Characterization of the inclusion limiting membrane of anaplasma marginale by immunoferritin labeling.

Purified anti-erythrocytic membrane antibody (PAMA) was prepared from rabbit anti-bovine erythrocyte serum by an adsorption and elution technique, utilizing bovine erythrocytes. Lysed and washed anaplasma-infected erythrocytes were incubated with PAMA or control reagents. Specimens were then subjected to immunoferritin labeling with ferritin antiglobulin conjugate. Upon examination by electron microscopy, specimens incubated with PAMA showed heavy ferritin labeling of erythrocytic membranes and also the limiting membranes of anaplasmal inclusions. Anaplasmal initial bodies freed from their inclusion membranes were not labeled. Negative control specimens, incubated with normal rabbit serum or PAMA which had been absorbed with erythrocytes, did not show specific ferritin labeling. Intact bovine erythrocytes, which were used as a positive control of anti-bovine erythrocytic membrane specificity, were heavily ferritin-labeled. Avian erythrocytes, a negative control of specificity, remained unlabeled. The results of this study indicate that the limiting membrane of the anaplasmal inclusion is derived from the erythrocytic membrane.     

Bar-structure in bovine erythrocytes infected with Theileria sergenti

The purpose of this study is to elucidate some morphological characteristics of the bar-structure in bovine erythrocytes infected with Theileria sergenti. The bar-structure, the veil or the both were observed in infected erythrocytes. Infected bovine erythrocytes were classified into four types according to the included structures. Infected cells containing bar-structures increased with the progress of parasitemia. In Giemsa-stained blood preparations, bar-structures appeared purplish-red and measured a mean of 1.6 micron in length and up to 0.1 micron in width. Bar-structures were usually straight, sometimes S-shaped, and located in the periphery of infected erythrocytes. In the direct fluorescent antibody test with T. sergenti-positive bovine serum both piroplasms and bar-structures exhibited fluorescence. However, in the indirect fluorescent antibody test with monoclonal antibodies against piroplasms only piroplasms showed a highly specific fluorescence. Electron microscopy revealed that bar-structures were vesicular in structure, surrounded by a double membrane connected to the host cell membrane.     

Demonstration of the inclusion appendage of Anaplasma marginale in nymphal Dermacentor andersoni

Dermacentor andersoni nymphs were placed in stockinettes and allowed to feed on a splenectomized calf with experimentally induced anaplasmosis when the parasitemia was 3%-5%. Nymphs were selected on each of the 6 days of feeding and every 5 days from repletion through molting to the adult stage (25 days postrepletion); they were killed and midgut tissues were processed and examined by light and electron microscopies. No stages of A marginale were seen in tissues of feeding ticks. Visualization of individual components of gut contents was difficult owing to presence of the concentrated, electrondense blood meal containing hemoglobin. Inclusion appendages were observed in midgut tissues of nymphs at 5 and 10 days postrepletion, but not at 20 or 25 days. The morphology of the appendages was similar to that described for inclusion appendages commonly associated with anaplasmal inclusions in bovine erythrocytes. Some appendages were free in the lumen of the midgut and occurred either alone or with clusters of small vesicular particles. Occasionally, initial bodies like those generally found in bovine erythrocytes were seen with the appendage, but most of them were swollen and appeared to be degenerating. Frequently, inclusion appendages were observed attached to the luminal surface of the midgut cell membrane by a blunt, electron-dense attachment complex. The attachment of the appendage appeared to be extracellular, with the pointed end extending into the lumen. Often, small particles were observed immediately across the cell membrane from where the appendages were attached; the small particles appeared to be generated from the appendage itself and to have passed through the membrane of the midgut cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of anti-Babesia gibsoni heat shock protein 70 antibody and anti-canine heat shock protein 70 antibody in sera from Babesia gibsoni-infected dogs

