Comparative Immunology, Microbiology and Infectious Diseases 38 (2015) 41–46
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Lack of transplacental transmission of Bartonella bovis S. Chastant-Maillard a,b,c , H.-J. Boulouis d , K. Reynaud a,b , S. Thoumire a,b , C. Gandoin d , C. Bouillin d , N. Cordonnier e , R. Maillard c,d,∗ a b c d e
INRA, UMR 1198 Biologie du Développement et Reproduction, F-78350 Jouy-en-Josas, France ENVA, UMR 1198 Biologie du Développement et Reproduction (BDR), 7 Avenue du Général de Gaulle, F-94700 Maisons-Alfort, France Université de Toulouse, INP, ENVT, 23 Chemin des Capelles, BP 87614, 31076 Toulouse Cedex 03, France Université ParisEst, INRA, Anses, ENVA, UMR BIPAR, 23 avenue du Général de Gaulle, 94706 Maisons-Alfort, France ENVA, Histology – Histopathology, 7 Avenue du Général de Gaulle, F-94700 Maisons-Alfort, France
a r t i c l e
i n f o
Article history: Received 8 October 2013 Received in revised form 7 November 2014 Accepted 21 November 2014 Keywords: Bartonella bovis Placenta Foetus Transmission Cattle Bacteraemia
a b s t r a c t Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Bartonella spp. are Gram negative, hemotropic bacteria which are emerging vector-borne pathogens in various domestic and wild species (rodents, felids, canids, cervids, bovids), with a worldwide distribution [3,6,8,12,26,30,34]. In cattle, infection with Bartonella bovis is highly prevalent, infecting nearly 80% of a North Carolina beef cattle herd [12] and 60% of a dairy herd in France [26]. As for most Bartonella species, the mode of transmission of B. bovis has not been precisely elucidated [6]. By similarity with the epidemiology of Bartonella henselae [13], Bartonella bacilliformis and Bartonella quintana [9], it is suspected that the
∗ Corresponding author at: INP, ENVT, 23 Chemin des Capelles, 31076 Toulouse Cedex 03, France. Tel.: +33 5 61 19 23 16; fax: +33 5 61 19 38 34. E-mail address: [email protected] (R. Maillard). http://dx.doi.org/10.1016/j.cimid.2014.11.002 0147-9571/© 2014 Elsevier Ltd. All rights reserved.
transmission between reservoirs is ensured horizontally by a blood-suckling arthropod [22]. B. bovis DNA has already been amplified from biting flies (Haematobia sp., Lipoptena cervi), cattle tail louse and ticks [4,10,14,15,18,21,22,33,38]. Domestic felids and canids bites have also been suspected for B. henselae transmission [19]. Some cases of direct transmission of some Bartonella have also been described, horizontal in the human ocular Parinaud’s syndrome [16], as well as vertical, from the mother to foetus in horse and rodents [6,23,25]. The ways of transmission of B. bovis in cattle are still unknown: arthropod transmission is suspected [26], but the phylogenetic vicinity of the genera Bartonella and Brucella [24], the second being a well-known placental pathogen in cattle, leads to address the question of the materno-foetal transmission for B. bovis. The objective of this study was to examine the possibility of contamination of the foeto-placental unit in B. bovis-infected cows and of their offspring.
