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1.
Background: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
Objectives: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses.
Animals: Fifteen horses experimentally infected with EHV-1.
Methods: Experimental study : Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed.
Results: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI ( P < .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86–100) and 27% (95% CI: 20–35).
Conclusions and Clinical Importance: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C T values are provided as well as justification of a minimum 10-day quarantine period.  相似文献   

2.
In this report we examined the presence of specific antibodies against equine herpesvirus type 1 (EHV-1), and equine herpesvirus type 4 (EHV-4) in several equidae, including mules, donkeys, horses. The presence of EHV-1 and EHV-4 in respiratory diseases of equids, and ability of multiplex nested polymerase chain reaction (PCR) screening in simultaneous diagnosis of horses acutely infected by EHV-1 and EHV-4 were also investigated. Sera from 504 horses, mules and donkeys sampled were tested for the presence of EHV-1 and EHV-4 specific antibodies. Blood samples taken from 21 symptomatic horses and nasal swabs taken from 40 symptomatic horses were tested for the presence of EHV-1 and EHV-4 by a multiplex nested PCR. A total of 14.3% (3/21) of buffy coat samples and 32.5% (13/40) nasal swab samples were found to contain EHV-1 DNA, while 19% (4/21) buffy coat samples and 22.5% (9/40) nasal swab samples were found to be positive for EHV-4 DNA. By species, 14.5% of horses, 37.2% of mules and 24.2% of donkeys tested were EHV-1 seropositive. EHV-4 specific antibodies were detected in 237 (81.7%) of 290 horse sera tested. Results from this investigation demonstrate that EHV-1 and EHV-4 are prevalent throughout the equid population, and that donkeys and mules might also represent an important source of infection for other equids. We also showed that the multiplex nested PCR assay might be useful for diagnosis of mixed respiratory infections in horses due to EHV-1 and EHV-4.  相似文献   

3.
Equine respiratory viral infections cause significant worldwide disease and economic loss. Common causes include equine influenza virus (EIV) and equine herpesviruses-1 and -4 (EHV-1 and -4), and risk of exposure to these agents may be highest in young horses commingling at sales and competitive events. A surveillance study was conducted at two horse shows and two Thoroughbred sales to determine whether horses shed EHV-1, EHV-4, or EIV on arrival, or 2-4 days later, and whether shedding was associated with identifiable risk factors. Real-time polymerase chain reaction assays were used to detect EHV-1, EHV-4, and EIV nucleic acid in nasal swabs obtained from 369 horses at the four events. In response to evidence of clinical disease, 82 additional horses were sampled at two farms providing horses for one of the sales. On arrival at the events, shedding of EHV-1 was detected in 3.3%, EHV-4 in 1.1%, and EIV in 0.8% of horses. EHV-1 was detected at low levels, and EHV-1 and EHV-4 detection was not associated with clinical disease. EIV was detected only in horses at a Thoroughbred sale, in association with an outbreak of respiratory disease traced back to regional farms. On arrival at events, horses younger than 2 years had a significantly greater risk of shedding EHV-1 compared with older horses; no other significant risk factors associated with viral shedding were identified. Thus, there is a risk of exposure to EIV, EHV-1, and EHV-4 at equine events, and horses and events should be managed to mitigate this risk.  相似文献   

4.
Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.  相似文献   

5.
AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.  相似文献   

6.
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7.
The immunotherapeutic effect of low-dose human alpha interferon on viral shedding and clinical disease was evaluated in horses inoculated with equine herpesvirus-1 (EHV-1). Eighteen clinically healthy weanling horses, 5 to 7 months old, were allotted to 3 equal groups. Two groups were treated orally with human alpha-2a interferon (0.22 or 2.2 U/kg of body weight), on days 2 and 1 before inoculation with EHV-1, the day of inoculation, and again on postinoculation day 1. The horses of the remaining group were given a placebo orally on the same days. The horses were monitored daily for changes in body temperature and for clinical signs of respiratory tract disease. Blood and nasal swab specimens were collected daily for virus isolation. Blood was also collected at intervals throughout the monitoring period for evaluation of CBC, serum IgG and IgM concentrations, and antibody titers to EHV-1. Febrile responses, nasal discharge, viral shedding, changes in CBC, and an increase in antibody titers to EHV-1 were noticed in all horses after inoculation. There was no significant difference (P greater than 0.05) in mean values of the factors measured between treatment and control groups.  相似文献   

