Tritrichomonas foetus

Synonyms: Trichomonas uterovaginalis vitulae, T. bovis, T. genitalis, T. bovinus, T. mazzanti.

Disease: Bovine trichomonad abortion, bovine genital trichomonosis.

Hosts: Ox, zebu, possibly pig, horse, roe deer.

Location: Genital tract.

Geographic Distribution: Worldwide. Morgan and Beach (1942) mapped the geographic distribution of bovine trichomonosis.

Prevalence: Altho T. foetus is known to be widely distributed, few studies have been made of its incidence. It is especially common in southern Germany and Switzerland, up to 30% of the cattle having been found infected in some areas. In the U.S., it is probably third to brucellosis and leptospirosis as a cause of abortion in cattle. In a survey of 383 beef bulls in Utah, Idaho and Colorado, Fitzgerald et al. (1958) found 6% to be infected.

The USDA Agricultural Research Service (1954) estimated that bovine trichomonosis causes an annual loss of $750,000 in the United States. This figure is probably low. Fitzgerald et al. (1958) estimated that losses in production because of reproductive disorders in cows bred by infected western range bulls may amount to about $800 annually per infected bull. On this basis, the 23 infected bulls they found in their survey would cost their owners about $18,000 a year. The annual loss due to an infected bull in an artificial insemination ring would be considerably greater than this.

Tritrichomonas foetus

Morphology: The morphology of T. foetus has been studied by Wenrich and Emmerson (1933), Kirby (1951) and Ludvik (1954), among others. The body is spindle- to pear-shaped, 10 to 25 u long and 3 to 15 u wide. It has 3 anterior flagella and a posterior flagellum which trails as a free flagellum about as long as the anterior flagella. The undulating membrane extends almost the full length of the body and has an accessory filament along its margin. The costa is prominent. The axostyle is thick and hyaline, with a capitulum containing endoaxostylar granules and a chromatic ring at its point of emergence from the posterior end of the body. The parabasal body is sausage- or ring-shaped. A cytostome is present. There is no pelta.

Pathogenesis: A great deal has been written about bovine trichomonosis. Morgan's (1946) review listed 447 references, and many more papers have been published since then. Among more recent papers on its pathogenesis are those of Morgan (1947), Bartlett (1947), Bartlett and Dikmans (1949), Bartlett, Moist and Spurrell (1953), Laing (1956) and Gabel et al. (1956).

Bovine trichomonosis is a venereal disease, transmitted by coitus. It can also be transmitted by artificial insemination. Non-venereal transmission is very rare under natural conditions. After infection of the female, the trichomonads multiply at first in the vagina, causing a vaginitis. They are most numerous here 14 to 18 days after infection (Hammond and Bartlett, 1945). They invade the uterus thru the cervix. They may disappear from the vagina or they may remain there, producing low-grade inflammation and catarrh.

Early abortion is characteristic of bovine trichomonosis. Abortion usually occurs 1 to 16 weeks after breeding. The foetus is often so small that it is not observed by the owner, and he does not realize that abortion has occurred, believing that the animal failed to conceive and that its heat periods are irregular. Morgan and Hawkins (1952) knew of only 6 reports in the literature of abortion due to T. foetus after 6 months gestation.

If the placenta and fetal membranes are completely eliminated following abortion, the animal usually recovers spontaneously. This is the most common course. If, however, part of the placenta or membranes remain, a chronic catarrhal or purulent endometritis results which which may cause permanent sterility.

Sometimes the animal does not abort, but the fetus dies and becomes macerated in the uterus. Pyometra results, and the uterus may contain several quarts of a thin, greyish white fluid swarming with trichomonads. In the absence of bacteria, this fluid is almost odorless. The cervical seal may remain intact or it may allow a small amount of fluid to escape when the animal is lying down. Animals with pyometra seldom come in heat, and the owner may believe them to be pregnant. In longstanding cases, the trichomonads may disappear from the uterine fluid. Occasionally normal gestation and calving may occur in an infected animal, but this is rare.

In the bull, the most common site of infection is the preputial cavity, altho the testes, epididymis and seminal vesicles may sometimes be involved. Spontaneous recovery is rare; bulls remain infected permanently unless treated. The numbers of trichomonads fluctuate, the intervals between peaks being 5 to 10 days according to Hammond et al. (1950).

