Neospora hughesi: experimental infections in mice, gerbils, and dogs

Neospora hughesi is a recently described cause of equine protozoal myeloencephalitis (EPM). A rodent model for pathogenicity would facilitate development of therapies to be used in horses. In the present study, we examined the susceptibility of BALB/c gamma-interferon gene knockout (gamma-INFKO), BALB/c, CD-1, and C57BL/6 strains of mice and gerbils to infection with tachyzoites of the Nh-A1 strain of N. hughesi isolated from a horse from AL, USA. Only the gamma-IFNKO mice developed severe clinical disease following infection with N. hughesi and died 19-25 days after infection and exhibited severe cardiac lesions. In contrast, experimental infection of gamma-INFKO mice with tachyzoites of the NC-1 or NC-Liverpool strains of Neospora caninum resulted in deaths 8-10 days after infection. The most severe lesions were in the livers, spleens, and lungs of these mice. Gerbils inoculated with N. hughesi did not develop clinical disease, had few microscopic lesions, but did seroconvert. Two dogs fed the brains of mice, shown to contain N. hughesi tissue stages by cell culture and gamma-IFNKO mouse bioassay, did not shed N. caninum-like oocysts over a 23 days observation period. The marked difference in pathogenicity between the two species of Neospora in gamma-IFNKO mice, and lack of oocyst excretion by dogs fed N. hughesi infected mice provide additional evidence that the species distinction between N. caninum and N. hughesi is valid.     

Exploring the life cycle of Besnoitia besnoiti - experimental infection of putative definitive and intermediate host species

The biology of Besnoitia besnoiti, the cause of bovine besnoitiosis, is poorly understood. Its definitive host is unknown, and information on potential intermediate hosts is scarce. In order to investigate potential definitive and intermediate hosts for European isolates of B. besnoiti, domestic dogs, cats, rabbits, guinea pigs (Cavia porcellus), gerbils (Meriones unguiculatus), common voles (Microtus arvalis) and NMRI-mice were inoculated with B. besnoiti isolated from naturally infected German cattle. Dogs and cats were fed 5×10(6)B. besnoiti tachyzoites (isolate Bb-GER1), or tissue cysts containing at least 2×10(7)B. besnoiti bradyzoites obtained from the skin of a naturally infected Limousin cow from the same herd where strain Bb-GER1 was isolated. Rodents and rabbits were subcutaneously inoculated with either 5×10(5) Bb-GER1 tachyzoites or 5×10(5) bradyzoites. Groups of 2-4 non-inoculated animals of each species were monitored as negative controls. Feces from all dogs and cats were daily examined by a sedimentation-flotation technique for at least 11 weeks after inoculation but no B. besnoiti oocysts were identified. Cats fed tachyzoites and dogs did not seroconvert, but specific antibodies to B. besnoiti tachyzoites were detected by IFAT (titer≥100) in 2 out of 3 cats fed tissue cysts since 5-7 weeks post infection. By immunoblot, these two cats exhibited a reaction pattern against tachyzoite antigens similar to that observed in naturally infected cattle. Antibodies against B. besnoiti tachyzoites were detected in all inoculated rodent species and rabbits by both, IFAT and immunoblot since 3 weeks post-inoculation. Rabbits and rodents, subcutaneously inoculated with same doses of inactivated bradyzoites remained serologically negative (IFAT titer<50). Clinical signs observed in the inoculated rabbits included fever, serous conjunctivitis and transient swelling of the testes. No clinical abnormalities were noticed in the other tested animal species. Voles developed pneumonia as observed by histological examination. B. besnoiti-DNA was detected by PCR in blood from rabbits, gerbils and voles at 9 days post-infection, and in skin, heart, lung, striated muscle and kidney tissues from voles at 19-21 weeks post-infection. Domestic dogs and cats could not be shown to be definitive hosts of B. besnoiti, but cats seroconverted after feeding on B. besnoiti tissue cysts indicating that B. besnoiti stages had invaded the cats' tissues. The molecular and serological results from this study indicate that European B. besnoiti isolates may infect cats, rabbits, guinea pigs, gerbils, mice and voles; however a persistence of the parasite could be demonstrated only in voles.     

Duration of strain 2308 infection and immunogenicity of Brucella abortus lipopolysaccharide in five strains of mice

A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as between vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.     