8.
AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.  相似文献   

9.
The polymerase chain reaction (PCR) is a sensitive technique used to detect DNA of viral pathogens. We have applied the technique to the detection of Equid herpesviruses-1 and -4 (EHV-1 and EHV-4) DNA within nasopharyngeal swab samples from horses. Ninety-eight samples from suspected field cases and in-contact horses were analysed. The assays were conducted blind and later decoded and compared with virus isolation data. Our results indicate that PCR is a sensitive and rapid technique for the diagnosis of EHV-1 and EHV-4 infection.  相似文献   

10.
OBJECTIVE: To determine the incidence of equine herpesvirus-1 (EHV-1) infection among Thoroughbreds residing on a farm on which the virus was known to be endemic. DESIGN: Prospective cohort study. ANIMALS: 10 nonpregnant mares, 8 stallions, 16 weanlings, 11 racehorses, and 30 pregnant mares and their foals born during the 2006 foaling season. PROCEDURES: Blood and nasopharygeal swab samples were collected every 3 to 5 weeks for 9 months, and placenta and colostrum samples were collected at foaling. All samples were submitted for testing for EHV-1 DNA with a PCR assay. A type-specific EHV-1 ELISA was used to determine antibody titers in mares and foals at birth, 12 to 24 hours after birth, and every 3 to 5 weeks thereafter. RESULTS: Results of the PCR assay were positive for only 4 of the 1,330 samples collected (590 blood samples, 590 nasopharyngeal swab samples, 30 placentas, and 30 colostrum samples), with EHV-1 DNA detected in nasal secretions from 3 horses (pregnant mare, stallion, and racehorse) and in the placenta from 1 mare. Seroconversion was detected in 3 of 27 foals during the first month of life. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that there was a low prevalence of EHV-1 infection among this population of Thoroughbreds even though the virus was known to be endemic on the farm and that pregnant mares could become infected without aborting. Analysis of nasopharyngeal swab samples appeared to be more sensitive than analysis of blood samples for detection of EHV-1 DNA.  相似文献   

11.
AIMS: To determine which viruses circulate among selected populations of New Zealand horses and whether or not viral infections were associated with development of respiratory disease.

METHODS: Nasal swabs were collected from 33 healthy horses and 52 horses with respiratory disease and tested by virus isolation and/or PCR for the presence of equine herpesviruses (EHV) and equine rhinitis viruses.

RESULTS: Herpesviruses were the only viruses detected in nasal swab samples. When both the results of nasal swab PCR and virus isolation were considered together, a total of 41/52 (79%) horses with respiratory disease and 2/32 (6%) healthy horses were positive for at least one virus. As such, rates of virus detection were significantly higher (p<0.001) in samples from horses with respiratory disease than from healthy horses. More than half of the virus-positive horses were infected with multiple viruses. Infection with EHV-5 was most common (28 horses), followed by EHV-2 (27 horses), EHV-4 (21 horses) and EHV-1 (3 horses).

CONCLUSIONS: Herpesviruses were more commonly detected in nasal swabs from horses with respiratory disease than from healthy horses suggesting their aetiological involvement in the development of clinical signs among sampled horses. Further investigation to elucidate the exact relationships between these viruses and respiratory disease in horses is warranted.

CLINICAL RELEVANCE: Equine respiratory disease has been recognised as an important cause of wastage for the equine industry worldwide. It is likely multifactorial, involving complex interactions between different microorganisms, the environment and the host. Ability to control, or minimise, the adverse effects of equine respiratory disease is critically dependent on our understanding of microbial agents involved in these interactions. The results of the present study update our knowledge on the equine respiratory viruses currently circulating among selected populations of horses in New Zealand.  相似文献   

12.
The aim of the present study was to investigate abortion storms that occurred in the Marmara region of Turkey in 2008-2009 using a real-time PCR. Two aborted foetuses were necropsied and histo-pathological findings reported herein. Ten lungs, 3 brains and one nasal swab from 10 aborted foetuses, 6 nasal swabs and 3 vaginal swabs from aborting mares were included in this study. EHV-1 was isolated from the lung, liver and brain of 1 aborted foetus. EHV-1 DNA was detected in the lungs, livers and spleens of 2 necropsied foetuses and in 3 lungs from 10 foetuses submitted for diagnosis. A brain from one of the aborted foetuses was also positive for EHV-1 DNA. EHV-4 DNA was detected only in a nasal swab of one of the tested foetuses. Neither EHV-1 nor EHV-4 DNA was detected in the swabs of aborting mares. Sequence analysis of the glycoprotein B of the strains was performed and a phylogenetic tree was generated. The results indicated that 4 of the 5 Turkish EHV-1 strains (TR02, TR03, TR04 and TR05) clustered together; the fifth strain (TR01) was slightly removed from the group and clustered with other EHV-1 from various origins. Single nucleotide polyporphism (SNP in ORF30) associated with neuropathogenesis was not detected in any of the strains. At necropsy, sub-milier focal necrosis in the liver and spleen was observed. Microscopically, focal coagulation necrosis and marked eosinophilic intranuclear and intracytoplasmic inclusion bodies in the hepatocytes localised around the necrotic areas in the liver. Severe coagulation necrosis in white pulp of the spleen was also observed.  相似文献   