Immunology: Cows or heifers which recover from infection are usually relatively immune, altho reinfections can occur. A number of investigators have studied various immunological responses to trichomonad infection. Kerr and Robertson (1945) showed that there is more than one serological strain of T. foetus. McEntegart (1956) found that T. foetus var. belfast and T. foetus var. manley differed serologically from each other and from T. vaginalis. Menolasino and Hartman (1954) were unable to distinguish T. foetus from T. vaginalis serologically, but McDonald and Tatum (1948) and Schoenherr (1956) were able to do so. Both also found serological differences between T. foetus and Pentatrichomonas hominis, and the latter between T. foetus and Trichomonas gallinae. Sanborn (1955) found that T. foetus differed serologically from the large pig cecal trichomonad, T. suis and from the pig nasal trichomonad.

Kerr and Robertson (1941, 1943) and Pierce (1947) studied the agglutination test in cattle, and Feinberg (1952) described a capillary agglutination test. Kerr (1943) felt that his test was positive in about 60% of all infected cattle, but Morgan (1943a) considered it impractical. The wide distribution in the animal kingdom of nonspecific antibodies against T. foetus was brought out by Morgan (1944), who showed that the sera of the carp, horned lizard and leopard frog agglutinated T. foetus at 1:2, those of the gold fish, pigeon and domestic rabbit at 1:4, those of the guinea fowl and chicken at 1:8, those of the turkey and sheep at 1:16, those of deer and goat at 1:32, that of the cow at 1:128 and that of the horse at 1:1024.

Nakabayasi (1952) distinguished between agglutination and agglomeration. With immune rabbit and infected guinea pig sera, agglomeration reached its maximum within 30 minutes and then decreased gradually as the agglomerated individuals separated. On the other hand, the agglutination reaction reached its maximum within about an hour and did not reverse. Levine et al. (unpublished) have seen agglomeration of T. foetus following mixture with fresh culture media containing inactivated serum.

Kerr and Robertson (1954, 1956) found "normal agglutinin" in the blood of calves which they apparently acquired in the colostrum; this agglutinin disappeared after 17 to 55 days. Injection of calves less than 4 weeks old did not induce antibody formation, but instead caused impairment of antibody production (immunological paralysis) which persisted for about 2 years.

Complement fixation and precipitin reactions have been studied, but with unsatisfactory results (Svec, 1944; Morgan, 1948).

Kerr (1944) developed an intradermal test, using a trichloracetic acid-precipitated extract of T. foetus called "tricin". Positive reactions appear in 10 minutes, reach their peak within 30 minutes and disappear in about 6 hours. Fifty of 592 cows at an abattoir were positive to this test, and trichomonads were found in 11 of them on direct examination. Trichomonads were also found in 11 of 34 bulls which were positive to the skin test. Morgan (1948) obtained negative results with skin tests with a number of different antigens. Kerr, McGirr and Robertson (1948) found that cattle could be desensitized to the skin test by injecting antigen intramuscularly, instilling it into the uterus of non-pregnant cows, or by injecting adreno-cortical hormone or sphingomyelin at parturition. Absorption of antigen from acute uterine infections also desensitized the animals.

A local immune reaction takes place in the vaginal mucosa of infected animals. In addition, the uterine mucosa is sensitized (Kerr and Robertson, 1953). The presence of agglutinins in the vaginal mucus prompted the development of a mucus agglutination diagnostic test by Pierce (1947a, 1949) and Florent (1947, 1948, 1957). This test is considerably better than the blood agglutination test, but, according to Pierce (1949), must still be regarded as only a herd test because a number of infected animals fail to react. Unsatisfactory results are obtained with estral and post-estral vaginal mucus and with purulent uterine mucus containing trichomonads. Mucus from pregnant animals sometimes gives a false positive reaction. Schneider (1952), too, considered the mucus agglutination test simply an adjunct to other means of diagnosis.

Morgan (1947a) found that a series of 16 intramuscular or intravenous injections with living T. foetus over a period of 3 months apparently protected heifers temporarily against genital infection, but 6 intramuscular injections over a period of 3 weeks did not. This does not appear to be a practical method of prevention.

Epidemiology: Bovine trichomonosis is a venereal disease transmitted at coitus. T. foetus is known to occur in cattle, but whether it is also present in other animals and whether it may be transmitted from them to cattle by a non-venereal route remain to be determined. With the introduction of the technic of preserving bovine semen by freezing in the presence of glycerol, the question arose whether T. foetus would survive in frozen semen. Several investigators have studied the problem, and have found that the protozoa may or may not survive freezing in the presence of glycerol, depending on the conditions (see Levine, Mizell and Houlahan, 1958 for a review of the literature). They survive in some media but not in others. Rapid freezing and high salt concentration are deleterious (Levine and Marquardt, 1955; Levine, Mizell and Houlahan, 1958). The stage of the population growth curve is important, the protozoa being much more sensitive to injury when frozen during the initial and logarithmic phases than at the peak of the curve and for some time thereafter (Levine, McCaul and Mizell, 1959). Temperature fluctuation during storage is deleterious (Fitzgerald and Levine, 1961).

