Can Ureaplasma diversum be transmitted from donor to recipient through the embryo? Two case reports outlining U. diversum losses in bovine embryo pregnancies

Abstract

Two bovine embryo recovery results are outlined from different herds. Both cases involve significant late gestational loss from embryos relating back to a single donor. Ureaplasma diversum was confirmed in 3 of 4 cases submitted for postmortem examination. Natural infection originating from the donor and transmitted to the recipient has not previously been documented.

Mycoplasmas and ureaplasmas are recognized as 2 of the few potential pathogens that are not removed from embryos through the recommended International Embryo Transfer Society (IETS) washing procedures (1,2). Ureaplasma diversum is a widespread commensal and pathogenic bacterium associated with vulvitis, endometritis, abortion, and infertility in cattle (3,4). When endemic within a herd, the typical clinical presentation of U. diversum infection includes infertility and vulvitis (5). This article reports pregnancy results from 2 herds experiencing late gestational losses due to U. diversum, in which both herds had the same underlying and unique history of the affected pregnancies all originating from 1 embryo donor. This clinical presentation suggests that it may be possible for this bacterium to be transmitted by embryo transfer. Furthermore, this route of infection and the insidious nature of this bacterium may be more likely to result in late term pregnancy loss. Ureaplasma diversum is likely an unrecognized factor contributing to gestational and neonatal losses in pregnancies from embryo transfer.

Case descriptions

Herd 1: Donor 1A, 1B, and 1C

The first case occurred in a herd of approximately 60 lactating Holstein cows in Nova Scotia. This herd was under regular veterinary care and had a comprehensive vaccination protocol in place for protection against infectious bovine rhinotracheitis (IBR) and bovine viral diarrhea (BVD) viruses. Frozen embryos from 3 donors (Donors 1A, 1B, and 1C) were purchased from a single herd in Ontario. The frozen embryos from each donor were from single collection procedures and therefore the same donor-sire matings. Frozen-thawed embryos were transferred into virgin heifer recipients. Transfers took place between September 2012 and May 2013 and occurred on 5 separate occasions when recipients were synchronized. Recipients that were not pregnant after embryo transfer were bred by artificial insemination (AI) and did not receive a second embryo. The transferred embryos from Donor 1A had an initial pregnancy rate of 7/7, or 100%. Embryos from Donor 1B had an initial pregnancy rate of 4/9, or 44%. Embryos from Donor 1C had an initial pregnancy rate of 5/7, or 71%. The pregnancy outcomes for embryos from Donor 1A are outlined in Table 1. Pregnancies resulting from embryos from Donors 1B and 1C were normal and all calves survived. One of those 9 births occurred early at 255 d; despite this, the calf did survive. Only 1 of the 7 pregnancies from Donor 1A resulted in a full-term surviving calf. The remaining 6 pregnancies were associated with pregnancy loss, stillbirths, premature births, and neonatal loss (Table 1). Each case was either suspicious of, or confirmed, as having U. diversum infection. During this time period, pregnancies from AI in heifers from the same groups as the recipients were normal and none aborted, were stillborn, or experienced neonatal loss.

Table 1.

Details and outcomes from the 7 embryos transferred that originated from Donor 1A in Herd #1. All 7 embryos resulted in pregnancies.

Embryo number	Transfer date	Gestation length	Outcome	Postmortem diagnoses
1	November 15, 2012	Full term	Stillborn	None performed
2	November 15, 2012	250 days	Aborted	None performed
3	February 6, 2013	270 days	Survived 4 days	None performed
4	February 6, 2013	250 days	Aborted	PM #1 — Placentitis and fetal pneumonia, no U. diversum growth, no U. diversum PCR performed
5	February 6, 2013	268 days	Dysmature bull calf euthanized	PM #2 — Placentitis, conjunctivitis, lung pathology, U. diversum confirmed by culture (heavy growth), no PCR performed
6	February 6, 2013	Full term	Live bull calf	N/A
7	March 5, 2013	250 days	Aborted	PM #3 — No placenta submitted, conjunctivitis, lung pathology, U. diversum confirmed by culture (few colonies) and PCR positive

The first 3 losses from Donor 1A were not submitted for necropsy. From the 3 calves submitted for necropsy, all polymerase chain reaction (PCR) tests for BVD, IBR, and Neospora caninum were negative and all aerobic bacterial cultures were negative. In addition, when fungal cultures, Coxiella burnetti PCR tests, or Chlamydia spp. PCR tests were performed, all were negative. Samples were submitted for mycoplasma/ureaplasma culture from all 3 calves and there was growth in 2 of the 3 cases. In the case with no mycoplasma/ureaplasma growth (Table 1; Embryo #4), a PCR test for U. diversum was not performed. In the case of Embryo #7 (Table 1), in which only a few colonies were detected by culture, the PCR test for Ureaplasma diversum was also positive.

