Salivary IgA as a useful non-invasive marker for restraint stress in pigs.

In this study, we investigated the expression of immunoglobulin A (IgA) in porcine salivary gland and its relationship with restraint stress in pigs. IgA was expressed in plasma cells in pig salivary gland, as confirmed by immunohistochemical staining. IgA was also detected in pig saliva itself by ELISA, and salivary IgA levels were increased by a restraint stress. Moreover, there was a circadian rhythm of IgA over the course of a day. These results are the first evidence of IgA expression related to stress in the pig saliva and may make IgA useful as a non-invasive biological marker to evaluate acute stress condition in the pigs.

Serological survey of Toxoplasma gondii in wild waterfowl in Chukotka, Kamchatka, Russia and Hokkaido, Japan.

Antibodies to Toxoplasma gondii were assayed by ELISA in 22 experimentally inoculated domestic ducks. In addition, a serological assay was carried out at Obihiro, Hokkaido, Japan, in 2004 and 2005, on 221 wild ducks of 5 species: Anas platyrhynchus (n = 111); A. poecilorhyncha (n = 27); A. acuta (n = 58); A. penelope (n = 16); and A. crecca (n = 9). Assays were also conducted using sera from 197 wild geese of 2 species, i.e., Anser albifrons (n = 162) and Ans. fabalis (n = 35). Birds were collected between 2003 and 2005 from 3 different areas: Lake Miyajima-numa, Hokkaido, Japan, regions around Anadyr city of Chukotka autonomous okrug, and Lake Makobetukoe, Kamchatka oblast, Russia. The ELISA cutoff value (OD) was > or =0.395 based on results from uninfected ducks; the final dilution ratio recognized as positive was represented by the end titer. The end titer in the experimentally infected ducks ranged from 1:400 to 1:3,200. Antibodies to T. gondii were found in 49 of the 221 wild duck samples from Japan: A. platyrhynchus (22/74); A. poecilorhyncha (2/15); A. penelope (3/16); A. acuta (4/58); and A. crecca (0/9), all in 2004. In 2005, T. gondii was found in A. platyrhynchus (13/37); and A. poecilorhyncha (5/12). Thirty-two of 197 wild goose samples were seropositive, i.e., Ans. albifrons (7/51) in 2004 and (11/72) in 2005 in Miyajima-numa, Japan and 9/39 in Chukotka, Russia as well as in Ans. fabalis (5/35) in Kamchatka, for which the end titer ranged from 1:100 to 1:3,200. In immunoblotting, the A. platyrhynchus samples showed specific IgG antibody binding to several antigens in the T. gondii lane, i.e., at 30 and 43 kDa, but not in the Neospora caninum lane. No specific bands were noted in samples for which antibody activity was not detected. These results suggest that wild waterfowl inhabiting Hokkaido, Chukotka, and Kamchatka may be exposed to T. gondii.

Proliferative enteropathy involving Lawsonia intracellularis infection in rabbits (Oryctlagus cuniculus).

Five rabbits suffering from diarrhea were diagnosed with proliferative enteropathy (PE). Histopathology revealed a thickened mucosa consisting of hyperplastic intestinal epithelium and infiltration of inflammatory cells mainly consisted of macrophages. In the affected epithelial cytoplasm, numerous curved bacillus-like organisms were observed in the Warthin-Starry silver stain and electron microscopy observation. In polymerase chain reactions, Lawsonia intracellularis-specific DNA fragment were amplified from affected ileal tissue extracted DNA in each case and present 5 cases were confirmed to be L. intracellularis infection. Serum collected from the affected rabbit was immunohistochemically reactive with L. intracellularis in tissue sections from pigs with porcine proliferative enteropathy, as well as with tissue sections from the five affected rabbits. Thus, serum obtained from the affected rabbit may be applicable to immunohistochemical detection for L. intracellularis infection in other species.

A survey of abortifacient infectious agents in livestock in Luzon, the Philippines, with emphasis on the situation in a cattle herd with abortion problems.

