Point-of-care platelet function testing results in a dog with Bernard-Soulier syndrome.

Bernard-Soulier syndrome (BSS), also known as hemorrhagiparous thrombocytic dystrophy (OMIA 002207-9615), is a rare defect in platelet function recognized in both dogs and humans. It is caused by a deficiency in glycoprotein 1b-IX-V, the platelet surface protein which acts as a receptor for the von Willebrand factor. The characteristic features of BSS in humans and dogs include macrothrombocytes and mild-to-moderate thrombocytopenia with a bleeding tendency. This condition has previously been reported in European Cocker Spaniel dogs; however, the results of platelet function tests in these animals have not been reported. This case report describes a European Cocker Spaniel dog with spontaneously occurring Bernard-Soulier syndrome and the results of point-of-care platelet function tests, including a prolonged buccal mucosal bleeding time (>8 min), prolongation (>300 s) of PFA-200 COL/ADP, COL/EPI, and P2Y closure times, and reduced aggregation (15%-48%) with Plateletworks ADP, but with normal aggregation (92%) with Plateletworks AA. This is the first description of the results of platelet function tests in canine Bernard-Soulier syndrome.

Compartment 1 acidosis associated with hemorrhagic diathesis causing mortalities among racing camels in Oman.

Camels are adapted to digestion of dry rough forages for their nutrition, and sudden change to highly digestible feed during the racing season causes digestive disorders. The current study investigated the cause of death among racing dromedary camels within 3-7 days following a sudden onset of fever ≈ 41 °C, colic with tarry feces, and enlarged superficial lymph nodes. Marked leukopenia, low RBC count and thrombocytopenia, deranged liver and renal function tests, and prolonged coagulation profiles were reported. Compartment 1 fluid revealed a pH of 4.3-5.2 with absence or few ciliated protozoa and Gram-positive microbial flora. Widespread petechial to ecchymotic hemorrhages were observed in various organs including the gastrointestinal tract (compartment 3 and colon), lungs, and heart. Fibrin thrombi in arterioles, capillaries, venules, and medium-sized veins were observed especially in the pulmonary interstitium, submucosa of the large intestine (ascending colon), deep dermis, and renal cortex. Furthermore, widespread hemorrhages and necrosis were constant histopathological lesions in parenchymatous organs. Based on clinical signs, hematology, blood biochemistry, and gross and microscopical findings, the cases were diagnosed as compartment 1 acidosis associated with hemorrhagic diathesis and endotoxicosis. Finally, compartment 1 acidosis associated with hemorrhagic diathesis is a serious fatal disease on the Arabian Peninsula in racing dromedaries causing multi-organ dysfunction and coagulopathy and disseminated hemorrhages.

Primary Infection May Be an Underlying Factor Contributing to Lethal Hemorrhagic Disease Caused by Elephant Endotheliotropic Herpesvirus 3 in African Elephants (Loxodonta africana).

Distinct but related species of elephant endotheliotropic herpesviruses (EEHVs) circulate within Asian and African elephant populations. Primary infection with EEHVs endemic among Asian elephants can cause clinical illness and lethal EEHV hemorrhagic disease (EEHV-HD). The degree to which this occurs among African elephants has not been fully established. Recent cases of EEHV-HD caused by the EEHV3 species in African elephants housed in North American zoos has heightened concern about the susceptibility of this elephant species to EEHV-HD. In this study, we utilize the luciferase immunoprecipitation system (LIPS) to generate a serological assay specific for EEHV3 in African elephants by detecting antibodies against the EEHV3 E34 protein. The results showed that the majority of tested elephants from four separate and genetically unrelated herds, including five elephants that survived clinical illness associated with EEHV3, were positive for prior infection with EEHV3. However, African elephants who succumbed to EEHV3-HD were seronegative for EEHV3 prior to lethal infection. This supports the hypothesis that fatal EEHV-HD caused by EEHV3 is associated with primary infection rather than reactivation of latent virus. Lastly, we observed that African elephants, like Asian elephants, acquire abundant anti-EEHV antibodies prenatally and that anti-EEHV3 specific antibodies were either never detected or declined to undetectable levels in those animals that died from lethal disease following EEHV3 infection. IMPORTANCE Prior to 2019, only five cases of clinical disease from EEHV infection among African elephants had been documented. Since 2019, there have been at least seven EEHV-HD cases in North American zoos, resulting in three fatalities, all associated with EEHV3. Evidence is accumulating to suggest that EEHV-associated clinical illness and death among Asian elephants is due to primary infection and may be associated with waning anti-EEHV antibody levels in young elephants. The development of the EEHV3 serological test described in this study enabled us to confirm that similar dynamics may be contributing to EEHV-HD in African elephants. The ability to screen for EEHV immune status in African elephant calves will have a major impact on managing captive African elephant herds and will provide new tools for investigating and understanding EEHV in wild populations.

