571993 research-article2015

VDIXXX10.1177/1040638715571993Coxiella burnetii infection patterns in young damsSerrano-Pérez et al.

Full Scientific Report

Coxiella burnetii total immunoglobulin G, phase I and phase II immunoglobulin G antibodies, and bacterial shedding in young dams in persistently infected dairy herds

Journal of Veterinary Diagnostic Investigation 2015, Vol. 27(2) 167­–176 © 2015 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638715571993 jvdi.sagepub.com

Beatriz Serrano-Pérez,1 Sonia Almería, Joan Tutusaus, Isabel Jado, Pedro Anda, Eva Monleón, Juan Badiola, Irina Garcia-Ispierto, Fernando López-Gatius

Abstract. The current study examines Coxiella burnetii infection patterns in young dairy dams around the calving period in persistently infected high-producing dairy herds. Infection patterns were determined in terms of total immunoglobulin G (IgG) and phase-specific IgG antibodies by enzyme-linked immunosorbent assay and bacterial shedding by real-time polymerase chain reaction (qPCR). On days 171–177 of gestation, at parturition, and on days 15–21 and 91–97 postpartum, 7 firstparity cows and 7 second-parity cows were sampled for serology and qPCR. Total phase-specific I (PhI) and II (PhII) IgG antibodies were detected in 2 animals at days 171–177 of gestation. Four additional animals underwent seroconversion on days 91–97 postpartum. Three of 6 seropositive dams according to total IgG, showed a PhI+/PhII+ profile, whereas dams that seroconverted exhibited a PhI–/PhII+ (2/6) or PhI+/PhII– (1/6) profile. An indirect fluorescent antibody test for PhI and PhII immunoglobulin M (IgM) was performed on plasma samples from the shedding dams, confirming seropositivity in a first-parity dam that seroconverted, and detecting a sudden spike of PhI-IgM antibodies in 1 further dam. No relationship was detected in young C. burnetii–infected animals between total IgG, PhI and/or PhII antibodies, and bacterial shedding throughout the study period. The highest bacterial load measured by qPCR was recorded in a second-parity dam. This animal presented abnormal peripheral blood counts, which would be an indication of severe peripheral blood alterations in some infected cattle. This study suggests that young shedder cows are mostly seronegative in early stages of infection. Key words: Cattle; hematology; phase-specific antibodies; polymerase chain reaction; Q fever.

Introduction Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes Q fever, a zoonotic illness showing a worldwide distribution.24 Although livestock species are considered one of the most common sources of human infection,3 studies have shown that coxiellosis in cattle is caused by a widespread specifically cattle-adapted strain not linked to human outbreaks in Europe of Q fever.20,45 Yet, human infection through C. burnetii–contaminated aerosols or products from cattle should not be underestimated.5,14 Infected cattle shed high numbers of C. burnetii in milk, vaginal mucus, feces,14 urine,6 and, importantly, birth products,17 and generally exhibit subclinical symptoms.1 However, reproductive disorders such as placental damage, abortion, stillbirth, placental retention, or infertility have been linked to C. burnetii infection in dairy herds.12,15,23 To control C. burnetii infection at the herd level, treatment with antibiotics (tetracycline) or vaccination may reduce, but not prevent, bacterial shedding.44 In Spain, seroprevalence of C. burnetii in dairy herds ranges from 67% to 74%.4,29 Financial consequences of infection can be substantial at the herd

level,32 but production losses and costs have not been properly assessed in cattle. The identification of asymptomatic infected animals at the farm level relies on laboratory methods such as enzymelinked immunosorbent assay (ELISA) to detect an antibody response, and polymerase chain reaction (PCR) to detect bacterial DNA.21,22,26 However, pathogen detection in cattle is usually challenging because bacterial shedding occurs intermittently and through multiple routes such as birth prod-

