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Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle.

To compare clinicopathological findings in first and second passage chronic wasting disease (CWD(mule deer)) in cattle, six calves were inoculated intracerebrally with brain tissue derived from a first-passage CWD-affected calf in an earlier experiment. Two uninoculated calves served as controls. The inoculated animals began to lose both appetite and weight 10-12 months later, and five subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months, all cattle had been subjected to euthanasia because of poor prognosis. None of the animals showed microscopical lesions of spongiform encephalopathy (SE) but PrP(res) was detected in their CNS tissues by immunohistochemistry (IHC) and rapid Western blot (WB) techniques. Thus, intracerebrally inoculated cattle not only amplified CWD PrP(res) from mule deer but also developed clinical CNS signs in the absence of SE lesions. This situation has also been shown to occur in cattle inoculated with the scrapie agent. The study confirmed that the diagnostic techniques currently used for diagnosis of bovine spongiform encephalopathy (BSE) in the US would detect CWD in cattle, should it occur naturally. Furthermore, it raised the possibility of distinguishing CWD from BSE in cattle, due to the absence of neuropathological lesions and to a distinctive multifocal distribution of PrP(res), as demonstrated by IHC which, in this study, appeared to be more sensitive than the WB technique.

Animals↗

Molecular analysis of the proviral DNA of equine infectious anemia virus in mules in Greece.

Molecular analysis of the regulatory and structurally important genetic segments of equine infectious anemia virus (EIAV) in mules is presented. We have previously reported clinicopathological and laboratory findings in mules infected with EIAV, both naturally and after experimental inoculation. In this study the fragment coding for integrase, gp90, tat and the fusion domain of gp45 of the proviral genome from these animals was sequenced and compared with one another and with that of EIAV strains already published in the literature. Significant variations were observed mainly in the sequences of the gp90 surface protein. In the two wild type sequences, there were substitutions in the V5 hypervariable domain of this protein. In the sequences of the experimentally inoculated animals and the donor strain, variations were due to insertions/duplications in the V3 principal neutralizing domain (PND) and substitutions in the V5 hypervariable domain. Finally, when compared with the already published strains, the wild type sequences had single amino acid substitutions across the whole protein and multiple substitutions in the V4-V6 variable domains. In general, the two Greek wild type sequences were closer to two of the American strains (WSU5 and Massachusetts), than to the two Japanese (V26 and V70) or the third American strain (Wyoming_wi) used in this study.

Amino Acid Sequence↗

Identification of goose (Anser anser) and mule duck (Anasplatyrhynchos x Cairina moschata) foie gras by multiplex polymerase chain reaction amplification of the 5S RDNA gene.

Polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene has been used for the identification of goose and mule duck foie gras. Two species-specific reverse primers were designed and used in a multiplex reaction, together with a forward universal primer, to amplify specific fragments of the 5S rDNA in each species. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose and mule duck foie gras samples. This genetic marker can be useful for detecting fraudulent substitution of the duck liver for the more expensive goose liver.

Animals↗

Identification of goose, mule duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction.

A specific Polymerase Chain Reaction (PCR) has been developed for the identification of goose (Anser anser), mule duck (Anas platyrhynchos x Cairina moschata), chicken (Gallus gallus), turkey (Meleagris gallopavo), and swine (Sus scrofa domesticus) in foie gras. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose, mule duck, chicken, turkey, and swine in foie gras. Analysis of experimental mixtures demonstrated that the detection limit of the assay was approximately 1% for each species analyzed. This genetic marker can be very useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in foie gras.

Animals↗

Refined genome-wide comparative map of the domestic horse, donkey and human based on cross-species chromosome painting: insight into the occasional fertility of mules.

We have made a complete set of painting probes for the domestic horse by degenerate oligonucleotide-primed PCR amplification of flow-sorted horse chromosomes. The horse probes, together with a full set of those available for human, were hybridized onto metaphase chromosomes of human, horse and mule. Based on the hybridization results, we have generated genome-wide comparative chromosome maps involving the domestic horse, donkey and human. These maps define the overall distribution and boundaries of evolutionarily conserved chromosomal segments in the three genomes. Our results shed further light on the karyotypic relationships among these species and, in particular, the chromosomal rearrangements that underlie hybrid sterility and the occasional fertility of mules.

