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Results for “Tissue Antigens”

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USGS1974

Fish sporozoa: Extraction of antigens from Myxosoma cerebralis spores which mimic tissue antigens of rainbow trout (Salmo gairdneri)

This is the first report of an antigenic preparation from the spore stage of any histozoic sporozoan parasite. The method for successful extraction of the antigens is presented. This antigenic preparation is active in rabbits, but not salmonid fish. The detection of asymptomatic carrier fish using the spore antigens was not successful and evidence is presented to show that the parasite is successful because its antigens mimic those of trout. Immunoelectrophoresis indicates the parasite and trout antigens are different proteins.

Journal of the Fisheries Research Board of Canada↗

USGS2006

Cell proliferation detected with flow cytometric cell cycle analysis and immunohistochemical detection of Proliferating Cell Nuclear Antigen (PCNA) from somatic tissues of Eastern Oysters, Crassostrea virginica

Two novel biomarker of response assays were developed and compared for use with bivalves. Bivalve mollusks are often used as bioindicators to monitor contaminant body burdens and are employed globally in pollution monitoring and as sentinels of environmental quality. The prevalence of proliferating cells in tissues of the eastern oyster, Crassostrea virginica , (n = 10) was investigated by using immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and by flow cytometric analysis of DNA in cell cycle phases. The PCNA protein is highly conserved and central to DNA synthesis in dividing cells. This protein was detected by using a commercial antibody in the histological staining of gills, mantle, digestive gland, stomach, and labial palps. Cells obtained from these same organs and from the hearts of the oysters were used for flow cytometry (FCM). The proportions of nuclei in the S plus G2/M and G2/M fractions were compared with percentages of PCNA-positive cells scored through microscopy. Proliferation responses generated by microscopy and FCM (S plus G2/M) were equal for digestive gland, gills, stomach, and mantle. The proliferation response in labial palps was significantly higher by PCNA microscopy than by DNA FCM (S plus G2/M). For PCNA microscopy results, a significantly higher proliferation response was noted both for labial palps as compared to gills and mantle and for stomach and digestive gland as compared to gills (P < 0.005). For DNA FCM results, the percentages of proliferating cells in G2M fractions were significantly higher for labial palps and heart when compared to gills, mantle, and digestive gland. By FCM (S plus G2M), the percentages of proliferating cells followed a similar trend, although no significant differences were found between organs. The choice of a relatively highly proliferative tissue, such as labial palps as determined by this study, has utility for developing a cell line, for examining proliferation as a biomarker due to stressor impacts in feral oysters, and for detecting impacts of compounds on proliferative mechanisms.

Environmental Bioindicators↗

USGS1990

Multiplication of infectious hematopoietic necrosis virus in rainbow trout following immersion infection: whole-body assay and immunohistochemistry

The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and in the anterior internal tissues below the epidermis. Subsequently, extensive virus replication and tissue destruction were observed in the spleen, dorsal adipose tissues, ventricle, and pseudobranch. Replication in the liver, the muscularis layers of the digestive tract, and the general body musculature followed later. These infection experiments indicated that the epidermis and gills of fish constitute important sites of early IHNV replication.

Journal of Aquatic Animal Health↗

USGS2003

Recombinant raccoon pox vaccine protects mice against lethal plague

Using a raccoon poxvirus (RCN) expression system, we have developed new recombinant vaccines that can protect mice against lethal plague infection. We tested the effects of a translation enhancer (EMCV-IRES) in combination with a secretory (tPA) signal or secretory (tPA) and membrane anchoring (CHV-gG) signals on in vitro antigen expression of F1 antigen in tissue culture and the induction of antibody responses and protection against Yersinia pestis challenge in mice. The RCN vector successfully expressed the F1 protein of Y. pestis in vitro. In addition, the level of expression was increased by the insertion of the EMCV-IRES and combinations of this and the secretory signal or secretory and anchoring signals. These recombinant viruses generated protective immune responses that resulted in survival of 80% of vaccinated mice upon challenge with Y. pestis . Of the RCN-based vaccines we tested, the RCN-IRES-tPA-YpF1 recombinant construct was the most efficacious. Mice vaccinated with this construct withstood challenge with as many as 1.5 million colony forming units of Y. pestis (7.7×10 4 LD 50 ). Interestingly, vaccination with F1 fused to the anchoring signal (RCN-IRES-tPA-YpF1-gG) elicited significant anti-F1 antibody titers, but failed to protect mice from plague challenge. Our studies demonstrate, in vitro and in vivo, the potential importance of the EMCV-IRES and secretory signals in vaccine design. These molecular tools provide a new approach for improving the efficacy of vaccines. In addition, these novel recombinant vaccines could have human, veterinary, and wildlife applications in the prevention of plague.