From the Centre for Research in Agrotechnology, Animal Production Department, University of Lleida, Lleida, Spain (Serrano-Pérez, Tutusaus, Garcia-Ispierto, López-Gatius); the Centre for Research on Animal Health, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain (Almería); the Department of Bacteriology, National Microbiology Centre, Carlos III Institute of Health, Majadahonda, Madrid, Spain (Jado, Anda); and the Centre for Research on Transmissible Spongiform Encephalopathies and Emergent Diseases, University of Zaragoza, Zaragoza, Spain (Monleón, Badiola). 1 Corresponding Author: Beatriz Serrano-Pérez, Animal Production Department, University of Lleida, Avenue Alcalde Rovira Roure, 191, E-25198, Lleida, Spain. [email protected]

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ucts, milk, vaginal fluids, or feces.11,13 Additionally, bacterial shedding and antibody production are not always concomitant, and many shedding animals are diagnosed as seronegative.14,34 Defense against C. burnetii requires both strong cellular immunological mechanisms and a humoral response.39 The humoral response is conditioned by antigenic variation or phase variation, when the bacterium shifts from a virulent phase (phase I) to an avirulent phase (phase II) due to changes in lipopolysaccharide composition.47 The study of phasespecific serological responses allows for a better understanding of protective immunology against C. burnetii infection both in human beings24 and ruminants.7,35,42 In cattle, as in people, phase II antibodies have been associated with acute infection and bacterial shedding, while phase I antibody production is a reflection of chronic infection.7 Newly pregnant animals exhibit a higher susceptibility to infection.7,28 After an initial clinical or subclinical infection, coxiellosis persists over time.18,46 Despite the presence of an adequate cellular immune response, the subversion of microbicidal functions of macrophages, targets of C. burnetii, guarantees bacterial survival in the animal.8 The physiological immunosuppressive status of pregnancy and parturition makes this a critical period of increased susceptibility to C. burnetii infection,18,36 and a high bacterial load has been recorded at parturition in cattle.11 The present study examines the dynamics of C. burnetii infection and seroconversion from young dams in 2 highproducing dairy herds persistently infected with C. burnetii in northeastern Spain. The main objectives were to establish interactions between total antibodies, phase-specific serology, and bacterial shedding in young dams (first-parity and second-parity dams) during the late gestation, parturition, and postpartum periods. In addition, factors affecting phase-specific serology and hematological profiles were investigated.

Materials and methods Cattle and herd management The study was performed on 2 commercial, high-producing Holstein–Friesian dairy herds in northeastern Spain. Data recorded from October 2010 to November 2011 showed that the 2 herds were comprised of 625 and 125 lactating cows with mean annual milk production of 11,343 kg and 8,846 kg and annual culling rate of 29% and 23%, respectively. The cows calved all year round and were fed complete rations. Feeds consisted of cotton-seed hulls, barley, corn, soybean, and bran. Mainly corn, barley, or alfalfa silages and alfalfa hay were provided as roughage. Rations were in line with National Research Council recommendations.27 All cows were bred by artificial insemination using semen from bulls of proven fertility. Clinical examinations of the reproductive tract during the postpartum period were performed on a weekly basis with ultrasound.50

Both herds had, in the past, returned positive real-time PCR (qPCR) results for C. burnetii in the bulk tank milk samples with excretion higher than 104 Coxiella/mL10 and had seroprevalence levels of 46% and 53% for herds 1 and 2, respectively.23 The characterization of the C. burnetii genotype in positive PCR placental tissues was performed through a multiplex IS1111-based endpoint PCR coupled with hybridization by reverse line blotting that classifies C. burnetii isolates into 8 genomic groups.20 Detection of acute disease antigen A (adaA), associated with acute Q fever–causing strains, was included.20 Based on previous serological analysis, qPCR analysis of bulk tank milk samples, and genotyping of placental tissues,20 both herds were classified as chronically infected with C. burnetii genomic group III strain adaA positive.11,20,23 Only animals free of clinical disease were included in the study to reduce variation in the general health status of the animals. The final data analyzed corresponded to 7 firstparity dams and 7 second-parity dams that delivered live calves. Cows bearing twin pregnancies were not included in the study in order to discard possible postpartum reproductive disorders derived from twin pregnancies.23

