Brucellosis should not be omitted entirely from a discussion on this subject. Terpstra (1956) apparently regards venereal Brucellosis as a fairly important cause of infertility in heifers. This opinion might not be widely shared. We know, however, that an infected bull can transmit "C.ab," through either natural or artificial insemination; and, further, that mechanical transmission from cow to cow via the bull can occur.
We know also that Brucellosis is extremely widespread in New South Wales, and accounts for a large proportion of recognised abortions. Blood (1957) reported that 50% of more than 50 consecutive abortion cases investigated at the University Farm Clinic were due to this disease, and that as a general rule 50% of cows tested were positive.
Increasing interest in Trichomoniasis and Vibriosis unfortunately seems often to be accompanied by a reduction in attention to the diagnosis of Brucellosis. Unless there is good reason for their omission, serum samples should always be included among specimens submitted from problem herds for laboratory examination. They can, incidentally, be tested for leptospiral as well as for Brucella antibodies if required.
Trichomoniasis also is known to be widespread and an important cause of bovine abortion and infertility in this State. Some indication of this may be gained from the facts that we recovered T. foetus from 16 sets of specimens in the first 16 weeks of the current financial year (1957/58), and that the Interstate Disease Notification for January, 1958, alone listed 35 "Tricho" herds in 16 Shires and Municipalities in New South Wales,
Venereal transmission is the rule in this case. Unlike Brucellosis, again, it is not communicable to man, the various trichomonads often found in humans being of different types.
As with Brucellosis, most cows soon "learn to live with" Trichomoniasis: heifers and susceptible introduced females being mainly affected when the infection has been present in a herd for some time. This should be borne in mind when selecting individuals for examination and "sampling". Whereas Cows which have "recovered" from Brucellosis remain "carriers", however, and may discharge as many of the organisms at full-term calving as they would have done had they aborted (Gregory, 1956), the majority of those recovering from Trichomoniasis soon throw off their infection. Also, their acquired "immunity" is rather transient, in the absence of further contacts. While an infected bull remains in a herd, he provides such contacts. Unfortunately, unless mating is very carefully controlled, he serves as a source of infection for heifers, other susceptible females, and indirectly for other bulls.
Trichomoniasis may appear suddenly in a herd to which there have been no recent introductions, and its source may seem something of a mystery if neighbouring farmers report no significant breeding trouble in their own herds. It might be found, however, that one of these has a "dry run", to which heifer to which heifer calves are sent after weaning, there to remain until nearly ready to calve themselves. In one such instance recently investigated in the area of the District Veterinary Officer, Grafton, it was found that the heifers were usually about 3 years of age before coming in from the dry run, which was heavily timbered; and the cattle on it received little supervision. In such a case it could quite well be that the heifers conceived, aborted, developed an immunity, conceived again to an infected bull and held. The immunity being reinforced by subsequent service after each calving, herd breeding results might be satisfactory, the owner thinking only perhaps that his stock were comparatively slow in reaching sexual maturity. In such case, too, incidentally, attempts to control the disease might lead to increased trouble if they were not thorough, owing to an increase in the number of susceptible animals before the infection had been completely eradicated from the herd.
Artificial insemination, where practicable, is highly desirable. In the unfortunate event of an infected bull gaining entry to an A.I. centre some cows could become infected through insemination, though the semen dilution factor alone would ensure that the number was not large. The consequences would not be very serious in a herd in which there were no natural services, but could be calamitous where a bull was used in addition to A.I. There would be little to fear in the case of frozen semen, provided it contained not less than about 10% glycerol in a yolk/citrate diluent and had been subjected to a pre-freezing equilibrium period of at least 6 to 8 hours (Beattie, 1957). In the case of chilled semen, 15% glycerol should kill any trichomonads present, within a few hours, without causing appreciable harm to the spermatozoa. In 1949 we observed that T. foetus could remain viable in ordinary y-c semen diluent in a refrigerator for at least 4 or 5 days. Later we found them to persist in culture media containing 4% glycerol, but not 10%. In due course freezing trials were carried out, in which some were recovered and sub-cultured on thawing after freezing to -79 C. in the presence of various concentrations of glycerol; the pre-freezing equilibrium period being omitted (Blackshaw & Beattie, 1955). It is stressed, however, that very careful precautions, including test-mating, are taken to prevent the entry of infected bulls to A.I. centres in N.S.W.
