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.     

Spread of Coxiella burnetii infection in a flock of sheep after an episode of Q fever

In October 1998, two abortions associated with Coxiella burnetii occurred in a group of 34 pregnant ewes in the sheep flock belonging to INRA Tours-Nouzilly. The flock was kept in groups of approximately 40 ewes, which were housed together in the same accommodation. The prevalence of C burnetii infection in the groups was investigated by using ELISA and PCR tests, which revealed a high prevalence of C burnetii. The ewes were treated with oxytetracycline to reduce the shedding of C burnetii and to prevent further abortions. Nevertheless, five abortions attributed to C burnetii occurred in January and March 1999 in three groups of ewes, and 24 of the ewes still shed the bacteria into their vaginal tracts. In addition, a serological study was carried out during the first year of life of the female lambs born in 1999 and 2000; 12 per cent of 113 lambs born in 1999 were seropositive for C burnetii by ELISA, and half of the ELISA-positive lambs were born either to serologically positive ewes or to dams that excreted the pathogen into their vaginal tracts. However, all the 150 lambs born in 2000 were ELISA-negative, suggesting that the preventive measures undertaken had suppressed both the abortions and the shedding of C burnetii, and reduced the transmission of the agent.     

Studies of Coxiella burnetii infections in dairy herds with special regard to infections in men

Investigations of 1167 dairy cows out of 105 herds with fertility disorders on secretion of Coxiella burnetii (C. burnetii) by the genitals as well as serological studies of these animals using ELISA demonstrated that C. burnetii infections are significantly associated with abortions but not with repeated inseminations without success or vaginal excretions. The demonstration of an increased risk of infections for persons caring for those dairy herds could be shown by a total of 253 serological studies. A comparison of these studies of farmers caring for dairy herds suffering from abortions a seroprevalence of > or = 20% showed that these persons revealed significantly more frequent antibodies against C. burnetii than farmers of the group compared with. Further studies showed that in herds suffering from abortions a seroprevalence of > or = 20% means an additional risk of infections of farmers. Vice versa abortions of the cows in herds with a seroprevalence of > or = 20% imply an additional infectious risk.     

Goats may experience reproductive failures and shed Coxiella burnetii at two successive parturitions after a Q fever infection

Q fever is a zoonosis caused by the obligate intracellular bacterium, Coxiella burnetii. Aborting domestic ruminants are the main source of human infection. In January 2003, an abortion episode occurred in a dairy caprine herd where 18/60 (30%) goats experienced reproductive problems: 4/60 (7%) aborted and 14/60 (23%) had stillbirths. Serological screening for abortion-related infectious diseases suggested Q fever. The diagnosis of C. burnetii infection was confirmed with PCR based on the occurrence of C. burnetii shedding into vaginal mucus, faeces and colostrums taken after kidding from the affected animals. The pregnancy following this episode resulted in one abortion and four stillbirths; three of those goats had already experienced reproductive failure during the previous kidding season. The seroprevalence of C. burnetii infection and the bacteria shedding were investigated using both ELISA and PCR assays, respectively, during the course of the initial and subsequent kidding seasons. Serological testing, performed on the whole herd 6 weeks after the abortion episode, showed 48/60 (80%) of ELISA positive goats. PCR assay performed on both vaginal swab and milk samples showed that the bacterium was shed for almost four months after the outbreak. C. burnetii DNA was also amplified from vaginal swab and milk samples taken from goats after the second kidding season. Furthermore, the bacteria were found into 14 vaginal swabs and 12 milk samples taken from infected females at both kidding seasons.     

Shedding of Coxiella burnetii in ewes in two pregnancies following an episode of Coxiella abortion in a sheep flock.

