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1.
Humoral immune response to Rhodococcus equi in experimentally infected foals was studied with the enzyme-linked immunosorbent assay (ELISA) method. Class-specific antibodies were measured by ELISA in the sera of foals after intratracheal or oral inoculation with R. equi ATCC 6939 or T 48 and in the lung washings of a foal after intratracheal inoculation or of normal horses. After intratracheal or oral inoculation with R. equi, serum antibodies were first detected in immunoglobulin G (IgG) followed by IgM and IgA classes, but significant levels of IgM and IgA developed only in the foal infected intratracheally with R. equi T 48. Only the foal infected intratracheally with T 48 developed pneumonia. Anti-R. equi IgG and IgA antibodies appeared in lung washings of the intratracheally infected foal. There were differences in the antibody response to R. equi among the intratracheally infected foals, the orally infected foal and the naturally infected foal. These results suggest that the humoral immune response to R. equi may be affected by the type of R. equi strain and the route and extent of R. equi exposure.  相似文献   

2.
An enzyme-linked immunosorbent assay (ELISA) was developed for use in horses to determine serum titers of antibodies of the immunoglobulin classes IgA, IgG, and IgM to Streptococcus equi M-like protein and culture supernatant protein antigens. Serum antibodies were determined in 28 adult horses, including 9 horses with recent S. equi infections, 17 horses without known exposure to S. equi, but without a history of respiratory disease in the preceding 4 months, and 2 horses with clinical purpura hemorrhagica. Serum IgA titers to culture supernatant protein antigen were highest in recently infected horses (P less than 0.001). Serial determinations of antibody titers in the horses with purpura showed that IgG antibodies to both S. equi M-like protein and culture supernatant protein antigens were undetectable initially, but later rose coincidental with clinical recovery from the disease. Possible mechanisms for these findings are discussed.  相似文献   

3.
Nineteen horses with no prior experience with equine arteritis virus (EAV) were inoculated IM with an avirulent live-virus vaccine against equine viral arteritis; the vaccinal virus had been passaged serially 131 times in primary cell cultures of equine kidney, 111 times in primary cell cultures of rabbit kidney, and 16 times in an equine dermis cell line (EAV HK-131/RK-111/ED-16). Three or 4 of the vaccinated horses each, along with appropriate nonvaccinated controls, were inoculated nasally with virulent EAV at each of months 3, 6, 9, 12, 18, and 24 after they were vaccinated. The following was concluded: Vaccination did not induce clinical signs of disease in any horse and, thus, seemed safe for use in the field. All vaccinated horses (n = 19) developed serum-neutralizing antibodies to EAV. Fourteen of the vaccinated horses were completely protected from clinical arteritis when exposed to large doses of virulent EAV. Four were partially protected, and one had little or no protection. Six of 13 nonvaccinated horses died of acute arteritis, and the remaining 7 horses experienced severe signs of disease, but survived the infection. All horses (n = 32), whether vaccinated or not, became infected when inoculated nasally with virulent EAV. Virus was recovered from 17 of the 19 vaccinated horses, and all 19 had a secondary humoral immune response. The duration and severity of thermal reaction and persistence of virus were more transitory in vaccinated horses than in the nonvaccinated controls. Protection afforded by this vaccine can persist for at least 24 months, the maximal time after horses were vaccinated that immunity was challenged in the present study.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

4.
For diagnosis of equine granulocytic ehrlichiosis (EGE) serological testing of antibodies to Ehrlichia equi is frequently used. An elevated antibody level is often misinterpreted as confirmative of active infection and results in treatment with antibiotics. If only seropositivity is considered as the diagnostic criterium, many horses showing convalescence titres will be treated. This study was undertaken to obtain information about the kinetics of antibodies during the course of infection and, for this purpose, 45 horses with clinical signs of EGE and confirmed ehrlichiaemia were monitored serologically and clinically over time. For a correct handling of cases with suspected EGE, the following results should be helpful: (i) 44% of the horses in the acute ehrlichiaemic stage were found to be serologically positive to E. equi; (ii) all horses showed a rapid increase in antibody titre, reaching maximum value within a month after the ehrlichiaemic stage; (iii) when 8 months had passed, titres had decreased, but 18 of 24 examined horses were still serologically positive; (iv) after 12-15 months most of the horses (n = 10) were serologically negative; and (v) the period required for complete clinical recovery varied from one day up to 6 months after antibiotic treatment.  相似文献   

