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1.
Leptospirosis was identified to be the possible cause of premature birth in a foal on a farm with a history of repeated abortions. Using an appropriate polymerase chain reaction (PCR) assay, the presence of Leptospira kirschneri was detected in the tissues of the prematurely born foal. Further confirmation of L. kirschneri was obtained by nucleotide sequence analysis of the PCR-amplified DNA fragment and the partial 16S ribosomal RNA gene sequence. This report further supports mounting evidence that a PCR assay capable of detecting L. kirschneri should be included in routine diagnostic investigations in which Leptospira spp. infection is suspected.  相似文献   

2.
A LightCycler real-time PCR hybridization probe-based assay that detects a conserved region of the16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n=180) and aborted pig foetuses (n=24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n=7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n=30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents.  相似文献   

3.
A Leptospira interrogans serogroup australis serovar lora infection in a stud farm is reported. During three successive years (1984-1986) clinical leptospirosis with a severe often rapid, fatal course was seen in 12 foals. Clinical examination revealed severe respiratory distress, depression and pyrexia. Other symptoms were diarrhea (2), jaundice (1), and an unsteady gait (1). Morphological characteristics of the disease were massive pulmonary haemorrhage and haemorrhagic-thrombotic or extracapillary glomerulonephritis with tubulonephrosis and interstitial oedema. In most foals high or increasing MAT titres to serovar bratislava were found; from one foal Leptospira interrogans serovar lora was isolated. Serological examination of all 56 mares at the farm (August 1986) revealed antibodies to serovar bratislava in 64 per cent of the animals. These findings support the idea that Leptospira interrogans serovar bratislava and closely related strains (in this study serovar lora) may be adapted to and maintained by the horse population.  相似文献   

4.
A multiplex polymerase chain reaction (PCR) method using primer sets of G1/G2 and B64-I/B64-II was validated to detect pathogenic leptospira serovars from canine urine samples. The PCR method was found to be specific and sensitive with a detection limit of 100 cells of Leptospira icterohaemorrhagiae per milliliter of urine. The primer set previously designated and erroneously transcribed B64-I/B64-II amplified a DNA fragment of 352 base pairs from Leptospira grippotyphosa and Leptospira sejroe but not from Leptospira autumnalis, Leptospira bratislava, Leptospira canicola, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona. From 100 diagnostic canine urine samples, 5 were found positive for Leptospira grippotyphosalsejroe with a PCR product of 352 base pairs and 6 were positive for other pathogenic leptospira serovars with a PCR product of 285 base pairs. One 285-base pair product was sequenced and found to be 99.3% homologous to the G1/G2 PCR fragment sequence reported previously. All 352-base pair PCR products of clinical samples and pure cultures of L. grippotyphosa and L. sejroe were sequenced. The 352-base pair fragment sequences of L. grippotyphosa and L sejroe were identical. Only 2 base pairs were found different between the sequences from pure cultures and those from clinical samples. Serum samples from 3 positive cases that generated a PCR product of 352 base pairs were tested by the microscopic agglutination test, and 2 were found to be positive for L. grippotyphosa (1:10,240 and 1:5,120), 1 was positive for L. grippotyphosa (1:320) or L. icterohaemorrhagiae (1:320). The results of this study suggest that the multiplex PCR with the primer set G1/G2 and the erroneously transcribed B64-I/B64-II may be able to differentiate L. grippotyphosa or L. sejroe from other pathogenic leptospira serovars commonly tested for in Canadian diagnostic laboratories.  相似文献   

5.
This paper confirms the important role of rodents to be maintenance hosts of leptospires. Their role is related to renal carriage and shedding of leptospires into urine, thus contaminating fresh water. Serological and carriage of feral rodents trapped in France were determined by MAT and hap1PCR specific for pathogenic leptospires. In same areas, fresh water samples were analyzed by hap1PCR. The overall seroprevalence was 44% in 649 rodents and was similar regardless of the species. Seroprevalence for leptospirosis is about 20-53% according to species. hap1PCR (516 kidneys) showed that renal carriage was higher in brown rats (34.7%) and muskrats (15.8%) than in coypus (3.3%). hap1PCR demonstrates a significative difference (P-value > 10(-12)) for the renal carriage between the different species: muskrats and rats are more efficient maintenance hosts than coypu but all infect water. Moreover 5/38 water samples associated with human cases were hap1PCR positive and 1/113 in controlled waters.  相似文献   

