Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris.

BACKGROUND: Species of Plasmodium (Haemosporida, Plasmodiidae) are remarkably diverse haemoparasites. Information on genetic diversity of avian malaria pathogens has been accumulating rapidly, however exo-erythrocytic development of these organisms remains insufficiently addressed. This is unfortunate because, contrary to Plasmodium species parasitizing mammals, the avian malaria parasites undergo several cycles of exo-erythrocytic development, often resulting in damage of various organs. Insufficient knowledge on the exo-erythrocytic development in most described Plasmodium species precludes the understanding of mechanisms of virulence during avian malaria. This study extends information on the exo-erythrocytic development of bird malaria parasites. METHODS: A roadkill fieldfare (Turdus pilaris) was sampled in Switzerland and examined using pathologic, cytologic, histologic, molecular and microbiologic methods. Avian malaria was diagnosed, and erythrocytic and exo-erythrocytic stages of the parasite were identified using morphologic characteristics and barcode DNA sequences of the cytochrome b gene. The species-specific characteristics were described, illustrated, and pathologic changes were reported. RESULTS: An infection with Plasmodium matutinum lineage pLINN1 was detected. Parasitaemia was relatively low (0.3%), with all erythrocytic stages (trophozoites, meronts and gametocytes) present in blood films. Most growing erythrocytic meronts were markedly vacuolated, which is a species-specific feature of this parasite's development. Phanerozoites at different stages of maturation were seen in leukocytes, macrophages, and capillary endothelial cells in most organs examined; they were particularly numerous in the brain. Like the erythrocytic meronts, growing phanerozoites were markedly vacuolated. Conspicuous exo-erythrocytic development and maturation in leucocytes suggests that this fieldfare was not adapted to the infection and the parasite was capable to escape from cellular immunity. CONCLUSIONS: This is the first report of exo-erythrocytic development of the malaria parasite lineage pLINN1 during single infection and the first report of this lineage in the fieldfare. The findings of multiple phanerozoites in brain, skeletal muscle, and eye tissue in combination with signs of vascular blockage and thrombus formation strongly suggest an impaired vision and neuromuscular responsiveness as cause of the unexpected collision with a slowly moving car. Further studies on exo-erythrocytic stages of haemosporidian parasites are pivotal to understand the true level of populational damage of avian malaria in wild birds.

Left shift and toxic change in heterophils and neutrophils of non-mammalian vertebrates: A comparative review, image atlas, and practical considerations.

Heterophils and neutrophils are important first cellular responders to inflammatory conditions. In addition to quantitative shifts in the numbers of these cells in blood, inflammatory disease states often have accompanying increases in immature precursor stages (left shift) and/or evidence of toxic change on blood film evaluation. Recognition of left shift and toxic change morphologies is a salient diagnostic finding with clinical relevance across species. The objectives of this report are to (a) review heterophil and neutrophil function and structure across the vertebrate animal kingdom, (b) compare morphologic features of left shift and toxic change in heterophils and neutrophils of non-mammalian vertebrates (NMV) to mammals, (c) provide an image guide demonstrating the breadth of morphologic diversity of heterophil and neutrophil lineages in health and disease across taxa, and (d) discuss practical considerations for clinical pathologists and other professionals involved in the recognition and interpretation of observations in the inflammatory leukogram of NMV.

Health monitoring in birds using bio-loggers and whole blood transcriptomics.

Monitoring and early detection of emerging infectious diseases in wild animals is of crucial global importance, yet reliable ways to measure immune status and responses are lacking for animals in the wild. Here we assess the usefulness of bio-loggers for detecting disease outbreaks in free-living birds and confirm detailed responses using leukocyte composition and large-scale transcriptomics. We simulated natural infections by viral and bacterial pathogens in captive mallards (Anas platyrhynchos), an important natural vector for avian influenza virus. We show that body temperature, heart rate and leukocyte composition change reliably during an acute phase immune response. Using genome-wide gene expression profiling of whole blood across time points we confirm that immunostimulants activate pathogen-specific gene regulatory networks. By reporting immune response related changes in physiological and behavioural traits that can be studied in free-ranging populations, we provide baseline information with importance to the global monitoring of zoonotic diseases.

Haemoproteus minutus is highly virulent for Australasian and South American parrots.

