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PubMed2005

Cloning and expression of ostrich trypsinogen: an avian trypsin with a highly sensitive autolysis site.

One of ostrich (Struthio camelus) trypsinogen genes was cloned from pancreatic cDNA. Its amino acid sequence compared to known trypsin sequences from other species shows high identity and suggests that it is a member of the phylogenetically anionic trypsinogen I subfamily. After cytoplasmic over expression in Escherichia coli and renaturation, the activation properties of ostrich trypsinogen were studied and compared to those of human trypsinogen 1 (also called as human cationic trypsinogen). Ostrich trypsinogen undergoes bovine enterokinase activation and autoactivation much faster than human trypsinogen 1 and exhibits on a synthetic substrate a somewhat higher enzymatic activity than the latter one. The most interesting property of ostrich trypsin is its relatively fast autolysis that can be explained via a mechanism different from the common mechanism for rat and human 1 trypsins. The latter proteases have a site, Arg117-Val118, where the autolysis starts and then goes on in a zipper-like fashion. This is absent from ostrich trypsin. Instead it has a couple of cleavage sites within regions 67-98, including two unusual ones, Arg76-Glu77 and Arg83-Ser84. These appear to be hydrolysed fast in a non-consecutive manner. Such an autolysis mechanism could not be inhibited by a single-site mutation which in humans is proposed to lead to pancreatitis.

Amino Acid Sequence↗
PubMed2006

Scorpion digestive lipase: a member of a new invertebrate's lipase group presenting novel characteristics.

Unlike classical digestive lipases, the scorpion digestive lipase (SDL) has a strong basic character. The SDL activity's optimal pH, when using tributyrin or olive oil as substrate, was 9.0. Added to that, the estimated isoelectric point of the native SDL using the electrofocusing technique, was found to be higher than 9.6. To our knowledge, this is the first report of an animal digestive lipase having such a basic character. When olive oil was used as substrate, SDL was shown to be insensitive to the presence of amphiphilic proteins such as bovine serum albumin (BSA). Furthermore, the hydrolysis was found to be specifically dependent on the presence of Ca(2+) ions, since no significant SDL activity was detected in the presence of ions chelator such as EDTA. Nevertheless, the SDL does not require Ca(2+) to trigger the hydrolysis of tributyrin emulsion. Interestingly Zn(2+) and Cu(2+) ions act as strong inhibitors of SDL activity when using tributyrin as substrate. An internal chymotryptic cleavage of SDL generated two fragments of 28 and 25 kDa having the same N-terminal sequence. This sequence of 19 residues does not share any homology with known animal and microbial lipases. Polyclonal antibodies directed against SDL (pAbs anti-SDL) failed to recognise ostrich pancreatic and dog gastric lipases (OPL and rDGL). Moreover, both pAbs anti-OPL and anti-rDGL failed to immunoreact with SDL. These immunological as well as distinct biochemical properties strengthen the idea that SDL appears to belong to a new invertebrate's lipase group.

Animals↗
PubMed2004

Effects of periodate and chondroitin 4-sulfate on proteoglycan stabilization of ostrich pericardium. Inhibition of calcification in subcutaneous implants in rats.

Chemical modification of biological materials used in the manufacture of cardiac valves tends to reduce the relatively high degree of biodegradation and calcification of the implanted bioprostheses. The most widely used treatment to reduce biodegradability of the valves is glutaraldehyde fixation. However, this treatment is potentially toxic and induces tissue calcification. In order to minimize these undesirable effects, we have analyzed the effect of a pre-fixation of endogenous proteoglycans and exogenous glycosaminoglycans, as well as the borohydride reduction influence on the different modified ostrich pericardium implants after subcutaneous implantation in rats. The presence of calcific deposits was detected in all implanted GA-fixed samples; however, calcification was highly reduced in both groups of periodate-prefixed materials, which showed also a very low Ca/P molar ratio. Borohydride post-treatment of these biomaterials resulted in a significant increase in calcium phosphate precipitation, with the appearance of calcium deposits mainly in an amorphous form even though X-ray diffraction allowed the detection of brushite- and apatite-like crystals. Regarding tissue stability, no significant differences were found among the borohydride-untreated implants but higher levels of matrix metalloproteinases were observed by gelatin zymography in the periodate pre-fixed materials. This increase was partially reduced by pre-fixation of exogenous chondroitin 4-sulfate. On the other hand, borohydride post-treatment not only increased calcification, but also reduced tissue stability and increased the presence of matrix-degrading activities.