5.
Equine cell-mediated immune response to Rhodococcus (Corynebacterium) equi   总被引：4，自引：0，他引：4  
A lymphocyte blastogenic assay was developed to serve as an in vitro correlate of cell-mediated immunity to Rhodococcus (Corynebacterium) equi (R equi) in the equine species. Lymphocytes obtained from a group of experimental ponies showed no response in cell culture to R equi heat extract or lysozyme extract antigens. Ponies were assigned to groups for experimental inoculation. Three ponies were inoculated subcutaneously with live R equi, 3 were given live R equi by intranasal and intratracheal routes, and 4 ponies were left untreated. Lymphocytes from all inoculated ponies had a mitogenic response to R equi antigens in lymphocyte blastogenic assays performed between the 7th and 40th days after inoculation. Lymphocytes from noninoculated control ponies remained unresponsive to R equi antigens. Delayed-type hypersensitivity reactions developed in all experimentally exposed ponies after intradermal administration of the R equi antigen preparations. In a 2nd phase of experimentation, blastogenesis assays were performed on lymphocytes from horses in herds with endemic R equi infections. Results indicated that many of the animals had significant (stimulation index greater than 2) cell-mediated responses to the bacterium, but there was no distinct correlation between the immune response and clinical history. These data indicated that cell-mediated immunity is involved in the interaction of the equine immune system with R equi.  相似文献   

6.
Equine humoral immune response to Rhodococcus (Corynebacterium) equi   总被引：7，自引：0，他引：7  
An enzyme-linked immunosorbent assay was developed to test equine serum for the presence of antibodies to Rhodococcus (Corynebacterium) equi. Experimental ponies had no detectable antibody to R equi before exposure to the bacterium. After experimental inoculation, animals in groups that received live R equi subcutaneously or intranasally/intratracheally developed high titers to R equi. Noninoculated controls remained seronegative. Serum was also collected from horses of various ages that were naturally exposed to R equi. There was a wide range of anti-R equi titers in these horses. Because experimentally infected horses seroconverted when some naturally infected foals did not seroconvert, the function of antibody in resistance to R equi infection remains unknown.  相似文献   

7.
OBJECTIVE: To determine susceptibility of cattle to infection with Ehrlichia equi and the agent of human granulocytic ehrlichiosis (HGE). DESIGN: Experimental disease and prevalence survey. ANIMALS: 6 cattle, 2 horses, and 2,725 serum samples from healthy cattle. PROCEDURE: 2 cattle and 1 horse were inoculated with E equi, 2 cattle and 1 horse were inoculated with the HGE agent, and 2 cattle served as sham-inoculated controls; inoculated animals were evaluated via clinical, hematologic, serologic, and real-time polymerase chain reaction tests. Prevalence of antibodies against E equi in 2,725 healthy cattle was determined by use of an indirect immunofluorescent technique. RESULTS: No abnormal clinical or hematologic findings or inclusion bodies within granulocytes were observed in the cattle after inoculation, and results of all polymerase chain reaction tests were negative. Seroconversion in inoculated cattle developed 10 to 12 days after inoculation (reciprocal titers, 160). Both horses developed clinical signs of ehrlichiosis. Five of 2,725 (0.18%) cattle were seropositive for E equi, with titers ranging from 20 to 80. All seropositive cattle originated from the same tick-rich region in the Sierra Nevada foothills. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle are not susceptible to infection with E equi or the agent of HGE and that prevalence of exposure to E equi in healthy cattle is low. Therefore, E equi and the agent of HGE are likely of negligible importance for cattle in North America.  相似文献   

