Citrobacter koseri related abortion and fetal septicemia in cattle

Abstract

A 31-month-old Holstein dairy cow aborted at 224 days of gestation with ejection of cheese-like lochia. Citrobacter koseri, which commonly exists in the normal flora of human and animal digestive tracts, was isolated from aborted fetal tissues (liver, spleen, kidney, heart, lung, cerebrum, and skeletal muscle) and fetal membranes. Histopathological examination revealed suppurative fibrinous meningoencephalitis of the cerebrum, cerebellum, and brainstem; suppurative bronchopneumonia; suppurative chorioamnionitis; and fibrous splenic serositis. Numerous gram-negative bacilli were detected in the cytoplasm of macrophages and/or neutrophils in these lesions. Bacteriological investigation and immunohistochemical staining identified the bacilli as C. koseri. This is the first report of cattle abortion caused by C. koseri infection in dairy cattle.

Citrobacter koseri (formerly known as Citrobacter diversus) is a gram-negative bacillus belonging to the family Enterobacteriaceae, which is common in the flora of digestive tracts of healthy humans and animals [3, 6, 8, 16, 18, 20]. Among the 11 Citrobacter spp., C. koseri, C. freundii, and C. amalonaticus are believed to be harmful to humans, although they seldom cause clinical diseases in adult humans [1, 4, 11, 19]. However, it was reported that C. koseri occasionally causes severe septicemia and meningitis in neonatal infants and in immunosuppressed adults [7]. Cerebral abscesses were also detected in 77% of the patients [7]. The infection may cause sudden death (30%) and serious neurological disorders (50%) in affected patients [5, 7, 13, 18, 22]. Horizontal transmission from the hands of the hospital staff is frequently observed in the neonatal wards of medical hospitals, and vertical transmission with mothers transmitting bacteria to their children in utero or at birth has been reported [7, 9, 10, 21, 23]. Vertical transmission cause prematurity (gestation of <37 weeks), sepsis, and meningitis commonly; however, abortion has not been reported so far [7, 9, 10, 21, 23].

In contrast to human cases, there are only a few reports of animal cases. In one report of C. koseri infection in two 12-week-old boxer siblings with severe myocarditis [3], bacteria were isolated from the heart of one puppy. Histological examination revealed rod-shaped gram-negative bacilli in the cytoplasm of macrophages from myocarditis lesions of both puppies [3]. In their report, the authors attributed the clinical consequences to the immunological incompetence of the animals due to immaturity, vaccine-related immunosuppression, adventitious parvovirus infection, or hereditary characteristics [3].

In livestock animals, to our knowledge, only one published report described septicemia and meninges in C. koseri infected 4-day-old Holstein cattle [16]. In that report, C. koseri was isolated from the meninges, ocular fluid, synovial fluid, spleen, and small intestine [16]. Suppurative and histiocytic meningitis with rod-shaped bacilli, endophthalmitis, suppurative splenitis, and multiple renal micro-abscesses were confirmed. The authors suggested that insufficient transfer of maternal antibodies due to delayed colostrum feeding might account for the development of such severe manifestations [16].

In this report, we describe a case of abortion in a dairy cow in Japan at 224 days of gestation after artificial insemination. We conducted a pathological investigation of the ejected body and fetal membranes and attempted to isolate the causal agents from these samples. For the immunohistochemical examination, we prepared mouse antisera using a taxonomically defined isolate of the species and detected specific antigens in the suppurative lesion areas of the collected specimens. To our knowledge, this is the first case report to indicate the ability of C. koseri to cause abortion in pregnant cattle.

A female Holstein cow was artificially inseminated with beef cattle semen in May 2020. In December, at 224 days’ gestation, the cow suddenly aborted with the ejection of cheese-like lochia in the pasture. The following day, necropsy of the aborted fetus (male) and the fetal membrane was performed. The cow was vaccinated with two doses of a vaccine containing four different arbovirus antigens including Akabane virus (AKAV), Aino virus (AINOV), Chuzan virus (CHUV) and Peaton virus (PEAV). After the first abortion, artificial insemination became inconceivable and the cow aborted afterwards. (The second aborted fetus was not examined.)

No visible malformations were observed in the aborted fetuses on external examination. After removing the skull, thickened meninges of the cerebrum, cerebellum, and brainstem with opacities were observed (Fig. 1a). Hemorrhagic pericardial effusion and atelectatic lungs were detected, but no significant changes were observed in the other organs on visual inspection.

