Footpad reaction induced by Neospora caninum tachyzoite extract in infected BALB/c mice

Little information is available regarding a delayed type hypersensitivity (DTH) reaction in neosporosis. In this study, we examined the elicitation of a DTH reaction in mice infected with Neospora caninum by inoculation of the footpad with tachyzoite antigens. The footpads of BALB/c mice infected with N. caninum and those of non-infected were injected with either the tachyzoite extract, or paraformaldehyde-fixed tachyzoites. In mice inoculated with N. caninum antigens on day 7 p.i. swelling peaked at 6h after injection of the tachyzoite extract. In mice inoculated on days 14, 28 and 56, swelling was observed between 6 and 72 h afterwards. Mice immunized with the tachyzoite extract plus adjuvant showed peak footpad swelling at 6h post injection, and the swelling had decreased at 24h or later. In contrast, mice injected before infection showed no specific swelling. In sections of footpads injected with the tachyzoite extract, exudate had accumulated at 6h post injection and clusters of infiltrated lymphocytes were observed at 48 h post injection. In mice administered anti-CD4+ cell monoclonal antibodies swelling had decreased at 24h post injection of the extract. These results indicate that mice infected with N. caninum produce a DTH reaction, which is a good indicator of the development of type 1 immune responses.     

Relationship between type 1/type 2 immune responses and occurrence of vertical transmission in BALB/c mice infected with Neospora caninum

To examine the relationship between occurrence of vertical transmission and type 1/type 2 immune responses induced by Neospora caninum infection in BALB/c mice, pregnant (group 1 p) and non-pregnant mice (group 1 np) were inoculated with 2 x 10(6) of the N. caninum parasites and then we examined the vertical transmission rate and production of IFN-gamma and IL-4. We also studied chronically infected mice, which were bred at 4 weeks or more after infection (group 2), and mice inoculated during pregnancy and re-bred at 4 weeks or more after delivery (group 3). In groups 1p, 2 and 3, vertical transmission was observed in 27.4, 41.4, and 50% of the offspring, respectively. The serum IFN-gamma level increased on days 1 and 5 post-inoculation (p.i.) in groups 1 p and 1 np, while no increase level was observed in groups 2 and 3 during pregnancy or after delivery. When the mice in groups 2 and 3 were re-inoculated, all mice showed a transient increase in serum IFN-gamma on day 1 post-re-inoculation. The serum IL-4 level in both of groups 1p and np increased in a similar manner following infection. In group 3, the serum IL-4 level was somewhat higher than that in group 2 after re-inoculation. The anti-N. caninum antibody IgG1 titer in group 3 increased on day 10 post-re-inoculation. These results suggest that the mice infected during pregnancy may acquire a weaker immune response to the parasite than mice infected when they are not pregnant, and that mice infected during pregnancy may show an enhanced type 2 immune response in the recrudescence of the infection.     

Neospora caninum: adoptive transfer of immune lymphocytes precipitates disease in BALB/c mice

Neospora caninum is a recently described apicomplexan parasite first isolated from a dog in 1988 and has subsequently been shown to infect a wide range of mammals. In mice, Neospora can cause primary pneumonia, myositis, encephalitis, radiculoneuritis, and pancreatitis. Whereas, certain aspects of the host immune response to Toxoplasma gondii have been well studied, not as much is known about the full immune response to Neospora. This paper examines whether or not immune splenocytes are able to adoptively transfer protection against N. caninum infection in BALB/c mice. Mice receiving immune enriched CD8+ cells had severe neurological signs by 19 days post infection. Mice receiving immune enriched CD4+ cells had mild neurological signs on day 22 post infection. It would appear that additional immune cells can precipitate disease in the presence of circulating lymphocytes.     

Vertical transmission of Neospora caninum in BALB/c mice in both acute and chronic infection

To examine the frequency of congenital infection by Neospora caninum, BALB/c mice were inoculated intraperitoneally with tachyzoites of N. caninum either during pregnancy (Group 1) or 4 weeks or more before pregnancy (Group 2). Further, the mice inoculated during pregnancy were bred at 4 weeks or more after delivery to form Group 3. Congenital transmission was observed in 76% of the neonates of the mice in Group 1 and in 50% of the neonates of the mice in Group 2. Interestingly, congenital transmission was observed in 86% of the neonates from Group 3. These results suggest that chronically-infected BALB/c mice efficiently transmit N. caninum infection to their offspring.     

