Christiane Girard

Toxic Compounds and Health and Reproductive Effects in St. Lawrence Beluga Whales

An epidemiologic study was carried out over a period of 9 years on an isolated population of beluga whales (Delphinapterus leucas) residing in the St. Lawrence estuary (Quebec, Canada). More than 100 individual deaths were aged, and/or autopsied and analyzed for toxic compounds, and the population was surveyed for size and structure. Arctic belugas and other species of whales and seals from the St. Lawrence were used for comparison. Population dynamics: Population size appeared to be stable and modeling showed this stable pattern to result from low calf production and/or low survival to adulthood. Toxicology: St. Lawrence belugas had higher or much higher levels of mercury, lead, PCBs, DDT, Mirex, benzo[a]pyrene metabolites, equivalent levels of dioxins, furans, and PAH metabolites, and much lower levels of cadmium than Arctic belugas. In other St. Lawrence cetaceans, levels of PCBs and DDT were inversely related to body size, as resulting from differences in metabolic rate, diet, and trophic position, compounded by length of residence in the St. Lawrence basin. St. Lawrence belugas had much higher levels than predicted from body size alone; levels increased with age in both sexes, although unloading by females through the placenta and/or lactation was evidenced by overall lower levels in females and very high burdens in some calves. No PCDDs and only low levels of some PCDFs were detected in St. Lawrence belugas, while proportions of toxic non-ortho (coplanar) PCBs were low relative to proportions seen in other species. At least ten different PCB methylsulphone metabolites were detected in St. Lawrence belugas. Levels of B[a]P adducts to DNA in St. Lawrence beluga brain and liver approached those associated with carcinogenis in small laboratory animals. Pathology: St. Lawrence belugas were not emaciated, and major findings were: a high prevalence of tumors (40% of animals) including eight malignant neoplasms; a high incidence of lesions to the digestive system (53%), to the mammary glands (45% of adult females), and to other glandular structures (11%); some evidence of immuno-suppression; frequent tooth loss and periodontitis. Two animals had severe ankylosing spondylosis and another was a true bilateral hermaphrodite. No such lesions were observed in 36 necropsies of Arctic belugas and of seals and cetaceans from the St. Lawrence.

Pathology and toxicology of beluga whales from the St. Lawrence Estuary, Quebec, Canada. Past, present and future

Abstract An indigenous population of 450–500 beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence Estuary has been exposed chronically for more than 50 years to a complex mixture of industrial pollutants including organochlorinated compounds (OC), polycyclic aromatic hydrocarbons (PAH) and heavy metals. From 1983 to 1990, we have necropsied 45 well preserved carcasses out of a total of 120 beluga whales reported dead over this period. Of these 45 animals, nine were affected by 10 malignant neoplasms. Fifteen animals (33%) were affected by pneumonia. Milk production was compromised in eight of 17 mature females (41%), by inflammatory changes (seven animals) and cancer (one animal) which affected the mammary glands. Opportunistic bacteria were found in pure culture, and/or in significant amounts in at least two organs in 20 belugas (44%). The concentrations of both total PCBs and highly chlorinated PCB congeners were much higher in St. Lawrence animals than in Arctic beluga whales. OC-induced immunosuppression has been repeatedly demonstrated in a wide variety of animal species. Therefore, it is probable that the immune functions of St. Lawrence beluga whales are impaired. Benzo[α]pyrene adducts were detected in 10 of the 11 St. Lawrence beluga whales of which tissues (six livers, 10/11 brains) were analyzed by a method based on HPLC. No such adducts were found in four Arctic animals. Since benzo[α]pyrene is one of the most potent chemical carcinogens known to man, these compounds might be responsible for some of the cancers observed in that population. Overall, our findings contrast vividly with those of others who found that cancers are exceedingly rare in free-ranging odontocete populations and that the major causes for mortalities in these populations are bacteria, parasites, and trauma.

The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rabbit.