Fig. 1.

a. The meninges is thickened with opacity (arrows) in the cerebrum. Bar=2 cm. b. Diffuse and moderate meningitis (arrows) in the cerebrum with moderate fibrin deposition. Hematoxylin and eosin staining. Bar=500 µm. c. Severe perivascular cuffing is seen in the cerebrum. Perivascular inflammation spread into the surrounding neuropil. Hematoxylin and eosin staining. Bar=100 µm. d. Suppurative bronchopneumonia. Neutrophils and macrophages infiltrate into the bronchiolar and alveolar spaces. Hematoxylin and eosin staining. Bar=200 µm. e. Numerous gram-negative bacilli (arrows) were detected in the cytoplasm of macrophages and/or neutrophils of the cerebrum. Gram staining. Bar=10 µm. f. Positive reactions for Citrobacter koseri antiserum are detected in the cytoplasm of macrophages infiltrate around blood vessels in the cerebrum. Immunostaining. Bar=10 µm.

Tissue samples collected from the fetus (liver, spleen, kidney, heart, lung, brain, rumen, abomasum, duodenum, jejunum, mesenteric lymph nodes, and skeletal muscle) and fetal membranes were subjected to histopathological examination using standard procedures. To investigate the presence of bacilli, Gram staining was performed on all tissue sections.

Sulcal dilatation was clearly observed in the cerebrum. Diffuse to moderate suppurative histiocytic meningitis and meningoencephalitis were confirmed with moderate fibrin deposition and mild hemorrhage in the cerebrum, cerebellum, and brainstem (Fig. 1b). Severe perivascular cuffing (Fig. 1c) was detected in the necrotic vessels of the brain. Some spheroids were observed around the blood vessels. Moderate suppurative bronchopneumonia (Fig. 1d) containing meconium, localized suppurative and edematous chorioamnionitis (Fig. 1e), mild fibrinous splenic serositis with increased numbers of macrophages in the red pulp, and intrasinusoidal leukocytosis in the liver were detected. Mild inflammatory lesions are observed in the renal medulla, epicardium, mucosa of the rumen, abomasum, duodenum, and jejunum. No lesions were detected in the other organs.

Gram-negative bacilli were detected in the brain, lungs, spleen, liver, abomasum, duodenum, and jejunum of the aborted fetus and the fetal membrane. Numerous gram-negative bacilli were identified in the cytoplasm of macrophages and neutrophils in the cerebrum.

Bacteria were isolated from the liver, spleen, kidney, heart, lung, cerebrum, skeletal muscle, and pericardial fluid of the fetus. The specimens were plated onto deoxycholate-hydrogen sulfide-lactose (DHL) agar and 5% enriched sheep blood-containing agar plates (Nissui, Tokyo, Japan) and then incubated at 37°C in 5% CO₂ and 95% air. After 24 hr of incubation, bacterial colonies that appeared reddish on DHL agar plates and white-to cinnamon-colored in blood-containing agar were isolated from all the samples examined. The gram-negative bacilli were examined using commercially available identification kits (ID32E, BioMérieux, Tokyo, Japan). The results of the identification with profile number 34574517331 indicated that the bacteria were 99.9% identical to C. koseri. One isolate was selected for further examination using molecular biological identification based on 16S ribosomal RNA gene (16S rRNA) sequence identity. Sequencing analysis of 16S rRNA was performed as previously described [12]. The obtained data were compared with the 16S rRNA sequences of other bacterial species using EzBioCloud Version 20210707 (https://www.ezbiocloud.net/) [24]. The determined sequence was 99.73% identical (1,453 / 1,457 bp) to the C. koseri type strain LMG 5519 (accession no. HQ992945).

The same set of tissue samples from the fetuses and paired sera from the mother cow, were subjected to the detection for arbovirus antigens by PCR, and serological assays for the presence of neutralizing antibodies for AKAV, AINOV, CHUV, Ibaraki virus (IBAV), bovine epidemic fever virus (BEV), PEAV, Sathuperi virus (SATV), Shamonda virus (SHAV), D’Aguilar (DAV) and bovine viral diarrhea virus (BVDV) type I (BVDV1) and II (BVDV2) were performed. All these abortion-related viruses were negative in paired blood samples and aborted materials. In contrast, specific antibodies for these viruses were detected in the blood sample of the mother cow, while the titers remained stable between pre-and post-sera.

For the immunohistochemical investigation, we prepared mouse antisera against a defined isolate of C. koseri. Purely cultured bacteria fixed in 20% phosphate-buffered formalin were mixed with Freund’s Complete Adjuvant (Fujifilm Wako, Osaka, Japan) and then inoculated into ICR mice twice at two-week intervals. Blood was taken one week after the last injection, and serum was collected and used in this study. The specificity of the serum against C. koseri was confirmed using chicken liver sections containing mechanically injected bacteria. In addition, it was confirmed that the serum did not react with other gram-negative bacteria, such as Salmonella typhimurium (O4, R2-70 strain) and Escherichia coli (O119, R2-73 strain).

