Isolation of Arcobacter species and other neglected opportunistic agents from aborted bovine and caprine fetuses

Background Infectious abortion in ruminants is a problem in animal husbandry worldwide. It is important to obtain a diagnosis, to make sure that proper control measures can be instituted, but most abortion cases remain without an etiologic diagnosis. This report describes the presence of Arcobacter species and several neglected opportunistic abortifacient agents in ruminant abortion cases showing or not co-infections among at least one of the major recognized protozoal, fungal, bacterial and viral abortifacient agents. Results A total of 67 fetuses (55 cattle and 12 goats) and just one placenta (cattle) were considered. Among the most common abortive agents, Neospora caninum (19,4%), followed by Chlamydophila abortus (4,5%), Listeria monocytogenes 1/2a (2,98%), Bovine Viral Diarrhea Virus type 1b (2,98%), Bovine herpesvirus 4 (2,98%), and Aspergillus spp. (2,98%) were detected. The isolated neglected opportunistic bacteria include Escherichia coli, Acinetobacter lwoffii, Staphylococcus spp., Streptococcus spp., Streptococcus uberis, Streptococcus suis, Trueperella pyogenes, Mannheimia haemolytica, Bacillus cereus and Nocardia spp. Other bacterial species, not associated with abortion by literature, but described as causes of diseases occurring sporadically both in humans and animals, were also detected. Three Arcobacter strains, namely two A. skirrowii and one A. cryaerophilus, were isolated from 3 bovine aborted fetuses, and A. butzleri was isolated from the placenta. Conclusions A not negligible isolation of Arcobacter species and other neglected abortifacient agents has to be mentioned, with prevalences that seem to be emerging and replacing or co-placing the major infectious players in bovine and caprine reproductive failure due to abortion disease, even if further studies investigating the aetiological power and transmission routes are needed in order to define the role of these microrganisms in ruminant abortion.


Background
Infectious abortion in ruminants is a problem in animal husbandry worldwide, and abortion in cattle, with an estimated rate from 2 to 5%, is widely acknowledged to cause considerable economic losses and, despite extensive histopathologic, microbiologic, and molecular investigations, most bovine abortion cases remain without an etiologic diagnosis [1]. The major infectious agents involved in ruminant abortion are Neospora caninum, Bovine pestiviruses, Bovine herpesvirus 1 and 4, Parainfluenza virus type 3, Brucella spp., Chlamydophila abortus, Coxiella burnetii, Campylobacter fetus, Salmonella sp., Listeria spp., and Aspergillus spp.. However, most bacterial causes of abortion are opportunistic pathogens. These organisms are not infectious, and are common inhabitants of the host or its environment and might include Arcanobacterium pyogenes and Bacillus spp. followed by Escherichia coli, Histophilus somni, Pasteurella spp., Staphylococcus spp., Streptococcus spp., and any other bacteria that can find their way into the bloodstream can cause sporadic abortion [2]. Moreover Arcobacter species have been reported to be involved in animals abortion [3,4].
The first report of bacteria now known as arcobacters was originally described by Ellis et al. (1977) [5] as an isolation of "Spirillum/Vibrio-like organisms" from placenta and internal organs of bovine fetuses; these microorganisms were initially assigned to the genus Vibrio, but, considering their failure in fermenting carbohydrates, they were subsequently transferred into the genus Campylobacter as an atypical and heterogeneous group of aerotolerant campylobacters [6]. In 1991, the genus Arcobacter was created by Vandamme and Colleagues to accommodate these two atypical campylobacters, Campylobacter nitrofigilis [7] and Campylobacter cryaerophila [6] and, in 1992, the genus was enlarged with the reclassification of Campylobacter butzleri, isolated from human stools but also from animals [8], as Arcobacter butzleri, and with the inclusion of a new species, Arcobacter skirrowii, isolated predominantly from preputial fluids of bulls or from bovine, porcine, and ovine aborted fetuses, as well as from diarrhoeic feces [9]. Hitherto, a total of 27 Arcobacter species have been described in a wide variety of ecological niches and from various hosts and environments, mainly described from water-related environments and shellfish [10]. Arcobacters are considered emerging bacterial zoonotic agents: A. butzleri, A. cryaerophilus and A. skirrowii are the species most commonly described in clinical conditions, namely associated with human and animal disease. Furthermore, as the Arcobacter organisms are commonly isolated from fecal samples of healthy animals and humans, and they are unable to satisfy the Koch's postulate, their pathology and pathogenicity are under dispute [11]. In animals, arcobacters may play a role in animal reproductive disorders, such as infertility and late-term abortions in cattle, pigs, sheep [12], equine and alpaca [13]. In literature, the species involved in abortions are A. cryaerophilus, A. skirrowii, A. butzleri, A. thereius [3] and A. porcinus [4]. However, since the first isolation, arcobacters were reported in bovine abortions only in three cases [14][15][16] and, differently from pigs, no studies in literature have clearly identified the role of the genus Arcobacter as abortifacient pathogen and as causative agent of disease in cattle.
This report is to describe the isolation of Arcobacter species, long time since the last description in literature, and other neglected opportunistic abortifacient agents from the abomasum's content of aborted fetuses both of cattle and small ruminants coming from a passive Bovine Brucellosis (BB) Surveillance National plan performed in Piedmont, an officially BB-free Italian region. The aim of this study is presenting important and inspiring data on the isolation of Arcobacter species and other bacterial agents that seem to be emerging and replacing or co-placing the major infectious players in bovine reproductive failure due to abortion disease.