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2. Materials and methods 2.1. Animals Fifty-six healthy pregnant cows from various herds and breeds were delivered within the last two weeks from term by elective caesarean section (standing left paralumbar appoach) 24 h after an injection of dexamethasone (21isonicotinate salt, Voren® Solution, Boehringer Ingelheim, Reims, France; 0.05 mg/kg intra-muscular) [11,31]. After birth, the calf was housed in its mother’s paddock, 1.5 L of colostrum from the dam was administrated orally by calf-drencher, and then the calf was allowed to suckle ad libitum. 2.2. Cotyledons During caesarean section, after delivery of the calf, one cotyledon close to the uterine opening was excised after ligation of the caruncular vessels. The cotyledon was then brought to the laboratory within 5 min and processed under sterile conditions. Two cubes (1 cm × 1 cm) were cut in the zone of materno-foetal contact. One was fixed into 10% phosphate-buffered formalin for histology and one was frozen for immuno-histochemistry in Tissue Tek® III VIP medium (Sakura Finetek, Villeneuve d’Asq, France). Foetal chorion was then separated and frozen at −20 ◦ C for bacterial culture and PCR. 2.3. Blood collection Blood was collected on EDTA (4 mL; 18-gauge needle, vacuum tubes) from the coccygeal vein of each cow before the surgery and frozen (−20 ◦ C) until culture. Blood was also collected from all newborns immediately after delivery and before colostrum intake: two samples (4 mL) were obtained by puncture at the jugular vein, one on EDTA for Bartonella culture and the second on dry tube for serology. Eight (14.3%) calves were selected at random and collected at one week of age. 2.4. Culture and identification of B. bovis After blood centrifugation, pellets were cultured on brain heart infusion agar (Difco, Detroit, MI, USA) containing 5% defibrinated rabbit blood and incubated at 35 ◦ C in a 5% CO2 atmosphere. The plates were kept for six weeks and were examined daily for the first three weeks and then once a week. The number of CFU (Colony Forming Unit) per mL was registered as an evaluation of blood bacterial load [26]. Bacterial identification was performed on cultivated colonies by genus-specific PCR and species-specific PCR for B. bovis identification–amplification of citrate synthase and ITS portions [29,32]. 2.5. Serology Serological testing was performed by ELISA, as previously described [26]. Briefly, plates (Dynatech® Immulon
1B, Dynex Technologies Inc., Chantilly, VA, USA) were sensitized with 300 ng per cup of B. bovis antigen (Type strain 91-4, CIP # 106692T) [27]. Serology was performed at birth before colostral intake on all calves, and at day seven on eight calves selected at random from bacteremic dams. 2.6. PCR on cotyledons After DNA extraction (DNeasy® Blood and Tissue kit, Qiagen, Courtaboeuf, France), a fragment of the citrate synthase gene (gltA) was amplified, as previously described [32]. In order to detect Taq Polymerase inhibitors, two PCR were realized for each extract: one with Bartonella birtlesii DNA added to the extracts (inhibitor detection control) and one without additional B. birtlesii DNA (Test). 2.7. Histology Cotyledons from the 56 cows, fixed in 10% phosphatebuffered formalin, were dehydrated and embedded in paraffin wax at 56 ◦ C, sectioned at 5 m and stained with haematoxylin–eosin-safran (HES). Analysis for histopathologic changes (especially signs of inflammation and/or placental modifications) was performed in a blind manner, without knowing the infectious status of the cows. 2.8. Immunohistochemistry Polyclonal antibodies were obtained from two SPF rabbits immunized with two subcutaneous injections two weeks apart of a bacterial suspension (two loops of one week old B. bovis culture in 2 mL of sterile NaCl 0.9%) added with 2 mL of adjuvant (complete Freund adjuvant at first injection, incomplete Freund adjuvant at the second). Sera were collected one week before and three weeks after the first injection of antigen. Immunization was performed against B. bovis RFLP-type 1 (one rabbit) and against B. bovis RFLP-type 3 (one rabbit). Specificity of antibodies was checked by Western blot-SDS-PAGE (Fig. 1). Frozen sections (7 m) from 7 bacteremic cows (2cows >2500 CFU/mL, 2 cows with 1 CFU/mL and one cow of each group between 1 and 2500 CFU/mL) and from one non bacteremic cow (negative control) were used for immunohistochemistry. After rehydration in PBS (Sigma, St Quentin Fallavier, France), sections were incubated 20 min in a blocking solution (10% pig serum in PBS, Interchim, Montluc¸on, France). Slides were then incubated overnight at 4 ◦ C with the primary antibody (total serum obtained from immunized rabbit) diluted at 1/1000 in PBS. Immunostaining was revealed by streptavidin–biotin according to the recommendations of the manufacturer (LSAB kit, DAKO S.A., Trappes, France). Briefly, after two rinsings in PBS (5 min each), sections were incubated with a biotinylated anti-rabbit secondary antibody for 20 min at room temperature (dilution 1/2 in PBS), then, with 3% H2 O2 during 5 min and with peroxidase-conjugated streptavidin for 20 min. After two extra washings in PBS, sections were incubated with the streptavidine horseradish peroxydase solution diluted ½ in PBS. After 3 washings in PBS, AEC solution (AminoEthylCarbazole) was used as a chromogen to reveal presence of peroxidase. Sections were
S. Chastant-Maillard et al. / Comparative Immunology, Microbiology and Infectious Diseases 38 (2015) 41–46
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Fig. 1. Western blot with (A) non immune serum (B) immune sera against Bartonella bovis (RFLP 1) and (C) immune sera against Bartonella chomelii. Lane 1: antigen Bartonella bovis (RFLP type 1). Lane 2: antigen Bartonella bovis (RFLP type 2). Lane 3: antigen Bartonella chomelii (RFLP type 3)
counterstained with Harris haematoxylin (Sigma) during 1 min and mounted with a water-soluble medium (Immumount; Thermo scientific, Courtabeuf, France). Slides were observed under a light microscope (BX50, Olympus, Rungis, France).