BACKGROUND: Haemoproteus and Plasmodium species are widespread avian blood parasites. Several Plasmodium species are known for their high virulence and have caused significant declines in naïve bird populations. The impact of closely related Haemoproteus parasites is largely unknown. Recently we reported a lethal disease in two parrot aviaries caused by Haemoproteus parasites. RESULTS: Here we show that the causative pathogen Haemoproteus minutus is responsible for further 17 lethal outbreaks in parrot aviaries in Denmark, Germany and Great Britain. All affected parrots are endemic to Australasia and South America. We sequenced the cytochrome b gene from megalomeront-infected muscle tissue of 21 parrots and identified the two lineages TUPHI01 and TURDUS2 as causative agents, commonly naturally infecting the common blackbird (Turdus merula) and the song thrush (Turdus philomelos), respectively, in the Palaearctic. No intraerythrocytic parasite stages were found in any of the parrots. We failed to detect H. minutus in invasive Indian ring-necked parakeets (Psittacula krameri) in Germany. Together this suggests that abortive infections with two virulent lineages of H. minutus are lethal for naïve parrot species from Australasia and South America. We asked whether we could detect H. minutus in New Zealand, where its Turdus hosts were introduced in the 1800s. We therefore tested invasive blackbirds and song thrushes, and the co-existing endemic red-fronted parakeet (Cyanoramphus novaezelandiae) population on three New Zealand islands. No Haemoproteus spp. DNA was detected in all blood samples, indicating absence of transmission. CONCLUSIONS: The results of this study show that captive parrots in Europe are threatened by two lineages of an otherwise benign parasite of Turdus spp. Aviary collections of parrots should be protected from Culicoides spp. vectors in Europe. Animal trade and climate changes extending the current vector and parasite distribution have to be considered as potential risk factors for the introduction of the disease in naïve parrot populations.

CLINICAL SIGNS, DIAGNOSIS, AND TREATMENT OF LEAD INTOXICATION IN AN ELECTRIC EEL ( ELECTROPHORUS ELECTRICUS).

An adult, wild-caught electric eel ( Electrophorus electricus), weighing 18 kg and measuring 2 m in length, presented with bilateral swellings behind the pectoral fins, lethargy, and anorexia for 2 days. Anesthesia was performed with immersion in tricaine methanesulphonate and supplemented with 0.11 mg/kg medetomidine and 2.2 mg/kg ketamine intramuscularly. Endoscopy revealed blood in the oral and gastric cavity. The stomach was grossly enlarged, flaccid, and contained a lead wire which was removed manually. Blood lead values were severely elevated. The fish was treated with 28 mg/kg calcium disodium ethylenediaminetetraacetate intramuscularly every 72 hr for 5 doses, which resulted in an improved clinical condition. Because lead values had not decreased to normal values within 4 wk of initial presentation, 35 mg/kg dimercaptosuccinic acid was given orally twice weekly for 3 wk. The electric eel made a full recovery.

New Haemoproteus parasite of parrots, with remarks on the virulence of haemoproteids in naive avian hosts.

Haemoproteus infections can cause fatal disease in parrots (Psittaciformes), one of the most endangered groups of birds. The great diversity of parrots in tropical and subtropical ecosystems has been markedly understudied in terms of their parasite diversity. Only two psittacine Haemoproteus species have been described. Here we report a new Haemoproteus parasite, H. (Parahaemoproteus) homohandai n. sp. (lineage hARCHL01) found in erythrocytes of a Red-and-green macaw Ara chloropterus. We morphologically and genetically characterize the parasite based on a segment of the mitochondrial cytochrome b gene, which can be used for identification and diagnosis of infection. This is the first Haemoproteus species described from South American parrots and the first genetically characterized psittacine Haemoproteus sp. Haemoproteus homohandai n. sp. can be readily distinguished from other haemoproteids by its growing circumnuclear and close to circumnuclear macrogametocytes, which are strictly associated with erythrocyte nuclei, but do not touch the erythrocyte envelope along their entire margin and do not fill erythrocytes up to their poles. Illustrations of growing and mature gametocytes of the new species are given, and a phylogenetic analysis identifies the position of this parasite lineage in relation to other Haemoproteus parasites. Importantly, H. homohandai n. sp. and all other Haemoproteus lineages reported from parrots cluster with species of the subgenus Parahaemoproteus, indicating the transmission by Culicoides biting midges.

PLASMA BIOCHEMISTRY AND HEMATOLOGY REFERENCE VALUES OF CAPTIVE PANTHER CHAMELEONS (FURCIFER PARDALIS) WITH SPECIAL EMPHASIS ON SEASONALITY AND GENDER DIFFERENCES.

