Application of quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types to examine associations between severity of anaemia and parasitaemia in bovine anaemia outbreaks

AIMS: To use quantitative PCR assays to detect Theileria orientalis Ikeda type in cattle presumed infected with T. orientalis, to examine the relationship between theilerial piroplasm count and haematocrit (HCT), and the relationship with quantification cycle threshold (Cq) values.

METHODS: Blood samples in EDTA (n=1,024), derived from herds affected by anaemia associated with T. orientalis infection (TABA) between April and October 2013, were submitted for testing using quantitative PCR (qPCR) assays for T. orientalis and Ikeda type. Nucleotide sequencing of the major piroplasm surface protein (MPSP) gene was performed on 16 samples to identify T. orientalis types. Blood smear and/or HCT results were supplied with most samples. For data analysis, the number of theilerial piroplasm per 1,000 erythrocytes counted was categorised as negative (0), low (1–9), moderate (10–100) or high (>100). HCT was categorised as severely anaemic (<0.15 L/L), mildly anaemic (0.15–0.24 L/L) or not anaemic (>0.24 L/L). Differences between categories in proportion of samples positive for Ikeda type or mean Cq value were examined using χ² tests or analysis of variance, respectively.

RESULTS: Of 1,022 samples containing amplifiable DNA, 916 (90%) were positive for T. orientalis and 789 (77%) were positive for Ikeda type. Nucleotide sequencing of MPSP amplicons also identified the presence of Chitose and Buffeli types in 11 samples without Ikeda. Ikeda was detected in a greater proportion of severely anaemic (288/302; 95%) than mildly anaemic (227/252; 90%) cattle (p=0.02). In non-anaemic cattle, 344/406 (85%) were positive for T. orientalis and 247/406 (60%) were positive for Ikeda type. In samples from cattle that were piroplasm-positive, a greater proportion of anaemic (483/505, 96%) than non-anaemic (211/307; 69%) cattle were positive for Ikeda type (p<0.001). In piroplasm-negative cattle, 20/37 (54%) anaemic and 25/78 (32%) non-anaemic cattle were Ikeda-positive (p<0.05). The distributions of Cq values differed between piroplasm count and HCT categories (p<0.001). Mean Cq differed between high and negative, and low piroplasm categories (p<0.001), but not between high and moderate categories (p=0.81), and differed between severely anaemic and mildly anaemic (p<0.001), and non-anaemic categories (p<0.001).

CONCLUSIONS: The Ikeda type was found in a high proportion of cattle during outbreaks of TABA in New Zealand. Analysis of Cq values suggested a relationship of Ikeda parasitaemia with severity of anaemia, but further investigation is required to better understand the role of parasitaemia in the pathogenesis of TABA.     

血清1型鸭甲型肝炎病毒实时定量PCR检测方法的建立与初步应用

为建立了一种可检测血清1型鸭甲型肝炎病毒（DHAV-1）的实时定量PCR方法,根据GenBank中DHAV-1 5,非编码区的保守区,设计合成1对引物和1条TaqMan探针,以构建的重组质粒作为标准品,绘制标准曲线,并对所建立方法进行了特异性、敏感性和可重复性试验以及临床病料检测初步应用。结果,该方法与血清3型鸭甲型肝炎病毒、鸭瘟病毒、新城疫病毒、禽流感病毒、呼肠孤病毒、传染性支气管炎病毒等无交叉反应性;最低可以检测到10copies/μL;组内和组间变异系数均小于3%;临床病料的检测结果与测序检测结果一致。结果表明,所建立的实时定量检测方法具有特异、敏感、稳定等优点,可用于DHAV-1的快速检测与定量分析。     

