Bovine abortion associated with Staphylococcus aureus infection – characterization of S. aureus strain isolated from fetal tissues

Staphylococcus aureus is a gram-positive bacterium, commonly found colonizing the skin and mucous membranes of humans and animals. This report describes a case of fetal loss associated with S. aureus infection in a cow. A six-month old, crossbred male bovine fetus from a beef farm was submitted for necropsy. At gross examination fibrinous pleuropneumonia was observed. Histologically, lesions were restricted to the lungs and consisted of marked multifocal to coalescing areas of inflammatory infiltrate of neutrophils, abundant fibrin exudation, necrosis of bronchiolar epithelium and numerous aggregates of coccoid bacteria. Lung and abomasal fluid bacterial culture yielded pure culture of S. aureus, which was characterized as a multidrug resistant strain. Molecular analysis indicated that the studied strain presented several genes of virulence factors including toxic shock syndrome toxin-1 (tst), staphylococcal enterotoxin type A (sea), Panton– Valentine leukocidin (pvl), alpha-hemolysin (hla) and delta-hemolysin (hld). This report documents an infrequent case of fetal loss in cattle due to infection with a highly virulent S. aureus strain.

Staphylococcus aureus is a gram-positive, catalase-positive bacterium which is commonly reported colonizing the skin, mucous membranes and other sites of healthy carriers, and may be associated with a wide range of clinical conditions in animals and humans (WERTHEIM et al., 2005;PETON & LOIR, 2014). S. aureus may carry numerous genes encoding virulence factors, which have been associated to its capacity to evade host immune response and ultimately cause disease (FOSTER, 2005). In cattle, such bacterium is commonly associated with cases of chronic mastitis, leading to significant economic losses in the dairy industry (RAINARD et al., 2018).
Abortion in cattle due to S. aureus has been infrequently described, and such event is classified as a sporadic cause of fetal loss in the bovine species Henker et al. (CORBELLINI et al., 2006). Thorough pathological descriptions of S. aureus-induced abortions are scarce. Additionally, data characterizing the virulence factors of S. aureus involved in these cases are exceedingly limited. Therefore, the objective of this work was to describe the gross, histopathological, microbiological and molecular findings of a case of bovine abortion associated with a highly virulent S. aureus strain.
In August 2018, a crossbred male bovine fetus was referred for postmortem examination at the Setor de Patologia Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS). The fetus was referred from a cow-calf operation beef farm, in the state of Paraná, southern Brazil. Herd was composed of 2,500 cows raised in grassland, and only sporadic abortions had been previously documented. The aborting animal was a 6-year-old, multiparous Zebu cow (forth calving), with no history of health problems or treatments, which did not show any clinical signs before or after abortion. In the referred farm, vaccination against the main reproductive pathogens used to be routinely conducted, including Bovine herpesvirus type I, Bovine Viral Diarrhea Virus and Leptospira spp., and the herd overall health status was good. Reproductive management used to be performed with artificial insemination, and natural service was only used in empty cows.
Fetus crown-rump length measured 57cm, compatible with six months of gestation. External examination revealed no abnormalities. Fetal membranes were not available for evaluation. At the necropsy, gross lesions were restricted to the lungs and were characterized by moderate fibrin deposition covering the visceral pleura surface in the pulmonary cranioventral area ( Figure 1A). Samples of various organs were collected and fixed in 10% formalin. Fixed tissues were routinely processed, embedded in paraffin wax, and sections (3 -4 µm) were stained by hematoxylin and eosin. In addition, lung sections were stained with Brown-Hopps method.
Histologically, marked multifocal to coalescing areas characterized by inflammatory infiltrate of neutrophils, fewer lymphocytes and macrophages, as well as abundant fibrin exudation, necrosis, accumulation of cell debris and numerous aggregates of 0.5 -1µm coccoid bacteria were observed affecting alveolar spaces, bronchioles and bronchi ( Figure 1B-C). Also, interlobular septa were markedly expanded by fibrin exudation and edema, and abundant fibrin deposition associated with inflammatory infiltrate of neutrophils was seen covering the visceral pleura. No microscopic lesions were detected in other organs. Brown-Hopps staining showed that bacterial aggregates observed in the lungs were gram-positive cocci ( Figure 1D).
