The Veterinary Forensic Necropsy: A Review of Procedures and Protocols.

Investigation of animal-related crime, and therefore submission of forensic cases to veterinary pathology facilities, is increasing, yet many veterinary pathologists are unfamiliar and often uncomfortable with involvement in the forensic necropsy. This article discusses various aspects of the forensic necropsy without specific attention to any particular species group or crime. General advice is given on procedures, documentation, and recording of the examination, and the article indicates how these features may differ from those used in investigation of natural disease. It also discusses evidence management, including recordkeeping, identification of evidence, labeling of photographs, and use of standard operating procedures and protocols. Various written and visual methods for documentation of the forensic necropsy are covered, and adjunctive topics such as sample collection, assessment, and description of wounds and taphonomy are included. Cause, mechanism, and manner of death are defined, and guidance to the use of these terms is given. The aim of this article is to offer guidance on procedural aspects of the forensic necropsy that will help those developing their forensic services, contribute to standardization of the provision of forensic veterinary pathology, and build the confidence of the "uncomfortable" forensic veterinary pathologist.

European Forensic Veterinary Pathology Comes of Age.

During the mid-1700s, development of the veterinary profession was largely focussed on equine medicine and surgery. Subsequently, rather erratic development encompassed other species and eventually led to specialization in different disciplines. Teaching of veterinary pathology was well established in Europe and North America by the late 19th century. Specialization in this discipline was boosted in the 1940s by the formation, in the USA, of the Register of Veterinary Pathology and American College of Veterinary Pathologists. National societies followed soon afterwards in Europe. The European Society of Veterinary Pathology evolved during this period and the European College of Veterinary Pathologists (ECVP) was created in 1995 to promote high standards in the discipline. As an accrediting body, its emphasis is on training and harmonization across Europe. There is an increasing demand for high-grade forensic veterinary pathology reports which address the requirements of the legal system, but so far only a few countries have defined protocols for these reports. In recognition of the need for a specific qualification that benchmarks the competences and experience expected of forensic veterinary pathologists, the ECVP recently launched the Certificate in Forensic Veterinary Pathology.

Differential effects of acute and chronic social defeat stress on hypothalamic-pituitary-adrenal axis function and hippocampal serotonin release in mice.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) stress axis and disturbances in serotonin (5-HT) neurotransmission have been implicated in the pathogenesis of depressive disorder. Repeated social defeat of male NMRI mice has been shown to induce increases in core body temperature and corticosterone, indicative of a state of chronic stress in subordinate animals. The present study further characterised the HPA axis response to social defeat stress, and also examined hippocampal extracellular 5-HT release during the stress. Exposure to an acute social defeat elicits increases in plasma adrenocorticotrophic hormone and corticosterone levels, peaking at 15 and 30 min, respectively, and enhances corticotrophin-releasing factor (CRF) mRNA, but not arginine vasopressin (AVP) mRNA within the medial parvocellular division of the hypothalamic paraventricular nucleus. A concomitant increase in hippocampal corticosterone and 5-HT levels is observed. By contrast, although chronic social defeat is associated with greatly elevated corticosterone levels, the predominant drive appears to be via parvocellular AVP rather than CRF. Furthermore, subordinate animals allowed to recover for 9 days after chronic social defeat display an increase in immobility in the forced swimming model of depression, indicating that animals previously exposed to the homotypic defeat stress are sensitised to the behavioural effects of a novel stressor. These results demonstrate that social defeat induces prolonged activation of the HPA axis and alterations in 5-HT neurotransmission that could be of relevance to some of the pathological abnormalities observed in clinical depression.

Responses of magnocellular neurons to osmotic stimulation involves coactivation of excitatory and inhibitory input: an experimental and theoretical analysis.