A particularly interesting fact is that glycerol appears to be toxic at refrigerator temperatures but not at either sub-freezing or incubator (37° C) temperatures (Joyner, 1954; Joyner and Bennett, 1956; Fitzgerald and Levine, 1961). It may be possible to develop a technic for freezing semen which would be sure to kill the protozoa, but at present the use of frozen semen from infected bulls cannot be recommended.

Many different laboratory animals can be infected experimentally in various ways with T. foetus (see Morgan, 1946 for review). Leaving aside other routes of infection, successful vaginal infections with T. foetus have been established in the rabbit by Witte (1933) and others, in the guinea pig by Riedmiiller (1928) and several others, in the golden hamster by Kradolfer (1954) and Uhlenhuth and Schoenherr (1955), in the dog by Trussell and McNutt (1941), in the goat by Wittfogel (1935) and Hammond and Leidl (1957), in the sheep by Wittfogel (1935) and Andrews and Rees (1936), and in the pig by Hammond and Leidl (1957). The golden hamster is the laboratory animal of choice for experimental vaginal infections. Abortions were produced in some of the infected guinea pigs. Laboratory mice and rats are refractory to vaginal infection.

Kiist (1936) found trichomonads similar to T. foetus in the genital tract and aborted fetuses of swine and horses in Germany. Petersen (1937) cultured trichomonads resembling T. foetus from the genital tracts of 13 mares with pyometra. He also found an infected stallion which had transmitted trichomonads to mares. Schoop and Oehlkers (1939) also found trichomonads in the genital tract of horses. Schoop and Stolz (1939) found trichomonads resembling T. foetus in the uteri of 4 out of 5 roe deer in Germany. The infections were associated with sterility, and the trichomonads produced vaginitis in guinea pigs. Schoop (1940) suggested that if the trichomonads from deer were T. foetus, deer might be a source of infection for cattle.

The relation of T. foetus to the trichomonads of swine still remains to be elucidated. The pig nasal trichomonad, Tritrichomonas suis, greatly resembles T. foetus morphologically (Buttrey, 1956) and in metabolic characteristics (Doran, 1957, 1959), and vaginal infections were readily established in cattle with it by Switzer (1951) and Fitzgerald et al. (1958). The infection reported by Switzer lasted 3 weeks and was accompanied by a mild catarrhal vaginitis. Those reported by Fitzgerald et al. lasted 46 to 133 days, and some infections appeared to interfere with breeding efficiency.

Vaginal infections of cattle with trichomonads from the cecum and stomach of swine have also been readily established (Switzer, 1951; Hammond and Leidl, 1957a; Fitzgerald et al., 1958), and the latter two authors reported that bulls became infected by breeding infected heifers and then transmitted their infections to other heifers. The bulls later recovered spontaneously in both studies. Kerr (1958), too, infected heifers intravaginally with trichomonad from swine, using both strains obtained from Hammond and a strain of T. suis isolated in England. He found that the vaginal mucus agglutination test of heifers infected with porcine trichomonads was positive with T. suis and Belfast strain T. foetus antigens but not with Manley strain T. foetus antigen. In the other direction, Fitzgerald et al. (1954) produced cecal infections with T. foetus in young pigs.

Robertson (1960) made a serologic comparison of the Belfast and Manley strains of T. foetus and Strains S2 and 414 of T. suis, isolated by Hammond and Leidl from the ceca of pigs in Germany and Utah, respectively. Using both the tube agglutination and precipitin tests and especially the gel diffusion precipitin test, she found considerable cross-reaction between the 4 strains. All had the same major protein antigens, but they shared their major polysaccharide antigens only partially. The 2 bovine strains were readily distinguished from each other, while the 2 porcine strains were very closely related but not identical. The porcine strains were more closely related to the Belfast than to the Manley strain of T. foetus. Robertson concluded that the serologic distinctions between the 4 strains did not justify separating them into 2 species, and she called them all T. foetus.

Diagnosis: Altho the mucus agglutination test and a number of other serological procedures have been suggested for diagnosing T. foetus infections, the only sure method is to demonstrate the protozoa microscopically either directly or in culture. Diagnostic procedures have been described by Hammond and Bartlett (1945), Morgan (1945), Bartlett (1949), Fitzgerald et al. (1952) and Thorne, Shupe and Miner (1955), among others.

