A new method for rapid detection of the mutant allele for Chediak-Higashi syndrome in Japanese black cattle.

Chediak-Higashi syndrome (CHS) is an autosomal recessive hereditary disorder in Japanese Black cattle, caused by a mutation of the Lyst gene. So far, the mutation has been detected by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. However, this method is disadvantaged by its low-throughput performance. Here, we report an alternative method involving real-time PCR with TaqMan minor groove binder probes, which shortens the total assay time by more than 120 min, analyzing 10 samples in a duplicated manner. Using this method, we examined 102 Japanese Black cattle and found that 8.8% of the cattle were CHS-carriers. These data indicate that our technique is useful for routine diagnostic testing for CHS in Japanese Black cattle.

Alteration of release and role of adenosine diphosphate and thromboxane A2 during collagen-induced aggregation of platelets from cattle with Chediak-Higashi syndrome.

OBJECTIVE: To compare the interaction of endogenous ADP with collagen and thromboxane A(2) (TXA(2)) during collagen-induced platelet aggregation between platelets from healthy cattle and those with Chediak-Higashi syndrome (CHS). POPULATION SAMPLE: Platelets harvested from blood samples from healthy Japanese Black cattle and those with CHS. PROCEDURES: Aggregation of gel-filtered platelets; release of ATP-ADP; and generation of thromboxane B(2) (TXB(2)), a metabolite of TXA(2), were measured. RESULTS: The potency of collagen to induce aggregation in platelets of cattle with CHS (ie, CHS platelets) was less than a tenth of that in platelets of healthy cattle (ie, control platelets). Platelet aggregation induced by collagen at an intermediate concentration depended on the coexistence of ADP and TXA(2), suggesting that released ADP cannot cause platelet aggregation by itself. Collagen-induced ADP release was markedly decreased, whereas TXB(2) production was slightly low in CHS platelets, compared with that in control platelets. A combination of subthreshold amounts of ADP and 9,11-dideoxy-9alpha, 11alpha-methano-epoxy-prostaglandin F(2) (U46619), a TXA(2) analogue, caused platelet aggregation. Similarly, a combination of subthreshold amounts of collagen and ADP caused platelet aggregation, whereas collagen and U46619 were not synergistic. CONCLUSIONS AND CLINICAL RELEVANCE: Deficient ADP release ensuing from the delta-storage pool deficiency in platelets from cattle with CHS resulted in reduction of collagen-induced platelet aggregation, through attenuation of synergism between TXA(2) and ADP and between ADP and collagen. Furthermore, results of the study reported here indicated that TXA(2) was important for aggregation of bovine platelets.

Radiographic evaluation of destructive periodontal disease in blue mink in relation to age and blood morphology.

In this study, blood samples and jaws were collected from 2 genotypes of blue mink (n = 289) in order to examine phenotypic expression of specific characteristics of Chediak-Higashi Syndrome (C-HS). Blood samples were subjected to differential counts to assess the proportion of abnormal polymorphonuclear leukocytes characteristic for CH-S (C-HS-leukocytes). Abnormal leukocytes with characteristic signs of C-HS were found in blood smears from all mink included in this study. Four teeth in one half of the mandible (P3, P4, M1, M2) were subjected to quantitative radiographic evaluation of alveolar bone loss and tooth loss. There was a high prevalence of destructive periodontal disease among blue mink included in this study. Mild to moderate periodontal disease (defined by less than 50% alveolar bone loss related to 1 or more teeth) affected 73.7% of young mink (age = 7 mo) and 67.9% of older animals (age > or = 19 mo). Severe periodontal disease (defined by more than 50% bone loss related to one or more teeth) was not detected in mink aged 7 mo, but affected 15.3% of mink aged 19 mo and 39.6% of mink aged 31 mo. The positive relationship between age and periodontal disease was statistically significant (P < 0.01). The prevalence of tooth loss was found to be high among blue mink aged > 19 mo (21.6%) and was also significantly related to age (P < 0.01). A significant positive interaction between alveolar bone loss and tooth loss (P < 0.01), implies that the highly prevalent tooth loss in the mink was related to and possibly caused by destructive periodontal disease. There was no significant difference in the prevalence of periodontal disease between the 2 genotypes and age was found to be the only statistical predictor of poor production results (P < 0.01) in blue mink.

