Morphometric and immunohistochemical study of the rumen of red deer during prenatal development

Abstract A detailed study of the ontogenesis of deer stomach has not been undertaken to date, and our aim was to sequence several histological phenomena that occur during the ontogenesis of one of the gastric compartments, the rumen. Histomorphometric and immunohistochemical analyses were carried out on 50 embryos and fetuses of deer from the initial stages of prenatal life until birth. For the purposes of testing, the animals were divided into five experimental groups: group I, 1.4-3.6 cm crown-rump length, 30-60 days, 1-25% of gestation; group II, 4.5-7.2 cm crown-rump length, 67-90 days, 25-35% of gestation; group III, 8-19 cm crown-rump length, 97-135 days, 35-50% of gestation; group IV, 21-33 cm crown-rump length, 142-191 days, 45-70% of gestation; and group V, 36-40 cm crown-rump length, 205-235 days, 75-100% of gestation. The rumen of the primitive gastric tube was observed at approximately 60 days. At 67 days the rumen consisted of three layers: internal or mucosal, middle or muscular, and external or serosal layer. The stratification of the epithelial layer was accompanied by changes in its structure with the appearance of ruminal pillars and papillae. The outline of the ruminal papillae began to appear at 142 days of prenatal development as evaginations of the basal zone toward the ruminal lumen, pulling with it in its configuration the stratum basale, the lamina propria and the submucosa. From the pluripotential blastemic tissue at 60 days we witnessed the histodifferentiation of the primitive tunica muscularis, composed of two layers of myoblasts with a defined arrangement. It was also from the pluripotential blastemic tissue, at 97 days, that the lamina propria and the submucosa were differentiated. The serosa showed continuity in growth as well as differentiation, already detected in the undifferentiated outline phase. The tegumentary mucosa of deer rumen was shown without secretory capacity in the initial embryonic phases; neutral mucopolysaccharides appeared from 67 days. The presence of neuroendocrine cells (non-neuronal enolase) in the ruminal wall of deer during development was not detected until 97 days. The glial cells were detected at 142 days for glial fibrillary acidic protein and at 67 days for vimentin. The immunodetection of neuropeptides vasointestinal peptide and neuropeptide Y progressively increased with gestation period, starting from 97 days. In terms of the structure of the rumen of the primitive gastric tube, our observations revealed that the deer is less precocious than small and large domestic ruminants. Thus its secretory capacity, detected by the presence of neutral mucopolysaccharides, and its neuroendocrine nature, determined by the presence of positive non-neuronal enolase cells, were evident in more advanced stages of prenatal development than those detected in the sheep, goat and cow.     

Extrachromosomal deer fibromavirus DNA in deer fibromas and virus-transformed mouse cells

The non-virus-producing fibromatous portions of five deer fibromas were examined for deer fibromavirus (DFV) DNA sequences. Liquid-phase hybridization revealed 100 to 330 copies per cell of the virus genome. Southern blot analysis of undigested deer tumor DNA preparations indicated that most of the DFV DNA was present as monomeric, unintegrated genomes; however, restriction enzyme digestion patterns suggest a small population of resistant DFV sequences. DFV DNA was also present in virus-transformed NIH/3T3 mouse cells as multiple, extrachromosomal genomes.     

Localisation of actin in the liver fluke,Fasciola hepatica

Adult and 3-week-old juvenileFasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1% (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.     

Phosphatidate phosphatase activity in the blood fluke, Schistosoma mansoni

Phosphatidate phosphatase activity was found in tissue homogenates and a multilamellar body fraction of Schistosoma mansoni. The identity of the enzyme activity was established on the basis of its pH optimum and inhibition by Mg²⁺, Ca²⁺ and Tween 20. A seven-fold enrichment of enzyme activity occurred in response to fractionation of the multilamellar bodies. The importance of this enzyme is discussed in terms of its being a marker enzyme and its central role in phospholipid biosynthesis.     

Intrahepatic biliary cysts in deer

The gross and histological appearances of intrahepatic cysts in red and roe deer are described. It is suggested that the structures arise as anomalous developments of the biliary tree. Although not of clinical significance, these cysts may pose a problem at meat inspection.     

Intestinal fluke infection as a result of eating sushi

Severe diarrhea in a female outpatient was caused by an intestinal fluke, identified as Heterophyes heterophyes, a natural parasite of humans and domesticated and wild fish-eating mammals. This parasite is endemic in the Orient and the Middle East. A detailed case history revealed that the woman had never traveled outside the continental United States but became infected while eating raw fresh-water fish (sushi) that had been served at a local Japanese restaurant. The restaurant specialized in serving a great variety of fresh-water and salt-water fish that were flown in from the Orient and other parts of the world. The authors' findings indicate that a person does not have to travel to an endemic area to become infected with this organism.     

