Post mortem findings and their relation to AA amyloidosis in free-ranging Herring gulls (Larus argentatus).

Since the late 1990s, high mortality and declining populations have been reported among sea birds including Herring gulls (Larus argentatus) from the Baltic Sea area in Northern Europe. Repeated BoNT type C/D botulism outbreaks have occurred, but it remains unclear whether this is the sole and primary cause of mortality. Thiamine deficiency has also been suggested as a causal or contributing factor. With this study, we aimed to investigate gross and microscopic pathology in Herring gulls from affected breeding sites in Sweden in search of contributing diseases. Herring gulls from Iceland served as controls. Necropsies and histopathology were performed on 75 birds, of which 12 showed signs of disease at the time of necropsy. Parasites of various classes and tissues were commonly observed independent of host age, e.g. oesophageal capillariosis and nematode infection in the proventriculus and gizzard with severe inflammation, air sac larid pentastomes and bursal trematodiasis in pre-fledglings. Gross and microscopic findings are described. Notably, amyloidosis was diagnosed in 93 and 33% of the adult birds from Sweden and Iceland, respectively (p<0.001), with more pronounced deposits in Swedish birds (p<0.001). Gastrointestinal deposits were observed in the walls of arteries or arterioles, and occasionally in villi near the mucosal surface. Amyloid was identified within the intestinal lumen in one severely affected gull suggesting the possibility of oral seeding and the existence of a primed state as previously described in some mammals and chickens. This could speculatively explain the high occurrence and previously reported rapid onset of amyloidosis upon inflammation or captivity in Herring gulls. Amyloid-induced malabsorbtion is also a possibility. The Herring gull SAA/AA protein sequence was shown to be highly conserved but differed at the N-terminus from other avian species.

GASTROINTESTINAL PARASITIC INFECTIONS IN DEAD OR DEBILITATED MOOSE ( ALCES ALCES) IN SWEDEN.

: Our objectives were to determine prevalence and intensity of gastrointestinal parasites of dead or euthanized emaciated moose in central and southern Sweden ( n=50) and to assess parasite intensity as a major contributing factor in the poor condition of these moose. All animals were infected and most had gastrointestinal nematodes. Seven parasite species were found in the abomasa and 10 species were found in the small intestine. Coinfections were commonly found in the abomasum ( Ostertagia antipini and Mazamastrongylus dagestanica) and in the small intestine ( Nematodirella alcidis and Trichostrongylus capricola). Moose had many nematodes; the highest numbers were 224,400 and 11,760, in the abomasum and in the small intestine, respectively. Fourteen moose had more than 40,000 gastrointestinal nematodes (excluding large intestine nematodes represented by Trichuris spp. and Oesophagostomum venulosum). Additionally, a moderate prevalence (36%) of protostrongylid larvae (dorsal spine larvae) and a low prevalence (2-4%) of protozoal infections were identified at microscopic examination of feces. Adult moose had significantly more parasites than did subadults. The results did not show parasite load as a single or a major cause of the moose mortality, but they provided an update on the species composition of helminth parasites in moose in Sweden and illustrated the extreme infection intensities that free-ranging moose can have.

Thiamine deficiency impairs common eider (Somateria mollissima) reproduction in the field.

The Baltic Sea population of the common eider (Somateria mollissima) has declined dramatically during the last two decades. Recently, widespread episodic thiamine (vitamin B1) deficiency has been demonstrated in feral birds and suggested to contribute significantly to declining populations. Here we show that the decline of the common eider population in the Baltic Sea is paralleled by high mortality of the pulli a few days after hatch, owing to thiamine deficiency and probably also thereby associated abnormal behaviour resulting in high gull predation. An experiment with artificially incubated common eider eggs collected in the field revealed that thiamine treatment of pulli had a therapeutic effect on the thiamine status of the brain and prevented death. The mortality was 53% in untreated specimens, whereas it was only 7% in thiamine treated specimens. Inability to dive was also linked to brain damage typical for thiamine deficiency. Our results demonstrate how thiamine deficiency causes a range of symptoms in the common eider pulli, as well as massive die-offs a few days after hatch, which probably are the major explanation of the recent dramatic population declines.

