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Bycatch is considered one of the most significant threats affecting cetaceans worldwide. In the North Sea, bottom-set gillnets are a specific risk for harbor porpoises ( Phocoena phocoena). Methods to estimate bycatch rates include on-board observers, remote electronic monitoring, and fishermen voluntarily reporting; none of these are systematically conducted. Additionally, necropsies of stranded animals can provide insights into bycatch occurrence and health status of individuals. There are, however, uncertainties when it comes to the assessment of bycatch in stranded animals, mainly due to the lack of diagnostic tools specific for underwater entrapment. We conducted a literature review to establish criteria that aid in the assessment of bycatch in small cetaceans, and we tested which of these criteria applied to harbor porpoises retrieved from gillnets in the Netherlands ( n = 12). Twenty-five criteria were gathered from literature. Of these, “superficial incisions,” “encircling imprints,” and “recent ingestion of prey” were observed in the vast majority of our confirmed bycatch cases. Criteria like “pulmonary edema,” “pulmonary emphysema,” and “organ congestion” were also frequently observed, although considered unspecific as an indicator of bycatch. Notably, previously mentioned criteria as “favorable health status,” “absence of disease,” or “good nutritional condition” did not apply to the majority of our bycaught porpoises. This may reflect an overall reduced fitness of harbor porpoises inhabiting the southern North Sea or a higher chance of a debilitated porpoise being bycaught, and could result in an underestimation of bycatch rates when assessing stranded animals.

Human babesiosis in Europe has been attributed to infection with Babesia divergens and, to a lesser extent, with Babesia venatorum and Babesia microti, which are all transmitted to humans through a bite of Ixodes ricinus. These Babesia species circulate in the Netherlands, but autochthonous human babesiosis cases have not been reported so far. To gain more insight into the natural sources of these Babesia species, their presence in reservoir hosts and in I. ricinus was examined. Moreover, part of the ticks were tested for co-infections with other tick borne pathogens. In a cross-sectional study, qPCR-detection was used to determine the presence of Babesia species in 4611 tissue samples from 27 mammalian species and 13 bird species. Reverse line blotting (RLB) and qPCR detection of Babesia species were used to test 25,849 questing I. ricinus. Fragments of the 18S rDNA and cytochrome c oxidase subunit I (COI) gene from PCR-positive isolates were sequenced for confirmation and species identification and species-specific PCR reactions were performed on samples with suspected mixed infections. Babesia microti was found in two widespread rodent species: Myodes glareolus and Apodemus sylvaticus, whereas B. divergens was detected in the geographically restricted Cervus elaphus and Bison bonasus, and occasionally in free-ranging Ovis aries. B. venatorum was detected in the ubiquitous Capreolus capreolus, and occasionally in free-ranging O. aries. Species-specific PCR revealed co-infections in C. capreolus and C. elaphus, resulting in higher prevalence of B. venatorum and B. divergens than disclosed by qPCR detection, followed by 18S rDNA and COI sequencing. The non-zoonotic Babesia species found were Babesia capreoli, Babesia vulpes, Babesia sp. deer clade, and badger-associated Babesia species. The infection rate of zoonotic Babesia species in questing I. ricinus ticks was higher for Babesia clade I (2.6%) than Babesia clade X (1.9%). Co-infection of B. microti with Borrelia burgdorferi sensu lato and Neoehrlichia mikurensis in questing nymphs occurred more than expected, which reflects their mutual reservoir hosts, and suggests the possibility of co-transmission of these three pathogens to humans during a tick bite. The ubiquitous spread and abundance of B. microti and B. venatorum in their reservoir hosts and questing ticks imply some level of human exposure through tick bites. The restricted distribution of the wild reservoir hosts for B. divergens and its low infection rate in ticks might contribute to the absence of reported autochthonous cases of human babesiosis in the Netherlands.

We detected infection with highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b in 2 red fox (Vulpes vulpes) cubs found in the wild with neurologic signs in the Netherlands. The virus is related to avian influenza viruses found in wild birds in the same area.

Scoliosis is a deformation of the spine that may have several known causes, but humans are the only mammal known to develop scoliosis without any obvious underlying cause. This is called ‘idiopathic’ and is the most common type. Recent observations showed that human scoliosis, regardless of its cause, has a relatively uniform three-dimensional anatomy. We hypothesize that scoliosis is a universal compensatory mechanism of the spine, independent of cause and/or species. We had the opportunity to study the rare occurrence of scoliosis in a whale (Balaenoptera acutorostrata) that stranded in July 2019 in the Netherlands. A multidisciplinary team of biologists, pathologists, veterinarians, taxidermists, radiologists and orthopaedic surgeons conducted necropsy and imaging analysis. (Dorso-)lateral blunt traumatic injury to two vertebrae caused an acute lateral deviation of the spine, which had initiated the development of compensatory curves in regions of the spine without anatomical abnormalities. Three-dimensional analysis of these compensatory curves showed strong resemblance with different types of human scoliosis, amongst which idiopathic. This suggests that any decompensation of spinal equilibrium can lead to a uniform response, regardless of underlying cause or species. The unique biomechanics of the upright human spine, with significantly decreased rotational stability, explains why only in humans this universal mechanism of scoliosis can occur without an obvious cause, and is thus still called ‘idiopathic’.

