ABSTRACT Protection against the avian influenza (AI) H5N1 virus is suspected to be mainly conferred by the presence of antibodies directed against the hemagglutinin (HA) protein of the virus. A single electroporation delivery of 100 or 250 μg of a DNA vaccine construct, pCAG-HA, carrying the HA gene of strain A/Hanoi/30408/2005 (H5N1), in chickens led to the development of anti-HA antibody response in 16 of 17 immunized birds, as measured by a hemagglutination inhibition (HI) test, competitive enzyme-linked immunosorbent assay (cELISA), and an indirect ELISA. Birds vaccinated by electroporation (n = 11) were protected from experimental AI challenge with strain A/chicken/Pennsylvania/1370/1/1983 (H5N2) as judged by low viral load, absence of clinical symptoms, and absence of mortality (n = 11). In contrast, only two out of 10 birds vaccinated with the same vaccine dose (100 or 250 μg) but without electroporation developed antibodies. These birds showed high viral loads and significant morbidity and mortality after the challenge. Seroconversion was reduced in birds electroporated with a low vaccine dose (10 μg), but the antibody-positive birds were protected against virus challenge. Nonelectroporation delivery of a low-dose vaccine did not result in seroconversion, and the birds were as susceptible as those in the control groups that received the control pCAG vector. Electroporation delivery of the DNA vaccine led to enhanced antibody responses and to protection against the AI virus challenge. The HI test, cELISA, or indirect ELISA for anti-H5 antibodies might serve as a good predictor of the potency and efficacy of a DNA immunization strategy against AI in chickens.

A needle-free delivery system was assessed as a route for providing quick, safe, and effective vaccination against avian influenza (AI). Two groups of chickens were vaccinated with a commercially available inactivated H5N3 virus vaccine delivered either with a needle-free device or with the conventional syringe-and-needle method recommended by the vaccine manufacturer. The kinetic aspects of seroconversion, peak antibody levels, and antibody titers were measured by a combination of an indirect enzyme-linked immunosorbent assay and the hemagglutination-inhibition test and were all found to be similar in the 2 groups of chickens. We conclude that the needle-free delivery system could result in effective immunization against H5N1 AI epidemics and pandemics in chickens.

Cattle infected with the tapeworm cyst, Taenia saginata metacestode (synonym: Cysticercus bovis) are a source of human infection if affected beef is eaten raw or undercooked. Control measures targeted at individual cattle rather than all animals in a T. saginata-exposed herd should help reduce costs and alleviate current constraints associated with managing an outbreak. To that end, we have developed a reliable diagnostic test for use in live animals that would enable veterinary regulators to focus disease control strategies. The test detects bovine anti-T. saginata immunoglobulin G1 antibodies using an enzyme-linked immunosorbent assay (ELISA) which relies on the excretory-secretory antigens of T. saginata. Animals were inoculated with 10, 100 or 1000 viable T. saginata eggs in order to simulate the parasite burden of field-infected animals (parasite load = 1–86; n = 28). By testing sera obtained from the inoculated animals 84 days post-inoculation, test sensitivity was estimated to be 92.9% (95% confidence interval or CI = 83.4–100.0%). Another 17 animals inoculated with 5000 or 10,000 viable eggs of T. saginata and shown to harbour metacestodes at post-mortem, all tested positive in the ELISA. Test specificity estimated from a herd of field animals with no historical, epidemiological, or post-mortem evidence of infection was 90.6% (95% CI = 87.0–94.2%; n = 256 field cattle). Using the test on samples (n = 347) from a T. saginata-infected feedlot, the Bayesian approach estimate of seroprevalence was 4.6% (95% probability intervals = 0.5–10.3%). The test performance characteristics of the ELISA suggest that it will be adequate for field application in bovine cysticercosis outbreaks.

