Coinfection by avirulent Rhodococcus equi and Klebsiella oxytoca as a cause of atypical abortion in a thoroughbred mare

Received 30 January 2014 Accepted 18 July 2014 Department of Preventive Veterinary Medicine, Universidade Federal de Santa Maria – UFSM, 97105-900, Santa Maria, State of Rio Grande do Sul, Brazil Department of Pathology Veterinary Medicine, Universidade Federal de Santa Maria – UFSM, 97105-900, Santa Maria, State of Rio Grande do Sul, Brazil Department of Veterinary Hygiene and Public Health, Universidade Estadual Paulista UNESP, Box 560, 18618-970, Botucatu, State of Sao Paulo, Brazil


Introduction
The majority of placental infections are caused by opportunistic bacteria migrating into the uterus from the caudal reproductive tract (Macpherson, 2005).The most commonly isolated bacteria in equine placentitis and/or abortion include Streptococcus equi subsp.zooepidemicus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and nocardioform species (Giles et al., 1993).Fungal and viral organisms can also infect the placentas of mares (Macpherson, 2005).
Rhodococcus equi is a Gram-positive facultative intracellular bacterium that is associated with pyogranulomatous infections in animals and humans (Prescott, 1991).Three virulence levels of R. equi are recognized, named virulent, intermediately virulent and avirulent, based on the presence or absence of virulence-associated antigens and plasmids (Vap).Virulent isolates contain a large plasmid (80-90 kb) that contains a gene encoding a virulence-associated antigen (15-17 kDa) called VapA, which is subdivided into 11 classical types with different geographical distributions (Ribeiro et al., 2005).Virulent R. equi strains (vapA positive) are the major cause of bronchopneumonia and enteritis in foals (Van Der Kolk et al., 1999).Intermediately virulent (VapB) isolates present the vapB gene and are commonly found in the lymph nodes of swine and HIV-positive humans (Takai et al., 2003).Avirulent isolates show no evidence of vapA or vapB genes and are found in HIV-positive patients, livestock environments and soil from parks (Takai, 1997).R. equi is widespread in soil, farm manure and the faeces of healthy herbivores, especially foals.R. equi from soil samples is usually vapA negative.However, Makrai et al. (2006) reported vapA positivity in 20 % of isolates, highlighting that soil isolates are an important source of Abbreviation: EHV-1, equine herpesvirus type 1.
virulent R. equi for horses.Nevertheless, R. equi-associated abortions in mares are unusual (Hines, 2007).The first description of placentitis and abortion as a result of R. equi infection was described with a virulent strain (Patterson-Kane et al., 2002), although there is a paucity of data regarding the virulence-plasmid profile of R. equi isolates from mares that have experienced abortion.In contrast, Nakamura et al. (2007) reported an abortion in a thoroughbred mare caused by an avirulent R. equi strain.
Like R. equi, species of the Klebsiella genus are unusual causative agents of abortion in mares (Hines, 2007).The first case of Klebsiella oxytoca infection inducing the abortion of a 10-month-old female equine was described in Hungary (Szeredi et al., 2008).In an aetiological study of 72 cases of abortion in mares from Brazilian farms, bacterial infections were observed in 36.1 % (Pereira et al., 2012).From these, K. pneumoniae was diagnosed in only 5.6 % of the fetuses.
To the best of our knowledge, there have been no reports of R. equi and K. oxytoca coinfection in mares experiencing placentitis and abortion.The present report describes a rare case of abortion caused by R. equi and K. oxytoca coinfection in a thoroughbred mare in Brazil, in which the plasmid-virulence profile of R. equi was investigated.

Case report
A 6-year-old thoroughbred mare, in her first pregnancy, showed increased uteroplacental junction and suspected placentitis on transabdominal ultrasound.The mare was treated with trimethoprim/sulfamethoxazole and antiinflammatories, and given a daily progesterone supplement.However, the prognosis was guarded and the mare aborted a 10-month-old female fetus.After the abortion, a field necropsy was performed by the attending veterinarian and hydrallantois was diagnosed.This case occurred on a stud farm in the State of Rio Grande do Sul, Brazil.

Investigations
Necropsy revealed that the whole placenta was thickened, and oedema and nodular areas (2 cm in diameter) without villi was was seen in the pregnant horn, which had a ruptured area of about 5 cm in diameter.Amnion showed vascular congestion, which is suggestive of placentitis.Fragments of the heart, lung, liver, spleen, kidney, adrenal gland, small and large intestine, mesenteric lymph nodes and brain were collected for histopathological examination.Simultaneously, swab samples from the peritoneal and abdominal cavities, trachea, stomach and aspirate of stomach contents were subjected to microbiological culture for fungi and bacteria.Virological analysis was carried out using samples of the stomach contents.
The tissues fragments were fixed in 10 % buffered formalin and embedded in paraffin to perform haematoxylin and eosin staining.In addition, Gomori's methenamine silver stains for fungi were performed using liver fragments.The samples collected for bacterial cultures were inoculated on defibrinated sheep blood agar (5 %) and MacConkey agar, and incubated under aerobic conditions at 35 u C for 48 h.Bacteria were identified on the basis of conventional phenotypical properties (Quinn et al., 2011).The presence of R. equi colonies was confirmed by species-specific multiplex PCR, and a virulence profile analysis according to the presence of the vapA gene was performed as described previously (Krewer et al., 2008).Virological tests were performed on RK13 (rabbit kidney cell line) to identify equine herpesvirus type 1 (EHV-1) and equine arteritis virus.Samples were grown in minimal essential medium containing penicillin (1.6 mg l 21 ), streptomycin (0.4 mg l 21 ) and nystatin (0.02 mg l 21 ) supplemented with 5 % fetal bovine serum for virus isolation.The supernatant of homogenized and centrifuged fetal stomach content was inoculated in RK13 cell monolayers and the cultures were incubated at 37 u C and 5 % CO 2 , and monitored for four consecutive passages of 96 h each to check for a cytopathic effect.