How does a neuron, challenged by an increase in synaptic input, display a response that is independent of the initial level of activity? Here we show that both oxytocin and vasopressin cells in the supraoptic nucleus of normal rats respond to intravenous infusions of hypertonic saline with gradual, linear increases in discharge rate. In hyponatremic rats, oxytocin and vasopressin cells also responded linearly to intravenous infusions of hypertonic saline but with much lower slopes. The linearity of response was surprising, given both the expected nonlinearity of neuronal behavior and the nonlinearity of the oxytocin secretory response to such infusions. We show that a simple computational model can reproduce these responses well, but only if it is assumed that hypertonic infusions coactivate excitatory and inhibitory synaptic inputs. This hypothesis was tested first by applying the GABA(A) antagonist bicuculline to the dendritic zone of the supraoptic nucleus by microdialysis. During local blockade of GABA inputs, the response of oxytocin cells to hypertonic infusion was greatly enhanced. We then went on to directly measure GABA release in the supraoptic nucleus during hypertonic infusion, confirming the predicted rise. Together, the results suggest that hypertonic infusions lead to coactivation of excitatory and inhibitory inputs and that this coactivation may confer appropriate characteristics on the output behavior of oxytocin cells. The nonlinearity of oxytocin secretion that accompanies the linear increase in oxytocin cell firing rate reflects frequency-facilitation of stimulus-secretion coupling at the neurohypophysis.

Rapid diagnosis of intravascular catheter-related sepsis.

The use of Gram-stained "impression smears" of the external surface of intravascular catheters for rapid detection of catheter-associated infection was studied. Gram's stain results of 322 catheters were correlated with clinical episodes of systemic sepsis and semiquantitative cultures of the catheters. Organisms were seen on Gram's stain of 82 catheters, 37 of which were positive on semiquantitative cultures (greater than or equal to 15 colonies per plate). Catheter-related bacteremia occurred on three occasions. All three catheters showed numerous organisms on Gram's stain, although one was negative on semiquantitative culture. All five catheters, in place during bacteremic episodes that were unrelated to catheter infection, were negative on Gram's stain. If the presence of any organisms on Gram's stain was taken as a positive test result, the sensitivity of Gram's stain in predicting the result of semiquantitative culture was 83%, the specificity was 81%, and the predictive value of a positive and negative culture was 44% and 96%, respectively. Slides took two to five minutes to examine microscopically. Gram-stained impression smears of intravenous catheters can be made by a simple, inexpensive, and rapid technique that is accurate in diagnosing catheter-related infection. However, in this study in which a relatively low prevalence of catheter-related bacteremia occurred, the positive predictive value of the Gram's stain result in the diagnosis of catheter-related bacteremia, in contrast to catheter colonization, was low. Only in a patient group with a high prevalence of catheter-related bacteremia would the test be likely to have a high positive predictive value. Thus, selectivity should be exercised in the application of this method.

hUBC9 associates with MEKK1 and type I TNF-alpha receptor and stimulates NFkappaB activity.

hUBC9, an E2 ubiquitin conjugating enzyme, was identified by yeast two-hybrid screening and coprecipitation studies to interact with MEKK1 and the type I TNF-alpha receptor, respectively. Because both of these proteins regulate NFkappaB activity, the role of hUBC9 in modulating NFkappaB activity was investigated. Overexpression of hUBC9 in HeLa cells stimulated the activity of NFkappaB as determined by NFkappaB reporter and IL-6 secretion assays. hUBC9 also synergized with MEKK1 to activate NFkappaB reporter activity. Thus, hUBC9 modulates NFkappaB activity which, at least in part, can be attributed to its interaction with MEKK1 and the type I TNF-alpha receptor.

Sustained bleeding after a leech bite in the apparent absence of hirudin.

The bite of the medicinal leech bleeds for many hours. For decades it has been assumed that the remarkably prolonged bleeding time of a leech bite wound is due to hirudin, a specific anti-thrombin secreted by the leech during feeding. By measuring haematological parameters of blood oozing from a leech bite wound on 15 different occasions in 7 human volunteers, we demonstrate that the hirudin-sensitive coagulation parameters, including thrombin-induced platelet aggregation, are prolonged for only 15 min, after which they return to normal. This suggests that excess hirudin secreted by the leech is washed out during this period. However, bleeding from the leech bite wound persists for a mean of 10 h. Platelets in smears of exuding blood show no evidence of spontaneous aggregation, but in vitro platelet aggregation can be induced by exogenous collagen at any time. In view of sustained bleeding in the apparent absence of hirudin, attention is focussed onto an unsuspected factor or factors which may better explain the prolonged bleeding phenomenon.