The meningeal worm (Parelaphostrongylus tenuis; Family: Protostrongylidae) of white-tailed deer (Odocoileus virginianus) in eastern North America is a cause of neurologic disease and mortality in co-habiting, native cervid species and certain livestock. The traditional method of identifying animals exposed to the parasite relies on detecting excreted larvae in feces and has been found inadequate. New enzyme-linked immunosorbent assays (ELISAs) using excretory-secretory antigens of the infective larvae of Parelaphostrongylus tenuis to detect antibodies in infected white-tailed deer, elk (Cervus elaphus), and moose (A Ices alces) have been developed. Subsequently, the tests were rigorously evaluated to assess their fitness-for-use as tools for preventing the spread of the parasite through animal translocation and for clinical diagnosis. For the purpose of developing reliable performance parameters for the ELISAs, serum samples obtained from the three cervids species, either naturally- or experimentally-infected, were tested. All three tests showed high sensitivity (>= 97%) and specificity (similar to 100%), good repeatability, and reproducibility, and are deemed useful for detecting cervids with either early exposure to P. tenuis or with long-standing infections. The tests are now commercially available for diagnostic use. The application of the ELISA test in a free-ranging elk showing characteristic clinical signs was instrumental in achieving the first antemortem laboratory diagnosis of P. tenuis infection.

Abstract Newly developed serological tests for diagnosing parelaphostrongylosis in cervids, using the excretory-secretory products (ES) of the infective larvae of Parelaphostrongylus tenuis in enzyme-linked immunosorbent assays (ELISAs), have demonstrable superiority over the traditional method of larval recovery and microscopic identification. To generate a source of ELISA antigen by genetic engineering, we created a complementary DNA (cDNA) expression library by the reverse transcription of mRNA of P. tenuis adult worms and by ligation with the vector λ-ZAP II. The library was screened using antisera produced in mice by immunization with a somatic antigen preparation of adult worms. Seventeen clones were isolated, sequenced, and checked for similarity to other DNA sequences in GenBank. A previously identified parasite gene encoding an aspartyl protease inhibitor (API) was isolated from the cDNA library, subcloned, and expressed using the pET expression vector to produce a glutathione S transferase (GST)–His-S-tag-P. tenuis API fusion protein (molecular weight = 63 kDa). An ELISA utilizing the API fusion protein as the coating antigen was used to serologically diagnose all white-tailed deer (WTD, Odocoileus virginianus; 10 out of 10) that had been inoculated with a range of 6–150 L3 P. tenuis, indicating that the antigen may be a useful serodiagnostic antigen for P. tenuis infection in this cervid species.

We evaluated the indirect fluorescent-antibody (IFA) test and complement-fixation (CF) test for diagnosis of equine piroplasmosis in the absence of a gold standard. Using Evan's blue, we estimated the specificity of the IFA test on a parasite-free, field horse population to be 98% (95% confidence interval=97, 99). We observed an excellent test agreement (kappa=0.83) between two collaborating laboratories when the IFA test was performed on identical samples from an endemic area. Using Bayesian analysis with informative prior probability distributions, we estimated the sensitivity of the IFA test to be 92% (95% probability interval, PI=81, 98), and specificity to be 95% (95% PI=88, 99). The CF test sensitivity and specificity estimates were 28% (95% PI=15, 47) and 99% (95% PI=96, 100), respectively. We found the IFA to be superior to the CF test, and the inclusion of Evan's blue in test protocol improved the performance of the IFA test. We conclude that the IFA test for Babesia caballi is a sensitive and specific test for the diagnosis of equine piroplasmosis.

Thirty-six moose (Alces alces) reported as acting abnormally were examined in north- western Ontario and adjacent northeastern Minnesota in 1986 - 2000. Thirty-four typically had little fear of humans, remained in an area for some time, and showed clinical signs of neuromotor inco- RUGLQDWLRQLQFOXGLQJZDONLQJLQFLUFOHV��VKRZLQJZHDNQHVVDQGG

The antemortem detection of a Parelaphostrongylus tenuis infection in a free-ranging wild elk (Cervus elaphus) in southern Ontario is documented. Postmortems on other free-ranging elk that died during 2000-2005 indicated that 59% (17/29) were infected with P tenuis, based on presence of lesions in the brain.

