Experimental infection of C3H/HeJ mice with the NY18 isolate of Anaplasma phagocytophilum

Human granulocytic anaplasmosis (HGA), an emerging disease of public health concern in many areas of the world, is caused by Anaplasma phagocytophilum. Small animal models of A phagocytophilum in laboratory mice have been developed and used to study the pathogenesis of HGA. In this study, we characterized the pathologic changes in acute infection of C3H/HeJ mice experimentally infected with the NY18 isolate of A phagocytophilum. Although no clinical signs were noted, acute infection was associated with gross splenomegaly, microscopic inflammatory lesions in the lung and liver, hyperplastic lesions on the spleen, and clinical pathology abnormalities including neutropenia and monocytosis. This study emphasizes the use of well-defined animal models as a valuable tool for the study of A phagocytophilum infections.     

Alopecia areata in C3H/HeJ mice involves leukocyte-mediated root sheath disruption in advance of overt hair loss

Alopecia areata (AA) can be induced in C3H/HeJ mice by grafting full-thickness AA-affected skin. An 8- to 12-week delay between surgery and overt hair loss onset provides an opportunity to examine disease pathogenesis. Normal haired C3H/HeJ mice were sham-grafted or grafted with AA-affected skin. Mice were euthanatized 2, 4, 6, 8, 10, and 12 weeks after surgery along with chronic AA-affected mice as a positive control. Until 6 weeks after grafting, inflammation was only evident around anagen-stage hair follicles in host skin adjacent to but not distant from the AA-affected graft. From 8 weeks on, AA-grafted but not sham-grafted mice exhibited a diffuse dermal inflammation at distant sites that progressively focused on anagen-stage hair follicles at 10 and 12 weeks. Perifollicular inflammation was primarily composed of CD4+ and CD8+ cells associated with follicular epithelium intercellular adhesion molecule -1 expression. Only CD8+ cells penetrated intrafollicularly by 12 weeks after surgery, although both CD4+ and CD8+ intrafollicular cells were observed in chronic AA-affected mice. Under electron microscopy, intrafollicular lymphocyte and macrophage infiltration associated with hair follicle dystrophy was prominent 10 weeks after surgery, primarily within the differentiating outer and inner root sheaths. This study shows that focal follicular inflammation develops some time in advance of overt hair loss and focuses on the differentiating root sheaths in C3H/HeJ mice. The severity of inflammation and the degree of hair follicle dystrophy induced by the infiltrate appear to reach a threshold level before overt hair loss occurs.     

Humoral antibody responses to different H5N1 and H5N2 vaccination regimes: implications for the development of autogenously based vaccines

Whereas H5N1 vaccine and several H5N2 vaccines are commercially available and are used to control H5N1 outbreaks in some endemic countries, infections hit many vaccinated flocks. The following study was conducted to compare the efficacy of such vaccines and to assess their potential induction of antibodies against the haemagglutinin of local H5N1 isolate after single vaccination. The possible beneficiary effect of booster dose at different intervals was screened for both H5N1 vaccine as well as a selected H5N2 candidate. Differences in the serological immune response among native and cross breeds were also screened. No significant variations were detected between available commercial H5N1 and H5N2 vaccines after single vaccination. Two vaccination shots using H5N1 but not H5N2 vaccine were found to be superior to a single vaccination scheme, where chicks developed more conceivable antibody titers than in single vaccination program. There was considerable variation among chicken lines in the immune response to H5N1 vaccine: native breeds possessed the highest antibody titers as compared to other breeds.     

Monoclonal antibody specific to HA2 glycopeptide protects mice from H3N2 influenza virus infection

Canine influenza virus (CIV) subtype H3N2 is a newly identified, highly contagious respiratory pathogen that causes cough, pneumonia and other respiratory symptoms in dogs. Data indicate that the virus is responsible for recent clinical cases of dog disease in China. However, therapeutic options for this disease are very limited. In this study, seven monoclonal antibodies (mAbs) against CIV JS/10 (an H3N2 subtype virus) were produced and characterized. Among them, mAb D7, which is specific for the HA2 glycopeptide (gp), induced the highest neutralization titers. The protection provided by mAb D7 was evaluated in BALB/c mice challenged with homologous or heterologous strains of H3N2 influenza virus, including two strains of CIV and one strain of swine influenza virus (SIV). The data show that mAb D7 protected the mice from infection with the three viral strains, especially the homologous strain, which was indicated by the recovery of body weight, reduction of viral load, and reduction of tissue damage. Moreover, the levels of IFN-γ and TNF-α in the lungs, as detected by ELISA, were reduced in the infected mice treated with the mAb D7 compared with those without mAb D7 treatment. Thus, our findings demonstrate, for the first time, that a mAb could reduce the release of IFN-γ and TNF-α associated with tissue damage by CIV infection and that the mAb might be of great therapeutic value for CIV infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0146-7) contains supplementary material, which is available to authorized users.     