Coxiella burnetii infection in pregnant sheep typically causes abortion or the birth of weak lambs. Two C. burnetii-related abortions in a group of 34 pregnant ewes were reported at their first lambing in our research institute. The seroprevalence of C. burnetii infection and bacteria shedding were investigated using an ELISA and PCR, respectively, during the course of two subsequent pregnancies. None of the ewes examined seroconverted from negative to positive at the time of the second and the third parturition and most of the ewes that were seropositive at the abortion episode remained positive throughout the investigation. The two successive pregnancies resulted in the birth of healthy lambs without PCR evidence of Coxiella infection from placenta and vaginal swabs taken postpartum. PCR assay performed on vaginal swabs taken from all animals 1, 5 or 12 days after the second lambing were also negative for Coxiella. However, one ewe that had previously experienced C. burnetii shedding at the first lambing excreted the bacteria in the genital tract after the third parturition. The bacteria could not be detected by PCR in milk and faecal samples taken up to 12 days after both parturitions.     

Diagnosis by ELISA of bovine abortion due to Campylobacter fetus

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antigen-specific secretory IgA antibody in bovine vaginal mucus after abortion due to Campylobacter fetus subsp venerealis. Abortions were diagnosed by isolating the organism from 8 foetuses and/or foetal membranes and by histopathology. Vaginal mucus was collected from 7 cows shortly after abortion. All showed a high level of IgA antibody in their vaginal mucus when they were compared with an uninfected control group. The new ELISA is simple and practical and provides a useful tool for diagnosis of bovine venereal campylobacteriosis.     

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.     

Serological screening for Coxiella burnetii infection and related reproductive performance in high producing dairy cows

The possible relationship between Coxiella-seropositivity and the reproductive performance of cows during the previous year to the serological screening was examined in three high producing dairy herds. The herds had a history of subfertility (<25% of pregnancies for the total number of AI), abortion (>18% abortions) and a positive polymerase chain reaction test for Coxiella burnetii in the bulk tank milk with an excretion higher than 10(4)Coxiella /ml for all three herds. Antibodies against C. burnetii were detected in 50.2% of the 781 parous cows analyzed. Coxiella seropositivity was linked to placenta retention, to changes in the interval from parturition to conception (with the lowest interval parturition-conception for cows with low level of seropositivity), early pregnancy (cows becoming pregnant before Day 90 postpartum) and maintenance of gestation during the early fetal period, while it failed to affect rates of abortion after Day 90 of gestation or stillbirth.     

Serological investigation of an outbreak of Neospora caninum-associated abortion in a dairy herd in southeastern United States

An outbreak of Neospora caninum-associated abortion occurred in a South Carolina dairy wherein greater than 10% of the herd aborted over a 4-month period. Of the total number of cows at mid-late gestation, nearly 40% (28/71) aborted while the remaining 60% (43/71) gave birth to normal calves. Immunohistochemical examination of brain tissue from a subset of aborted fetuses confirmed N. caninum as the causative agent of abortion in these animals. A variety of serological assays, including indirect fluorescence antibody test (IFAT), recombinant enzyme-linked immunosorbent assay (rELISA), ISCOM-ELISA, avidity ELISA, and Neospora agglutination test (NAT), were used to evaluate sera collected during the outbreak from 240 cows for antibodies to N. caninum. IFAT and ISCOM-ELISA testing showed that nearly 80% of the dairy cows had antibodies to N. caninum. NAT and rELISA had similar levels of seropositivity relative to IFAT and ISCOM-ELISA, but the percentage of positive sera was dependent on the cut-off value chosen. As indicated by kappa coefficient statistical analysis, ISCOM-ELISA and IFAT exhibited the highest level of agreement in identifying N. caninum-positive and -negative cows. A decrease in the percentage of seropositive cows as determined by ISCOM-ELISA and IFAT with increasing cow age was noted. However, no significant difference was observed between cow age and abortion status. In addition to these tests, an avidity ELISA was performed on all sera with high (> or =0.4) ISCOM-ELISA readings. Avidity index (AI) increased with time post-abortion suggesting that most abortions were due to recent N. caninum infection. Of the cows at risk for abortion, the mean serological AI of aborting cows was significantly lower (P<0.05) than mean serological AI of non-aborting cows.     

