Cloning and expression of complementary DNA encoding an antigen of Trichinella spiralis.

Complementary DNA (cDNA) encoding a 49-kDa antigen of Trichinella spiralis was cloned, characterized, and expressed in Escherichia coli by recombinant DNA methods. As a first step, 29 residues of N-terminal amino acid sequence were determined by direct analysis of highly purified antigen. Mixed-sequence primers based on the amino acid sequence were then synthesized and used as primers to generate a partial cDNA by the polymerase chain reaction (PCR). A nearly full-length cDNA was then obtained by PCR using a specific 5'-end primer and a non-specific 3'-end primer. Complete sequence of the 1133-bp cDNA was determined. Translation of this sequence predicts an N-glycosylated polypeptide of 322 amino acids. The cDNA was expressed as a fusion protein in bacteria which could be recognized in Western blots both by sera from mice immunized with purified native 49-kDa antigen and by sera from swine infected with the parasite. These findings suggest that recombinant P49 is a potentially valuable antigen both for vaccine development and immunodiagnosis.

Production of recombinant porcine tumor necrosis factor alpha in a novel E. coli expression system.

DNA sequences encoding porcine tumor necrosis factor alpha (TNF alpha) were reconstructed from a genomic-derived PCR product for expression in Escherichia coli. A synthetic DNA primer containing most of exon III was fused to exon IV sequences by means of PCR. The fused product was then inserted into the novel FLAG vector by restriction and ligation. This initial recombinant construct was propagated in single-strand form through use of a helper phage and subjected to oligonucleotide-directed mutagenesis for the purpose of introducing additional coding sequences from exons II and III. The final construct encoded a fusion protein consisting of the Omp-A signal peptide, a seven-amino acid FLAG peptide and the soluble form of porcine TNF alpha. Bacteria containing this construct produced a protein which was recognized by anti-FLAG monoclonal antibody in Western blots and which was purified by anti-FLAG immunoaffinity chromatography. The purified material was cleaved with enterokinase to remove the FLAG peptide. Both the enterokinase-cleaved form and the uncleaved form were shown to have TNF activity in a WEHI cell cytotoxicity assay.

A complementary DNA encoding an antigen from Trichinella spiralis muscle larvae and its analog from Trichinella T5 of bobcat origin: sequence, cloning and expressions.

Reverse transcription-polymerase chain reaction (RT-PCR) was employed to amplify a cDNA encoding an excretory-secretory (ES) antigen with mol. wt 45-50 kDa by SDS-PAGE from T. spiralis muscle larvae. The PCR product was purified by electrophoresis and sequenced by thermal cycle sequencing with primer walking. The cDNA is 890 bp long and encodes a polypeptide of 255 amino acid (AA) residues. Using the same methods, we also recovered a corresponding cDNA from Trichinella T5, which is 891 bp long and encodes 255 AAs. Comparison of the 2 Trichinella species indicates approximately 2.6% and 2.4% differences between the 2 cDNA sequences and between the 2 deduced AA sequences, respectively.

Immunopathogenesis of canine angistrongylosis: pulmonary effects of infection.

Deposits of immunoglobulins, complement, and fibrinogen were localized in the lungs of Angiostrongylus vasorum-infected dogs. In acutely infected dogs, significant deposits of IgA, IgG and IgM, complement, and fibrinogen were seen. The predominant immunoglobulin noted in infections of longer duration was IgG. The continued presence of fibrinogen as the predominant protein deposited suggested a continuation of intravascular coagulation. This was borne out by demonstrating luminal deposits of fibrinogen in many blood vessels. Histopathologic examination of the large revealed lesions typical of angiostrongylosis, as well as eggs of the worm as early as 35 days postinfection. This is the earliest report of egg production by A. vasorum on record.

Mitogen and antigen-specific induction of lymphoblast transformation in cats with subclinical toxoplasmosis.

Lymphoblast transformation in whole blood was assessed by 3H-thymidine incorporation after stimulation by concanavalin A and Toxoplasma gondii secretory and intracellular antigens in samples from cats with experimentally induced toxoplasmosis. Toxoplasma gondii-specific immunoglobulin M, immunoglobulin G, and circulating antigens were also measured throughout the study period. Lymphocytes from all cats were responsive to concanavalin A pre- and post-inoculation with T. gondii. Suppression of mitogen-stimulated lymphoblast transformation during the course of infection was not observed. Both the secretory and intracellular antigens stimulated lymphoblast transformation in many cats from Week 2 to Week 52 post-inoculation. Lymphoblast transformation was stimulated more frequently by intracellular antigens (66.25%) than by secretory antigens (48.75%). Lymphoblast transformation was not induced by T. gondii antigens in any cat prior to appearance of T. gondii-specific antibodies in serum or during the oocyst shedding period. Cats with persistent antigenemia had the most consistent lymphoblast transformation results induced by T. gondii-specific antigens.