3.3. Cotyledons
B. bovis was isolated from the blood of 29 (52%) cows, as confirmed by PCR. In bacteremic cows, the number of CFU varied from one to more than 2500 CFU/mL of blood. Four (14%) of the 29 bacteremic cows yielded a high bacterial load (>2500 CFU/mL). Similarly, four cows had a low bacteremia ( 2500 CFU/mL; Fig. 3) and whatever the immune serum used (anti B. bovis RFLP-1 or -3). By histopathological examination, no abnormal image could be observed at histology. No sign of placentitis was observed on the cotyledons taken from bacteremic cows, neither on the foetal nor on the maternal side of the placenta. No morphological difference distinguished the cotyledons issued from the bacteremic cows from the ones issued from the non bacteremic cows. Both maternal and foetal sides of all the cotyledons were classified as normal.
3.2. Calves
4. Discussion
At birth, before colostral intake, none of the 56 calves was bacteremic or seropositive, whatever the dam status (bacteremic or not bacteremic). Among the 29 calves issued from infected cows, eight calves were sampled again at random at seven days of age and none were bacteremic but they all were seropositive.
The dense vascularization intrinsic to placenta makes this organ a good candidate site for the concentration and multiplication of Bartonella, bacteria known for their tropism for erythrocytes and vascular endotheliums [17,35]. Nevertheless, occurrence of in utero infection of the foetus may depend on the histological organization of
3. Results All results are summarized in Table 1. 3.1. Cows
12
Number of cows
10 8 6 4 2 0 1-10
11-100
101-500
501-1000
CFU/ml Fig. 2. Number of Bartonella spp. in the blood of the 29 bacteremic cows.
>1000
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Table 1 Results for 56 dams (blood culture), their offsprings (serology, blood culture), and cotyledons (PCR/immunohistochemistry). Cows No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
Cows
Calves
Cotyledons
Bacteraemia (Range of CFU/mL)
Serology Day 0
Serology Day 8
Culture
PCR/H/IHC
1–10 11–100 101–500 1–10 101–500 11–100 11–100 1–10 1–10 1–10 501–1000 >2500 1–10 501–1000 1–10 11–100 501–1000 11–100 >2500 1–10 >2500 11–100 >2500 501–1000 1–10 11–100 1–10 1–10 11–100 N N N N N N N N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
P NT NT NT NT P NT NT NT NT NT P NT NT P NT P NT P NT NT NT NT P NT NT P NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
N/N/N N/N/NT N/N/N N/N/NT N/N/NT N/N/NT N/N/N N/N/NT N/N/NT N/N/NT N/N/NT N/N/N N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/N N/N/NT N/N/NT N/N/N N/N/N N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT
NT: non tested; N: negative; P: positive; H: histology; IHC: immunohistochemistry.
the placenta, variable between species: maternal circulation is closer from the foetal one in mouse (hemochorial placentation) than it is in cats (endotheliochorial) and in the bovine (epitheliochorial) and may also vary with the infecting Bartonella species. In cows, six cellular layers separate maternal and foetal circulation compare to only one in mice [2].
This study confirms the high prevalence of B. bovis blood circulation in pregnant cows, as 52% of the tested animals of various breeds and origins were bacteremic at the end of pregnancy. It is quite similar to the 68% detected in pregnant Holstein cows at different stages of pregnancy within one dairy herd [27] or similar to the 59% detected in dairy cattle in Israel [37]. No other species of Bartonella was
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Fig. 3. Immunohistochemistry on foetal side of one cotyledon.