Widespread episodic thiamine deficiency in Northern Hemisphere wildlife.

Many wildlife populations are declining at rates higher than can be explained by known threats to biodiversity. Recently, thiamine (vitamin B1) deficiency has emerged as a possible contributing cause. Here, thiamine status was systematically investigated in three animal classes: bivalves, ray-finned fishes, and birds. Thiamine diphosphate is required as a cofactor in at least five life-sustaining enzymes that are required for basic cellular metabolism. Analysis of different phosphorylated forms of thiamine, as well as of activities and amount of holoenzyme and apoenzyme forms of thiamine-dependent enzymes, revealed episodically occurring thiamine deficiency in all three animal classes. These biochemical effects were also linked to secondary effects on growth, condition, liver size, blood chemistry and composition, histopathology, swimming behaviour and endurance, parasite infestation, and reproduction. It is unlikely that the thiamine deficiency is caused by impaired phosphorylation within the cells. Rather, the results point towards insufficient amounts of thiamine in the food. By investigating a large geographic area, by extending the focus from lethal to sublethal thiamine deficiency, and by linking biochemical alterations to secondary effects, we demonstrate that the problem of thiamine deficiency is considerably more widespread and severe than previously reported.

A study on the effect of different rifle calibres in euthanisation of grey seals (Halichoerus grypus) in seal traps in the Baltic Sea.

BACKGROUND: In recent years, the euthanasia of seals has been discussed internationally and concern has been raised regarding the use of rifles, the effect of different calibres, and which calibres are sufficient for humane euthanasia. This study therefore investigated the effect of different firearm calibres on euthanasia of grey seals (Halichoerus grypus) in traps, and provides information for the development and refinement of regulations for hunting seals in the wild. FINDINGS: The effect of different calibres was studied in 19 seals shot in the head and neck at close range. All seals were necropsied and radiographed to characterize the injuries caused by the bullets. All tested calibres, 5.6 mm bullet diameter or larger, and .12 shotgun, were sufficiently effective to cause severe skull fractures, meningeal haemorrhages and instant death. CONCLUSIONS: Rifles with 5.6 mm bullet diameter or larger, and a .12 shotgun loaded with a slug fired at close range to the head and neck of grey seals all caused instant death and can therefore be recommended for hunting seals in the wild.

Potential application of serological tests on fluids from carcasses: detection of antibodies against Toxoplasma gondii and Sarcoptes scabiei in red foxes (Vulpes vulpes).

BACKGROUND: Serological surveys for disease investigation of wild animal populations require obtaining blood samples for analysis, which has logistic, ethic and economic difficulties. Applying serological test to fluids collected from dead animals is an alternative. The aim of this study was to assess if antibodies could be detected in two types of fluids collected from 56 carcasses of red foxes (Vulpes vulpes): pleural fluid and lung extract. FINDINGS: In 22 (39%) foxes antibodies against Sarcoptes scabiei were detected in both fluid types by ELISA and Western blot. In 46 (82%) foxes, antibodies against Toxoplasma gondii were detected in pleural fluid and in 41 (73%) in lung extract applying a Toxo-screen test (DAT). Antibodies were still detectable in the same fluids kept at room temperature for 28 days, although in fewer foxes (16 and 14 foxes tested for T. gondii in lung extract and pleural fluid respectively; and 1 and 4 tested for S. scabiei in lung extract and pleural fluid respectively. CONCLUSIONS: These results indicate the potential utility of using fluids from carcasses for antibody screening of wild animals at the population level.

Orthopoxvirus DNA in Eurasian lynx, Sweden.