Lipid metabolism and Sarcocystis miescheriana infection in growing swine.

Sixteen 2-month-old pigs were divided into four equal groups and infected with either 500,000, 1,000,000 or 3,000,000 sporocysts of Sarcocystis miescheriana. Four pigs served as uninfected controls. Pigs were bled weekly and serum was collected beginning 14 days prior to infection and continuing until 63 days after infection. Body fat composition, as measured by the specific gravity of the carcass, was not affected by infection. There were no significant effects of infection on serum concentrations of glucose, insulin, triglycerides, and total, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol. A slight depression in HDL cholesterol occurred during the acute phase of infection. Tumor necrosis factor (TNF) was not detected in serum from infected swine when assayed by a cytotoxicity assay using TNF-sensitive WEHI 164 clone 13 cells. Attempts to stimulate TNF production in RAW 264.7 cells with parasitic lysates gave mixed results. This study suggests that the disruption of lipid metabolism is not the primary cause of growth retardation in growing swine infected with S. miescheriana.

Prevalence of Toxoplasma gondii infection in cats in Georgia using enzyme-linked immunosorbent assays for IgM, IgG, and antigens.

Serologic prevalence of Toxoplasma gondii infection was determined using enzyme-linked immunosorbent assays detecting immunoglobulin M (IgM), immunoglobulin G (IgG), and circulating T. gondii antigens (Ag) in 81 healthy cats and 107 cats with clinical signs referable to toxoplasmosis. A higher prevalence of infection was detected using the three assays together in healthy cats, clinically ill cats, and combined healthy and clinically ill cats than when IgG class antibody detection alone was used. IgM titers greater than or equal to 1:256 and IgG titers greater than or equal to 1:512 were present more frequently in cats with clinical signs of disease. Prevalence of present or prior infection as defined by these three assays combined increased with advancing age in both groups of cats.

Alterations of serum insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) in swine infected with the protozoan parasite Sarcocystis miescheriana.

The effects of a Sarcocystis miescheriana infection on insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were investigated to determine possible mechanisms of growth retardation in growing pigs. Sixteen pigs averaging 14 kg body weight were divided into 4 groups of 4 pigs each and infected either with 0.5, 1.0, or 3.0 x 10(6) sporocysts of S. miescheriana. Four pigs were retained as non-infected controls; however, they became serologically positive during the course of the infection. Effects also were investigated in 2 groups of 3 pregnant sows. One group was infected with 0.5 x 10(6) sporocysts and the other group was retained as uninfected controls. Body weights of infected growing pigs were depressed as compared to controls following the acute phase 15 d after infection (dai). Serum concentrations of IGF-I dropped significantly (p < 0.05) during the acute phase of infection in all infected groups of growing pigs. Conversely, the amounts of unsaturated serum IGFBPs were elevated significantly (p < 0.05) during the acute phase of infection. Specifically, serum concentrations of IGFBP-1, IGFBP-2, and IGFBP-4 were elevated at this time, as determined by ligand blot analysis. There was no association between growth factor alterations and tissue damage as measured by serum creatinine kinase and aspartate aminotransferase levels. The extent of effects in growing pigs was related to the amount of the original parasite inoculum. During the acute phase of infection 2 of 3 pregnant sows aborted. The third sow went to term, but piglets were stillborn or died within 24 hr. Compared to uninfected controls, serum concentrations of IGF-I in infected pregnant sows were depressed during and after the acute phase of the infection. Levels of unsaturated serum IGFBPs in pregnant sows were not affected. These data suggest that decreased IGF-I levels and/or elevated levels of specific forms of IGFBPs may be a mechanism by which growth is affected in feeder pigs infected with S. miescheriana.

Didelphostrongylus hayesi gen. et sp. n. (Metastrogyloidea: Filaroididae) from the opossum, Didelphis marsupialis.

Didelphostrongylus hayesi gen. et sp. n. from the opossum, Didelphis marsupialis, is distinguished from other genera of the Angoistrongylinae by the structure of the dorsal and lateral rays, complex gubernaculum, and ovoviviparity. Third-stage larvae developed in Mesodon perigraptus and Triodopsis albolabris. The prepatent period was 22 days in experimentally infected opossums.

In vitro excystation and structure of Sarcocystis suicanis Erber, 1977, sporocysts.