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection. © Copyright © 2020 Ruetten, Steinmetz, Thiersch, Kik, Vaughan, Altamura, Muckenthaler and Gassmann.

In a practical feeding trial at Ouwehand Zoo, plasma concentrations of vitamin A1, calcidiol (D3), α-tocopherol (E), and B1 in 17 Humboldt penguins ( Spheniscus humboldti) were measured before and after supplementation to gain insight into the effect of supplementing these vitamins in animals being fed thawed frozen-fish diets. None of the penguins received vitamin supplements for at least 6 mo before the supplementation trial, which was conducted prior to their normal nesting and molting period. During the trial period, eight penguins received daily vitamin A1, D3, tocopheryl acetate, and B1 supplementation placed in their fish immediately prior to feeding and nine control penguins received no supplementation. Concentrations of vitamins A1, D3, α-tocopherol, and B1 were also measured in the thawed ready-to-feed fish. Concentrations of vitamins B1 and α-tocopherol were below the Association of Zoos and Aquariums (AZA) recommendations for penguin diets, while concentrations of vitamins A1 and D3 were far above AZA recommendations. At the start of the study and after 70 days of supplementation, plasma concentrations were determined for these vitamins. Vitamin B1 concentrations in plasma increased significantly ( P < 0.05) between Day 0 (mean 39.9 μg/L) and day 70 (mean 160.5 μg/L) in the supplemented group. Plasma vitamin D3 and α-tocopherol did not show a significant change. Vitamin A1 levels in the supplemented group decreased significantly from 1.65 mg/L on day 0 to 1.4 mg/L on day 70. In the control group no significant changes were observed. The results of the study support the necessity of supplementing vitamin B1 in penguins fed thawed frozen fish. Depletion of vitamin A and E concentrations in frozen food fish over time support recommendations to regularly measure vitamin concentrations in different batches of frozen fish.

Members of the genus Bartonella are fastidious Gram-negative facultative intracellular bacteria that are typically transmitted by arthropod vectors. Several Bartonella spp. have been found to cause culture-negative endocarditis in humans. Here, we report the case of a 75-year old German woman with prosthetic valve endocarditis due to Bartonella washoensis. The infecting agent was characterized by sequencing of six housekeeping genes (16S rRNA, ftsZ, gltA, groEL, ribC, rpoB) applying a multilocus sequence typing (MLST) approach. The 5097 bp of the concatenated housekeeping gene sequence from the patient were 99.0% identical to a B. washoensis strain from a red squirrel (Sciurus vulgaris orientis) from China. 39% (24/62) of red squirrel (S. vulgaris) samples from the Netherlands were positive for the B. washoensis gltA gene variant detected in the patient. This suggests that the red squirrel is the reservoir host for human infection in Europe.

Ranavirus is the second most common infectious cause of amphibian mortality. These viruses affect caudates, an order in which information regarding Ranavirus pathogenesis is scarce. In the Netherlands, two strains (CMTV-NL I and III) were suspected to possess distinct pathogenicity based on field data. To investigate susceptibility and disease progression in urodeles and determine differences in pathogenicity between strains, 45 adult smooth newts (Lissotriton vulgaris) were challenged via bath exposure with these ranaviruses and their detection in organs and feces followed over time by PCR, immunohistochemistry and in situ hybridization. Ranavirus was first detected at 3 days post infection (p.i.) in the oral cavity and upper respiratory mucosa. At 6 days p.i, virus was found in connective tissues and vasculature of the gastrointestinal tract. Finally, from 9 days p.i onwards there was widespread Ranavirus disease in various organs including skin, kidneys and gonads. Higher pathogenicity of the CMTV-NL I strain was confirmed by higher correlation coefficient of experimental group and mortality of challenged animals. Ranavirus-exposed smooth newts shed virus in feces intermittently and infection was seen in the absence of lesions or clinical signs, indicating that this species can harbor subclinical infections and potentially serve as disease reservoirs.