Influence of Neospora caninum infection in BALB/c mice during pregnancy in post-natal development

The influence of Neospora caninum infection during pregnancy on the post-natal period has been poorly investigated. In a previous study, we suggested that infection with N. caninum during pregnancy could affect the normal post-natal development of the offspring. For this reason, in the present work we evaluated the influence of N. caninum infection in pregnant BALB/c mice at days 0, 7 and 14 of gestation (groups A, B and C, respectively) on the post-natal development of the offspring from birth to day 60 post-partum (PP). Morbidity and mortality, vertical transmission, and histopathological lesions were investigated. The humoral immune response (IgG) of pups was also evaluated. Results showed that infection with N. caninum during pregnancy had fatal consequences for pups, especially during mid-gestation (day 7). Infection provoked a delay in the general development of neonates, clinical signs compatible with neosporosis and severe histopathological lesions. A high mortality rate was found in all infected groups. A 69% of mortality rate was found in group A, a 100% in group B and a 46% in group C. Necrotizing encephalitis and multifocal hepatocellular necrosis were the most severe lesions found. All neonates, except four animals from group C, had antibodies against N. caninum but the immune response was not sufficient to control parasite infection. We have demonstrated that extension of the observation period after N. caninum infection permits a more accurate study of vertical transmission, the major route of parasite transmission, and mortality rates. We propose that infection at mid-gestation (day 7) in BALB/c mice and its study during the post-natal period constitutes a valuable experimental model for testing new chemotherapeutic agents and vaccines designed to protect against congenital neosporosis, in order to select effective protocols before its use on bovine.     

猪圆环病毒2型感染裸鼠及BALB/c小鼠的研究

用猪圆环病毒2型（PCV2）经腹腔注射BALB/c小鼠及裸鼠各15只,每隔2周1次,共感染3次.BALB/c小鼠初次、再次感染后均未出现明显的临床症状,仅有2只小鼠出现病理变化;从血清中可检测到PCV2核酸及其ORF2抗体;同时淋巴细胞增殖反应显著增强,NK和CTL细胞杀伤率显著升高;CD4^＋、CD8^＋、CD3^＋T淋巴细胞及CD19^＋B淋巴细胞数量显著减少.裸鼠也未出现明显的临床症状,从血清中可检测到PCV2核酸,但未检测到PCV2 ORF2抗体;除NK细胞杀伤率显著升高外,其余免疫学指标无显著改变.结果表明,BALB/c小鼠比裸鼠对PCV2易感,各项免疫学指标变化与PCV2感染猪一致,但不表现临床症状,仅个别BALB/c小鼠出现病理变化,说明BALB/c小鼠对PCV2不如猪易感.     

Immune response and antigen recognition in non-pregnant ewes experimentally infected with Neospora caninum tachyzoites

The cellular and humoral responses as well as the antigen recognition during the acute stage of a Neospora caninum (NC) infection were investigated in non-pregnant ewes. The experimentally infected ewes developed specific lymphoproliferative and humoral responses within 2 weeks post-infection (PI). The magnitude of the cellular response showed large variations between animals. A significant decrease in the proliferative response to Con A mitogen and N. caninum, Toxoplasma gondii (TG) antigens was recorded on day 21 post-infection (PI). The humoral response and the pattern of antigen recognition were similar among infected ewes. Proteins of 44, 42, 40, 39 and 28 kDa were intensively recognized by the infected animals during the experiment. The 42 and 28 kDa antigens should be considered as useful for the diagnostic of N. caninum infection, as the intensity of recognition infection of the other antigens had decreased markedly 8 weeks post-infection. For some antigens a sequential recognition was recorded. The 59, 54 and 38-37 kDa proteins were frequently recognized by infected sera during the first weeks of the infection, but recognition of these antigens was absent or rare at the end of the experiment. These antigens could be related to the acute stage of the infection.     

Serological analysis of calves experimentally infected with Neospora caninum: a 1-year study

A serological study was conducted with calves experimentally infected with the protozoan parasite Neospora caninum. The animals were inoculated with either a low or high dose of N. caninum tachyzoites and temperature responses monitored daily for the first 2 weeks after inoculation. Blood samples were collected before inoculation, and at regular intervals thereafter for 1 year. Serological analysis was achieved using an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and an IgG avidity ELISA.Injection of Neospora produced a significant rise in rectal temperature in the high dose group. In addition, the lymph node draining the site of inoculation increased in size following injection in all animals, in both infected groups, before returning to normal by day 14 after injection. Both groups given N. caninum produced specific antibody that was detected by the IFAT and the ELISA, which remained elevated for the 12-month duration of the experiment. The specific Neospora antibodies produced did not cross-react in an IFAT for the detection of antibodies to Toxoplasma gondii. IgG avidity increased 2 weeks after inoculation, in both infected groups, until week 12 when infection was well established. There was a little difference between the two infected dose groups. This study demonstrates that the two different doses of N. caninum produced a similar antibody response, and that the higher dose also induced a febrile reaction. The IgG avidity ELISA was successful at distinguishing between recent and long-standing infection in this study. However, in both groups, there was fluctuation in the levels of specific antibody throughout the yearlong study, which accords with similar experiments in pregnant cattle, where it has been suggested that fluctuation may indicate periodic recrudescence of infection and a re-stimulation of antibody production by antigen.     

Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.     

Neospora caninum infected the alimentary tract of nude mice and was transmitted to other mice by intraperitoneal inoculation with the intestinal contents

Neospora caninum (BT-2 strain) that originated from the brain of a Holstein calf was serially passaged through 10 generations of BALB/c nude mice by intraperitoneal inoculation. Histological examination of the mice revealed that numerous clusters of tachyzoites appeared in the pancreas, stomach and small intestine as well as in the central nervous system (CNS) and skeletal muscles. Intestinal contents of the infected mice were inoculated intraperitoneally into uninfected nude mice and 3 of the 17 inoculated mice showed clinical signs at post inoculation days 3 to 10. The present experiments demonstrated a proliferation of N. caninum tachyzoites in the mucosa of the alimentary tract and pancreas of the nude mice and the intestinal contents of the mice were infective to other nude mice.     

Pathological and immunological study of goose embryos experimentally infected with duck plague virus

A total of 240 embryonated goose eggs obtained from two susceptible flocks were used. Half of the eggs were inoculated into the allantoic cavity with a virulent strain (7593) of duck plague virus isolated from an acute outbreak, and the other half were inoculated with the attenuated vaccine virus (KAPEVAC). Ten, 100 or 1000 CPU/0.1 ml virus were given on days 12 and 20 of incubation. Embryos that died and surviving embryos killed at 5-day intervals were examined by light and electron microscopy. The yolk and the serum of embryos that survived until hatching were assayed for antibody content. Lymphocytes separated from the blood were used for the immuno-rosette formation and lymphocyte stimulation tests. Pathomorphological changes indicative of virus replication occurred in the liver, kidney, myocardium, gizzard muscle and chorioallantoic membrane (CAM) of the embryos in the case of both virus strains. The time of onset and severity of these changes and the time and rate of embryonic mortality depended on the virulence of the strain used for inoculation, the virus dose and the time of inoculation. Virus-neutralizing (VN) antibodies were demonstrable neither in the yolk nor in the serum of goslings exsanguinated after hatching. The lymphocytes recognized the virus antigen in the in vitro cellular tests and responded to it with blastogenic transformation. As opposed to adult birds, in the embryos duck plague virus infection did not cause damage to the digestive tract mucosa and the lymphoid organs.     

Interaction of Tritrichomonas foetus with the reproductive tract of experimentally infected female BALB/c mice: ultrastructural evaluation

The interaction of Tritrichomonas foetus with its host is a complex process that involves colonisation, attachment and persistence. The goal of the present study was to describe the interaction of T. foetus with the genital tract using a model of non-oestrogenised female BALB/c mice which had been intravaginally infected with a suspension of T. foetus during oestrus. Animals were sacrificed after 10 weeks and the uteri fixed and processed for light and electron microscopy. Ultrastructural analysis showed that the attached protozoa interacted with the mucosa through a somal projection. With an amorphous secretion at the protozoa-host cell interface. There was no direct contact between the protozoal plasma membrane and the epithelial cell membrane. Our results demonstrated the participation of an active phagocytosis and the destruction of T. foetus by eosinophils.     

Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαβ⁺CD8⁺IFN-γ⁺ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαβ⁺CD4⁺ T cells were also found to increase in the infected mice, however later than CD8⁺ T cells. Interestingly, splenic and MLN CD4⁺CD25⁺ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαβ⁺CD8⁺ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.     

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.     

Influence of Neospora caninum intra-specific variability in the outcome of infection in a pregnant BALB/c mouse model

Previous assays in pregnant animals have demonstrated the effect of different host factors and timing of infection on the outcome of neosporosis during pregnancy. However, the influence of Neospora caninum isolate itself has been poorly investigated. Here, we compared the effects on clinical outcome and vertical transmission observed in a pregnant mouse model following infection with 10 different N. caninum isolates. The isolates in our study included the Nc-Liv isolate and nine N. caninum isolates obtained from calves. Female BALB/c mice were inoculated with 2 × 10⁶ tachyzoites at day 7 of pregnancy. Morbidity and mortality, in both dams and offspring during the course of infection, and transmission to progeny at day 30 postpartum were evaluated. The serum IgG1 and IgG2a production in dams were also examined. All dams showed elevated IgG1 and IgG2a responses, confirming N. caninum infection, although signs of disease were only exhibited in dams infected with 4 of the 10 isolates (Nc-Spain 4H, Nc-Spain 5H, Nc-Spain 7 and Nc-Liv). In neonates, clinical signs were observed in all N. caninum-infected groups, and neonatal mortality rates varied from greater than 95% with the isolates mentioned above to less than 32.5% with the other isolates. Vertical transmission rates, as assessed by parasite PCR-detection in neonate brains, also varied from 50% to 100% according to the isolate implicated. These results confirm the wide pathogenic and transmission variability of N. caninum. The intra-specific variability observed herein could help us explain the differences in the outcome of the infection in the natural host.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

