Analysis of differential protein expression in Actinobacillus pleuropneumoniae by Surface Enhanced Laser Desorption Ionisation--ProteinChip (SELDI) technology

Actinobacillus pleuropneumoniae (APP) is the aetiological agent of porcine pleuropneumonia. An increased understanding of its molecular basis of pathogenicity and vaccine development will be facilitated by the availability of sequence data from a complete genome which, by analogy to other bacteria, is predicted to encode many proteins in the molecular mass range 3-20kDa. However, conventional techniques to study bacterial protein expression, such as SDS-PAGE and 2-dimensional electrophoresis, typically focus on the 15-200kDa range. In this study we have evaluated Surface Enhanced Laser Desorption Ionisation-ProteinChip (SELDI) technology for the analysis of protein expression, in particular those of <20kDa, of APP grown under different environmental conditions. Cytoplasmic/periplasmic and outer membrane protein fractions were obtained from the APP wildtype serotype 1 strain 4074 grown in Brain Heart Infusion (BHI) broth (+different concentrations of NAD), BHI containing pig serum or defined medium. Optimum conditions for SELDI profiles included a sample size of 1 microg and the use of sinapinic acid as the energy absorbing matrix. In the <20kDa range, the SELDI profiles obtained from wild-type bacteria grown in rich medium plus 33-66% pig serum were most similar to those grown in defined medium. The SELDI profiles of extracts of the wild-type and of an rpoE mutant were similar although there were clear differences. The results suggest that SELDI is a useful complementary approach to conventional proteomic analytical methods with APP, and presumably other bacterial pathogens, being particularly suited for analysis of proteins in the <20kDa mass range.     

Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, Anaplasma marginale, causes bovine anaplasmosis and infects erythrocytes of the vertebrate host and undergoes a complex developmental cycle in ticks which serve as biological vectors. Infected cattle, wild ruminants and ticks can all serve as reservoirs of A. marginale. In this study, hunter killed Iberian red deer (Cervus elaphus hispanicus) from the region of Castilla-La Mancha in southwestern Spain were tested for Anaplasma infection. We found that 10% of the deer examined were seropositive for Anaplasma. Three A. marginale strains were subsequently obtained from salivary glands of Hyalomma marginatum that were removed from these deer, and the sequence of the major surface protein (msp)4 gene was determined for each strain and used for phylogenetic studies. Maximum parsimony analyses of msp4 sequences from H. marginatum ticks in comparison with New World cattle and bison isolates reported previously, suggested different origins for these Spanish A. marginale strains. The results of this study demonstrated that Iberian red deer are naturally infected with Anaplasma, and may therefore serve as a wildlife reservoir of the pathogen. Although the link between deer infection and the strains of A. marginale identified in ticks was not established, H. marginatum and Rhipicephalus bursa were identified as potential biological vectors for A. marginale in this region and may effect transmission of A. marginale between deer and cattle populations.     

Fatal intraoperative pulmonary fat embolism during cemented total hip arthroplasty in a dog

A 3-year-old, German shepherd dog died suddenly during cemented total hip arthroplasty. Gross necropsy findings included severe pulmonary edema and congestion as well as congestion of the liver and kidneys. Acute pulmonary embolism was suspected as the cause of death. Microscopic examination of hematoxylin and eosin-stained, formalin-fixed, and oil red O-stained frozen tissue sections confirmed the presence of large numbers of fat globules in blood vessels in the lungs, liver, and kidneys. Fat embolism during total hip arthroplasty is a common surgical complication in humans, but it is uncommon in veterinary cases and is rarely a cause of death.     

Ensuring that the competent are truly competent: an overview of common methods and procedures used to set standards on high-stakes examinations

Determining whether or not an examinee has met an adequate standard of performance constitutes a central task for licensure and certification bodies. Consequently, standard setting is a key activity for all certification and licensing testing programs. The purpose of this article is to provide an overview of methods that have been proposed for set a passing standard on an examination. First, the distinction between norm-referenced and criterion-referenced methods for setting a standard will be outlined. Then, both test-centered and examinee-centered methods for setting a passing standard will be explicated. The importance of factoring in the consequences of adopting a standard will also be illustrated via the Hofstee method. In the concluding section, important issues pertaining to the selection of panelists as well as the validation of the standard will be addressed briefly.     

Effect of strain and serotype of vesicular stomatitis virus on viral shedding, vesicular lesion development, and contact transmission in pigs

OBJECTIVE: To determine whether pigs can be infected with strains of vesicular stomatitis virus New Jersey (VSV-NJ) and vesicular stomatitis virus Indiana (VSV-I) isolated during recent vesicular stomatitis outbreaks that primarily involved horses in the western United States and determine the potential for these viruses to be transmitted by contact. ANIMALS: 128 pigs. PROCEDURE: Pigs were challenged with VSV-NJ or VSV-I from the 1995 and 1997 outbreaks of vesicular stomatitis in the western United States, respectively, or with VSV-NJ (OS) associated with vesicular stomatitis in feral pigs on Ossabaw Island, Ga. Pigs (3/group) were inoculated with each virus via 3 routes and evaluated for viral shedding, seroconversion, and the development of vesicular lesions. In another experiment, the potential for contact transmission of each virus from experimentally infected to na?ve pigs was evaluated. RESULTS: Infection of pigs was achieved for all 3 viruses as determined by virus isolation and detection of seroconversion. In inoculated pigs, all 3 viruses were isolated from multiple swab samples at concentrations sufficient to infect other pigs. However, compared with results obtained with the 2 VSV-NJ strains, viral titers associated with VSV-I were low and the duration of virus shedding was reduced. Results from the contact transmission trials were consistent with these results; virus transmission was detected most frequently with the VSV-NJ strains. CONCLUSIONS AND CLINICAL RELEVANCE: Pigs can be infected with VSV-NJ and VSV-I. Differences in the extent of viral shedding and potential for contact transmission were apparent between serotypes but not between the VSV-NJ strains investigated.     