Comparative infectivity of oocysts and bradyzoites of Toxoplasma gondii for intermediate (mice) and definitive (cats) hosts

Tachyzoites, bradyzoites (in tissue cysts), and sporozoites (in oocysts) are the three infectious stages of Toxoplasma gondii. The prepatent period (time to shedding of oocysts after primary infection) varies with the stage of T. gondii ingested by the cat. The prepatent period (pp) after ingesting bradyzoites is short (3-10 days) while it is long (18 days or longer) after ingesting oocysts or tachyzoites. The conversion of bradyzoites to tachyzoites and tachyzoites to bradyzoites is biologically important in the life cycle of T. gondii and it has been proposed that the pp can be used to study stage conversion. In the present study, infectivity of oocysts and bradyzoites released from tissue cysts of a recent isolate of T. gondii, TgCkAr23, to cats and mice was compared. Ten-fold dilutions of oocysts or bradyzoites were administered orally to cats, and orally and subcutaneously to mice. Of the 29 cats each fed 1-10 million oocysts only one cat shed oocysts and the pp was 23 days; all cats remained asymptomatic. In contrast, all mice administered the same 10-fold dilutions of oocysts either orally or subcutaneously died of toxoplasmosis. The results confirm that infectivity of the oocysts to cats is lower than for mice and that oocysts are non-pathogenic for cats. Of the 41 cats each fed 1-1,000 free bradyzoites, 15 shed oocysts with a short pp of 4-9 days, and all remained asymptomatic. The infectivity of bradyzoites to mice by the oral route was approximately 100 times lower than that by the subcutaneous route. The results confirm the hypothesis that the pp in cats is stage and not dose dependent, and that transmission of T. gondii is most efficient when cats consume tissue cysts (carnivory) or when intermediate hosts consume oocysts (fecal-oral transmission).     

Evaluation of cytokines concentration and percentage of survival of rabies virus-infected mice submitted to anti-rabies Vero-cell propagated vaccine and P. acnes

Previously, survival of rabies infection was shown to correlate with low IL-6 serum concentration in mice subjected to post-exposure treatment with the Fuenzalida Palacios rabies vaccine in conjunction with the immunomodulator Propionibacterium acnes, previously Corynebacterium parvum. Considering the substitution of the Fuenzalida Palacios rabies vaccine by the Vero cell raised anti-rabies vaccine in almost all countries, the objective of this work was to evaluate the survival and cytokine serum concentration of rabies virus-infected mice treated with P. acnes in conjunction with or the anti-rabies-VERO vaccine. For this, Swiss mice were experimentally infected with street rabies virus and subjected to vaccine and/or P. acnes following infection. Animals were killed at different times and serum was collected to evaluate cytokines. The greatest survival was observed in animals given one or two does of P. acnes in the absence of vaccination. Animals given anti-rabies VERO vaccine alone or with three doses of P. acnes had the second highest survival rate. The group that had the highest percentage of mortality also had the highest IL-6 concentration on the 10th day, a time correlating with clinical symptoms of the animals. The results reinforce the inefficacy of anti-rabies vaccine in only one dose as a post-exposure treatment irrespective of the type of vaccine used, the immunomodulation activity of P. acnes in rabies post-exposure treatment and suggest a role for IL-6 in rabies virus pathogenesis.     

Susceptibility of Djungarian hamsters (Phodopus sungorus) to Neospora caninum infection

Djungarian hamsters were examined for the susceptibility to Neospora caninum infection. After 29 Djungarian hamsters were intraperitoneally inoculated with 5 x 10(6) N. caninum tachyzoites of JPA1 strain, some animals showed symptoms such as ataxia, and many tissue cysts were detected in the brain and a cyst in the muscular tunics of stomach. Especially, more than 100 cysts per head were observed after 5 weeks post inoculation. It is suggested that the Djungarian hamster is a model useful to examine neosporosis.     

Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2 (PCV2)

Immunological impairment by porcine circovirus type 2 (PCV2) infection is well documented in pigs suffering from postweaning multisystemic wasting syndrome. Nonetheless, little is known about immune status of pigs that remain PCV2 subclinically infected. Thus, seven pigs successfully infected in an experimental inoculation and without developing disease and nine control non-inoculated pigs were examined. Serological, virological and immunological determinations were done throughout ten weeks post-infection (PI). At week 3 PI, inoculated animals presented the peak of viremia and produced higher levels of IL-10 than the controls; correlation between viral load and IL-10 amounts was observed (p<0.05). Also, the ratio IgM/IgG suffered a shift skewing IgM production towards an IgG response. By 10 weeks PI, levels of IL-10 disappeared and the viremia decreased. In summary, subclinically PCV2-infected pigs developed a transient PCV2-specific IL-10 response during the viremic phase of infection which coincided with the inversion of the IgM/IgG ratio.     