8.
OBJECTIVE: To determine whether horses living in tick-infested areas of northeastern United States with clinical signs of borreliosis or granulocytic ehrlichiosis had detectable serum antibodies to both Borrelia burgdorferi and Ehrlichia equi. DESIGN: Prospective study. ANIMALS: Serum samples from 51 clinically normal horses, 14 horses with clinical signs of borreliosis, and 17 horses with clinical signs of granulocytic ehrlichiosis. PROCEDURE: Serum B burgdorferi or E equi antibodies were measured by use of an ELISA, immunoblot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: Of the 82 serum samples tested, 37 (45.1%) and 13 (15.9%) had detectable antibodies to B burgdorferi or E equi, respectively. Test results indicated that 12 horses had been exposed to both agents, 11 of these horses had granulocytic ehrlichiosis. The ELISA regularly detected antibodies to the following recombinant protein (p) antigens of B burgdorferi: p29, p37, p39, and p41-G. The use of immunoblot analysis confirmed ELISA results by indicating antibody reactivities to antigens of whole-cell B burgdorferi having molecular masses of predominantly 31, 34, 37, 39, 41, 58, and 93 kd. CONCLUSIONS AND CLINICAL RELEVANCE: Horses living in areas where ticks (Ixodes scapularis) abound are sometimes exposed to multiple pathogens. Analyses for specific recombinant borrelial antibodies using an ELISA can help separate horses with borreliosis from those with granulocytic ehrlichiosis, even when antibodies to both etiologic agents are detected in serum samples. Analysis using immunoblots is sensitive, and along with ELISA or IFA procedures, is suitable for confirming a clinical diagnosis of each disease.  相似文献   

9.
The medical records of 53 horses with purpura haemorrhagica were reviewed. Seventeen of them had been exposed to or infected with Streptococcus equi, nine had been infected with Corynebacterium pseudotuberculosis, five had been vaccinated with S. equi M protein, five had had a respiratory infection of unknown aetiology, and two had open wounds; the other 15 cases had no history of recent viral or bacterial infection. The horses were between six months and 19 years of age (mean 8.4 years). The predominant clinical signs were well demarcated subcutaneous oedema of all four limbs and haemorrhages on the visible mucous membranes; other signs included depression, anorexia, fever, tachycardia, tachypnoea, reluctance to move, drainage from lymph nodes, exudation of serum from the skin, colic, epistaxis and weight loss. Haematological and biochemical abnormalities commonly detected were anaemia, neutrophilia, hyperproteinaemia, hyperfibrinogenaemia, hyperglobulinaemia and high activities of muscle enzymes. All of the horses were treated with corticosteroids; 42 also received non-steroidal anti-inflammatory drugs and 26 received antimicrobial drugs. Selected cases received special nursing care, including hydrotherapy and bandaging of the limbs. Most of the horses were treated for more than seven days and none of them relapsed. Forty-nine of the horses survived, one died and three were euthanased, either because their severe clinical disease failed to respond to treatment or because they developed secondary complications. Two of the four non-survivors had been vaccinated against S. equi with a product containing the M protein, one had a S. equi infection and the other had a respiratory infection of undetermined aetiology.  相似文献   

10.
The prevalence of antibodies to Ehrlichia equi in horses from the foothill regions of northern California and from the Sacramento valley (non-foothill area) was determined, using an indirect fluorescent antibody test. Horses from foothill regions had a higher prevalence of seropositivity (10.4%) and higher titer (1:10 to 1:80) than did those from non-foothill regions (3.1%; titer less than or equal to 1:10). Fifty percent of healthy horses on a foothill farm enzootic for E equi had titer to E equi, suggesting that infection with E equi can be subclinical. Six veterinarians surveyed from northern California diagnosed clinical E equi infection in 38 horses during 1985-1986 based on clinical signs of infection and observation of E equi inclusion bodies in neutrophils on blood smears.  相似文献   