Antibodies that recognized either Babesia gibsoni or canine red blood cell (RBC) 70-kilodalton (kDa) protein were detected in serum from acutely and chronically B. gibsoni-infected. In those sera, antibodies that reacted with recombinant B. gibsoni and canine heat shock protein 70 (rBgHsp70 and rcHsp70) were detected; therefore, B. gibsoni and canine RBC 70-kDa proteins seemed to be BgHsp70 and cHsp70, respectively. In infected dogs, the amounts of these antibodies increased after infection. Interestingly, polyclonal antibody raised against rBgHsp70 in two rabbits reacted not only with rBgHsp70 but also with rcHsp70 and native cHsp70 from canine RBCs. Because BgHsp70 showed high homology with cHsp70 (70.8%), anti-rBgHsp70 antibody might cross-react with cHsp70. Additionally, the localizations of both BgHsp70 and cHsp70 were observed by indirect fluorescence assay. As a result, cHsp70 was not found on the membrane surface of erythrocytes, suggesting that erythrocytes would not be targets of anti-cHsp70 antibody. Meanwhile, only exoerythrocytic parasites were stained by anti-rBgHsp70 antibody. This result showed that BgHsp70 would be expressed on the surface of parasites during the exoerythrocytic stage. These results indicated that BgHsp70 was a highly immunogenic protein in canine B. gibsoni infection, and that exoerythrocytic parasites might be targets of anti-BgHsp70 antibody.     

In vitro and in vivo association of African swine fever virus with swine erythrocytes

The association of African swine fever virus (ASFV) with swine erythrocytes in vivo, in high titers, was verified by inoculating 30 pigs with 17 ASFV isolates and assaying their plasma and washed erythrocyte fractions for residual virus. Viral antigens were specifically localized on the surface of in vitro and in vivo swine erythrocytes, using the fluorescent antibody technique and 3 monoclonal antibodies specific for ASFV. The same monoclonal antibodies immunoprecipitated virus-specific polypeptides of molecular weights 13 kd and 73 kd from ASFV-infected Vero cells. Erythrocytes from viremic swine infected with Lisbon-60, Dominican Republic, Badajoz-M98, or Cameroon isolates of ASFV were studied by transmission electron microscopy. Virus was found in membrane depressions at the surface of erythrocytes. These surface depressions resembled stages of smooth surfaced pits. Erythrocytes from viremic pigs were fragile osmotically.     

Ultrastructure of Anaplasma marginale after freeze-fracture.

Stained thin sections and freeze-fractured replicas of Anaplasma marginale-infected bovine erythrocytes were examined by electron microscopy. Freeze-fracture replication not only verified basic Anaplasma ultrastructure, but also allowed visualization of structures not previously reported. Because of the partial 3-dimensional views obtained with freeze-fracture replication, a new structure that appears as a protruding tip was discernible. Also, the surface of Anaplasma's limiting membrane was less granular than the fractured surface of host erythrocyte. A corrugated surface with a periodicity of 10.5 nm was seen when the limiting membrane was fractured.     

Ultrastructure of Anaplasma marginale with an inclusion appendage, isolated in Minas Gerais State, Brazil

This is the first report on the occurrence and isolation of a strain of Anaplasma marginale with an inclusion appendage in Brazil. The inclusion appendage presented longitudinal electron-dense striations and did not originate directly from the body of the rickettsia but from an electron-dense complex located at the junction of the inclusion membrane and inclusion appendage. The inclusion appendage remained in the host cell after the Anaplasma inclusion appeared to be leaving the red blood cell. Other ultrastructures of this rickettsia are described and its epidemiological importance is discussed.     

In vitro cultivation of Anaplasma marginale: invasion of and development in noninfected erythrocytes

Ovine erythrocytes infected with attenuated Anaplasma marginale organisms were cultured in a suspension of normal ovine erythrocytes and normal bovine erythrocytes for 42 days. In each system, the organism showed an initial period of rapid growth followed by a gradual decrease in the percentage of parasitized erythrocytes accompanied by cyclic peaks. The percentage of infection of ovine erythrocytes were not different when normal ovine or bovine erythrocytes were added to the cultures. In vitro transmission of the organism from infected ovine cells to normal bovine cells was demonstrated by use of a two-step direct fluorescent antibody method, which allowed for specific identification of the two cell types and the organism.     