Vaccine↗

USGS2006

Expressed MHC class II genes in sea otters (Enhydra lutris) from geographically disparate populations

The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters ( Enhydra lutris ) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA , DQB , DRA , and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA , DQB , and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide‐binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters.

Tissue Antigens↗

USGS1992

In vitro infection of salmonid epidermal tissues by infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus

The ability of two rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), to infect fish skin was investigated by in vitro infection of excised tissues. Virus replication was determined by plaque assay of homogenized tissue extracts, and the virus antigen was detected by immunohistology of tissue sections. Gill, fin, and ventral abdominal skin tissues of rainbow trout Oncorhynchus mykiss that had been infected in vitro with a virulent strain of IHNV (193–110) produced substantial increases in virus titer within 24 h. Titers continued to increase up until day 3 of incubation; by this time, virus had increased 1,000-fold or more. This increase in IHNV titer occurred in epidermal tissues of fingerlings and of older fish. In another experiment, IHNV replicated in excised rainbow trout tissues whether the fish had been subject to prior infection with a virulent strain of IHNV (Western Regional Aquaculture Consortium isolate) or whether the fish had been infected previously with an attenuated strain of the virus (Nan Scott Lake, with 100 passes in culture). A virulent strain of VHSV (23/75) replicated effectively in excised gill tissues and epidermal tissues of rainbow trout and chinook salmon O. tshawytscha ; however, the avirulent North American strain of VHSV (Makah) replicated poorly or not at all.

Journal of Aquatic Animal Health↗

USGS1994

Neutralization-resistant variants of infectious hematopoietic necrosis virus have altered virulence and tissue tropism

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes an acute disease in salmon and trout. In this study, a correlation between changes in tissue tropism and specific changes in the virus genome appeared to be made by examining four IHNV neutralization-resistant variants (RB-1, RB-2, RB-3, and RB-4) that had been selected with the glycoprotein (G)-specific monoclonal antibody RB/B5. These variants were compared with the parental strain (RB-76) for their virulence and pathogenicity in rainbow trout after waterborne challenge. Variants RB-2, RB-3, and RB-4 were only slightly attenuated and showed distributions of viral antigen in the livers and hematopoietic tissues of infected fish similar to those of the parental strain. Variant RB-1, however, was highly attenuated and the tissue distribution of viral antigen in RB-1-infected fish was markedly different, with more viral antigen in brain tissue. The sequences of the G genes of all four variants and RB-76 were determined. No significant changes were found for the slightly attenuated variants, but RB-1 G had two changes at amino acids 78 and 218 that dramatically altered its predicted secondary structure. These changes are thought to be responsible for the altered tissue tropism of the virus. Thus, IHNV G, like that of rabies virus and vesicular stomatitis virus, plays an integral part in the pathogenesis of viral infection.

Journal of Virology↗

USGS2017

The pathogenesis of clade 2.3.4.4 H5 highly pathogenic avian influenza viruses in Ruddy Duck (Oxyura jamaicensis) and Lesser Scaup (Aythya affinis)