The indirect fluorescent antibody (IFA) test for Theileria equi was evaluated to assess test's suitability for the serological diagnosis of equine piroplasmosis, to provide performance parameters for the purpose of test validation, and to compare it with the complement fixation (CF) test. Using a protocol that included Evan's blue, the specificity of the IFA test was estimated at 99.0% for T. equi by the classical method of analysis, and 96.6% by the Bayesian method. The use of Evan's blue in the test protocol increased test specificity and contributed to an excellent test agreement between two collaborating laboratories (kappa = 0.96). Using Bayesian analysis, the sensitivity estimate for the IFA test was 89.2%. The CF test sensitivity and specificity estimates for T. equi were 63.1 and 96.4%, respectively, as determined by Bayesian analysis. The IFA test was more sensitive than the CF test but the specificity estimates were similar.

Immunoglobulin M (IgM) antibodies to the variant surface glycoproteins (VSG) of African trypanosomes are the first and predominant class of anti-trypanosomal antibodies in the infected host. They are a major factor in controlling waves of parasitemia, but not in long-term survival. The macrophage receptor(s) that enables phagocytosis of IgM anti-VSG-coated African trypanosomes is unknown. We assessed whether complement receptor CR3 (CD11b/CD18) might be involved in mediating phagocytosis of Trypanosoma congolense. We show that murine complement C3 fragments are deposited onto T. congolense when the trypanosomes are incubated with IgM anti-VSG and fresh mouse serum. In the presence of fresh mouse serum, there is significantly and markedly less phagocytosis of IgM-opsonized T. congolense by CD11b-deficient macrophages compared to phagocytosis by wild-type macrophages (78% fewer T. congolense are ingested per macrophage). Significantly less tumor necrosis factor (TNF)-α (38% less), but significantly more nitric oxide (NO) (63% more) are released by CD11b-deficient macrophages that have engulfed trypanosomes than by equally treated wild-type macrophages. We conclude that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of T. congolense by macrophages. We further conclude that IgM anti-VSG-mediated phagocytosis of T. congolense enhances synthesis of disease-producing TNF-α and inhibits synthesis of parasite-controlling NO. We suggest that signaling of inhibition of NO synthesis is mediated via CR3.

A newly developed immunohistochemical test was used for the first time to demonstrate the presence of Taenia saginata (Cysticercus bovis) antigens in the lymph nodes of a heifer calf experimentally inoculated with Taenia saginata eggs. The new test should aid in the differential diagnosis of eosinophilic lymphadenitis in cattle.

A new method of diagnosing cysticercus or larval stage of the human tapeworm, Taenia saginata, also known as Cysticercus bovis, in formalin-fixed bovine tissue was developed using a monoclonal antibody to T. saginata and avidin–biotin complex immunohistochemistry. Grossly recognizable viable and degenerate cysts were identifiable after immunohistochemical staining and could be differentiated from Sarcocystis, Actinobacillus, or non-cyst, normal bovine structures. The new test should permit laboratory confirmation of suspected T. saginata cysticercus lesions.

A 3-day old female bison calf (Bison bison) was presented in lateral recumbency to the Université de Montréal Veterinary Teaching Hospital. The animal was severely depressed and dehydrated (10%) and died a few hours after admission. Prior to death, blood samples were obtained for CBC, clinical chemistry, and serology tests. Abnormal CBC findings included thrombocytopenia, lymphocytosis, mild monocytosis, and a toxic left shift. Abnormal serum clinical chemistry findings included marked azotemia, hyperphosphatemia, hypernatremia, hyperalbuminemia, hypoglobulinemia, and low gamma-glutamyltransferase activity. Serologic test results for bovine leukosis virus and bovine viral diarrhea virus were negative. Blood smear examination revealed numerous elongated organisms that were tapered at both ends and characterized by an undulating membrane and a long flagellum. The organisms ranged in length from 35 to 40 micro meter, excluding the flagellum, and were identified as Trypanosoma theileri. Postmortem examination revealed that the animal suffered from concurrent mycotic abomasitis and colisepticemia.

