Rapid virulence typing of Pasteurella multocida by multiplex PCR

The gram-negative bacterium Pasteurella multocida constitutes a heterogeneous species associated with wide range of disease in many animals. Isolates are classified into five groups based on capsular antigen (capA, B, D, E and F). Recently, a new valuable PCR-based method was introduced to determine the epidemiological correlation between P. multocida infection and existence of virulence genes including tbpA, pfhA, toxA and hgbB. However, this method is tedious and laborious. Thus, in the current study, we designed a reliable multiplex PCR method for rapid detection of virulence genes in P. multocida. Eighty seven strains of P. multocida isolated from various clinically healthy and infected hosts were examined by uniplex PCR method for each virulence associated genes. Based on our improved and simplified multiplex PCR method, rapid detection of four virulence genes was accomplished. It is proposed that its implementation may benefit the epidemiological investigations.     

Evaluation of a serum-based PCR assay for the diagnosis of canine monocytic ehrlichiosis

The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389 bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates (P < 0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs (P = 0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available.     

多重PCR同时检测猪圆环病毒2型,猪细小病毒,猪繁殖与呼吸综合征病毒和猪瘟病毒

本试验建立一种可同时检测猪圆环病毒2型(PCV-2),猪细小病毒(PPV),猪繁殖与呼吸综合征病毒(PRRSV),猪瘟病毒(CSFV)4种病毒的多重PCR方法.对于每一种特定的病毒,用4对寡核苷酸引物均能特异扩增其目的片段.以含有病毒目的片段的质粒为模板,测定了多重PCR的检测灵敏度,PRRSV和CSFV检测最低限是48 pg,而PPV和PCV-2为0.48 pg.利用建立的多重PCR方法对具有产自有繁殖障碍母猪的仔猪或具有呼吸障碍症状的76个仔猪样本进行检测.检出了4种病毒的存在,其中26个样本(34.2%)同时感染了2种以上病毒.结果表明多重PCR方法检测猪混合感染的病毒,是一种快速、灵敏、低成本、高效率的病原学诊断工具.     

Detection of Bartonella spp. in wild rodents in Israel using HRM real-time PCR

The prevalence of Bartonella spp. in wild rodents was studied in 19 geographical locations in Israel. One hundred and twelve rodents belonging to five species (Mus musculus, Rattus rattus, Microtus socialis, Acomys cahirinus and Apodemus sylvaticus) were included in the survey. In addition, 156 ectoparasites were collected from the rodents. Spleen sample from each rodent and the ectoparasites were examined for the presence of Bartonella DNA using high resolution melt (HRM) real-time PCR. The method was designed for the simultaneous detection and differentiation of eight Bartonella spp. according to the nucleotide variation in each of two gene fragments (rpoB and gltA) and the 16S–23S intergenic spacer (ITS) locus, using the same PCR protocol which allowed the simultaneous amplification of the three different loci. Bartonella DNA was detected in spleen samples of 19 out of 79 (24%) black rats (R. rattus) and in 1 of 4 (25%) Cairo spiny mice (A. cahirinus). In addition, 15 of 34 (44%) flea pools harbored Bartonella DNA. Only rat flea (Xenopsyla cheopis) pools collected from black rats (R. rattus) were positive for Bartonella DNA. The Bartonella sp. detected in spleen samples from black rats (R. rattus) was closely related to both B. tribocorum and B. elizabethae. The species detected in the Cairo spiny mouse (A. cahirinus) spleen sample was closely related to the zoonotic pathogen, B. elizabethae. These results indicate that Bartonella species are highly prevalent in suburban rodent populations and their ectoparasites in Israel. Further investigation of the prevalence and zoonotic potential of the Bartonella species detected in the black rats and the Cairo spiny mouse is warranted.     

Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays

The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n = 94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes 460, 1044, 657 and 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.     