11.
CASE DESCRIPTION: A 4-month-old American Paint Horse colt was evaluated because of acute onset of ataxia, left-sided head tilt, and fever and a recently noticed heart murmur. Upper respiratory tract infection caused by Streptococcus equi subsp equi had been diagnosed at 3 months of age. CLINICAL FINDINGS: Hematologic abnormalities included leukocytosis, mature neutrophilia, monocytosis, and mild anemia. Analysis of a CSF sample revealed high total protein concentration and total nucleated cell count; nucleated cells consisted mainly of degenerate neutrophils. Results of a real-time PCR assay were positive for S equi subsp equi, and a diagnosis of S equi subsp equi meningoencephalomyelitis was made. TREATMENT AND OUTCOME: Treatment included administration of potassium penicillin and fluids, but the foal developed uroperitoneum and was subsequently euthanized. Postmortem examination revealed meningoencephalomyelitis, and S equi subsp equi was cultured from a brain aspirate. Additional findings included suppurative cystitis with rupture and neutrophilic myocarditis. CLINICAL RELEVANCE: Findings suggest that S equi subsp equi meningoencephalomyelitis should be considered in the differential diagnosis for foals with neurologic signs that have a history of strangles or exposure to affected horses.  相似文献   

12.
Hobo S  Niwa H  Anzai T 《The Veterinary record》2008,162(15):471-474
The reactivity of the proline-glutamic acid-proline-lysine (PEPK) repetition peptide antigen in 3176 serum samples was investigated to evaluate its utility as an antigen for the serological diagnosis of strangles. The reactivity of the sera of horses infected with Streptococcus equi subspecies equi was high when the peptide had several PEPK repetitions. However, as the number of PEPK repetitions increased, the reactivity of the antigen with the sera of horses infected with Streptococcus equi subspecies zooepidemicus also increased. In horses infected experimentally with S equi, the reactivity of the PEPK antigen with five repetitions increased one week after inoculation and continued to increase during the following four weeks. The optical density (OD) values of test sera from horses infected experimentally with S equi and sera from horses that had recovered from strangles were high. The od values of sera from horses that had recovered from an experimental infection with S zooepidemicus and of sera from healthy horses were comparatively low.  相似文献   

13.
In order to assess the level of Rhodococcus equi infection in southeast Turkey, 679 sera from healthy foals and adult horses and 78 sera from donkeys were tested by indirect ELISA using a R. equi reference strain (ATCC 33701) as antigen. Eighty (11.7%) sera from horses and 9 (11.5%) sera from donkeys with titres >0.85 were positive. The prevalence of seropositive horses in Sanliurfa Province was higher than in Diyarbakir Province; 56 (13.9%) horses in Sanliurfa Province and 24 (8.7%) horses in Diyarbakir Province were defined as seropositive. In Sanliurfa Province 14.5% of female (n=343) and 10.1% of male (n = 59) horses tested were defined as seropositive, while in Diyarbakir Province more males (11.4%, n=114) were seropositive than females (6.7%, n=163). Horses 1 to 5 years of age were found to have the highest seropositivity rate in both provinces. A total of 78 sera from donkeys were investigated in Sanliurfa Province, of which 9 (11.5%) were positive by ELISA. Among the 9 positive sera, 6 (12.8%) were from donkeys 1-5 years old and 3 (13.6%) were from donkeys >5 years of age. No positive sera were found in donkeys less than 1 year old. Five (12.5%) sera of females and 4 (10.5%) sera of males tested were positive. These results indicate the existence of R. equi in the horse populations in Sanliurfa and Diyarbakir Provinces. Similar infection rates were found for donkeys in Sanliurfa. This suggests the importance of serological surveys to diagnose R. equi infection in the region and to prevent the zoonotic risk.  相似文献   