Confirming Parelaphostrongylus tenuis infection in moose (Alces alces) and other susceptible hosts is difficult. An enzyme-linked immunosorbent assay (ELISA) was developed using the excretory-secretory (ES) products of third-stage P. tenuis larvae (ES-ELISA) and the test applied to serum samples obtained from seven moose calves (5–9.5 mo old) given infective larvae (L3) in doses approximating those likely to be received in nature (3–30 L3). Anti-P. tenuis immunoglobulin G antibodies were detected in all seven inoculated moose during the course of infection until the termination of experiment 61–243 days post-inoculation (DPI). Five animals tested between 16–25 DPI had significant antibody levels, while a sixth animal did not test positive until 46 DPI. The seventh animal was not tested until 199 DPI. Antibody levels remained elevated in all five animals that harbored adult worms at the termination of the experiment. Whereas, antibody levels showed a gradual decline in the two remaining animals, presumably because of death of worms, and antibodies were undetected in one animal at the time of necropsy. The other animal displayed an anamnestic increase in antibody level following a challenge inoculation of infective larvae. Terminal and peak optical density (OD) values detected by ES-ELISA strongly correlated with inoculation dose (r=0.98, P=0.02 and r=0.95, P=0.04, respectively) among animals harboring adult worms (n=4) but not significantly with the number of worms recovered postmortem (peak OD, r=0.82, P=0.18; terminal OD, r=0.93, P=0.07). Unlike the ES products, use of somatic antigens of the adult worm in ELISA did not provide satisfactory results. Antibodies to P. tenuis were detectable by ES-ELISA in two of 21 free-ranging moose from an enzootic area but not from any of 23 animals from a non-enzootic area. The ES-ELISA appears to be a useful test for assessing exposure of moose to P. tenuis.

An appreciation of global features of immune regulation may lead to vaccination strategies effective in a genetically diverse population against a number of intracellular pathogens that cause chronic disease. Such global strategies appear more straightforward than strategies requiring a detailed knowledge of the specificity of ‵protective T-cells′. Global strategies may be effective against the virus, bacteria and protozoa that respectively cause AIDS, tuberculosis and the leishmaniases.

Elk infected with the meningeal worm, Parelaphostrongylus tenuis (Protostrongylidae), do not consistently excrete larvae in feces, making the current method of diagnosing live animals using the Baermann fecal technique unreliable. Serological diagnosis could prove more useful in diagnosing field-infected animals but depends on the identification and availability of good quality antigen. To mimic field infections, 2 elk were inoculated with 6 infective L3 larvae of P. tenuis, and another 2 with 20 L3 larvae. Fecal samples were examined for nematode larvae using the Baermann technique and serum samples taken were tested for anti-P. tenuis antibody with ELISAs by using the excretory-secretory (ES) products of L3, and sonicated adult worms as antigens. One animal passed first-stage larvae in its feces 202 days postinoculation, but passed none thereafter. The remaining 3 inoculated animals did not pass larvae. In contrast to parasite detection, antibodies against larval ES products were detected in all animals starting from 14 to 28 days postinoculation and persisted until the termination of the experiment on day 243 in 2 animals that harbored adult worms. Antibodies against somatic antigens of the adult worm were not detected until day 56 but also persisted until the end of the experiment in the animals with adult worms. In 2 elk that had no adult worms at necropsy, anti-ES antibodies were detected transiently in both, while anti-adult worm antibodies were present transiently in one. These findings confirm the superiority of P. tenuis larval ES products over somatic adult worm antigens as serodiagnostic antigens, as previously observed in studies of infected white-tailed deer, and extend the application of the newly developed ELISA test in diagnosing and monitoring cervids experimentally infected with P. tenuis.

Parasites are often found in a milieu that requires extensive preparation and labor-intensive cleaning before they are suitable for use in analytical procedures. Application of modern techniques in immunology and molecular biology demands pure yields of parasites. To purify first-stage (L1) larvae of Elaphostrongylus cervi, fecal suspensions from an infected red deer were processed by the Baermann method and embedded in a gel matrix with the objective of selectively trapping fecal debris. About half the number (50.9%) of embedded larvae migrated out of the gel within a 24-hr period and were collected as clean parasite suspensions, virtually free from fecal debris. The numbers of L1 emigrating from gels were inversely proportional to the fecal debris content and the thickness of the gel. Removal of fecal debris from Baermann fluid by sieving prior to gel embedment enhanced the yield of pure L1.