AIM The primary goal of this body of work is to suggest a standardized system for histopathological assessment of experimental surgical instability models of osteoarthritis (OA) in rabbits, building on past experience, to achieve comparability of studies from different centres. An additional objective is to review methodologies that have been employed in the past for assessing OA in rabbits with particular reference to the surgical anterior cruciate ligament transection (ACLT) model. METHODS A panel of scientists and clinician-scientists with recognized expertise in assessing rabbit models of OA reviewed the literature to provide a critical appraisal of the methods that have been employed to assess both macroscopic and microscopic changes occurring in rabbit joint tissues in experimental OA. In addition, a validation of the proposed histologic histochemical grading system was performed. RESULTS The ACLT variant of the surgical instability model in skeletally mature rabbits is the variation most capable of reproducing the entire range of cartilage, synovial and bone lesions recognized to be associated with OA. These lesions can be semiquantitatively graded using macroscopic and microscopic techniques. Further, as well as cartilage lesions, this ACLT model can produce synovial and bone lesions similar to that of human OA. CONCLUSIONS The ACLT variant of the surgical instability model in rabbits is a reproducible and effective model of OA. The cartilage lesions in this model and their response to therapy can be graded according to an adapted histological and histochemical grading system, though also this system is to some extent subjective and, thus, neither objective nor entirely reproducible.

Early degradation of type IX and type II collagen with the onset of experimental inflammatory arthritis

OBJECTIVE To determine whether following the onset of intraarticular inflammation, there is early damage to articular cartilage, specifically to types II and IX collagen, and the proteoglycan (PG) aggrecan, and whether measurement of the degradation products of these molecules in synovial fluid (SF) and serum may permit the detection of cartilage damage. METHODS A rabbit model of rheumatoid arthritis, antigen (ovalbumin)-induced arthritis, was studied. Articular cartilage samples were analyzed by immunoassays for total type II collagen content, its denaturation and cleavage by collagenases, and for type IX collagen content. PG content was determined by colorimetric assay. In serum and SF, total PG content and collagenase-generated peptides of type II collagen were measured. RESULTS After 6 days, both the PG content and the NC4 domain of type IX collagen were reduced in femoral and tibial cartilage, concomitant with the onset of arthritis. In only the tibial cartilage did this reduction in PG persist up to day 20. However, denatured type II collagen was increased in all cartilage samples, but only on day 20. In SF, the PG content was significantly reduced on day 20, and products of type II collagen cleavage by collagenase were significantly increased on both day 6 and day 20. CONCLUSION This study, which is the first of its kind examining changes in both types II and IX collagen and PG content, reveals early damage to both types of collagen as well as to PG in articular cartilage samples following induction of joint inflammation. SF analyses reveal this early damage and may be of value in the study and treatment of inflammatory arthritic diseases such as rheumatoid arthritis.

Non-neoplastic lesions in beluga whales (Delphinapterus leucas) and other marine mammals from the St Lawrence Estuary

In a 3-year (1988-1990) pathological study, 24 carcasses of beluga whales from the St Lawrence Estuary, Québec, Canada, showed numerous severe lesions, many of which had never been reported in cetaceans. The most common lesions were found in the digestive tract (21 animals) and consisted mainly of periodontitis and of erosions and ulcers in the oesophagus and the first two gastric compartments. Pneumonia, usually of parasitic origin, was also a common finding (12 animals). The adrenal glands often contained nodules (five animals) or cysts (seven animals), and mastitis was observed in five females. Overall, the incidence of degenerative, infectious, hyperplastic or necrotic lesions, in addition to numerous neoplasms described in another paper, was considerably higher than that found in marine mammals elsewhere or in other species of marine mammal from the same waters.