Results
A total of 67 fetuses (n = 55 cattle and n = 12 goats) and just one placenta (cattle) were collected and analyzed. The absence of gross lesions at necropsy was reported in almost all (n = 64) the fetuses. The findings of the diagnostic approach showed that: i) 25 fetuses (37,3%) were negative for any protozoal, fungal, bacterial and viral agents investigated; ii) 22 fetuses (32,8%), 19 bovine and 3 caprine, were positive only to routine bacteriological exam on blood agar plates (Table 1); iii) 10 fetuses (14, 9%), 8 bovine and 2 caprine, showed a single positivity for only one among the investigated pathogens, namely N. caninum (n = 6), C. abortus (n = 2), L. monocytogenes 1/2a (n = 1) and Bovine Viral Diarrhea Virus (BVDV) type 1b (n = 1); iv) 10 fetuses (14,9%) showed different coinfections among recognized protozoal, fungal, bacterial and viral abortifacient agents, and, three Arcobacter strains, two A. skirrowii and one A. cryaerophilus, were isolated from 3 among these 10 bovine aborted fetuses (Table 2). Moreover, a strain of A. butzleri was isolated from the placenta, in purity. Among the overall investigated Arcobacter virulence genes, only ciaB and mviN were detected and were present in all the isolates.

Discussion
Establishing a definitive cause of bovine abortion is a challenging problem faced by veterinary practitioners and diagnosticians [17] and the knowledge of the most common causes of bovine abortion is useful to set up interventions able to mitigate future fetal loss in the herd. A not negligible percentage of fetuses with negative results for infectious abortion agents and the almost overall absence of anatomopathological lesions have to be mentioned in this report, in line with the literature, and highlighting, once again, as these scenarios can interfere with the recovery of causative agents.
Among the 42 fetuses in which an infectious agent was identified, bacterial agents, followed by viral, protozoal and fungal agents were identified. The most common abortive agent detected resulted N. caninum (n = 13, 19,4%), followed by C. abortus (n = 3, 4,5%), L. monocytogenes 1/2a (n = 2, 2,98%), BVDV type 1b (n = 2, 2,98%), Bovine herpesvirus 4 (n = 2, 2,98%), and Aspergillus spp. (n = 2, 2,98%). However, several abortion cases with multiple possible etiologies were detected, both in the 10 cases with co-infections among the well-recognized abortifacient agents (Table 2) as well as in the 22 cases with positive bacterial cultures on blood agar plates that are not primary abortifacient agents (Table  1). In fact, whereas Neospora spp., C. abortus, L. monocytogenes, BVDV and BoHV4 are recognized as abortifacient agents, the other isolated bacterial species are usually considered pathogenic or opportunistic or commensal or environmental bacteria. Recent studies  indicate that uterus is not sterile during pregnancy and potentially pathogenic bacteria can be present in the placenta of term pregnancies without causing abortion or inflammation [18], and therefore, the bacterial species not described as primary abortifacient agents could play a role as opportunistic pathogens, components of the uterine microbiota, or contaminants of the post-abortive phase. In our study, several abortion cases contained different bacterial species, recovered from the routine bacteriological exam on blood agar plates, that could be classified as possible abortive bacterial agents, given they were previously reported by literature as opportunistic abortive bacteria, namely Escherichia coli (n = 9), Acinetobacter lwoffii In fact, it is often difficult to assess the presence of potentially infectious bacteria in cultures, particularly in cases where rare opportunistic agents are identified, as they tend to be overlooked as contaminants, but, similarly, complementary pathological analyses are needed to avoid overinterpretation of mere detection of the opportunistic pathogens [22]. Regarding the association of Arcobacter spp. with infertility and abortion in livestock animals, it has been reported worldwide but its role in abortions was investigated predominantly in pigs, in which the transmission of Arcobacter species was demonstrated from carrying sows to their offspring, both vertically and horizontally [37]. On and Colleagues in 2002 [38], reported, in one hand, a high Arcobacter prevalence in Danish pig abortions, in cases in which no established abortifacient agent is detected, but in the other, they recommend that other studies are needed to define the role of each Arcobacter species, especially where co-infections with other agents are present, suggesting that some Arcobacter strains play a primary role in abortion while others may be opportunistic pathogens colonizing the fetus.
In our report, Arcobacter species resulted the most isolated bacteria in bovine fetuses among the 10 cases with co-infections, with N. caninum in two cases and BVD type 1b in one other case; in addition, in two of these cases A. skirrowii and A. cryaerophilus resulted the only bacterial species isolated. To note that Arcobacter species were never isolated from blood agar plates performed during routine microbiological exam with incubation temperature of 37°C; in case of other microbial contamination, Arcobacter species are masked by the other populations that overgrow on plates, or the higher incubation temperature could hinder the Arcobacter growth, and, therefore, a selective bacteriological approach should be the better choice in order to evaluate the presence of the genus Arcobacter. The true incidence and data on Arcobacter species on animal abortion are presumed to be underreported and probably greatly underestimated.
This report revived the presence of Arcobacter spp. in bovine abortion and described as A. cryaerophilus and A. skirrowii could have had an active but not exclusive role in the observed fetal deaths and, consequently, in the observed abortion cases. Differently from what observed in sows by On et al. (2002) [38], the isolation of A. cryaerophilus and A. skirrowii was reported in all the described cases in addition to other organisms frequently associated with bovine abortion (N. caninum and BVDV), and therefore, authors supposed an opportunistic role, if there was, or at least superinfection of Arcobacter spp.. A synergic effect could have occurred on the pathogenesis of the abortion in which several determinants have been explicated their pathogenicity: on the basis of the recovery of A. cryaerophilus and A. skirrowii in the abomasum's content, it is possible to hypothesize an exceeding of the placental barrier, thanks to the injurious or immunosuppressive state induced by other pathogens (N. caninum and BVDV), otherwise, since Arcobacter species have been isolated from several environments and hosts [39,40], an ascending infection by vaginal pathway or a post-fetal ascendant invasion by Arcobacter spp. could also be hypothesized. The isolation of A. butzleri in the only one placenta submitted to the laboratory immediately after its expulsion, has to be described, even if no specific conclusions could be extrapolated. Collection and submission of the placenta should be recommended, if it can be sampled, because it is equally important and could contribute with the entire fetus to abortion diagnosis, but a whole, intact placenta is often rarely received [2], as confirmed by our report.
In our study, no carcasses showed gross lesions, suggesting an indirect action of pathogens on the fetus by impairment of the maternal-fetal circulation (N. caninum) or the mother's immune system failure (BVDV) [2]. The breakable conservation of abortion products leads often to receive specimens in poor condition not suitable for histopathology purposes, and, consequently, a lack of data about the presence and the type of microscopic lesions could limit the definition of the role of each determinant involved in abortion cases.