identified in our population [29,36], which is similar to the study of Cherry et al. [12], reporting that 115 of 117 Bartonella isolates in beef cattle were confirmed to be B. bovis. As in previous epidemiological observations [27,28], blood bacterial load was highly variable among individuals. Not only Bartonella infection seems frequent in pregnant cows, but blood bacterial loads dramatically increase during the last third of pregnancy [26]. Gestation also amplified the bacteremia levels in mice infected with B. birtlesii [5]. Despite an increase load of bacteria in pregnant cows, we were not able to detect in utero transfer of B. bovis to calves. B. bovis was not detected in maternal and foetal parts of placental cotyledons by any of three complementary approaches, i.e. culture (multiplication ability), PCR (DNA) and immunochemistry (protein). Moreover, no indirect sign of the presence of the bacteria in the cotyledons, such as placentitis, was observed. No sign of bacterial evidence was detected, even in cows with high Bartonella bacteremia levels. In naturally infected rodents, Bartonella spp. was identified in placentas of infected mothers. Placental infection was a relatively efficient phenomenon, since it was observed in 10 (30%) of 33 placentas [25]. Histological signs of bacterial infection of placentas, i.e. vasculitis with moderate necrosis, have been observed in mice experimentally infected with B. birtlesii [5] and in an aborted foal infected with B. henselae [23]. These lesions might be responsible for perturbations of the materno-foetal exchanges, responsible for decreased foetal weight and finally, for resorption/foetal death and abortion [5,23]. Our results support that B. bovis does not cross the placental barrier, despite high bacteremia levels in the dams. In other species, vertical transmission to fetuses was demonstrated in rodents experimentally infected with B. birtlesii [5], during natural infection with Bartonella spp. in cottons rats [25] and in a recent equine case [23]. Infected fetuses were either looking healthy [25] or presenting abnormalities, such as delayed growth [5] or even death [5,23]. Recently, infection with B. henselae and/or Bartonella vinsonii subsp. berkhoffii has been suspected to be responsible for a case of newborn death in humans [7]. Infertility (no
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pregnancy or pregnancy obtained after numerous matings or assisted reproduction) has been associated with Bartonella infection in females, including B. henselae in cats and co-infection with B. henselae and B. vinsonii subsp. berkhoffii in humans, probably due to embryonic or foetal resorption [7,20]. However, Bartonella infection was not detected in viable fetuses at the end of pregnancy or newborns from infected mothers, either in mice [5], cats [1,20] or calves (this study). One exception was the detection of seven bacteremic neonates among 19 neonates in naturally infected wild rodents [25]. The absence of in utero transmission of B. bovis in cattle was also supported by the absence of circulating anti-B.bovis antibodies in calves before colostrum intake, indicating the absence of exposition to the pathogen during pregnancy. As no bacteraemia was detected in newborn calves, the hypothesis of immunotolerance for explining the lack of antibodies connot be supported. Maillard et al. [26] in a longitudinal study, without the knowledge of the dam’s status, also observed no B. bovis bacteremia (by PCR) in calves until 8 months of age. Finally, our study also suggests that the transmission of B. bovis by colostrum and milk is of low probability, since none of the five calves allowed to suckle their infected dam were bacteremic after one week. The risk of contamination through colostrum and milk was only evaluated in cats milk/colostrum from B. henselae infected queens [20]. The colostrum and milk were negative by culture and by PCR and no kitten became infected until weaning. 5. Conclusions Contamination of placenta and vertical transmission of calves with B. bovis was not demonstrated and may be an incidental (if any) transmission route for B. bovis in cattle. The post-natal transmission of B. bovis within cattle populations may rely only on a horizontal and likely vectorial mode. However, the implicated vector(s) remain to be identified, even though many arthropods have been suspected. Acknowledgments We thank warmly Professor Bruno Chomel (UC Davis, California, USA) for correcting this manuscript. This work was funded by a grant of French Ministry of Agriculture (DGER). References [1] Abbott RC, Chomel BB, Kasten RW, Floyd-Hawkins KA, Kikuchi Y, Koehler JE, et al. Experimental and natural infection with Bartonella henselae in domestic cats. Comp Immunol Microbiol Infect Dis 1997;20:41–51. [2] Benirschke K. Comparative placentation, Ithaca NY; 2007. http://www.ivis.org (last updated: 01.08.07). [3] Bermond D, Boulouis HJ, Heller R, Van Laere G, Monteil H, Chomel BB, et al. Bartonella bovis Bermond et al. sp. nov. and Bartonella capreoli sp. nov., isolated from European ruminants. Int J Syst Evol Microbiol 2002;52:383–90. [4] Billeter SA, Levy MG, Chomel BB, Breitschwerdt EB. Vector transmission of Bartonella species with emphasis on the potential for tick transmission. Med Vet Entomol 2008;22:1–15.