Cowpox virus, which has been used to protect humans against smallpox but may cause severe disease in immunocompromised persons, has reemerged in humans, domestic cats, and other animal species in Europe. Orthopoxvirus (OPV) DNA was detected in tissues (lung, kidney, spleen) in 24 (9%) of 263 free-ranging Eurasian lynx (Lynx lynx) from Sweden. Thymidine kinase gene amplicon sequences (339 bp) from 21 lynx were all identical to those from cowpox virus isolated from a person in Norway and phylogenetically closer to monkeypox virus than to vaccinia virus and isolates from 2 persons with cowpox virus in Sweden. Prevalence was higher among animals from regions with dense, rather than rural, human populations. Lynx are probably exposed to OPV through predation on small mammal reservoir species. We conclude that OPV is widely distributed in Sweden and may represent a threat to humans. Further studies are needed to verify whether this lynx OPV is cowpox virus.

Pathology of natural highly pathogenic avian influenza H5N1 infection in wild tufted ducks (Aythya fuligula).

Highly pathogenic avian influenza (HPAI) subtype H5N1 is an infectious systemic viral disease that results in high morbidity and mortality in poultry, and has been reported in a wide range of wild bird species during the last few years. An outbreak of HPAI H5N1 occurred in wild birds in Sweden in 2006 that affected several duck species, geese, swans, gulls, and raptors. Tufted ducks (Aythya fuligula) accounted for the largest number of positive cases and, therefore, were selected for more in-depth histologic and immunohistochemical evaluations. The main histologic lesions associated with the presence of avian influenza antigen were found in the brain, pancreas, and upper respiratory tract. Other tissues in which influenza antigen was variably found included liver, lung, adrenal glands, kidneys, and peripheral nerve ganglia. The current study describes the pathology and viral tissue targeting of H5N1 by using histology, polymerase chain reaction, and immunohistochemistry, and highlights the range and variation in the presentation of the natural disease in tufted ducks.

Wild birds of declining European species are dying from a thiamine deficiency syndrome.

Wild birds of several species are dying in large numbers from an idiopathic paralytic disease in the Baltic Sea area. Here, we demonstrate strong relationships between this disease, breeding failure, and thiamine (vitamin B(1)) deficiency in eggs, pulli, and full-grown individuals. Thiamine is essential for vertebrates, and its diphosphorylated form functions as a cofactor for several life sustaining enzymes, whereas the triphosphorylated form is necessary for the functioning of neuronal membranes. Paralyzed individuals were remedied by thiamine treatment. Moreover, thiamine deficiency and detrimental effects on thiamine-dependent enzymes were demonstrated in the yolk, liver, and brain. We propose that the mortality and breeding failure are part of a thiamine deficiency syndrome, which may have contributed significantly to declines in many bird populations during the last decades.

Mycotic proventriculitis in gray partridges (Perdix perdix) on two game bird farms.

Proventriculitis and chronic respiratory disease were diagnosed in two flocks of gray partridges (Perdix perdix) on unrelated Swedish game bird farms. Affected birds showed loss of condition, respiratory signs, and flock mortality rates of 50 and 98%, respectively. The proventricular lesions were associated closely with fungal organisms that were microscopically indistinguishable from the ascomycetous yeast Macrorhabdus ornithogaster (former provisional name "megabacterium"). At necropsy, the proventriculi were swollen and hyperemic, and viscous mucus adhered to the mucosa. Proventricular hemorrhages were commonly detected, and one bird had proventricular rupture and peritonitis. Microscopically, mild to severe subacute to chronic lymphoplasmacytic proventriculitis, microabscesses, necrosis, epithelial metaplasia, disrupted koilin, ulcers, and hemorrhages were observed. Transmission electron microscopy of the proventricular microorganisms revealed a membrane-bound nucleus, vacuoles, ribosomes, microtubules in parallel arrays, and a two-layered cell wall but no mitochondria. Scanning electron microscopy of the proventricular epithelium demonstrated masses of organisms with occasional constrictions in parallel arrangement. Many of the birds also suffered from concurrent respiratory bacterial infections and/or gastrointestinal candidiasis. The clinical course and gross and microscopic proventricular lesions were similar to those described in psittacine and passerine pet birds colonized by M. ornithogaster-like microorganisms but differed from published case reports and experimental infections of chickens in which the clinical signs and lesions have been considerably milder. The findings presented in this paper suggest that mycotic proventriculitis, presumably associated with M. ornithogaster, may be a serious but possibly opportunistic, although unusual, disease problem in gray partridges on game farms.