Effect of strains of mice and challenge dose on the infectivity and virulence of Trypanosoma vivax

Factors influencing the infectivity and virulence of clone-derived Trypanosoma vivax for mice were investigated using a titration technique capable of recognizing differences in magnitude of about 10-fold. Marked differences were observed in the susceptibility and duration of infection in four inbred strains of mice after low dose (10(2.2)ID63) challenge. Only mild differences in survival period were observed when challenge doses of 10(3.2) and 10(4.2)ID63 per mouse were used. In all tests, Balb/C mice were most susceptible while CBA mice were most resistant. Long survival of infected mice was highly correlated with frequent occurrence of remission of parasitaemia. Infectivity of T. vivax isolates varied during the course of an infection, being high at the early stages of the infection and very low at later stages.     

Natural Besnoitia sp. infection in domestic rabbits from Argentina

Besnoitia sp. are apicomplexan coccidian parasites affecting several species of mammals and cold-blooded animals in several countries. Besnoitia sp. tissue cysts were seen in several tissues of five rabbits from a rabbit breeder in La Plata, Argentina. Bradyzoites released from macroscopic tissue cysts were inoculated onto bovine monocytes, and into interferon gamma gene knockout (KO) mice. Besnoitia sp. tachyzoites were seen in the peritoneal exudate of KO mice on day 10 pi and these tachyzoites were infective to other KO mice. Tachyzoites grown in cell culture were infective to gerbils (Meriones unguiculatus). This is the first report of Besnoitia sp. infection in any host in Argentina.     

Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus

To investigate cytokine alterations in pigs infected in-utero with porcine reproductive and respiratory syndrome virus (PRRSV), constitutive mRNA expression by peripheral blood mononuclear cells (PBMCs) was measured. PBMC from in-utero PRRSV-infected pigs displayed significantly increased IL-6, IL-10, and IFN-gamma mRNA expression at 0 and 14 days of age compared with age-matched control pigs. There were no significant differences in IL-2, IL-4, and IL-12 mRNA expression between in-utero PRRSV-infected and control pigs. However, the IL-10/IL-12 ratio was significantly increased in in-utero PRRSV-infected pigs at 0 and 14 days of age, suggesting the imbalance of IL-10 and IL-12 mRNA production. The abnormal mRNA expression of cytokines in in-utero PRRSV-infected pigs occurred concurrently with a significant decrease in the CD4(+)/CD8(+) T-cell ratio in peripheral blood. PRRSV was not isolated from the sera of pigs at 9 weeks of age that had been viremic at 0 and 14 days old. Delayed type hypersensitivity (DTH) responses to Tuberculin and analysis of cytokine mRNA expression by PBMC showed that cell-mediated immune response and cytokine message profiles in pigs infected in-utero with PRRSV had returned to levels similar to those of control pigs by 9 weeks of age. We conclude that in-utero infection with PRRSV results in significant alteration of cytokine mRNA expression that may cause transient immunomodulation. However, at 10 weeks of age the pigs' immune responses seemed to recover. This may help to understand the immunopathogenesis of in-utero PRRSV infection and the increased susceptibility to secondary bacterial pathogens in neonatal piglets.     

Interleukin-2 and interleukin-10 gene expression in calves experimentally infected with Fasciola gigantica

The gene expression of interleukin-2 and interleukin-10 in calves with a primary infection of Fasciola gigantica was studied. Five calves were infected orally with a dose of 1000 viable metacercariae of F. gigantica and five calves served as uninfected control animals. Expression of two cytokine genes i.e. IL-2 and IL-10 (Th1 and Th2) was measured at 10, 30 and 75 days post-infection (PI) by real-time polymerase chain reaction with the double stranded DNA-binding dye SYBR Green. Interleukin-2 was not detected in the peripheral blood mononuclear cells (PBMCs) of infected or control animals at 10, 30 and 75 days PI, however, IL-10 was present in detectable levels in PBMCs of infected animals at 10, 30 and 75 days PI, with no expression of this cytokine in the control animals. With an increased expression of IL-10 and no expression of IL-2 cytokine gene, the present study suggests that F. gigantica infection in calves evoked Th2 immune response.     

Antibody reactivity in mice and cats to feline enteroepithelial stages of Toxoplasma gondii.

The reactivity of antibodies in mice and cats to feline enteroepithelial stages of Toxoplasma gondii was examined by means of an indirect immunofluorescent antibody test. Mice immunized with feline enteroepithelial stage (FES) parasites produced antibodies not only against FES, but also against tachyzoites, sporozoites/oocysts, tissue cysts and one part of the infected feline enterocytes. After absorption with tachyzoites, the titer of antibodies reactive to enterocytes was significantly reduced. In contrast, the titer of antibodies reactive to FES remained unchanged. The antibodies from cats immunized with FES, reacted specifically to FES, but not to tachyzoites, tissue cysts or enterocytes. These results suggest that FES parasites may have stage-specific antigen(s).     

