Multiplex PCR for the diagnostic detection of Coxiella burnetii in cow's milk

A multiplex PCR based assay was developed for the highly sensitive and specific detection of Coxiella (C.) burnetii in cow's milk. The assay simultaneously amplifies a diagnostic target within the C. burnetii IS1111 sequence and a control target within the bovine CD18 gene. The internal PCR amplification control allows the discrimination of false negative results (single tube reaction failures) from negative results due to true absence of target sequences. In order to maximize the sensitivity of the assay, a sample preparation method including a centrifugation step to concentrate the bacterium was developed. In milk samples artificially contaminated with serial dilutions of C. burnetii, about four particles per ml could reproducibly be detected. The sensitivities of both assays, multiplex PCR and PCR with only a single pair of primers ('simplex' PCR), were observed to be similar.     

Coxiella burnetii shedding by dairy cows

While shedding routes of Coxiella burnetii are identified, the characteristics of Coxiella shedding are still widely unknown, especially in dairy cattle. However, this information is crucial to assess the natural course of Coxiella burnetii infection within a herd and then to elaborate strategies to limit the risks of transmission between animals and to humans. The present study aimed at (i) describing the characteristics of Coxiella burnetii shedding by dairy cows (in milk, vaginal mucus, faeces) in five infected dairy herds, and at (ii) investigating the possible relationships between shedding patterns and serological responses. A total of 145 cows were included in a follow-up consisting of seven concomitant samplings of milk, vaginal mucus, faeces and blood (Day 0, D7, D14, D21, D28, D63, D90). Detection and quantification of Coxiella burnetii titres were performed in milk, vaginal mucus and faeces samples using real-time PCR assay, while antibodies against Coxiella were detected using an ELISA technique. For a given shedding route, and a given periodicity (weekly or monthly), cows were gathered into different shedding kinetic patterns according to the sequence of PCR responses. Distribution of estimated titres in Coxiella burnetii was described according to shedding kinetic patterns. Coxiella burnetii shedding was found scarcely and sporadically in faeces. Vaginal mucus shedding concerned almost 50% of the cows studied and was found intermittently or sporadically, depending on the periodicity considered. Almost 40% of cows were detected as milk shedders, with two predominant shedding patterns: persistent and sporadic, regardless of the sampling periodicity. Significantly higher estimated titres in Coxiella burnetii were observed in cows with persistent shedding patterns suggesting the existence of heavy shedder cows. These latter cows were mostly, persistently highly-seropositive, suggesting that repeated serological testings could be a reliable tool to screen heavy shedders, before using PCR assays.     

Improved detection of Mycobacterium avium subsp. paratuberculosis In milk by immunomagnetic PCR

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centrifugation (2500 g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100 degrees C for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3)CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 10(5)CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (10(6)CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS-PCR and conventional IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.     

Assessing the within-herd prevalence of Coxiella burnetii milk-shedder cows using a real-time PCR applied to bulk tank milk

Thirty-seven bulk tank milk (BTM) and individual milk samples of all contributing cows were tested for Coxiella burnetii detection by a real-time PCR assay and used to assess the relationship between the BTM PCR-response and (i) the within-herd prevalence of milk-shedder cows and (ii) the proportion of heavy milk-shedder cows. The within-herd prevalence of milk-shedder cows (i) was found to be significantly higher in herds with a positive BTM and (ii) increased significantly with the estimated titre in Coxiella burnetii obtained in positive BTM. The proportion of heavy milk-shedder cows among the milk-shedder cows increased significantly with an increased estimated titre in Coxiella burnetii in positive BTM. Therefore, a real-time PCR assay applied to BTM samples collected repeatedly over time appears to be a valuable tool to assess on a larger scale the status of herds towards Coxiella shedding, and to evaluate the efficiency of control actions aimed at controlling and/or preventing Coxiella shedding in dairy herds.     

Shedding routes of Coxiella burnetii in dairy cows: implications for detection and control

Reliable detection of Coxiella burnetii shedders is a critical point for the control of the spread of this bacterium among animals and from animals to humans. Coxiella burnetii is shed by ruminants mainly by birth products (placenta, birth fluids), but may also be shed by vaginal mucus, milk, and faeces, urine and semen. However, the informative value of these types of samples to identify shedders under field conditions is unknown. Our aim was then to describe the responses obtained using a real-time PCR technique applied to milk, vaginal mucus and faeces samples taken from 242 dairy cows in commercial dairy herds known to be naturally infected with Coxiella burnetii, and to assess their putative associations. Positive results were found in all types of tested samples even in faeces. No predominant shedding route was identified. Among the shedder cows, 65.4% were detected as shedders by only one route. By contrast, cows with positive results for all three samples were scarce (less than 7%). Testing a cow based on only one type of biological sample may lead to misclassify it with regards to its shedding of Coxiella burnetii and thereby underestimate the risk of bacterial spread within a herd.     