Spatial Analysis of the 2017 Outbreak of Hemorrhagic Disease and Physiographic Region in the Eastern United States.

Hemorrhagic disease (HD) is considered one of the most significant infectious diseases of white-tailed deer in North America. Investigations into environmental conditions associated with outbreaks suggest drought conditions are strongly correlated with outbreaks in some regions of the United States. However, during 2017, an HD outbreak occurred in the Eastern United States which appeared to be associated with a specific physiographic region, the Appalachian Plateau, and not drought conditions. The objective of this study was to determine if reported HD in white-tailed deer in 2017 was correlated with physiographic region. There were 456 reports of HD from 1605 counties across 26 states and 12 physiographic regions. Of the 93 HD reports confirmed by virus isolation, 76.3% (71/93) were identified as EHDV-2 and 66.2% (47/71) were from the Appalachian Plateau. A report of HD was 4.4 times more likely to occur in the Appalachian Plateau than not in 2017. Autologistic regression models suggested a statistically significant spatial dependence. The underlying factors explaining this correlation are unknown, but may be related to a variety of host, vector, or environmental factors. This unique outbreak and its implications for HD epidemiology highlight the importance for increased surveillance and reporting efforts in the future.

In vivo characterization of target cells for acute elephant endotheliotropic herpesvirus (EEHV) infection in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is a dangerous viral infectious disease in young Asian elephants. Despite hypotheses underlying pathogenesis of the disease, it is unclear which cell types the virus targets during acute or persistent infections. This study investigated the tissues and target cells permissive for EEHV infection and replication in vivo. Rabbit polyclonal antibodies against the non-structural proteins of EEHV, DNA polymerase (EEHV DNAPol), were generated and validated. These were used to examine EEHV infection and replication in various tissues of acute EEHV-HD cases and compared to an EEHV-negative control. The results indicated that viral antigens were distributed throughout the epithelia of the alimentary tract and salivary glands, endothelia and smooth muscle cells, and monocytic lineage cells of the EEHV-infected elephants. Moreover, EEHV DNAPol proteins were also found in the bone marrow cells of the EEHV1A-HD and EEHV1A/4-HD cases. This study demonstrated for the first time the target cells that favor in vivo EEHV replication during acute infection, providing a promising foundation for investigating EEHV propagation in vitro.

Bleeding diathesis in canine multiple myeloma and prognostic implications: A cohort study in 156 dogs.

Multiple myeloma (MM) is a tumor of plasma cells representing approximately 1% of all canine tumors. Clinical evident bleeding is often referred to as the main finding. The aim of the study was to evaluate the occurrence of clinical bleedings in dogs with MM and its prognostic implications compared to a population of dogs not affected by MM. Two groups of dogs (# 78 each) individually matched for breed, age and gender were considered. Group-1 (exposed) was affected by MM and group-2 (unexposed) was affected by other diseases. They were compared for bleeding and mortality at 90 days after diagnosis (relative risk, RR; attributable risk, AR). Among group-1, bleeding patients (B) were compared with non-bleeding patients (NB) in terms of mortality at 90 days (RR, AR). Incident cases of MM were 78/57,694 (0.13%). Signs of bleeding up to 30 days before the referral presentation were found in 33 (42.3%) group-1 dogs in comparison to 6 (7.7%) group-2 dogs (RR, 5.50, CI 95% 2.55-12.3, p = 0.0001; AR, 0.34, CI 95% 0.22-0.47, p = 0.0001). Epistaxis was the most frequent sign of bleeding recorded. Nineteen dogs from group-1 (24.3%) and eight from group-2 (10.2%) were non-survivors (RR = 2.37, CI 95% 1.14-5.06, p = 0.01; AR = 0.14, CI 95% 0.02-0.26, p = 0.01). Among the group-1, the B dogs, 4/33 (12.1%) were non-survivors, while 15/45 NB dogs (33.3%) were non-survivors (RR = 2.75, CI 95% 1.08-7.44, p = 0.03; AR = 0.21, CI 95% 0.20-0.38, p = 0.03). Epistaxis at diagnosis was frequent in MM dogs, and signs of bleeding were associated with a more favorable 90-day prognosis.