Immunologic surface markers on nonhuman primate lymphocytes.

Peripheral blood lymphocytes from 37 healthy rhesus macaques (Macaca mulatta) and thymocytes from 10 fetal and neonatal rhesus macaques were studied for membrane characteristics. Spontaneous rosette formation with sheep erythrocytes, a characteristic of human T lymphocytes, was evaluated. The presence of membrane-bound immunoglobulin and surface receptors for fixed complement was measured, using fluorescent antibody techniques and erythrocyte-antibody-complement rosettes, respectively. The mean percentages +/- 1 standard error of the lymphocyte markers in the peripheral blood lymphocytes from the macaques were: spontaneous rosettes, 63 +/- 1.0; erythrocyte-antibody-complement rosettes, 14.9 +/- 1.2; and membrane immunoglobulin-positive cells, 21.9 +/- 2.2. These values are very similar to values reported for human beings.     

Effects of treatment with cobra venom factor on experimentally induced feline leukemia

Five- to six-month-old specific-pathogen-free cats were exposed to cobra venom factor (CVF) alone (4 cats), Rickard feline leukemia virus (FeLV; 9 cats), or CVF and FeLV (6 cats). Host-virus relationships were evaluated by monitoring the development of viremia, production of antibody against feline oncornavirus-associated cell membrane antigen, and amount of circulating immune complexes (CIC). Exposure to CVF induced complement depletion, which lasted 8 to 15 days. However, complement depletion did not promote the development of persistent viremia nor alter the production of antibody to feline oncornavirus-associated cell membrane antigen or CIC. Results indicated that the complement system did not protect cats during their initial exposure to FeLV and that an intact complement system was not necessary for the development of antibody against feline oncornavirus-associated antigen or for the formation of CIC.     

Surface ultrastructure of pyruvate kinase-deficient erythrocytes in the Basenji dog.

The scanning electron microscope was used to study the surface morphologic features of erythrocytes from a Basenji dog with hereditary hemolytic anemia due to a pyruvate kinase-deficient erythrocytes (RBC). Cells from this dog were compared with RBC from normal dogs and from dogs with regenerative anemias unrelated to pyruvate kinase deficiency. Demonstration of spherical RBC with uniform spicules on their surface (spheroechinocytes) may provide a morphologic explanation for the shortened erythrocytic life-span previously reported in congenital hemolytic anemia of Basenji dogs. Spherical, spiculated RBC were not found in blood from normal dogs or from anemic dogs with reticulocytoses. The surface of reticulocytes from all dogs with regenerative anemia was roughened, with pronounced folding of the cell membrane.     

Antibody responses of ponies to initial and challenge infections of Strongylus vulgaris

An indirect fluorescent antibody assay (IFA) was developed using Strongylus vulgaris third stage larvae (L3) as antigens. Observations using the IFA indicate that a species-specific antibody response to S. vulgaris L3 develops in S. vulgaris-infected ponies and that some surface L3 antigens are shared by adult worms. Sequential antibody levels against S. vulgaris were measured in strongyle-naive and in immune ponies following initial and challenge infections using the IFA and an indirect hemagglutination assay (IHA). Antibody levels measured by IFA increased faster following initial infections than did levels measured by IHA. Antibody levels appear to increase following challenge infections of immune ponies when measured with the IFA, but not with the IHA. Significant differences in antibody titers were not seen between ponies which developed colic following challenge infections and those that did not develop colic. Antibodies were not detectable in ponies unexposed to larval migrations, but which received surgical implantation of S. vulgaris adults into the cecum.     

Description of Aegyptianella botuliformis n. sp. (Rickettsiales: Anaplasmataceae) from the helmeted guineafowl, Numida meleagris.