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Contents lists available at ScienceDirect
Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Lack of transplacental transmission of Bartonella bovis S. Chastant-Maillard a,b,c , H.-J. Boulouis d , K. Reynaud a,b , S. Thoumire a,b , C. Gandoin d , C. Bouillin d , N. Cordonnier e , R. Maillard c,d,∗ a b c d e
INRA, UMR 1198 Biologie du Développement et Reproduction, F-78350 Jouy-en-Josas, France ENVA, UMR 1198 Biologie du Développement et Reproduction (BDR), 7 Avenue du Général de Gaulle, F-94700 Maisons-Alfort, France Université de Toulouse, INP, ENVT, 23 Chemin des Capelles, BP 87614, 31076 Toulouse Cedex 03, France Université ParisEst, INRA, Anses, ENVA, UMR BIPAR, 23 avenue du Général de Gaulle, 94706 Maisons-Alfort, France ENVA, Histology – Histopathology, 7 Avenue du Général de Gaulle, F-94700 Maisons-Alfort, France
a r t i c l e
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Article history: Received 8 October 2013 Received in revised form 7 November 2014 Accepted 21 November 2014 Keywords: Bartonella bovis Placenta Foetus Transmission Cattle Bacteraemia
a b s t r a c t Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Bartonella spp. are Gram negative, hemotropic bacteria which are emerging vector-borne pathogens in various domestic and wild species (rodents, felids, canids, cervids, bovids), with a worldwide distribution [3,6,8,12,26,30,34]. In cattle, infection with Bartonella bovis is highly prevalent, infecting nearly 80% of a North Carolina beef cattle herd [12] and 60% of a dairy herd in France [26]. As for most Bartonella species, the mode of transmission of B. bovis has not been precisely elucidated [6]. By similarity with the epidemiology of Bartonella henselae [13], Bartonella bacilliformis and Bartonella quintana [9], it is suspected that the
∗ Corresponding author at: INP, ENVT, 23 Chemin des Capelles, 31076 Toulouse Cedex 03, France. Tel.: +33 5 61 19 23 16; fax: +33 5 61 19 38 34. E-mail address: [email protected] (R. Maillard). http://dx.doi.org/10.1016/j.cimid.2014.11.002 0147-9571/© 2014 Elsevier Ltd. All rights reserved.
transmission between reservoirs is ensured horizontally by a blood-suckling arthropod [22]. B. bovis DNA has already been amplified from biting flies (Haematobia sp., Lipoptena cervi), cattle tail louse and ticks [4,10,14,15,18,21,22,33,38]. Domestic felids and canids bites have also been suspected for B. henselae transmission [19]. Some cases of direct transmission of some Bartonella have also been described, horizontal in the human ocular Parinaud’s syndrome [16], as well as vertical, from the mother to foetus in horse and rodents [6,23,25]. The ways of transmission of B. bovis in cattle are still unknown: arthropod transmission is suspected [26], but the phylogenetic vicinity of the genera Bartonella and Brucella [24], the second being a well-known placental pathogen in cattle, leads to address the question of the materno-foetal transmission for B. bovis. The objective of this study was to examine the possibility of contamination of the foeto-placental unit in B. bovis-infected cows and of their offspring.