Confirmation of diagnosis of Trichomoniasis rests upon the recovery and identification of the casual protozoon, Trichomonas foetus, with its undulating membrane, rotation on its long axis during progression, and single posterior and three anterior flagella. (NOTE: Contaminated specimens may contain other protozoa. In this connection it is of interest that Pavlov & Dimitrov (1955) considered "Trichomonas foetus, sp.n." in the intestines of calves, in Bulgaria, to be a cause of gastro-enteritis, with fever and haemorrhagic diarrhoea; the calves being mainly 5 to 7 days but cccasionally up to 4 months of age.
Sulphaguanidine at the rate of 4 x 1 gm. daily for 2 to 4 days was found useful in treatment). These organisms usually abound in the foetal organs and uterine fluids at the time of a trichomonad abortion, found in specimens secured about 48 hours later, though reappearing in the vagina at various intervals subsequently.
A fresh foetus, or pipettes of its stomach contents, and uterine fluid taken within about 24 hours after abortion, are thus suitable for laboratory examination. No preservative should be used, though adding portion of any suitable specimen to a T.f. culture medium—e.g. 2% glucose broth—is an advantage if more than a day or two is likely to elapse before the examination can be undertaken.
Microscopic examination on the spot is desirable, as trichomonads sometimes die and become unrecognisable within 2 or 3 days. In some discharges, etc., however, they may remain viable for up to 5 or 6 days at atmospheric temperatures, and longer if kept cooler. Recently we found some trichomonads still active in a sample of pyrometron pus on warming after 13 days in an open pipette which had been lightly wrapped in cotton wool and stored in a refrigerator.
Packing with ice for long journeys may be helpful with some specimens, though dry ice would freeze and kill any T. foetus organisms present. Where culture medium containing antibiotics to control contaminants is used, a warm environment is preferable; encouraging multiplication of the protozoa. Only in rare instances, however, have we found T. foetus in field-inoculated media without detecting them also in the "straight" specimen, given the opportunity. Occasionally we find them in the latter, but not in the former. Thus it would be reasonable in many cases to send a single jar of culture medium containing mucus from several cows, plus individual samples without medium or other additive. The latter could be left in the plastic A.I. pipettes, where these are used, which can be cut short enough to fit in a standard specimen box and plugged at each end with clean cotton wool and/or covered with cellulose tape.
In some cases, Trichomonas infection in a bull can be demonstrated fairly readily by means of preputial washings or scrapings, though test-mating is a far surer method. Washings can be secured with the aid of a flutter-valve apparatus fitted with a 6-inch length of half-inch glass tubing. The sheath orifice is held tightly around the latter to reduce leakage while sterile normal saline, buffered saline, or broth is run in from a 6 or 8 oz. bottle and back again after vigorous massage. A preliminary cleansing of the area with water, preferably warm, often induces urination; reducing the likelihood of this occurring during the operation.
Other outfits designed for this purpose which are in use to-day include the following:
An American telescopic tube in 3 sections. This is used with a plastic bulb on one end and a thin rubber stall on the other. It is a little awkward to manage, and involves some risk of injuring the bull. The end of the first tube is inserted into the sheath. The second tube is forced through the first, breaking the rubber cover, and avoiding contact with the most contaminated portion of the sheath. The innermost, third section is then protruded through the second, to scrape the glans and sheath lining while alternatively squeezing and releasing the bulb. The third and second tubes are then retracted in turn so that both are covered by the first before withdrawing the ingenious contraption from the sheath.
The Hammond-Bartlett preputial pipette is of glass tubing and thus is rather fragile and risky for the job. About 20 inches long, it has a slight bend about 31 inches from the free or scraping end, which is flattened. A rubber bulb is employed to draw an inch or so of smegma and scrapings into the tube. Plastic tubing would be preferable, but we have not located a source same of suitable diameter and which would retain the required shape.