肺炎克雷伯氏菌强毒株的分离鉴定及16-23SrRNAITS序列分析

为确诊疑似仔猪肺炎克雷伯氏菌(K.pneumonia)感染,并研究其病原的致病性、耐药性、16-23SrRNA ITS系统进化特征,本研究从云南因肺炎、腹泻而大量死亡的仔猪中分离到1株革兰氏阴性短粗杆菌,命名为KP14013,对其进行生化鉴定、16SrRNA鉴定,研究其对小白鼠和仔猪的致病性,并对其16-23SrRNA ITS基因进行测序和遗传进化分析。结果显示,KP14013分离株生化特征与肺炎克雷伯氏菌相符,其16SrRNA与GenBank中23株肺炎克雷伯氏菌代表株之间的同源性均为99%,将KP14013鉴定为肺炎克雷伯氏菌。KP14013对小白鼠半数致死量(LD50)为3×101.8 CFU,腹腔注射3×108 CFU可使仔猪100%致死。16-23SrRNA ITS系统进化关系结果表明,KP14013与GenBank中收录的15株肺炎克雷伯氏菌形成进化树的一个分支,属于同一个亚群,它们之间的核苷酸同源性为98.4%~99.2%。本研究证实了肺炎克雷伯氏菌是该起仔猪腹泻大量死亡的病原;KP14013分离株为毒力极强菌株,具有多重耐药性,其16-23SrRNA ITS与GenBank中收录的肺炎克雷伯氏菌代表株之间核苷酸存在差异,可用于肺炎克雷伯氏菌菌株间的鉴别。     

水貂奇异变形杆菌的分离鉴定及16S rRNA基因序列分析

从辽宁某貂场发病水貂中分离到1株致病菌,命名为PMSD株,通过形态学观察和生化试验等常规鉴定发现符合奇异变形杆菌特性,进一步经VITEK 2Compact 30全自动细菌鉴定及药敏分析系统鉴定该株细菌为奇异变形杆菌。药敏试验结果显示PMSD株对氨基糖苷类药物、喹诺酮类药物等敏感,而对β-内酰胺类药物和磺胺类药物等不敏感。以细菌16SrRNA基因为模板应用通用引物进行PCR扩增,得到PMSD株的16SrRNA基因序列,长约1 504bp,提交到GenBank中,登录号:KM229530。将该序列与GenBank中序列进行BLAST比对,结果发现与其匹配度最高的均是奇异变形杆菌各株系的16SrRNA序列,均高达99%以上。选取其中前20株作为参考序列,运用生物学软件构建系统发育树并进行同源性比对,结果表明,分离菌PMSD株与20个代表菌株的同源性为98.9%~99.7%,其中与BB2000株、HI4320株、B1株和FCC141株同源性最高,为99.7%。本研究为预防和控制奇异变形杆菌引起的水貂疾病奠定了一定的基础。     

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.     

Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S-23S rRNA intergenic spacer region sequences

In the present study, the 16S-23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.     

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.     

Interference of intradermal tuberculin tests on the serodiagnosis of paratuberculosis in cattle

In this experiment 63 animals from a paratuberculosis (PTB) and tuberculosis-free herd were tested by Intradermal Tuberculin Tests (ITT) and blood samples were collected before PPD inoculation and on days 3, 15, 30, 60 and 90 post-inoculation (p.i.). Sera were tested for PTB-specific antibodies by ELISA-PPA and confirmed by a commercial ELISA. Three (4.76%) animals were positive by ELISA-PPA and five (7.93%) in the commercial ELISA, between days 30 and 90 p.i. These results suggest that ITT can interfere in the reliability of ELISAs and that serological testing for PTB should be avoided for 90 d after PPD inoculation.     

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 ± 0.09 and 0.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 ± 0.21 and 1.65 ± 0.07 log₁₀ CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.     

IFN-g response to vaccination against tuberculosis in dairy heifers under commercial settings

The purpose was to determine IFN-g release as a response to vaccination against tuberculosis in dairy heifers under commercial settings. Four-hundred pregnant heifers from ten herds were randomly allocated into four groups: (1) unvaccinated, (2) BCG vaccinated, (3) BCG vaccinated plus a CFPP400 μg + polygen boost, and (4) BCG vaccinated plus a CFP200 μg + polygen boost, under a completely randomized blocks design. A dose of 106 CFU of BCG was delivered SC in the neck, then blood samples were taken at days 0, 30, 120, 210, 300 and 720 to estimate IFN-g release in response to bovine-PPD antigen. No significant difference (P > 0.05) was observed in IFN-g release between groups at days 0 and 120. At days 30 and 210, vaccinated groups show higher IFN-g release than the control group but only difference of group 3 was significant (P < 0.05). At day 300, group 1 showed significantly higher IFN-g release. No significant difference was observed at day 720. Using IFN-g release as a surrogate for vaccine efficacy, BCG plus a boost with CFP or CFPP combined with an adjuvant that improves cellular immune response has the potential to protect cattle against tuberculosis for moderate periods of time in vaccinated cattle under commercial settings.     