Aegyptianella botuliformis n. sp. (Rickettsiales: Anaplasmataceae) isolated from helmeted guineafowls Numida meleagris from the Kruger National Park is described. The rickettsia occurs within a membrane-bound vacuole in the cytoplasm of erythrocytes with up to 8 organisms in a mature inclusion. The initial body resembles that of Aegyptianella pullorum. The tightly packed, sausage-shaped intermediate forms are a distinctive morphological feature, seen as irregular, pleomorphic forms under light microscopy. While more larvae and nymphs of Amblyomma hebraeum and Amblyomma marmoreaum were found on the birds than larvae of an Argas sp., it is believed that the latter are the vectors of A. botuliformis n. sp. In addition to the Kruger National Park, positive blood smears were obtained from guineafowls at other localities in the Transvaal.     

Immunologic reactions of pigs regrouped at or near weaning

Using 64 pigs, 2 experiments (32 pigs each) were conducted to evaluate the effects of regrouping nonlittermate pigs at weaning or 2 weeks after weaning on mitogen-induced lymphocyte blastogenesis, intradermal reactions to phytohemagglutinin, and primary antibody responses to sheep erythrocytes. Plasma cortisol concentrations were determined in all pigs and behavior of regrouped pigs was monitored. Compared with control values, plasma cortisol concentrations were higher in nonlittermate pigs regrouped at weaning (P less than 0.001) or 2 weeks after weaning (P less than 0.01). However, regrouping pigs at weaning or 2 weeks after weaning did not influence lymphocyte blastogenesis, phytohemagglutinin skin-test responses, or antibody titers to sheep erythrocytes. Plasma cortisol concentrations were not related to agonistic behavior in regrouped pigs or to lymphocyte blastogenic or phytohemagglutinin skin-test responses; however, higher plasma cortisol concentrations were related (P less than 0.05) to lower sheep erythrocyte antibody titers. These data indicate that regrouping nonlittermate pigs at weaning or 2 weeks after weaning is an acute stressor that does not detrimentally affect mitogen-induced lymphocyte blastogenesis, intradermal reactions to phytohemagglutinin, or primary antibody responses to sheep erythrocytes.     

Recombinant galectins of male and female Haemonchus contortus do not hemagglutinate erythrocytes of their natural host

Recombinant galectins of female and male adult worms of Haemonchus contortus were expressed in Escherichia coli and their hemagglutinating activities to human and different animal erythrocytes were analyzed. The results showed that female and male galectins could be highly expressed in E. coli using a temperature-sensitive plasmid, with the recombinant protein being mainly appeared in inclusion bodies. Hemagglutinating activity assays showed that both of the galectins hemagglutinated human A, B, O type, dog, rabbit, chicken and mouse erythrocytes at the high concentration of 40 microg/well, but did not hemagglutinate erythrocytes of the natural host of H. contortus, the goat. Sugar inhibition assays confirmed that, out of eight sugars tested, only lactose was effective to inhibit agglutination of human type B erythrocytes by the recombinant galectins.     

Preparation of monoclonal antibodies to Theileria sergenti and their reactivity to antigens in experimentally infected cattle

Two distinct monoclonal antibodies (3-H and 11-D) were produced against Theileria sergenti. These two new products, together with monoclonal antibody 1-G obtained in a previous study, were used to detect the parasites in experimentally infected cattle. During the first period of dexamethasone treatment, which was carried out to increase parasitemia in the infected cattle, the number of erythrocytes detected by 3-H, 11-D and 1-D increased in two experimentally infected calves. During the second period of dexamethasone treatment, the number of infected erythrocytes detected by 3-H and 11-D were similarly increased, but the number of infected erythrocytes detected by 1-G did not increase and infected erythrocytes in one calf were not detected by 1-G.     

Assessment of anti-bovine IL4 and IFN gamma antibodies to label IL4 and IFN gamma in lymphocytes of the koala and brushtail possum