Evaluation of outcome of otitis media after lavage of the tympanic bulla and long-term antimicrobial drug treatment in dogs: 44 cases (1998-2002)

OBJECTIVE: To evaluate the outcome of otitis media in dogs after video-otoscopic lavage of the tympanic bulla and long-term antimicrobial drug treatment. DESIGN: Retrospective study. ANIMALS: 44 dogs with otitis media treated in an academic referral practice. PROCEDURE: Medical records were reviewed for signalment, duration of ear canal disease, previous medical treatments, dermatologic diagnosis, results of cytologic examination and microbial culture of ear canal exudate, findings during video-otoscopy, medical treatment, days to resolution, and maintenance treatments prescribed. Four independent variables (age, duration of ear canal disease prior to referral, use of corticosteroids in treatment regimens, and infection with Pseudomonas aeruginosa) were evaluated statistically for potential influence on time to resolution. RESULTS: Mean +/- SD (range) duration of ear canal disease prior to referral was 24.9 +/- 21.6 (3 to 84) months. Otitis media in 36 dogs resolved after lavage of the tympanic bulla and medical management; mean +/- SD (range) time to resolution was 117 +/- 86.7 (30 to 360) days. Time to resolution was not significantly influenced by any variable evaluated. Three dogs were lost to follow-up, and 4 dogs eventually required surgical intervention. Seven of 36 dogs in which otitis had resolved relapsed; 4 required additional lavage procedures. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that lavage of the tympanic bulla combined with medical management is an effective and viable option for treatment of otitis media in dogs.     

Chronic wasting disease in Canada: Part 1

The purpose of part 1 is to provide an overview of published literature (1980-2002) on chronic wasting disease (CWD) to inform Canadian readers about the disease and to explain Canadian regulatory approaches to the surveillance and control of CWD. Much of the scientific information is drawn from American publications obtained from internet searches in PubMed and Medline databases. The following keywords were used: chronic wasting disease, prion, diagnosis, transmissible spongiform encephalopathies, CWD and deer, CWD and elk, and CWD and environment. The article also presents information from Canadian publications and unpublished observations, Canadian Food Inspection Agency (CFIA) documents, and both government and nongovernment internet Web sites. The article highlights some different features of CWD in Canada, as compared with the situation in the United States, and mentions public health implications of the disease. It also describes the basis for development of Canada's surveillance and control program. Part 2 will detail the activities and results of the surveillance and control program during 2000 to 2002 and discuss factors that will influence the feasibility of eradicating CWD. Chronic wasting disease appears to have been introduced into Canada through the importation of infected farmed elk from the United States in the late 1980s and early 1990s, at a time when little was known about the disease. Since then, eradication efforts in Canada have led to the control of the spread of CWD in the farmed elk industry. Still, management of this disease, especially in free-ranging cervids, is a challenge.     

Viability of Iberian x Meishan F2 newborn pigs. II. Survival analysis up to weaning

Iberian x Meishan F2 piglet's preweaning survivability was analyzed using categorical data regression procedures within the proportional hazards assumption. A frailty sire model was assumed with the litter effect treated as an additional random source of variation. Moreover, the relative birth weight within litter and the litter effect were considered time-dependent covariates that changed their values in the second day of life due to cross fostering carried out to standardize litters. Six variables had a significant effect on survivability: birth weight (P < 0.01), relative birth weight within litter (P < 0.001), rectal temperature 60 min after birth (P < 0.01), type of presentation at birth (P < 0.05), presence of stillbirths (P < 0.001), and presence of mummified fetuses (P < 0.001). Small piglets (<0.98 kg) suffered a high hazard ratio (6.57; P < 0.001), with this variable being clearly lower for the rest of birth weight categories. Piglets that were small in relation to their siblings (relative birth weight within litter) also suffered an increased death risk, with a hazard ratio of 1.81 (P < 0.05), which was similar to animals with posterior presentations at birth (hazard ratio = 1.80; P < 0.05). Piglets with a rectal temperature lower than 35.4 degrees C 60 min after birth showed the highest hazard ratio (7.18; P < 0.01). Furthermore, the presence of mummified fetuses decreased the survivability of the remaining siblings, with a hazard ratio of 2.03 (P < 0.01), as did the presence of stillbirths (hazard ratio = 3.55; P < 0.001). The inclusion of the two random effects allowed us to estimate the mode of the joint posterior density of the sire variance (0.08) and the litter variance (1.98). The estimated heritability of preweaning survival reached a value of 0.03. We conclude that piglet survival involves several systematic influences related to birth weight, thermoregulatory ability, and injuries suffered during gestation and farrowing. The genetic variance was small compared with those generated by the common environment, for which the genetic improvement of piglet survival seems difficult.     

Intraocular osteosarcoma in a dog

A 10-year-old German shepherd dog was presented with unilateral uveitis and hyphaema. Treatment was unsuccessful and the eye was enucleated. Intraocular osteosarcoma was diagnosed by histological examination.