Hemorrhagic diathesis and bone marrow aplasia secondary to lomustine overdose in a dog.

A 9-year-old mixed breed 13 kg spayed female dog was presented for evaluation of two masses in the right abdominal mammary gland region. Surgery was conducted to excise the masses. A grade I complex mammary gland carcinoma and high grade (grade III) mast cell tumor with an inguinal lymph node metastasis were diagnosed. Forty-seven days after the surgical procedure, the mast cell tumor relapsed, and neoadjuvant treatment with lomustine (81 mg/m2 ) was prescribed. Thirteen days from initiation of lomustine therapy, the dog was re-presented to the hospital with bloody diarrhea, hematemesis, epistaxis, an elevated rectal temperature, depression, severe dehydration, and marked dyspnea. The CBC showed severe thrombocytopenia and leukopenia. According to the owner, lomustine (45mg per os [PO]) was mistakenly administered daily for 10 consecutive days (total dose, 810 mg/m2 ). The dog died and a necropsy was performed. The main gross lesions consisted of severe multifocal hemorrhages in multiple organs, especially in the digestive system. Histopathologic evaluation revealed disseminated hemorrhages, as well as marked bone marrow aplasia. This report describes the clinical, hematologic, gross, and histologic findings in a fatal case of lomustine overdose in a dog.

Experimental infection of Japanese macaques with simian retrovirus 5.

Recently, a large number of Japanese macaques (Macaca fuscata) died of an unknown hemorrhagic syndrome at Kyoto University Primate Research Institute (KUPRI) and an external breeding facility for National Institute for Physiological Sciences (NIPS). We previously reported that the hemorrhagic syndrome of Japanese macaques at KUPRI was caused by infection with simian retrovirus 4 (SRV-4); however, the cause of similar diseases that occurred at the external breeding facility for NIPS was still unknown. In this study, we isolated SRV-5 from Japanese macaques exhibiting thrombocytopenia and then constructed an infectious molecular clone of the SRV-5 isolate. When the SRV-5 isolate was inoculated into two Japanese macaques, severe thrombocytopenia was induced in one of two macaques within 22 days after inoculation. Similarly, the clone-derived virus was inoculated into the other two Japanese macaques, and one of two macaques developed severe thrombocytopenia within 22 days. On the other hand, the remaining two of four macaques survived as asymptomatic carriers even after administering an immunosuppressive agent, dexamethasone. As determined by real-time PCR, SRV-5 infected a variety of tissues in Japanese macaques, especially in digestive and lymph organs. We also identified the SRV-5 receptor as ASCT2, a neutral amino acid transporter in Japanese macaques. Taken together, we conclude that the causative agent of hemorrhagic syndrome occurred at the external breeding facility for NIPS was SRV-5.

Extended genotypic evaluation and comparison of twenty-two cases of lethal EEHV1 hemorrhagic disease in wild and captive Asian elephants in India.

Thirteen new lethal cases of acute hemorrhagic disease (HD) with typical histopathogical features were identified in young Asian elephants (Elephas maximus indicus) in India between 2013 and 2017. Eight occurred amongst free-ranging wild herds, with three more in camp-raised orphans and two in captive-born calves. All were confirmed to have high levels of Elephant Endotheliotropic Herpesvirus type 1A (EEHV1A) DNA detected within gross pathological lesions from necropsy tissue by multi-locus PCR DNA sequencing. The strains involved were all significantly different from one another and from nine previously described cases from Southern India (which included one example of EEHV1B). Overall, eight selected dispersed PCR loci totaling up to 6.1-kb in size were analyzed for most of the 22 cases, with extensive subtype clustering data being obtained at four hypervariable gene loci. In addition to the previously identified U48(gH-TK) and U51(vGPCR1) gene loci, these included two newly identified E5(vGPCR5) and E54(vOX2-1) loci mapping far outside of the classic EEHV1A versus EEHV1B subtype chimeric domains and towards the novel end segments of the genome that had not been evaluated previously. The high levels of genetic divergence and mosaic scrambling observed between adjacent loci match closely to the overall range of divergence found within 45 analyzed North American and European cases, but include some common relatively unique polymorphic features and preferred subtypes that appear to distinguish most but not all Indian strains from both those in Thailand and those outside range countries. Furthermore, more than half of the Indian cases studied here involved calves living within wild herds, whereas nearly all other cases identified in Asia so far represent rescued camp orphans or captive-born calves.