Animals↗

Benign fibrous histiocytoma of prepuce in the mule.

Here we present a case of preputial benign fibrous histiocytoma in a 7-year-old mule. This case was treated surgically. Pathologic analysis and diagnosis of these lesions were based on a combination of light microscopy and immunohistochemistry. Histologically, the tumour was composed primarily of a storiform pattern of fibroblastic and poligonal cells. In immunohistochemistry, except for consistent immunoreactivity for vimentin, nearly all tumour cells were positive for CD68, S-100 protein, pan-cytokeratin, alpha-smooth muscle actin, and desmin. Benign fibrous histiocytoma of the prepuce in mule is rarely reported in veterinary literature.

Animals↗

Cytological and biochemical correlation of late X-chromosome replication and gene inactivation in the mule.

The correlation between late X-chromosome replication and the quantitation of different X-linked glucose-6-phosphate dehydrogenase electrophoretic forms was studied in a natural hybrid, the female mule. In all four animals examined, a significant deviation from the expected 1: 1 ratio of random X-chromosome inactivation was observed, with the donkey X-chromosome the more frequently late-replicating one. The relative amounts of horse and donkey enzymes activities in lysates of mule skin fibroblasts and in peripheral blood were in agreement with this finding: the donkey enzyme was the minor component. Although random expression of the enzymes from the two parent species was not observed, sampling, selection, or adaptation may actually be responsible for the apparent "preferential inactivation". These studies support the hypothesis that late DNA replication indicates genetic inactivation.

Acetates↗

Force-feeding procedure and physiological indicators of stress in male mule ducks.

1. The effects of the force-feeding procedure and its different components on various physiological indicators of acute and chronic stress were investigated in male mule ducks before and during a 12-d cramming period. 2. The highest concentration of corticosterone were measured after injection of ACTH agonist, during the pre-experimental period when the ducks were still housed in collective floor pens and at the time of transfer. 3. During the cramming period, corticosterone measured before and after force-feeding did not differ significantly though there was a non-significant trend towards an increase in certain cases. 4. The different components of force-feeding, including manipulation, intubation, force-feeding with a standard or a large amount of food had no significant effect upon corticosterone concentrations. 5. There was no indication from ACTH agonist challenge either of a change in adrenal sensitivity or a change in its responsiveness. 6. The heterocyte-lymphocyte ratio measured before and at the end of the cramming period did not differ significantly. 7. In conclusion, we observed no significant indication that force-feeding is perceived as an acute or chronic stress by male mule ducks, in our experimental conditions. Nevertheless, it remains to be shown that their adrenocorticotropic axis is responsive to acute stressors.

Adrenocorticotropic Hormone↗

Effects of age and sex on the structural, chemical and technological characteristics of mule duck meat.

1. The aim of the study was to analyse the effect of age and sex on the chemical, structural and technological characteristics of mule duck meat. 2. Ten males and 10 females were weighed and slaughtered at 8, 10, 11, 12 and 13 weeks of age. Weight, pH value, colour, tenderness and juice loss of breast muscle were determined. 3. The activities of 3 enzymes (citrate synthase, beta-hydroxyacyl CoA dehydrogenase, lactate dehydrogenase) which indicate muscular metabolic activity were assayed. 4. Chemical composition (moisture, lipids, proteins, minerals, lipid and phospholipid classes, fatty acid composition) of breast muscle was analysed. 5. Fibre type, fibre type percentage and cross-sectional areas were determined using histochemistry and an image analysis system. 6. For growth performance and muscular structure, the ideal slaughter age of mule ducks is 10 weeks of age. Chemical and technological analysis indicated that muscular maturity in Pectoralis major was reached at 11 weeks of age, but, at this age, breast lipid content is high. Moreover, after 10 weeks of age, food costs rapidly increased. 7. Lastly, sexual dimorphism for body weight is minor. In this study, at any given age, no significant differences between males and females were shown. Thus, it is possible to rear both sexes together and to slaughter them at the same age.

