Presumptive Toxoplasma gondii abortion in a sheep

Abstract

A primiparous ewe aborted in mid-gestation. Toxoplasma gondii was suspected as the cause of abortion and a presumptive diagnosis of T. gondii abortion was based on histological lesions of the placenta.

A primiparous North Country Cheviot ewe aborted a single lamb in mid-gestation a month after the rest of the flock had finished lambing. On the morning of the abortion, the ewe, which had been considered not pregnant, was found with the aborted fetus, isolated from the flock in an empty claiming pen. There had been no known disease or exceptional degree of stress in the flock or in this ewe in the previous 5 mo.

A gross necropsy was performed on the fetus and placenta. Fetal crown rump length was approximately 20 cm. The abdominal cavity was filled with blood-tinged fluid. All tissues were otherwise grossly normal, with adequate stores of perinephral and pericardial adipose tissue. The eponychium was intact and the lungs were not inflated. The placenta was diffusely pale and purple-gray, and the cotyledons were friable and pale tan, with a gritty texture as though mineralized. Rumen and abdominal fluid samples were collected into sterile vacuum tubes (Vacutainers, Becton Dickinson, Oakville, Ontario), and samples of fetal thymus, lung, heart, liver, kidney, spleen, skeletal muscle, umbilicus, and placenta, including several cotyledons, were collected aseptically and submitted in 10% buffered formalin solution to a veterinary diagnostic laboratory (Histovet Surgical Pathology, Guelph, Ontario).

Mineralization of the cotyledons is characteristic of Toxoplasma gondii abortion. Other differential diagnoses included Chlamydia abortus (enzootic abortion), pestivirus (border disease), Campylobacter fetus subsp. fetus and C. jejuni (vibriosis), Salmonella spp., and Coxiella burnetii (Q fever). Stress or concurrent subclinical disease was considered a possible but unlikely differential diagnosis.

The aborted ewe was one of a flock of 37 sheep comprising 13 North Country Cheviot ewes (4 pregnant with their first lambs), 2 North Country Cheviot rams, and 22 commercial sheep. The 15 North Country Cheviots, which had arrived at the farm 8 mo to 1.5 y after the 22 commercial sheep, had been isolated for 1 mo before introduction to the resident flock.

The reproductive history of the flock prior to this abortion suggested that other sheep might have been affected. A 50% incidence of stillbirth in the North Country Cheviot ewes was sufficiently severe to suggest an infective agent. The cause of the poor reproductive performance in the North Country Cheviots had not been investigated. There had been no abortions or stillbirths among the commercial crossbred ewes.

In October 2000, the North Country Cheviot ewes had been put out to pasture with an 11-month-old ram in his first breeding season. After all 13 ewes had been marked, they were pastured with the commercial flock, which included 2 commercial cross rams. The conception rate was 92.3% (12/13), and the 12 ewes that lambed 147 d postmarking, between February 21 and April 24, produced 20 lambs, for a prolificacy rate of 1.67 (20/12). However, 10 of the 20 lambs were stillborn. Of the 10 that were born alive, 2 were poor doers. The poor-doing lambs had improved during the spring and appeared normal, including fleece and pigmentation. Necropsies had not been performed on the stillborn lambs, but the producer had noted no gross abnormalities. The ewe that aborted was marked to lamb in March. When she did not produce a lamb at the expected time, the producer considered her open, but did not reassess her reproductive status, and she was subsequently exposed to 3 rams.

The flock had access to 100 acres of pasture from April to November. The sheep were confined overnight, sharing the barn with approximately 20 free-range laying hens and 2 adult male cats. No litter boxes were provided for the cats and feces had been found in the hay fed to the sheep. Blood was collected from the aborted ewe, the 2 ewes that had produced the 2 weak lambs, and the lambs, and serum was submitted to a veterinary diagnostic laboratory (Animal Health Laboratory, University of Guelph, Guelph, Ontario) for serological testing. Serum from the aborted ewe tested negative for border disease. The standardized Toxoplasma gondii titer was 1:1024. Titers ≥ 1:512 are considered positive for toxoplasmosis. Serum samples from the 2 ewes and their weak lambs were negative for toxoplasmosis.

Microscopic examination of tissues from the aborted fetus and placenta showed evidence of necrotic placentitis. The placenta was diffusely edematous and contained a population of mononuclear and segmented leukocytes throughout the amnion. Necrosis along the chorion and basophilic granular debris, believed to be mineralization, was characteristic of toxoplasmosis; however, as no organisms were seen, a definitive diagnosis was not possible.

The same tissues were submitted for immunohistochemical testing for T. gondii (Prairie Diagnostic Services, Saskatoon, Saskatchewan). Brain, considered to be the most diagnostic sample, was not submitted. A definitive diagnosis was not made, as there was no immunohistochemical evidence of T. gondii in the tissues submitted.

