The use of non-prion biomarkers for the diagnosis of Transmissible Spongiform Encephalopathies in the live animal

Scrapie and bovine spongiform encephalopathy (BSE) are major global concerns and the emergence of variant Creutzfeldt-Jakob disease (vCJD) has caused turmoil for blood transfusion services and hospitals worldwide. Recent reports of iatrogenic CJD (iCJD) cases following blood transfusions from Transmissible Spongiform Encephalopathies (TSE)-infected donors have fuelled this concern. Major diagnostic tests for BSE and scrapie are conducted post-mortem from animals in late stages of the disease. Although the lymphoreticular system is involved in the earlier pathogenesis of some forms of sheep scrapie and vCJD, which presents great opportunity for diagnostic development, other TSE diseases (some strains of scrapie, sporadic CJD (sCJD) and bovine BSE) do not present such a diagnostic opportunity. Thus, there is an urgent need for pre-mortem tests that differentiate between healthy and diseased individuals at early stages of illness, in accessible samples such as blood and urine using less invasive procedures. This review reports on the current state of progress in the development and use of prion and non-prion biomarkers in the diagnosis of TSE diseases. Some of these efforts have concentrated on improving the sensitivity of PrPSc detection to allow in vivo diagnosis at low abundances of PrPSc whilst others have sought to identify non-prion protein biomarkers of TSE disease, many of which are still at early stages of development. In this review we comment upon the limitations of prion based tests and review current research on the development of tests for TSE that rely on non-prion disease markers in body fluids that may allow preclinical disease diagnosis.     

Unexpected newcastle disease virus in day old commercial chicks and breeder hen

Newcastle disease virus (NDV) specific antigen in the gut contents and NDV specific antibody in blood circulation were seen in day old chicks belonging to nine different commercial hatcheries of Tamil Nadu, India. Antigen disappeared by 4th week and antibody by 6th week of age. Fourteen NDV isolates obtained from the gut contents of day old chicks of different commercial hatcheries, one NDV isolate from dead in shell eggs and one NDV isolate from breeder hen were characterized and grouped under velogenic, mesogenic and lentogenic pathotypes. Four isolates were grouped under F and another four isolates were grouped under E based on reaction with monoclonal antibodies (Mabs) but found to be velogenic based on pathogenicity tests. In one particular flock velogenie NDV was isolated from breeder hen, dead in shell embryos and day old chicks and they all belong to Mabs group E. Vertical transmission of velogenic, mesogenic and lentogenic NDVs and role of NDVs in the gut contents have been discussed.     

Species of Staphylococcus isolated from animal infections

One hundred randomly selected clinical strains of staphylococci were identified by species using a commercial micromethod system. Eight species of staphylococci were identified. Staphylococcus intermedius was the most frequent (n = 74) species identified and accounted for 70/74 (94.6%) of the coagulase-positive strains and 70/79 (88.6%) of the total isolates from dogs. Other species identified, in order of their frequency, included S. epidermidis (8), S. aureus (7), S. simulans (4), S. sciuri (2), S. xylosus (2), S. hyicus (2) and S. saprophyticus (1). These results show that at least 8 different species of staphylococci can be recovered from animal infections and that coagulase-positive species such as S. intermedius may be more common than S. aureus. The relative significance of these other species in animal infections needs to be assessed.     

Development and validation of species-specific primers that provide a molecular diagnostic for virus-vector longidorid nematodes and related species in German viticulture

The nematode species Longidorus attenuatus, L. elongatus, L. macrosoma and Paralongidorus maximusare economically important pests to the viticulture industry due to their ability to vector two nepoviruses (Raspberry Ringspot Virus and Tomato Black Ring Virus) to grapevines. In Germany, these species occur in vineyard soil with other non-vector but morphologically similar longidorid species, L. helveticus, L.profundorum and L. sturhani. Species-specific primers were designed from ribosomal DNA for all seven species to facilitate taxonomic identification for non-specialists. Primers were assessed for their reliability by screening, where possible, a number of populations of each species. Furthermore, their selectivity and sensitivity were determined when challenged with closely related longidorid species and general nematode communities typical of vineyard soil. A multiplex approach using a common forward primer combined with species-specific reverse primers enabled three target nematode species to be detected in the same PCR reaction. All primers were highly specific, detecting all nematode developmental forms from disparate populations and were sufficiently sensitive to detect a single target nematode within a whole nematode community typical of a vineyard soil comprising of a range of non-target species. Given their specificity, sensitivity and reliability, these diagnostic primers should be of great benefit to both phytosanitary/quarantine services related to the viticulture industry and also as a decision management tool for growers.     