We assess anti-bovine IL4 and IFN gamma (IFNg) antibodies for their ability to label IL4 and IFNg in koala (Phascolarctos cinereus), common brushtail possum (Trichosurus vulpecula) and mountain brushtail possum (Trichosurus caninus) lymphocytes using flow cytometry and immunohistochemistry to determine their applicability to studies of host response to intracellular pathogens. Anti-IFNg labelled a product of PMA-ionomycin stimulated sheep, koala and possum lymphocytes. High intensity labelling was not reduced by blocking non-specific binding with 10% FCS; and non-permeabilised koala lymphocytes labelled less, demonstrating that the labelled product was intracellular. The anti-IL4 antibody labelled variably more cells than the irrelevant antibody in some stimulated and non-stimulated preparations in all species but intensity of this labelling was similar to that of cells labelled with the irrelevant antibody. In this study, the antibodies did not label frozen or formalin-fixed tissues in a range of immunohistochemical techniques. We expect the anti-IFNg antibody to be effective in evaluating Th1 responses of koalas and possums exposed to various host, pathogen and environmental factors and add to the limited tools available for investigating the pathogenesis of marsupial diseases, especially those caused by intracellular organisms, such as tuberculosis of brushtail possums and chlamydial disease of koalas.     

Reproduction of inclusion body hepatitis in conventionally raised chickens inoculated with a New Zealand isolate of avian adenovirus

Conventionally raised chickens were inoculated with a local isolate of serotype 8 of avian adenovirus by an oral or intraperitoneal route, or were exposed to the infection by contact. Fatal hepatitis, resembling inclusion body hepatitis, occurred in 30% and 45% of the birds inoculated by the oral and intraperitoneal routes respectively, and severe growth depression was recorded in survivors and in birds in contact. Birds which had maternally derived virus neutralising antibody titres of 64 or greater at the time of viral exposure did not succumb to fatal infection, but their growth rates were significantly depressed.     

Detection of antibodies to platelets and erythrocytes during infection with haemorrhage-causing Trypanosoma vivax in Ayrshire cattle

Ayrshire cattle, which were infected with a stock of Trypanosoma vivax from Galana, Kenya, which produced haemorrhagic disease, were examined for the presence of antibodies to erythrocytes and platelets. Antibodies to normal erythrocytes and platelets were detected in the plasma of infected animals using the enzyme-linked immunosorbent assay (ELISA). The antibodies were detectable following the first peak of parasitaemia (10-15 days after infection) and antibody activity was maximal 30-35 days after infection. Plasma from cattle, taken 32 days after infection, precipitated radiolabelled proteins from autologous platelets and, less efficiently, from autologous erythrocytes. Fluorescence-activated cell sorter (FACS) assays demonstrated that erythrocytes and platelets from infected cattle bound IgM and IgG in vivo, and that both normal blood cell types could adsorb these antibodies following incubation in plasma from infected animals. Complement (C3) was similarly adsorbed to erythrocytes during infection. Antibodies adsorbed to infected erythrocytes could be eluted and the eluted antibodies bound to normal erythrocytes, as detected by immunofluorescence, but they did not react with the infecting trypanosome. It is hypothesised that although anti-blood cell antibodies may not be the primary cause of the severe anaemia and thrombocytopaenia which accompany the haemorrhagic syndrome, they could play an important role in the maintenance of these signs of disease, adversely affecting the outcome of T. vivax-associated haemorrhagic disease in the field.     

大熊猫犬冠状病毒的分离与鉴定

以犬肾传代细胞(MDCK)从1只病死大熊猫肝脏中分离到1株病毒,命名为DXMV。电镜观察磷钨酸负染的病毒细胞培养物，发现冠状病毒样粒子，超薄切片上可见细胞浆内病毒包涵体。理化学和生物学鉴定结果表明，DXMV核酸类型为RNA，对氯仿、乙醚敏感，可耐受1％胰蛋白酶的作用，具有一定的耐酸性和耐热性，可在MDCK、FCWF、F81细胞中增殖，无血凝性和血吸附特性。血清中和试验结果表明，DXMV可被犬冠状病毒阳性血清中和。采用犬冠状病毒间接荧光抗体法染色DXMV细胞飞片，可在细胞浆内见到特异性荧光。以冠状病毒通用引物和犬冠状病毒特异性引物，可从大熊猫肝脏和不同代次的病毒细胞培养物中扩增出与预期值251bp和515bp大小相同的核苷酸片段。由此说明，该病毒为犬冠状病毒，大熊猫可被犬冠状病毒感染。