Characterization of spontaneous dermatitis in beige rats with IgE hyperproduction.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease characterized by chronic recurrent eczematous lesions, but its exact etiology and mechanism are unclear. We found that beige rats (DAbg/bg), a mutant model of Chediak-Higashi syndrome, develop skin lesions characterized by pruritus, excoriation, erosion and alopecia. We describe the beige rat and examine its possible usefulness as an AD model. METHODS: Beige rats of 4, 8, 13, 16, 26 and 52 weeks were used. Histological analysis of the skin was performed. Plasma IgE and cytokines were measured. Th1 and Th2 cytokines and RANTES mRNA expression of skin and lymph nodes were evaluated. Passive cutaneous anaphylaxis (PCA) reactions were examined, and maximization tests were conducted. RESULTS: Skin lesions begin to develop with increases in serum IgE levels and the expression of IL-4 mRNA in the lymph node and skin. Histologically, skin lesions are characterized by acanthosis, ulceration and inflammatory cell infiltration in the dermis. Inflammatory cells consist of CD3+, CD4+, ED1+, ED2+ and I-A+ mononuclear cells, eosinophils, degranulated mast cells and neutrophils accompanying interleukin (IL)-4, interferon (IFN)-gamma and RANTES mRNA expressions of the skin. Inflammatory cells are reduced during chronification with decreased expressions of IL-4, IFN-gamma and RANTES mRNA. In addition, the rats show a high sensitivity to PCA reactions and maximization tests. CONCLUSIONS: Our results show that some of the skin lesions of beige rats are morphologically similar to human AD, being characterized by inflammatory cell composition in the acute phase, and increased IgE and RANTES levels. However, the inflammatory process and cytokine expression pattern are different from those in human AD.

Platelet dysfunction in Chediak-Higashi syndrome-affected cattle.

A serious symptom of cattle affected with Chediak-Higashi syndrome (CHS) is a bleeding tendency. This diathesis is characterized by insufficient platelet aggregation as a result of depressed response to collagen. One possible cause for the depression is a decrease in contribution of endogenous agonists such as ADP or thromboxane A(2), which are released following collagen stimulation. However, these endogenous agonists play only a minor role in collagen-induced aggregation of bovine platelets. More importantly, activation of phospholipase C as a result of a direct action of collagen is depressed, leading to a depression of Ca(2+) mobilization, in platelets from CHS-affected cattle. Several types of collagen receptor are proposed to work in concert to induce aggregation. Among them, glycoprotein VI (GPVI) and GPIa/IIa (integrin alpha2 beta1) have been supposed to play dominant roles in collagen-induced aggregation. However, there are arguments about the role of each receptor, especially the role of GPIa/IIa, and the crosstalk between receptors. Recently, we reported that the Ca(2+) signaling produced by rhodocytin, which had been first reported to be an agonist for the collagen receptor GPIa/IIa, produced much less Ca(2+) signaling in CHS platelets than in normal ones, whereas that produced by GPVI activators was normal. These suggest that GPIa/IIa or the rhodocytin-associated pathway is impaired in CHS platelets. CHS platelets are valuable to reassess the mechanism of collagen-dependent signal transduction system and to delineate the inter-relationship among collagen receptors.

A defect in collagen receptor-Ca2+ signaling system in platelets from cattle with Chediak-Higashi syndrome.

Decreased platelet aggregation to collagen is a cause for bleeding diathesis of Chediak-Higashi syndrome (CHS). We investigated whether the collagen receptor-Ca2+ signaling system was impaired in platelets from cattle affected with CHS. A collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) was depressed in CHS platelets, which was accompanied by a decrease in the production of inositol 1,4,5-trisphosphate. When the influences of endogenous arachidonic acid metabolites and ADP were excluded, convulxin or collagen-related peptide, which are specific agonists for the collagen receptor GPVI, increased [Ca2+]i in both normal and CHS platelets. In contrast, rhodocytin, which was thought to activate another collagen receptor GPIa/IIa, increased [Ca2+]i in CHS platelets to a lesser extent than in normal ones. Cytochalasin D, an actin polymerization inhibitor, depressed the response to collagen or rhodocytin but not the response to convulxin. Adhesion of CHS platelets to acid soluble type I collagen, which was mediated by GPIa/IIa, was similar to that of normal platelets. These results suggest that a defect in the rhodocytin-sensitive pathway is responsible for decreasing the response to collagen in CHS platelets. It remains to be determined which receptor is associated with the mechanism.