Experimental infection of Swiss webster mice with four rat bartonella strains: host specificity, bacteremia kinetics, dose dependent response, and histopathology

Groups of Swiss Webster outbred mice were each inoculated with one of four bartonella strains originally isolated from Rattus spp. at doses ranging from 10¹ to 10⁷ bacteria per mouse. One strain, Rn1691yn (Bartonella coopersplainensis-like), infected mice and produced bacteremias at levels up to 10⁵ bacteria/ml of blood and from 3 to 8 weeks duration. A dose dependent response was also observed with differing proportions of mice bacteremic following inoculation at different doses. In addition weeks-to-months long lags in bacteremia manifestation occurred following lower dose exposures. The possibility of bacterial transmission from bacteremic mice to uninfected cagemates was assessed and no naïve mice became infected from contacts with infected mice. Finally, a subset of bacteremic mice inoculated with high doses of Rn1691yn were examined histopathologically and multifocal, granulomatous lesions were detected in both liver and kidneys. The host specificity and infectivity of the strains is discussed in relation to their potential for zoonotic transmission to incidental hosts.     

Role of the suppressor of cytokine signaling 2 (SOCS2) during meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5)

The role of suppressors of cytokine signaling (SOCS) in meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5) was evaluated by intracranial infection in C57BL/6 wild-type mice (WT) and SOCS2 deficient mice (SOCS2^−/−). Both infected groups presented weight loss, ruffled fur and hunched posture. Additionally, infected SOCS2^−/− mice showed swollen chamfer and progressive depression. Infected WT animals developed mild meningitis, characterized by infiltration of mononuclear cells. Moreover, viral DNA was detected in liver and lung from infected WT group. This group also showed elevated brain levels of IFN-γ, IL-10, CXCL1 and CCL5, when compared with non-infected WT animals. Brain inflammation was exacerbated in infected SOCS2^−/− mice with widespread distribution of the virus and increased brain levels of TNF-α, IFN-γ, IL-10, IL-12, CXCL1 and CCL5, when compared with WT infected mice. Moreover, infected SOCS2 deficient mice exhibited reduced brain mRNA expression of IFNα and IFNβ and increased expression of mRNA of SOCS1, compared with infected WT mice. Taken together, our study provides an insight into the role of SOCS2 in modulating the immune response to BoHV-5 infection.     

山豆根多糖对感染PRRSV小鼠脾脏细胞因子水平的影响

探讨山豆根多糖对PRRSV感染小鼠脾淋巴细胞分泌细胞因子水平的影响。将70只昆明种小鼠随机分为7组(A组、B组、C组、D组、E组、F组、G组),每组10只,雌雄各半,依据前期已经建立氧化应激模型的感染条件,D组、E组、F组、G组小鼠于试验第1、2、3天分别采用口服、滴鼻和腹腔注射3种途径联合感染PRRSV病毒原液1.0mL/只,A组、B组、C组给予生理盐水1.0mL/只。第4、5、6天,A、D组小鼠分别腹腔注射生理盐水,0.2mL/10g。B组小鼠腹腔注射5.0mg/kg的脂多糖(LPS)溶液,C组、E组、F组、G组小鼠分别腹腔注射不同剂量的山豆根多糖(200、50、100、200mg/kg)。供试小鼠均于第14天处死,并取其脾脏制备匀浆。采用ELISA检测脾匀浆中TNF-α、IL-1β、IL-6、IL-8、IL-10和MCP-1等细胞因子的水平。结果显示,PRRSV感染小鼠后能升高小鼠脾脏匀浆内TNF-α、IL-1β、IL-6、IL-8、IL-10和MCP-1水平,50mg/kg~100mg/kg剂量的山豆根多糖能降低上述细胞因子的水平。结果表明,山豆根多糖能有效降低PRRSV感染小鼠脾脏细胞因子的水平。     

Gerbil model of acute neosporosis

Experimental infections with the NC-1 strain of Neospora caninum were conducted in gerbils (Meriones unguiculatus) to determine their acute responses to experimental intraperitoneal infection. Five groups of five female gerbils were used and they were intraperitoneally infected with 1x10(6), 2x10(6), 3x10(6), 4x10(6) or 5x10(6) tachyzoites. Gerbils in all groups developed clinical signs of neosporosis which consisted of inactivity 4-5 days post-inoculation. Morbidity and mortality were observed in all groups. Grossly there was a clear fibrinous exudate in the abdominal cavity and adhesions of the spleen and pancreas to the stomach in gerbils suffering from acute neosporosis. The LD50 was calculated as 9.3x10(5) tachyzoites per gerbil. The results indicate that gerbils can be used as a suitable model of acute neosporosis. This model can be used to screen candidate treatments and to test the efficacy of vaccines for neosporosis without the need to use histology or PCR to demonstrate treatment efficacy.     