6.
A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.  相似文献   

7.
OBJECTIVE: To evaluate the efficacy of a novel monoclonal antibody (MAb)-based dot-blot ELISA for detection of Leptospira antigens in urine samples of cattle. SAMPLE POPULATION: Blood and urine samples of 45 test cattle from 5 farms in Chonburi province and 20 control cattle from 2 farms in Khon Kaen province in Thailand. PROCEDURE: Blood and urine samples were assayed (microscopic agglutination test and urine antigen test) for Leptospira infection by use of an MAb-based dot-blot ELISA, and results for the ELISA were compared with those for dark-field microscopy (DFM), microbial culture, and a polymerase chain reaction (PCR) assay. RESULTS: All urine samples with positive results for DFM, microbial culture, PCR assay, or > 1 of these tests also had positive results when tested by use of the MAb-based dot-blot ELISA, except for 1 sample that had positive results only for the PCR assay. Detection limits of the dot-blot ELISA were 10(3) leptospires/mL of urine and 9.3 ng of Leptospira homogenate. Comparison revealed that the diagnostic sensitivity, specificity, efficacy (accuracy), positive predictive value, and negative predictive value for the ELISA were in agreement with results for DFM (100%, 72.72%, 80%, 57.14%, and 100%, respectively), microbial culture (100%, 61.54%, 66.62%, 28.57%, and 100%, respectively), and PCR assay (95.45%, 100%, 91.77%, 100%, and 95.83%, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The MAb-based dot-blot ELISA is suitable as a tool for detecting leptospires in urine samples of cattle.  相似文献   

8.
Clostridium perfringens type C is one of the most important agents of enteric disease in newborn foals. Clostridium difficile is now recognized as an important cause of enterocolitis in horses of all ages. While infections by C. perfringens type C or C. difficile are frequently seen, we are not aware of any report describing combined infection by these two microorganisms in foals. We present here five cases of foal enterocolitis associated with C. difficile and C. perfringens type C infection. Five foals between one and seven days of age were submitted for necropsy examination to the California Animal Health and Food Safety Laboratory. The five animals had a clinical history of acute hemorrhagic diarrhea followed by death and none had received antimicrobials or been hospitalized. Postmortem examination revealed hemorrhagic and necrotizing entero-typhlo-colitis. Histologically, the mucosa of the small intestine and colon presented diffuse necrosis and hemorrhage and it was often covered by a pseudomembrane. Thrombosis was observed in submucosal and/or mucosal vessels. Immunohistochemistry of intestinal sections of all foals showed that many large bacilli in the sections were C. perfringens. C. perfringens beta toxin was detected by ELISA in intestinal content of all animals and C. difficile toxin A/B was detected in intestinal content of three animals. C. perfringens (identified as type C by PCR) was isolated from the intestinal content of three foals. C. difficile (typed as A(+)/B(+) by PCR) was isolated from the intestinal content in 3 out of the 5 cases. This report suggests a possible synergism of C. perfringens type C and C. difficile in foal enterocolitis. Because none of the foals had received antibiotic therapy, the predisposing factor, if any, for the C. difficile infection remains undetermined; it is possible that the C. perfringens infection acted as a predisposing factor for C. difficile and/or vice versa. This report also stresses the need to perform a complete diagnostic workup in all cases of foal digestive disease.  相似文献   