Biliary sepsis. Reviewing treatment options.

Bactobilia is a frequent accompaniment of obstruction in the biliary tract, organisms present being normal intestinal aerobes and anaerobes. Bacterial colonisation of the bile may occur asymptomatically, may predispose to infection postoperatively, or may be associated with an attack of acute cholecystitis, occurring secondary to obstruction. The choice of an antimicrobial regimen for biliary infection should take into account the expected antibiotic sensitivities of organisms colonising bile, whether biliary obstruction or bacteraemia is present, and the activity of the antibiotic in bile. Often, high biliary concentrations of an antibiotic cannot be achieved due to obstruction, and in many cases high blood and tissue concentrations are of greater importance. Surgical prophylaxis should be reserved for patients at high risk of bactobilia (e.g. the elderly), when obstruction is present, for immunosuppressed patients, and those with artificial heart valves. A single perioperative dose of a 'first' or 'second generation' cephalosporin, gentamicin, or co-trimoxazole is effective. Antibiotic therapy for acute cholecystitis should be instituted if there is evidence of systemic toxicity, when surgery is to be delayed, or in patients with identified risk factors for bactobilia. Ampicillin or a cephalosporin may be appropriate in less severe disease, while in seriously ill patients, an aminoglycoside or cephalosporin with metronidazole or clindamycin is appropriate. Oral regimens include amoxycillin, an oral cephalosporin, or co-trimoxazole, in combination with metronidazole. In acute cholangitis, systemic therapy similar to that recommended for acute cholecystitis is indicated. Patients with recurrent cholangitis may have relatively antibiotic-resistant bacteria and efforts should be made to obtain a bacteriological diagnosis. Long term suppressant therapy with oral agents such as amoxycillin, cephalexin, or co-trimoxazole may be tried.

Chronic pain, chronic stress and depression: coincidence or consequence?

Chronic pain and depressive illness are debilitating disease states that are variably resistant to currently available therapeutic agents. Animal models of chronic pain are associated with activation of the hypothalamo-pituitary-adrenal (HPA) axis, upon which chronic pain acts as an inescapable stressor. Inescapable stress is also associated with 'depressive-like' symptoms in experimental animals. Based on reports of the comorbidity between chronic pain and depressive illness in human patients, it is possible that these disease states are linked, via chronic stress-induced HPA dysfunction. Here, we discuss the possible involvement of the HPA axis in the aetiology of both chronic pain and clinical depression, and suggest a strategy for the development of novel pharmacotherapies.

Evaluation of lysis filtration as an adjunct to conventional blood culture.

A lysis filtration system was used in conjunction with conventional broth culture for 1112 blood cultures. The system, which entailed collection of 5 ml of blood into bottles containing 50 ml isotonic phosphate buffer, Tween 20, and Rhozyme with subsequent filtration using a 0.45 micron Millipore field monitor, was simple and economical to use. Positive results were obtained earlier than those obtained with conventional broth cultures, and almost twice as many fungi and yeasts were isolated. Some fastidious organisms such as Haemophilus influenzae and Streptococcus pneumoniae however, were not recovered from the lysis system, and contaminants in lysis cultures were three times as common as in conventional culture. The number of positive cultures was also adversely influenced by incubation of the blood lysis mixture overnight before filtration. We conclude that this lysis filtration system is useful as an adjunct to conventional broth culture in selected patients in cases in which filtration can be carried out soon after collection.

Is the antimicrobial removal device a cost-effective addition to conventional blood cultures?

Two hundred and thirty-four blood cultures from 140 patients receiving antibiotics were processed using the antimicrobial removal device (ARD) in parallel with conventional blood cultures. One hundred and seventy cultures were obtained from patients suspected to have bacteraemia and 64 from patients known to have a positive conventional blood culture within the preceding three days. A total of 38 (16.2%) ARD-processed cultures were positive, compared with 21 (8.9%) conventional cultures (p less than 0.0001, Fisher's exact test). No instances of positive conventional cultures and negative ARD-processed cultures were identified. Thirty-three of 38 ARD-processed cultures became positive within 24 h, compared with 14 parallel conventional cultures (p less than 0.0001 Fisher's exact test). Although the yield and rapidity of isolation of bacteria from blood were improved by ARD processing, in only one of 140 patients did this information alter treatment. As the use of an ARD is associated with a sixfold increase in the cost of blood cultures, we conclude that, in our hands, general use of the device in patients receiving antibiotics is not cost-effective. Considerable care should be taken in selecting patients for ARD-processing of blood cultures.