In heavy infections, particularly of females, the trichomonads can be seen by direct examination of mucus or exudate from the vagina or uterus, amniotic or allantoic fluid, fetal membranes, placenta, fetus stomach contents, oral fluid or other fetal tissues, or, in bulls, of washings from the preputial cavity and rarely seminal fluid or semen. If trichomonads cannot be found on direct microscopic examination, cultures should be made in CPLM, BGPS or Diamond's media (see below).

Samples can be obtained from the vagina by washing with physiological salt solution in a bulbed douche syringe. They can be obtained from the preputial cavity with a cotton swab or, better, by washing with physiological salt solution in a bulbed pipette or syringe. The washings should be allowed to settle for 1 to 3 hours or centrifuged before examination.

The external genitalia should be cleaned thoroughly before the samples are taken in order to avoid contamination with intestinal or coprophilic protozoa which might be mistaken for T. foetus. Among these are Tritrichomonas enteris, Monocercomonas ruminantium, Protrichomonas ruminantium, Bodo foetus, B. glissans, Spiromonas angusta, Cercomonas crassicauda, Polytoma uvella, Monas obliqua and Lembus pusillus. In identifying T. foetus, it must be distinguished from these.

Trichomonads are most numerous in the vagina 2 to 3 weeks after infection. Their numbers fluctuate in bulls, the interval between peaks being 5 to 10 days (Hammond et al., 1950).

A single examination is not sufficient to warrant a negative diagnosis. A cow can be considered uninfected if, after at least 3 negative examinations, she has 2 normal estrus periods and subsequently conceives and bears a normal calf; she should be bred by artificial insemination to avoid infecting the bull. A bull can be considered negative if, after at least 6 negative examinations at weekly intervals, he is bred to 2 or more virgin heifers and they remain negative.

Cultivation: T. foetus can be readily cultivated in a number of media. Among them are CPLM (cysteine-peptone-liver extract-maltose-serum) medium (Johnson and Trussell, 1943), BGPS (beef extract-glucose-peptone-serum) medium (Fitzgerald, Hammond and Shupe, 1954) and Diamond's (1957) trypticase-yeast extract-maltose-cysteine-serum medium. T. foetus was first cultivated in tissue culture by Hogue (1938). It was cultivated in the chorioallantoic sac of chicken embryos by Nelson (1938) and independently by Levine, Brandly and Graham (1939) and Hogue (1939).

Treatment: Since trichomonosis is ordinarily self-limiting in females, treatment is unnecessary. No satisfactory treatment is known for these infections. Many investigators have reported on treatment of T. foetus infections in bulls, including Bartlett (1948), Bartlett, Moist and Spurrell (1953), Mahoney, Christensen and Steere (1954), Thorne, Shupe and Miner (1955), Gabel et al. (1956) and Brodie (1960). Treatment is expensive, tedious and time-consuming; unless a bull is exceptionally valuable, it is best to sell it. Bartlett (1948) found that the German proprietary preparation, Bovoflavin-Salbe, cured 7 out of 8 infected bulls, and later workers have confirmed its effectiveness. This salve, which contains trypaflavine and surfen in an ointment base, is rubbed into the penis and prepuce following pudendal nerve block or relaxation of the retractor penis muscles with a tranquilizer. Brodie (1960) injected 200 to 1000 mg promazine hydrochloride intravenously for the latter purpose, and found that its ease of administration and quieting effect made it preferable to nerve block.

Massage is continued for 15 to 20 minutes, using 120 ml of the ointment. In addition, 30 ml of 1% acriflavine solution is injected into the urethra. Repeated treatment may be necessary. If the epididymis or testis are affected, this treatment will be ineffective.

For reasons which have not been determined, American bulls are much more refractory to treatment and much more difficult to infect experimentally than European bulls. Treatment with silver nitrate or by injecting 10 1 of 3% hydrogen peroxide into the preputial cavity under pressure with the apparatus described by Hess (1949), which is successful in Germany and Switzerland (Jondet and Guilhon, 1957), has been found unsatisfactory in the United States.

Control: Control of bovine trichomonosis depends on proper herd management. Most infected bulls should be slaughtered. Infected cows should be given breeding rest, and should then be bred by artificial insemination to avoid infecting clean bulls. Proper management of bulls used for artificial insemination is especially important, since they may spread the infection widely. They should be examined for T. foetus before purchase, and the herds from which they originated should be studied at the same time. In addition, they should be examined repeatedly while in use (Bartlett, Moist and Spurrell, 1953). Freezing the semen in the presence of glycerol cannot be expected to kill the trichomonads.