9.
Listeria monocytogenes septicemia was diagnosed in a 6-day-old Thoroughbred foal. Primary clinical signs included fever, depression, diarrhea, and respiratory distress. Hematologic abnormalities included leukopenia, neutropenia, degenerative left shift, and hyperfibrinogenemia. Clinical chemistry and blood gas abnormalities included metabolic acidosis, hypoxemia, hypocapnia, hypoglycemia, and hyponatremia. Despite aggressive therapeutic intervention and intensive care, the foal died within 12 hours of admission. A postmortem examination was performed, and the primary gross lesion was bilaterally severe, focally extensive bronchopneumonia. Histopathology revealed severe subacute multifocal suppurative bronchopneumonia with necrotizing vasculitis and intralesional coccobacilli. Cultures of blood collected at admission and immediately prior to death were positive for L. monocytogenes, as were cultures obtained from lung and liver at necropsy. Immunohistochemical examination of formalin-fixed tissues revealed abundant intra- and extracellular L. monocytogenes antigen within the lung and intravascularly in multiple organs.  相似文献   

10.
Leptospirosis is the most widespread zoonosis worldwide, and it can cause reproductive failures in livestock, while in humans may vary from a mild fever to multi-organ failure and death. Due to this, in this study, we evaluated the usefulness of the segment encoding LigB C-terminus region, only present in pathogenic as target for a diagnostic PCR.This new PCR yielded a 100 % positivity for pathogenic Leptospira species and no cross-reactivity was found with intermediate or non-pathogenic species, or with other microorganisms, demostrating its high analytical specificity.The estimated analytical sensitivity was higher in serum samples than in blood or urine samples (6−9 × 102 lept/mL and 6−9 × 105 and 6−9 × 106 lept/mL, respectively). Multiple sequence alignment of the target region from different pathogenic Leptospira species confirmed that this gene region is highly conserved among these species, with few single nucleotide polymorphisms.The ligb-ct PCR here developed appears as a useful tool for the molecular diagnosis of leptospirosis.  相似文献   

11.
A nested polymerase chain reaction (PCR) using primers from the LipL32 sequence of Leptospira spp. was used to detect shedding of pathogenic leptospires in urine from naturally infected cattle. Amplicons (497bp) were obtained from 21 pathogenic reference serovars belonging to four species (L. interrogans, L. borgpetersenii, L. santarosai, L. kirschneri). DNA was amplified from 26/30 urine samples taken from cattle with suspected leptospirosis and from leptospires cultivated from 10 of these samples. The limit of detection of DNA in the clinical samples was 200pg and the nested PCR detected all pathogenic reference serovars of Leptospira spp. tested. No PCR products were amplified using DNA from other common bacterial species from the bovine urogenital tract or urine, or from the non-pathogenic L. biflexa Andamana serovar. The nested PCR exhibited high specificity and sensitivity for detection of pathogenic serovars in urine from cattle.  相似文献   

12.
为建立特异、敏感、快速的二温式PCR诊断方法,根据牛环形泰勒虫裂殖子表面抗原(tams1)基因,设计了一对特异性引物,扩增出大小为154bp基因片段,经克隆、测序分析,与已知基因序列的相似性为96%。用建立的牛环形泰勒虫病二温式PCR诊断方法,对从新疆牛环形泰勒虫病流行地区采集的50份全血样品进行诊断,阳性率为88%,而血涂片检出的阳性率只有58%。经试验验证,该方法具有特异性高、敏感性强、重复性和稳定性好等优点。表明本试验所建立的二温式PCR诊断方法可用于牛环形泰勒虫病的临床诊断、隐性感染检测和流行病学调查。  相似文献   

13.
Equine piroplasmosis due to Theileria equi and Babesia caballi is endemic in Trinidad. A case of equine piroplasmosis due to T. equi was diagnosed in a thoroughbred foal at 10h post-partum. A high parasitaemia (63%) of piroplasms was observed in a Wright-Giemsa(?) stained thin blood smear from the foal. In addition, the 18S rRNA gene for Babesia/Theileria was amplified from DNA extracted from the blood of the foal and the mare. Amplified products were subjected to a reverse line blot hybridization assay (RLB), which confirmed the presence of T. equi DNA in the foal. The mare was negative by RLB but was positive for T. equi using a nested PCR and sequence analysis. In areas where equine piroplasmosis is endemic, severe jaundice in a post-partum foal may be easily misdiagnosed as neonatal isoerythrolysis. Foals with post-partum jaundice should be screened for equine piroplasmosis, which may be confirmed using molecular methods if available.  相似文献   

