Erythropoiesis and erythrocytic survival in dogs with cyclic hematopoiesis.

Erythrocytic survival and ferrokinetic studies were carried out in dogs with cyclic hematopoiesis. The serum iron values varied during different phases of the cycle. The highest values occurred when the bone marrow predominantly contained granulocytic cells. The erythrocytic survival and the remainder of the ferrokinetic variables were comparable to those in normal dogs of a similar age.     

Reproducible cloning assays for in vitro growth of canine hematopoietic progenitor cells and their potential applications in investigative hematotoxicity

A variety of in vitro cloning assays have been used for studying hematopoiesis in mice and human beings. However, these techniques have had limited use in dogs, a species used extensively as a model for hematopoietic research, particularly hematotoxicity. We have adopted cloning assays for in vitro growth of canine colony-forming unit-erythroid (CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitor cells, using modified microplasma clot and soft agar culture systems, respectively. Marrow mononuclear cells separated by density-gradient centrifugation were added to the aforementioned culture systems. Erythroid colonies were stimulated with sheep plasma erythropoietin and incubated at 37 C in 5% CO2 for 2 days. The CFU-E colonies were fixed with 5% glutaraldehyde, stained with benzidine, counted, and expressed as a mean of 8 replicates. The CFU-GM colonies were stimulated with pooled serum from endotoxin-treated dogs and incubated for 8 days at 37 C in 10% CO2. Using an inverted microscope, the CFU-GM colonies were counted and expressed as a mean of 6 replicates. The number of colonies was proportional to the plated cell concentrations. The addition of 10% autologous serum to CFU-GM cultures increased the number of colonies by 80 to 100%, but markedly reduced the size and number of CFU-E colonies. The marrow cloning capacity among dogs of comparable age was similar, and little variation was noticed when bone marrow cells from the same dogs were cultured repeatedly over a period of 3 to 4 months. We concluded that these cloning assays are fast, reliable, and reproducible and that they allow quantitative determination of canine hematopoietic progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of platelet-bound antibodies in beagle dogs after artificial infection with Ehrlichia canis

Six dogs were infected with Ehrlichia canis by intravenous injection of heavily infected DH82 cells. All dogs developed typical signs of canine monocytic ehrlichiosis. Using flow cytometric technology, platelet-bound IgG (PBIgG) were detected in 5 of the 6 dogs after experimental infection with E. canis over a period of 3-10 days post infection (PI). The first detection of PBIgG was made as early as day 3 PI in 2 out of 6 dogs, and on day 5 PI in 1 dog. On day 7 PI, PBIgG was detected in 2 dogs, and on day 10 PI in 3 out of 6 dogs. This is the first report documenting the presence of PBIgG following E. canis infection in dogs. This finding further supports the theory that the thrombocytopenia seen in canine monocytic ehrlichiosis has an immunological component and that exposure to an infectious agent, in this case the rickettsia E. canis, can trigger autoimmune mechanisms. Due to the heterogeneous appearance of PBIgG among the infected dogs it was concluded that other non-immunological mechanisms are probably also involved in the pathogenesis of the thrombocytopenia seen in canine monocytic ehrlichiosis.     

Long-term bone marrow culture systems: normal and cyclic hematopoietic dogs.

A continuous long-term liquid culture in both a micro and macro system that incorporates bone marrow cells from normal and cyclic hematopoietic dogs is described. An adherent layer composed of fibroblasts, endothelial cells, mononuclear phagocytic cells, and fat-containing cells is essential for continuous hematopoiesis. Hematopoiesis was measured by the recovery of the nonadherent cells and the generation of committed granulocyte-monocyte progenitor cells for a period of seven weeks. Optimum growth factors include the use of horse serum, fetal bovine serum, dog serum, hydrocortisone, a 33 degrees C incubation temperature and feeding twice a week. As is true for both human and murine marrow liquid cultures, horse serum and hydrocortisone are essential for development and maintenance of fat-containing cells in the described systems. Both factors are important in hematopoiesis but their respective roles have not been defined. Normal and cyclic hematopoietic dogs bone marrow cells are comparable in their ability to establish long-term cultures. The micro-method (Linbro-well culture) gave similar results in maintaining hematopoiesis as did a macromethod (flask culture).     