Humoral immune responses to Mycoplasma hyopneumoniae in sows and offspring following an outbreak of mycoplasmosis

Previously healthy sows, seropositive to Mycoplasma hyopneumoniae, developed clinical signs of mycoplasmosis, as well as increasing amounts of antibodies to M. hyopneumoniae during an outbreak of the disease in a herd. During the early phase of the outbreak, young piglets (2 weeks) with maternal antibodies remained healthy while older seronegative piglets (4–7 weeks) developed the disease. The duration of the maternal antibodies to M. hyopneumoniae varied between litters and was related to the amount of antibodies in the serum of the dam. In sows, the level of serum antibodies decreased continuously from 4 weeks ante partum to partus, and the level of antibodies in the whey of colostrum was comparable to that in serum 4 weeks ante partum. After loss of maternal antibodies to M. hyopneumoniae, seropositive animals were not found among piglets younger than 9 weeks. Therefore peripheral blood mononuclear cells (PBMC) were collected from various age categories of piglets in order to measure the ability to produce antibodies to M. hyopneumoniae in vitro. PBMC obtained from piglets aged 1 and 3 weeks produced few antibodies to M. hyopneumoniae. Significantly higher levels of antibodies to M. hyopneumoniae were produced by PBMC obtained from pigs aged 5–9 weeks. Thus, the ability of PBMC to produce antibodies to M. hyopneumoniae in vitro seemed to be age-dependent.     

Evaluation of antibody response and nonspecific lymphocyte blastogenesis following inoculation of a live attenuated bluetongue virus vaccine in goats

OBJECTIVE: To evaluate vaccine safety, antibody response, and nonspecific lymphocyte blastogenesis following inoculation of a commercial monovalent live attenuated bluetongue virus (BTV) serotype 2 vaccine in goats. ANIMALS: 12 nonpregnant and nonlactating Saanen goats. PROCEDURE: 6 goats were inoculated with the monovalent live attenuated BTV serotype 2 vaccine, which has been widely used in Italy during the proceding 2 years. The other 6 goats were unvaccinated and represented negative controls. Nonspecific lymphocyte blastogenesis was evaluated 14 and 7 days before and 7, 21, and 49 days after vaccination by measuring DNA synthesis in peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin, concanavalin-A, and pokeweed mitogen. On the same days as lymphocyte blastogenesis, blood samples were taken to determine serum concentrations of anti-BTV antibodies. RESULTS: During the 7 weeks following vaccination, PBMCs obtained from vaccinated goats had a significantly decreased response to mitogens in terms of DNA synthesis, compared with PBMCs from the same goats before vaccination. Conversely during the experiment, no significant change was found in the response of the PBMCs obtained from unvaccinated goats. Starting from 21 days after vaccination, serum from vaccinated goats had anti-BTV antibodies. No anti-BTV antibodies were detected in the serum from unvaccinated goats. CONCLUSIONS AND CLINICAL RELEVANCE: Inoculation of goats with the monovalent live attenuated BTV serotype 2 vaccine described herein resulted in a profound depression of nonspecific lymphocyte blastogenesis, which might compromise the resistance of vaccinated goats to pathogens.     

Serosurvey of Babesia canis, Babesia gibsoni and Ehrlichia canis in pound dogs in California, USA

The seroprevalence of three canine tick-transmitted parasites, Babesia gibsoni, Babesia canis and Ehrlichia canis, was estimated in selected regions of California. Blood smears and sera were obtained from 971 dogs in seven animal shelters: four in Los Angeles County, one in Yolo County, one in El Dorado County in California and one in Minden, Nevada. Seroprevalence in Los Angeles County shelters were 0–13%, 0–2.6% and 0% for B. canis, B. gibsoni and E. canis, respectively. Seroprevalences of the same three parasites in Yolo County and El Dorado County Shelters were 0% except for a 1% seroprevalence of B. canis in dogs from Yolo County Shelter.

