Estimating pregnancy rate from blubber progesterone levels of a blindly biopsied beluga population poses methodological, analytical and statistical challenges.

Beluga (Delphinapterus leucas) from the St. Lawrence Estuary, Canada, have been declining since the early 2000s, suggesting recruitment issues as a result of low fecundity, abnormal abortion rates or poor calf or juvenile survival. Pregnancy is difficult to observe in cetaceans, making the ground truthing of pregnancy estimates in wild individuals challenging. Blubber progesterone concentrations were contrasted among 62 SLE beluga with a known reproductive state (i.e. pregnant, resting, parturient and lactating females), that were found dead in 1997 to 2019. The suitability of a threshold obtained from decaying carcasses to assess reproductive state and pregnancy rate of freshly-dead or free-ranging and blindly-sampled beluga was examined using three statistical approaches and two data sets (135 freshly harvested carcasses in Nunavik, and 65 biopsy-sampled SLE beluga). Progesterone concentrations in decaying carcasses were considerably higher in known-pregnant (mean ± sd: 365 ± 244 ng g-1 of tissue) than resting (3.1 ± 4.5 ng g-1 of tissue) or lactating (38.4 ± 100 ng g-1 of tissue) females. An approach based on statistical mixtures of distributions and a logistic regression were compared to the commonly-used, fixed threshold approach (here, 100 ng g-1) for discriminating pregnant from non-pregnant females. The error rate for classifying individuals of known reproductive status was the lowest for the fixed threshold and logistic regression approaches, but the mixture approach required limited a priori knowledge for clustering individuals of unknown pregnancy status. Mismatches in assignations occurred at lipid content < 10% of sample weight. Our results emphasize the importance of reporting lipid contents and progesterone concentrations in both units (ng g-1 of tissue and ng g-1 of lipid) when sample mass is low. By highlighting ways to circumvent potential biases in field sampling associated with capturability of different segments of a population, this study also enhances the usefulness of the technique for estimating pregnancy rate of free-ranging population.

Pathologic Findings and Trends in Mortality in the Beluga (Delphinapterus leucas) Population of the St Lawrence Estuary, Quebec, Canada, From 1983 to 2012.

An isolated population of beluga (Delphinapterus leucas) inhabits the St Lawrence Estuary, Quebec, Canada. This population has failed to recover despite the prohibition of hunting >30 years ago, suggesting the presence of other limiting factors. The authors summarize the reported causes of death and propose risk factors to explain the lack of recovery of this population. From 1983 to 2012, a total of 472 beluga were found stranded. Complete necropsies were carried out on 222 beluga, including 178 adults, 25 juveniles, and 19 newborn calves. Infectious diseases, the most prevalent cause of mortality in this population, accounted for the death of one-third of all beluga (32%). Verminous pneumonia was the cause of mortality of 13 juvenile beluga (52% of juvenile beluga). A total of 39 malignant neoplasms, diagnosed in 35 beluga, caused the death of 31 beluga (20% of beluga >19 years old). Median age at diagnosis of cancer was 48 years (range, 30-61 years). Dystocia and postpartum complications were the cause of death in 18 beluga, accounting for 19% of the females >19 years old examined. The occurrence of parturition-associated complications, as well as mortality of calves <1 year old, have increased recently in this population and may be the probable cause of the recent decrease in the size of this population. One of the hypotheses proposed to explain the unusually high occurrence of some of the pathologic conditions observed in this population is chronic exposure to environmental contaminants.

Intestinal volvulus in cetaceans.

Intestinal volvulus was recognized as the cause of death in 18 cetaceans, including 8 species of toothed whales (suborder Odontoceti). Cases originated from 11 institutions from around the world and included both captive (n = 9) and free-ranging (n = 9) animals. When the clinical history was available (n = 9), animals consistently demonstrated acute dullness 1 to 5 days prior to death. In 3 of these animals (33%), there was a history of chronic gastrointestinal illness. The pathological findings were similar to those described in other animal species and humans, and consisted of intestinal volvulus and a well-demarcated segment of distended, congested, and edematous intestine with gas and bloody fluid contents. Associated lesions included congested and edematous mesentery and mesenteric lymph nodes, and often serofibrinous or hemorrhagic abdominal effusion. The volvulus involved the cranial part of the intestines in 85% (11 of 13). Potential predisposing causes were recognized in most cases (13 of 18, 72%) but were variable. Further studies investigating predisposing factors are necessary to help prevent occurrence and enhance early clinical diagnosis and management of the condition.