14.
Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.  相似文献   

15.
The purpose of this study was to determine the presence of stx genes in avian pathogenic Escherichia coli (APEC). We examined 97 APEC isolates: 34 from lesions of avian cellulitis, 31 from avian septicemia, 13 from swollen head syndrome (SHS) in chickens, and 19 from diseased turkeys. We also examined five isolates from the feces of healthy chickens. All 102 E. coli isolates were tested for the presence of stx genes by PCR amplification and by colony blots using probes specific for stx1 and stx2. Fifty-three percent (52) of the 97 APEC carried stx gene sequences: one isolate carried stx2 sequences, two carried both stx1 and stx2 sequences, and the remaining 49 isolates carried only stx1 sequences. Twenty-six isolates were positive by both hybridization and PCR amplification, 10 were positive by PCR only, and 16 were positive by hybridization only. All the stx-positive isolates were negative by PCR for the eae and E-hlyA genes. The five isolates from healthy chickens were all negative for stx. All 13 SHS isolates were positive for the stx1 gene and had low titres for cytotoxicity in the Vero cell assay (VCA). Other stx-positive isolates were negative in the VCA. The stx1 gene from one SHS E. coli isolate was cloned and sequenced and shown to be identical to that of the stx gene of Shigella dysenteriae. The observations indicate that stx1 gene sequences are widespread among APEC but that cytotoxicity on Vero cells is uncommon.  相似文献   

16.
A 49-kg (107.8-lb) sexually intact male Arabian foal was evaluated at 3 days of age because of profuse watery diarrhea, anorexia, and signs of abdominal pain. Physical examination findings were unremarkable except for evidence of diarrhea. A catheter was placed in the right jugular vein for administration of antimicrobials and lactated Ringer's solution. The foal was discharged with instructions to the owner to continue antimicrobial administration and fluid therapy; at home, the owner inadvertently cut the catheter at the level of the hub during attempted removal, and the catheter fragment migrated distally in the jugular vein and subsequently lodged in the pulmonary artery. The foal was readmitted to the hospital for retrieval of the fragment, using a percutaneous retrieval technique. Catheter fragmentation is a well-recognized risk of catheterization in horses. Catheter fragments can be retrieved somewhat easily from the jugular vein; however, if the fragment migrates to the heart or pulmonary artery, imaging the fragment to locate and retrieve it can be difficult. Complications associated with catheter fragmentation include septicemia, endocarditis, lung abscesses, pulmonary embolism, dysrhythmias, cardiac perforation, pulmonary or caval thrombosis, and death. To our knowledge, this is the first report of successful retrieval of a catheter fragment from the pulmonary artery in a horse.  相似文献   

17.
OBJECTIVE: To develop a polymerase chain reaction (PCR) assay that detects and differentiates the Ohio strain of Haemobartonella felis (H. felis-OH) and the California strain of H. felis (H. felis-CA) and to apply the assay to blood samples from cats with and without suspected haemobartonellosis (suspect and control cats, respectively). SAMPLE POPULATION: 220 blood samples were examined; 82 were from suspect cats, and 138 were from control cats. PROCEDURE: A PCR assay was designed to detect and differentiate H. felis-OH and H. felis-CA. RESULTS: On the basis of PCR assay results, the overall prevalence of H. felis infection was 19.5% (43/220). Suspect cats (28.0%; 23/82) were significantly more likely than control cats (14.5%; 20/138) to be H. felis infected. Significantly greater numbers of suspect cats were H. felis-OH infected (12.2%, 9/82) or H. felis-OH and H. felis-CA infected (4.9%, 4/82) than control cats (0% [0/138] and 0.7% [1/138], respectively). Significantly more anemic cats were H. felis-OH infected (14.3%; 4/28) or H. felis-OH and H. felis-CA infected (7.1%; 2/28) than nonanemic cats (2.3% [3/128] and 0.8% [1/128], respectively). The PCR assay was more accurate than cytologic examination for detection of H. felis. CONCLUSIONS AND CLINICAL RELEVANCE: Haemobartonella felis infections are more common in cats than previously recognized. Haemobartonella felis-OH is apparently more pathogenic than H. felis-CA. The PCR assay is more accurate than cytologic examination for detection of H. felis infection and is an effective clinical tool for the detection and differentiation of both H. felis strains known to infect cats.  相似文献   