Diagnosis
Microscopically, significant lesions were observed only in the lungs and liver.In the lungs, alveolar spaces were filled with large numbers of foamy macrophages, epithelioid cells, neutrophils and multinucleated Langhans giant cells (Fig. 1).Bronchioles were obliterated by large numbers of neutrophils.In the liver, there were multifocal areas of necrosis, neutrophil infiltration and haemorrhage (Fig. 2).Both Gram-negative intracytoplasmic bacilli and Grampositive intracytoplasmic coccobacilli were observed.The K. oxytoca was isolated from all of the samples except aspirate of stomach content, while R. equi was isolated only from the stomach contents.Species-specific multiplex PCR for R. equi confirmation amplified a single 458 bp fragment, confirming the presence of R. equi, although samples were negative for the vapA gene.Virological tests did not demonstrate virus isolation from the fetal samples.

Discussion
Hydrallantois is a rare clinical manifestation in horses (Vandeplassche et al., 1976).Affected mares are usually multiparous, and the condition generally becomes apparent at 7-10.5 months' gestation (Koterba et al., 1983) in mares aged 5-21 years (Blanchard et al., 1987).Different pathogeneses have been suggested to explain this pathological condition, including excessive umbilical cord torsions, immunological incompatibility (Ru ¨sse & Grunert, 1993) and hereditary factors (Waelchli & Ehrensperger, 1988).Although it is generally accepted that placental dysfunction is the reason for this disorder in mares, only a few reports have described inflammatory changes in the placenta (Koterba et al., 1983;Stich & Blanchard, 2003;Shanahan & Slovis, 2011).In the case reported, any dysfunction cited above was observed in the mare or fetus, then hydrallantois was probably a result of ascending placentitis.
Inhalation and consumption of contaminated water and pastures appears to be the major source of transmission of R. equi in horses, particularly foals (Prescott, 1991).However, routes of R. equi transmission to mares as an agent of abortion remain controversial, although transmission probably occurs by septicaemia or genital contamination from the farm environment (Hines, 2007).The common presence of avirulent R. equi strains in the soil and faeces of stud farms (Bell et al., 1998), associated with the immunosuppression that occurs during pregnancy, may have contributed to the opportunistic infection by R. equi in the mare described in the current case.R. equi survives and replicates within macrophages through an ability to prevent the phagosome-lysosome fusion, resisting clearance by the organism's defences (Kanaly et al., 1993).The lesions observed in the abortion described here are similar to those in the first case of abortion as the result of an avirulent R. equi isolate described in a thoroughbred mare (Nakamura et al., 2007).
In contrast to the common presence of R. equi in the environment, K. oxytoca is found on the mucosal surfaces of horses (Podschun & Ullmann, 1998), and can occasionally cause placental infection and abortion.Although K. oxytoca is considered to be an extracellular pathogen, Szeredi et al. (2008) reported numerous intraalveolar neutrophils, macrophages and multinucleated Langhans giant cells containing Gram-negative intracytoplasmic bacilli in a mare with K. oxytoca-associated abortion.In the current case, both Gram-negative intracytoplasmic bacilli and Gram-positive intracytoplasmic coccobacilli, suggestive of enterobacteria and actinomycetes, were observed; these were later diagnosed as K. oxytoca and R. equi coinfection, respectively.
Several aetiological agents can be involved in cases of placentitis and abortion in mares.Hong et al. (1993) noted a high prevalence of Leptospira species and nocardioform actinomycetes in a 2-year study.Abortions related to Leptospira species and nocardioform actinomycetes in mares are associated with diffuse placentitis, jaundice and chronic placentitis at the junction of the body and horns (Hong et al., 1993;Pereira et al., 2012).In the present report, the macroscopic lesions observed were not suggestive of infection by Leptospira species or nocardioform actinomycetes.Moreover, there was no history of Leptospira species infection from serological tests performed on the stud farm.
EHV-1 is the major viral cause of abortion in mares (Leon et al., 2012).In late-term abortions, gross lesions found in fetuses consist of multiple foci of acute necrosis, randomly distributed on the affected organs, especially the liver.In the lung, pneumonia with fibrin has been observed in the tracheas of fetuses (Foster, 2009).In the present case, although some areas of liver necrosis were observed (Fig. 2), there were no lesions characteristic of EHV-1 in other organs and no organisms were grown in cell culture.In addition, this virus typically causes abortion earlier in gestation (Marenzoni et al., 2012).This is the first report of K. oxytoca and R. equi coinfection causing an uncommon abortion in a mare.Although the role of R. equi virulence in the pathogenicity of abortion in mares remains unclear, the combined infection of R. equi and K. oxytoca in the placenta and uterus probably potentiated the pathogenic effect of these micro-organisms, resulting in abortion.