The first postmortem (PM) examination (#1) was performed on a fresh premature fetus with a placenta that was aborted at 250 d gestation (PM #1, Table 1). This postmortem examination revealed focal placentitis and fetal pneumonia. The fetal pneumonia consisted of inflammatory cells in the alveoli as well as small lymphoid cell aggregates seen near small bronchioles and blood vessels. The final diagnosis was an abortion of infectious etiology with lesions consistent with a ureaplasma infection. In this case, however, mycoplasmas and ureaplasmas were not isolated from the lung that was submitted for culture and a PCR test for ureaplasmas was not performed.

The second postmortem examination (PM #2) was performed on a fresh bull calf that was born alive at 268 d gestation. It was small for its gestational age and was euthanized (PM #2, Table 1). This postmortem examination revealed placentitis, conjunctivitis, and lung pathology, which was described as an immune-mediated response but not a bronchopneumonia. Lung histopathology showed significant infiltrations and accumulations of small lymphocytes throughout the lung near the vessels and bronchioles as well as in alveolar tissue. Sections of eyelid had extensive non-suppurative conjunctivitis with severe goblet cell metaplasia in the epithelium. In this case, heavy growth (4+) of U. diversum was cultured from the placenta and lung tissue.

The 3rd postmortem examination (PM #3) was performed on a frozen-thawed fetus that aborted at 250 d gestation. The placenta was not submitted with this fetus (PM #3, Table 1). Histological findings included lymphocytic infiltration in the lung near the bronchioles and a moderate to severe conjunctivitis. Ureaplasma diversum (few, 2+) was cultured from the fetus and was also detected by PCR. The final diagnosis was antigenic stimulation in the lung, conjunctivitis, and late term ureaplasma abortion.

Herd 2: Donor 2

The second case occurred in a herd of approximately 100 lactating Holstein cows in Prince Edward Island. This herd was also under regular veterinary care and had a comprehensive vaccination protocol in place for protection against IBR and BVD. Pregnancy diagnosis typically occurred between 28 and 42 d and again between 60 and 75 d after embryo transfer. On this farm, embryo collections from various donors were regularly performed (1 to 2/month). Embryo transfers and the resulting pregnancies were generally successful and uneventful.

Although this herd rarely purchased new animals, the owners purchased a virgin heifer (Donor 2) at 11 mo old and entered her into the herd in April 2014 with the intent of having her become an embryo donor. Three embryo recoveries were performed between May and September 2014 (Table 2; Flush #1 to #3). A significant proportion of frozen embryos from each of these flushes was sold and the outcomes associated with these embryos are unknown. Fresh embryos were transferred into synchronized recipient heifers from the same housing group as the donor. Although not all the pregnancy outcomes are precisely known, 5/9 pregnancies resulting from fresh embryos from the first 3 flushes were lost after pregnancy diagnosis, with 3 occurring late in gestation (Table 2). Only 1 postmortem examination was conducted from these losses. During this same time period, heifers in the same group as the recipients that received embryos from other donors or were bred by AI did not experience gestational losses.

Table 2.

Details and outcomes from the embryos originating from Donor 2 in Herd #2.

	Total viable embryos	Number pregnant/Number fresh transfers	Fresh embryo pregnancy losses/Number pregnant	Loss at 30 to 75 days	Loss at 75 days to full term
Flush 1
May 27, 2014	22	2/5	1/2	0	1 (150 days)
Flush 2
July 3, 2014	12	3/6	1/3	0	1 (Stillborn)
Flush 3
September 9, 2014	13	4/7	3/4	2a	1 (250 days)b
Flush 4
September 11, 2015	5	0/1	N/A	N/A	N/A
Flush 5
November 5, 2015	12	1/1	N/A	N/A	N/A
Flush 6
December 9, 2015	1	1/1	1/1	N/A	1 (100 days)
Totals	65	11/21	6/11	2	4

^aPregnancies were thought to be lost in this time frame because there was no record of live births or late term abortions from these recipients.

^bCalf submitted for necropsy (see PM report).

N/A — Not available.

The specimen submitted from herd 2 for postmortem examination was a fresh female fetus, 250 d gestation with the placenta. The final diagnosis was placentitis and bronchopneumonia. The moderate to severe placentitis was described as necrosuppurative, multifocal, and acute. The moderate to severe bronchopneumonia was described as neutrophilic, diffuse, and acute. Lesions were consistent with a bacterial infection, but bacterial cultures were non-diagnostic (mixed growth), fungal culture was negative and viral PCR tests were all negative. At the submitting clinician’s request, samples were sent for ureaplasma evaluation, which required diagnosis at an outside laboratory and cost approximately an additional $200. Ureaplasma diversum was cultured from the fetal lungs (few, 2+) and the fetal abomasal fluid (few, 2+). These findings explained the inflammatory changes observed in the placenta and fetal lungs, confirming that U. diversum was the cause of abortion in this case.

Donor 2 delivered a full term viable calf in June 2015 and was flushed 3 more times during her first lactation (Table 2; Flush #4 to #6). There were fewer fresh transfers with these flushes, yet 1 out of 2 pregnancies resulted in a mid-gestation loss.