Ⅰ型鸭肝炎病毒SYBR-Green实时荧光定量PCR检测方法的建立

为建立一种快速、特异的Ⅰ型鸭肝炎病毒（DHV-Ⅰ）SYBR GreenⅠ荧光定量PCR检测方法,根据GenBank已登录的DHV-Ⅰ型疫苗株C80（DQ864514.3）的非编码区基因序列设计1对特异性引物,经RT-PCR扩增出230bp的靶序列,并克隆到pMD18-T载体上,构建重组质粒;将纯化的重组质粒10倍梯度稀释后作为标准阳性模板,进行SYBR GreenⅠ荧光定量PCR扩增并建立标准曲线,对其敏感性、特异性和重复性进行评价。结果显示,建立的标准曲线的循环阈值与模板浓度呈现良好的线性关系（R2=0.998 2）,产物Tm值为87.2～87.9℃,检测灵敏度为71.5拷贝/μL。对55份疑似病料进行检测,荧光定量检测48份为阳性,而用常规PCR方法只能检出39份阳性,ELISA方法只检出30份阳性。这表明所建立的DHV-Ⅰ的SYBR GreenⅠ荧光定量PCR检测方法具有特异、灵敏、快速和重复性良好等优点,可用于临床DHV-Ⅰ感染的快速检测,为DHV的分子诊断、流行病学调查及定量分析奠定了基础。     

Dynamics of Moraxella bovis infection and humoral immune response to bovine herpes virus type 1 during a natural outbreak of infectious bovine keratoconjunctivitis in beef calves

Infectious bovine keratoconjunctivitis (IBK) is an acute disease caused by Moraxella bovis (Mb). Several factors may predispose animals to an IBK outbreak; one commonly observed is infection with bovine herpes virus type 1 (BHV-1). The aim of this study was to investigate the dynamics of BHV-1 virus infection and its relation with clinical cases of IBK in weaned calves from a beef herd with a high prevalence of lesions caused by Mb. Sampling was carried out in six stages and included conjunctival swabs for isolating Mb as well as blood samples for identifying antibodies specific for BHV-1. A score for IBK lesions after observing each eye was determined. The findings of this study showed a high prevalence of BHV-1 virus infection (100% of animals were infected at the end of the trial); 67% of animals were culture-positive for Mb, but low rates of clinical IBK (19% of calves affected) were detected at the end of the trial. These results suggest that infection with BHV-1 did not predispose these animals to IBK, and that Mb infection produced clinical and subclinical disease in the absence of BHV-1 co-infection.     

The use of quantitative PCR to detect Felis catus papillomavirus type 2 DNA from a high proportion of queens and their kittens

Squamous cell carcinomas are common feline skin cancers that have been associated with infection with Felis catus papillomavirus type 2 (FcaPV-2). Currently, little is known about the epidemiology of FcaPV-2 infection. The aim of this study was to develop a real-time PCR assay to quantify FcaPV-2 DNA in plucked hairs and skin swabs from 11 healthy breeding queens and their kittens. Samples were taken prior to kittening and then 2, 7 and 28 days after kittening to determine the age at which the kittens were first exposed to the virus. FcaPV-2 DNA was amplified from all of the queens and from 91% of the kittens at 2 days of age. There was a wide range in the quantity of FcaPV-2 DNA detected, from 1 to 92,520 copies per swab, and from 0.01 to 234 copies per copy of reference gene DNA in the hair plucks. The quantity of FcaPV-2 DNA detected in samples collected from the kittens was strongly correlated to that of their respective queens and the mean viral DNA load was similar for cats within a household but varied significantly between households. This is the first time that quantitative PCR has been used to detect FcaPV-2 DNA and the results suggest that the virus is ubiquitous but there is a wide variation of viral DNA loads. Kittens appear to be exposed to FcaPV-2 early in life, presumably from direct contact with their queen. These results are important when determining if FcaPV-2 infection of cats is preventable.     

A PCR assay to specifically detect serovar 1a strains of Erysipelothrix rhusiopathiae and differentiate them from serovar 2 strains possessing an intact ERH_1440 gene

The Erysipelothrix rhusiopathiae ERH_1440 gene, which encodes CDP-glycerol:poly (glycerophosphate) glycerophosphotransferase, is conserved in serovar 1a strains. The gene is usually missing or truncated in other serovar strains and therefore has been used for PCR detection of serovar 1a strains. We have previously reported a rare case of an E. rhusiopathiae serovar 2 strain possessing an intact ERH_1440. In this study, we analyzed three additional serovar 2 strains with an intact ERH_1440 and developed a new PCR assay for the specific detection and differentiation of serovar 1a strains from these serovar 2 strains. PCR with primers designed based on serovar 1a-specific gene sequences upstream of ERH_1440 showed 100% specificity for four hundred thirty Erysipelothrix strains isolated from extensive origins.     

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.