Comparative diagnostic potential of three serological tests for abortive Q fever in goat herds

Performances of an ELISA, an immunofluorescence assay (IFA) and a complement fixation test (CFT) were assessed for detecting antibodies against Coxiella burnetii after Q fever abortions in naturally infected goats. The goal of the study was to provide information useful for veterinary serodiagnosis in regard to categories of goats either experiencing Q fever abortion or not, blood sampling times and recommended cut-offs. The study was conducted on eight goat herds with evidence of C. burnetii abortions. In each herd, at least 5 goats that had aborted and 10 goats prior to parturition or at term were monitored 15, 30 and 60 days (D15, D30, D60) after the onset of Q fever abortion. The overall CFT results distribution did not differ between the two groups of goats and showed poor agreement with the ELISA results. In contrast, the ELISA and IFA results revealed comparable significant differences, but overall the ELISA test was slightly more sensitive than the IFA test. Seroprevalence, according to ELISA and IFA respectively, was higher in the aborting (88% and 82%) than in the non-aborting group (60% and 50%). High levels of serum antibodies were detected in goats post-abortion with an average of 114 %OD using ELISA and a log10(titer) of 2.4 using IFA. Strongly positive ELISA (%OD>80) and positive IFA results (log10(titers)>1.9) were significantly associated with abortion. Sampling on D15 gave the best association with ORs of 10 for ELISA and 6 for IFA. The practical interest of these results is discussed.     

A prospective study of the effect of Neospora caninum and BVDV infections on bovine abortions in a dairy herd in Arequipa, Peru

We used a prospective seroepidemiological approach to investigate endemic abortion in a dairy herd in Arequipa, Peru, and its association with Neospora caninum and bovine viral-diarrhoea virus (BVDV) infections. Between January 2002 and March 2004, 1094 pregnancies were confirmed in 538 cows. Of these, 137 pregnancies (13%) in 121 cows ended in abortion. The serological status to N. caninum was assessed using a single serological screening, whereas BVDV status was assessed at the herd level through consecutive samplings of young stock.

Cox proportional-hazards models were used to estimate the effect of N. caninum and BVDV on the hazard of early (between day 42 and day 100 in gestation), and late (after day 100) abortions, respectively. Serological status to N. caninum was included as a dichotomous variable, and the effect of BVDV estimated at the herd level, as a time-dependent seasonal effect. Because data from repeated pregnancies were included, we considered possible lack of independence between observations and included frailty effects into the models. Our models also considered the possible confounding by parity and animal origin.

Only multiparity was associated with the hazard of early abortion (HR = 2.8 compared to nulliparous heifers). N. caninum seropositivity significantly affected the hazard of late abortion, but interacted with parity. The HRs for Neospora-positive animals were 6.4, 3.7 and 1.9, respectively, for nulliparous heifers, first-lactation cows and multiparous cows. Evidence of BVDV circulating (or not) among the young stock was not associated with abortions, but few cows in this herd were susceptible to incident infection.     

Relationships between the shedding of Coxiella burnetii, clinical signs and serological responses of 34 sheep

Two abortions associated with Coxiella burnetii occurred in a group of 34 pregnant ewes. The seroprevalence of C. burnetii infection was studied by using an ELISA and the immunofluorescence (IF) assay was applied to the contents of vaginal swabs. In addition, a PCR assay, with primers based on a transposon-like repetitive region of the C. burnetii genome (trans-PcR), was used for the highly sensitive and specific detection of C. burnetii in vaginal swabs, milk and faeces. Of the 34 animals tested at parturition, eight (24 per cent) were positive by ELISA, 11 (32 per cent) were positive by IF, and 15 (44 per cent) were positive when the vaginal swab extract was subjected to the trans-PCR assay. C. burnetii was therefore detected by PCR in the vaginal swabs of seven seronegative ewes. However, five weeks after lambing, 16 (47 per cent) of the animals tested were ELISA positive but only two animals (6 per cent) were positive by PCR. Among the ELISA- and PCR-positive animals, eight (25 per cent) shed coxiella in their milk and six (18 per cent) did so in their faeces.     