Scrapings have the advantages, over washings, of no dilution and usually less contamination. Active spermatoza often present in either render direct examination for trichomonads tedious and difficult. Sometimes, too, ciliated epithelial cells may be present, and rather resemble protozoa at first glance; a pitfall for the unwary.
When the first method is used the bottle of washings can be sent as it is, or several mls. of cellular deposit may be pipetted from the bottom after it has been allowed to settle. A drop of this should be examined microscopically soon after collection, if practicable. It is desirable also to add 1 ml. or so of T.f. culture medium containing streptomycin and penicillin, to forward with the remainder of the reduced specimen. Such treatment, incidentally, renders this portion of the sample unsuitable for examination for Vibrio fetus. Any of the latter organisms present in the washings are likely to die within 24 hours or so, in any case, even if buffered saline (pH7) or broth is used. Thus smears also should be sent, and chilling of the washings is an advantage.
For test-mating, virgin heifers are best, preferably not less than 12 months of age. They should be examined, and "sampled" between the 10th and 18th days after service. If a group is available it may be helpful to select several in which full-sized corpora lutea can be felt and readily enucleated, with the object of inducing oestrus at a convenient time. About 9 to 14 days after heat is a suitable stage. Expression of the corpora should result in heat signs in at least one or two of the heifers 3 or 4 days later. If the feel of the ovaries and the results of manipulation P.R. leave much doubt as to the outcome, about 30 mgm. of stilboestrol may be administered. The latter alone will often induce heat, sometimes within 24 hours or so. The chances of ovulation and conception then will be very remote. Natural heats should follow at normal intervals, unless influenced by an infection such as Vibriosis: but stilboestrol might upset the cycle. Used in the presence of a well-developed corpus, in fact, it might lead to the ovary becoming cystic.
When dealing with a problem herd, any apparently abnormal discharge calls for examination, as does any female which has been served about 10 to 18 days previously, and any other whose history singles her out for attention. Great care should be taken to avoid risk of transferring infection from animal to animal via the hands or instruments. Soap, water and nail brush are useful for the hands, as also is methylated spirits. Special care is necessary with instruments, but any disinfectant used should be removed before the instrument is introduced into the breeding organs.
If looking mainly for trichomonads, and especially when miscroscopic examination is being carried out immediately, samples of vaginal material may be taken with the aid of a speculum alone, or even by hand if required, into a fairly wide-mouthed, clean jar.
Where Vibriosis is suspected, contamination should be avoided as far as possible; V. fetus being a slow growing organism requiring reduced oxygen tension, and easily swamped by others. Dried smears sent in addition to main samples may give a better indication of the original bacterial flora. Procedures at the laboratory will usually include plating out on blood agar and incubating under 10% CO2 for a week or two. Brucella organisms, if present, should show up in 2 days, Vibrio colonies seldom appear before the fifth day in original cultures. Contaminants such as coliforms grow profusely overnight. Some types are rapid spreaders, and will cover the entire surface of a plate in a very short time.
To minimise contamination when securing vaginal material, we generally use a sterile A.I. pipette, taking care to hold the vulval lips apart both when introducing and when withdrawing the pipette. Before commencing, a 10 ml. syringe is attached by means of about 6 inches of rubber tubing. This allows negative pressure to be applied while the free end of the pipette is up near the cervix, but the pressure should be brought to normal again, by depressing the plunger, before the pipette is withdrawn from the vagina; otherwise the mucus tends to rush up into the rubber tube and syringe, which means that the same ones with a fresh pipette cannot be used for the next cow.
Vaginal mucus is not always obtained readily, or in great volume. Usually, however, sufficient can be secured even when only a small amount of very viscid mucus is present, provided the pipette is rotated and moved back and forth over the vaginal floor perseveringly.
If difficulty is experienced often, pipettes of larger diameter may be preferred. These generally are more fragile. Further, they cannot normally be passed into or through the cervix, as may be desired in some instances. Being of glass, they are sterilised and used again many times, as can glass but not plastic A.I. pipettes.