Shedding and serological patterns of dairy cows following abortions associated with Coxiella burnetii DNA detection

To describe both shedding and serological patterns following abortions detected as being associated with Coxiella burnetii (Cb), 24 cows experiencing an abortion due to Cb were followed over a one month period. Samples taken on the day of abortion (D0) were followed 3-fold by weekly samplings from day 14 (D14) to D28 after the abortion. Milk and vaginal mucus were collected at each weekly sampling and tested using real-time PCR while blood samples were collected 2-fold on D21 and D28 and tested using ELISA. We found a very short duration of C. burnetii shedding in vaginal mucus after abortion, highlighting the need to collect samples as rapidly as possible following an abortion to avoid false negative results. In contrast with previous results, concomitancy of vaginal and mucus shedding was frequent, especially for cows shedding a high bacterial load on DO leading to the hypothesis that the clinical onset of the infection influences the modalities of Cb shedding. Lastly, serological results indicating a lack of sensitivity to detect Cb shedder cows (especially for cows for which Ct values were high) suggest that ELISA is not a useful tool to diagnose abortions at the individual level.     

Coxiella burnetii associated placental lesions and infection level in parturient cows

Cotyledons (n=170) from dairy cattle were analysed for Coxiella burnetii by real-time (rt) PCR targeting the IS1111a and icd genes. Positive cases (n=90) and a random selection of negative cases (n=20) were examined by histology, immunohistochemistry and, if infection level was high, by fluorescence in situ hybridisation. PCR results were compared to bulk tank milk (BTM) antibody levels. Placental infection was detected in cows from herds at all BTM antibody levels. However the likelihood of placental infection was generally higher in herds with intermediate or high BMT antibody levels than in herds with low antibody levels. Histological examination revealed a range of mostly mild cotyledonary changes; C. burnetii infection was only rarely associated with inflammation. This may explain why bovine Q fever is usually not clinically apparent. Nevertheless, infected cattle will shed C. burnetii at calving and this can occur even in herds without BTM antibodies.     

Prevalence of Coxiella burnetii infection in Dutch dairy herds based on testing bulk tank milk and individual samples by PCR and ELISA

A study using an ELISA and a real-time PCR assay based on the detection of the repetitive transposon-like gene of Coxiella burnetii revealed that infection with the bacterium was widespread among Dutch dairy herds, with antibodies detected in bulk tank milk (BTM) from 268 of 341 herds (78.6 per cent) and bacterial DNA detected in 193 of 341 herds (56.6 per cent). The BTM samples were taken in November and December 2007. Serological and molecular studies in young and adult cattle selected from 100 herds showed that antibodies were present in the blood of 470 of 2936 (16.0 per cent) lactating cows but only in 19 of 1831 (1.0 per cent) young animals. Bacterial DNA was detected in the milk of 254 of 2925 (8.7 per cent) lactating cows; bacterial DNA was not detected in any of the faecal samples obtained from youngstock. The blood and milk samples were taken from the cattle in the period January to April 2008.     

Molecular detection of Coxiella burnetii in horse sera in Iran

Coxiella burnetii is a zoonotic bacterium that can infect a wide range of animals including horses. However, its circulation dynamics in and through horses are still unclear. The aim of this study was to evaluate prevalence of C. burnetii and its genomic characteristics in horse sera samples in the North of Iran (Golestan Province). The samples were collected in 2018 and the age, sex, and breed of each animal were recorded. Nested-PCR was used to detect C. burnetii based on the presence of the transposable gene IS1111. The results showed that 7.50 % (P < 0.05; 95 % CI: 0.5 %–0.12 %) of the examined sera samples were positive for C. burnetii. Based on the resuls, prevalence of C. burnetii in the age groupof < Years 1–5 (p-value <0.05, 95 % CI: 1 %–8 %) was less than the age group of >6 years old (p-value <0.05, 95 %, CI: 7 %–19.8 %). In previous studies, it was concluded that the horses' population in Golestan Province should be considered as an important factor in the epidemiology of Q fever and consequently in public health. Further studies should be implemented to evaluate if horses may be relevant indicators of zoonotic risk in urban and suburban endemic areas.     

Definitive ability of Stamp-staining,antigen-ELISA,PCR and cell culture for the detection of Coxiella burnetii

Many assays are used for the detection of the aetiological agent of Q fever, Coxiella burnetii, i.e. staining according to the method of Stamp, capture ELISA, PCR or isolation by cell culture. In this study the results of these four assays are compared for their sensitivity and specificity. Staining smears according to the method of Stamp gave many false positive or false negative results. The capture ELISA seems to be a very sensitive assay for the detection of Coxiella burnetii but it has a lack in specificity. It is a useful test system for the screening of large scales of samples. Positive ELISA results should be confirmed by PCR, a very sensitive and specific method for the detection of Coxiella burnetii but more time consumptive than the ELISA. Isolation of the agent using cell cultures was not completely satisfactory because of its lack in sensitivity. Therefore it should only be used in special cases.     