Immunohistochemical and polymerase chain reaction studies in Neospora caninum experimentally infected broiler chicken embryonated eggs

The diagnostic characteristics of immunohistochemistry (IHC) and polymerase chain reaction (PCR) methods were studied in the tissues of broiler chicken embryos experimentally infected by Neospora caninum. An infection with N. caninum NC-1 isolate was conducted in 70 broiler chicken embryonated eggs randomly divided into seven equal groups. After 8 days of incubation, six groups were inoculated with 10, 10(2), 10(3), 10(4), 10(5), and 10(6) doses of tachyzoites/embryonated egg. The 7th group was considered as control. The mortality rate and pathological changes of the dead embryos and hatched chickens up to 60 days old were noticed. Consecutive sections to those used for histopathological examination including the liver, heart, brain, and chorioalantoic (CA) membrane were subjected to IHC. The intensity and distribution of the immunostaining was graded as highly to mildly positive. For PCR procedure, DNA was extracted from 50mg of the tissues and primer pair Np21/Np6 was used for amplification of the Nc-5 gene. The results of the immunosignaling ranged from variable degrees of mild to moderate staining as dark-brown to brown and coarsely to finely granular, mostly within the cytoplasm of infected cells such as the endothelial cells of blood vessels. The parasite aggregation was more predominant in the heart than other tissues. Immunoreactivity for N. caninum antigen was multifocally moderate positive in the heart, liver and CA of the 10(3) dose, and also heart, liver, brain and CA of the 10(4) dose. IHC showed mildly positive in the liver and heart of the chicken embryos infected with 10 and 10(2) tachyzoites, as well. The results of the PCR confirmed the existence of the parasite in all of the examined tissues from the 10(3) and 10(4) doses. In conclusion, the results indicate a good agreement between IHC and PCR in diagnosis of neospora antigen in the infected tissues.     

High susceptibility of Djungarian hamsters (Phodopus sungorus) to the infection with Babesia microti supported by hemodynamics

As the comparative study was carried out on the susceptibility by the pursuit of parasitemia among the Djungarian, Syrian, and Chinese hamsters as well as BALB/c mice infected with the Syrian hamster-adapted Babesia microti strain, and Djungarian hamsters showed the highest parasitemia among them. Then, the other hematological parameters were pursued in the Djungarian hamsters infected with the hamster-adapted B. microti strain. Remarkable symptoms observed were hemoglobinuria clinically, anemia hematologically, and splenomegaly macroscopically during all over the observation period for 24 weeks post infection (PI). Parasitemia began to rise at 2 weeks and peaked at 4 weeks PI. After that, parasitemia decreased gradually but was maintained with a level of about 10% on average until 24 weeks PI at the end of the experiment. A decrease in the RBC count, Hb, and PCV, and an increase in the reticulocyte and WBC counts due to the development of immature neutrophils, lymphocytes and monocytes were recognized together with a rise of parasitemia. The hamsters had macrocytic hypochromic anemia due to the increase of MCV and the decrease of MCHC in the growth phase of the parasite. It was considered that the Djungarian hamsters will be useful for the infection examination, isolation, maintenance, and passage of B. microti in laboratory.     

Immunologic, histopathologic, and bacteriologic responses of five strains of mice to Brucella abortus strain 2308

A study was conducted to establish baseline data on Brucella abortus infection induced in 5 strains of mice (CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ). The strains were compared on the basis of immunologic, histopathologic, and bacteriologic responses. There were 4 treatment groups for each strain of mice: (1) vaccinated with homologous lipopolysaccharide and challenge exposed to B abortus strain 2308; (2) not vaccinated but challenge exposed; (3) vaccinated and not challenge exposed; and (4) not vaccinated and not challenge exposed. Results indicated that mice can be used for comparative studies on the pathogenesis and immunogenesis of B abortus infections; strains of mice may vary in their responses to Brucella infection, regardless of their vaccination status. Bacteriologic and immunologic responses in mouse strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ, but not those of CBA/NJ, were extrapolative among strains.