In the Philippines, insufficient consideration has been given to the implementation of systematic control measures against major abortifacient infectious agents in livestock. To elucidate the epidemiology of abortifacient infectious agents in livestock, the prevalence of four abortifacient agents was assessed. Initially, a total of 96 cattle including 17 cows with history of abortion were examined in a herd in Luzon at the request of the farm owner. Six (35.3%) of the 17 aborting cows were found to be serologically positive for Neospora caninum (N. caninum), whereas the seroprevalence in non-aborting cows was 15.9% (10/63). Four of the 6 serologically positive aborting cows were also RT-PCR-positive for bovine viral diarrhea virus (BVDV). Two (12.5%) of the 16 bulls examined were also found to be infected with BVDV, suggesting a putative risk factor of transmission via semen. Based on sequence analysis, the isolates detected belong to BVDV type 1b group. Furthermore, an epidemiological survey of abortifacient infectious agents was conducted with various species of livestock from herds located in Luzon. Out of the 105 water buffalo samples collected, 4 (3.8%) were indicated positive to N. caninum, 2 (1.9%) to Toxoplasma gondii (T. gondii) and 2 (1.9%) to Trypanosoma evansi (T. evansi). The overall seroprevalence of N. caninum in goat and sheep were 23.6% (21/89) and 26.3% (10/38), respectively. BVDV was not detected in these herds. The findings of this exploratory study indicate a relationship between infection and bovine abortion and that a lager study is required to statistically confirm this relationship.

Toxoplasma gondii: detection of MIC10 antigen in sera of experimentally infected mice.

We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection.

C57BL/6 mice infected with Neospora caninum during administration of progesterone show bias toward type 2 immune response.

To clarify the role of progesterone in the development of immune responses during pregnancy against Neospora caninum infection, C57BL/6 mice were given a progesterone pellet, and measured on Interferon-gamma and interleukin-4 production following the infection. IFN-gamma production in the prescribed group was significantly lower than that in the intact group on day 40 post administration. IL-4 producing cell population in the prescribed group was larger than that in the intact group. These results suggest that progesterone may alter the balance of cytokine production, and that the bias toward type 2 immune response may remain for a certain period after the infection.

Possibility of Neospora caninum infection by venereal transmission in CB-17 scid mice.

CB-17 scid and BALB/c male mice were inoculated intraperitoneally with Neospora caninum to examine the possibility of its venereal transmission. Some of these mice were killed on days 7 and 20 post-inoculation to examine the genital organs for presence of the parasite. The remaining scid male mice were housed with non-infected female mice from day 7 p.i. and kept with them for 14 days. These scid mice died between days 28 and 35 p.i. N. caninum DNA was detected in the testis of mice on days 7 and 20 p.i. by PCR and tachyzoite viability was determined by bioassay conducted by means of mouse inoculation. Microscopically, fewer tachyzoites were detected in the testis obtained on day 20 p.i., than in other organs. The inoculated BALB/c male mice survived until the end of the experiment with no clinical signs and N. caninum DNA was detected in the testis on day 7 p.i. but not on day 14 p.i. Five of eight female scid mice housed with infected males became pregnant. Tachyzoites were detected in three of these mice and their neonates (n=3, 5 and 13, respectively). In three non-pregnant mice, no parasite was detected. Two of the four female BALB/c mice housed with infected male scid mice became pregnant but the parasite was not detected in them or in the neonates (n=3 and 13, respectively). These results indicate that the tachyzoites were present in the genital organs of the immunodeficient mice from day 7 p.i. and suggest that transmission may occur through mating with male mice.

Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential.