MRI CHARACTERISTICS AND HISTOLOGY OF BONE MARROW LESIONS IN DOGS WITH EXPERIMENTALLY INDUCED OSTEOARTHRITIS

Signal changes within the bone marrow adjacent to osteoarthritic joints are commonly seen on magnetic resonance (MR) images in humans and in dogs. The histological nature of these lesions is poorly known. In this study, we describe the MR imaging of bone marrow lesions adjacent to the stifle joints of dogs with experimental osteoarthritis over 13 months. Histology of the proximal tibia at the end of the study was compared with the last MR imaging findings. In five adult dogs, the left cranial cruciate ligament was transected. Post-operatively, MR imaging was performed at 1, 2, 3, 4, 6, 8, and 13 months. Dogs were euthanised after 13 months and histological specimen of the proximal tibia were evaluated. Bone marrow edema like MR imaging signal changes were seen in every MR examination of all dogs in one or more locations of the proximal tibia and the distal femur. Lesions varied in size and location throughout the whole study with the exception of constantly seen lesions in the epiphyseal and metaphyseal region at the level of the tibial eminence. On histology, hematopoiesis and myxomatous transformation of the bone marrow and/or intertrabecular fibrosis without signs of bone marrow edema were consistent findings in the areas corresponding to the MR imaging signal changes. We conclude that within the bone marrow, zones of increased signal intensity on fat suppressed MR images do not necessarily represent edema but can be due to cellular infiltration. Contrary to humans, hematopoiesis is seen in bone marrow edema-like lesions in this canine model of osteoarthritis.     

Anemia of Chronic Renal Failure in Dogs

Seventeen dogs with chronic renal failure (CRF) were studied to evaluate the incidence, type, and etiology of anemia in CRF. A nonregenerative, normochromic, normocytic anemia was seen in 12 of 17 dogs (70.6%). There was a direct correlation between the degree of anemia and the extent of CRF as assessed by serum creatinine concentrations (P = .0386, r = .50923). Erythrocyte concentrations of 2,3-diphosphoglycerate (DPG) were significantly increased in anemic animals and showed a close correlation to the degree of anemia. The high DPG concentrations may compensate for the anemia by decreasing the hemoglobin-oxygen affinity and thereby facilitating tissue oxygenation at low hematocrits. Serum concentrations of erythropoietin (Epo) were in the low to normal range, despite mild to moderate anemia, documenting a deficiency of Epo in dogs with CRF. The nonregenerative nature of the anemia supports impaired hematopoiesis as a significant etiologic factor. Other factors, such as increases in serum parathyroid hormone and phosphorus, were not found to correlate significantly with the degree of anemia, although there were significant differences between their concentrations in anemic compared with non-anemic dogs. There was no change in erythrocyte osmotic fragility with uremia. The documentation of a nonregenerative, normochromic, normocytic anemia, with failure of an appropriate increase in Epo production, supports the therapeutic use of Epo in the management of the anemia seen in CRF in the dog.     

Cyclic hematopoiesis in a colony of dogs.

A laboratory-maintained colony of dogs heterozygous for cyclic hematopoiesis (ch) has been established. The colony consists of Collies and Collie-Beagle mixed dogs. Matings between dogs heterozygous for ch resulted in 110 pups, 28 of which were homozygous for ch. Each of the 28 homozygous ch pups (15 females and 13 males) was gray and white. Ninetten of them survived the preweaning period and were found to have cyclic fluctuations of neutrophils, platelets, and reticulocytes.     

Clinical and pathological findings in dogs following supralethal total body irradiation with and without infusion of autologous long-term marrow culture cells.

We developed a canine model for autologous bone marrow transplantation (AuBMT) with long-term marrow culture (LTMC) cells. Marrow was harvested from nine normal dogs. Harvests from dogs 2-7 were placed into 21 day LTMC. Cells in LTMC from dogs 4-7 were labelled with the neomycin phosphotransferase gene neo. Dogs were given 60Co total body irradiation (TBI) and then infused with LTMC cells: dog 1 received 500 cGy TBI and 2.08 x 10(8)/kg uncultured marrow cells. Dogs 2-7 received 600-800 cGy TBI and 0.07-0.45 x 10(8)/kg LTMC cells. Dogs 8 and 9 received 600 and 800 cGy TBI, respectively, but no infusion of marrow or LTMC cells. For all dogs, profound myelosuppression developed during week 1 and pyrexia developed during week 2. Enrofloxacin was given from one day before TBI until a peripheral neutrophil count > 1.0 x 10(9)/L was achieved, which eliminated Escherichia coli from feces. Dogs 1, 2 and 5-9 also received gentamicin and/or combination beta-lactam antibiotics. Numerous platelet transfusions were needed to control hemorrhages in all dogs except dog 1. Dog 1 achieved neutrophils > 1.0 x 10(9)/L on day 15, while dogs 2 and 5-9 achieved this count on days 33-48. Dogs 3 and 4 died on days 17 and 18, respectively, of beta-hemolytic streptococcal sepsis and hemorrhage, with no evidence of hematopoiesis at necropsy. The marker gene, neo, was documented in lymphoid and myeloid cells of dogs 5-7 up to 21 months post-AuBMT. Our studies indicate that dogs can recover following supralethal TBI and can survive the delayed engraftment associated with AuBMT using LTMC cells, if they receive intensive platelet and antimicrobial therapy. Used prophylactically for such therapy, enrofloxacin achieved selective intestinal decontamination, but did not prevent sepsis when used as the sole antimicrobial agent during myelosuppression. Furthermore, our studies indicate that infused LTMC cells, at the above doses, can contribute to hematopoietic recovery, but are not essential for recovery following TBI, and do not shorten the period of prolonged profound myelosuppression induced by TBI.     