Experimental design Young dams in both herds were serologically tested on days 171–177 of gestation. Of these, 7 first-parity cows (F1–F7) and 7 second-parity cows (S1–S7) were tested at late gestation (days 171–177), parturition (day 0), and postpartum (days 15–21 and 91–97, as early and late postpartum periods, respectively). Five dams belonged to herd 1 (3 first-parity and 2 second-parity dams), and 9 dams to herd 2 (4 firstparity and 5 second-parity dams). Blood samples for plasma antibody testing, and feces and vaginal fluid for qPCR detection were collected throughout the study period. Colostrum and cotyledons at parturition, milk samples during the prepartum period in second-parity cows, and milk samples during the postpartum period in all of the animals were also collected for qPCR detection of the bacterium. For further analysis, 5 second-parity cows were sampled 8 times during the study period: on days 171–177 of gestation, on the day of parturition, and on days 1–7, 8–14, 15–21, 22–28, 29–35, and 91–97 postpartum. Besides milk, feces, vaginal fluid, colostrum, and cotyledon samples, additional blood samples were obtained for hematological tests in these cows.

Blood sampling Blood samples were collected from the coccygeal vein in 2 ethylenediamine tetra-acetic acid vacuum tubes.a One tube was centrifuged (10 min at 1,600 × g) within 30 min of collection and the plasma stored at −20°C until analysis, whereas the blood in the second tube was used for hematological analysis.

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Serological analyses Plasma samples were screened for total immunoglobulin G (IgG) and phase-specific I (PhI) and II (PhII) antibodies against C. burnetii by ELISA. A commercial indirect ELISAb and 2 phase-specific ELISAs (PhI- and PhII-ELISAs), were used to determine antibodies against C. burnetii in plasma samples. A cocktail of the PhI and PhII Nine Mile antigen strain of C. burnetii was used in the commercial ELISA kit to detect total anti–C. burnetii IgG. For phase-specific ELISAs, PhI and PhII antigens were coated individually onto ELISA plates under the same conditions and using the same concentrations as for the commercial ELISA. The tests were carried out according to the manufacturer’s instructions. For each sample, the sample-to-positive (S/P) ratio was calculated as follows: sample optical density (OD) – negative control OD/ positive control OD – negative control OD. The results were expressed as titers (titer = S/P × 100). A plasma sample was scored as positive when OD% was higher than 40%. According to phase-specific profiles, the animals were classed as PhI–/PhII–, PhI+/PhII+, PhI–/PhII+, or PhI+/PhII–.7 Phasespecific profiles were determined for each parity group: firstparity and second-parity dams. The sensitivity and specificity of the commercial ELISA in blood samples are 84% and 99%, respectively.31 Results were expressed as indicated above for total anti–C. burnetii IgG. The presence of immunoglobulin M (IgM) antibodies for both PhI and PhII antigens was analyzed by indirect fluorescent antibody test (IFAT) in the shedding animals as previously described.37 Titers ≥1:50 were considered positive. Phase I and phase II antigens were derived from the Nine Mile strain.c

Real-time polymerase chain reaction Samples for qPCR were collected and processed as previously described.11 The DNA was extracted from milk, feces, vaginal fluid, colostrum, and cotyledon samples using a DNA extraction kitd according to the manufacturer’s instructions. The presence of C. burnetii DNA was detected by qPCR using a commercial kit targeting the repetitive transposon-like region of C.burnetii e normalized by the reference housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), according to the manufacturer’s instructions. A standard curve based on 10-fold dilutions of an external positive control solution containing 105 C. burnetii/ mL was performed following the recommendations given by the kit manufacturer.f The negative control sample used was DNase- and RNase-free water. An endogenous control to monitor inhibition of the qPCR was included. Only the samples that showed a typical amplification curve with a threshold cycle (Ct) for the target gene below 40 were considered to be positive. Amplification of the GAPDH gene was required to rule out PCR inhibition in negative samples. If GAPDH was not amplified, DNA from those samples was

diluted 1:10, and the qPCR assays were run again. If inhibition still occurred on a second round of qPCR, new DNA was extracted from those samples and the testing repeated. Negative results were not considered valid until the GAPDH gene was detected.