Localization of the locus responsible for Chediak-Higashi syndrome in cattle to bovine chromosome 28.

Chediak-Higashi syndrome in Japanese black cattle is a hereditary disease with prolonged bleeding time and partial albinism. In the present study, we mapped the locus responsible for the disease (CHS) by linkage analysis using microsatellite genotypes of paternal half-sib pedigrees obtained from commercial herds. Analysis revealed significant linkage between the CHS locus and marker loci on the proximal end of bovine chromosome 28. The CHS locus was mapped on the region incorporating the microsatellite markers BMC6020, BM2892, and RM016 with recombination fraction 0 and lod score 4.9-11.2. We also assigned the bovine CHS1/LYST, the homologue of the gene responsible for human Chediak-Higashi syndrome, to bovine chromosome 28 using a bovine/murine somatic cell hybrid panel. These findings suggest that a mutation in the CHS1/LYST gene is likely to be responsible for Chediak-Higashi syndrome in Japanese black cattle.

Chediak-Higashi syndrome mutation and genetic testing in Japanese black cattle (Wagyu).

Chediak-Higashi Syndrome (CHS) is an autosomal recessive disorder that affects several species including mice, humans, and cattle. Evidence based on clinical characteristics and somatic cell genetics suggests that mutations in a common gene cause CHS in the three species. The CHS locus on human chromosome 1 and mouse chromosome 13 encodes a lysosomal trafficking regulator formerly known as LYST, now known as CHS1, and is defective in CHS patients and beige mice, respectively. We have mapped the CHS locus to the proximal region of bovine chromosome 28 by linkage analysis using microsatellite markers previously mapped to this chromosome. Furthermore, we have identified a missense A:T-->G:C mutation that results in replacement of a histidine with an arginine residue at codon 2015 of the CHS1 gene. This mutation is the most likely cause of CHS in Wagyu cattle. In addition, we describe quick, inexpensive, PCR based tests that will permit elimination of the CHS mutation from Wagyu breeding herds.

Cloning of bovine LYST gene and identification of a missense mutation associated with Chediak-Higashi syndrome of cattle.

An inheritable bleeding disorder with light coat color caused by an autosomal recessive gene has been reported in a population of Japanese black cattle. The disease has been diagnosed as Chediak-Higashi Syndrome (CHS) of cattle which correspond to a human inheritable disorder caused by mutation in LYST gene. To characterize the molecular lesion causing CHS in cattle, cDNAs encoding bovine LYST were isolated from a bovine brain cDNA library. The nucleotide and deduced amino acid sequences of bovine LYST had 89.6 and 90.2% identity with those of the human LYST gene, respectively. In order to identify the mutation within the LYST gene causing CHS in cattle, cDNA fragments of the LYST gene were amplified from an affected animal by RT-PCR and their nucleotide sequences were completely determined. Notably, a nucleotide substitution of A to G transition, resulting in an amino acid substitution of histidine to arginine (H2015R) was identified in the affected animal. The presence of the substitution was completely corresponding with the occurrence of the CHS phenotype among 105 members of pedigrees of the Japanese black cattle and no cattle of other populations had this substitution. These findings strongly suggested that H2015R is the causative mutation in CHS of Japanese black cattle.

Impaired cytosolic calcium mobilization and aggregation in response to collagen in platelets from Japanese black cattle with Chediak-Higashi syndrome.