Toxoplasma gondii is frequently implicated in ovine reproductive failure. The organism, an obligate intracellular protozoal parasite with a 2-stage life cycle, is capable of producing infection in humans (1). In the cat, which is the single definitive host for T. gondii, the organism undergoes both sexual and asexual life cycles. Mammals and birds are intermediate hosts in which T. gondii undergoes only the asexual life cycle (2). The sexual cycle is initiated when a susceptible cat ingests oocysts or tissue cysts. In the latter case, bradyzoites excyst from the tissue, penetrate the epithelial cells of the small intestine, and disseminate to other tissues, such as striated muscle and that of brain, where they encyst (3). In the enteroepithelial cells of the cat, the oocysts undergo gametogony, and the gametocytes are released into the lumen of the intestine and pass out in the feces. Sporulation occurs in the environment within 1 to 5 d, with each oocyst containing 8 infectious sporozoites. Four days after ingesting tissue cysts, the cat is capable of shedding millions of oocysts in the feces (3). Oocysts may be shed in smaller numbers for a few days, or not at all, 20 d after a cat ingests oocysts or tachyzoites (3).

Ovine infection with T. gondii produces variable clinical disease, depending on the ewe's immune and reproductive status and stage of gestation (4). Sheep may ingest sporulated oocysts in contaminated feed or pasture. The oocysts excyst in the small intestine, invade the intestinal mucosa, and multiply as tachyzoites in regional lymph nodes. Hematogenous spread of the tachyzoites establishes a generalized infection. Host immune response is mounted, and the tachyzoites are cleared from the system or transformed into bradyzoites (4). Bradyzoites, sequestered in tissue cysts, maintain the infection and a lifelong immunity (4). A nonpregnant ewe exposed to T. gondii will thus be immune to the organism and is refractory to toxoplasma-induced abortion during subsequent pregnancies (4). The naive and pregnant ewe is susceptible to toxoplasma-induced reproductive losses when parasitemia occurs and tachyzoites invade the uterus and placenta (2). The privileged immune status of the fetal placental unit allows infection to progress unhindered (4). The sequence of pathological events associated with T. gondii is determined by the stage of gestation at the time of infection. Fetal resorption, fetal mummification, abortion, stillbirth, and birth of weak lambs characterize the spectrum of clinical toxoplasmosis (3).

Approximately 5 d after infection, pregnant ewes infected with T. gondii develop pyrexia that lasts for 4 d (5). Infectious placentitis occurs, and abortion and stillbirth may be attributed to placental insufficiency (5). Pyrexia may also induce abortion in the acute phase of toxoplasma infection (5).

Several diagnostic tools exist for diagnosis of fetal loss induced by T. gondii. Histologic examination of the cotyledon may reveal multifocal, mineralized, necrotic lesions, highly suggestive of T. gondii infection, as was demonstrated in this case. Rarely, the organism may be visible on the periphery of the necrotic focus or in a villus in the early stages of infection (3). When the organism is not identifiable in hematoxylin and eosin-stained sections of severely decomposed tissues, immunohistochemical staining may be used to test for T. gondii antigen (6). The peroxidase-antiperoxidase technique identifies the organism or antigen residues in sections of formalin-fixed, paraffin-embedded tissues (6). A negative result may be due to poor sampling technique and tissue selection. Serologic testing is also an important diagnostic tool, but it must be carefully interpreted (7). Serum antibody titers to T. gondii may be detected by a fluorescent-antibody test. Titers are long lasting and indicate exposure; however, exposure to T. gondii prior to conception results in a high serum antibody titer and immunity to the parasite. In that case, the high titer is not diagnostic of reproductive failure due to T. gondii (7).

The presence of feral or domestic cats parallels the epidemiology of toxoplasmosis in sheep. Control of T. gondii must focus on farm management: specifically, reducing the presence of felids or feline fecal contamination of feeds, including pasture and hay. Tom cats and spayed female cats are an alternative to no cats. Providing litter boxes is one step towards minimizing fecal contamination. Top bales of hay should not be fed to pregnant ewes.

A live, attenuated vaccine containing the S48 strain (Toxovax; MAF, New Zealand) has been developed against T. gondii (8). Although the efficacy and safety of the vaccine has been proven, it is not yet available in North America (8). In an effort to produce a vaccine for both human and ovine use, research continues to focus on stimulation of protective immunity without induction of chronic subclinical infection in the form of tissue cysts (8).

Pharmaceutical control of toxoplasmosis is efficacious. The anticoccidial drug decoquinate (Deccox; Alpharma, Mississauga, Ontario) reduces the effect of toxoplasmosis in experimentally infected pregnant ewes (9). In one study, feeding decoquinate at 2 mg/kg bodyweight (BW) daily, prior to exposure to T. gondii, was associated with less placental damage attributed to toxoplasmosis, a longer mean gestation period, and increased birth weight and number of live lambs (9). Monensin (Bio Agri Mix, Mitchell, Ontario), a less expensive coccidiostat sometimes used in sheep, is equally effective against T. gondii, but due to its low margin of safety in sheep, it should not be used for this purpose (1). Decoquinate may be added as a premix to commercial rations, on the basis of calculated dry matter intake, to provide an effective anticoccidial dose of 2 mg/kg BW. If treatment begins by mid-gestation, ewes challenged with T. gondii become persistently infected, but reproductive losses associated with toxoplasmosis are reduced (1).

The autolytic changes characteristic of T. gondii infection may make field diagnosis of ovine abortion difficult. Farm and flock history, histopathologic examination, and serologic testing may or may not further diagnostic efforts, as the complex host-pathogen interactions depend on the host's reproductive status, immunity, and stage of gestation. Immunohistochemical testing may provide a costly but accurate diagnosis in spite of autolytic changes in the samples submitted. Samples of cotyledon and brain are the most important to submit when T. gondii abortion is suspected. In this case, a presumptive diagnosis of toxoplasmosis was made.