Infections with Philometra sp. associated with mortalities in wild-hatched captive-raised striped bass, Morone saxatilis (Walbaum).

The striped bass, Morone saxatilis (Walbaum), once represented an important resource for fisheries in the St Lawrence River (Quebec, Canada). A restoration programme, involving captive propagation, was implemented with the objective of restocking the population, which had disappeared in the late 1960s. An unusually high rate of mortality was observed during the winter of 2006 in captive-raised fingerlings that were originally collected from the Miramichi River (New Brunswick, Canada) the previous summer. Post-mortem examinations revealed extensive granulomatous and hyperplastic peritonitis associated with numerous nematodes of the genus Philometra. Given the severity of the lesions, high intensity of infection by Philometra sp. was presumed to be the primary factor in the unusual mortalities reported that winter. Observations suggest that this nematode, which was acquired in the wild, cannot establish itself in a captive environment, most likely because of the absence of the obligate intermediate host. Examination of archived specimens of striped bass showed that this parasite was probably present in the St Lawrence River population prior to its extirpation. Consequently, the introduction of infected fish into this ecosystem should not be a concern. Nevertheless, infection-related mortalities of fingerlings might affect dynamics of wild striped bass populations.

Comparative pathology of nocardiosis in marine mammals.

Nocardia spp. infections in mammals cause pyogranulomatous lesions in a variety of organs, most typically the lung. Members of the Nocardia asteroides complex are the most frequently recognized pathogens. Nine cases of nocardiosis in free-ranging pinnipeds and 10 cases of nocardiosis in cetaceans were evaluated. Host species included the hooded seal (Cystophora cristata, n = 8), leopard seal (Hydrurga leptonyx, n = 1), Atlantic bottlenose dolphin (Tursiops truncatus, n = 4), beluga whale (Delphinapterus leucas, n = 4), and killer whale (Orcinus orca, n = 2). The most common presentation of nocardiosis in both pinnipeds and cetaceans was the systemic form, involving 2 or more organs. Organs most frequently affected were lung and thoracic lymph nodes in 7 of 9 cases in pinnipeds and 8 of 10 cases in cetaceans. Molecular identification and bacterial isolation demonstrated a variety of pathogenic species. N. asteroides, N. farcinica, N. brasiliensis, and N. otitisdiscaviarum are pathogenic for pinnipeds. In cetaceans N. asteroides, N. farcinica, N. brasiliensis, N. cyriacigeorgica, and N. levis are pathogenic. Hematoxylin and eosin and acid fast staining failed to reveal bacteria in every case, whereas modified acid fast and Grocott's methenamine silver consistently demonstrated the characteristic organisms. In both pinnipeds and cetaceans, juvenile animals were affected more often than adults. Hooded seals demonstrated more cases of nocardiosis than other pinnipeds.

ACTuDB, a new database for the integrated analysis of array-CGH and clinical data for tumors.

In recent years, an increasing number of projects have investigated tumor genome structure, using microarray-based techniques like array comparative genomic hybridization (array-CGH) or single nucleotide polymorphism (SNP) arrays. The forthcoming studies have to integrate these former results and compare their findings to the existing sets of copy number data for validation. These sets also form the basis from which many comparative retrospective analyses can be carried out. Nevertheless, exploitation of this mass of data relies on a homogeneous preparation of copy number data, which will make it possible to compare them together, and their integration into a unified bioinformatics environment with ad hoc analysis tools and interfaces. To our knowledge, no such data integration has been proposed yet. Therefore the biologists and clinicians involved in cancer research urgently need such an integrative tool, which motivated us to undertake the construction of a database for array-CGH and other DNA copy number data for tumors (ACTuDB). When available, the associated clinical, transcriptome and loss of heterozygosity data were also integrated into ACTuDB. ACTuDB contains currently about 1500 genomic profiles for tumors and cell lines for the bladder, brain, breast, colon, liver, lymphoma, neuroblastoma, mouth and pancreas, together with data for replication timing experiments. The CGH array data were processed, using ad hoc algorithms (probe mapping, breakpoint detection, gain or loss status assignment and visualization) developed at Institut Curie. The database is available from http://bioinfo.curie.fr/actudb/ and can be browsed with a user-friendly interface. This database will be a useful resource for the genomic profiling of tumors, a field of highly active research. We invite research groups involved in tumor genome profiling to submit their data to ACTuDB.