This annual report present the results of post-mortem examinations of porpoises (Phocoena phocoena) in 2017. One of the main objectives of the research is to quantify human-induced causes of death. In 2017, 55 dead harbour porpoises were examined: 25 males and 30 females, including 22 adults, 26 juveniles and 7 neonates. Most of the harbour porpoises examined died as a result of infectious diseases (36%), bycatch (20%) or grey seal attacks (18%). The proportion of animals dying of infectious diseases was higher in 2017 than in previous years. In dit jaarrapport worden de resultaten gepresenteerd van pathologisch onderzoek aan bruinvissen (Phocoena phocoena) in 2017. Eén van de hoofddoelen van het onderzoek is het kwantificeren van sterfte van bruinvissen door menselijk toedoen. In 2017 zijn 55 dode bruinvissen onderzocht; 25 mannelijke en 30 vrouwelijke, 22 volwassen, 26 juveniel en 7 neonaat. De meeste van de onderzochte bruinvissen waren gestorven door infectieziekten (36%), bijvangst (20%) en aanvallen van grijze zeehonden (18%). De sterfte door ziekte lag in 2017 hoger dan in voorgaande jaren.

SUMMARY In a breeding group of Inca terns (Larosterna inca), 14 birds died without antemortem signs of illness. Other than a poor body condition and a bloody cloaca, no symptoms were observed. Gross necropsy revealed severe segmental hemorrhagic enteritis with intralesional trematodes in most birds. Histopathologic examination revealed infiltration of lymphocytes, plasma cells, and granulocytes in the lamina propria of the duodenum and cross-sections of trematodes in the lumen. The parasites were identified as Ichthyocotylurus erraticus, a trematode of fish-eating birds. The cause of the infestation most likely was the feeding of unfrozen fresh fish. We describe the first case of a lethal I. erraticus infestation in Inca terns.

A 2-year-old, male castrated ferret ( Mustela putorius furo ) was presented with progressive abdominal distention and loss of muscle mass despite normal appetite. Physical examination findings included pale mucous membranes, a prolonged capillary refill time, a pulse rate greater than 300 beats/min, and severe abdominal distention. Abdominal ultrasound showed free abdominal fluid and an enlarged liver with distended hepatic veins and caudal vena cava. During the echocardiographic examination, abnormalities observed included a 2-mm-diameter left-to-right shunting atrial septal defect (ASD) with concurrent severe dilatation of the right atrium and eccentric hypertrophy of the right ventricle with mild pulmonary hypertension. All other echocardiographic measurements were within normal limits. The owner declined treatment, and the ferret was euthanized. Postmortem examination confirmed the ultrasonographic findings. The free abdominal fluid (200 mL) was a non-septic fibropurulent exudate. Decompensated right-sided heart failure due to ASD and exudative peritonitis of undetermined origin were the final diagnoses. To our knowledge, this is the first report of an ASD in a ferret.

The ranaviruses are an emerging group of viruses that infect amphibians, fish, and reptiles. Although ranaviruses have not been linked to extinctions, emergence in amphibian communities has resulted in population declines for some species. We present the results of ranavirus-associated mortality in a Dutch national park in the aftermath of an outbreak associated with a common midwife toad virus (CMTV)-like ranavirus. We monitored five bodies of water across Dwingelderveld National Park, the Netherlands, in 2011-13. Dead and live amphibians were counted weekly July-September and every 2 wk in June and October. Dead amphibians were collected and tested for ranavirus infection. In addition, we measured biologic, chemical, and physical site characteristics to test for a correlation with ranavirus-associated mortality. Ranavirus infection was widespread in our study area and we observed nearly continuous presence of dead, ranavirus-infected amphibians in the presence of asymptomatic, live amphibians throughout our study. Fatalities occurred in larval, subadult, and adult amphibians. Ranavirus infection prevalence (based on fatal cases) was significantly associated with increasing fractions of adults and subadults compared to juveniles and larvae in the population, but was unrelated to any other measured site characteristics. Our findings showed that a CMTV-like ranavirus can persist long term in an ecosystem, affecting a diversity of amphibian species and life stages for a prolonged period. This study illustrates the importance of monitoring the modes of spread for ranaviruses and their impact on amphibian populations.