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2. Materials and methods 2.1. Animals Fifty-six healthy pregnant cows from various herds and breeds were delivered within the last two weeks from term by elective caesarean section (standing left paralumbar appoach) 24 h after an injection of dexamethasone (21isonicotinate salt, Voren® Solution, Boehringer Ingelheim, Reims, France; 0.05 mg/kg intra-muscular) [11,31]. After birth, the calf was housed in its mother’s paddock, 1.5 L of colostrum from the dam was administrated orally by calf-drencher, and then the calf was allowed to suckle ad libitum. 2.2. Cotyledons During caesarean section, after delivery of the calf, one cotyledon close to the uterine opening was excised after ligation of the caruncular vessels. The cotyledon was then brought to the laboratory within 5 min and processed under sterile conditions. Two cubes (1 cm × 1 cm) were cut in the zone of materno-foetal contact. One was fixed into 10% phosphate-buffered formalin for histology and one was frozen for immuno-histochemistry in Tissue Tek® III VIP medium (Sakura Finetek, Villeneuve d’Asq, France). Foetal chorion was then separated and frozen at −20 ◦ C for bacterial culture and PCR. 2.3. Blood collection Blood was collected on EDTA (4 mL; 18-gauge needle, vacuum tubes) from the coccygeal vein of each cow before the surgery and frozen (−20 ◦ C) until culture. Blood was also collected from all newborns immediately after delivery and before colostrum intake: two samples (4 mL) were obtained by puncture at the jugular vein, one on EDTA for Bartonella culture and the second on dry tube for serology. Eight (14.3%) calves were selected at random and collected at one week of age. 2.4. Culture and identification of B. bovis After blood centrifugation, pellets were cultured on brain heart infusion agar (Difco, Detroit, MI, USA) containing 5% defibrinated rabbit blood and incubated at 35 ◦ C in a 5% CO2 atmosphere. The plates were kept for six weeks and were examined daily for the first three weeks and then once a week. The number of CFU (Colony Forming Unit) per mL was registered as an evaluation of blood bacterial load [26]. Bacterial identification was performed on cultivated colonies by genus-specific PCR and species-specific PCR for B. bovis identification–amplification of citrate synthase and ITS portions [29,32]. 2.5. Serology Serological testing was performed by ELISA, as previously described [26]. Briefly, plates (Dynatech® Immulon
1B, Dynex Technologies Inc., Chantilly, VA, USA) were sensitized with 300 ng per cup of B. bovis antigen (Type strain 91-4, CIP # 106692T) [27]. Serology was performed at birth before colostral intake on all calves, and at day seven on eight calves selected at random from bacteremic dams. 2.6. PCR on cotyledons After DNA extraction (DNeasy® Blood and Tissue kit, Qiagen, Courtaboeuf, France), a fragment of the citrate synthase gene (gltA) was amplified, as previously described [32]. In order to detect Taq Polymerase inhibitors, two PCR were realized for each extract: one with Bartonella birtlesii DNA added to the extracts (inhibitor detection control) and one without additional B. birtlesii DNA (Test). 2.7. Histology Cotyledons from the 56 cows, fixed in 10% phosphatebuffered formalin, were dehydrated and embedded in paraffin wax at 56 ◦ C, sectioned at 5 m and stained with haematoxylin–eosin-safran (HES). Analysis for histopathologic changes (especially signs of inflammation and/or placental modifications) was performed in a blind manner, without knowing the infectious status of the cows. 2.8. Immunohistochemistry Polyclonal antibodies were obtained from two SPF rabbits immunized with two subcutaneous injections two weeks apart of a bacterial suspension (two loops of one week old B. bovis culture in 2 mL of sterile NaCl 0.9%) added with 2 mL of adjuvant (complete Freund adjuvant at first injection, incomplete Freund adjuvant at the second). Sera were collected one week before and three weeks after the first injection of antigen. Immunization was performed against B. bovis RFLP-type 1 (one rabbit) and against B. bovis RFLP-type 3 (one rabbit). Specificity of antibodies was checked by Western blot-SDS-PAGE (Fig. 1). Frozen sections (7 m) from 7 bacteremic cows (2cows >2500 CFU/mL, 2 cows with 1 CFU/mL and one cow of each group between 1 and 2500 CFU/mL) and from one non bacteremic cow (negative control) were used for immunohistochemistry. After rehydration in PBS (Sigma, St Quentin Fallavier, France), sections were incubated 20 min in a blocking solution (10% pig serum in PBS, Interchim, Montluc¸on, France). Slides were then incubated overnight at 4 ◦ C with the primary antibody (total serum obtained from immunized rabbit) diluted at 1/1000 in PBS. Immunostaining was revealed by streptavidin–biotin according to the recommendations of the manufacturer (LSAB kit, DAKO S.A., Trappes, France). Briefly, after two rinsings in PBS (5 min each), sections were incubated with a biotinylated anti-rabbit secondary antibody for 20 min at room temperature (dilution 1/2 in PBS), then, with 3% H2 O2 during 5 min and with peroxidase-conjugated streptavidin for 20 min. After two extra washings in PBS, sections were incubated with the streptavidine horseradish peroxydase solution diluted ½ in PBS. After 3 washings in PBS, AEC solution (AminoEthylCarbazole) was used as a chromogen to reveal presence of peroxidase. Sections were
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Fig. 1. Western blot with (A) non immune serum (B) immune sera against Bartonella bovis (RFLP 1) and (C) immune sera against Bartonella chomelii. Lane 1: antigen Bartonella bovis (RFLP type 1). Lane 2: antigen Bartonella bovis (RFLP type 2). Lane 3: antigen Bartonella chomelii (RFLP type 3)
counterstained with Harris haematoxylin (Sigma) during 1 min and mounted with a water-soluble medium (Immumount; Thermo scientific, Courtabeuf, France). Slides were observed under a light microscope (BX50, Olympus, Rungis, France).