3-Hydroxyacyl CoA Dehydrogenases↗

Horse, ass, and mule chromosomes.

Karyotypes of the horse with 64 chromosomes, the ass with 62 chromosomes, and the mule with 63 chromosomes are presented. The chromosome complements of each species and their mule hybrid are analyzed and compared.

Animals↗

Oral treatment of mule ducks with arsenicals for inducing fatty liver.

The aim of this study was to determine the dosage and the compounds of arsenic that induce fatty liver in mule ducks and also to investigate their effects on tissue residues. One hundred four ducks, 8 wk old, were randomly selected for one of six dietary treatments in Trial 1 or one of seven dietary treatments in Trial 2. Different levels of roxarsone were administrated: 0, 10, 20, 30, 40, or 50 mg/d, respectively, in Trial 1. In Trial 2, the experimental treatments were of the same level (11.36 mg/d) with different sources of arsenic that included the control without As, roxarsone (3-nitro-4-hydroxyphenylarsonic acid), arsanilic acid, phenylarsonic acid, O-nitro-phenylarsonic acid, As2O3, or As2O5. Both trials lasted 3 wk, with 1 wk on the treatment followed by 2 wk of withdrawal. Results in Trial 1 showed that a dose of 40 mg roxarsone/d increased liver weight and caused fatty liver, whereas administration of 50 mg/d was lethal. In Trial 2, administration of arsenic (11.36 mg/d) for 1 wk significantly depressed feed intake in the roxarsone, As2O3, and As2O5 groups (P < 0.05), whereas the treatment significantly decreased only live weight gain in the roxarsone group (P < 0.05). Administration of roxarsone alone increased (P < 0.05) serum cholesterol (CHOL), albumin (ALB), and total protein (TP), whereas only As2O3 among treatments significantly decreased serum triacylglycerol (TG) concentration (P < 0.05). In the roxarsone, arsanilic acid, and phenylarsonic acid groups, serum high density lipoprotein (HDL) decreased to a greater extent (P < 0.05), and arsanilic acid treatment significantly increased the very low density lipoprotein (VLDL) (P < 0.05). After 2 wk of withdrawal, liver weights and relative liver weights were heavier in the treatment groups of roxarsone, As2O3, and As2O5 as compared to the control (P < 0.05). Levels of CHOL, TG, TP, and ALB were significantly higher in the groups treated with As2O3 or As2O5 as compared to the control (P < 0.05). The roxarsone and arsanilic acid treatments significantly decreased HDL and increased VLDL in plasma (P < 0.05). The creatine kinase (CK) level in the roxarsone, As2O3, and As2O5 groups was significantly higher compared to the control group (P < 0.05). Among the As sources, roxarsone, As2O3, and As2O5 caused fatty liver in mule ducks.

Administration, Oral↗

A glucokinase-like enzyme induced in Mule duck livers by overfeeding.

The Mule duck develops a fatty liver in response to overfeeding, which results from a dramatic increase in de novo liver lipogenesis, and thus raises questions regarding the role of glucokinase (GK), a key enzyme regulating carbohydrate metabolism in mammals. However, the presence of GK in avian species is still a matter of debate. The aim of the present study was to characterize a GK-like protein (using an immunological technique) and a GK-like activity (using an enzymatic assay) in duck liver and to measure their respective variations during various stages of overfeeding. Duck liver protein cross-reacted with antibodies directed against mammalian GK yielding a band at 50 kDa, i.e., the same molecular weight as mammalian GK. The intensity of the signal varied significantly between overfed and control ducks but in opposing ways according to the GK antibodies used, which suggests the presence of 2 isoforms of GK in the duck liver as in mammals. Enzymatic analysis demonstrated the presence of glucose phosphorylation activity sensitive to high and low glucose concentrations (high/low ratio between 1.7 and 3.7) in the soluble and particulate fractions of liver homogenates. Glucokinase-like activity per milligram protein was strongly induced by overfeeding, and plasma insulin levels increased concomitantly. More than 80% of total GK-like activity was concentrated in the soluble component from 1 to 13 d of overfeeding. These results suggest that a GK-like enzyme may actively contribute to glucose disposal throughout the overfeeding period in Mule ducks fed a carbohydrate-rich diet.