Lepeophtheirus acutus Heegaard, 1943 (Caligidae, Siphonostomatoida, Copepoda), was collected from or observed on four of six elasmobranch species held at Burgers Zoo (Arnhem, The Netherlands). Circumstantial evidence suggested that a zebra shark, Stegostoma fasciatum (Hermann), from the wild carried the infection into the facility, where copepods reproduced and colonized additional hosts. Copepods typically attached on and about the eyes, in the mouth and occasionally about the cloaca and on the claspers. Severe ocular lesions were associated with infections on zebra sharks, a grey reef shark, Carcharhinus amblyrhynchos (Bleeker), whitetip reef

Red-mark syndrome (RMS), a disease seen mostly in rainbow trout, Oncorhynchus mykiss, is of unknown aetiology. The research presented here indicates the presence of an intracellular bacterium in RMS-affected fish. A positive reaction was observed using immunohistochemistry (IHC) with skin lesions, liver, kidney and spleen of affected fish sampled from several locations within the United Kingdom using two different polyclonal antisera raised against Piscirickettsia salmonis. The same reaction was also seen with a number of different anti-P. salmonis monoclonal antibodies (MAbs). A disease with similar clinical signs to RMS, referred to as strawberry disease (SD), has been reported in the USA. A Rickettsia-like organism (RLO) has recently been associated with SD based on analysis of 16S rDNA sequences. Using the same panel of anti-P. salmonis antibodies used to screen the RMS samples, similar staining was obtained in tissue of SD-affected fish by IHC. A polymerase chain reaction (PCR) using RLO-specific primers was also performed on RMS-affected fish from the United Kingdom, and the samples were positive for the RLO 16S rRNA sequence. These findings suggest that the same aetiological agent may be responsible for RMS in the United Kingdom and SD in the USA.

Objective— To characterize biologic behavior, clinical outcome, and effect of histologic grade on prognosis for dogs with appendicular chondrosarcoma treated by amputation alone. Study Design— Case series. Animals— Dogs (n=25) with appendicular chondrosarcoma. Methods— Medical records were searched to identify dogs with appendicular chondrosarcoma treated by limb amputation alone. Information recorded included signalment, anatomic location, radiographic appearance, and development of metastasis. Histopathologic diagnosis was confirmed and graded (1, 2, or 3). Survival curves were generated by the Kaplan–Meier method and the association between covariates (gender, age, weight, and tumor grade) and survival were evaluated using the univariate proportional hazards model. Results— Histopathology slides were available for 25 dogs. Rates of pulmonary metastasis were as follows: grade 1–0%, grade 2–31%, and grade 3–50%. Overall median survival time (MST) was 979 days. Age, weight, and sex were not significantly associated with survival (P=.16; .33; and .31, respectively). Survival was significantly associated with tumor grade (P=.008), with dogs with tumor grade of 1, 2, and 3 having MSTs of 6, 2.7, and 0.9 years, respectively. Conclusion— Canine appendicular chondrosarcoma can be treated effectively with amputation alone. Low to intermediate grade chondrosarcoma has a good prognosis, whereas high-grade tumors appear to behave aggressively. Clinical Relevance— The overall prognosis for appendicular chondrosarcoma is better than that of appendicular osteosarcoma treated by amputation alone or in combination with chemotherapy.

Objective— To investigate mutations of the TP53 gene in canine osteosarcoma (OS). Study Design— Clinical historic cohort study. Animals— Client-owned dogs. Methods— OS (n=59) were screened for mutations of the complete TP53 gene using polymerase chain reaction and the mutation was analyzed by single-strand conformational polymorphism. Clinical outcome of dogs with TP53-mutated OS were compared with dogs with OS without a mutation after complete surgical excision of the primary tumor. Results— TP53 gene mutations were observed in 24 of 59 (40.7%) OS; 3 mutated OS had 2 mutations. The alterations consisted mainly of point mutations (74%). Dogs with mutated OS had a significantly shorter survival time (ST) after surgery than dogs with normal tumor TP53 gene expression (P=.03). Other significant prognosticators for ST and disease-free interval included elevated serum alkaline phosphatase (P

Three mixed-bred raptors (Falco rusticolus x Falco cherrug) from a German falcon breeder were presented with a history of respiratory distress. In one bird a laryngeal stridor was noted, and oral examination revealed an epiglottal swelling. In the other two birds, nasal discharge and sneezing were the main clinical symptoms. Nasal flushing samples and biopsies were collected for pathologic, bacteriologic, and parasitologic examination. Results confirmed a cryptosporidial infection. Polymerase chain reaction (PCR) and DNA analysis identified the causative agent to be Cryptosporidium baileyi. No cryptosporidia were detected in fecal samples, indicating the infection was confined to the respiratory system. Analysis of prey animals (pigeons, quail) failed to identify the source of infection. Treatment was initiated with paromomycin in all three birds, whereas in two birds an additional therapy with azithromycin was given. However, no clinical improvement was seen after several weeks of treatment, and the birds either died or were euthanatized. To the authors' knowledge, these are the first confirmed cases of disease caused by cryptosporidia in the order of Falconiformes.