Hematology In 5 cows, hematological tests were performed on blood samples collected on days 171–177 of gestation, and on days 1–7, 8–14, 15–21, 22–28, 29–35, and 91–97 postpartum. Blood samples were automatically analyzed on the day of collection using a hematology analyzerg for total and differential (neutrophils, lymphocytes, monocytes, and eosinophils) leukocyte counts. The device was pre-adjusted for use with cow blood as specified by the manufacturer.

Data collection and statistical analyses Herd, parity, C. burnetii total and phase-specific antibodies, phase-specific profiles, and bacterial shedding during late gestation (days 171–177 of gestation), at parturition, and early and late postpartum (days 15–21 and 91–97 postpartum) were recorded for each animal. In the subset of 5 dams, bacterial shedding, in milk, feces, and vaginal fluid on days 1–7, 8–14, 22–28, and 29–35 postpartum, and peripheral blood cell populations were recorded. When at least 1 sample (milk, feces, vaginal fluid, colostrum, and/or cotyledon) collected on at least 1 sampling day proved PCR-positive, the cow was scored as a positive shedder. Also recorded were postpartum reproductive disorders such as placental retention (retention of the fetal membranes >24 hr) or primary metritis (acute puerperal metritis diagnosed during the first or second week postpartum in cows not suffering placental retention). Coxiella burnetii–infected animals were defined as either seropositive and/or shedders.16 The effects of sampling time, bacterial shedding on sampling, herd, parity (first-parity vs. second-parity cows), and plausible interactions of paired factors on C. burnetii total and phase-specific PhI and PhII antibody titers were assessed by general linear models repeated measures analysis of variance. All statistics procedures were performed using commercial software.h Significance was set at P < 0.05. Values are expressed as the mean ± standard deviation.

Results Total immunoglobulin G antibodies At the first sampling time (days 171–177 of gestation), 1 first-parity cow (F1) and 1 second-parity cow (S1; 2/14, 14.3%) were already C. burnetii seropositive. The highest percentage of seropositive animals (6/14, 42.8%) was observed on days 91–97 postpartum in 3 first-parity (F1–F3) and 3 second-parity dams cows (S1–S3; Table 1). At this

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Table 1.  Animals testing positive for Coxiella burnetii according to total immunoglobulin G (IgG) antibodies, seroprevalences, and phase-specific I (PhI) and II (PhII) IgG profiles by enzyme-linked immunosorbent assay of first-parity dams (F1–F7) and second-parity dams (S1–S7) and bacterial shedders according to parity and date of sampling.* Dam Date of sampling/Parity Prepartum (day 171–177)   First parity (n = 7)   Second parity (n = 7) Parturition   First parity (n = 7)   Second parity (n = 7) Early postpartum (day 15–21)   First parity (n = 7)   Second parity (n = 7) Late postpartum (day 91–97)   First parity (n = 7)   Second parity (n = 7)

Total IgG

Shedding†

PhI–/PhII–

PhI+/PhII–

PhI–/PhII+

F1 (14.3) S1 (14.3)

0 (0) S5 (14.3)

F2–F7 (85.7) S2–S7 (85.7)

0 (0) 0 (0)

0 (0.0) S1, S2 (28.6)

F3–F6 (57.1) S5 (14.3)

F1–F7 (100) S3–S7 (71.4)

0 (0) 0 (0)

0 (0) S2 (14.3)

0 (0) S1 (14.3)

F1 (14.3) S1, S2 (28.6)

F5 (14.3) S5 (14.3)

F2–F7 (85.7) S3, S5–S7 (57.1)

0 (0) 0 (0)

0 (0) S2, S4 (28.6)

F1 (14.3) S1 (14.3)

F1–F3 (42.9) S1–S3 (42.9)

F6 (14.3) 0 (0)

F4–F7 (57.1) S4–S7 (57.1)

F2 (14.3) S2 (14.3)

F1, F3 (28.6) S1 (14.3)

0 (0) S3 (14.3)

0 (0) 0 (0)

PhI+/PhII+ F1 (14.3) S1 (14.3)

* Numbers in parentheses are percentages. † The percentage of bacterial shedders is based on the number of available samples from dams for real-time polymerase chain reaction analysis with respect to their serological profile.

time point, the seroconversion (28.6%) of 4 dams previously testing seronegative (2 first-parity cows [F2–F3] and 2 second-parity cows [S2–S3]) was observed. Thus, 4 first-parity cows (F4–F7) and 4 second-parity cows (S4–S7) remained seronegative during the entire study period. One seropositive animal (F1) lost its seropositivity at parturition but recovered this status during the postpartum period.