18.
A 3-day-old Quarter Horse colt was examined because of signs of severe depression, discomfort, and abdominal straining. The foal seemed disoriented, and the abdomen was tense and distended ventrally. The differential diagnoses included ruptured urinary bladder, retained meconium, septicemia/bacteremia, and neonatal maladjustment syndrome. Serum biochemical analysis revealed marked hyponatremia, hypochloremia, and moderate hyperkalemia, as well as mildly high urea, creatinine, and phosphorus concentrations. The primary differential diagnosis at this time was ruptured urinary bladder. Abdominocentesis was performed to confirm this diagnosis. Microscopic examination of abdominal fluid revealed calcium carbonate crystals, which originated from the urine of the foal. Biochemical analysis also confirmed the diagnosis of ruptured urinary bladder, because the ratio of peritoneal fluid creatinine to serum creatinine was 2.8:1. The foal died before surgical correction could be attempted.  相似文献   

19.
The purpose of this study was to determine the incidence of neonatal mortality in a large, extensively managed mare herd and what risk factors were involved in foal mortality. For a 6 wk period between April 18, 1994, and May 31, 1994, 334 foals were born, of which 74 died before reaching 10 d of age, giving an overall mortality of 22% for this period. Seventy four percent of the foal deaths occurred within 48 h of parturition. The major causes of foal mortality included starvation/exposure 27%, septicemia 26%, and dystocia 20%. Weekly incidences varied significantly, ranging from 67% for week 1 to 14% for week 5 (P < 0.01). Other risk factors that were associated with foal death included failure of passive transfer (P < 0.0001), poor mothering ability (P < 0.0001), the presence of dystocia (P < 0.0001), low birth weight (p < 0.05), lack of rainfall (P < 0.01), and low temperatures (P < 0.1). The effect of sire, mare age, mare body condition, and foal sex were not significant risk factors for foal survival (P > 0.1). Further studies are required to determine if changing management procedures will be effective in reducing the incidence of neonatal foal mortality in this extensively managed herd.  相似文献   

20.
Streptococcus suis is an important pathogen of swine, causing meningitis, arthritis, polyserositis, septicemia, and sudden death in weaning piglets as well as fattening pigs. Recently, 3 molecular tests have been developed in our laboratory: a multiplex polymerase chain reaction (m-PCR) assay for the detection of S. suis species and serotypes 2 and 1/2, and 2 molecular typing methods, pulsed-field gel electrophoresis and an approach based on PCR amplification of a fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S rDNA intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis (ISR-RFLP). In the present study, we used these tests to analyze tonsil samples from clinically healthy pigs and to identify individual isolates of S. suis during epidemiologic investigations of 8 related herds with a history of septicemia caused by S. suis serotype 2. Capsular typing showed that 58% of the strains were nontypable. Of the 17 serotypes present, serotype 22 was the most prevalent. In the 7 farms without clinical signs on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, in less than 5% of the pigs by m-PCR or by bacteriologic culture. In the 8th farm, on which 2 pigs had clinical signs of septicemia on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, by m-PCR in the tonsils of 40% of fattening pigs (21 wk old) that lacked symptoms. Molecular typing of the serotype 2 strains showed a common origin of contamination in these herds, given that 1 pattern (C1) was detected in the isolates from 6 of the 8 herds. However, up to 4 patterns were associated with septicemia and sudden death. Several patterns of S. suis serotype 2 can be responsible for disease in the same herd. These molecular tools may be useful for confident studies of the transmission of S. suis, thereby contributing to the control of S. suis infection.  相似文献   

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