OBJECTIVE: To examine whether impaired aggregation of platelets from Japanese Black cattle with Chediak-Higashi syndrome (CHS) was attributable to mobilization of cytosolic Ca2+. ANIMALS: 4 healthy Japanese Black cattle and 3 Japanese Black cattle with CHS. PROCEDURE: Aggregation and mobilization of cytosolic Ca2+ in response to various receptor agonists was measured in platelets from healthy cattle and cattle with CHS. Involvement of endogenous ADP and arachidonic acid in collagen-induced responses was examined. Cytosolic Ca2+ concentration was measured after platelets were loaded with the Ca2+ indicator fura-PE3. Platelet aggregation was measured with an aggregometer. RESULTS: Collagen (3 to 15 micrograms/ml)-induced increases in cytosolic Ca2+ concentration and aggregation were markedly impaired for platelets from cattle with CHS, compared with values for platelets from healthy cattle. Although aggregation and the sustained phase of the cytosolic Ca2+ response to ADP were also decreased in platelets from cattle with CHS, these decreases were small, compared with those in response to collagen. A cyclooxygenase inhibitor and a phospholipase A2 inhibitor did not have any effect on peak cytosolic Ca2+ concentration or collagen-induced aggregation of platelets from healthy cattle. Responses to a P2 tau-purinoceptor antagonist suggested that decreased release of endogenous ADP was only partially involved in the impaired response to collagen among platelets from cattle with CHS. CONCLUSIONS AND CLINICAL RELEVANCE: Marked inhibition of collagen-induced Ca2+ mobilization, rather than decreased release of endogenous substances, appeared to be the major cause of impaired platelet response to collagen and the hemorrhagic tendency in cattle with CHS.

Clinical, morphologic, and biochemical characteristics of Chediak-Higashi syndrome in fifty-six Japanese black cattle.

OBJECTIVE: To characterize Chediak-Higashi syndrome (C-HS) in Japanese Black cattle. ANIMALS: 56 of 200 cattle with a bleeding disorder and giant granules in leukocytes. PROCEDURE: Clinical observation, CBC, hemostatic screening test, platelet aggregometry, electron microscopy, platelet constituent analysis, and ophthalmoscopic examination were done. RESULTS: Affected Japanese Black cattle had increased bleeding tendency and abnormal granules in their leukocytes. Susceptibility to infection was not increased. Cutaneous albinism was evident in 6 new-born calves, but not in most affected cattle. In all affected cattle, the tapetal fundus was pale and the nontapetal fundus was almost devoid of pigment. By electron microscopy, a remarkable decrease in the number of dense granules in platelets was observed. Functionally, collagen-induced platelet aggregation was markedly reduced. CONCLUSIONS: This bleeding disorder was diagnosed as C-HS. With regard to susceptibility to infection, albinism, and mortality, clinical manifestations of C-HS in Japanese Black cattle were moderate, compared with C-HS in human beings and Hereford cattle. CLINICAL RELEVANCE: Because an autosomal recessive mode of inheritance was documented and recessive homozygotes could be easily detected, C-HS in Japanese Black cattle can be controlled.

Spontaneous skin lesions in beige rats (Chediak-Higashi syndrome of rats).

Beige rats, a model animal of Chediak-Higashi syndrome (CHS), frequently developed the skin lesions consisting of crust formations and alopecia in the skin around the neck from about 4 months of age. Erosion and ulceration were also observed in advance of the skin lesions. In severe cases, the lesions spread to all of the dorsum of the trunk. Skin tissues with or without lesions were studied histopathologically in 41 beige rats comparing with normal skin from 26 age-matched DA rats. Microscopically, epidermal lesions consisted of spongiosis, pustules and erosions with crust. Inflammatory cells in pustules consisted predominantly of eosinophil, and colonization of gram-positive cocci was occasionally observed in the surface area. Mites on the epidermis were also seen in some cases. Dermal lesions were superficial perivascular inflammatory cell infiltrations of eosinophils, neutrophils and mastocytes, and edema under the epidermal lesions. Follicles in the alopecic area showed resting stage and atrophic hair germ, but inflammatory changes were slight. Morphologic characters were very similar to those of chronic eosinophilic dermatitis or spongiotic dermatitis.

Inhibition of collagen-induced platelet aggregation in Japanese black cattle with inherited platelet disorder, Chediak-Higashi syndrome.