Bacterial shedding Five dams, 4 first-parity cows (F3–F6) and 1 second-parity cow (S5), shed the bacterium in at least 1 sample during the study period (35.7%; Table 1). One seronegative secondparity cow (S5) was a shedder on days 171–177. Parturition was the period showing the highest incidence of bacterial shedding (5 shedders, F3–F6 and S5), and these animals were seronegative at that time (Table 1). The only shedder that underwent seroconversion was second-parity cow F3 on days 91–97 postpartum. The presence of C. burnetii DNA was recorded in 18 samples during the study period (Table 2). Parturition was the period in which the highest number of samples proved positive for this test (10/18). During parturition, C. burnetii DNA was simultaneously detected in colostrum, vaginal mucus, and feces samples in 2 animals (Table 2). Coxiella burnetii DNA was not identified in any of the cotyledon samples collected at parturition. The highest bacterial load was observed in the single second-parity cow shedder (S5) on days 171– 177, with a Ct value of 29.9 in milk, and at parturition, a Ct value of 27.5 in feces. The shedder animal (S5) scored positive by qPCR in 9 out of all samples collected (50%), and shed the bacterium in at least 1 sample on days 171–177 of

gestation, at parturition, and on days 15–21 postpartum (Table 2).

Phase-specific I and/or II antibodies Seropositive animals according to total IgG antibody titers were also consistently positive for PhI and PhII antibodies. Only cow S4, which was seronegative based on total IgG antibodies, was PhI–/PhII+ at early postpartum. The PhI+/ PhII+ profile was observed in 3 animals (S1, F1, and F3). This profile was consistently observed throughout the study period in animals S1 and F1, with the exception of F1 at parturition when this animal also lost its seropositivity for total IgG antibodies. Animal F3 was the only shedder that seroconverted for total IgG antibodies showing the PhI+/PhII+ profile on days 91–97 postpartum (Table 1). Profile PhI–/ PhII+ was observed in 3 animals (F2, S2, and S4) during the early postpartum period (Table 1). Of these, S2 seroconverted for total IgG antibodies at parturition, while S4 seroconverted to PhI–/PhII+ transiently during early postpartum and was not considered seropositive for total IgG antibodies throughout the study period. Profile PhI+/PhII– was only observed in animal S3 during late postpartum (Table 1). Four of the 5 shedding dams showed PhI–/PhII– phase-specific profiles throughout the study. Two shedder cows (F6 and S5) exhibited increasing PhI and PhII antibody titers during the postpartum period but did not reach a seropositive status (data not shown). In the 5 shedding animals IgM antibodies were evaluated by IFAT, and immunoglobulin M (IgM) detection was recorded in 2 animals. Animal F3, which seroconverted in the ELISA, presented PhII-IgM and PhI-IgM on days 91–97

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Table 2.  Threshold cycle values in positive real-time polymerase chain reaction samples (vaginal mucus, milk or colostrum, or feces) recorded for each sampling period in 5 dams (4 first-parity animals [F3–F6] and 1 second-parity animal [S5]).* Prepartum Dam F3 F4 F5 F6 S5

Vaginal mucus >40 >40 >40 >40 >40

Parturition

Milk Feces — — — — 29.9

>40 >40 >40 >40 >40

Vaginal mucus 28.9 29.6 >40 26.9 31.3

Early postpartum Vaginal mucus

Milk

Feces

Vaginal mucus

>40 >40 >40 >40 35.7 (1); 35.8 (5)