Renal tubular-cell neoplasms in black-footed ferrets (Mustela nigripes)--38 cases.

Thirty-eight cases of renal tubular cell neoplasms were diagnosed in 184 captive, adult (>1-year-old), black-footed ferrets (Mustela nigripes) examined from 1985 to 1996. This prevalence (20.7%) is one of the highest reported for this neoplasm in a population of animals. These tumors rarely metastasized (1/38), and usually were incidental postmortem findings, associated clinical disease being present in only 3 (8%) of the 38 cases. The prevalence of renal tubular cell neoplasms found at postmortem examination increased linearly with age, up to 67% in ferrets >8 years old. Both males (prevalence = 19%) and females (prevalence = 24%) were affected. Multiple renal tumors were common, and seven ferrets (18.4% of affected animals) had bilateral tumors. The cause of this neoplastic syndrome could not be determined. Since most of the animals affected by this condition were in their postreproductive years of life, the impact of this neoplastic syndrome on the captive propagation of this species is negligible.

Pharmacodynamics of flunixin and ketoprofen in mallard ducks (Anas platyrhynchos).

Flunixin (FLX) and ketoprofen (KET) are potent nonsteroidal anti-inflammatory drugs (NSAIDs) used to alleviate pain and decrease inflammation. These drugs block access of arachidonic acid to its binding site on the cyclooxygenase enzyme, thus preventing conversion to thromboxane A2 and subsequent degradation to thromboxane B2 (TBX). Consequently, plasma TBX may be used to estimate duration of NSAID action. Sixteen adult mallard ducks (Anas platyrhynchos) were randomly assigned to three treatment groups: control (n = 4), FLX 5 mg/kg (n = 6), or KET 5 mg/kg (n = 6). Blood samples were taken 1 hr prior to and just before (0 hr) injection and 0.25, 0.5, 1, 2, 4, 6, 12, 24, 36, and 48 hr after injection. Plasma samples were analyzed for corticosterone and TBX. The feces were tested for the presence of hemoglobin and the ducks were euthanized for complete necropsy at the end of the study. Samples of muscle, kidney, liver, proventriculus, and intestine were taken for histologic analysis. Thromboxane was suppressed significantly in all birds following administration of either FLX or KET for 4 hr and decreased for approximately 12 hr compared with baseline samples (-1 and 0 hr). In the control group, TBX gradually declined over time. None of the ducks showed evidence of gastrointestinal bleeding, but the FLX group had muscle necrosis present at injection sites. FLX and KET likely exert pharmacological effects for at least 12 h. Although degree of TBX inhibition cannot be correlated absolutely with degree of analgesia or anti-inflammatory effects, it is possible that these effects are present during this time. This work suggests that FLX and KET can potentially be used as anti-inflammatory and analgesic agents in waterfowl. However, because of muscle necrosis at the injection site, we do not recommend parenteral use of FLX in ducks.

Evaluation of a human immunometric assay for the determination of thyroid-stimulating hormone in nonhuman primates.

Sera from nine species of clinically healthy nonhuman primates were assayed for T4 and thyroid-stimulating hormone (TSH) using human immunoassays (a fluorescence polarization immunoassay for T4, a microparticle enzyme immunoassay for TSH). The T4 levels ranged from 20 to 132 nmol/L (x +/- SD = 62.8 +/- 24.7 nmol/L). Levels of TSH were detected only in western lowland gorillas (Gorilla gorilla gorilla) and Sumatran orangutans (Pongo pygmaeus abelii) (range, 0.4-10.8 mIU/L; detectable limit = 0.01 mIU/L). The results suggest that the antibodies used in the commercial TSH immunoassay assessed in this study cross-react with gorilla and orang-utan TSH but not with TSH of primates of the genera Macaca, Papio, Erythrocebus, Ateles, Leontopithecus, and Lemur.

Respiratory nematodiases in raptors in Quebec.

