assay.

A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method. The cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement (kappa = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Antibody responses to La Sota strain vaccines of Newcastle disease virus in ostriches (Struthio camelus) as detected by enzyme-link ed immunosorbent assay.

Because of the fact that South Africa is a Newcastle disease virus (NDV)-endemic country, major concerns exist that the export of ostrich meat could transmit velogenic strains of this disease. The ability to transmit the virus could be reduced by effective vaccination of South African ostriches. In this study, two vaccination trials were conducted to assess serum antibody production in response to vaccination with La Sota strain NDV vaccines. To this end, a commercially available chicken anti-NDV enzyme-linked immunosorbent assay (ELISA) was modified for the detection of anti-NDV antibodies in ostrich serum. The results obtained with this ELISA were verified by comparison with an indirect ELISA. In the first trial, ostriches were immunized subcutaneously four times with different volumes of an inactivated vaccine and their immune response was determined from 2.5 mo up to the ideal slaughter age of 14 mo. Results indicated that ostriches responded in a dose-dependent manner and gave support for the vaccination schedule currently recommended to South African farmers. In a second trial, immunization by eyedrop with a live La Sota vaccine of 5-wk-old ostriches did not elicit a humoral immune response. The results indicate that it is highly unlikely that ostriches that have been vaccinated according to the recommended vaccination schedule can transmit the virus.

Serologic survey of slaughter-age ostriches (Struthio camelus) for antibodies to selected avian pathogens.

Serum samples from 163 slaughter-age ostriches (Struthio camelus) in Ohio and Indiana were tested for antibodies to avian influenza virus (AIV), Newcastle disease virus (NDV), paramyxovirus (PMV) 2, PMV3, PMV7, infectious bursal disease virus (IBDV), Bordetella avium, Mycoplasma synoviae, Mycoplasma gallisepticum, Ornithobacterium rhinotracheale, Salmonella pullorum, Salmonella gallinarum, and Salmonella typhimurium. One ostrich had antibodies to AIV H5N9, 57% of the ostriches had antibodies to NDV, four ostriches had antibodies to both NDV and PMV2, and one ostrich had antibodies to NDV, PMV2, PMV3, and PMV7. None of the ostriches had antibodies to IBDV, B. avium, M. synoviae, M. gallisepticum, O. rhinotracheale, S. pullorum, S. gallinarum, and S. typhimurium. This is the first report of antibodies to avian influenza and PMV7 in ostriches in the United States.

Forssman penta- and tetraglycosylceramide are xenoantigens of ostrich kidney and liver.

The heterophile antigens Galalpha1-->3Gal and N-glycolylneuraminic acid are the major obstacle to grafting mammal organs, especially from pig, to man. Lack of expression of these common xenoantigens by birds has raised interest in ostrich as a potential organ donor for xenotransplantation. Glycosphingolipids of ostrich liver and kidney were investigated for their carbohydrate determinants. Both organs were found similar in their glycolipid composition with three major species, mono-, di-, and pentaglycosylceramide. The pentaglycosylceramide was characterized as the Forssman antigen. In both organs, the ceramide portion was highly hydroxylated with prevalence of alpha-hydroxylated fatty acids, C18 phytosphingosine in kidney and C18 sphingosine in liver Forssman glycolipid. These data indicate that hydroxylation of kidney glycosphingolipids, which is found in mammals, has been maintained since the divergence of birds from other vertebrates. Characterization of a minor glycolipid as a Forssman tetraglycosylceramide built on the galabiosylceramide core indicates that the Forssman tetraglycosylceramide also exists in vivo. Its precursors, galactosyl- and galabiosylceramide, were characterized in kidney and liver. The Forssman antigen is the third heterophile antigen against which man raises natural antibodies. Its localization in the vascular endothelium and connective tissue makes ostrich an unpromising organ or cell donor for xenotransplantation to man.

Haematological and immunological variables in a domesticated and wild subspecies of ostrich (Struthio camelus).

1. Domesticated ostriches have been selected rigorously for productive traits with little concern for immunological responses, in contrast to wild ostriches. 2. We hypothesised that the immunological responses of wild and domesticated ostriches would differ. Total leucocyte counts, differential counts, heterophil: lymphocyte ratios, phagocytic activity, lysosome levels and anti-sheep red blood cell (SRBC) antibody titres (total, IgG, IgM) were compared between domesticated (n=3) and wild (n=3) ostrich subspecies. 3. Total leucocytes, lymphocytes and heterophils were similar in the 2 subspecies, but basophils and eosinophils were lower in the wild than in the domesticated ostriches. Lysosome concentrations and phagocytic activities were higher in the wild ostriches. 4. Total and IgM antibody titres to SRBC reached peak values quicker in the domesticated than in wild ostriches. IgG development patterns were similar. 5. The results suggest that a stronger non-specific immune response was shown by the wild ostriches (higher phagocytosis and lysozymes) whereas a stronger specific immune response was shown by the domesticated ostriches (peak values of anti-SRBC antibody titres were reached more quickly).

Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein.

A competitive enzyme-linked immunosorbent assay (C-ELISA) employing a baculovirus-expressed recombinant nucleoprotein and a monoclonal antibody was developed for the detection of antibodies to type A influenza virus nucleoprotein. The performance of the C-ELISA was evaluated by testing 756 chickens, 1123 turkeys, 707 emus, and 1261 ostriches, for a total of 3847 serum samples. Relative to the agar gel immunodiffusion (AGID) test, the C-ELISA had a sensitivity of 100% for all four species. The C-ELISA's sensitivity relative to the hemagglutination-inhibition (HI) test results was 100% for chicken, turkey, and emu and 96.2% for the ostrich serum samples. More than 90% of the AGID-negative/C-ELISA-positive serum samples were found positive by HI for at least one influenza serotype. The specificity of C-ELISA relative to AGID ranged from 85.5% to 99.8% for sera collected from these species. These results indicated that the C-ELISA was more sensitive and more specific than the AGID test and as sensitive and as specific as the HI test. The C-ELISA has the potential to replace the AGID test for screening sera from avian species, including ratites, for detection of antibodies to type A influenza virus.