A combination of antigenic regions of microneme proteins have been previously reported as being protective against chronic toxoplasmosis. In this work, we evaluated immune responses induced by immunizing BALB/c and C57BL/6 mice intradermally with plasmid DNA encoding the protein sequences of Toxoplasma gondii AMA1, MIC2, M2AP and BAG1. Mice immunized with the AMA1 gene developed high levels of serum IgG2a and c antibodies as well as cellular immune responses associated with IFN-gamma synthesis suggesting a modulated Th1 type of response. Immunization with the AMA1 gene resulted in a partial but significant protection against the acute phase of toxoplasmosis compared to MIC2, M2AP and BAG1 genes. Therefore, the AMA1 gene appears to generate a strong specific immune response and also provides effective protection against toxoplasmosis more than the MIC2, M2AP and BAG1 genes.

Acute restraint stress enhances calcium mobilization and proliferative response in splenic lymphocytes from mice.

Calcium (Ca2+ ) plays an essential role in lymphocyte activation and maturation. Acute and chronic stress has been shown to modulate the lymphocyte immune response; but the relationship between cytosolic free Ca2+ concentration ([Ca2+ ]i) and the immune response in lymphocytes following exposure to stress has not been examined. In the present study, we investigated the effects of acute restraint stress on [Ca2+ ]i and the proliferation of splenic lymphocytes from mice. We observed that 2 h of restraint significantly increased plasma corticosterone levels in mice. On examining [Ca2+ ]i and the proliferation ex vivo of splenic lymphocytes isolated from restraint-stressed mice using fura-2 and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, respectively, we found that acute restraint stress caused a significant increase in resting [Ca2+ ]i and significantly enhanced the ability of concanavalin A (Con A; a T-cell-selective mitogen) to increase [Ca2+ ]i but not that of lipopolysaccharide (LPS; a B-cell-selective mitogen). In addition, acute restraint stress significantly enhanced Con A-stimulated but not LPS-stimulated lymphocyte proliferation. Overall, there was a positive correlation between [Ca2+ ]i and T-cell proliferation following acute restraint stress. The enhancements of [Ca2+ ]i and T-cell proliferation were completely suppressed by verapamil (a Ca2+ channel blocker). These results suggest that acute restraint stress enhances Con A-stimulated T-cell proliferation by increasing [Ca2+ ]i via stimulation of Ca2+ entry.

A serological survey of Brucella spp. in free-ranging wild boar (Sus scrofa leucomystax) in Shikoku, Japan.

Brucella, a causative agent of brucellosis and facultative intracellular pathogen, has been isolated recently from a variety of wild mammals. In this study, serum samples from 115 Japanese wild boar (Sus Scrofa leucomystax) killed by hunters in the 4 Prefectures of Shikoku, Japan were tested for antibodies to Brucella spp. by means of the tube agglutination test (TAT) and enzyme-linked immunosorbent assay (ELISA) using antigens extracted with n-lauroylsarcosine. In 9 of the 115 samples (7.8%) antibodies to Brucella spp. were detected by TAT and ELISA. These results suggest that wild boar in Shikoku may be exposed to Brucella spp. or other cross-reactive pathogen infection.

Antibodies to Brucella spp. in Pacific bottlenose dolphins from the Solomon Islands.

Brucella spp. have been recently isolated from a variety of marine mammals. Serum samples from 58 Pacific bottlenose dolphins (Tursipa aduncus) from the Solomon Islands were tested for antibodies to Brucella spp. by the tube agglutination test (TAT), enzymed-linked immunosorbent assay (ELISA), and immunoblotting. Anti-Brucella spp. antibodies were detected by TAT and ELISA in 31 and 40 of 58 samples, respectively. These results suggest that Pacific bottlenose dolphins from the Solomon Islands are infected with Brucella spp. or a Brucella-like organism.

Investigation for presence of Neospora caninum, Toxoplasma gondii and Brucella-species infection in killer whales (Orcinus orca) mass-stranded on the coast of Shiretoko, Hokkaido, Japan.