Existence of bovine neonatal pancytopenia before the year 2005? Retrospective evaluation of 215 cases of haemorrhagic diathesis in cattle.

Haemorrhagic diathesis (HD) in cattle is a relatively rare syndrome that can have many different causes. With the occurrence of bovine neonatal pancytopenia (BNP) in 2007, the number of cases of HD in cattle has increased. This led to an enhanced interest in diseases presenting with bleeding disorders. The possible causes of HD in cattle, the clinical findings, and the course of various diseases are described and evaluated. Furthermore, we determined whether cases of BNP occurred before the introduction of the vaccine Pregsure BVD since its widespread use was associated with the syndrome. Records of 215 cases of HD in cattle that had been referred to the Clinic for Ruminants with Ambulatory and Herd Health Services at the Centre for Clinical Veterinary Medicine, Ludwig Maximilian University, Munich, between 1982 and 2014 were evaluated. The two most commonly diagnosed diseases were BNP (n = 95) and septicaemia (n = 35), with fatality rates of 82% and 66%, respectively. In 27 (13%) cases, no clear cause for the HD could be designated. Statistically significant differences were found with regard to the course of the various disorders and the clinical findings. A receiver operating characteristic analysis of thrombocyte counts of affected animals at the time of arrival at the clinic did not provide any predictive information on disease outcome. Two cases of HD occurred before the introduction of Pregsure BVD (1989, 1991). In both cases, clinical, haematological, and pathological findings were identical to BNP. The cause of HD in these two cases could not be determined retrospectively.

Genome Sequence of Canine Herpesvirus.

Canine herpesvirus is a widespread alphaherpesvirus that causes a fatal haemorrhagic disease of neonatal puppies. We have used high-throughput methods to determine the genome sequences of three viral strains (0194, V777 and V1154) isolated in the United Kingdom between 1985 and 2000. The sequences are very closely related to each other. The canine herpesvirus genome is estimated to be 125 kbp in size and consists of a unique long sequence (97.5 kbp) and a unique short sequence (7.7 kbp) that are each flanked by terminal and internal inverted repeats (38 bp and 10.0 kbp, respectively). The overall nucleotide composition is 31.6% G+C, which is the lowest among the completely sequenced alphaherpesviruses. The genome contains 76 open reading frames predicted to encode functional proteins, all of which have counterparts in other alphaherpesviruses. The availability of the sequences will facilitate future research on the diagnosis and treatment of canine herpesvirus-associated disease.

Review of Elephant Endotheliotropic Herpesviruses and Acute Hemorrhagic Disease.

More than 100 young captive and wild Asian elephants are known to have died from a rapid-onset, acute hemorrhagic disease caused primarily by multiple distinct strains of two closely related chimeric variants of a novel herpesvirus species designated elephant endotheliotropic herpesvirus (EEHV1A and EEHV1B). These and two other species of Probosciviruses (EEHV4 and EEHV5) are evidently ancient and likely nearly ubiquitous asymptomatic infections of adult Asian elephants worldwide that are occasionally shed in trunk wash secretions. Although only a handful of similar cases have been observed in African elephants, they also have proved to harbor their own multiple and distinct species of Probosciviruses-EEHV2, EEHV3, EEHV6, and EEHV7-found in lung and skin nodules or saliva. For reasons that are not yet understood, approximately 20% of Asian elephant calves appear to be susceptible to the disease when primary infections are not controlled by normal innate cellular and humoral immune responses. Sensitive specific polymerase chain reaction (PCR) DNA blood tests have been developed, routine monitoring has been established, the complete large DNA genomes of each of the four Asian EEHV species have now been sequenced, and PCR gene subtyping has provided unambiguous evidence that this is a sporadic rather than epidemic disease that it is not being spread among zoos or other elephant housing facilities. Nevertheless, researchers have not yet been able to propagate EEHV in cell culture, determine whether or not human antiherpesvirus drugs are effective inhibitors, or develop serology assays that can distinguish between antibodies against the multiple different EEHV species.

Hemorrhagic diathesis in avian species following intramuscular administration of polysulfated glycosaminoglycan.