Waterfowl are the natural hosts of avian influenza virus (AIV) and disseminate the virus worldwide through migration. Historically, surveillance and research efforts for AIV in waterfowl have focused on dabbling ducks. The role of diving ducks in AIV ecology has not been well characterized. In this study, we examined the relative susceptibility and pathogenicity of clade 2.3.4.4 H5 highly pathogenic AIV (HPAIV) in two species of diving ducks. Juvenile and adult Ruddy Duck ( Oxyura jamaicensis ) and juvenile Lesser Scaup ( Aythya affinis ) were intranasally inoculated with A/Northern Pintail/WA/40964/2014 H5N2 HPAIV. Additional groups of juvenile Lesser Scaups were inoculated with A/Gyrfalcon/WA/41088/2014 H5N8 HPAIV. The approximate 50% bird infectious doses (BID 50 ) of the H5N2 isolate for adult Ruddy Ducks was <10 2 50% egg infectious doses (EID 50 ) and for the juvenile Lesser Scaups it was <10 4 EID 50 . There were insufficient juvenile Ruddy Ducks to calculate the BID 50 . The BID 50 for the juvenile Lesser Scaups inoculated with the H5N8 isolate was 10 3 EID 50 . Clinical disease was not observed in any group; however, mortality occurred in the juvenile Ruddy Ducks inoculated with the H5N2 virus (three of five ducks), and staining for AIV antigen was observed in numerous tissues from these ducks. One adult Ruddy Duck also died and although it was infected with AIV (the duck was positive for virus shedding and AIV antigen was detected in tissues), it was also infected with coccidiosis. The proportion of ducks shedding virus was related to the dose administered, but the titers were similar among dose groups. The group with the fewest ducks shedding virus was the adult Ruddy Ducks. There was a trend for the Lesser Scaups to shed higher titers of virus than the Ruddy Ducks. No virus shedding was detected after 7 d postinoculation in any group. Similar to dabbling ducks, Lesser Scaups and Ruddy Ducks are susceptible to infection with this H5 HPAIV lineage, although they excrete lower titers of virus.

Journal of Wildlife Diseases↗

USGS1985

Detection of Vibrio anguillarum antigen by the dot blot assay

The dot blot assay, modified and adapted for detection of antigens from Vibrio anguillarum in fish tissues, was specific for V. anguillarum and did not react with antigens of V. ordalii , Pseudomonas sp., or Yersinia ruckeri . The blot assay enabled detection of as little as 2.3 ng of a mixture of protein antigens obtained from cell-free extracts of V. anguillarum ; it was about 100 times more sensitive than either the indirect fluorescent antibody technique or bacterial isolation for detecting V. anguillarum in fish tissues.

Journal of Wildlife Diseases↗

USGS1998

Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

Ovarian fluid samples from naturally infected chinook salmon ( Oncorhynchus tshawytscha ) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 10 9 cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 10 4 cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum , 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

Journal of Veterinary Diagnostic Investigation↗

USGS2011

Experimental challenge and pathology of highly pathogenic avian influenza virus H5N1 in dunlin ( Calidris alpina ), an intercontinental migrant shorebird species

Background Shorebirds (Charadriiformes) are considered one of the primary reservoirs of avian influenza. Because these species are highly migratory, there is concern that infected shorebirds may be a mechanism by which highly pathogenic avian influenza virus (HPAIV) H5N1 could be introduced into North America from Asia. Large numbers of dunlin ( Calidris alpina ) migrate from wintering areas in central and eastern Asia, where HPAIV H5N1 is endemic, across the Bering Sea to breeding areas in Alaska. Low pathogenic avian influenza virus has been previously detected in dunlin, and thus, dunlin represent a potential risk to transport HPAIV to North America. To date no experimental challenge studies have been performed in shorebirds. Methods Wild dunlin were inoculated intranasally and intrachoanally various doses of HPAIV H5N1. The birds were monitored daily for virus excretion, disease signs, morbidity, and mortality. Results The infectious dose of HPAIV H5N1 in dunlin was determined to be 10 1.7 EID 50 /100 μl and that the lethal dose was 10 1.83 EID 50 /100 μl. Clinical signs were consistent with neurotropic disease, and histochemical analyses revealed that infection was systemic with viral antigen and RNA most consistently found in brain tissues. Infected birds excreted relatively large amounts of virus orally (10 4 EID 50 ) and smaller amounts cloacally. Conclusions Dunlin are highly susceptible to infection with HPAIV H5N1. They become infected after exposure to relatively small doses of the virus and if they become infected, they are most likely to suffer mortality within 3–5 days. These results have important implications regarding the risks of transport and transmission of HPAIV H5N1 to North America by this species and raises questions for further investigation.