This is a retrospective study on wild raptors submitted to the Université de Montréal (Quebec, Canada) from 1989 to 1996. Cyathostoma spp. (Nematoda: Syngamidae) adults and/or eggs were found in air sacs, lungs, bronchi, and trachea of 12 raptors (Falconiformes and Strigiformes) from Quebec, Canada, belonging to eight different species, five of which are first host records for this parasite: barred owl (Strix varia), snowy owl (Nyctea scandiaca), northern harrier (Circus cyaneus), northern goshawk (Accipiter gentilis), and broad-winged hawk (Buteo platypterus). The infection was considered fatal in four birds, while no significant clinical signs were observed in the other cases. Major pathologic changes included diffuse pyogranulomatous air sacculitis, pneumonia, and bronchitis. A few unidentified larval nematodes embedded in a granuloma were found in the lungs of an additional Coopers' hawk (Accipiter cooperii); they were not considered clinically significant. A dead nematode, surrounded by necrotic inflammatory cells, was found in the air sac of a northern goshawk. The presence of nematodes in air sacs or lungs should be considered in wild raptors demonstrating respiratory problems.

Diagnosis of hypothyroidism in a western lowland gorilla (Gorilla gorilla gorilla) using human thyroid-stimulating hormone assay.

Primary hypothyroidism was diagnosed in a 26-yr-old female western lowland gorilla (Gorilla gorilla gorilla) on the basis of serum levels of thyroxine (T4), free T4, and thyroid-stimulating hormone (TSH) measured by human immunoassays. Compared with clinically normal gorillas, the TSH level (107 mlIU/L) was markedly elevated, and T4 (<14.0 nmol/L) and free T4 (5.0 pmol/L) levels were decreased. Thyroid hypofunction could explain the weight gain, unsettled appetite, anxious behavior, lethargy, and poor intraspecies interactions shown by this gorilla. The antibodies in the commercial immunoassay used in this study apparently cross-reacted with gorilla TSH. Supplementation with levothyroxine sodium was initiated and was followed by a marked decrease in circulating TSH and a noticeable improvement in the animal's physiologic status and activity level.

Uterine adenocarcinoma with abdominal carcinomatosis in a beluga whale.

A case of uterine adenocarcinoma is reported in a 26-yr-old, free-ranging beluga whale (Delphinapterus leucas) from the St. Lawrence estuary (Quebec, Canada). This neoplasm appeared as a segmental stenotic thickening of the left uterine horn composed of well differentiated, but disorganized and infiltrative, glandular structures surrounded by an extensive scirrhous stroma. Abdominal carcinomatosis was observed on the mesosalpinx and on the serosal aspect of the gastric compartments. This is the first report of a malignancy originating in the uterus of a cetacean.

Adrenal hyperplastic and degenerative changes in beluga whales.

Thirty stranded beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary (Quebec, Canada) population and five animals from the Hudson Bay aboriginal hunt (North-west Territories, Canada) were examined. Twenty one animals from the St. Lawrence Estuary had mild to severe adrenal lesions and four whales from the Hudson Bay population were affected by minimal adrenal changes. Cortical hyperplasia was observed in 24 adult beluga whales all from the St. Lawrence Estuary. Bilateral cortical cysts and cellular vacuolar degeneration were observed in the adrenal glands of 19 beluga whales from both populations. The cysts, filled with a cortisol-rich liquid, were present in both sexes. Beluga whales with adrenal cysts were significantly older than animals without cysts, and the severity of the lesions increased with age. Nodular hyperplasia of the medulla was observed in seven of the beluga whales, all from the St. Lawrence Estuary population. All lesions could be part of a normal aging process. The adrenocortical lesions might be due to stress or adrenocorticolytic xenobiotics, while the medullary hyperplasia might be caused by hypoxia or exposure to estrogenic xenobiotics.

Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter.