Patterns of resistance to anaerobic organisms in Australia.

This study investigated patterns of resistance in anaerobic organisms isolated at Westmead Hospital in Sydney, Australia, during the years 1987 to 1988. Minimal inhibitory concentrations (MICs) to ampicillin, sulbactam/ampicillin, metronidazole, clindamycin, and cefoxitin were determined by agar dilution for 200 anaerobes from clinically significant infections. Antibiotics active against nearly all of these anaerobes included metronidazole and sulbactam/ampicillin, which demonstrated good activity against beta-lactamase producing Bacteroides spp. with the exception of Bacteroides distasonis. Resistance in non-beta-lactamase producing anaerobes was similar to that seen with ampicillin. As expected, ampicillin resistance was common in the Bacteroides fragilis group where beta-lactamase production was frequent. In addition, beta-lactamase was detected in 33% of other Bacteroides spp. Ampicillin resistance was also seen in 5 to 15% of additional anaerobes that did not produce beta-lactamase. Clindamycin resistance occurred in 4 to 18% of the B. fragilis group. Clindamycin resistance was also seen in 7 to 8% of Clostridium spp. and anaerobic Gram-positive cocci. Resistance to cefoxitin was variable in the B. fragilis group with the highest levels of resistance occurring in the indole-negative subgroup. Resistance in other anaerobes was not commonly seen.

Slow-acting antirheumatic drugs. Drug interactions of clinical significance.

The slow-acting antirheumatic drugs (SAARDs) are being used in an increasing proportion of patients with rheumatoid arthritis (RA). The potential toxicity of each drug is well recognised. Many patients with RA will be on other medications and the potential for adverse drug interactions with SAARDs is not so well publicised. There have, over the years, been numerous reports of possible drug interactions with SAARDs but few of these are clinically relevant. It is, however, vitally important that the physician is aware of a number of potentially life-threatening interactions, particularly those associated with methotrexate. The SAARDs are a very useful group of drugs for the treatment of RA and, by being aware of their potential toxicity and drug interactions, hopefully they can be used safely and effectively.

Aeromonas septicemia: relationship of species and clinical features.

Thirteen patients with Aeromonas species septicemia were seen at Westmead Hospital between 1983 and 1987. In 10 patients (77%) septicemia was caused by A. sobria, and in 3 (23%) by A. hydrophila, A. caviae was not isolated. Chronic underlying illness was present in 10 patients (77%), hematological malignancy being the most common (46%). The average age of the patients was 65.8 years. Seven patients (54%) presented with clinical sepsis, diarrhea and abdominal pain. The biliary tract, lung and soft tissue were other primary sites of infection. Nosocomial infection occurred in 5 patients (38%). Cross-infection was not seen. Contact with fresh water or fish was observed in only 2 patients (15%). The majority of cases occurred in the warmer months of the year. The overall mortality was 46%. All isolates of Aeromonas species were resistant to ampicillin 8 mg/L. Ten isolates were resistant to tobramycin 1 mg/L, and 3 to tobramycin 4 mg/L. In contrast, 3 isolates were resistant to gentamicin 1 mg/L, and none to 4 mg/L. The isolates showed a varied pattern of resistance to other antibiotics tested, but all were susceptible to piperacillin 64 mg/L, imipenem 4 mg/L, ciprofloxacin 1 mg/L, and amikacin 16 mg/L.

M-6 endocarditis: report of an Australian case.

Neisseria elongata subsp. nitroreducens (formerly CDC group M-6) is a newly-recognized cause of particularly destructive endocarditis, frequently requiring valve replacement. We describe an Australian case of endocarditis caused by this organism, summarizing the clinical and microbiological features of this rarely isolated subspecies.