Citrate synthase from the liver fluke Fasciola hepatica

Citrate synthase catalyses the first step of the Krebs’ tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K _m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.     

Evaluation of medicinal plant extracts against ticks and fluke

The present study was based on assessments of the antiparasitic activities to determine the efficacies of leaf hexane, chloroform, ethyl acetate, acetone and methanol extracts of Aegle marmelos (Linn.) Correa ex Roxb, Andrographis lineata Wallich ex Nees., Andrographis paniculata (Burm.f.) Wallich ex Nees., Cocculus hirsutus (L.) Diels, Eclipta prostrata L., and Tagetes erecta L. against the adult cattle tick Haemaphysalis bispinosa Neumann 1897 (Acarina: Ixodidae), the larvae of Rhipicephalus (Boophilus) microplus Canestrini 1887 (Acari: Ixodidae) and sheep fluke Paramphistomum cervi Zeder 1790 (Digenea: Paramphistomatidae). All plant extracts showed moderate toxic effect on parasites after 24 h of exposure; however, the highest parasitic activity was found in leaf ethyl acetate extract of A. lineata, methanol extract of A. marmelos, A. paniculata, and C. hirsutus against H. bispinosa (LC₅₀ = 395.27, 358.45, 327.21 and 420.50 ppm); ethyl acetate extract of A. paniculata, C. hirsutus, methanol extracts of A. marmelos, A. lineata, and E. prostrata against the larvae of R. microplus (LC₅₀ = 207.70, 258.61, 134.09, 206.00, and 274.33 ppm); hexane extract of A. lineata, ethyl acetate extract of A. paniculata, E. prostrata, acetone extracts of T. erecta, methanol extracts of A. marmelos and C. hirsutus against P. cervi (LC₅₀ = 254.23, 451.17, 425.73, 253.60, 542.71, and 360.17 ppm), respectively. The present study is the first report on the veterinary parasitic activity of plant extracts from Southern India.     

Calbindin-immunoreactive neurones in the ovine rumen

In small laboratory animals, such as guinea pigs, immunoreactivity for the calcium-binding protein calbindin (CALB) can be used to distinguish functionally different classes of myenteric neurones. The rumen of sheep is a highly specialized gastrointestinal region, and the control of its functions requires specific intrinsic innervation patterns. The aim of this study was to neurochemically identify and characterize CALB-positive myenteric neurones of the ovine rumen. Therefore, we performed quadruple immunohistochemistry against CALB, substance P (SP), vasoactive intestinal peptide (VIP), and nitric oxide synthase (NOS) using whole-mount preparations of the ruminal myenteric plexus. On average, 3 +/- 2 and 1 +/- 0.4 myenteric neurones/ganglion were CALB-immunoreactive in suckling lambs and adult sheep, respectively. These neurones had Dogiel type-I morphology. Most of them (89.2% +/- 8.7% and 71.7% +/-44.8% in suckling lambs and adult sheep, respectively) did not colocalize any of the other antigenes. Since it has been shown in previous studies that ruminal myenteric neurones are immunoreactive for either choline acetyltransferase (ChAT) or NOS, we defined neurones which were CALB-positive and NOS-negative as CALB/ChAT. The other CALB-positive neurones were encoded CALB/NOS/+/-VIP (10.3% +/- 9.3% and 26.7% +/- 46.2% in suckling lambs and adult sheep, respectively) or CALB/ChAT/SP (0.5% +/- 1.0% and 1.7% +/- 1.9% in suckling lambs and adult sheep, respectively). We used cryostat sections of the ruminal wall to analyze the projections of the CALB-positive neurones. CALB-immunoreactive somata were exclusively located within the myenteric plexus. CALB-immunoreactive nerve fibers were found primarily in the lamina propria of the ruminal papillae. We conclude that CALB-positive myenteric neurones within the ovine rumen project to the epithelium; however, their functional role remains to be investigated.     

Characterization of papillomaviruses isolated from cutaneous fibromas of white-tailed deer and mule deer

Deer fibromavirus (DFV), a member of the papillomavirus genus, was isolated from cases of fibromatosis in white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus). SDS-polyacrylamide gel electrophoresis analysis of virions indicated no molecular weight differences in the major structural protein. DFV agglutinated mouse erythrocytes and the reaction could be inhibited by both DFV-specific and BPV-1-specific antisera. Although analysis of restriction endonuclease digestion products indicated minor differences in cleavage patterns, the DFV DNAs were indistinguishable by liquid phase hybridization and restriction enzyme cleavage maps indicated most of the sites in common. Comparison of DFV DNA to BPV-1 and BPV-2 DNA under stringent liquid-phase hybridization conditions indicated that 3 to 9% DNA sequence homology could be detected between the genomes of these viruses. Blot-transfer hybridization revealed that DFV DNA reannealed to the same restriction fragments of BPV-2 DNA under stringent conditions that DNA from other papillomaviruses hybridize to under nonstringent conditions.     