Targeted sequencing of candidate gene regions for myelofibrosis in dogs

BackgroundMyelofibrosis often lacks an identifiable cause in dogs. In humans, most primary myelofibrosis cases develop secondary to driver mutations in JAK2, CALR, or MPL.ObjectivesTo determine the prevalence of variants in JAK2, CALR, or MPL candidate regions in dogs with myelofibrosis and in healthy dogs.AnimalsTwenty‐six dogs with myelofibrosis that underwent bone marrow biopsy between 2010 and 2018 and 25 control dogs matched for age, sex, and breed.MethodsCross‐sectional study. Amplicon sequencing of JAK2 exons 12 and 14, CALR exon 9, and MPL exon 10 was performed on formalin‐fixed, decalcified, paraffin‐embedded bone marrow (myelofibrosis) or peripheral blood (control) DNA. Somatic variants were categorized as likely‐benign or possibly‐pathogenic based on predicted impact on protein function. Within the myelofibrosis group, hematologic variables and survival were compared by variant status (none, likely‐benign only, and ≥1 possibly‐pathogenic). The effect of age on variant count was analyzed using linear regression.ResultsEighteen of 26 (69%) myelofibrosis cases had somatic variants, including 9 classified as possibly‐pathogenic. No somatic variants were detected in controls. Within the myelofibrosis group, hematologic variables and survival did not differ by variant status. The number of somatic variants per myelofibrosis case increased with age (estimate, 0.69; SE, 0.29; P = .03).Conclusions and Clinical ImportanceSomatic variants might initiate or perpetuate myelofibrosis in dogs. Our findings suggest the occurrence of clonal hematopoiesis in dogs, with increasing incidence with age, as observed in humans.     

Effects of amitraz on nerve conduction and neuromuscular transmission in anaesthetised dogs

Ataxia is an occasional side effect of amitraz when used as a wash to treat dogs with demodectic mange. In the present study, successive doses of 0.5, 2, 5 and 10 mg kg-1 amitraz were given intravenously at intervals of nine minutes to thiopentone/methoxyflurane/oxygen anaesthetised dogs. The amplitude of the evoked muscle action potential to electrical stimulation of the right ulnar nerve and the muscle refractory period were unchanged by increasing doses of amitraz but there was a progressive and significant decrease in nerve conduction velocity. The minimum recorded nerve conduction velocity (50.7 +/- 1.5 m s-1) was still within an adequate range. From these results it appears that the ataxia following amitraz is unlikely to be attributable to peripheral mechanisms. The concurrent amitraz-induced rise in mean arterial pressure and bradycardia was consistent with previous findings in which alpha 2-adrenoceptors were shown to be the major mediators.     

钴的动物营养学功能

钴作为动物体内一种必需的微量元素,虽然动物对其需要量并不大,但钴对动物的造血机能、免疫机能、生长发育及繁殖,特别是对反刍动物瘤胃微生物合成VB12等起着重要的作用,钴与其他微量元素合用会有更加明显的效果。钴缺乏会引起动物生产性能的下降,易给畜牧业带来很大经济损失,因此,随着科学研究的深入,钴的重要营养作用也逐渐被发现并受到人们的重视。就钴的生物学作用、钴的营养学作用机制、钴缺乏的发病机制、与其他营养素之间的关系及其在动物营养中的重要作用进行了阐述。     

Increased HAS2-driven hyaluronic acid synthesis in shar-pei dogs with hereditary cutaneous hyaluronosis (mucinosis)