13.
The prevalence of equine respiratory virus infections among a suspected population of race horses was examined using polymerase chain reaction (PCR). One or more of five equine respiratory viruses were detected in the nasal swabs of 45 of 89 horses (50.6%), and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 5 (EHV-5), equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) were 5.6%, 7.9%, 39.0%, 2.2%, and 6.7%, respectively. Among the 45 infected horses, 7 were co-infected with EHV and/or equine rhinitisvirus (ERV). Equine influenzavirus and equine arteritisvirus were not detected in any samples. Specific antibodies to EHV-1 and/or EHV-4 were detected in 59 of 73 tested sera (80.8%), using a virus neutralization test. This investigation suggests that equine respiratory viruses are endemic at Seoul Race Park and that the impact of viral infections on race horses’ health in Republic of Korea should be evaluated.  相似文献   

14.
REASONS FOR PERFORMING STUDY: Currently, there is no recommended immunoprophylaxis against febrile respiratory diseases due to equine herpesvirus-1 (EHV-1) and -4 (EHV-4) in horses below age 5-6 months. This is because of interference by maternally-derived antibody (MDA) of vaccines. OBJECTIVE: Unweaned equine foals are an important reservoir of EHV-1 transmission; therefore, we experimentally assessed the efficacy of a live EHV-1 vaccine in foals age 1.4-3.5 months with MDA. METHODS: Following vaccination and challenge, parameters assessed were virus shedding in nasal mucus, leucocyte-associated viraemia, circulating virus neutralising antibody activity and clinical reactions. RESULTS: Controlled challenge showed that a single intranasal dose of the vaccine afforded partial but significant protection against febrile respiratory disease, virus shedding and viraemia due to EHV-1 infection, despite virus-neutralising MDA. CONCLUSIONS AND POTENTIAL RELEVANCE: The prospective vaccine would be a significant step forward in reducing the incidence of the disease caused by EHV-1 infection.  相似文献   

15.
A PCR assay for the diagnosis of respiratory disease induced by equine herpesvirus type 1 (EHV-1) was performed at the clinical laboratory in the Racehorse Clinic of the Ritto Training Center of the Japan Racing Association from December 2007 to March 2008. The assay was performed without the trouble of contamination throughout the study and its turnaround time was approximately 6 hr. The PCR detection rates of EHV-1 among seroconverted horses were 22.2% for nasal swabs and 33.3% for blood samples. However, EHV-1 DNA was also detected in horses without seroconversion at a low rate. These results indicated that the PCR assay should be used as an adjunct method, but would help to make an early diagnosis of EHV-1 infection.  相似文献   

16.
A group of three horses was experimentally infected with equine herpesvirus type 1 (EHV-1) and showed clinical signs characterised by a biphasic febrile response, leucopenia and cell associated viraemia accompanied by virus shedding from the nasopharynx. A second exposure to the virus 18 days later resulted in the isolation of virus from the nasopharynx of one horse. This and a further group of three EHV-1 seropositive horses were subsequently infected with equine herpesvirus type 4 (EHV-4) 147 days after the initial EHV-1 infection and virus was shed from the nasopharynx in the absence of clinical disease. Following the first EHV-1 infection, virus specific immunoglobulin M (IgM) was present by day 5 and remained high until the second exposure at day 18 at which point levels decreased. In contrast, EHV-1 specific IgG, detected at day 6 peaked at day 18, after which time levels remained high. Virus neutralising antibodies and antibodies able to mediate antibody-dependent cellular cytotoxicity were present by day 10. The immune response to EHV-1 is discussed with reference to the disease.  相似文献   