DNA samples from a variety of New World monkeys were screened by using a broad-spectrum PCR targeting the VP1 gene of polyomaviruses. This resulted in the characterization of the first polyomavirus from a New World primate. This virus naturally infects squirrel monkeys (Saimiri sp.) and is provisionally named squirrel monkey polyomavirus (SquiPyV). The complete genome of SquiPyV is 5075 bp in length, and encodes the small T and large T antigens and the three structural proteins VP1, VP2 and VP3. Interestingly, the late region also encodes a putative agnoprotein, a feature that it shares with other polyomaviruses from humans, baboons and African green monkeys. Comparison with other polyomaviruses revealed limited sequence similarity to any other polyomavirus, and phylogenetic analysis of the VP1 gene confirmed its uniqueness.

A 30-yr-old blue-fronted Amazon parrot (Amazon aestiva aestiva) was presented to the clinic with a history of sneezing more often during the last 2 mo. Physical examination revealed only a mild nasal discharge. Complete hematologic and plasma biochemical examination showed no abnormalities. Computerized tomography (CT) of the complete bird showed generalized lung alterations consistent with lung fibrosis. Two lung biopsies were taken. The results of the histologic examination of the biopsies confirmed the tentative CT diagnosis of pulmonary interstitial fibrosis. To our knowledge this is the first reported case of chronic pulmonary interstitial fibrosis diagnosed by means of a lung biopsy in an avian species. The histologic characteristics are discussed and compared with those of human idiopathic pulmonary fibrosis.

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity is described. In a zoo in The Netherlands 5 adult seals died within 12 days. At necropsy all animals showed signs of acute septicaemia, consistent with acute leptospirosis. Serological examination of one animal was positive for antibodies against Leptospira interrogans serovar Icterohaemorrhagiae and the serologically closely related serovar Copenhageni. Polymerase chain reaction was positive in one other animal. 8 nutria (Myocastor coypus) were examined, serologically, through bacteriological culture and PCR. 81,8% (9/11) were serologically positive for Leptospira. The seals and nutria were housed in the same water system.

Reptile cardiology is in its infancy. Veterinarians treating reptiles should develop a basic knowledge of reptile cardiovascular anatomy and physiology. Cardiology is vital to interpreting the results of various diagnostic tests and planning an effective therapeutic plan for a case. This article will provide a review of the anatomy and physiology of the reptilian cardiovascular system, the common diagnostic tests used to assess cardiac function, and the common disease presentations associated with the cardiovascular system.

The present study describes cartilage-free areas on the ulnar trochlear notch and the humeral condyle of eight very young golden retrievers with otherwise healthy elbow joints. Remarkably, the youngest dog with full-thickness cartilage-free areas was only 8 weeks old. The younger dogs showed no macroscopic abnormalities on the locations that were affected in the older dogs. Two kinds of cartilage modifications were found. Cartilage-free areas at the edges of the articular cartilage layer were present on the humeral capitulum and on two locations of the ulna, (the medial and lateral at the base of the anconeal process, and the trochlear notch near the lateral coronoid process, which was fractured in two cases). Histological examination showed that these cartilage-free areas were filled with dense supportive tissue. Synovial cells covered this tissue as well as the surrounding hyaline cartilage. The synovial membrane covering the areas was macroscopically enlarged, but histological examination revealed no signs of inflammation. The second type of modification consisted of discoloration of the articular surface at the humeral trochlea. Histological examination revealed that in this area the articular surface was composed of fibrocartilage instead of hyaline cartilage. Apparently, there are locations within the elbow joint in which articular cartilage is not necessary for normal joint functioning. The presence of fibrocartilage on the articular surface of the humeral condyle is a surprising finding, for which no explanation has yet been found.

Malignant lymphoma is a common malignancy in birds. Paraneoplastic syndromes, which are commonly observed in domestic animals, have not been reported in association with lymphoma in birds. Hypercalcemia and hyperglobulinemia were found on plasma chemistry in two Amazon parrots, which were presented with aspecific symptoms. In both cases radiography and ultrasound demonstrated signs of hepatomegaly, which proved to be due to malignant lymphoma on postmortem examination. The hypercalcemia was found to be most consistent with a paraneoplastic effect of the malignant lymphoma in these birds. The exact origin of the hyperglobulinemia remains unclear.