>40 >40 27.0 (3) >40 35.4 (5)

>40 >40 >40 >40 34.9 (4); 33.0 (5)

>40 >40 >40 >40 >40

Colostrum Feces 36.4 >40 36.4 >40 33.2

35.2 >40 36.7 >40 27.5

Late postpartum Milk Feces >40 >40 >40 >40 >40

>40 >40 >40 35.4 >40

* All cotyledon samples were negative. The positive samples recorded in the early postpartum period are coded by a number in parentheses that indicates as follows: 1 = days 1–7; 2 = days 8–14; 3 = days 15–21; 4 = days 22–28; 5 = days 29–35. Dash (—) indicates no milk samples.

Figure 1.  Mean total and phase-specific I (PhI) and phase-specific II (PhII) Coxiella burnetii antibody titers recorded throughout the prepartum (gestation days 171–177), parturition, and postpartum (days 15–21 and 91–97 postpartum) periods.

postpartum. In addition, animal F5 was positive for PhI-IgM antibodies on days 1–7 postpartum.

Factors affecting C. burnetii antibody status and antibody dynamics Given that only 1 cow (S1) suffered metritis after parturition, reproductive disorders were not included in the statistical analyses. Using general linear models repeated measures analysis of variance, no effects of herd, parity, or bacterial shedding at parturition on C. burnetii antibody status were detected. Significant effects on total IgG antibodies were observed for the day of sampling (within subject effects).

Toward parturition, antibody titers declined while a significant increase in total antibodies was observed in the postpartum period (P = 0.015). A trend toward significance was also observed for increasing PhII antibody levels at postpartum (P = 0.053; Fig. 1).

Peripheral white blood cell counts For a subgroup of 5 second-parity dams, hematological data was available. This subgroup included the C. burnetii–seronegative shedder cow (S5), 2 C. burnetii–seronegative nonshedding (S4 and S6) cows, and 2 C. burnetii–seropositive non-shedding second-parity cows (according to serology on

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Figure 2.  Neutrophil concentrations (×103/μL) recorded in a Coxiella burnetii–seronegative shedding second-parity dam (S5) from gestation days 171–177 to days 91–97, versus non–C. burnetii shedding second-parity dams (S1, S2, S4, S6).

days 91–97: S1 and S2). The non-shedding animals showed peripheral total and differential leukocyte concentrations within physiological ranges.40 However, abnormal total and differential leukocytes were observed in the shedder cow. This animal showed neutrophilic and eosinophilic leukocytosis and lymphopenia on days 171–177 of gestation and during the early postpartum period (days 15–28; Figs. 2, 3, and 4, respectively). Higher lymphocyte levels were recorded in the shedder animal at all other sampling periods compared to the non-shedders (Fig. 4). Monocytes were within the physiological range but greater numbers were observed during the study period in the shedder animal (Fig. 5).

Discussion This report describes the dynamics of C. burnetii total IgG antibodies, PhI and PhII IgG antibodies, and infection in young pregnant dams (first-parity and second-parity cows) across the late gestation, peripartum, and postpartum periods. One of the most relevant findings was that no relationship was detected in young C. burnetii–infected animals between total IgG, PhI and/or PhII antibodies, and bacterial shedding throughout the study period. Of the 5 young shedder animals examined (35.7% of all the animals analyzed), 4 remained seronegative to IgG antibodies up to days 91–97 postpartum, although 1 of the 4 showed IgM by IFAT. This information is consistent with the detection of intermittent or sporadic seronegative shedding animals in previous studies performed in cattle14,34 and points to the difficulty in diagnosing infected animals based on serological analysis alone.