That designed by Bartlett is sealed over at one end, but has a hole in the side near that end, and is used with a rubber or plastic bulb on the other extremity. These are difficult to clean properly after use, and are more expensive than plain tubing to replace.
Pierce's vaginal pipette has a slight bend near the free end, which is not facilitate good contact (sic) with the vaginal floor. Instead of a rubber bulb on the outer end, Pierce attached a length of rubber tubing with a terminal, flanged glass mouth-piece. Negative pressure is then applied by the operator's mouth, which is protected by a cotton wool plug in the tube. This outfit, also, can be sterilised and used many times.
Generally speaking, voluminous samples of fluid mucus are most readily obtained at or near oestrus. Such samples are suitable for examination for vibrio organisms, but not for mucus agglutination tests. Mucus for the latter should not be secured within a day or so before or after oestrus, should not contain appreciable amounts of pus or blood, and should be of reasonable volume—say 1 ml. or more. (NOTE: While discussing trichomoniasis, Dennis (1958) drew attention to the fact that non-specific agglutinins found in all cattle sera may give rise to false positive reactions in the case of mucus samples containing blood).
Owing to the difficulty sometimes experienced in securing mucus per pipette between heats, tampons are often preferred in the field, though the pipette sample seems superior from the point of view of the test and its interpretation. Having been deposited in the anterior chamber of the vagina, preferably by being pushed by means of a rod through a clean metal, glass, or even cardboard (used once only) cylinder, the tampon should be left there for 20 to 30 minutes, the attached string or tape being folded in, to reduce contamination, or cut off afterwards. Each tampon is usually forwarded in a separate jar containing a measured volume of formol saline. This means that there will be no trouble due to bacterial growth en route to the laboratory. At the same time, of course, it renders the sample useless for cultural examination; and some of the samples might otherwise have yielded more valuable information from cultures than from the agglutination test.
If the mucus agglutination test is to be of any value, a number of samples is required, not less than 10 or 12 for preference, and several visits may be necessary (Murnane, 1957). The samples should be from carefully selected animals in carefully selected herds for which a full and reliable breeding history is available.
At Glenfield, at present large numbers of mucus samples could not be submitted to agglutination test without serious interference with other infertility work. To be swamped with samples, the majority of the tests of which would be virtually meaningless, as past experience suggests could easily happen, would be most undesirable. The main weight of the diagnostic spade work still rests on the broad shoulders of the veterinarian in the field.
With a view to undertaking some mucus agglutination tests, on as sound a basis as possible, special investigations are in progress; Dr. Jakovljevic in particular specialising on this work at Glenfield. One aim is to learn more regarding what vibrios are present in this State, and their pathogenicity and antigenicity. Simply to use one or more imported strains, such as the English "1980" or "Longsdon", for preparation of antigen, without more information of this type, does not appeal.
In December last, we recovered a vigorous vibrio from a vaginal sample from a cow in a herd under quarantine for proven Trichomoniasis, which had aborted 3 days previously. It might be mentioned that various authorities warn that especial care is called for in interpreting vibriosis agglutination tests in areas in which Trichomoniasis also might exist. Further non-pathogenic vibrios are stated to be present not uncommonly in bovine vaginae (and preputial cavities).
Differentiation between pathogenic and non-pathogenic vibrios rests partly upon laboratory criteria. No test is infallible. We do not even know for certain that an apparently harmless vibrio could not become "pathogenic" in a different environment; or that sheep or some other animals could not, in certain circumstances, serve as reservoirs of infection for cattle. (Note: Easterbrooks et al (1955) state that vibriosis is a genital disease of cattle, sheep, goats and possibly humans, adding that horses and pigs also may be susceptible). It might be mentioned further, that it is surprising how frequently some other factor also is found in vibrio positive herds, e.g., Trichomoniasis, or Brucellosis, or in South Africa, the virus (?) of "epivag".
On the laboratory side, since the well-known work of Frank and Bryner (1955), it has been usual to consider catalase positive and hydrogen sulphide negative vibrios as pathogenic and catalase negative, H2S positive ones as nonpathogenic; with particular emphasis on the catalase test.