Sarcosporidia of the cloven-hoofed game. III. Sarcosporidia in red deer and fallow deer]

The incidence of Sarcosporidia among red-deer and fallow-deer was investigated. The fallow-deer is less discussed because the low number of investigations. The red-deer shows with 15% the lowest incidence among the red-deer, fallow-deer and wild-boar investigated. The Sarcosporidia of the red-deer are smaller than those of the roe, therefore the identification in the trichinoscopic picture is more difficult. With regard to this it is recommended to name the Sarcosporidia of the red-deer Sarcocystis cervi (VON HESSLING, 1854), because their form is more similar to the cattle and not those of the roe. To be sure a greater number of investigations is necessary to detect all connections.     

Cytochrome P450 in fluke Opisthorchis felineus: identification and characterization

Infection with the human liver fluke Opisthorchis felineus is a serious public health problem in Russia and other Eastern Europe countries. The aim of this work was to identify and sequence cytochrome P450 mRNA from O. felineus and to analyze its expression at different developmental stages. We found only one cytochrome P450 in O. felineus. It contains a conserved Pfam00067 domain which was typical of the CYP450 II eukaryotic microsomal type, and a putative transmembrane domain. Additionally, we identified a high degree of homology between a 3D model of O. felineus CYP450 and mammalian CYP2 structures. The level of O. felineus CYP mRNA expression in maritae (adult stage in definitive mammal host) is significantly higher than in metacercaria. This fact indicates an important role of this biotransformation enzyme in the biochemistry of the parasite at the maritae stage.     

Enterocytozoon bieneusi in sika deer (Cervus nippon) and red deer (Cervus elaphus): deer specificity and zoonotic potential of ITS genotypes

As the most common cause of the human microsporidiosis, Enterocytozoon bieneusi has been found in a wide variety of animal hosts. Deers are the ruminant mammals living in a variety of biomes, and the distribution of deer species differ by geography. To understand the prevalence of natural infection of E. bieneusi in deer and to assess their epidemiological role in the transmission of microsporidiosis caused by E. bieneusi, 91 fecal specimens were collected from 86 sika deers and five red deers in the northeast of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an average infection rate of 31.9 % (29/91) was observed in deer, with 32.6 % (28/86) for sika deer, and 20 % (1/5) for red deer. Six ITS genotypes were identified: one known genotype BEB6 (n?=?20) and five novel genotypes HLJD-I to HLJD-IV (one each) and HLJD-V (n?=?5). A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that genotypes HLJD-II and HLJD-III fell into group 1 of zoonotic potential, while the other genotypes (BEB6, HLJD-I, HLJD-IV, HLJD-V) were clustered into so-called bovine-specific group 2. This is the first report of E. bieneusi in deer in China. The observation of genotype BEB6 in humans previously and in deer here and also the findings of the two novel genotypes (HLJD-II to HLJ-III) belonging to potential zoonotic group 1 suggested the possibility of deer in the transmission of E. bieneusi to humans.     

Amino acid composition of the indian liver fluke Fasciola indica

Summary Eighteen free amino acids have been found in Fasciola indica by the chromatographic method (two-dimensional ascending). Besides methionine, taurine and histidine, not reported earlier in the liver fluke, two non-protein amino acids, - and -amino-butyric acid have also been found. Valine, reported earlier in F. hepatica by Daugherty (1952), has not been found in F. indica.     

Expression of VEGF and pleiotrophin in deer antler

Deer antlers represent a unique model of mammalian regeneration in that they cast and fully regenerate every year. The deer antler thus provides a fascinating model of both rapid angiogenesis and chondrogenesis and the opportunity to investigate unique growth regulatory processes. One such phenomenon is the presence of vascularized cartilage in the growing antler tip-unlike other cartilage, which is typically avascular. The mechanisms by which blood vessels grow in the cartilage as well as the factors that drive antler extension at approximately 1 cm a day have been hitherto largely unknown. The aim of this study was to determine the expression of VEGF and pleiotrophin within the growing antler tip. We isolated cervine VEGF121 and VEGF165 from deer antler and found that mRNA is produced for VEGF in the precartilage and cartilage regions. By in situ hybridization, we examined whether the VEGF receptors Flt-1 and KDR are present in deer antler and found only KDR mRNA within the endothelial cells of the precartilage region. This finding is compatible with VEGF having an angiogenic effect within antler. Pleiotrophin mRNA was found in the vascular smooth muscle cells of the dermis, thus supporting a possible role in vascular growth. High levels of pleiotrophin mRNA were also detected in the precartilage region with possible implications for both angiogenesis and chondrogenesis. This is the first report of cervine angiogenic growth factors within the growing antler tip.