Prevalence of and carcass condemnation from maedi–visna, paratuberculosis and caseous lymphadenitis in culled sheep from Quebec, Canada

We determined the prevalence of lung and mammary gland lesions associated with maedi-visna (MV) infection, the prevalence of paratuberculosis (PTB), and the prevalence and lesions distribution of caseous lymphadenitis (CL) in culled sheep. Total of 451 ewes and 34 rams were selected randomly from two slaughterhouses in Quebec, Canada. MV serostatus was determined by recombinant ELISA test. PTB diagnosis was based on characteristic histological lesions in the terminal ileum, ileocecal lymph node and/or ileocecal valve and CL by gross detection of abscesses and isolation of Corynebacterium pseudotuberculosis. Seroprevalence of MV was 44% (95% CI: 40, 48). Seropositivity increased with age and was higher in ewes than in rams. The percentages of lung and mammary gland lesions in seropositive sheep were 14 and 40%, respectively, but mammary gland lesions lack specificity. The prevalence of PTB was 3% (95% CI: 2, 5). PTB increased with age and was lower among sheep with abscesses. The prevalence of CL was >/=21% (95% CI: 17, 24). The most-prevalent site of caseous lymphadenitis lesions was the thoracic cavity. The risk of carcass condemnation was significantly associated with region, body score and abscesses. Only the presence of abscesses was associated with an increase in trimming of carcasses.

Cartilage matrix changes in the developing epiphysis: early events on the pathway to equine osteochondrosis?

REASONS FOR PERFORMING STUDY The earliest osteochondrosis (OC) microscopic lesion reported in the literature was present in the femorotibial joint of a 2-day-old foal suggesting that OC lesions and factors initiating them may arise prior to birth. OBJECTIVE To examine the developing equine epiphysis to detect histological changes that could be precursors to OC lesions. METHODS Osteochondral samples from 21 equine fetuses and 13 foals were harvested from selected sites in the scapulohumeral, humeroradial, metacarpophalangeal, femoropatellar, femorotibial, tarsocrural and metatarsophalangeal joints. Sections were stained with safranin O and picrosiruis red to assess cartilage changes and structural arrangement of the collagen matrix. RESULTS Extracellular matrix changes observed included perivascular areas of paleness of the proteoglycan matrix associated with hypocellularity and, sometimes, necrotic chondrocytes. These changes were most abundant in the youngest fetuses and in the femoropatellar/femorotibial (FP/FT) joints. Indentations of the ossification front were also observed in most specimens, but, most frequently, in scapulohumeral and FP/FT joints. A cartilage canal was almost always present in these indentations. The vascular density of the cartilage was higher in the youngest fetuses. In these fetuses, the most vascularised joints were the metacarpo- and metatarsophalangeal joints but their cartilage canals regressed quickly. After birth, the most vascularised cartilage was present in the FP/FT joint. Articular cartilage differentiated into 4 zones early in fetal life and the epiphyseal cartilage also had a distinct zonal cartilage structure. A striking difference was observed in the collagen structure at the junction of the proliferative and hypertrophic zones where OCD lesions occur. CONCLUSION Matrix and ossification front changes were frequently observed and significantly associated with cartilage canals suggesting that they may be physiological changes associated with matrix remodelling and development. The collagen structure was variable through the growing epiphysis and a differential in biomechanical properties at focal sites may predispose them to injury.

Relationship between cartilage and subchondral bone lesions in repetitive impact trauma-induced equine osteoarthritis