To investigate the tissue localization of C. koseri in aborted fetuses and fetal membranes, immunostaining was performed using C. koseri-specific antiserum. Serial histological sections were incubated with serum (1:50,000) and enzyme-labeled secondary antibody (Histofine simple stain MAX-PO (MULTI) Kit, Nichirei Corp., Tokyo, Japan) after antigen retrieval in a microwave at 500 W for 15 min in citric acid buffer and the removal of endogenous peroxidase with 3% H₂O₂. Antigens were visualized using a commercial kit (Histofine Simple Stain SAB-PO (M) Kit (Nichirei Corp.)). All sections were lightly counterstained with hematoxylin and assessed using light microscopy.

Positive reactions were detected in the cytoplasm of macrophages and/or neutrophils in the brain (Fig. 1f), lung, abomasum, spleen, and liver of the fetus, and fetal membrane. Positive reactions were also detected in the lumen of the digestive tract (duodenum and jejunum) of the fetus.

The present results indicate that C. koseri infection may cause septicemia and suppurative chorioamnionitis in the fetuses of pregnant cattle, leading to subsequent abortion. The fetus appeared to have been delivered stillborn, because no air inclusions were found in the lungs at necropsy. In this study, no clinical signs were observed in the mother cow. We did not examine the umbilical cord and placenta; hence, we could not conclude that fetal infection was hematogenously established. However, a few changes were found in the cow, such as widespread inflammation in the fetal membrane and suppurative lesions with C. koseri in the fetal lungs and gastrointestinal tract, and the ejection of cheese-like lochia suggesting that the disease developed by the ascending progression of infection through the vagina of the mother cow. Presumably, the infection caused chorioamnionitis, and subsequently, the fetus was infected with the bacteria via the oral route through the ingestion of contaminated fetal fluids. Notably, the presence of neutralizing antibodies against several abortion-related viruses (AKAV, AINOV, CHUV, IBAV, BEV, PEAV, SATV, BVDV1 and 2) was confirmed in the mother. Because the animal was vaccinated with a combined vaccine containing inactivated AKAV, AINOV, CHUV, and PEAV, it was difficult to specify which factors contributed to the increase in antibody titers: vaccination or natural infection. The reason for the increase in antibodies against IBAV, BEV, SATV, and BVDV1 and 2 is unknown, but it is presumably due to spontaneous infections by field viruses before abortion.

Meningoencephalitis and brain abscess formation are commonly observed in newborns with postnatal nosocomial C. koseri in humans [7]. The 32 kD outer membrane protein of C. koseri is considered a virulence factor for suppurative lesions in the brain [14, 15, 17, 18]. Citrobacter spp. invade and replicate in the brain vascular endothelial cells, which constitute the blood-brain barrier [2, 18, 21]. In infants, the barrier function is immature and cannot completely prevent the penetration of C. koseri into the central nervous system [2, 18, 21]. In addition, as C. koseri persists in the cytoplasm of macrophages, such as Salmonella spp., the disease becomes chronic [2, 16, 17, 22]. Once C. koseri enters the host macrophages, the fusion of phagolysosomes is arrested, allowing the bacteria to survive longer within the cells [15, 16, 21]. Citrobacter spp. also infiltrate into vascular endothelial cells in the brain through blood vessels and form abscess lesions [2, 21]. When neonatal rats were experimentally inoculated with C. koseri via the intraperitoneal or intranasal route, acute bacteremia occurred within 24 hr and ventriculitis developed 72 hr post-inoculation. At the late stage of infection (8–10 days post inoculation), brain abscesses form [15, 21]. In the present study, we found that numerous bacilli accumulated in the cytoplasm of macrophages in the aborted fetal brain and that the inflammatory response extended from the blood vessels to the surrounding neuropil. These findings suggest that if the fetus survives longer during gestation, similar abscess lesions may form in the brain.

To our knowledge, there are no reports that describe C. koseri-induced abortions in cattle. We demonstrated that the lesions formed in the aborted fetus in the present report were similar to those observed in a previously reported calf with clinical septicemia [16]. Taken together, we propose that C. koseri causes clinical diseases in cattle and that the involvement of this bacterial species in the development of clinical inflammation, especially in vulnerable immature animals, should be carefully considered.

Conflicts of Interest

The authors declare no conflicts of interest.