Alaska↗

USGS1958

The isolation and identification of Trypanosoma cruzi from raccoons in Maryland

Five raccoons trapped at Patuxent Research Refuge, Laurel, Maryland, were found to have trypanosomes in the blood which were morphologically indistinguishable from Trypanosoma cruzi on stained smears. The organism grew well in culture. It developed and reproduced in Triatoma protracta, T. infestans, T. phyllosoma , and Rhodnius prolixus . Experimental infections were produced in raccoons, opossums, mice, rats, and monkeys by inoculation of blood, culture, and triatome forms. Typical leishmaniform bodies were found in tissue sections of cardiac muscle fibers from naturally and experimentally infected animals. Cross agglutinations carried out with living cultural forms and rabbit antisera demonstrated a close antigenic relationship between the raccoon trypanosome and T. cruzi (Brazil strain). On the basis of (1) morphology, (2) presence of leishmaniform tissue stages, (3) development in triatomes, (4) infectivity to a variety of mammals, (5) culture characteristics, and (6) cross reactions in serological tests, this parasite is considered conspecific with Trypanosoma cruzi (Chagas, 1909) the causative agent of American human trypanosomiasis.

Maryland↗

USGS1997

Renibacterium salmoninarum in spring-summer Chinook salmon smolts at dams on the Columbia and Snake Rivers

We evaluated Renibaeterium salmoninarum infection in smolts of hatchery and wild spring-summer Chinook salmon Oncorhynchus tshawytscha sampled during most of the outmigration at Little Goose (1988) and Lower Granite dams (1988–1991) on the Snake River and at Priest Rapids and McNary dams on the Columbia River (1988–1990). We sampled 860–2,178 fish per dam each year. Homogenates of kidney–spleen tissue from all fish were tested for the presence of R. salmoninarum antigens by the enzyme-linked immunosorbent assay (ELISA), and homogenates from 10% of the fish were examined by the fluorescent antibody technique (FAT). Although only 1–11% of fish sampled at a given dam during any l year exhibited lesions characteristic of bacterial kidney disease, 86–100% of the fish tested positive for R. salmoninarum antigen by ELISA, whereas 4–17% of the fish tested positive by the FAT. During most years, a majority (68–87%) of fish testing positive by the ELISA had low R. salmoninarum antigen levels, but in 1989, 53% of positive fish from Lower Granite Dam and 52% from McNary Dam showed medium-to-high antigen levels. For most years, the highest mean antigen levels were measured in fish sampled after 75% of the total out-migrants had passed a given dam. When the largest numbers of fish were being collected for bypass or downriver transportation, mean antigen levels were relatively low.

Washington↗

USGS2006

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Journal of Veterinary Diagnostic Investigation↗

USGS1991

In vivo and in vitro transfer of trout spleen sections for early analysis of the immune response

To determine the earliest time after in vivo immunisation that the spleen could be excised and held in vitro to detect an immune response, lake trout ( Salvelinus namaycush ) were exposed to DNP-Ficoll or Yersinia ruckeri O antigen administered by intraperitoneal injection or by bath. The spleens were excised from the fish at selected times after immunisation, placed in vitro and held in tissue culture media at 14°C until 10 days after the in vivo immunisation. They then were analysed for an immune response by counting the numbers of plaqueforming cells (PFC). PFC were first detected in samples taken 3 days after injection with either antigen. With bath immunisation, however, PFC were first seen 6 days post-immunisation when using the Y. ruckeri O antigen and no PFC above background levels appeared in fish bathed in the DNP-Ficoll solution. On day 10 after immunisation, the spleens taken directly from immunised fish always produced more PFC than when the spleens were taken through in vitro culture. A unique feature of this method, is that an immune response to an antigen may be detected by excising the spleen and holding it in in vitro culture without necessarily holding the fish for long periods of time after antigen injection or bath. This technique also adds more information on how rapidly the bacterins or antigens are processed by fish to initiate an immune response.