Methods previously described for artificially excysting coccidia were applied to oocysts and sporocysts of Sarcocystis suicanis recovered from the intestinal mucosa of experimentally infected dogs. Observations with a scanning electron microscope showed that sporocysts consisted of 4 plates, each with a series of accessory veinations; plate margins joined at obtuse angles; plates separated at their margins and curled inward, allowing the sporozoites to escape. Transmission electron microscopy suggested that the sporocyst wall is ultrastructurally similar to that of other coccidia.

A dot-ELISA mimicry western blot test for the detection of swine trichinellosis.

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody affinity chromatography was developed for detecting Trichinella spiralis infection in swine. The test was as sensitive as an ELISA using excretory-secretory products as antigen and western blot analysis, and nearly as specific as the western blot. The dot-ELISA detected all of 20 low infections (0.08-4.74 larvae per gram of diaphragm), most of them by 5-6 wk postinfection. Sera from 1,960 farm-reared swine were tested by conventional ELISA, dot-ELISA, and western blot. Of the 1,960 sera, 262 (13.4%) were considered positive on conventional ELISA, 16 (0.82%) by dot-ELISA, and 15 (0.77%) by western blot. The improved specificity was achieved by employing species-specific denatured antigens. More importantly, the dot-ELISA was much simpler to perform than western blot analysis. The principles employed in this test can be adapted to other infectious diseases, such as AIDS.

Isolation of Trichinella-specific antigens for diagnosis by gradient monoclonal antibody affinity chromatography.

Monoclonal antibodies were generated for the isolation of specific antigens from Trichinella spiralis. Two monoclonal antibodies (7G6-2 and 10B6-1) of class IgG2b and IgG1 were selected according to their reactivities in an enzyme-linked immunosorbent assay and western blot. Clone 7G6-2 reacted with an antigen with molecular mass of approximately 60 kDa, and clone 10B6-1 bound to multiple antigens ranging from 49 to 62 kDa on western blot. Antibodies of each clone were purified partially from mouse ascites fluid by ammonium sulfate precipitation and were coupled to CNBr-activated Sepharose 4B. Antigens with molecular masses of 49 kDa and 57 kDa (P49/57), 52-62 kDa (P52/62), and 60 kDa (P60) were isolated from larval excretory-secretory products and crude worm extract with column 10B6-1 and column 7G6-2, respectively, in part by changing the pH of elution buffers. These antigens were mostly glycoproteins, strongly immunogenic, and specific to the parasite.

Repeated low-level infection of white-tailed deer with Parelaphostrongylus andersoni.

Two white-tailed deer (Odocoileus virginianus) were given 5 infective larvae of Parelaphostrongylus andersoni each weekday for 13 weeks. At 23 weeks one of these deer and a control were challenged with single doses of 200 third-stage larvae. Repeated low-level infection of P. andersoni resulted in a sustained leukocytosis with an absolute eosinophilia which declined only after administration of larvae ceased, partial failure of worms to become established in the musculature, reduced numbers and reduced viability of eggs in the lungs, and an apparent active immunity which enabled the deer to resist challenge. The results of this study suggest that wild deer become infected with P. andersoni by isolated chance encounters with infected gastropods.

Apteragia pursglovei sp. n. (Trichostrongyloidea: trichostrongylidae) from the white-tailed deer, Odocoileus virginianus.

Two species of Apteragia were found in white-tailed deer (Odocoileus virginianus) from 152 counties in 13 southeastern states. Specimens previously reported as Skrjabinagia odocoilei were reidentified as belonging to 2 similar species of the genus Apteragia, A. odocoilei, and A. pursglovei sp. n. Apteragia pursglovei sp. n. is differentiated primarily by the length, conformation, and degree of sclerotization of the spicules. Of the 824 deer, A. odocoilei occurred in 76.5%, A. pursglovei in 13.8%, both species in 5.0%, and neither in 4.7%. Reassessment of distribution data revealed that only A. odocoilei was present in deer from 99 counties, only A. pursglovei in deer from 25 counties, and both species in deer from 28 counties. Both A. odocoilei and A. pursglovei were found in Alabama, Arkansas, Florida, Georgia, Kentucky, Louisiana, Mississippi, North Carolina, South Carolina, and Virginia. Apteragia odocoilei also occurred in Maryland, Tennessee, West Virginia, Texas, Oklahoma, New Jersey, and the Virgin Islands.

Tetrameres (Tetrameres) tinamicola sp. n. from the crested tinamou, Eudromia elegans, with comments on the subgenus Petrowimeres (Nematoda: Tetrameridae).

Tetrameres (Tetrameres) tinamicola sp. n. is described from the crested tinamou, Eudromia elegans, of Argentina. The new species is most similar to other alate species of the subgenus Tetrameres, but it can be differentiated on the basis of its large size, length and structure of the left spicule, and arrangement of the 5 subventral and 3 sublateral pairs of postanal papillae. A review of the subgenus Petrowimeres is presented including the reassignment of several species to the subgenus Tetrameres.