Detection of fluoroquinolone resistance level in clinical canine and feline Escherichia coli pathogens using rapid real-time PCR assay

Fluoroquinolones are used to treat infections caused by Escherichia coli in canine and feline veterinary patients, particularly those infecting the urinary tract. The gyrA gene is a primary target causing fluoroquinolone resistance in Gram negative coliforms, with mutations in codons 83 and 87 generally associated with high-level of resistance E. coli clinical isolates. We have developed a fluorescence resonance energy transfer (FRET) quantitative PCR to identify enrofloxacin-resistance in clinical E. coli isolates that carry mutations in codons 83 and 87 of gyrA. This real-time quantitative PCR assay is rapid, economical, and sensitive compared with cultured antimicrobial susceptibility testing. The assay identified as few as four genome copies per reaction from culture and 19 genome copies in urine. For the 70 isolates tested, the sensitivity was 87.5% (95% CI = 75–95.3%) (n = 42/48), specificity was 100% (95% CI = 87.3–100%) (n = 22/22), whereas accuracy was 91.4% (95% CI = 82.3–97%) (n = 64/70). Furthermore, we were able to accurately differentiate between the wild type and mutants E. coli directly from infected canine urine samples (n = 5) within 2 h. These results were confirmed by sequence alignments of the PCR products and comparison with the susceptibility testing. The FRET-PCR assay appears to have promising clinical application as an early diagnostic tool for rapid and sensitive detection and differentiation of the level of fluoroquinolone resistance among clinical E. coli isolates that may facilitate design of the dosing regimen.     

A novel multiplex PCR assay to detect and distinguish between different types of Paenibacillus larvae and Melissococcus plutonius,and a survey of foulbrood pathogen contamination in Japanese honey

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FI763267","term_id":"224923825"}}FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.     

Occurrence and characterization of gastric Helicobacter spp. in naturally infected dogs

Helicobacter-like organisms are frequently observed in the stomach of dogs but the relationship between these microorganisms and gastric pathology has not been clearly established. Different species of helicobacters are known to be present in the canine stomach but their specific prevalence in naturally infected dogs is unknown. The aims of this study were to isolate and characterize helicobacters in canine gastric biopsies, to compare the commonly used tests for the identification of Helicobacter spp. and to determine the occurrence of these species in dogs. Twenty-three out of 25 dogs (92%) were positive for Helicobacter-like organisms in cytological screening. Culture was successful from biopsies of 5/25 dogs. The isolates were analyzed by electron microscopy, biochemical and physiological tests, whole protein analysis and 16S rDNA sequencing. Helicobacter felis was identified in four samples and Helicobacter bizzozeronii in one sample. Only the whole protein analysis in combination with electron microscopy was able to clearly discriminate the two species. Compared to the high prevalence of Helicobacter-like organisms, the occurrence of H. felis and H. bizzozeronii, was low (17 and 4%, respectively). No Flexispira rappini-like organisms or H. salomonis were detected. Electron microscopy revealed that H. bizzozeronii-like microorganisms were present in three additional biopsies where we were unable to culture any Helicobacter-like organisms. These observations indicate that in the stomach of dogs not all helicobacters are culturable. The unculturable bacteria appeared to be the prevalent ones and may represent different spiral organisms. The presence of distinct helicobacters with different characteristics can reflect different roles in the pathogenesis of canine gastric disease.     

Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M

Brucellosis causes serious economic losses to goat farmers by way of reproductive losses in the form of abortions and stillbirths. Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. In this study, we evaluated the ability of DNA vaccine encoding the omp31 protein of Brucella melitensis 16M to induce cellular and humoral immune responses in mice. We constructed eukaryotic expression vectors called pTargeTomp31, encoding outer membrane protein (omp31) of B. melitensis 16M. pTargeTomp31 was injected intramuscularly three times, at 3-week intervals in groups of mice 6 weeks of age. pTargeTomp31 induced good antibody response in ELISA . pTargeTomp31 elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the omp31 antigen or B. melitensis 16M extract. We also demonstrate that animals immunized with this plasmid elicited a strong and long-lived memory immune response. Furthermore, pTargeTomp31 elicited a typical T-helper 1-dominated immune response in mice, as determined by immunoglobulin G isotype analysis. This vaccine also provided the moderate degree of protection to the mice. This study for the first time focuses on DNA immunization of a gene from B. melitensis. These results may lead to the development of a DNA-based vaccine for the control of brucellosis in goats.     