Conclusions
Although N. caninum resulted the most common agent of bovine abortions, demonstrating a consistent presence of the organism in cattle abortion cases and, therefore confirming in line with literature that it is still a cattle problem, a not negligible isolation of Arcobacter species has to be mentioned, with prevalence in line with the major abortifacient agents, as well as the isolation of other opportunistic, commensal or environmental bacteria, even if with lower occurrence levels. The frequent isolation of these bacteria should be taken into account in order to assess if the once-major abortion players could be or are being replaced by an increase in these opportunistic pathogens that are identified as possible abortive agents but that remain in the dark zone of knowledge and that seem to be emerging and needing immediate attention. Further studies are needed in order to both identify these emerging opportunistic bacteria, whose incidence in ruminant abortion is likely underestimated, and investigate the transmission routes and the specific role of Arcobacter in bovine abortion.

Methods
Aborted fetuses coming from a passive BB Surveillance plan in the Piedmont region, in Italy, were included in this report. All abortion products, collected from bovine and caprine herds between October 2017 to July 2018 in the area covered by the Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d' Aosta (IZSPLV), that includes three Italian regions (Piedmont, Liguria, and the Aosta Valley) of the Northwest Italy, were received at IZSPLV laboratory. Samples and analyses included in the present study were performed in accordance with current Regulation concerning of Maintenance of Bovine Brucellosis free Health Status (Directive 97/12/CE, adopted by Italian Law 196/199). Firstly, the carcasses were submitted to necropsy and then followed the application of the routine diagnostic protocol for the detection of the major infectious agents of abortion, namely Salmonella sp., Listeria spp., Brucella spp., C. fetus, C. abortus, C. burnetii, N. caninum, Bovine pestiviruses, Bovine herpesvirus 1 and 4, Parainfluenza virus type 3, fungi and yeasts. See Table 3 for more details and references [41][42][43][44][45][46][47]. In addition, a routine bacteriological examination on blood agar plates (Columbia agar, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) incubated at 37°C was performed for each fetus sample considered in the study from the abomasum's content. This protocol has been extended with a selective bacteriological exam for the isolation of Arcobacter spp. [48]. In detail, 1 ml of this specimen was enriched in Preston broth (Microbiol & C. snc, Cagliari, Italy) under Carbon Dioxide-enriched atmosphere condition at 30°C; every 24 h for the following 3 days, broth culture were subcultured, under the same conditions, on Charcoal Cefoperazone Deoxycholate Agar (CCDA; Oxoid, Milan, Italy) added with CAT supplement, cefoperazone (8 mg l-1), amphotericin (10 mg l-1), teicoplanin (4 mg l-1) (Oxoid, Milan, Italy). CAT-CCDA plates were observed for 5 days and on suspected colonies Gram staining was performed. According to the protocols described by Douidah (2010Douidah ( , 2012 [49,50], the strains identified morphologically as Arcobacter spp. were processed with two end-point multiplex PCR in order to identify the species and to detect