Animals↗
PubMed2006

Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich, and turkey.

Transferrins form an important class of iron-binding proteins widely distributed in the physiological fluids of vertebrates and invertebrates. In vertebrates they are present mostly in serum as serotransferrins. In birds and reptiles transferrins are also found in eggs as ovotransferrins. However, until now only chicken and duck ovotransferrin sequences have been published. This paper presents data on the purification, biochemical analysis, cloning and sequencing of ovotransferrins from red-eared turtle, African ostrich and turkey, revealing their significant homology with other known ovotransferrin sequences. The proteins were purified by size-exclusion and anion-exchange chromatography. Isoelectric points, iron-saturated and iron-free spectra, as well as the mRNA nucleotide sequences of 2,409 nt (ORF: 2,106 nt encoding a 701-amino-acid polypeptide; ), 2,418 nt (ORF 2,118 nt encoding a 705-amino-acid polypeptide; ), and 2,397 nt (ORF: 2,118 nt encoding a 705-amino-acid polypeptide; ) were determined for ostrich (OtrF), red-eared turtle (TtrF), and turkey (MtrF) ovotransferrin, respectively.

Amino Acid Sequence↗
PubMed2005

Differential effects of detergents, fatty acids, cations and heating on ostrich skeletal muscle 20S proteasome.

The 20S proteasome, the catalytic core of the 26S proteasome, has previously been isolated, purified and partially characterised from ostrich skeletal muscle (Thomas, A.R., Oosthuizen, V., Naude, R.J., Muramoto, K. 2002. Biol. Chem. 383, 1267-1270). Due to the apparent latency of the 20S proteasome purified from various sources, this study focuses on further characterising the ostrich enzyme in terms of the effects of selected detergents, fatty acids and cations, as well as heating at 60 degrees C, on four of its activities. Results showed that ostrich skeletal muscle 20S proteasome was affected in a non-concentration-dependent manner by the selected detergents and fatty acids. Monounsaturated fatty acids, unlike unsaturated fatty acids, showed no major effects on the activities of the ostrich enzyme. The enzyme did not show sensitivity towards monovalent cations and the only divalent cations that showed a relevant effect were Ca2+ and Mg2+. Heating at 60 degrees C for 1-2 min had a substantial activating effect only on the peptidylglutamylpeptide-hydrolase (PGPH) and caseinolytic activities. In conclusion, many of the effects by the abovementioned reagents and conditions were noticeably different to those shown on different sources of the enzyme, further demonstrating the unique kinetic characteristics of the ostrich skeletal muscle 20S proteasome.

Animals↗
PubMed2006

The relation between ultrasonographic observations in the oviduct and plasma progesterone, luteinizing hormone and estradiol during the egg laying cycle in ostriches.

In this study we investigated the temporal relationship between ovulation, egg formation, oviposition and the changes in plasma concentrations of progesterone, luteinizing hormone and estradiol-17beta during the egg laying cycle in farmed ostriches. In 10 egg-producing birds, transcutaneous ultrasound scanning was performed at 3h intervals and blood sampling at hourly intervals during a period of at least 48h (one egg laying cycle). In hens (n=8) that ovulated during the observational period, the ovulated egg was first detected 2h after oviposition; thus, ovulation occurred shortly after oviposition in all birds. During the period between two consecutive ovipositions, the developing egg remained for 9h in the proximal part (infundibulum, magnum or isthmus) and for 39h in the distal part of the oviduct (uterus). In ovulating hens, plasma progesterone concentrations showed a characteristic and consistent profile: from basal levels of around 0.1ng/ml concentrations started to increase 12h before oviposition, reached an average maximum of 3.5ng/ml at 3h before oviposition and returned to basal levels 3h and 30min after oviposition. Changes in plasma luteinizing hormone and estradiol-17beta concentrations showed comparable patterns of elevation and decline relative to the timing of oviposition and ovulation. However, variation in their individual basal concentrations was generally larger and peak values were less conspicuous than those of progesterone. In non-ovulating hens (n=2) neither progesterone, nor luteinizing hormone nor estradiol-17beta showed elevations to peak concentrations before oviposition. These data demonstrate that during the egg laying cycle of ostriches, events such as ovulation, egg development and oviposition evolve according to a rather strict time schedule, and that progesterone, luteinizing hormone and estradiol-17beta reach peak concentrations shortly before ovulation. Additionally, our findings also show that on-farm ultrasound scanning is a useful technique to discriminate between ovulating and non-ovulating hens.