14.
Equine protozoal myeloencephalitis (EPM) is generally caused by Sarcocystis neurona and can produce substantial economic losses on equine production in America. The aims of the present study were to evaluate the seroprevalence of S. neurona in the main horse-production area of Argentina and associate it with the occurrence of neurologic disorders. Serum samples were collected from 640 horses in nine Argentinean provinces. Most of the samples correspond to animals ≥1.5-year-old from different breeds (n = 628); 12 samples were from younger horses. Further seroprevalence comparison was conducted from the older animals grouped with (n = 148) or without neurologic signs (n = 480). Immunoblot: proteins from 2 × 107S. neurona merozoites were used as antigen on each membrane. Reactivity to antigens with relative mobility of 7, 10, and 16 kDa was considered specific for antibodies against S. neurona; reactivity at 30 kDa was recorded separately. The overall seroprevalence for S. neurona was 26.1% (167/640), and all the provinces had positive horses. Seroprevalence of animals with neurologic signs was greater (P < .001) than what was observed in normal horses (39.2% vs. 22.1%), with an odds ratio of 2.27. Reactivity at 30 kDa was detected in 71% of all samples. This study identified a wide distribution of S. neurona–positive animals in Argentina and horses with neurologic signs having a greater seroprevalence than normal horses. Sarcocystis neurona infection should be considered for early differential diagnosis and treatment of animals with neurologic disorders to decrease the economic impact of EPM in Argentina.  相似文献   

15.
Rhodococcal pneumonia is an important disease of young horses that is not seen in immunocompetent adults. Since all foals are normally exposed to Rhodococcus equi in their environment, we hypothesized that most develop protective immune responses. Furthermore, these antigen-specific responses were hypothesized to operate throughout adult life to prevent rhodococcal pneumonia. A better understanding of the mechanisms of immune clearance in adult horses would help define the requirements for an effective vaccine in foals. Adult horses were challenged with virulent R. equi by intrabronchial inoculation into the right lung, and pulmonary immune responses were followed for 2 weeks by bronchoalveolar lavage. Local responses in the inoculated right lung were compared to the uninfected left lung and peripheral blood. Challenged horses rapidly cleared R. equi infection without significant clinical signs. Clearance of bacteria was associated with increased mononuclear cells in bronchoalveolar lavage fluid (primarily lymphocytes) and inversion of the normal macrophage:lymphocyte ratio. There was no significant increase in neutrophils at 7 days post-challenge. Flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that clearance correlated with significant increases in pulmonary T-lymphocytes, both CD4+ and CD8+. Prior to challenge, most adult horses demonstrated low proliferative responses when pulmonary lymphocytes were stimulated with soluble R. equi ex vivo. However, clearance was associated with marked increases in lymphoproliferative responses to soluble R. equi antigen and recombinant VapA, a virulence associated protein of R. equi and candidate immunogen. These results are compatible with previous work in mice which showed that both CD4+ and CD8+ T-cells play a role in immune clearance of R. equi. Recognition of VapA in association with clearance lends further support to its testing as an immunogen. Importantly, the cellular responses to R. equi challenge were relatively compartmentalized. Responses were more marked and the sensitivity to antigen dose was increased at the site of challenge. The blood, including peripheral blood mononuclear cells, was an insensitive indicator of local pulmonary responses.  相似文献   

16.
REASONS FOR PERFORMING STUDY: Streptococcus equi is the cause of strangles in horses. To improve diagnostic sensitivity, development and evaluation of DNA-based methods are necessary. OBJECTIVES: To evaluate diagnostic methods and observe the pattern of bacterial shedding during natural outbreaks. METHODS: Two herds with natural outbreaks of strangles were visited over a period of 15 weeks and 323 samples originating from 35 horses investigated. The diagnostic use of a nested PCR test was evaluated using a collection of 165 isolates of Lancefield group C streptococci (species specificity) and swabs from nasal passages or from abscesses from horses infected with S. equi (diagnostic sensitivity). RESULTS: All 45 S. equi isolates tested positive in the nested PCR, whereas no amplicon was formed when testing the other 120 Lancefield group C isolates. A total of 43 samples were collected from 11 horses showing clinical signs of strangles during the study period. The diagnostic sensitivity for PCR test was 45% and 80% for samples from the nasal passages and abscesses, respectively; the corresponding diagnostic sensitivity for cultivation was 18% and 20%. The diagnostic sensitivity was significantly higher for PCR than for bacterial cultivation. Furthermore, the shedding of S. equi in 2 infected horse populations was evaluated. An intermittent shedding period of S. equi of up to 15 weeks was recorded in this part of the study. It was also shown that shedding of S. equi occurred both from horses with and without clinical signs. CONCLUSIONS AND POTENTIAL RELEVANCE: The nested PCR test represents a species-specific and -sensitive method for diagnosis of S. equi from clinical samples. It may, however, be desirable in future to develop detection methods with high diagnostic sensitivity and specificity without the potential problems inherent in nested PCR.  相似文献   