Experimental Coxiella burnetii infection in pregnant goats: excretion routes

Q fever is a widespread zoonosis caused by Coxiella burnetii. Infected animals, shedding bacteria by different routes, constitute contamination sources for humans and the environment. To study Coxiella excretion, pregnant goats were inoculated by the subcutaneous route in a site localized just in front of the shoulder at 90 days of gestation with 3 doses of bacteria (10(8), 10(6) or 10(4) i.d.). All the goats aborted whatever the dose used. Coxiella were found by PCR and immunofluorescence tests in all placentas and in several organs of at least one fetus per goat. At abortion, all the goats excreted bacteria in vaginal discharges up to 14 days and in milk samples up to 52 days. A few goats excreted Coxiella in their feces before abortion, and all goats, excreted bacteria in their feces after abortion. Antibody titers against Coxiella increased from 21 days post inoculation to the end of the experiment. For a Q fever diagnostic, detection by PCR and immunofluorescence tests of Coxiella in parturition products and vaginal secretions at abortion should be preferred to serological tests.     

Serology of a Neospora abortion outbreak on a dairy farm in New Zealand: a case study

AIM: To describe the kinetics of serological titres after an abortion outbreak in April-May 1995 due to Neospora caninum affected 17 dairy cows in a herd of 320. METHODS: Thirty-five cows, that had either aborted, carried mummified calves, were not pregnant or calved normally were: bled several times at regular intervals and the sera tested for Neospora antibodies in the indirect fluorescence antibody test (IFAT). RESULTS: Maximal IFAT titres of up to 1:4000 occurred within 6 weeks of the abortion outbreak, decreased over the next 2 months to < or = 1:200 and remained at this level until the next scheduled bleed a further 2 months later. A rise in titres was subsequently observed in the cows that had aborted or were not pregnant (at the time of the abortions) or had carried mummified foetuses. Seroconversion was also observed in some of the control cows, which had, up until then, remained seronegative. A dog and cat in contact with the cows in the herd investigated were, however, negative in the IFAT. CONCLUSIONS: Maximal serological titres in Neospora abortions are observed within weeks of the abortion event and then quickly return to very low levels. Subsequently, a recrudescence of titres can be observed in infected cows during the next pregnancy, without it being associated with repeat abortions.     

Seroprevalence of Neospora caninum in aborting dairy cattle in the Czech Republic

A serological survey for antibodies against Neospora caninum in aborting cattle was carried out in the Czech Republic. Serum samples from 463 aborting dairy cows originated from 137 farms from different parts of the Czech Republic were tested for presence of N. caninum antibodies by use of an enzyme-linked immunosorbent assay (ELISA) and an indirect fluorescent antibody test (IFAT). Antibodies (> or = 1:640) to N. caninum were found in 18 (3.9%) of 463 aborting cows. Farm prevalence in aborting cows was 12.4% (17/137). The antibody titres of cows were 1:200 (9 cows), 1:640 (7 cows), 1:1280 (3 cows), 1:2560 (3 cows), 1:5120 (3 cows), 1:10,240 (2 cows) and 1:20,480 (0 cow). A case-control study was conducted to estimate the association of N. caninum infection and abortion. For this 407 serum samples were collected from cows on five dairy farms with repeated occurrence of endemic and sporadic abortion of unidentified etiology. These samples were obtained from aborting cattle (n=44) and normally calving cattle (control group; n=363) and tested for N. caninum antibodies by an immunofluorescent antibody test (IFAT). Overall, 3.19% (13/407) of cows sampled had positive N. caninum fluorescence with a cut-off titre of 1:200. The prevalence of N. caninum was significantly higher (P<0.05) in the aborting group (13.64%; 95% confidence interval (CI): 5.2, 27.4) than in the control group (1.93%; 95% CI: 0.8, 3.9). A strong association between seropositivity and abortion was found, with seropositive cows being eight times more likely to abort than seronegative cows (odds ratio=8; 95% CI: 2.6, 25.1). This first report on the serological prevalence of N. caninum in cows in the Czech Republic verified a strong association between N. caninum infection and abortions in five dairy farms. Thus, the neosporosis should be considered in differential diagnosis of bovine abortion.     