Detection of gastric Helicobacter species in free-ranging lynx (Lynx lynx) and red foxes (Vulpes vulpes) in Sweden.

Specimens of gastric mucosa and liver of 25 free-ranging Eurasian lynx (Lynx lynx), and four red foxes (Vulpes vulpes) shot in Sweden during 1999-2000, were investigated for the presence of Helicobacter species. Histopathology, bacteriologic culture and urease test, Helicobacter genus-specific 16S rDNA PCR analysis, and DNA sequence analysis were applied. Numerous Helicobacter-like organisms were observed histologically in the gastric mucosa of one fox. Helicobacter spp. were detected in the stomach by PCR analysis in 17 (68%) of the lynx and in three (75%) of the foxes. Seven of the positive lynx were also positive in the urease test. PCR fragments, amplified from lynx and foxes, were sequenced and compared with those of known Helicobacter species. PCR products from lynx were closely related (>or=98% homology) to H. heilmannii, and PCR fragments from foxes demonstrated close homology to H. heilmannii and H. salomonis. No Helicobacter spp. or Helicobacter-like organisms could be cultured. The PCR analysis of the liver was negative for all animals. The pathologic significance of the presence of Helicobacter spp. in the stomach of free-ranging lynx and foxes remains uncertain.

Identification of a novel Mannheimia granulomatis lineage from lesions in roe deer (Capreolus capreolus).

Eight atypical Mannheimia isolates were isolated from lesions in roe deer (Capreolus capreolus). Traditional classification based on morphologic and physiologic traits showed that they belong to a distinct biogroup (taxon) within genus Mannheimia. Extensive phenotypic characterization suggested that the isolates should be classified as M. granulomatis, although the presence of distinct traits justified their classification into a separate biogroup within this species. Phylogenetic analyses based on 16S rRNA sequences from two roe deer isolates and 41 other Mannheimia strains supported that the roe deer isolates form a monophyletic group within M. granulomatis. The lktA genotype was present in all roe deer isolates based on Southern blot analysis, whereas the corresponding beta-hemolytic phenotype was absent in one of these isolates.

Necrotizing encephalitis of unknown cause in Fennoscandian Arctic foxes (Alopex lagopus).

A neurologic disease affected a colony of endangered Fennoscandian arctic foxes (Alopex lagopus) kept in captivity for breeding purposes. Several outbreaks of disease occurred between 1994 and 2004. The clinical signs included ataxia, indications of anosmia, blindness, and abnormal behavior. The disease was characterized by severe necrotizing encephalitis affecting mostly the cranial cerebrum, basal ganglia, and olfactory bulbs. Investigations to identify the etiology of the disease included testing for several infectious agents known to cause encephalitis in carnivores. Tests for Toxoplasma gondii, Encephalitozoon cuniculi, Neospora caninum, canine distemper virus, rabies, adenovirus type 1, Borna disease virus, and Listeria monocytogenes were negative. The colony was closed, and the cause of the disease remains undetermined.

Evaluation of a serological test (indirect ELISA) for the diagnosis of sarcoptic mange in red foxes (Vulpes vulpes).