3.3. Cotyledons
B. bovis was isolated from the blood of 29 (52%) cows, as confirmed by PCR. In bacteremic cows, the number of CFU varied from one to more than 2500 CFU/mL of blood. Four (14%) of the 29 bacteremic cows yielded a high bacterial load (>2500 CFU/mL). Similarly, four cows had a low bacteremia ( 2500 CFU/mL; Fig. 3) and whatever the immune serum used (anti B. bovis RFLP-1 or -3). By histopathological examination, no abnormal image could be observed at histology. No sign of placentitis was observed on the cotyledons taken from bacteremic cows, neither on the foetal nor on the maternal side of the placenta. No morphological difference distinguished the cotyledons issued from the bacteremic cows from the ones issued from the non bacteremic cows. Both maternal and foetal sides of all the cotyledons were classified as normal.
3.2. Calves
4. Discussion
At birth, before colostral intake, none of the 56 calves was bacteremic or seropositive, whatever the dam status (bacteremic or not bacteremic). Among the 29 calves issued from infected cows, eight calves were sampled again at random at seven days of age and none were bacteremic but they all were seropositive.
The dense vascularization intrinsic to placenta makes this organ a good candidate site for the concentration and multiplication of Bartonella, bacteria known for their tropism for erythrocytes and vascular endotheliums [17,35]. Nevertheless, occurrence of in utero infection of the foetus may depend on the histological organization of
3. Results All results are summarized in Table 1. 3.1. Cows
12
Number of cows
10 8 6 4 2 0 1-10
11-100
101-500
501-1000
CFU/ml Fig. 2. Number of Bartonella spp. in the blood of the 29 bacteremic cows.
>1000
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Table 1 Results for 56 dams (blood culture), their offsprings (serology, blood culture), and cotyledons (PCR/immunohistochemistry). Cows No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
Cows
Calves
Cotyledons
Bacteraemia (Range of CFU/mL)
Serology Day 0
Serology Day 8
Culture
PCR/H/IHC
1–10 11–100 101–500 1–10 101–500 11–100 11–100 1–10 1–10 1–10 501–1000 >2500 1–10 501–1000 1–10 11–100 501–1000 11–100 >2500 1–10 >2500 11–100 >2500 501–1000 1–10 11–100 1–10 1–10 11–100 N N N N N N N N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
P NT NT NT NT P NT NT NT NT NT P NT NT P NT P NT P NT NT NT NT P NT NT P NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
N/N/N N/N/NT N/N/N N/N/NT N/N/NT N/N/NT N/N/N N/N/NT N/N/NT N/N/NT N/N/NT N/N/N N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/N N/N/NT N/N/NT N/N/N N/N/N N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT N/N/NT
NT: non tested; N: negative; P: positive; H: histology; IHC: immunohistochemistry.
the placenta, variable between species: maternal circulation is closer from the foetal one in mouse (hemochorial placentation) than it is in cats (endotheliochorial) and in the bovine (epitheliochorial) and may also vary with the infecting Bartonella species. In cows, six cellular layers separate maternal and foetal circulation compare to only one in mice [2].
This study confirms the high prevalence of B. bovis blood circulation in pregnant cows, as 52% of the tested animals of various breeds and origins were bacteremic at the end of pregnancy. It is quite similar to the 68% detected in pregnant Holstein cows at different stages of pregnancy within one dairy herd [27] or similar to the 59% detected in dairy cattle in Israel [37]. No other species of Bartonella was
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Fig. 3. Immunohistochemistry on foetal side of one cotyledon.