The Chinese shar-pei dog is known for its distinctive feature of wrinkled and thickened skin, defined as primary or hereditary cutaneous mucinosis. In a recent report, we identified the mucinous material deposited in the shar-pei skin as the polysaccharide hyaluronan (HA). In the present work, the molecular and cellular mechanisms underlying this phenotype have been identified in dermal fibroblasts isolated from shar-pei dogs. The production of HA, which appeared to be mainly associated with cell membrane protrusions and also intracellular, was higher in shar-pei fibroblasts than in control cells. The HA accumulation is related to a higher mRNA expression of the isoform HAS2 of the HA-synthesizing enzyme family, hyaluronan synthases (HAS). The higher expression of HAS2 in shar-pei fibroblasts was confirmed at the protein level. The other HAS isoenzymes, HAS1 and HAS3, and the HA-degrading enzymes, Hyal1 and Hyal2, were not differentially expressed in shar-pei fibroblasts compared with cells from control dogs. Fibroblasts from shar-pei dogs and from control dogs are morphologically different as observed by transmission electron microscopy. Scanning electron microscopy revealed a large number of cellular protrusions with associated globular deposits. Electron microscopy after labelling with biotinylated HA-binding protein confirmed an increased HA content in shar-pei fibroblasts, which could be localized in several subcellular structures. The authors propose the name hereditary cutaneous hyaluronosis (HCH) for affected dogs, because it better defines the cutaneous mucinosis of shar-pei dogs.     

Identification of anti-idiotypic antibody in dogs

There is general agreement that idiotype/anti-idiotype (id/anti-id) networks are important mechanisms of immune regulation, based primarily on studies conducted using inbred laboratory animals. To determine if anti-idiotypic antibody (anti-id) could be induced during an immune response in outbred dogs, the dogs were immunized to the hapten-carrier combination dinitrophenol-ascaris (DNP-ASC) and subsequently immunized with autologous antibody in complete Freund's adjuvant (CFA). Auto-anti-id was detected in three of five dogs during the DNP-ASC response. A cross-reactive anti-id was detected in dogs immunized with autologous antibody when a mouse monoclonal antibody was used as the id. These experiments further suggest that the regulation of the immune response via network interaction, as first illucidated in inbred animals, may occur in dogs.     

Altered spontaneous and histamine-induced in vitro suppressor-cell function in dogs with atopic dermatitis.

Spontaneous, histamine-induced and Concanavalin A (Con A)-induced suppression of Con A mitogenesis of autologous responder cells was studied in normal dogs and in dogs with atopic dermatitis. Histamine-induced suppression was significantly decreased in the atopic dogs, as was the Con A-induced suppression, at supraoptimal concentration of Con A, to a lesser extent. Total numbers of histamine type 1 or type 2 receptors were not different for cells from atopic or normal dogs. The spontaneous suppression was significantly greater for the atopic dogs and this was not accounted for by the effect of non-specific dermatitis, increased macrophage-induced suppression or increased induction by mitogenic factors in the culture medium. Some possible mechanisms for these results are discussed, and the similarities to suppressor cell function in humans with atopic disease are noted.     

Bone marrow contamination of canine cerebrospinal fluid

Bone marrow cells were identified in cytocentrifuge preparations of cerebrospinal fluid (CSF) obtained by lumbar punctures from two dogs. CSF analyses were characterized by normal or increased total cell counts, normal or increased total protein concentration and the presence of erythrocytes. Hematopoietic cells present included both erythroid and myeloid precursors and, in one case, an erythroblastic island was seen. Peripheral blood smear examination confirmed that the hematopoietic cells observed were not the result of blood contamination. Neither case was associated with a difficult tap procedure, nor with a specific disease process. Contamination may have resulted from actual marrow penetration or from extramedullary hematopoiesis in the dura mater. Accurate interpretation of CSF cytology requires the consideration of possible bone marrow contamination when hematopoietic cells are observed.     

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.     

Efficacy of intrasinusal administration of bifonazole cream alone or in combination with enilconazole irrigation in canine sino-nasal aspergillosis: 17 cases

This study evaluated the effect of 1% bifonazole cream in the treatment of canine sino-nasal aspergillosis (SNA). The cream was instilled through perendoscopically placed catheters into the frontal sinuses and was used either as single therapy after debridement (DC) or as adjunctive therapy after 2% enilconazole infusion (DEC). Twelve dogs were treated initially with DEC: 7 and 3 of these dogs were free of disease after 1 and 2 procedures, respectively, while 2 dogs were cured after DC was used as a second procedure. Five dogs were treated with DC only: in 3 dogs with moderate disease, cure was obtained after a single procedure while, in 2 debilitated patients, cure could not be confirmed. Topical administration of 1% bifonazole cream appears as an effective therapy in SNA, either as an adjunctive therapy to enilconazole infusion or as sole therapy in moderately affected patients.