Potential risk factors (breed, age, sex and evidence of ticks on the dogs) for B. canis seropositivity were evaluated. Dogs 3 years of age or older had a significantly higher risk (odds ratio 5.04) of being seropositive to B. canis compared with dogs less than 1 year old. Breed, sex and evidence of ticks were not associated with seropositive reactions to B. canis. Of 29 coyotes captured in Los Angeles County, three (10.3%) were seropositive for B. gibsoni, with titers of 1280 to 2560. This study indicated that dogs in Los Angeles County were at higher risk of being seropositive and potentially infected with canine babesial parasites than dogs in Yolo and El Dorado Counties. Movement of chronically infected dogs from Los Angeles County into other areas could contribute to the spread of these important pathogens.     

Humoral and cell-mediated immune responses in non-pregnant heifers following infection and vaccination with Brucella abortus

Humoral and cell-mediated-immune responses to Brucella abortus were observed in non-pregnant heifers following infection alone; infection followed by vaccination; vaccination followed by infection; and vaccination alone. The humoral responses, as measured by the Rose Bengal test (RBT), complement fixation test (CFT), indirect haemolysis test (IHLT) and the enzyme-linked immunosorbent assay (ELISA) tended to be immediate and transient following infection alone, infection following vaccination and vaccination alone. However, when vaccination was superimposed on infection, reactions were maintained for at least 2 years. The cell-mediated-immune (CMI) responses were assessed by the lymphocyte stimulation test. The responses occurred after the humoral responses had peaked and were present for periods of 6-22 weeks. However, the level of stimulation was greater following infection than following vaccination, and the response when vaccination was superimposed on infection was present for less than 6 months.     

Serum and mucosal antibody responses against Mycoplasma hyopneumoniae following intraperitoneal vaccination and challenge of pigs with M hyopneumoniae

Pigs were immunised intraperitoneally when six weeks old and again at about 10 weeks old with killed Mycoplasma hyopneumoniae antigen prepared in an oil adjuvant. The pigs were challenged with live M hyopneumoniae (Beaufort strain) at between 11 and 15 weeks old. Antigen specific antibody levels for both IgG and IgA classes in serum and respiratory tract secretion were monitored over time. In serum anti-M hyopneumoniae antibody was detected shortly after the second intraperitoneal vaccination and was largely IgG. In respiratory tract secretion the response was observed after challenge, and was primarily IgA. Anti-M hyopneumoniae antibody-containing cells and their immunoglobulin class specificity were monitored in lung and tracheal lamina propria. In lung the majority of anti-M hyopneumoniae-containing cells were IgG, whereas in the tracheal lamina propria the majority were IgA. These results are discussed in terms of the use of intraperitoneal vaccination for the control of M hyopneumoniae infection.     

鸭源H5N5和H5N1亚型禽流感病毒BALB/c鼠感染试验

比较鸭源H5N5亚型禽流感病毒(A/Duck/Changchun/01/2010)和鸭源H5N1亚型禽流感病毒(A/Duck/Liaoning/N/2011)对BALB/c鼠的致病性。以106EID50/50μL剂量鼻腔感染6周龄BALB/c鼠,攻毒后3,5,7,10,14 d取小鼠的肺、脑和肝脏,处理后接种10日龄SPF鸡胚做病毒回收试验,取死亡鸡胚的尿囊液进行RT-PCR检测;分别取接种病毒后5 d小鼠的脑、肝脏、肺脏、脾脏、肾脏进行病理组织学检测。结果显示,小鼠接种H5N5和H5N1亚型禽流感病毒后,均无明显的临床症状,肝脏中分别于接种后3,5 d分离到病毒,肺脏中于接种后5 d分离到病毒,脾脏、肾脏和粪便中均未分离到病毒。病理组织学检测发现,病毒对小鼠的脏器组织产生了不同程度的病理损伤,以肺脏、脑和肝脏较为明显,且H5N1亚型禽流感病毒引起小鼠脑和肝脏的病理损伤比H5N5亚型更严重。这表明2株鸭源禽流感病毒对小鼠均有一定的致病性,且H5N1亚型强于H5N5亚型。     

Effect of influenza A/equine/H3N8 virus isolate variation on the measurement of equine antibody responses.