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.     

Non specific serological reactions in the diagnosis of bovine brucellosis: experimental oral infection of cattle with repeated doses of Yersinia enterocolitica O:9

Eight heifers were orally infected with 4 x 10(9) colony forming units of a field cattle strain of Yersinia enterocolitica O:9 in a capsule, 5 days a week, for about 9 weeks (day 0-day 64 (D0-D64). The faecal shedding of Y. enterocolitica O:9 began on D5 for seven out of the eight challenged cattle with a high level of excretion during the first month, followed by a decrease till the day of slaughter (D76). Y. enterocolitica O:9 was not isolated from organs collected at slaughter. No clinical symptoms were observed. Hyperplasia of intestinal lymph formations was the sole microscopic lesions observed. Five animals showed a serological reaction against Brucella antigens in at least one of the following tests: Rose-Bengal test, complement fixation test, tube agglutination test or indirect ELISA (iELISA) tests. Only one animal showed a high level of serological response and a positive reaction in the dithiothreitol-microagglutination test. The observed variability in terms of individual sensitivity to the Y. enterocolitica O:9 infection is in agreement with the low individual prevalence rate and the transient serological reaction and faecal Y. entercolitica O:9 shedding observed in herds showing false positive serological reactions in brucellosis.     

Occurrence, distribution, and role in abortion of Coxiella burnetii in sheep and goats in Sardinia, Italy

Between 1999 and 2002, 9349 sera and 517 aborted samples (422 foetuses and 95 placenta) were analysed from 675 sheep and 82 goat farms distributed all over the island of Sardinia. After abortion notification, sera collected at random from adult animals were examined to detect antibodies specific to Coxiella burnetii by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 255 (38%) sheep farms and in 39 (47%) goat herds whereas 40 ovine (10%) and 3 (6%) caprine foetuses were C. burnetii PCR-positive. Although C. burnetii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. Seroprevalence analysis indicates that C. burnetii distribution in sheep and goats is very high, but PCR results demonstrate that C. burnetii has a relatively low role in abortion, especially in goats.     

Diagnosis of bovine venereal campylobacteriosis by ELISA

SUMMARY An enzyme-linked immunosorbent assay (ELISA) measuring IgA antibodies in the vaginal mucus was used to diagnose bovine venereal campylobacteriosis in 241 herds with infertility and abortions. The presence of the disease was confirmed on 64 farms (34.8%) and it was suspected on a further 27 farms (11.2%). The specificity of the ELISA was found to be 98.5% but in the absence of a reliable comparative test sensitivity can not be estimated. Vaccination against campylobacteriosis will not interfere with the IgA ELISA because only IgG is present in the vaginal mucus of vaccinates. Because of the possibility of false reactions caused by antibody fluctuations in individual cattle, the ELISA is best used as a herd test. It appears that at present the vaginal mucus IgA ELISA is the test of choice for the diagnosis of bovine venereal campylobacteriosis.     

Enzyme-linked immunosorbent assay for antibodies to Campylobacter fetus in bovine vaginal mucus

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to Campylobacter fetus subspecies venerealis in bovine vaginal mucus. The results of testing 168 samples from experimentally infected, field cases and control cows showed that the ELISA was more sensitive than the vaginal mucus agglutination test and also detected antibodies in earlier stages of infection.