A possible explanation for these results could be that seronegative shedders had been born infected in utero and did not produce antibodies due to immunotolerance. Transplacental infection of the fetus in utero by C. burnetii is possible, but its consequences are still unknown.3 If infection occurs in early gestation, the fetus could be immunotolerant as occurs in calves persistently infected with Bovine viral diarrhea virus.25 In a study in dairy herds in northeastern Spain, no detectable C. burnetii precolostral antibody response was observed in calves born to dams with cotyledons positive for C. burnetii by qPCR, and seroconversion was only observed in calves born to seropositive dams after colostrum intake.49 Taken together, these results could indicate that seronegative shedders may have been infected in utero. In these animals, infected macrophages and dendritic cells could act as “Trojan horses” in tissues, mainly in placental and adipose tissue.2 If immunotolerance to the organism is confirmed, it may be critical in the pathogenesis of C. burnetii infection. Another possibility is that seronegative shedder animals could become seropositive at periods of time later than those examined in the present study. A lack of sensitivity in commercial ELISA kits for veterinary diagnosis has been proposed to explain the seronegativity of shedding animals.34 While there is no gold standard test in Q fever diagnosis, the ELISA kit used in this study, based on tick-borne (Nine Mile)-derived antigen, has been described as highly sensitive for detection of IgG responses in cattle.22,31 The antibody dynamics observed herein are in agreement with the results of previous studies.11,28,50 Once antibodies were detected, total IgG antibodies remained highly stable during the study

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Figure 3.  Eosinophil concentrations (×103/μL) recorded in a Coxiella burnetii–seronegative shedding second-parity dam (S5) from gestation days 171–177 to days 91–97, versus non–C. burnetii shedding second-parity dams (S1, S2, S4, S6).

Figure 4.  Lymphocyte concentrations (×103/μL) recorded in a Coxiella burnetii–seronegative shedding second-parity dam (S5) from gestation days 171–177 to days 91–97, versus non–C. burnetii shedding second-parity dams (S1, S2, S4, S6).

course. Antibodies diminished at parturition, probably due to their loss through colostrum.19 After parturition, there was a significant increase in total IgG antibody levels in several

dams (first-parity dam F2, and second-parity dams F2, F3), including 1 shedder animal (F3) that seroconverted in late parturition. Seroconversion after parturition and the fact that

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Figure 5.  Monocyte concentrations (×103/μL) recorded in a Coxiella burnetii–seronegative second-parity dam (S5) from gestation days 171–177 to days 91–97, versus non–C. burnetii shedding second-parity dams (S1, S2, S4, S6).

another shedder animal showed a sudden spike of IgM antibodies, but lacked IgG antibodies, could suggest a new infection in those animals. Since shedding in seronegative animals took place mainly at parturition, the excretion of bacteria could have led to the infection of more animals in the herd at this time. The possibility that these seroconverted animals will be shedders in future gestations remains to be determined. In previous studies, PhII antibodies have been related to acute infections and PhI antibodies to chronic infections.7 However, contrary to previous studies,7,42 no correlation between ELISA PhII IgG antibodies and bacterial shedding was detected in the current study, and, indeed, most shedders showed PhI–/PhII– profiles throughout the study. Only a first-parity animal, F3, which shed the bacterium concomitantly via 3 routes at parturition, underwent seroconversion on days 91–97 to a PhI+/PhII+ IgG profile by ELISA, and was confirmed with IFAT. One additional seronegative shedding dam, S5, exhibited increased ELISA PhI and PhII IgG antibody titers during the late postpartum period although the levels did not reach seropositivity up to days 91–97. In human beings, diagnostic methods depend on the stage of infection. Bacterial DNA detection in serum is accompanied by the production of PhII IgM antibodies, while seroconversion to PhI IgM antibodies and PhI and PhII IgG antibodies entails undetectable bacterial DNA.38 In the present work, IgG humoral response in shedding animals also involved negative qPCR results. Newly infected young animals could