Prevalence of BSE in cattle found dead, euthanased or emergency slaughtered on farms in western France in 2000, 2001 and 2002

The overall trend and the trend within birth cohorts of the prevalence of bovine spongiform encephalopathy (BSE) in cattle found dead, euthanased or emergency slaughtered on farms in the Bretagne, Basse Normandie and Pays de la Loire regions of France, during the periods from August 7 to December 22 in 2000, 2001 and 2002, were analysed by non-conditional logistic regression, adjusted for the region and for the type of animals. The overall prevalence of BSE during these three periods decreased from 2.71 per 1000 in 2000 to 1.41 per 1000 in 2001 and 0.42 per 1000 in 2002. The prevalence within birth cohorts started to decrease for the cohort born between July 1, 1995 and June 30, 1996 (cohort 95/96) and the trend was reinforced for cohort 96/97, suggesting that the exposure of animals to the BSE agent had started to decrease for animals born after July 1995, that is, one year before the ban on specified risk materials in meat and bone meal was implemented in France. However, considering that most of the animals would have been infected at between six and 18 months of age, the decrease may have been due, at least partly, to this control measure.     

Proteome analysis of the sarcoplasmic fraction of pig semimembranosus muscle: implications on meat color development

Two-dimensional electrophoresis was used to investigate sarcoplasmic protein expression in pig Semimembranosus muscles sampled 20 min after slaughter. Two groups (light and dark) of 12 animals were selected from 1000 pigs, based on meat L values measured 36 h postmortem. Twenty-two proteins or fragments (p < 0.05) were differentially expressed. Muscles leading to darker meat had a more oxidative metabolism, indicated by more abundant mitochondrial enzymes of the respiratory chain, hemoglobin, and chaperone or regulator proteins (HSP27, alphaB-crystallin, and glucose-regulated protein 58 kDa). Conversely, enzymes of glycolysis were overexpressed in the lighter group. Such samples were also characterized by higher levels of glutathione S-transferase omega, which can activate the RyR calcium channels, and higher levels of cyclophilin D. This protein pattern is likely to have severe implications on postmortem metabolism, namely, acceleration of ATP depletion and pH fall and subsequent enhanced protein denaturation, well-known to induce discoloration.     

Comparison of biopsy samples obtained using standard endoscopic instruments and the harmonic scalpel during laparoscopic and laparoscopic-assisted surgery in normal dogs

Objectives— To compare tissue specimens of canine abdominal organs collected by standard techniques (ST) and harmonic scalpel (HS) and to determine the effect of each technique on wound healing.
Study Design— Experimental.
Animals— Twelve dogs.
Methods— Paired biopsy samples (ST, HS) were collected from liver, spleen, adrenal gland, pancreas, stomach, jejunum, and bladder using laparoscopic or laparoscopic-assisted methods. Hemorrhage at biopsy sites was assessed (present or absent). Specimens were evaluated for diagnostic quality and histologic changes. Dogs were necropsied at 4 or 14 days postoperatively.
Results— HS incision caused less frequent hemorrhage of all organs except spleen. HS specimens had more coagulation necrosis than ST specimens, but both techniques yielded specimens that were sized similarly and were of diagnostic quality. At necropsy, HS biopsy sites of the stomach, jejunum, bladder, adrenal gland, and pancreas were more hyperemic than ST sites. There were more adhesions at jejunal and pancreatic ST biopsy sites. HS biopsy sites had more coagulation necrosis at days 4 and 14 postoperatively. Differences in fibrin deposition, inflammation, and fibrosis were present at biopsy sites of some organs at days 4 or 14 and in comparisons between days 4 and 14.
Conclusions— Diagnostic quality biopsy specimens were obtained with HS and ST. Although HS-induced gross and histologic changes during the first 2 postoperative weeks, no clinical complications were observed.
Clinical Relevance— Both HS and ST can yield specimens with minimal hemorrhage and HS resulted in no apparent postoperative problems in normal dogs. Although HS caused more inflammation and adhesions at biopsy sites of the pancreas, adrenal gland, and jejunum, no clinical complications occurred.