Fish and Shellfish Pathology↗

USGS2006

Characterization of the host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy, and immunological techniques

The tissue response of Salmo gairdneri Richardson, against the myxosporean parasite. Ceratomyxa shasta (Noble), was investigated using histological techniques, scanning electron microscopy and immunological methods. The progress of infection in C. shasta ‐susceptible and resistant steelhead and rainbow trout was examined by standard histological techniques and by indirect fluorescent antibody methods using monoclonal antibodies directed against C. shasta antigens. Trophozoite stages were first observed in the posterior intestine and there was indication that resistance was due to the inability of the parasite to penetrate this tissue rather than to an inflammatory response. Examination of a severely infected intestine by scanning electron microscopy showed extensive destruction of the mucosal folds of the posterior intestine. Western blotting and indirect fluorescent antibody techniques were used to investigate the immunological component of the host response. No antibodies specific for C. shasta were detected by either method.

Journal of Fish Diseases↗

USGS2017

Natural infections with pigeon paramyxovirus serotype 1: Pathologic changes in Eurasian collared-doves (Streptopelia decaocto) and rock pigeons (Columba livia) in the United States

Pigeon paramyxovirus serotype 1 (PPMV-1) is a globally distributed, virulent member of the avian paramyxovirus serotype 1 serogroup that causes mortality in columbiformes and poultry. Following introduction into the United States in the mid-1980s, PPMV-1 rapidly spread causing numerous mortality events in Eurasian collared-doves ( Streptopelia decaocto ) (ECDOs) and rock pigeons ( Columba livia ) (ROPIs). The investigators reviewed pathological findings of 70 naturally infected, free-ranging columbiforms from 25 different mortality events in the United States. Immunohistochemistry targeting PPMV-1 nucleoprotein was used to determine the tissue distribution of the virus in a subset of 17 birds from 10 of the studied outbreaks. ECDOs (61 birds) and ROPIs (9 birds) were the only species in which PPMV-1-associated disease was confirmed by viral isolation and presence of histologic lesions. Acute to subacute tubulointerstitial nephritis and necrotizing pancreatitis were the most frequent histologic lesions, with immunolabeling of viral antigen in renal tubular epithelial cells and pancreatic acinar epithelium. Lymphoid depletion of bursa of Fabricius and spleen was common, but the presence of viral antigen in these organs was inconsistent among infected birds. Hepatocellular necrosis was occasionally present with immunolabeling of hypertrophic Kupffer cells, and immunopositive eosinophilic intracytoplasmic inclusion bodies were present in hepatocytes of 1 ECDO. Immunopositive lymphocytic choroiditis was present in 1 ECDO, while lymphocytic meningoencephalitis was frequent in ROPIs in absence of immunolabeling. This study demonstrates widespread presence of PPMV-1 antigen in association with histologic lesions, confirming the lethal potential of this virus in these particular bird species.

Veterinary Pathology↗

USGS2014

Environmental conditions associated with lesions in introduced free-ranging sheep in Hawai‘i

Wildlife species which have been translocated between temperate and tropical regions of the world provide unique opportunities to understand how disease processes may be affected by environmental conditions. European mouflon sheep (Ovis gmelini musimon) from the Mediterranean Islands were introduced to the Hawaiian Islands for sport hunting beginning in 1954 and were subsequently hybridized with feral domestic sheep (O. aries), which had been introduced in 1793. Three isolated mouflon populations have become established in the Hawaiian Islands but diseases in these populations have been little studied. The objective of this study was to evaluate and compare gross and histologic lesions in respiratory, renal, and hepatic systems of free-ranging sheep in two isolated volcanic environments on Hawai‘i Island. Tissue and fecal samples were collected in conjunction with population reductions during February 2011. We found gross or histologic evidence of lungworm infection in 44/49 sheep from Mauna Loa which were exposed to gaseous emissions from Kīlauea Volcano. In contrast, only 7/50 sheep from Mauna Kea had lesions consistent with lungworm, but Mauna Kea sheep had significantly more upper respiratory tract inflammation and hyperplasia consistent with chronic antigenic stimulation, possibly associated with exposure to fine airborne particulates during extended drought conditions. We hypothesize that gasses from Kīlauea Volcano contributed to severity of respiratory disease principally associated with chronic lungworm infections at Mauna Loa; however, there were numerous other potentially confounding environmental factors and interactions that merit further investigation.

Hawai'i↗