Sarcoptic mange occurs in many parts of the world and is common in populations of domestic and wild canids, including red foxes (Vulpes vulpes). In recent years, an indirect antibody enzyme-linked immunosorbent assay (ELISA), with higher sensitivity and specificity than traditional diagnostic methods, has been successfully applied in the diagnosis of sarcoptic mange in dogs. The same ELISA has also demonstrated specific antibodies to Sarcoptes scabiei in experimentally infected red foxes. The aim of this study was to evaluate the indirect ELISA when used to detect antibodies to S. scabiei in field sera from Swedish red foxes. One cohort of both infected and non-infected red foxes (cohort 1; n = 88), and one cohort of apparently non-infected foxes (cohort 2; n = 67) were examined for skin lesions and presence of S. scabiei by thorough visual examination at autopsy and skin scrapings. Samples of blood-tinted body liquid from the abdomen or thorax cavity were collected and analysed by the indirect ELISA. The relative sensitivity and specificity of the ELISA at different cut-offs (OD values) were estimated by comparing the test results to the infection status as determined by examination and skin scrapings. The highest combination of relative sensitivity and specificity, calculated based on cohort 1, was 95.4 and 100.0%, respectively. These estimates were constant for cut-offs 0.150-0.225, which included the cut-off based on the mean plus three standard deviations of test results from cohort 2 (0.165). It is concluded that this test can be useful in diagnosis and epidemiological studies of S. scabiei infection in red foxes.

Serological survey of Toxoplasma gondii infection in free-ranging Eurasian lynx (Lynx lynx) from Sweden.

To investigate the prevalence of Toxoplasma gondii infection in free-ranging Eurasian lynx (Lynx lynx) in Sweden, serosanguinous fluids and feces were collected from 207 carcasses of lynx killed or found dead from 1996 to 1998. Sera were tested for antibodies against T. gondii by the direct agglutination test, and 156 (75.4%) of the sera tested positive at antibody titers>or=40. Antibody prevalence was significantly lower in lynx originating from the northern parts of Sweden than in lynx from the more southern regions that are more densely populated by humans. Age-related differences also were found, with a significantly lower prevalence (55%) in juvenile (<1-yr-old) than in subadult and adult animals (82%). There was no significant difference in seroprevalence between males and females. Oocysts typical of T. gondii were not detected in any of the fecal samples.

Diseases and mortality in free-ranging brown bear (Ursus arctos), gray wolf (Canis lupus), and wolverine (Gulo gulo) in Sweden.

Ninety-eight brown bears (Ursus arctos), 20 gray wolves (Canis lupus), and 27 wolverines (Gulo gulo), all free-ranging, were submitted to the National Veterinary Institute, Uppsala, Sweden, during 1987-2001 for investigation of diseases and causes of mortality. The most common cause of natural death in brown bears was infanticide. Infanticide also was observed in wolverines but not in wolves. Traumatic injuries, originating from road or railway accidents, were the most common cause of death in wolves and occurred occasionally in brown bears. Most wolverines were submitted as forensic cases in which illegal hunting/poaching was suspected. Sarcoptic mange was observed in several wolves but not in brown bears or wolverines. Sarcoptic mange most likely was acquired from infected red foxes (Vulpes vulpes) that were killed by wolves. Other parasites and infectious diseases were only found sporadically.

Epizootiologic investigations of selected infectious disease agents in free-ranging Eurasian lynx from Sweden.

Serum samples from 106 Eurasian lynx (Lynx lynx) from across Sweden, found dead or shot by hunters in 1993-99, were investigated for presence of antibodies to feline parvovirus (FPV), feline coronavirus, feline calicivirus, feline herpesvirus, feline immunodeficiency virus, Francisella tularensis, and Anaplasma phagocytophila, and for feline leukemia virus antigen. In addition, tissue samples from 22 lynx submitted in 1999 were analyzed by real-time polymerase chain reaction (PCR) to detect nucleic acids specific for viral agents and A. phagocytophila. Except for FPV antibodies in one lynx and A. phagocytophila in four lynx, all serology was negative. All PCR results also were negative. It was concluded that free-ranging Swedish lynx do not have frequent contact with the infectious agents considered in this study.