Fresh samples were collected aiming to perform microbiological diagnostic tests. Lung fragments and abomasal fluid were inoculated on 5% sheep blood agar and MacConkey agar and incubated at 37 °C for 72 h in aerobic and microaerophilic atmosphere. The isolated bacterium was identified by MALDI-TOF mass spectrometry (MS), using Microflex LT instrument and MALDI Biotyper 3.1 software (Bruker Daltonik, Bremen, Germany), and antimicrobial susceptibility test was performed using Kirby-Bauer method accordingly to the Clinical and Laboratory Standard Institute guidelines (CLSI, 2018).
Methicillin resistance was verified by conventional PCR for mecA gene and by the Kirby-Bauer method using cefoxitin and oxacilin.
Pure white, bright, hemolytic, medium size colonies grew in the blood agar in aerobic and microaerophilic conditions, in both inoculated samples, while no growth was observed from MacConkey agar. The isolated bacterium was classified as a catalase and coagulase positive grampositive coccus, which was identified by MALDI-TOF as S. aureus. This bacterium showed resistant phenotype to tetracycline, gentamicin, ceftazidime, ciprofloxacin, erythromycin, and chloramphenicol, characterizing the isolate as a multidrug resistant (MDR) bacterium. Otherwise, the strain was susceptible to sulfamethoxazole+trimethoprim, penicillin, imipenem and amoxicillin. Besides that, the S. aureus showed intermediary susceptibility to oxacillin and cefoxitin. However, the strain was negative to mecA gene.
Genomic DNA from the isolated bacterium was extracted and a molecular characterization was conducted by PCR assays to search the following virulence marker genes: toxic shock syndrome toxin-1 (tst), staphylococcal enterotoxin type A (sea), Panton-Valentine leukocidin (pvl), alpha-hemolysin (hla) and delta-hemolysin (hld), as previously described (ROSSATO et al., 2018). All the referred virulence marker PCR assays yielded positive results, providing information to assume the highly virulent character of the S. aureus studied. In addition, fresh kidney and liver samples were tested for Leptospira spp. through PCR as previously described (AHMED et al., 2012), and thymus and spleen samples were tested for pestivirus (BVDV) (VILCEK et al., 1994), both of which yielded negative results.
Abortion is considered a significant cause of economic losses in livestock systems worldwide and frequently represents a diagnostic challenge (CABELL, 2007). The main differential diagnosis in the present case should include other causes of pleuropneumonia and bronchopneumonia in bovine aborted fetuses, mainly B. abortus infection (POESTER et al., 2013), as well as sporadic bacterial agents (ANDERSON et al., 1990). However, based on histological and microbiological examinations, the aforementioned agents may be easily differentiated. Species in the genus Staphylococcus that have been implicated with abortion in cattle include Staphylococcus lugdunensis (ARDIGÒ et al., 2014) and S. aureus (CLOTHIER & ANDERSON, 2016). S. aureus infection has been associated mainly with bronchopneumonia, and rarely with skin lesions in bovine fetuses (CORBELLINI et al., 2006).
In the present case, the lungs were the only affected organs, and large numbers of coccoid aggregates were seen inside bronchioles and bronchi, which may be associated with inhalation of bacteria present in the amniotic fluid (MILLER, 1977). Bacterial infections leading to abortion may be a result of systemic bacterial spread, as well as reproductive tract ascending infections (PARTHIBAN et al., 2015). In the present case, however, it was not possible to infer the route of fetal infection, since no previous disease that could justify fetal spread was detected in the aborting dam.
S. aureus isolated was a methicillin sensitive strain (MSSA), but resistant to several antibiotics. S. aureus may express several virulence factors which have been associated with adhesion to host cells, tissue invasion and damage, host immune system escape, disease promotion, cytokine production, and systemic inflammation (FOSTER,

2005
). Although well characterized and described in human isolates, to the best of our knowledge, no information regarding virulence factors present in animal abortion associated-S. aureus strains is currently available. The identified genes from the S. aureus here described encoding toxins, tst, sea, pvl, hla and hld, have been described as some of the most frequent virulence genes of S. aureus (ROSSATO et al., 2018). The expression of these virulence factors is coordinated by cell-communication system (quorumsensing) in response to population density (KONG et al., 2016).
In conclusion, S. aureus should be considered as a sporadic cause of bacterial abortion in cattle, mainly in fetuses presenting fibrinosuppurative pleuropneumonia and bronchopneumonia. In the present case, the isolate was a multidrug resistant strain which presented several genes of virulence factors, indicating that these may play a role in S. aureus-induced abortion in cattle.