Aggregation properties of platelets were examined in Japanese Black cattle with Chediak-Higashi syndrome (CHS) and normal control cattle. Platelet aggregation induced by collagen was decreased in platelets of the cattle with CHS, but not ADP (10-20 microM), thrombin (0.5-1.0 U/ml) and phorbol-12-myristate 13-acetate (PMA, 3.2 microM). The aggregation response induced by collagen in CHS platelets lacks the change in shape which usually occurs in normal platelets. Simultaneous stimulation by collagen (10 micrograms/ml) + ADP (10 microM) is effective in restoring collagen-induced aggregating response in CHS platelet, although pretreatment of ADP (10 microM) could not restore the collagen (10 micrograms/ml)-induced aggregating response, suggesting that there is a certain threshold of stimulation intensity above which collagen-induced aggregation of CHS platelet can begin. Control normal platelets, previously exposed to ADP (10 microM) and collagen (10 micrograms/ml), showed no further response to exposure to a third aggregating agent (arachidonic acid, 5 mM). On the other hand, the final agent was able to elicit aggregating responses in CHS platelets, suggesting that the arachidonate aggregating system may be suppressed in CHS cattle, but fully activated in control animals. Furthermore, normal platelets showed a significant decrease in aggregating response to collagen when pretreated with a cyclooxygenase inhibitor, indomethacin (10(-5) M), whereas CHS platelet was insensitive to indomethacin. This indomethacin-treated normal platelet mimicked the CHS collagen-induced aggregation pattern. These data suggest that a signal transduction process from receptor-operated events to arachidonate metabolism is suppressed in collagen-induced CHS platelet aggregation.

Inheritance of Chediak-Higashi syndrome in Japanese black cattle.

Fifty-six Japanese black cattle affected with Chediak-Higashi syndrome (C-HS) have been referred to Miyazaki University Veterinary Teaching Hospital during the past 12 years, and 44 pedigree records were collected. In pedigree analysis, the parents had no clinical sign, the affected dams had clinically normal calves, and approximately equal numbers of males and females were affected, we therefore considered this syndrome to be an autosomal recessive trait. Quantitative genetic analyses were then made in a restricted area. Segregation analysis by the a priori method in 8 families showed that C-HS was a simple autosomal recessive trait. Furthermore, 36 dams and their 257 offspring (including 8 C-HS affected cattle) were analyzed using population genetics in the same area. The result was the same as in the former analysis.

Primary immunodeficiencies of food animals.

Although there are few, well-characterized PIDs of food animals, these diseases are important because they tend to be severe and with no cure. Most animals with PID do not receive the intensive and aggressive care required for survival: Veterinarians may be consulted only when the animals are in the terminal stages of illness; it is generally not economically practical for livestock producers or practitioners to pay for the exhaustive laboratory tests required to detect and characterize these anomalies. Another reason for the small numbers of characterized clinical cases of PID is that they are rare. It is possible, however, that intensive artificial insemination and embryo transfer could select for heterozygous carriers of these autosomal traits. As seen with bovine leukocyte adhesion deficiency, as the frequency of an allele increases in the population, the numbers of affected animals increase. Furthermore, other immunodeficient syndromes are likely to exist. Veterinarians therefore should be aware of these disorders and should seek laboratory assistance to arrive at a correct diagnosis. Because of the inheritable nature of PID, livestock producers need assistance from veterinarians to identify carriers and establish sound breeding and control programs. One positive outcome from studies of PID is that research scientists and veterinarians learn much about immune systems from these afflicted animals. In fact, these animals may become models for gene therapy or marrow reconstruction procedures.

Defective in vitro motility of polymorphonuclear leukocytes of homozygote and heterozygote Chediak-Higashi cats.

The in vitro migratory responses of neutrophils of homozygote and heterozygote Chediak-Higashi cats were defective in an under-agarose assay when compared to the behavior of phagocytes of control cats. The linear distances traversed by the leading front of migrating Chediak-Higashi neutrophils toward streptococcal culture supernatant, zymosan-activated serum or buffer were reduced and smaller numbers of Chediak-Higashi phagocytes populated the resulting migration areas than did cells of control animals. The relative migration parameters of the Chediak-Higashi phagocytes, however, did not differ from the corresponding parameters of control neutrophils in the presence of streptococcal culture supernatant. Therefore, phagocytes of homozygote and heterozygote Chediak-Higashi cats recognized and responded equally well to the bacterial stimuli as did cells of control animals but traveled shorter distances primarily because of a reduced inherent motility. Similar results were also obtained when the feline phagocytes were attracted by zymosan-activated serum. In addition the relative migration parameters of the neutrophils of homozygote Chediak-Higashi cats were reduced and the normalized spatial distributions of their migrating cells were significantly different in the presence of 100% and 20% zymosan-activated serum when compared to the corresponding migration parameters of carrier and control animals. Defective recognition or responses to the higher concentrations of these host-derived attractants complicated, therefore, the already reduced inherent motility of the phagocytes of homozygote Chediak-Higashi cats.