OBJECTIVE To correlate degenerative changes in cartilage and subchondral bone in the third carpal bone (C3) of Standardbred racehorses with naturally occurring repetitive trauma-induced osteoarthritis. DESIGN Fifteen C3, collected from Standardbred horses postmortem, were assessed for cartilage lesions by visual inspection and divided into Control (CO), Early Osteoarthritis (EOA) and Advanced Osteoarthritis (AOA) groups. Two osteochondral cores were harvested from corresponding dorsal sites on each bone and scanned with a micro-computed tomography (CT) instrument. 2D images were assembled into 3D reconstructions that were used to quantify architectural parameters from selected regions of interest, including bone mineral density and bone volume fraction. 2D images, illustrating the most severe lesion per core, were scored for architectural appearance by blinded observers. Thin sections of paraffin-embedded decalcified cores stained with Safranin O-Fast Green, matched to the micro-CT images, were scored using a modified Mankin scoring system. RESULTS Subchondral bone pits with deep focal areas of porosity were seen more frequently in AOA than EOA but never in CO. Articular cartilage damage was seen in association with a reduction in bone mineral and loss of bone tissue. Histological analyses revealed significant numbers of microcracks in the calcified cartilage of EOA and AOA groups and a progressive increase in the score compared with CO bones. CONCLUSION The data reveal corresponding, progressive degenerative changes in articular cartilage and subchondral bone, including striking focal resorptive lesions, in the third carpal bone of racehorses subjected to repetitive, high impact trauma.

Maedi-visna impact on productivity in Quebec sheep flocks (Canada)

An epidemiological study was conducted to determine the impact of maedi-visna (MV) seropositivity on productivity in commercial sheep flocks of the province of Quebec, Canada. A total of 1734 ewes and 220 rams were selected randomly from 29 flocks distributed in the Bas-St-Laurent and Estrie regions. Serostatus was determined with an enzyme-linked immunosorbent assay (ELISA) using recombinant proteins.Flock-specific, animal-level seroprevalence varied from 3 to 70% (median=29%). Seroprevalence increased with age and size of the flock, and was higher in ewes relative to rams (but was not associated with body score). A decrease of 0.94 kg per lamb in weaning weight was seen only for lambs raised by seropositive ewes >/=4 years old, and seropositivity in ewes of any age was associated with an increase in 0-30 days lamb-mortality (OR: 1.65). The impact of MV infection on weaning weight and lamb mortality did not vary between flocks, and seropositivity in ewes was not associated with litter size or lambs birth weight.

Transcriptional profiling of Actinobacillus pleuropneumoniae during the acute phase of a natural infection in pigs

BackgroundActinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. Many virulence factors are involved in the pathogenesis, namely capsular polysaccharides, RTX toxins, LPS and many iron acquisition systems. In order to identify genes that are expressed in vivo during a natural infection, we undertook transcript profiling experiments with an A. pleuropneumoniae DNA microarray, after recovery of bacterial mRNAs from serotype 5b-infected porcine lungs. AppChip2 contains 2033 PCR amplicons based on the genomic sequence of App serotype 5b strain L20, representing more than 95% of ORFs greater than 160 bp in length.ResultsTranscriptional profiling of A. pleuropneumoniae recovered from the lung of a pig suffering from a natural infection or following growth of the bacterial isolate in BHI medium was performed. An RNA extraction protocol combining beadbeating and hot-acid-phenol was developed in order to maximize bacterial mRNA yields and quality following total RNA extraction from lung lesions. Nearly all A. pleuropneumoniae transcripts could be detected on our microarrays, and 150 genes were deemed differentially expressed in vivo during the acute phase of the infection. Our results indicate that, for example, gene apxIVA from an operon coding for RTX toxin ApxIV is highly up-regulated in vivo, and that two genes from the operon coding for type IV fimbriae (APL_0878 and APL_0879) were also up-regulated. These transcriptional profiling data, combined with previous comparative genomic hybridizations performed by our group, revealed that 66 out of the 72 up-regulated genes are conserved amongst all serotypes and that 3 of them code for products that are predicted outer membrane proteins (genes irp and APL_0959, predicted to code for a TonB-dependent receptor and a filamentous hemagglutinin/adhesin respectively) or lipoproteins (gene APL_0920). Only 4 of 72 up-regulated genes had previously been identified in controled experimental infections.ConclusionsThese genes that we have identified as up-regulated in vivo, conserved across serotypes and coding for potential outer membrane proteins represent potential candidates for the development of a cross-protective vaccine against porcine pleuropneumonia.