Blood samples of 86 captive panther chameleons (Furcifer pardalis) were collected in January and August from the ventral coccygeal vein in order to establish reference intervals of clinical healthy individuals under similar husbandry conditions for plasma biochemistry and hematology for this species. Significant differences were found in phosphorus, glucose, total protein, albumin, and white blood cell count between males and females in both seasons. Calcium, alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase varied in only one season between genders. Significant differences between summer and winter values were present in both genders for uric acid, calcium, phosphorus, glucose, total protein, creatine kinase, and albumin. Additionally, females showed seasonal variations for alanine aminotransferase and aspartate aminotransferase whereas packed cell volume varied in males. Gravid females had significantly higher body weights and increased values for uric acid, calcium, phosphorus, alanine aminotransferase, total protein, and albumin. Cytomorphologic characteristics of blood cells in stained blood films were evaluated to serve as additional parameters for hematology.

Vertebral Osteomyelitis and Septic Arthritis Associated With Staphylococcus hyicus in a Juvenile Peregrine Falcon ( Falco peregrinus ).

A 6-week-old, parent-reared peregrine falcon ( Falco peregrinus ) was presented with spastic hypertonus of its hind limbs of unknown origin and duration. Radiologic examination revealed smooth periosteal reactions ventrally at thoracic vertebrae 5 to 7. Contrast-enhanced computed tomography identified the swelling as inflammation; antibiotic, antimycotic, anti-inflammatory, and analgesic treatments were initiated, and vitamins and minerals were supplemented. Because the bird's condition did not improve after 10 days, it was euthanatized and submitted for postmortem examination. On histopathologic examination, chronic, active osteomyelitis was diagnosed in thoracic vertebrae 5 to 7, and chronic, active arthritis was present in both the right shoulder and left elbow joints. Staphylococcus hyicus was isolated from these 3 locations, as well as from lungs and liver, indicating a chronic septic staphylococcosis. Although infections with Staphylococcus species are occasional causes of vertebral osteomyelitis in juvenile poultry with active growth plates, it is only sporadically reported in raptors and companion birds. This case report is the first description of the clinical features and diagnostic and pathologic findings in a juvenile peregrine falcon with hematogenous osteomyelitis and arthritis associated with septicemia caused by S hyicus.

Analysis of heterophil to lymphocyte ratios in laying hens kept in a small group housing system.

The objective of the present investigation was to assess the level of stress imposed on two different layer lines kept in a small group housing system Eurovent German with two group sizes and three tiers. A total of 615 Lohmann Selected Leghorn (LSL) and 633 Lohmann Brown (LB) hens were examined in four consecutive trials. Based on differential white blood cell counts, the heterophil to lymphocyte ratio (H/L-ratio) was calculated as an indicator of stress. The H/L-ratios significantly differed among the two layer lines, with 2.5-fold higher H/L-ratios in LB than in LSL. No significant differences across and within layer lines could be found between the different group sizes. A significant 0.7-fold decrease of the H/L-ratio could be shown in LSL layers when the space per hen was increased from 828 to 920 cm2.

A rapid high-precision flow cytometry based technique for total white blood cell counting in chickens.

The automated analysis of total white blood cell count and white blood cell differentials is routine in research and clinical diagnosis in mammalian species. In contrast, in avian haematology these parameters are still estimated by conventional microscopic procedures due to technical difficulties associated with the morphological peculiarities of avian erythrocytes and thrombocytes. Both cell types are nucleated and fairly resistant to cell lysis, a prerequisite for automated leukocyte quantification and differentiation by commercial instruments. By using an anti-CD45 monoclonal antibody in combination with selected subset specific markers we have established a simple (no-lyse no-wash single-step one-tube) flow cytometry based technique for high precision chicken blood cell quantification. EDTA-blood samples are diluted, spiked with fluorescence beads and incubated with a mixture of fluorochrome conjugated chicken leukocyte specific antibodies. We demonstrate that total leukocyte numbers as well as thrombocyte, monocyte, T-cell, B-cell and heterophilic granulocyte numbers can be determined by flow cytometry in a single step without prior cell lysis, cell separation or cell washing steps. Importantly, we also show that blood samples can be fixed prior to cell staining which enables shipping of samples making the technology widely available. Comparison of this technique with conventional microscopy revealed superior precision. By comparing leukocyte differentials of two chicken populations and during immune system development after hatch we demonstrate that large sample numbers can be analysed within hours. This technique will help to overcome previous restrictions in immune status analysis in chickens in experimental systems, during vaccine testing and health status monitoring in chicken flocks. Advances in avian genomics should facilitate the development of appropriate tools for other avian species in the future which will make this technique broadly applicable.