To the Editor: Toxoplasma gondii, a zoonotic protozoan parasite for which felids are the only definitive hosts, can infect humans and other warm-blooded animals. Transmission usually occurs orally from oocysts shed by felids in water and on food, through tissue cysts in undercooked meat, or transplacentally. In particular, young cats shed oocysts that can sporulate and become infectious within a day, depending on temperature and humidity. Sporulated oocysts can survive in moist soil for months to years (1). In September 2014, the number of dead squirrels reported to the Dutch Wildlife Health Centre and the Dutch Mammal Society increased suddenly. The red squirrel (Sciurus vulgaris) is the only species of squirrel endemic to the Netherlands. Members of the public claimed that squirrels were “dropping dead from trees.” Subsequently, the public was encouraged to report and submit dead squirrels. A total of 187 animals were reported through October 2014, of which 37 were submitted for necropsy. Necropsy included macroscopic examination; cytologic analysis of liver, spleen, lungs, and intestinal contents stained with hemacolor (Merck, Darmstadt, Germany); and histologic examination of samples of various organs fixed in formalin, embedded in paraffin, cut into 4-μm sections, and stained with hematoxylin and eosin. For 8 adult animals, body condition (based on degree of fat storage and muscle development) was good; 12 juveniles were in poor condition. Typically, the trachea contained foam, and lungs were hyperemic and edematous. The liver was enlarged and pale, and the spleen was enlarged. In 13 animals, numerous small crescent-shaped organisms, with eccentrically placed nuclei consistent with tachyzoites of T. gondii, were identified by cytology in lung, liver, and spleen (2). Main histopathologic findings were pulmonary interstitial lymphoplasmocytic and neutrophilic infiltrates with edema and numerous intra-alveolar macrophages (17/20) and multifocal lymphoplasmocytic infiltrates with necrosis in the liver (13/20). Extensive splenic necrosis was occasionally observed (4/20). Intestines contained mild plasmacytic infiltrates. Numerous tachyzoites consistent with T. gondii were present in alveolar macrophages and epithelial cells, splenic macrophages, and hepatocytes. Duplicate slides were stained immunohistochemically by using polyclonal antibodies against T. gondii following a standard ABC protocol (3). Organisms stained for T. gondii in liver, spleen, lungs, and intestine. Toxoplasma was not detected in any brain. DNA was isolated (DNeasy Blood and Tissue Kit; QIAGEN, Hilden, Germany) from tissues of 14 squirrels and tested by quantitative PCR (1); T. gondii DNA was detected in 13. We successfully sequenced the T. gondii GRA6 gene for 11 squirrels and identified sequences to clonal type II T. gondii previously identified in sheep from the Netherlands (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU325790","term_id":"283580620","term_text":"GU325790"}}GU325790) (4). Incidental findings in the animals tested were encephalitis (2/20), coccidiosis (5/20), trauma (6/20), myocarditis (4/20), nephritis (1/20), lymphadenitis (1/20), and intestinal (3/20) and external (5/20) parasites. The remaining 17 animals showed >1 of the following pathologic conditions: hemorrhages consistent with trauma (12/17), mild to severe intestinal coccidiosis (12/17), pneumonia (3/17), splenitis (1/17), Taenia martis cysticerci (1/17), and external parasites (8/17). Immunohistochemistry results for all 17 were negative for T. gondii. On the basis of necropsy and molecular findings, we conclude that 20 of 37 examined squirrels died of disseminated T. gondii type II infection. These animals included adults and juveniles and were not restricted to specific geographic areas (Figure). The remaining animals died of trauma (12/17) or other causes (5/17). Figure Spatial distribution of wild red squirrels (Sciurus vulgaris) investigated for Toxoplasma gondii and classified by cause of death, the Netherlands, 2014. Red squirrels are susceptible to T. gondii, and infection can lead to death. However, in our sample, the proportion of squirrels that died of toxoplasmosis (>50%) was higher than in other studies (≈16%) (5–7). The apparent increase in squirrel deaths and unexpectedly high proportion of fatal T. gondii infections suggests a toxoplasmosis outbreak among red squirrels. Possible explanations for this surge in cases include increased exposure to the parasite, increased susceptibility to infection, or increased virulence of the pathogen. Clonal T. gondii type II, the strain most frequently involved in human cases and endemic to Europe and North America, was identified. An increased virulence of the pathogen could not be proven (8). On the basis of lymphoid hyperplasia in the spleen and lymph nodes, affected squirrels had no signs of immunosuppression. Thus, the most likely explanation is increased exposure to the parasite. Sources of infection for red squirrels are not known; however, oocysts shed in cat feces may contaminate the nuts, fungi, shoots, and berries that constitute the diet of the squirrel. Stray, unspayed cats are common in the Dutch countryside. More than 3 million domestic cats (Felis domesticus) exist in the Netherlands, including several tens of thousands of free-roaming cats that reproduce (9). Determining the exact source of infection is important because humans also harvest wild fruits, nuts, and fungi from these areas. This outbreak highlights that contamination of the environment with T. gondii oocysts is of concern not only from a public health viewpoint but from a biodiversity perspective as well (1,10).