We report the cloning and sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP) from the yeast Pichia pastoris. The gene is predicted to encode a 35.4-kDa protein with significant sequence similarity to glyceraldehyde-3-phosphate dehydrogenases from other organisms. Promoter studies in P. pastoris using bacterial beta-lactamase as a reporter showed that the GAP promoter (P(GAP)) is constitutively expressed, although its strength varies depending on the carbon source used for cell growth. Expression of beta-lactamase under control of P(GAP) in glucose-grown cells was significantly higher than under control of the commonly employed alcohol oxidase 1 promoter (P(AOX1)) in methanol-grown cells. As an example of the use of P(GAP), we showed that beta-lactamase synthesized under transcriptional control of P(GAP) is correctly targeted to peroxisomes by addition of either a carboxy-terminal or an amino-terminal peroxisomal targeting signal. P(GAP) has been successfully utilized for synthesis of heterologous proteins from bacterial, yeast, insect and mammalian origins, and therefore is an attractive alternative to P(AOX1) in P. pastoris.

Hindlimb deformities (ectromelia, ectrodactyly) in free-living anurans from agricultural habitats.

High prevalences of hindlimb deformities were recorded in wild-caught green frogs (Rana clamitans), northern leopard frogs (Rana pipiens), American toads, (Bufo americanus), and bullfrogs (Rana catesbeiana) from agricultural sites exposed to pesticide runoff in the St. Lawrence River Valley of Québec, Canada, between July and September 1992 and 1993. Of 853 metamorphosing anurans examined in 14 farmland habitats, 106 (12%; range 0 to 69%) had severe degrees of ectromelia and ectrodactyly, compared to only two (0.7%; range 0 to 7.7%) of 271 in 12 control sites. However, the variation in the proportion of deformities among sites was too large to conclude that there was a significant difference between control and pesticide-exposed habitats. Clinical signs varied and were characterized by segmental hypoplasia or agenesis of affected limbs. Conspicuous abnormalities interfered with swimming and hopping, and likely constituted a survival handicap. Because of circumstances and the frequency of these malformations in nine distinct habitats, and in three different species from one of our study sites, we propose a teratogenic action of exogenous factors. Despite the fact that many biotic and abiotic agents are potentially harmful to limb development, agricultural contaminants were suspected as primary aggressors. Thus, clinical examination and frequency of deformities in anurans might be an economical screening tool to assess ecosystem health and the presence of environmental contaminants.

Myeloencephalitis associated with a viridans group Streptococcus in a colony of Japanese macaques (Macaca fuscata).

Five weaned immature Japanese macaques (Macaca fuscata), bred in captivity, showed nervous signs over a 12-month period. Hemorrhagic cerebral infarcts with vasculitis were detected in four necropsied animals. The distribution and nature of the lesions were consistent with bacterial embolism, and a Streptococcus isolate, biochemically similar to S. salivarius, was recovered from the cerebral lesions from three of the four necropsied macaques. Treatment with antibacterial agents (enrofloxacin) improved the clinical condition of the surviving affected animal. These observations strongly suggest that this Streptococcus spp., member of the viridans group, is responsible for this outbreak. Dental pulpitis, present in two of the four macaques, probably served as the entry for this bacterium.

A single temperature amplification technique applied to the detection of citrus tristeza viral RNA in plant nucleic acid extracts.

A procedure for the successful detection of citrus tristeza virus (CTV) RNA in total crude nucleic acid extracts of infected citrus whole leaves and bark is described. The method requires the isolation and precipitation of total nucleic acids from either infected whole leaf or bark tissue. The CTV viral RNA is then specifically amplified using a single temperature RNA Self-Sustained Sequence Replication technique (3SR) performed at 42 degrees C for 60 minutes. The amplified negative-sense viral RNA product can subsequently be detected by fixing a portion of the reaction mixture onto a nylon membrane and hybridizing with positive sense tristeza specific DNA oligonucleotide probes. Central California isolates of CTV were readily detected by this method. Denatured viral specific dsRNA was also a suitable template for the specific detection of CTV.

Thymocyte reconstitution of athymic and athymic-asplenic mice: graft rejection and antibody synthesis.

Hereditarily athymic and athymic-asplenic mice were reconstituted with syngeneic thymocytes at various times prior to and after tumor challenge. These mice were able to reject heterotransplanted human tumors in as little as 19 days after thymocyte transfer but were unable to do so when the thymocytes were given 7 days after tumor implantation. The humoral immunity of athymic nude mice was fully restored as determined by the primary immune response to sheep erythrocytes (SRBC), a thymocyte-dependent antigen. Athymic-asplenic mice failed to respond to SRBC after reconstitution and this was attributed to the absence of the spleen in these mice.