Animals↗

Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus).

Mule deer fawns (Odocoileus hemionus) were inoculated orally with a brain homogenate prepared from mule deer with naturally occurring chronic wasting disease (CWD), a prion-induced transmissible spongiform encephalopathy. Fawns were necropsied and examined for PrPres, the abnormal prion protein isoform, at 10, 42, 53, 77, 78 and 80 days post-inoculation (p.i.) using an immunohistochemistry assay modified to enhance sensitivity. PrPres was detected in alimentary-tract-associated lymphoid tissues (one or more of the following: retropharyngeal lymph node, tonsil, Peyer's patch and ileocaecal lymph node) as early as 42 days p.i. and in all fawns examined thereafter (53 to 80 days p.i.). No PrPres staining was detected in lymphoid tissue of three control fawns receiving a control brain inoculum, nor was PrPres detectable in neural tissue of any fawn. PrPres-specific staining was markedly enhanced by sequential tissue treatment with formic acid, proteinase K and hydrated autoclaving prior to immunohistochemical staining with monoclonal antibody F89/160.1.5. These results indicate that CWD PrPres can be detected in lymphoid tissues draining the alimentary tract within a few weeks after oral exposure to infectious prions and may reflect the initial pathway of CWD infection in deer. The rapid infection of deer fawns following exposure by the most plausible natural route is consistent with the efficient horizontal transmission of CWD in nature and enables accelerated studies of transmission and pathogenesis in the native species.

Animals↗

Low frequency of PrP genotype 225SF among free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease.

The prion protein (PrP) gene was characterized in 1482 free-ranging mule deer (Odocoileus hemionus) from Wyoming and Colorado. Using DNA sequences from 363 deer, dimorphisms at codons 20 (aspartate/glycine) and 225 [serine (S)/phenylalanine (F)] were found; silent changes occurred at codons 131 (tyrosine) and 247 (isoleucine). The remaining samples were surveyed for codon 225 genotype and all were characterized for chronic wasting disease (CWD) infection status. A total of 112 deer with the genotype 225SF or FF were found, of which one was CWD-positive; 1370 were 225SS, with 289 positive for CWD. Among CWD-negative deer, the frequency of 225SF/FF genotypes was 9.3 % but among CWD-positive deer it was only 0.3 %. For all samples combined, CWD status was not independent of codon 225 genotype (P<0.0001). The odds that a deer of the 225SS genotype was CWD-infected were 30 times greater (95 % confidence intervals=4-213) than for a 225SF deer. The proportion of 225SF animals in sampled subpopulations varied from 0 to 18 %; the CWD prevalence varied from 0 to 25 %. However, no relationship was observed between genotype frequency and CWD prevalence in different areas. The PrP sequences of experimentally infected mule deer were analysed from pre-existing projects and 10 animals were found with 225SF genotypes, all of which were positive for CWD. Data available from some of these animals suggest that the 225SF genotype could be associated with longer incubation periods in CWD infection compared with the 225SS genotype.

Animals↗

Patterns of PrPCWD accumulation during the course of chronic wasting disease infection in orally inoculated mule deer (Odocoileus hemionus).

Patterns of abnormal prion protein (PrP) accumulation during the course of chronic wasting disease (CWD) infection were studied and the distribution and timing of disease-associated PrP (PrP(CWD)) deposition and lesions in 19 mule deer (Odocoileus hemionus) 90-785 days after oral inoculation were described. PrP(CWD) deposition occurred relatively rapidly and widely in lymphoid tissues, later in central and peripheral nervous tissues and sporadically in a variety of tissues and organs in terminal disease stages. Development of spongiform encephalopathy lagged behind PrP(CWD) deposition in the central nervous system (CNS), but occurred in the same neuroanatomical locations. PrP(CWD) deposition in the lymphatic and nervous systems tended to be consistent and progressive in specific organs and tissues. Locations of PrP(CWD) deposition were similar between deer of two PrP genotypes (225SS and 225SF), but the time course differed between genotypes: in 225SF deer, PrP(CWD) accumulated more slowly in lymphatic tissues than in 225SS animals, but that disparity was small in comparison to the disparity between genotypes in timing of deposition in CNS tissue. These data confirm retropharyngeal lymph node and medulla oblongata at the level of the obex as early sites of PrP(CWD) accumulation in mule deer with CWD. Data on the relative time frames for and genetic influences on PrP(CWD) accumulation may also offer insights about epidemic dynamics and potential control strategies.