Fallot's tetralogy in a European brown bear (Ursus arctos).

A congenital heart malformation was diagnosed in a free-ranging adult female European brown bear (Ursus arctos) found dead due to intraspecific violence. At necropsy, the heart had all four features of Fallot's tetralogy. No further malformations were identified in the animal, which was of normal size and weight for its age, in normal body condition, and had probably borne young. The heart condition was considered to have contributed to death during an attack by another bear.

Health monitoring and conservation of wildlife in Sweden and Northern Europe.

Monitoring of wildlife diseases started in Scandinavia as sporadic postmortem examinations in the early 20th century. In 1945 a monitoring program for wildlife health was initiated. The program is today an integrated part of the National Environmental Monitoring programs in Sweden. The total material today comprises more than 80000 recorded investigations. There are similar programs in Denmark, Finland, and Norway, but no comparable program exists in other Northern European countries. The program has led to discoveries of new diseases in many mammals and birds, and demonstration of several environmental pollutants, such as mercury, lead, and cadmium, in wildlife. The success of the program is due to several factors such as: a rich wildlife with economically important game species; location of the program at the National Veterinary Institute with excellent facilities and competent staff, good cooperation with hunter and conservation organizations, stable financial support, a cool climate, and a successful relationship with media. Current work also includes more focused investigations on different specific pathogens and pollutants, as well as investigations in single animal species.

Molecular identification and prevalence of Dictyocaulus spp. (Trichostrongyloidea: Dictyocaulidae) in Swedish semi-domestic and free-living cervids.

Lungs of 102 roe deer (Capreolus capreolus), 136 moose (Alces alces), 68 fallow deer (Dama dama), and six red deer (Cervus elaphus) were examined during hunting seasons from 16 September 1997 to 1 March 2000. The aim was to determine the species composition and prevalence of Dictyocaulus lungworms in these hosts in Sweden. Worms were identified following polymerase chain reaction (PCR) amplification of the internal transcribed spacer of ribosomal DNA (ITS2), followed by hybridization with four species-specific oligonucleotides. In addition, 50 lungworms from five reindeer (Rangifer tarandus) from Norway were similarly analyzed. A total of 399 worms were recovered and analyzed representing a range of 29-128 worms per host species. All specimens from roe deer were identified as Dictyocaulus capreolus, whereas those from red deer and reindeer were identical with D. eckerti. From moose, 73 (81.1%) of the worms were identified as D. capreolus whereas 17 (18.9%) were D. eckerti. The ITS2 sequence of fallow deer lungworms differed significantly when compared with the ITS2 of D. viviparus, D. capreolus, and D. eckerti. This indicated that fallow deer in Sweden may be infected with a new genotype of Dictyocaulus spp. Consequently, a specific probe designed for the ITS2 from this Dictyocaulus sp. hybridized exclusively with samples from lungworms of fallow deer. Interestingly, no D. viviparus were found in any of these hosts. The prevalence of infection in each host was as follows: D. capreolus in roe deer (14.7%) and moose (10.6%); D. eckerti in moose (0.7%) and red deer (33.3%); and Dictyocaulus sp. in fallow deer (10.3%). Regardless of lungworm species, the overall prevalence of Dictyocaulus spp. in these hosts was 12.2%. Prevalence between male and female animals and among the different age groups did not differ significantly. Finally an enzyme linked immunosorbent assay (ELISA) specific for patent D. viviparus infection in cattle was utilized to analyze lung tissue fluids from infected animals. All samples from roe deer, red deer, and fallow deer were negative in the ELISA. However, three out of twelve (25%) samples from moose and 17 of 40 (43%) samples from cattle were positive. This indicated that moose anti-D. capreolus antibodies recognized the D. viviparus antigen and that anti-cattle immunoglobulin cross-reacted with moose antibodies.