Phenotypic and molecular characterization of Brachyspira spp. isolated from laying hens in different housing systems

Several species of intestinal spirochaetes, Brachyspira (B.) alvinipulli, B. intermedia and B. pilosicoli, may cause reduced egg production and faecal staining of eggshells in chickens. The aim of this study was to characterize potentially pathogenic and presumably non-pathogenic Brachyspira spp. from commercial laying hens. Selective culture, phenotyping, PCR and 16S rRNA gene sequencing were used and clinical data were collected. Phenotypic profiles were obtained for 489 isolates and 351 isolates obtained after subculture, and 30 isolates were selected for molecular characterization. Seven isolates were positive by a B. intermedia-specific PCR based on the nox gene, and two were positive in a B. hyodysenteriae-specific 23S rRNA gene based PCR. By comparative phylogenetic analysis in combination with PCR and phenotyping, seven isolates were identified as B. intermedia, eight isolates as B. innocens, five as B. murdochii, and three isolates each as B. alvinipulli and "B. pulli". The remaining four isolates could not be assigned to any presently recognized species. Co-infection with several species or genetic variants of Brachyspira spp. were detected in some flocks and samples, suggesting a high level of diversity. Organic flocks with access to outdoor areas were at higher risk (RR=2.3; 95% CI 1.5-3.6) for being colonized than chickens in other housing systems. No significant differences between colonized and non-colonized flocks were found regarding clinical parameters, i.e. mortality, egg production, faecally contaminated eggshells, and wet litter. Our results show that a combination of traditional laboratory diagnostics, molecular tests and phylogeny is needed for identification of Brachyspira sp. from chickens.     

Clinical features associated with seroconversion to Anaplasma marginale, Babesia bigemina and Theileria parva infections in African cattle under natural tick challenge

A longitudinal study was conducted in Southeast Uganda for 14 months on 640 Zebu cattle kept under natural tick challenge, with a view to identifying clinical features for prediction of seroconversion to Anaplasma marginale, Babesia bigemina and Theileria parva infections. Physical examination, condition scoring and tick counts were undertaken on all cattle every 4 weeks. In addition, 5300 sera were collected and analysed for antibodies against A. marginale, B. bigemina and T. parva infections using the enzyme-linked immunosorbent assay (ELISA). The major clinical features compiled included weight loss, fever (rectal temperature), anaemia (packed cell volume), pallor of mucous membranes, lymph node enlargement, staring coat, diarrhoea and lacrymation. The risk factors included tick challenge at village level, sex, age, Rhipicephalus spp. density and Boophilus spp. density on individual animals. Using a binary logistic regression model, the clinical features and risk factors were analysed. The results suggest that increasing rectal temperature was associated with increased probability for seroconversion to A. marginale, while high level of Rhipicephalus spp. density and increasing packed cell volume (PCV) were significantly associated with reduced probability of seroconversion. Although statistically significant, none of the factors had large effects, with odds ratios (OR) of 0.87, 1.15 and 0.98 for Rhipicephalus spp. density, rectal temperature and PCV, respectively. For B. bigemina infection, a high level of Boophilus spp. density, anaemia and staring coat were significantly associated with increased probability of seroconversion (OR 1.50, 1.78, 1.37, respectively). Presence of lacrymation and old age were associated with reduced probability of seroconversion (OR 0.52, 0.86 respectively). For T. parva infection, lymph node enlargement (OR 1.30) was associated with increased probability of seroconversion, while high Rhipicephalus spp. density and increasing packed cell volume (PCV) were associated with reduced probability of seroconversion (OR 0.68 and 0.98, respectively). In conclusion, presence and intensity of the respective tick vectors for tick-borne diseases, age and clinical features such as anaemia, fever, staring coat, lymph node enlargement and lacrymation are indicators for seroconversion to A. marginale, B. bigemina and T. parva infections in cattle. These indicators for seroconversion could be exploited in the development of decision support tools for clinical diagnosis of tick-borne diseases.     

Detection of Brucella species in the milk of infected cattle,sheep, goats and camels by PCR

One hundred and three milk samples were collected from 52 cows, 21 ewes, 18 goats and 12 camels. The animals tested positive to at least one of the following: (1) standard tube agglutination test (SAT); (2) Rose Bengal plate test (RBPT); (3) milk ring test (MRT). All milk samples were examined by culture and single-step polymerase chain reaction (PCR) techniques for detection of Brucella species. The PCR assay amplified Brucella-DNA from 29 bovine milk samples, 10 from sheep, 13 from goats and one from a camel. The direct culture method detected Brucella organisms from 24 samples of cows' milk, 12 from sheep, 10 from goats and failed to detect any Brucella organisms from camels' milk. PCR detected up to 100 colony forming units (CFU) of B. abortus per millilitre of milk in 100% of diluted milk samples, and 1000 CFU of B. melitensis from 70% of milk samples. Although the overall sensitivity of the PCR was higher than the culture method, it should be possible to increase the sensitivity to detect lower numbers of Brucella organisms in field samples. The speed and sensitivity of the PCR assay suggest that this technique could be useful for detection of Brucella organisms in bovine milk, as well as in sheep, goat, and camels milk.