Trichinella spiralis (T1) and Trichinella T5: a comparison using animal infectivity and molecular biology techniques.

We compared Trichinella T5 of bobcat (Lynx rufus) origin with Trichinella spiralis (T1) by using animal infectivity and molecular biology techniques. Swine, SD rats, and CF1 mice were highly resistant to infection with Trichinella T5 but sensitive to T. spiralis, whereas deer mice (peromyscus maniculatus) had similar sensitivity to both parasites. The fecundity of Trichinella T5 in deer mice was 10-35-fold higher in comparison to the fecundity in laboratory rodents (SD rats and CF1 mice). Fecundity of T. spiralis was approximately the same in both groups. A western blot, using excretory-secretory proteins (ESP) from first-stage larvae of T. spiralis as antigen, showed similar banding patterns in the pigs infected with either T. spiralis or Trichinella T5, however, the homologous reaction was stronger than the heterologous reaction. Antibodies were detectable in swine sera commencing 3 or 5 wk postinfection with T. spiralis or Trichinella T5, respectively. Complementary DNAs encoding the 46-, 49/43-, or 53-kDa ESP showed 3.54, 1.94, and 5.91% differences, respectively, between the 2 parasites. Deduced amino acid sequences of the 3 cDNAs were different at 7.20, 5.08, and 8.55%, respectively. All recombinant proteins of the 3 cDNAs from both parasites could detect antibodies in positive sera. The sequences of cDNAs encoding the 46-, 49/43-, or 53-kDa ESP from T. spiralis are also compared to the previously reported sequences, and the differences are discussed.

Exposure of swine to Trichinella spiralis antigen as determined by consecutive ELISAs and western blot.

Exposure of swine to Trichinella spiralis was evaluated using a combination of 3 consecutive enzyme-linked immunosorbent assays (ELISAs) based on larval T. spiralis excretory-secretory antigen as screening test and western blot analysis as confirmatory test. Ninety-three of 32,693 domestic swine sera collected in Georgia over a 5-yr period contained antibodies specific to T. spiralis (prevalence of exposure = 0.28%). The highest prevalence (0.52%) of exposure to T. spiralis was in samples from stockyards and salebarns. Prevalence of exposure in samples from cull sows from 1 slaughter house was 0.38% compared with 0.17% in samples obtained from farms. Pepsin-HCl digestion of diaphragms from 49 swine from 6 seropositive farms revealed 0.01 larvae/g in 4 swine from 3 farms. Determination of T. spiralis infection status of farms appears to be accurately determined with this combination of exploratory ELISAs and confirmatory western blot analysis.

Intramuscular Sarcocystis sp. in two cats and a dog.

Sarcocystis sp. was diagnosed in the skeletal muscle of a cat and myocardium of a dog and cat. The cysts were similar in size and structure when examined by light and transmission electron microscopy. The 3 animals were debilitated and probably immunocompromised due to pancytopenia or terminal neoplasia.

Procyonostrongylus muelleri gen. et sp. n. (Metastrongyloidea: Angiostrongylidae) from the raccoon (Procyon l. lotor).

Procyonostrongylus muelleri gen. et sp. n. from under the epithelium of the epiglottis of Procyon l. lotor is distinguished by 1) the absence of lips; 2) a highly developed reinforced bursa with a deep terminal incision; 3) highly developed toothlike dorsal rays; 1) greatly reduced externodorsal rays; and 5) the terminal or subterminal anus and vulva in the female.

Development of Sarcocystis suicanis Erber, 1977 in the pig.

Twenty-eight weanling pigs inoculated with sporocysts from an isolate of Sarcocystis suicanis from Georgia were examined at intervals ranging from 2 to 90 days postinoculation (DPI). Merogony was first observed histologically within the heart muscle 12 DPI and within 23 of 35 tissues examined 13 DPI. Most infected cells were "floating" in extravascular spaces and were near intact endothelial cells. In some cases, the infected cell clearly was an endothelial cell comprising a portion of the capillary wall. Immature sarcocysts containing metrocytes were observed in striated muscle 27 DPI, and bradyzoites were detected by digestion techniques 52 DPI. Sarcocysts matured between 27 and 80 DPI, after which thickness of the cyst wall and morphology of bradyzoites changed little. Dissolution of sarcocysts was detected as early as 38 DPI and was accompanied by ingress of plasma cells, lymphocytes, and occasionally, eosinophils. Based on information presented herein, feeder pigs reared on pasture may become infected, and infections mature well within the 100-day period usually considered necessary for production of marketable swine.