Histologic grade is an important determinant in clinical outcome of human osteosarcoma (OS). In this study, the histologic characteristics of primary and metastatic canine OS were evaluated using a new classification system. Histologic characteristics were classified in 166 primary and 34 metastatic canine OS. Prognostic variables for clinical outcome were determined using multivariate analysis. Most OS were histologically characterized by severe to extreme cellular pleomorphism, a variable number of mitoses, small to moderate amounts of matrix, a high percentage of tumor cells, and minimal to moderate amounts of necrosis. Tumor invasion into vessels was present in 117/152 (71%) tumors, and 12/50 (24%) of the regional lymph nodes had evidence of metastasis. Classification of the 166 tumors resulted in seven (4%) grade I, 34 (21%) grade II, and 125 (75%) grade III OS. In the multivariate analysis, histologic grade III OS and elevated pretreatment plasma alkaline phosphatase (AP) levels were independent predictors of clinical outcome. Dogs with high-grade tumors and elevated AP should be carefully evaluated for the presence of metastatic disease before starting adjunctive therapy protocols.

In 2010, a mass die-off of over 1000 wild water frogs (Pelophylax spp.) and at least 10 common newts (Lissotriton vulgaris) occurred in a pond in The Netherlands. Haemorrhagic disease with hepatomegaly and splenomegaly was evident. Microscopically, multiple organs presented cells with multifocal intracytoplasmic inclusion bodies, in which ranavirus-like particles were demonstrated ultrastructurally. All specimens examined tested positive for ranavirus by PCR. The sequence obtained showed a 100% identity with the one deposited for common midwife toad virus (CMTV). This is the first report of ranavirus-associated mortality in wild amphibian populations in The Netherlands. It is also the first time CMTV or a CMTV-like virus has been reported in these two species in the adult stage and outside of Spain.

A Grey parrot and a Yellow-crowned Amazon were presented with severe respiratory distress and weight loss, and collapse and melena, respectively. Despite supportive therapy, both parrots quickly succumbed and died, with no ante-mortem diagnosis made. Postmortem examinations yielded a diagnosis of a follicular and papillary form of thyroid adenocarcinoma. Although thyroid neoplasia has been reported in birds, these two cases are the first described thyroid neoplasia in these parrot species and the first reported papillary form of thyroid adenocarcinoma in birds.

A 16-year-old male white cockatoo was presented with lethargy and a decreased appetite. Auscultation between the second and third sternal rib revealed a heart murmur, which was confirmed by electrocardiographic and phonocardiographic examination to be systolic, with a shift of the heart axis to -152 degrees. Radiographs showed lack of detail in the cranial part of the abdominal coelom, indicative of ascites and an enlarged cardiac shadow, while ultrasonographic examination revealed pericardial effusion and fluid accumulation in the cavitas peritonealis hepatica. An extra fluid-filled cavity was found at the atrioventricular junction in the right cardiac wall and colour Doppler examination demonstrated a turbulent jetstream of blood into the cavity, originating directly above the aortic valve. Non-selective angiocardiography confirmed the ultrasonographic observations. Findings were indicative of an aneurysm of the a. coronaria dextra (right coronary artery). This was confirmed by necropsy which revealed atherosclerosis to be the underlying cause.

Mammalian articular cartilage serves diverse functions, including shock absorption, force transmission and enabling low-friction joint motion. These challenging requirements are met by the tissue’s thickness combined with its highly specific extracellular matrix, consisting of a glycosaminoglycan-interspersed collagen fiber network that provides a unique combination of resilience and high compressive and shear resistance. It is unknown how this critical tissue deals with the challenges posed by increases in body mass. For this study, osteochondral cores were harvested post-mortem from the central sites of both medial and lateral femoral condyles of 58 different mammalian species ranging from 25 g (mouse) to 4000 kg (African elephant). Joint size and cartilage thickness were measured and biochemical composition (glycosaminoclycan, collagen and DNA content) and collagen cross-links densities were analyzed. Here, we show that cartilage thickness at the femoral condyle in the mammalian species investigated varies between 90 µm and 3000 µm and bears a negative allometric relationship to body mass, unlike the isometric scaling of the skeleton. Cellular density (as determined by DNA content) decreases with increasing body mass, but gross biochemical composition is remarkably constant. This however need not affect life-long performance of the tissue in heavier mammals, due to relatively constant static compressive stresses, the zonal organization of the tissue and additional compensation by joint congruence, posture and activity pattern of larger mammals. These findings provide insight in the scaling of articular cartilage thickness with body weight, as well as in cartilage biochemical composition and cellularity across mammalian species. They underscore the need for the use of appropriate in vivo models in translational research aiming at human applications.