Discussion

The cases described in this report suggest that there is the potential for Ureaplasma diversum to infect embryos and then for these embryos to result in gestational losses in the recipient heifers. The postmortem results were conclusive for U. diversum infection as the cause of the abortion in 3 of the 4 submitted cases (Tables 1 and 2). Although U. diversum most commonly presents as vulvitis, endometritis, and infertility, it has been shown to cause sporadic abortions at any stage of gestation (4), but most commonly mid to late gestation (5). Ureaplasma diversum infection also results in stillborn and weak calves (4,6). Based on studies of aborted fetuses, the prevalence of ureaplasma abortion is approximately 1.7% to 3% (7,8). The 2 herds described in this report experienced a cluster of losses among the embryo pregnancies that were suspicious of or confirmed as U. diversum infection in 67% (12/18) of the losses. In these 2 herds, infertility was not the presenting complaint among the recipient heifers, rather pregnancy loss and stillbirths were the prominent clinical problems. It is possible that if embryos are the source of the U. diversum infection, abortions, stillbirths, and weak neonates could be how the disease is manifested. By the time the problem was recognized in these 2 cases, the embryos had been dispersed and it was too late to evaluate the embryos themselves as a source of the disease cluster.

Embryo processing techniques have been developed to reduce the risk of transfer of infectious disease by in vivo-derived bovine embryos (9). The standard International Embryo Transfer Society (IETS) recommended washing procedure includes at least 10 washes with serial 1:100 dilutions of embryo holding medium and 2 trypsin treatments (10). This protocol removes almost all of the important pathogenic viruses and bacteria (11). However, mycoplasmas and ureaplasmas are not removed from embryos with this protocol (12). In addition, standard antibiotics present in embryo processing media are not effective against ureaplasmas and mycoplasmas (11). Previously published data on the prevalence of U. diversum has identified the organism in vulvo-vaginal swabs in 38.8% of cows and heifers with granular vulvitis (13), 44% of beef heifers (14), and 11% to 100% of cows’ vaginas (4). Therefore, it is possible that U. diversum present on the vulva may contaminate the flush catheter or solutions and exist in bovine embryo recoveries at a similar prevalence.

Typical characteristics of an abortion caused by U. diversum include a relatively fresh fetus and a placenta that is frequently retained and exhibits a placentitis (15). Lung pathology of the fetus and conjunctivitis are often reported as well (6). The only postmortem examination in this report that did not result in a positive U. diversum culture did have a diagnosis of placentitis and fetal pneumonia (Table 1), which is highly suggestive that U. diversum infection was the cause of the abortion. A PCR test for ureaplasmas was not part of this postmortem examination. A PCR assay is a more sensitive test than culture because U. diversum requires special media to grow and it can be hard to isolate from clinical samples. In addition, the sample needs to be received at the laboratory quickly after collection and examined by personnel with expertise in ureaplasma and mycoplasma diagnoses (16). Most veterinary diagnostic laboratories have a standard diagnostic protocol for bovine specimens for postmortem investigation, but PCR testing for ureaplasmas is not usually included as a routine test. This occurred in the case of Herd 2, in which a PCR test for ureaplasmas had to be requested in addition to the standard postmortem examination. Therefore, it is possible that U. diversum abortions are underreported.

Despite the standard procedures put in place to prevent disease transmission, there is a still a risk of introducing U. diversum from a donor’s vulva into her uterus during artificial insemination. In fact, donor cows often undergo multiple inseminations after being super-ovulated. If this bacterium was transmitted into the uterus at the time of breeding, the uterus may have an established infection by the time of embryo collection resulting in adherence of the U. diversum to the developing embryos. Also, certain donor cows may carry a higher load of this bacterium than others, as evidenced in a study that cultured ureaplasmas from cows’ vulvas for up to 3 mo following a herd outbreak of granular vulvitis (17). This would potentially explain why even if the donor cow appeared clinically normal at the time of insemination, she could still have U. diversum present in her vulva, which could therefore contaminate the embryos and embryo flush fluid during embryo recovery. It is also theoretically possible that these clusters are connected due to a cause other than the donor cow, such as through a contaminated embryo transfer (ET) gun. However, if the technician’s ET gun was contaminated with this pathogen in these cases, there should have been an equal chance of infection among all embryos transferred, not only those from a single donor. In addition, in the case of Herd 2, different practitioners transferred the embryos on different locations with the losses spread across both locations. Intravaginal device applicators could be another potential source of transmission among a herd, but the cows would most likely present with a vaginitis and possible infertility if these devices were the source. In the cases reported, intravaginal devices were not used. In both of these herds, the reproductive losses could all be linked to 1 donor cow, so contamination from ET guns or intravaginal devices was unlikely to be the cause.

In conclusion, this case report suggests that practitioners need to consider U. diversum as a possible infectious cause of bovine abortion in embryo pregnancies and pursue this differential diagnosis despite additional costs and testing. To the authors’ knowledge, these cases are unique in that there have been no other published reports describing a single donor cow as a potential cause of natural U. diversum infection originating from her transferred embryos. This report indicates the need to continue investigation into the potential for natural infection of U. diversum by adherence to the embryo. CVJ