Animals↗

A comparison of injectable anaesthetic regimens in mules.

Three combinations of injectable anaesthetic agents were compared in nine adult mules. The combinations were xylazine/ketamine (X/K), xylazine/butorphanol/ketamine (X/B/K), and xylazine/tiletamine-zolazepam (X/T). Measured variables were heart rate, respiratory rate, systolic blood pressure, arterial blood pH, PCO2 and PO2, recumbency time and number of attempts to stand. Quality of induction and recovery, muscle relaxation and response to stimulus were evaluated subjectively. Recumbency time was significantly (P < 0.05) longer with X/B/K and X/T than with X/K. Mules required significantly more attempts to stand under the influence of X/T than X/K or X/B/K. No statistically significant (P < 0.05) differences in heart rate, respiratory rate, blood pressure or arterial pH, PCO2 and PO2 were detected between groups.

Anesthesia, Intravenous↗

A survey for antibodies to equine arteritis virus in donkeys, mules and zebra using virus neutralisation (VN) and enzyme linked immunosorbent assay (ELISA).

A seroepidemiological survey of donkeys in South Africa (n = 4300) indicated a wide distribution and increasing prevalence of antibodies to equine arteritis virus (EAV). Donkey sera inhibited equine arteritis virus infection in virus neutralisation (VN) tests and in ELISA specifically bound to a recombinant antigen derived from the Bucyrus isolate of EAV. These results suggest that donkeys have been exposed to the same serotype of this virus as circulates among horses. A good correlation existed between EAV neutralising antibody titres and ELISA absorbance values (0.8631); the ELISA was sensitive and specific (99.2% and 80.3% respectively) for donkey sera when compared to the VN test and the recombinant ELISA antigen did not cross-react with sera positive for common African equine pathogens. VN+ ELISA+ donkeys were also found in Morocco and Zimbabwe and seropositive mules in both South Africa and Morocco. No seropositive zebra (n = 266) were detected from game reserves or zoos in 9 countries. The results confirm that in addition to horses and donkeys, mules are naturally infected with EAV.

Animals↗

Neuropathology of chronic wasting disease of mule deer (Odocoileus hemionus) and elk (Cervus elaphus nelsoni).

The pathology of the central nervous system of nine mule deer (Odocoileus hemionus) and six elk (Cervus elaphus nelsoni) with chronic wasting disease, a spongiform encephalopathy of mule deer and elk, was studied by light microscopy. Lesions were similar in both species and were characterized by spongiform transformation of gray matter, intracytoplasmic vacuolation of neurons, neuronal degeneration and loss, astrocytic hypertrophy and hyperplasia, occurrence of amyloid plaques, and absence of significant inflammatory response. Distribution and severity of lesions were evaluated at 57 locations; there were only minor differences between deer and elk. Consistent, severe lesions occurred in olfactory tubercle and cortex, hypothalamus, and the parasympathetic vagal nucleus of deer, and sections examined from these regions would be sufficient to establish a diagnosis of chronic wasting disease. Lesions were milder in these locations in elk but were sufficiently apparent to be of diagnostic value. Other differences included increased severity of lesions in some thalamic nuclei in elk in contrast to deer, the occurrence of amyloid plaques demonstrable by hematoxylin and eosin and histochemical stains in deer in contrast to elk, and the presence of mild white matter lesions in elk but not in deer. Lesions of chronic wasting disease were qualitatively comparable to those of scrapie, bovine spongiform encephalopathy, transmissible mink encephalopathy, and the human spongiform encephalopathies. Topographic distribution and lesion severity of chronic wasting disease were most similar to those of scrapie and bovine spongiform encephalopathy. Duration of clinical disease did not significantly influence lesion distribution or severity in either species.

Animals↗