Animals↗
PubMed2006

Identification of three novel ostricacins: an update on the phylogenetic perspective of beta-defensins.

Three new beta-defensins, ostricacins-2, 3 and 4 (Osp-2, 3 and 4), have been successfully purified and characterised from ostrich heterophils in addition to ostricacin-1 (Osp-1). These peptides are composed of 36-42 amino acids with a molecular weight range of 4.70-4.98 kDa. In vitro, Osp-1, 3 and 4 were active against Escherichia coli O157:H7 and Staphylococcus aureus 1056 MRSA, whilst Osp-2 was active against bacterial strains plus the yeast Candida albicans 3153A. Minimal inhibitory concentrations of the three ostricacins ranged from 0.96 microg/mL to 12.03 microg/mL. Comparison with the known beta-defensins from mammalian and other avian species revealed that the four ostricacins shared eight conserved residues (six cysteines and two glycines), identified as the 'beta-defensin core motif'. Comparisons of the sequence also indicated that beta-defensins could have originated from a common beta-defensin-like ancestor that occurred before avian and mammalian lines diverged.

Animals↗
PubMed2005

Osteoinductivity of partially purified native ostrich (Struthio camelus) bone morphogenetic protein: comparison with mammalian species.

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily. They are capable of inducing ectopic bone formation. Until now, the main interest has been focused on mammalian osteoinductive BMPs, and there are no reports of native BMP extracts of birds. In this study, we isolated and characterized native BMPs of ostrich (Struthio camelus) and compared them with identically isolated native bovine (cow) and reindeer BMPs with regard to BMP pattern and osteoinductive capacity. The ostrich BMP pattern differed markedly from that of cow and reindeer BMP in non-reduced SDS-PAGE, reduced SDS-PAGE and Western blot. The differences in isoelectric focusing analysis were smaller. However, the ostrich BMP extract had a peak at pH 5.1, clearly differing from the BMPs of cow and reindeer. The osteoinductive capacity and density of ectopic bone, induced by BMP extracts in a mouse thigh muscle pouch, were determined radiographically. The ostrich BMP extract displayed significantly lower osteoinductive capacity and density of induced bone than the bovine and reindeer BMP extracts. In conclusion, our results indicate that the BMP pattern of birds differs considerably from that of mammals, and that the osteoinductive capacity of BMPs and the density of induced bone are lower in birds than in mammals. They also suggest that the bone metabolism of birds is adapted to make light bones suitable for flying.

Animals↗
PubMed2006

Female-specific DNA sequences in ostriches.

Ostrich absence of heteromorphic sex chromosomes, unique sequences or markers located in the ostrich W-chromosome. Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in ostrich. One hundred and forty random primers were used for random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). One of these primers, OPAJ-13, produced a sex-specific band only found in tested females, which was isolated and constructed into plasmids for nucleotide sequencing. A 760bp novel female-specific sequence was obtained. Two primers (OstSexOPAJ13-F and -R) were designed according to the cloned female sequence to amplify the female-specific fragment from genomic DNA of ostriches for sexing by PCR. The sex-specific band was represented in females but none were found in the males. This result showed that the sex of ostrich could be easily and effectively identified using the female-specific primers for PCR technique.

Animals↗
PubMed2004

Immunohistochemical localization of the progesterone and oestrogen receptors in the shell gland of sexually immature ostriches (Struthio camelus) with active or inactive ovaries.

The immunohistochemical localization of progesterone and oestrogen receptors was studied in the shell gland of the immature ostrich (Struthio camelus) during periods of ovarian activity and inactivity. In birds with active ovaries moderate to strong immunostaining for the progesterone receptor was observed in the surface epithelium and tubular glands. In contrast faint progesterone receptor immunostaining was observed in the surface epithelium of the shell gland in ostriches with inactive ovaries. In addition, bud-like invaginations of the surface epithelium, which signaled tubular gland development, were negative for the progesterone receptor. Oestrogen receptor immunostaining, which was seen only in birds with active ovaries, was weak and restricted to nuclei of the surface epithelium. These results suggest that steroid hormones secreted by the active ovary regulate the differentiation of the shell gland. Furthermore, the influence of these hormones on the shell gland appears to be mediated predominantly through the activation of the progesterone receptor.