Zusammenfassung Der Einflus von Phaseolus vulgaris-Isolektinen auf die Struktur und Funktion der Mucosa des Dunndarms beim Ferkel, beobachtet mit Hilfe einer sicb selbst entleerenden blinden Darmschleife Spezifisch pathogen freie Ferkel wurden operativ mit einer sich selbst entleerenden blinden Darmschleife im Jejunum versehen. Am Blindende der Darmschleife wurde eine Kanule eingesetzt. Durch die Kanule wurden Biopsien der jejunalen Mucosa entnommen. Die Biopsien wurden wahrend der Operation und dann 10 Tage post operationem in regelmasigen Abstanden 18 Tage vor und nach der Verabreichung einer einmaligen Dosis von Phaseolus vulgaris-Isolektin- E 4 und-L 4 durch die Kanule entnommen. Dann wurde die Morphologie der Mucosabiopsien licht- und elektronenmikroskopisch untersucht. Auserdem wurde die Aktivitat der Sucrase-Isomaltase bestimmt. In den ersten 18 Tagen nach der Operation konnten keine Veranderungen hinsichtlich Morphologie und Funktion der jejunalen Mucosa festgestellt werden. Nach der Verabreichung von Phaseolus vulgaris-Isolektin- E 4 fiel das Villus: Krypten-Verhaltnis ab. Die Glycocalyx war verdickt und die Microvilli zeigten sich verkurzt und waren unregelmasig angeordnet. Die Vesikel-Cluster waren von den Microvilli abgerissen. 48 h nach der Verabreichung des Isolektins waren diese Effekte noch sichtbar. Die Aktivitat der Sucrase-Isomaltase zeigte grose Schwankungen. Die Effekte nach der Verabreichung von Phaseolus vulgaris-Isolektin-L 4 waren ahnlich denen von -E 4 aber nicht so ausgepragt.

To the Editor: A Q fever epidemic among humans started in the Netherlands in 2007 and peaked in 2009 (1). Epidemiologic evidence linked the epidemic to abortions and deliveries among Coxiella burnetii–infected dairy goats and dairy sheep (1,2). However, questions arose about whether C. burnetii infection in free-living wildlife might be another source of Q fever in humans. C. burnetii has a wide host range (3), but to our knowledge no studies had addressed its occurrence in nondomestic animals in the Netherlands (4). The main objective of this study was to look for evidence of C. burnetii infection in carcasses of free-living roe deer (Capreolus capreolus) in the Netherlands, where C. capreolus is the most common species of wild ruminant. Additional objectives were to 1) analyze characteristics, location, and time of death of case-animals for more information on the infection in roe deer and 2) determine the genotype of C. burnetii strains from roe deer and compare them with the genotype of strains from domestic animals and humans for evidence of spillover. The sample consisted of 79 roe deer that were euthanized or found dead in 9 of the 12 provinces in the Netherlands during January 2008–May 2010. All animals had undergone postmortem examination, and tissue samples were frozen until testing. Tissues tested were lung (n = 46), spleen (n = 50), bone marrow (n = 50), liver (n = 74), and kidney (n = 75), as available. We extracted DNA by using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). A duplex quantitative PCR targeting the IS1111a element was used with an internal control gene, as described (2). Tissues with cycle threshold (Ct) values 1 year of age, 15 (30%) had positive results, compared with 2 (15%) of 13 deer

Objectives: To describe partial pancreatectomy using a bipolar vessel-sealing device (BVSD) and compare this novel technique to the conventional suture-fracture (SF) method for canine insulinoma. Methods: Pre-, intra- and postoperative data of eight dogs with insulinoma, which underwent resection using the BVSD (LigaSure V), were prospectively collected and compared with those of eight randomly selected case-matched patients that underwent resection using the conventional SF technique. Results: Mean surgical time was significantly (P=0·022) shorter in the BVSD (107 ±9 minutes) than in the SF (135 ±22 minutes) group. The BVSD technique was negatively associated with surgical time and duration of the hospitalisation period. Neither technique caused intraoperative complications, such as bleeding, collateral damage to adjacent tissues or problems with sealing or suturing the pancreatic tissue. Three dogs in the SF group and none in the BVSD group developed postoperative clinical signs associated with pancreatitis. Clinical Significance: BVSD is a safe and viable alternative to conventional methods of pancreatectomy for canine insulinoma. It provides the possibility to remove insulinomas in the pancreatic limbs and corpus with relative ease. BVSD pancreatectomy in dogs with insulinoma significantly decreases operative and hospitalisation times and is not associated with more clinical complications than SF pancreatectomy.