Polysulfated glycosaminoglycans (PSGAGs) have been used for decades in a variety of species for the management of osteoarthritic pain. However, reports on the use of PSGAGs in avian species are scarce. In domestic cats and dogs, PSGAG injections have caused prolongation of clotting times but are considered to be an efficacious drug with a wide margin of safety. This publication documents four cases of fatal coagulopathies in different avian species (one coraciiforme, two raptors, and one psittacine) following the intramuscular administration of PSGAG. All affected birds received varying dosages and dosing intervals of PSGAG. Three of the four birds experienced fatal hemorrhage into the pectoral muscle, while the fourth bled continuously from the injection site. Only one bird had chronic, severe pre-existing disease; the remainder were being managed for osteoarthritis. This report highlights the importance of species-specific dosing of PSGAG and warrants further investigation into the etiopathogenesis of this process.

Haematological analysis of calves with bovine neonatal pancytopenia.

Blood was obtained from 61 neonatal Holstein calves originating from a farm in Germany with a high incidence of bovine neonatal pancytopenia (BNP). In order to detect alterations that might be related to BNP, selected haematological analytes were determined. Haematological examinations demonstrated alterations in at least two of the three cell lineages in 10 calves (16.39 per cent). Six animals (9.84 per cent) developed a bleeding disorder indicative of BNP at approximately two weeks of age. None of these animals showed alterations in complete blood cell count at sampling in the first week of life. In weeks when calves with BNP were born, an increase in the number of apparently healthy calves demonstrating decreases in blood cell counts was observed.

Haemorrhagic diathesis in a ram with Anaplasma phagocytophilum infection.

A 10-month-old ram with fever, inappetence and haemorrhagic diathesis had petechiae and ecchymoses at various body sites and was infested by ticks. Haematological examination revealed pancytopenia, while serum biochemistry indicated hepatic dysfunction. Blood smears were negative for Ehrlichia spp. and other haemoparasites. Paired sera revealed infection by Anaplasma phagocytophilum, but testing by polymerase chain reaction was negative. Treatment with oxytetracycline was effective. This is the first reported clinical case of ovine anaplasmosis in Greece caused by A. phagocytophilum.

Effect of desmopressin on immune-mediated haemorrhagic disorders due to canine monocytic ehrlichiosis: a preliminary study.

To evaluate the possible use of desmopressin acetate (DDAVP) in haemorrhagic disorders consequent to canine monocytic ehrlichiosis (CME), three dogs infected by Ehrlichia canis, with a history of thrombocytopenia and recent bleeding, were studied. The dogs were administered desmopressin (1 µg/kg b.w. s.c.) every 24 h on three occasions. Blood samples were collected immediately before, and after 2 and 48 h the first DDAVP administration, to assess haematological, clinical chemistry and clotting time parameters. Spontaneous bleeding stopped within 1 h after the first DDAVP injection. Buccal mucosa bleeding time (BMBT) was shortened from 9.6 to 2.3 min within 2 h after the treatment. A statistically significant increase in platelet PLT count and fibrinogen, and a statistically significant decrease of PT and aPTT were observed after DDAVP administration. The haemorrhagic disorders caused by CME appear to be quickly corrected by DDAVP administration, giving the clinician the time necessary to administer appropriate chemotherapy.

Haemorrhagic diathesis in neonatal calves: an emerging syndrome in Europe.

In 2008 and 2009 a large number of cases of haemorrhagic diathesis (HD) in neonatal calves were reported in different European countries. In Flanders, 84 cases of neonatal HD in 30 herds were reported in this period. The disease typically affects calves younger than 1 month old from different breed and gender. Prominent clinical signs are cutaneous bleeding, petechiae on all mucosae, melena and often high fever. Early in the disease, the mental state of the animals is uncompromised. The typical haematological finding is pancytopenia, with severe to complete thrombocytopenia being the cause of the increased susceptibility to bleeding. In seven of the affected herds blood samples of calves of the same age group as the clinical case were collected and on six of those farms at least one subclinical case could be identified. Necropsy findings were generalized petechiae, ecchymoses or haemorrhages and variable lymphadenopathy. Histopathology of haemorrhagic lesions revealed multifocal extravasation of red blood cells (haemorrhage) with preservation of tissue architecture and absence of other abnormalities. Total bone marrow aplasia and depletion of all lymphoid tissue was the most prominent finding on histology. Activated macrophages and haemophagocytosis were seen on bone marrow cytology from two live calves. Polymerase chain reaction for bovine viral diarrhoea virus, bluetongue and epizootic haemorrhagic disease virus was negative. Several attempts to isolate a viral agent were unsuccessful.