Our special vibrio proved strongly positive to both the above tests. It also reduced nitrates to nitrites, while our "1980" and "Longsdon" strain sub-cultures did not. Its ready and vigorous growth, both before and after lyophilisation, makes it suitable from that point of view for the preparation of antigen. Evidence to date suggests that such antigen would give a comparatively large proportion of positive reactions. It seems, however, that some such reactions might lead to the indictment of innocent bulls.
Terpstra (1956) recovered vibrios which were both catalase positive and H2S-positive from 35 bovine vibrionic abortion cases. While believing that such vibrios were responsible for sporadic abortions, he did not accept these as evidence that bulls in the herds concerned were responsible for enzootic infertility.
Among many things to be realised in connection with vibrio agglutination tests is that a cow giving a genuine positive reaction might by that stage have thrown off the infection and developed a fairly high degree of immunity and thus be a very good one to retain in the herd; whereas some of the negatively reacting samples might easily be from cows in early and highly infective stages of the disease.
Agglutinins generally appear between one and four months after primary vibrio infection, but in some cases are not demonstrable until later than this. They may persist in the mucus for 2 to 9 months, and occasionally for 5 years or more. Their presence does not necessarily indicate immunity. On the average, resistance seems to persist for from one to three years. Infection with one strain may not induce a resistance to other, antigenically different, strains. Evidence available to date suggests that an occasional cow will carry the infection through a full-term pregnancy and perhaps for a few months afterwards, but not through two pregnancies.
Numerous and serious though problems associated with bovine Vibriosis may be—and we have touched on only some of them there is a brighter side. Control measures instituted on suspicion of its presence can be very helpful quite rapidly to the farmer. Such measures usually would include drug treatments, and division into two herds so far as breeding is concerned, with the use of A.I. if practicable.
In his report for January, 1958, the District Veterinary Officer, Grafton, mentioned that in a Tweed-Lismore district herd, in which it was suspected fertility had been due to a venereal infection, all cows conceived following vaginal irritation by the owner with streptomycin and penicillin two hours before service. This spectacular result sounds too good to be true; but streptomycin in particular does seem to be remarkably effective prophylactically against Vibriosis, and quite helpful also in treatment. A good response often is taken, in fact, as evidence of the presence of Vibriosis; and not unreasonably. though such treatment may be effective also against various non-specific infertility infections. Similarly, positive agglutination reactions may be taken as suggestive evidence but not as proof of the presence of Vibriosis in the herd.
Twenty mls. of sterile water containing one gram of streptomycin, and perhaps also about half a million units of penicillin, administered within 24 hours of service, is very useful prophylactically. One-third may be infused into each horn of the uterus and one-third into the cervical canal and around the external os.
It seems that many though not all females in which the infection has become established can be cured by similar treatment; repeated, after 48 hours, or daily for 3 days, at any stage of the oestrous cycle. One gram of streptomycin, or 0.5 gram aurcomycin or terramycin, in either aqueous solution or oily suspension, may be used. Some proprietary preparations, also have shown promise. Treated animals still should be regarded with suspicion, even in spite of pregnancy, as clinical cure does not necessarily indicate absence of the organism.
Most infected bulls remain permanent carriers, though some cases of apparent spontaneous recovery have been recorded. Such bulls are thought not to be immune from re-infection, especially by different strains. Various forms of local treatment of bulls have been reported as successful in some instances, and a degree of success sometimes is claimed even for systemic treatment, e.g. 5 to 10 mgm. streptomycin per kilo. body weight daily for 3 days subcutaneously. Given sufficient time and care, treatment might succeed with some bulls. More often perhaps it would simply cause the infection temporarily to be non-demonstrable, possibly for several or many months. Tests, including mating with virgin heifers, would need to be continued for long periods, or repeated after a long interval, in order to be sure that treatment had been effective. Evidence suggesting the possibility of direct transmission from bull to bull creates further confusion. As in the case of Trichomoniasis, treatment of bulls for Vibriosis is not approved, or not encouraged, in New South Wales at the present time.