elicit a primary humoral IgM response, not detectable with commercial ELISA kits, with a subsequent delayed IgG humoral response.22 Indeed, PhI IgM antibody detection was observed in animal F5, and it was not correlated to subsequent IgG seroconversion. Commercial ELISAs capable of detecting anti-PhI and anti-PhII IgM antibodies could improve the detection of serum antibodies in early stages of infection.22 In the present study, most animals shed at parturition, and the highest number of positive samples was also detected at this time in agreement with previous studies conducted in this region of Spain.11 As with previous results,28 newly pregnant animals showed a higher susceptibility to C. burnetii infection, shown by a higher rate of shedding by first-parity dams at parturition. This situation could be more the consequence of adaptation to new metabolic demands48 or restraint stress43 than impaired immune function in heifers.30 However, maternal immune suppression at parturition could promote infection recrudescence.36 In dairy cattle, the risk of infection could be particularly high due to metabolic impairment linked to milk production, a factor known to induce infectious diseases in the early postpartum period.41 In agreement with this, the current study found that the late postpartum period correlated with a lower number of qPCR-positive samples for C. burnetii. This is possibly explained by the fact that, during the late postpartum period, the cow has recovered from delivery and the immune system is again competent.11 The shedder animals found in the current study did not

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Coxiella burnetii infection patterns in young dams show a dominant route of bacterial shedding as appears to be common in cattle,13 and the bacterium was observed simultaneously in fecal, vaginal, and milk and/or colostrum samples in some animals. This could indicate severe immunosuppressive status of these animals. Further studies are needed to establish the exact time of placental infection in cattle, as C. burnetii DNA was detected in vaginal mucus but not in cotyledon samples of shedding animals. In a previous study, C. burnetii DNA was detected in the placental cotyledons of seropositive dams by the same technique as the present study.49 Therefore, although placental cotyledon samples were negative in young animals, it is possible that C. burnetii disrupted the maternal–fetal barrier at older ages. Normal leukocyte counts have been reported in seropositive animals.33 In the present study, the seronegative “super-shedder” second-parity cow that shed the bacterium in prepartum, parturition, and early postpartum periods also featured abnormal hematological variables during the study period, possibly as a result of trafficking of peripheral immune cells to infected tissues. This cow simultaneously shed the bacterium via 3 routes at parturition and in the early postpartum period, yet its peripheral blood cell kinetics differed depending on sampling time. Neutrophils and eosinophils decreased sharply at parturition and increased again on days 15–21, while lymphocytes increased at parturition but decreased sharply on days 22–28 postpartum. Augmented lymphocyte numbers were not associated with systemic antibodies, pointing to a Th1-immune response. In agreement with these results, higher concentrations of monocytes, responsible for clearing C. burnetii infection from tissues,18 were also observed. Studies have shown how bacteria could use neutrophils as an evasive strategy to infect macrophages.9 The C. burnetii strain that circulates in Spain represents a low risk for Q fever transmission in zoonotic terms.5,20,23 However, further studies designed to examine the cellular response are needed to clarify the pathogenesis of coxiellosis during acute infection in cattle. In conclusion, the results of the present study indicate that serology serves to reflect recent contact with C. burnetii in dairy cattle herds, but shedder animals were mostly seronegative. Thus, we hypothesize that young dams that are seronegative but shedding might have been infected in utero or may become seropositive in periods of time later than those examined in the present study. For this reason, it is important to test for C. burnetii DNA as well as antibodies for the detection of exposed and/or infected animals, in particular in newly infected young animals. The hematological variables detected in a prolonged high shedder animal point to severe peripheral blood alterations in some infected cattle. Acknowledgments The authors thank Ana Burton for assistance with the English translation; the owners and staff of the farms for their cooperation; Carmina Nogareda and Irene López-Helguera for technical assistance; and Christoph Egly and Ana Manso from IDEXX for providing the enzyme-linked immunosorbent assay kits.

Sources and manufacturers a. Vacutainer, BD, Plymouth, United Kingdom. b. IDEXX Q fever Ab test, IDEXX Switzerland AG, Bern, Switzerland. c. Institute of Virology, Bratislava, Slovakia. d. QIAamp DNA mini kit, Qiagen S.A., Courtaboeuf Cedex, France. e. LSI Taqvet Coxiella burnetii, Laboratoire Service International, Lissieu, France. f. UR INRA IASP, Nouzilly, France g. HEMAVET HV-950FS multispecies hematology system, Drew Scientific Inc., Dallas, TX. h. SPSS package version 18.0, SPSS Inc., Chicago, IL.

Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article

Funding This work received the financial support of IDEXX who provided the ELISA kits used in the study.

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