Animals↗
PubMed2004

Pharmacokinetics of marbofloxacin after intravenous and intramuscular administration to ostriches.

The pharmacokinetics of marbofloxacin was investigated after intravenous (IV) and intramuscular (IM) administration, both at a dose rate of 5 mg/kg BW, in six clinically healthy domestic ostriches. Plasma concentrations of marbofloxacin was determined by a HPLC/UV method. The high volume of distribution (3.22+/-0.98 L/kg) suggests good tissue penetration. Marbofloxacin presented a high clearance value (2.19+/-0.27 L/kgh), explaining the low AUC values (2.32+/-0.30 microgh/mL and 2.25+/-0.70 microgh/mL, after IV and IM administration, respectively) and a short half life and mean residence time (t(1/2 beta)=1.47+/-0.31 h and 1.96+/-0.35 h; MRT=1.46+/-0.02 h and 2.11+/-0.30 h, IV and IM, respectively). The absorption of marbofloxacin after IM administration was rapid and complete (C(max)=1.13+/-0.29 microg/mL; T(max)=0.36+/-0.071 h; MAT=0.66+/-0.22 h and F (%)=95.03+/-16.89).

Absorption↗
PubMed2006

Detection of thermotolerant Campylobacter in ostriches (Struthio camelus) in Italy.

During the period January 2004 to December 2005 four different ostrich farms were investigated to evaluate the presence of thermotolerant Campylobacter. A total of 150 ostriches were examined and all were found to be clinically healthy. Campylobacter spp. were isolated from 60/150 cloacal swabs. Among the isolates, 48 were identified as Campylobacter jejuni whereas the remainder were identified as Campylobacter coli. Campylobacter lari was not isolated. The ostriches, although apparently in a healthy body condition, can be considered, theoretically, as potential Campylobacter carriers.

Animals↗
PubMed2004

Type 2 heat-labile enterotoxin (LT-II)-producing Escherichia coli isolated from ostriches with diarrhea.

The culture supernatant of Escherichia coli, isolated from ostriches with diarrhea in Brazil, caused elongation in Vero cell, rounding in Chinese hamster ovary (CHO) cells and a cytoplasmic vacuolation in ostrich embryo fibroblasts (OEF), but it was not cytotoxic for chicken embryo fibroblasts (CEF). These effects were not neutralized by antiserum to cholera toxin. Polymerase chain reaction assays showed that the ostrich E. coli contained the gene encoding (eltII-A), but not those for type 1 heat-labile enterotoxin (eltA), heat-stable enterotoxins (estA, estB), verocytotoxins (stx-I, stx-II), or cytotoxic necrotizing factors (cnf 1, cnf 2). All isolates belonged to serotype O15:H8. The enteropathogenic relevance of LT-II in ostrich diarrhea remains undetermined.

Animals↗
PubMed2005

Identification of three novel mycoplasma species from ostriches in South Africa.

Mycoplasmas have been implicated in certain clinical syndromes in ostriches and are associated with upper respiratory tract infections. As these infections result in production losses, they are of considerable economic importance to the South African ostrich industry. Although poultry mycoplasmas have been shown to infect ostriches, the existence of unique ostrich-specific mycoplasmas has been suggested. In this study, mycoplasmas were isolated from ostriches in the Klein Karoo, Central Karoo and Garden Route areas of the Western and Northern Cape Provinces of South Africa and identified using 16S rRNA gene sequencing. These sequences indicated that ostriches in these areas carry three unique mycoplasmas and were not infected with chicken mycoplasmas. Phylogenetic analysis of the 16S rRNA sequences of the three isolated ostrich mycoplasmas showed them to be quite divergent and to fall into two distinct phylogenetic groupings. Unique sequences within the 16S rRNA gene of the ostrich mycoplasmas were subsequently used for the development of specific primers for the detection and diagnosis of mycoplasma infections in ostriches. Chickens kept in close proximity to infected ostriches were not infected with these ostrich mycoplasmas.