Twelve killer whale (Orcinus orca) were hemmed in by ice floes, and nine died on the Aidomari coast in the Nemuro Strait in Rausu, Shiretoko, Hokkaido, Japan on 8 February 2005. Tissue samples collected from 8 whales were tested for Neospora caninum, Toxoplasma gondii, and Brucella species DNA by polymerase chain reaction (PCR) assay. Gamma-globulin isolated from blood samples by ammonium sulfate precipitation was tested for antibodies to these pathogens by means of agglutination tests and immunoblotting. None of the 8 tissue samples had antibodies to the pathogens, when subjected to agglutination tests. In immunoblotting, one sample (sample No.5) showed antibody binding to N. caninum antigens. In the PCR assay, none of the samples was positive. Further study is necessary to examine the prevalence of the pathogens in marine mammals inhabiting this area.

Prevalence of Toxoplasma gondii and Neospora caninum in sika deer from eastern Hokkaido, Japan.

Brain and serum were collected from 120 and 12 free-ranging sika deer (Cervus nippon yesoensis), respectively, from six regions in eastern Hokkaido during controlled hunts in the autumn of 2003. Brains were tested for Neospora caninum and Toxoplasma gondii DNA by polymerase chain reaction (PCR) assays. Antibodies to Toxoplasma gondii were measured by means of a latex agglutination test. No brain tested positive for either type of DNA, and no antibody to Toxoplasma gondii was detected in serum, suggesting a low prevalence of infection with these organisms in free-ranging sika deer from eastern Hokkaido. Further examination of multiple tissues by PCR and serologic surveys will be necessary to confirm this.

Detection of Lawsonia intracellularis using immunomagnetic beads and ATP bioluminescence.

Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in various animals. The detection of L. intracellularis in clinical and environmental samples is necessary for the diagnosis of infection and epidemiological investigations. For the detection of L. intracellularis in fecal samples, we have developed an immunological method using immunomagnetic separation and ATP bioluminescence. Magnetic beads were coated with an anti-Lawsonia surface antigen (LsaA) antibody in order to capture the L. intracellularis in fecal samples from infected rabbits and the bacteria captured by the immunomagnetic beads were assayed by means of ATP bioluminescence. Our results showed that L. intracelluraris was detected by immunomagnetic separation of bacteria-holding magnetic beads and ATP-based bioluminescence, suggesting that our methods could be useful for the diagnosis of proliferative enteropathy.

Antibodies against Toxoplasma gondii in the Pacific bottlenose dolphin (Tursiops aduncus) from the Solomon Islands.

Serum samples from 58 Pacific bottlenose dolphins (Tursiops aduncus) from the Solomon Islands were tested for the IgG antibody to Toxoplasma gondii by the latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. The ELISA cut-off value was taken as OD > or = 0.276, and the final dilution ratio, recognized as positive, was represented by the end titer. In 25 of 58 samples, no antibody activity was detected by LAT and ELISA. In 8 of 58 samples, anti-T. gondii IgG antibodies were detected by both LAT and ELISA, with titers of greater than 1 : 64 and 1 : 160, respectively. By immunoblotting, the 8 serum samples producing higher titers showed specific antibody IgG binding to several antigens on the T. gondii lane, but not on the Neospora caninum lane. No specific bands were noted on the lanes for either parasite in the 25 serum samples for which no antibody activity was detected. The specific binding of IgG antibodies to T. gondii antigens observed for serum samples producing higher titers suggests that Pacific bottlenose dolphins from the Solomon islands are exposed to T. gondii.

Enzyme-linked immunosorbent assay to detect Lawsonia intracellularis in rabbits with proliferative enteropathy.

Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in domestic and experimental animals. Antiserum against synthetic peptides of the Lawsonia surface antigen (LsaA) well recognized L. intracellularis in infected ileum by immunohistochemistry. The synthetic peptides in LsaA showed strong reaction with serum from rabbits infected with L. intracellularis by enzyme-linked immunosorbent assay. These results suggest that ELISA used synthetic peptides in LsaA and anti-LsaA serum might be useful to diagnose for proliferative enteropathy.