17.
The age-specific attack rates of Streptococcus equi infections of the upper respiratory tract and lymph nodes (strangles) in horses for the different age groups were 17.6% for broodmares, 47.5% for 1-year-old horses, and 37.5% for foals. Streptococcus equi was isolated from nasal, pharyngeal, or lymph node specimens in 31 (60.8%) of 51 sick horses. A male 1-year-old horse, shipped from Kentucky to farm A, was considered to be the index case. Six (19.4%) of 31 horses with strangles remained as shedders of S equi after clinical signs of the disease had ended. Shedders of S equi were not identified from horses that were exposed to infected horses but never developed strangles.  相似文献   

18.
Four Quarter Horses (9 months to 7 years of age) with submandibular lymphadenopathy and firm muscles (palpation of which elicited signs of pain) were evaluated; in general, the horses had a stiff gait, and 3 horses became recumbent. Streptococcus equi was cultured from aspirates of lymph nodes or samples of purulent material collected from the auditory tube diverticula. Once the horses were recumbent, their condition deteriorated rapidly despite aggressive antimicrobial and antiinflammatory treatment, necessitating euthanasia within 24 to 48 hours. One horse did not become recumbent and recovered completely. Among the 4 horses, common clinicopathologic findings included neutrophilia, hyperfibrinogenemia, and high serum activities of creatine kinase and aspartate aminotransferase. Necropsies of the 3 euthanatized horses revealed large, pale areas most prominent in the semimembranosus, semitendinosus, sublumbar, and gluteal muscles that were characterized histologically by severe acute myonecrosis and macrophage infiltration of necrotic myofibers. Streptococcus equi was identified in sections of affected muscle by use of immunofluorescent stains for Lancefield group C carbohydrate and S. equi M protein. In the 4 horses of this report, acute severe rhabdomyolysis without clinical evidence of muscle atrophy or infarction was associated with S. equi infection; rhabdomyolysis was attributed to either an inflammatory cascade resembling streptococcal toxic shock or potentially direct toxic effects of S. equi within muscle tissue.  相似文献   

19.
The prevalence and epidemiology of important viral (equine influenza virus [EIV], equine herpesvirus type 1 [EHV-1] and EHV-4) and bacterial (Streptococcus equi subspecies equi) respiratory pathogens shed by horses presented to equine veterinarians with upper respiratory tract signs and/or acute febrile neurological disease were studied. Veterinarians from throughout the USA were enrolled in a surveillance programme and were asked to collect blood and nasal secretions from equine cases with acute infectious upper respiratory tract disease and/or acute onset of neurological disease. A questionnaire was used to collect information pertaining to each case and its clinical signs. Samples were tested by real-time PCR for the presence of EHV-1, EHV-4, EIV and S equi subspecies equi. A total of 761 horses, mules and donkeys were enrolled in the surveillance programme over a 24-month study period. In total, 201 (26.4 per cent) index cases tested PCR-positive for one or more of the four pathogens. The highest detection rate was for EHV-4 (82 cases), followed by EIV (60 cases), S equi subspecies equi (49 cases) and EHV-1 (23 cases). There were 15 horses with double infections and one horse with a triple infection. The detection rate by PCR for the different pathogens varied with season and with the age, breed, sex and use of the animal.  相似文献   

20.
Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79-100kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10(6) and 10(9) cells of R. equi IMV strain A5, respectively. The foal infected with 10(9) cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10(6) cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10(5) or 10(6) of virulent R. equi ATCC 33701 having 15-17kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10(9) bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.  相似文献   

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