Animals↗
PubMed2004

Entamoeba struthionis n.sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus).

In the present work we identify the species of Entamoeba from ostriches (Struthio camelus). The complete sequence of the small subunit ribosomal RNA gene from this organism has been compared with those published for other species of the genus and clear differences have been found. These results confirm previous data which showed differences on parasite morphology and class of host with the other Entamoeba species. Taking all these data together, it can be concluded that the organism from ostriches is a new species whose proposed name is Entamoeba struthionis n.sp. This species probably infects rheas (Rhea americana), but genetic analysis of isolates from this host should be performed to confirm morphological data. Also, comparison of gene sequences with data from other authors on cysts recovered from human stool samples showed the possibility that this amoeba may affect humans. Further studies are needed to determine the risk of transmission of this new species to humans.

Animals↗
PubMed2006

Occurrence of Libyostrongylus sp. (Nematoda) in ostriches (Struthio camelus Linnaeus, 1758) from the north region of the state of Rio de Janeiro, Brazil.

Domestic production of ostrich in Brazil started in the beginning of the last decade, but its sanitary state has not been reported. Libyostrongylus sp. is an ostrich specific nematode whose parasitism can severely affect the birds. Thus, Libyostrongylus spp. larvae were identified in commercial ostriches in the north region of the state of Rio de Janeiro, Brazil. The EPG was determined and fecal cultivation was performed. The eggs presented typical characteristics of strongylid and were present in five out of six farms. The mean EPG varied from 22 to 2395 and Libyostrongylus spp. prevalence was from 0 to 100%, with adult birds more infected. Two types of infective larvae with tail finishing in a tipped spiny knob were distinguished. The first had a mean length of 848 microm (710-1010) with a long sheath tail of about 66 microm (52-112). The other had a mean length of 826 microm (620-940) with a short, more abruptly ending sheath tail of 32 microm (22-40) and a less rounded cephalic end. The differences between these larvae suggest two Libyostrongylus species.

Animals↗
PubMed2006

Molecular cloning and characterization of preproopiomelanocortin (prePOMC) cDNA from the ostrich (Struthio camelus).

To date proopiomelanocortin (POMC), the precursor protein for melanotropin (MSH), adrenocorticotropin (ACTH), lipotropins (LPH), and beta-endorphin (beta-END) in the pituitary gland, has been studied extensively over a wide spectrum of vertebrate classes. A paucity of information exists, however, with regard to POMC in the avian class, where to date POMC from only one species, the domestic chicken, appears to have been fully characterized. In the present study, we report the use of three clones of cDNA to provide the complete nucleotide sequence of ostrich prePOMC cDNA, consisting of 1072 bp (excluding the poly(A) tail). The deduced amino acid sequence of 253 amino acid residues includes the N-terminal signal peptide of 17 amino acid residues. The predicted amino acid sequence in the overall arrangement of its domains, conforms to that found in other tetrapods. Sequence domains for gamma-MSH, ACTH, alpha-MSH, gamma-LPH, beta-MSH, and beta-END are located at positions 74-85, 134-172, 134-146, 175-220, 203-220, and 223-253, respectively, in ostrich prePOMC, but some of them may not be released in the ostrich pituitary gland, despite the presence of nine potential processing sites consisting of 2-4 dibasic amino acids each. Substitution of glutamic acid for a dibasic amino acid at position 202 in ostrich prePOMC could prevent release of beta-MSH. To date the release of pro-gamma-MSH, beta-LPH, ACTH, gamma-LPH, and beta-END have been confirmed by direct isolation and characterization from ostrich pituitary extracts. In the present study, we have also identified ACTH, gamma-LPH and beta-END in a single frozen ostrich pituitary slice by means of MALDI-TOF mass spectrometry. When compared to a wide range of vertebrate prePOMC molecules, ostrich prePOMC revealed a high level of amino acid sequence identity (77%) with chicken prePOMC, which is the only other avian sequence available. As with other vertebrate classes, considerable intraclass differences were also evident between chicken and ostrich prePOMCs, which belong to different avian orders. Identity of ostrich prePOMC with non-avian tetrapod counterparts is only moderate (53-56%), whereas lower identities (20-49%) are evident over a range of fish prePOMCs.

Adrenocorticotropic Hormone↗