Restoration of neutrophil and platelet function in feline Chediak-Higashi syndrome by bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) was successfully performed in four Chediak-Higashi (CHS) syndrome affected cats. Preparatory regimens included selective intestinal flora decontamination, fractionated total body irradiation for myeloablation, and prophylactic treatment for graft-versus-host disease with cyclosporin A. Neutrophil chemotaxis under-agarose and whole-blood platelet aggregation/secretion were characterized prior to BMT and after engraftment of donor-origin marrow cells. Liver and kidney biopsies were obtained and evaluated by light and electron microscopy before, and at 6 months post-BMT to determine what effect BMT might have on abnormal lysosome fusion in hepatocytes and renal tubule cells. The platelet storage pool defect was resolved by day 40 post-BMT. In vitro neutrophil migration in all cats appeared to improve with time after BMT and complete restoration was evident by day 175 post-BMT. No apparent differences were evident in either the liver or the kidney at 6 months post-BMT. One cat developed seizures and one developed posterior paresis 5 months post-BMT; neurologic impairment ultimately resulted in death of two cats at 6 and 8 months post-BMT, respectively. Neurologic lesions in both cats were characterized by non-suppurative encephalitis. Allogeneic BMT successfully corrected the neutrophil migration defect and platelet storage pool deficiency but had no effect on lysosome distribution in liver and kidney cells of CHS cats.

Identification of dense granule specific membrane proteins in bovine platelets that are absent in the Chediak-Higashi syndrome.

Platelets from cattle with the Chediak-Higashi syndrome (CHS) have a platelet dense granule deficiency. One hypothesis for the platelet dense granule deficiency is that the granule is simply not formed in CHS megakaryocytes (MK). Alternative hypotheses include that the granule is assembled in CHS MK but a functional amino-nucleotide-cation storage complex cannot be formed or that the dense granule or its precursor fuses with other granules. This study was undertaken to determine if membrane proteins specific for platelet dense granules can be identified in membranes of other granules in CHS platelets. Platelets were disrupted; a mixed-granule fraction and alpha-granule enriched, mitochondrial-enriched, and dense granule-enriched subfractions were obtained. Membrane proteins in these intact granules were radiolabeled and the granule underwent hypotonic lysis. Membrane proteins were extracted from granule "ghosts", separated, and then visualized by autoradiography. Three major proteins were identified in platelet dense granule membrane subfractions. Two of these proteins could be identified in membrane extracts from the mixed-granule fraction from normal platelets. They could neither be identified in extracts from the mixed granule fraction of CHS platelets nor in membranes from alpha granule-enriched and mitochondrial-enriched subfractions. The absence of dense granule membrane proteins in membranes of other organelles within CHS platelets suggests that fusion of dense granules or its precursor with other granules cannot account for the platelet dense granule deficiency in CHS platelets.

Primary and secondary lysosomes in megakaryocytes and platelets from cattle with the Chediak-Higashi syndrome.

The ultrastructure of lysosomes from megakaryocytes (MK) and platelets of cattle with the Chediak-Higashi syndrome (CHS) was characterized using acid phosphatase histochemistry with beta-glycerophosphate as substrate and cerium as a capturing agent. Acid phosphatase was localized in the trans aspect of the Golgi complex and/or granules in MK at all stages of maturation. Morphometric analysis of the diameter of each lysosome was performed on MK from CHS cattle and compared to MK from normal cattle. Lysosomes in CHS MK were neither enlarged nor different with respect to classification as secondary lysosomes, which composed 35% of the lysosomes in CHS MK. Lysosomes were demonstrated in 22% of the CHS platelet sections and appeared similar to those from normal cattle, 56% of them being classified as secondary lysosomes. Why lysosomes are not enlarged in bovine CHS MK and platelets, whereas they are enlarged in most other cell types, remains unknown.