This study has tested the effect of using homologous or heterologous equine influenza A virus isolates to evaluate serum antibody levels to influenza A virus in vaccinated and naturally-infected horses. In addition, the potential effect of antigenic selection of virus variants in egg versus tissue culture propagation systems was studied. Serum antibody levels in samples from horses recently infected with a local influenza A virus isolate (A/equine 2/Saskatoon/1/90) or recently vaccinated with a prototype isolate (A/equine 2/Miami/1/63) were assessed by hemagglutination inhibition and by single radial hemolysis using cell or egg-propagated A/equine 2/Saskatoon/1/90, A/equine 2/Miami/1/63 or A/equine 2/Fontainebleau/1/79. There were no significant differences in hemagglutination inhibition or single radial hemolysis antibody levels obtained with homologous or heterologous isolates or between viruses propagated in either eggs or cell culture. However there was a trend to higher titers in the hemagglutination inhibition assay when cell-propagated virus was used. These results suggest that antigenic variation in equine influenza A virus isolates and host-cell selection of antigenic variants during virus propagation may not be of sufficient magnitude to influence serological evaluation of antibody responses by hemagglutination inhibition or single radial hemolysis.     

The effect of infectious bursal disease virus infection on local and systemic antibody responses following infection of 3-week-old broiler chickens

Broiler chickens infected at 3 weeks of age with infectious bursal disease virus (IBDV) were given Brucella abortus (BA) or sheep red blood cell (SRBC) antigens before, during, and after the acute phase of the infection. Gland of Harder (GH) extracts and serum samples were used to assay local and systemic antibody titer to each antigen 7 days after antigen was administered. Antibody titers to both BA and SRBC antigens were lower (P less than 0.05) in GH extracts and serum of IBDV-infected broilers than uninfected controls. The responses to BA, a thymus-independent antigen, took longer to become depressed than the responses to SRBC, a thymus-dependent antigen. The depression of antibody titers following IBDV inoculation suggests compromise of both local and systemic immune function, a finding of importance to the broiler industry.     

The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination

This study investigated the efficacy of a bivalent swine influenza virus (SIV) vaccine in piglets challenged with a heterologous H1N1 SIV isolate. The ability of maternally derived antibodies (MDA) to provide protection against a heterologous challenge and the impact MDA have on vaccine efficacy were also evaluated. Forty-eight MDA(+) pigs and 48 MDA(-) pigs were assigned to 8 different groups. Vaccinated pigs received two doses of a bivalent SIV vaccine at 3 and 5 weeks of age. The infected pigs were challenged at 7 weeks of age with an H1N1 SIV strain heterologous to the H1N1 vaccine strain. Clinical signs, rectal temperature, macroscopic and microscopic lesions, virus excretion, serum and local antibody responses, and influenza-specific T-cell responses were measured. The bivalent SIV vaccine induced a high serum hemagglutination-inhibition (HI) antibody titer against the vaccine virus, but antibodies cross-reacted at a lower level to the challenge virus. This study determined that low serum HI antibodies to a challenge virus induced by vaccination with a heterologous virus provided protection demonstrated by clinical protection and reduced pneumonia and viral excretion. The vaccine was able to prime the local SIV-specific antibody response in the lower respiratory tract as well as inducing a systemic SIV-specific memory T-cell response. MDA alone were capable of suppressing fever subsequent to infection, but other parameters showed reduced protection against infection compared to vaccination. The presence of MDA at vaccination negatively impacted vaccine efficacy as fever and clinical signs were prolonged, and unexpectedly, SIV-induced pneumonia was increased compared to pigs vaccinated in the absence of MDA. MDA also suppressed the serum antibody response and the induction of SIV-specific memory T-cells following vaccination. The results of this study question the effectiveness of the current practice of generating increased MDA levels through sow vaccination in protecting piglets against disease.     

Cell mediated immunity in equine herpesvirus type 1 infection I. In vitro lymphocyte blastogenesis and serum neutralization antibody in normal parturient and aborting mares.

Blastic transformation of peripheral blood mononuclear cells and serum neutralization antibody levels for equine herpesivurs type 1 were measured in 19 mares from three farms at the time of termination of their pregnancy by normal foaling or viral abortion. The stimulation indexes of lymphocytes obtained from the mares from two farms (Farm 1 and 2) which had virus abortions, ranged from 2.1 to 10.8. But there was no significant difference in stimulation index levels between the aborting and normal foaling mares on these two farms. Equine herpesvirus type 1 was isolated from the mononuclear cells of one mare (No. 5) about two months after she aborted. The stimulation index of lymphocytes from that mare was not significantly different from that of other mares on these farms. Stimulation index of lymphocytes from the mares on one farm (Farm 3) where there was no virus abortion or previous history of virus abortion but were exposed to virus antigen from vaccination, ranged from 1.6 to 2.9. The serum neutralization antibody levels were low in most mares ranging from 1/4 to 1/20 and in three mares these were higher. There was no direct correlation between the levels of serum neutralization antibody and stimulation index of lymphocytes from the mares on these farms.     