Survival, immune responses and tissue cyst production in outbred (Swiss white) and inbred (CBA/Ca) strains of mice experimentally infected with Neospora caninum tachyzoites

The present work compared inbred (CBA/Ca) and outbred (Swiss white) strains of mice for their capacity to cope with a Neospora caninum infection and to consistently produce tissue cysts. In each experiment Swiss white and CBA/Ca mice were given three different doses of NC-1 tachyzoites. Lymphoproliferative and humoral responses as well as cytokine production were evaluated eight weeks after infection (PI) whereas tissue cyst production and histopathology were assessed 4, 6 and 10 weeks PI in immunosuppressed mice. Tissue cysts were observed 10 weeks after infection only in CBA/Ca mice receiving the two highest inoculum doses. Furthermore this strain showed the highest specific lymphoproliferative response. A mixed cytokine response with elevated IFN-gamma and fairly low IL-4 and IL-10 secretion was recorded. In both strains, no lesions were observed in the tissues of infected mice. This study indicates that CBA/Ca female mice infected with 5 x 10(6) NC-1 tachyzoites represent a useful model for the study of specific maternal immune responses in pregnant animals.     

Comparative histopathology in BALB/c mice infected with virulent and attenuated strains of Brucella abortus

The course of infection in BALB/c mice of virulent Brucella abortus strain 2308 (S-2308) and attenuated strain 19 (S-19) varies markedly. Whereas S-19 is eliminated at an exponential rate beginning at 2 weeks post infection (p.i.), strain 2308 assumes a steady state or plateau during the first 6 weeks p.i. and thereafter is eliminated very slowly over a period exceeding 6 months. Here we compared the initiation and maintenance of inflammatory reactions in spleens and livers of mice infected with either of the two strains of B. abortus for the first 6 weeks p.i. Histological changes in the liver were similar in response to either strain and were characterized by the development of small granulomas and an influx of polymorphonuclear leukocytes (PMN) and monocytes. Tissue reactions in the spleen were similar at weeks 1 and 2 p.i. At 3 weeks p.i. and thereafter, focal granulomatous responses in S-2308-infected mice exceeded those in mice infected with S-19. Numbers of nonspecific esterase (NSE) positive mononuclear leukocytes in S-19-infected spleens had increased by 3 weeks p.i. and remained elevated. No comparable increase in NSE positive cells occurred in mice infected with S-2308, and numbers were significantly lower. At 4 weeks p.i. the influx of mature neutrophils and the intensity of extramedullary hematopoiesis were significantly greater in S-19-infected spleens. A profound depletion of periarteriolar lymphoid tissue was noted in both infections for the first 3 weeks p.i. However, repopulation of lymphoid sheaths in S-19-infected spleens became significantly greater by 4 weeks p.i. and continued to increase at significantly higher levels during the next 2 weeks. This study demonstrates quantitative differences in splenic inflammatory responses which are temporally related to the more rapid elimination of S-19. Based upon the lower susceptibility of strain 2308 to the protective effects of immune serum it is hypothesized that the different patterns of infection and inflammation displayed by the 2 strains may related to the differential capacities of antibody opsonized S-19 and S-2308 to survive in activated macrophages.     

Detection of bovine respiratory syncytial virus in experimentally infected balb/c mice

The present study used an RT-nested-PCR and an immunohistochemistry assay to detect bovine respiratory syncytial virus in tissues from experimentally infected balb/c mice. As a first step, Chicken Embryo Related (CER) cell monolayers infected with the BRSV-25-BR strain isolated in Brazil were used for antigen production. Then, the infected lung and tracheal tissues of female balb/c mice were collected on 3, 5, 7 and 10 days post-infection and submitted to both techniques. Primers specific to F and G genes that amplify fragments of 481 bp and 371 bp, respectively, were used. The BRSV detection was not successful in all of the animals tested. The genomic fragment of the G gene from the organs of some infected mice on all analyzed post-infection days was amplified. However, in the RT-nested-PCR corresponding to the F gene, it was not possible to observe any amplified fragment. This was probably due to the higher sensitivity of the developed technique to amplify the fragment corresponding to the G gene compared to the F gene. Moreover, only three of the lungs collected five days post-infection were positive by immunohistochemistry. To the author's knowledge, this is the first study reporting bovine respiratory syncytial virus detection in balb/c mice after experimental inoculation.