Ten cases of Mycobacterium avium subsp. hominissuis infections linked to equine abortions in Japan, 2018–2019

Abstract Bacterial placentitis in horses commonly results in abortion, premature birth or compromised neonatal foal health. Although mycobacterial infections are generally uncommon in horses, 10 equine abortion cases caused by Mycobacterium avium subsp. hominissuis (MAH) infections occurred between 2018 and 2019 in Japan. They occurred on seven Thoroughbred horse farms in the Hidaka district of Hokkaido, but direct contact among the mares on different farms was not recorded. Most cases were characterized by extensive pathological lesions of the placenta, which are not typical in cases of common pathogenic bacteria such as Streptococcus zooepidemicus and Escherichia coli. All abortions featured white–yellow exudates on the surface of the placenta. Mycobacterial granuloma formations were histologically found in the placenta and fetal organs, and acid‐fast bacteria were isolated from the placenta, fetal samples (heart, lung, liver, kidney, spleen and stomach contents) or uterine lavage fluid. The greatest number of bacteria was isolated from necrotic lesions on the placenta, which could be an important site for bacterial isolation in mycobacterial equine abortions. The isolates were identified as MAH based on internal genome sequences. In variable number tandem repeat analysis, all patterns of the strains were identical. Single nucleotide polymorphism analysis of the core genome grouped all strains in the II‐a/SC3 subcluster. Both results reveal that these strains share the same genetic background, suggesting that the horses had been infected by the same unknown contagious source.

bacterial agents are Streptococcus zooepidemicus, Escherichia coli and Pseudomonas aeruginosa (Bazanow et al., 2014;LeBlanc, 2010). A retrospective cohort study of 2,137 equine abortions placed bacterial infection as the second common infectious cause (7.3%) after viral infection (10.1%) in Thoroughbred horses in Japan (Murase et al., 2017). Of the bacteria, S. zooepidemicus and E. coli were the most frequently isolated in Japan, which is common with findings from other countries (Murase et al., 2017).
Horses are considered to be highly resistant to mycobacterial infections (Pavlik et al., 2004;Thorel et al., 1997). However, occasional cases are reported. Of the cases investigated, Mycobacterium avium complex (MAC) members were the most common pathogens (Pavlik et al., 2004). Mycobacterium avium subsp. hominissuis (MAH) is a member of the MAC and is frequently isolated from pigs and humans (Mijs et al., 2002). In horses, most MAH infections have taken the form of tubercular lesions in the liver, spleen, lung tissue, colon, lymph nodes and other organs (Pavlik et al., 2004). Five cases of equine abortion associated with fetal mycobacterial infections have been reported: one case of M. terrae in Australia in 1981 (Tasler & Hartley, 1981), one case of MAC infection in the USA in 1991 (Cline et al., 1991), one of MAC in Canada in 1996 (Helie & Higgins, 1996), one case of a novel Mycobacterium species in 2012 (Johnson et al., 2012) and one case of MAH in Japan in 2014 (Sano et al., 2014). In this paper, we report an outbreak of MAH infection resulting in 10 equine abortions in Japan.

| Cases of equine abortions
We describe 10 abortions in Thoroughbred horses caused by MAH infections that occurred in 2018 and 2019 (Table 1). The 10 mares (average age ± SD, 15.1 ± 4.0 years) were kept on seven horse farms located within a 30-km radius in the Hidaka district of Hokkaido, Japan. No evidence of direct contact between the mares was found.
The fetal ages at the time of the abortions ranged from 148 to 303 days (average age ± SD, 222.6 ± 46.1 days). All abortions except case no. 4, from which placental samples were not available, were further pathologically investigated. Equine herpesvirus type 1 was tested in fetal lung and thymus by using a loop-mediated isothermal amplification method (Nemoto et al., 2010). Uterine lavage was conducted in case no. 4 after the abortion and uterine lavage fluid was sampled for the following bacterial isolation.

| Bacterial isolation
For isolation of acid-fast bacteria, the placenta, fetal samples (heart, lung, liver, kidney, spleen and stomach contents) or uterine lavage fluid from each case were used. Except for stomach contents, 1 g of each specimen was homogenized in a 15-ml glass homogenizer. Two TA B L E 1 Detail information of aborted Thoroughbred horses Case no. volumes of N-acetyl-L-cysteine-sodium hydroxide (2%) was added to the bacterial suspensions of each specimen (except for stomach contents) and incubated at room temperature for 15 min. Phosphate buffer solution (PBS, pH 6.8) was added up to 15 ml and the mixture was centrifuged at 3,000 g for 20 min. The supernatant was discarded and the pellets were resuspended in PBS. Then 100 µl of the mixed suspensions or stomach contents were inoculated on Middlebrook 7H11 agar plates (Kyokuto Pharmaceutical Industrial

Date of abortion
Co., Ltd.) and incubated at 37°C for 2 weeks in a 5% CO 2 atmosphere. For standard bacterial isolation, cut surfaces of each organ were directly stamped onto sheep blood agar plates and deoxycholate hydrogen sulphide lactose agar plates, and the plates were incubated at 37°C for 2 days in a 5% CO 2 atmosphere.

| DNA extraction and bacterial identification
The genomic DNA of each MAH strain was obtained for whole-ge-

| RE SULTS AND D ISCUSS I ON
Ten cases of infectious placentitis that led to equine abortions oc-  To determine the genetic relatedness among the 10 isolated MAH strains, we conducted VNTR and SNP analyses. Although MATR-VNTR reportedly shows high resolution for genotyping MAH (Inagaki et al., 2009), all 10 MAH strains had the same VNTR pattern  (Uchiya et al., 2017;Yano et al., 2017). All 10 strains were categorized as II-a/SC3, being closest to human MAH strains from Taiwan (strain 11 and aviumMD30).
To the best of our knowledge, these MAH strains are the first II-a/ SC3 strains isolated in Japan. One case of equine abortion from MAH infection in Hokkaido was described in 2014 (Sano et al., 2014), but we could not find any epidemiological relationship between that case and ours. Detailed comparisons based on genetic information unfortunately could not be carried out owing to a lack of information on the previous strain.
In conclusion, although horses are normally resistant to mycobacterial infections (Pavlik et al., 2004), we observed 10 mycobacterial abortions on seven horse farms in two breeding seasons. The abortions occurred during mid to late gestation, and the characteristic features of the placentitis were extensive pathological lesions and white-yellow exudates, in addition to mycobacterial granuloma formations in fetal organs. All strains had the same MATR-VNTR pattern and were clustered together in II-a/SC3 based on core-genome SNPs, suggesting that the same unknown infectious source was involved in all cases. We believe that this report offers useful characteristic features of rare equine mycobacterial placentitis, and warns of the potential for future equine abortions caused by MAH.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. All relevant guidelines for the use of animals in scientific studies were followed. The study did not include any experimentation on animals or humans, and samples were taken from natural abortion cases or routine uterine lavage.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.411.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. Mycobacterium avium subspecies hominissuis disseminated infection F I G U R E 2 Phylogenetic tree of the 137 MAH strains. Five subclusters described in previous reports (Uchiya et al., 2017;Yano et al., 2017) were found here. Strains that were previously assigned to each subcluster and strains from this study are shown in the same colours as those of each subcluster. Sample origins are designated by the symbols shown