17.
OBJECTIVE: To evaluate a technique for identifying horses latently infected with neuropathogenic strains of equine herpesvirus-1 (EHV-1). ANIMALS: 36 adult mares, 24 of which were experimentally infected as weanlings with neuropathogenic or nonneuropathogenic EHV-1. PROCEDURES: Mandibular lymph node (MLN) tissue was obtained from each horse via biopsy during general anesthesia. Purified DNA from MLNs was tested for EHV-1 DNA by use of a magnetic bead, sequencecapture, nested PCR assay. For MLNs that contained EHV-1 DNA, the 256-bp DNA fragments amplified via sequence-capture nested PCR were sequenced to determine the nucleotide at the polymorphic site that determines pathotype (ie, neuropathotype [G(2254)] or non-neuropathotype [A(2254)]). RESULTS: Latent viral DNA was detected in 26 of the 36 (72%) mares tested. Neuropathogenic and nonneuropathogenic EHV-1 genotypes were detected in the latently infected horses. In each mare previously infected with known EHV-1 pathotypes, the open reading frame 30 genotype of latent EHV-1 was identical to that of the strain that had been inoculated 4 to 5 years earlier. Latent viral DNA was detected in 10 of the 12 mares that were inoculated as weanlings with neuropathogenic strains of EHV-1. The detection rate of the sequence-capture PCR method for EHV-1 latency was double that of conventional nested or realtime PCR assays performed on the same MLN DNA preparations. CONCLUSIONS AND CLINICAL RELEVANCE: The magnetic bead, sequence-capture, nested PCR technique enabled low-threshold detection of DNA from latent neuropathogenic strains of EHV-1 in MLN specimens from live horses. The technique may be used to screen horses for latent neuropathogenic EHV-1 infection.  相似文献   

18.
REASONS FOR PERFORMING STUDY: A silent cycle of equine herpesvirus 1 infection has been described following epidemiological studies in unvaccinated mares and foals. In 1997, an inactivated whole virus EHV-1 and EHV-4 vaccine was released commercially in Australia and used on many stud farms. However, it was not known what effect vaccination might have on the cycle of infection of EHV-1. OBJECTIVE: To investigate whether EHV-1 and EHV-4 could be detected in young foals from vaccinated mares. METHODS: Nasal and blood samples were tested by PCR and ELISA after collection from 237 unvaccinated, unweaned foals and vaccinated and nonvaccinated mares during the breeding season of 2000. RESULTS: EHV-1 and EHV-4 DNA was detected in nasal swab samples from foals as young as age 11 days. CONCLUSIONS: These results confirm that EHV-1 and EHV-4 circulate in vaccinated populations of mares and their unweaned, unvaccinated foals. POTENTIAL RELEVANCE: The evidence that the cycle of EHV-1 and EHV-4 infection is continuing and that very young foals are becoming infected should assist stud farms in their management of the threat posed by these viruses.  相似文献   

19.
Equineherpesvirustypes 2 and 5 (EHV-2andEHV-5)have a rather unclearpathogenicity and distribution within the equid population. In order to gain more information on the prevalence of these two viruses, type-specific PCR assays were developed to detect viral DNA in nasal specimens and in peripheral blood leukocytes (PBLs) of adult horses and foals from various regions of Europe, i.e. Sweden, Hungary and the United Kingdom. In adult horses, the prevalence of EHV-2 in PBLs was up to 68% in Sweden and 71% in the United Kingdom. EHV-2 DNA was detected in the PBLs from all the foals tested in all countries and most (93%) of the nasal specimens also yielded positive results. The prevalence of EHV-5 DNA in the PBLs of foals in Hungary was 15 and 24% in adult horses in the United Kingdom. This observation was among the very few reports of the presence of EHV-5 in horses. In summary, the specific PCR assays revealed important data on the occurrence and distribution of EHV-2 and EHV-5 in large horse populations. The findings indicated that infection with EHV-5 occurred later than EHV-2 in foals. This study may contribute to a better understanding of the etiological role of these gammaherpesviruses in equine diseases.  相似文献   

20.
The dissemination of equine herpesvirus types 1 (EHV-1) and 4 (EHV-4) among various horse populations in Japan was investigated through the isolation and typing of virus strains from horses with respiratory diseases. Type specific monoclonal antibody pools were used for the typing of isolates. The 42 strains of EHV-1 and 64 strains of EHV-4 were isolated from 4593 nasal swabs and/or blood plasma samples collected from 3326 horses during a period from 1979 to 1990. All the strains of EHV-1 were isolated from racehorses only and during the winter season exclusively, when the epizootic of respiratory diseases occurred among racehorse populations at two Training Centers of the Japan Racing Association. In contrast, the strains of EHV-4 were isolated from horses irrespective of the season, facility, or horse population; foals and yearlings in breeding farms and our institute, rearing horses in rearing farms, and racehorses. Especially, 4 strains of EHV-4 were isolated from plasma samples containing buffy coat cells. We believe this is the first reported case of EHV-4 cell-associated viremia in the world. All 87 strains isolated from aborted fetuses were identified as EHV-1. The results suggest that EHV-1 is responsible for epizootic respiratory diseases in racehorses in the winter and abortion among mares at the late stage of gestation, and that EHV-4 causes respiratory diseases throughout the year among all horse populations.  相似文献   

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