An evaluation of a rapid latex test for the diagnosis of Clostridium difficile-associated diarrhea.

We present here the results of an evaluation of a rapid latex test for detection of Cl. stridium difficile-associated in comparison with our standard cytotoxin assay and culture for C. difficile. Some 515 diarrheal stools were examined. C. difficile was cultured from 70 specimens (13.5%); 53 specimens (10.2%) were positive with the latex test, and 50 (9.6%) by cytotoxin assay. The latex test did not differ significantly from the cytotoxin assay in sensitivity or specificity compared to culture results. There was also no significant difference in the specificity of the latex test compared to cytotoxin assay in patients in whom the diagnosis of C. difficile-associated diarrhea was negative. Positive and negative predictive values of the latex test for C. difficile-associated diarrhea were similar to those of cytotoxin assay. The latex test thus appears to be a rapid and practical test for the laboratory diagnosis of C. difficile-associated diarrhea. To optimize specificity and sensitivity its use should be restricted to patients where the diagnosis is strongly suspected and a rapid answer is required. As it does not distinguish between toxigenic virulent C. difficile strains and non-toxigenic avirulent strains, it would seem prudent to confirm positive results subsequently by demonstrating in-vivo or in-vitro cytotoxin production.

Flavimonas oryzihabitans infection of a surgical wound.

An unusual case of a post operative wound infection involving Flavimonas oryzihabitans is described. This organism is rarely isolated from human sources. It can cause infections in patients having continuous ambulatory peritoneal dialysis. Our patient developed a wound infection 2 months after femoro-popliteal bypass grafting. The source of the organism was unknown.

Use of a selective enrichment broth for isolation of Campylobacter species from human feces.

Isolation of Campylobacter species from 1126 fecal specimens from patients with diarrhea was compared using direct plating and selective enrichment broth. The use of the enrichment broth did not increase the isolation rate, which was 4.2%. While a selective enrichment broth may have advantages where there is delay in transit to the laboratory, or where small numbers of organisms are sought, we do not recommend its use for clinical specimens from patients with diarrhea.

Susceptibility of anaerobic bacteria to antimicrobial agents.

The antimicrobial susceptibility of 1,117 clinical isolates of anaerobic bacteria was determined by the agar dilution technique. Metronidazole was the most active agent; only Propionibacterium acnes and Actinomyces sp. isolates were resistant. Clindamycin and chloramphenical were the next most effective agents. Beta-lactam antibiotics, with the exception of penicillin, were active against most anaerobes other than the Bacteroides fragilis group. At a breakpoint of 8 mg/l, 25% of Fusobacterium spp. and 30% of the non-fragilis Bacteroides spp. were resistant to penicillin. The highest resistance to beta-lactams was seen in the B. fragilis group. Within the indole-positive members of the group, resistance rates of 71% were seen for cefoxitin, 49% for moxalactam, 79% for cefotaxime, 22% for piperacillin and 89% for penicillin. We conclude that metronidazole has the most predictable in vitro activity against common clinical anaerobic isolates and that resistance to beta-lactams was frequent and of potential clinical importance as these latter agents are frequently used in the prophylaxis and therapy of mixed anaerobic infections.

Detection and serogroup determination of Neisseria meningitidis in CSF by polymerase chain reaction (PCR).

A PCR protocol for the detection and serogroup determination of Neisseria meningitidis in CSF from 85 cases of suspected meningitis was evaluated. Screening assays for both IS1106 and the ctrA gene were used to detect meningococcal DNA, and a further two assays using the siaD gene were performed to determine the serogroup. PCR results were compared with results of bacteriological culture and discrepant results resolved by analysis of clinical data and further laboratory test results. The resolved sensitivity and specificity of the PCR screening assay were 89 and 100%, and those of bacteriological culture were 37 and 100%, respectively. The siaD B/C PCR assay was able to determine a serogroup in 85% of cases positive by the PCR screening assay compared with 50% of cases where a serogroup was determined by traditional methods. PCR is a useful tool for diagnosis of meningococcal meningitis when Gram stain and culture tests are negative, a situation that may arise when antibiotic treatment has commenced prior to lumbar puncture.