identified in our population [29,36], which is similar to the study of Cherry et al. [12], reporting that 115 of 117 Bartonella isolates in beef cattle were confirmed to be B. bovis. As in previous epidemiological observations [27,28], blood bacterial load was highly variable among individuals. Not only Bartonella infection seems frequent in pregnant cows, but blood bacterial loads dramatically increase during the last third of pregnancy [26]. Gestation also amplified the bacteremia levels in mice infected with B. birtlesii [5]. Despite an increase load of bacteria in pregnant cows, we were not able to detect in utero transfer of B. bovis to calves. B. bovis was not detected in maternal and foetal parts of placental cotyledons by any of three complementary approaches, i.e. culture (multiplication ability), PCR (DNA) and immunochemistry (protein). Moreover, no indirect sign of the presence of the bacteria in the cotyledons, such as placentitis, was observed. No sign of bacterial evidence was detected, even in cows with high Bartonella bacteremia levels. In naturally infected rodents, Bartonella spp. was identified in placentas of infected mothers. Placental infection was a relatively efficient phenomenon, since it was observed in 10 (30%) of 33 placentas [25]. Histological signs of bacterial infection of placentas, i.e. vasculitis with moderate necrosis, have been observed in mice experimentally infected with B. birtlesii [5] and in an aborted foal infected with B. henselae [23]. These lesions might be responsible for perturbations of the materno-foetal exchanges, responsible for decreased foetal weight and finally, for resorption/foetal death and abortion [5,23]. Our results support that B. bovis does not cross the placental barrier, despite high bacteremia levels in the dams. In other species, vertical transmission to fetuses was demonstrated in rodents experimentally infected with B. birtlesii [5], during natural infection with Bartonella spp. in cottons rats [25] and in a recent equine case [23]. Infected fetuses were either looking healthy [25] or presenting abnormalities, such as delayed growth [5] or even death [5,23]. Recently, infection with B. henselae and/or Bartonella vinsonii subsp. berkhoffii has been suspected to be responsible for a case of newborn death in humans [7]. Infertility (no
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pregnancy or pregnancy obtained after numerous matings or assisted reproduction) has been associated with Bartonella infection in females, including B. henselae in cats and co-infection with B. henselae and B. vinsonii subsp. berkhoffii in humans, probably due to embryonic or foetal resorption [7,20]. However, Bartonella infection was not detected in viable fetuses at the end of pregnancy or newborns from infected mothers, either in mice [5], cats [1,20] or calves (this study). One exception was the detection of seven bacteremic neonates among 19 neonates in naturally infected wild rodents [25]. The absence of in utero transmission of B. bovis in cattle was also supported by the absence of circulating anti-B.bovis antibodies in calves before colostrum intake, indicating the absence of exposition to the pathogen during pregnancy. As no bacteraemia was detected in newborn calves, the hypothesis of immunotolerance for explining the lack of antibodies connot be supported. Maillard et al. [26] in a longitudinal study, without the knowledge of the dam’s status, also observed no B. bovis bacteremia (by PCR) in calves until 8 months of age. Finally, our study also suggests that the transmission of B. bovis by colostrum and milk is of low probability, since none of the five calves allowed to suckle their infected dam were bacteremic after one week. The risk of contamination through colostrum and milk was only evaluated in cats milk/colostrum from B. henselae infected queens [20]. The colostrum and milk were negative by culture and by PCR and no kitten became infected until weaning. 5. Conclusions Contamination of placenta and vertical transmission of calves with B. bovis was not demonstrated and may be an incidental (if any) transmission route for B. bovis in cattle. The post-natal transmission of B. bovis within cattle populations may rely only on a horizontal and likely vectorial mode. However, the implicated vector(s) remain to be identified, even though many arthropods have been suspected. Acknowledgments We thank warmly Professor Bruno Chomel (UC Davis, California, USA) for correcting this manuscript. This work was funded by a grant of French Ministry of Agriculture (DGER). References [1] Abbott RC, Chomel BB, Kasten RW, Floyd-Hawkins KA, Kikuchi Y, Koehler JE, et al. Experimental and natural infection with Bartonella henselae in domestic cats. Comp Immunol Microbiol Infect Dis 1997;20:41–51. [2] Benirschke K. Comparative placentation, Ithaca NY; 2007. http://www.ivis.org (last updated: 01.08.07). [3] Bermond D, Boulouis HJ, Heller R, Van Laere G, Monteil H, Chomel BB, et al. Bartonella bovis Bermond et al. sp. nov. and Bartonella capreoli sp. nov., isolated from European ruminants. Int J Syst Evol Microbiol 2002;52:383–90. [4] Billeter SA, Levy MG, Chomel BB, Breitschwerdt EB. Vector transmission of Bartonella species with emphasis on the potential for tick transmission. Med Vet Entomol 2008;22:1–15.
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