Needle-free delivery of an inactivated avian influenza H5N3 virus vaccine elicits potent antibody responses 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.     

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.     

Systemic and mucosal immune responses to H1N1 influenza virus infection in pigs

Influenza is a common respiratory disease in pigs, and since swine influenza viruses are zoonotic pathogens, they also pose human health risks. Pigs infected with influenza virus mount an effective immune response and are protected from subsequent challenge, whereas the currently available, inactivated-virus vaccine does not consistently confer complete protection to challenge. To develop and evaluate new vaccination strategies, it is imperative to fully understand the immune responses that are associated with protection following natural infection. Therefore, we have evaluated the phenotype and kinetics of immune responses to primary and re-challenge infection with H1N1 swine influenza virus in the pig. Through the use of isotype-specific antibody secreting cell ELISPOT assays, interferon-gamma ELISPOT assays and isotype-specific ELISAs on serum, nasal wash and bronchoalveolar lavage samples, we defined the humoral and cellular immune responses, both locally in the respiratory tract and systemically, to this viral infection. Virus-specific serum IgG, IgA, and HI titers all peaked 2-3 weeks after primary infection and did not substantially increase after re-challenge. The predominant virus-specific isotype in serum was IgG. Pigs responded with virus-specific IgG and IgA in both the upper (nasal washes) and lower (bronchoalveolar lavages) airways; IgA was the predominant isotype in both sites. Despite the fact that the pigs were completely protected from re-challenge, the antibody titers in the nasal washes increased. Results of the antibody-secreting cell ELISPOT assays demonstrated that the numbers of both IgG and IgA secreting cells in the nasal mucosa were dramatically higher than in any other tissue examined. In contrast, IFN-gamma secreting cells were predominantly localized to the spleen and tracheobronchial lymph nodes. These data will be helpful in the future development and evaluation of novel vaccines.     

A serological survey of canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in dogs in China

Influenza viruses have been isolated from dogs in China; however, the extent of influenza infection among dogs is not yet clear. Here, we examined the seroprevalence of avian-origin canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in pet dogs in China during January 2012 to June 2013. The seropositivity rate of canine H3N2, H1N1/09 and human H3N2 were 3.5%, 1.5%, and 1.2%, respectively. Dogs aged 2–5 years were most commonly seropositive to canine H3N2 virus. It is worth noting that two serum samples were positive against both canine H3N2 and H1N1/09 viruses, suggesting the possibility of coinfection with both viruses. Our findings emphasize the necessity for continued surveillance of influenza viruses in dogs in China.     

Concurrent response to challenge infection with Cryptosporidium parvum in immunosuppressed C57BL/6N mice

We investigated the response to challenge infection with Cryptosporidium parvum oocysts in immunosuppressed C57BL/6N mice. In the primary infection, fecal oocyst shedding and parasite colonization were greater in immunosuppressed mice than in nonimmunosuppressed mice. Compared with primary infection, challenge infection with C. parvum didn''t show any oocyst shedding and parasite colonization. Especially, oocyst shedding and parasite colonization from the mice infected with heat-killed oocysts were not detected. After challenge infection with C. parvum oocysts, however, these mice were shedding small numbers of oocysts and parasite colonization. Except normal control and uninfected groups, the antibody titers of other groups appear similar. Based on the fecal oocyst shedding, parasite colonization of ilea, and antibody titers in the mice, these results suggest that the resistance to challenge infection with C. parvum in immunosuppressed C57BL/6N mice has increased.     

Cellular and humoral immune responses during intrathoracic paracoccidioidomycosis in BALB/c mice

Paracoccidioidomycosis is a chronic infection that primarily affects the lungs. Here we investigated cellular and humoral immune responses after intrathoracic Paracoccidioides brasiliensis infection in BALB/c mice. P. brasiliensis-colony-forming units (CFUs), fungal DNA and granulomas in lungs increased progressively, peaking at day 90 postinfection (p.i.). IFN-γ production was highest on day 15 p.i., declining thereafter. The kinetics of the NO production was similar to that described for IFN-γ. In contrast, IL-10 increased from day 45 p.i. reaching a peak at day 90. Levels of serum IgG1 were higher than IgG2a between days 30 and 90 p.i. 30% of mice died by day 90 p.i. These data indicate that infection with P. brasiliensis by the intrathoracic route shows high IFN-γ and NO production at day 15 p.i., unable to control multiplication of fungi, which appears to be associated with a progressive increase in IL-10 and in the number and complexity of granulomas.