Association between Coxiella burnetii shedding in milk and subclinical mastitis in dairy cattle

The objective of this research was to explore the potential association between Coxiella burnetii shedding in milk and chronic subclinical mastitis in dairy cattle. In two separate studies, we identified an association between PCR-based detection of C. burnetii in milk and chronic subclinical mastitis in lactating dairy cows. These studies were conducted in a commercial dairy herd where there was ongoing intensive monitoring of subclinical mastitis by aerobic bacteriology, but no prior knowledge or management of C. burnetii infections. In a case-control study, quarter level C. burnetii status determined by real-time quantitative PCR (RT-qPCR) was strongly associated with chronic subclinical mastitis as measured by milk somatic cell counts. In a subsequent cross sectional study, 147 (45%) of 325 lactating cows were positive for C. burnetii by RT-qPCR of composite milk samples. In a generalized linear model, accounting for the effect of covariates including aerobic intramammary infection status, C. burnetii PCR status was a significant predictor of linear somatic cell count score. In agreement with a small number of previous reports, this research provides evidence that there may be mammary gland specific manifestations of C. burnetii infections in dairy cattle.     

Coxiella burnetii seropositivity is highly stable throughout gestation in lactating high-producing dairy cows

The aims of this study were to analyse, in high-producing dairy cows, plasma Coxiella burnetii antibody titres and seroconversion throughout gestation, along with possible factors affecting such titres. The study was performed on 65 lactating pregnant non-aborting animals in a commercial Holstein-Friesian dairy herd in northeastern Spain. Blood samples for antibody determinations were collected on days 40, 90, 120, 150, 180 and 210 of gestation. By General Linear Model (GLM) repeated measures analysis of variance, the effects of milk production and reproductive variables as well as Neospora caninum-seropositivity on C. burnetii antibody levels for all animals and for seropositive animals were established. Significant effects were observed of day of gestation, parity and N. caninum-seropositivity (between subject effects) on the C. burnetii antibody levels recorded for the whole population of animals throughout the gestation period. C. burnetii antibody levels were higher in primiparous than in multiparous cows, with titres in primiparous cows diminishing during the post-partum period. In seropositive cows, significant effects were observed of milk production and inseminating bull on gestational C. burnetii antibody levels. When the data were subjected to binary logistic regression considering C. burnetii-seropositivity as the dependent variable, the resultant odds ratios indicated that the likelihood of C. burnetii-seropositivity was lower in N. caninum-seropositive animals (OR 0.12) compared to N. caninum-seronegative animals, and in multiparous cows (OR 0.12) compared to primiparous cows. In conclusion, Coxiella-infected dams remained seropositive during the whole gestation period, though primiparous cows showed a drop in antibody titres post-partum. No seronegative cow suffered seroconversion. Presence of both, N. caninum and C. burnetii antibodies in the same animal, was associated with a decrease in antibody titres against C. burnetii, perhaps indicating some cross-protection in animals infected by both pathogens.     

Effectiveness of vaccination and antibiotics to control Coxiella burnetii shedding around calving in dairy cows

Effectiveness of phase 1 vaccine, combined or not with tetracycline, to control Coxiella burnetii vaginal shedding at calving in cows was assessed through a 13 months study in 22 Q fever clinically affected commercial dairy herds. Four medical strategies implemented at herd level but randomly assigned to cows (vaccination, vaccination and tetracycline, tetracycline, nothing) were compared. There was no significant interaction effect between vaccination and antibiotherapy. Tetracycline used once at drying off was associated with a lower risk of being detected shedder at calving (OR=0.40, CI 95% [0.21-0.75]), but had no significant effect on the bacterial load shed. Vaccination did not significantly prevent shedding but was significantly (OR=0.15, CI 95% [0.03-0.85]) associated with lower bacterial load shed. Thus, vaccination using a phase 1 vaccine and antibiotherapy using tetracycline is associated with a decrease in shedding in dairy cows and could contribute to reduce the bacterial load generated in the environment. To the best of our knowledge, this is the first study providing useful information for evidenced-based and rational use of medical strategy combining antibiotic and vaccination in infected dairy cattle herds.     

Q热贝纳柯克斯体TaqMan荧光定量PCR检测方法的建立

根据Q热贝纳柯克斯体(Coxiella burnetii)插入IS1111序列设计引物和探针,建立快速检测Q热的TaqMan实时荧光定量PCR方法。以梯度稀释含有目的扩增片段的重组质粒作为标准品,进行定量PCR反应。结果显示,该方法能够检测出10个拷贝数的阳性质粒;标准曲线相关系数为0.995,扩增效率为103%;结核分枝杆菌(M.tuberculosis)、衣原体(C.psittaci)、布鲁氏菌(Brucella.spp)及牛血液的核酸样本特异性检测结果均为阴性。本研究建立的TaqMan荧光定量PCR法灵敏度高、特异性好,对Q热的检测与鉴定中具有良好的应用前景。