G. E. Cottral

The epizootiological importance of foot-and-mouth disease carriers

1. Susceptible and immunized cattle were exposed to FMDV in varying amounts, and by different routes, and a high percentage of cattle became carriers after pharyngeal or nasal exposure. 2. The percentage of virus “takes” was not altered appreciably by the immune status of the cattle; however, as could be expected, the susceptible cattle more often became clinically ill. 3. If only minimal amounts of virus were inoculated, carriers were sometimes produced without overt disease. In immune cattle, this direct establishment of asymptomatic carriers appeared to be the rule. 4. In immunized cattle, the number of virus “takes” was somewhat reduced when low doses of virus were inoculated by the pharyngeal or nasal route. 5. If the virus was able to establish itself in the pharynx of immunized cattle, active virus multiplication took place in spite of preinfection serum antibody and the absence of clinical signs. Susceptible and immunized cattle were exposed to FMDV in varying amounts, and by different routes, and a high percentage of cattle became carriers after pharyngeal or nasal exposure. The percentage of virus “takes” was not altered appreciably by the immune status of the cattle; however, as could be expected, the susceptible cattle more often became clinically ill. If only minimal amounts of virus were inoculated, carriers were sometimes produced without overt disease. In immune cattle, this direct establishment of asymptomatic carriers appeared to be the rule. In immunized cattle, the number of virus “takes” was somewhat reduced when low doses of virus were inoculated by the pharyngeal or nasal route. If the virus was able to establish itself in the pharynx of immunized cattle, active virus multiplication took place in spite of preinfection serum antibody and the absence of clinical signs.

Improved techniques for the detection of foot-and-mouth disease virus in carrier cattle

A cup probang was used to collect oesophageal-pharyngeal fluid specimens from cattle carriers of foot-and-mouth disease (FMD) virus (Type A). The specimens were assayed for virus content by inoculation of primary calf kidney cell cultures in prescription bottles and in Povitsky bottles for assay of greater volumes. The efficiency of the techniques was improved by treating the specimens with trichlorotrifluoroethane (TTE) prior to assay. With the TTE-treatment, bacterial and fungal contaminants were virtually eliminated and FMD virus apparently was reactivated from neutralizing antibodies or other inhibitors. Aliquot specimens stored at −20° C for more than 2 months did not have an appreciable loss in titer when the TTE treatment was used. Specimens with 50% glycerin added maintained their original virus titers during prolonged storage at −20° C.

Foot-and-mouth disease virus in semen of bulls and its transmission by artificial insemination

Semen from 16 bulls experimentally infected with foot-and-mouth disease virus (FMDV) was examined for virus content and its ability to produce FMD in heifers by artificial insemination. FMDV appeared in semen of 2 bulls as early as 12 hours after inoculation and, thereafter, virus was found in 58 of 71 semen samples from 16 bulls for as long as 10 days. The highest titer in semen was 105.8 mouse LD50/ml and the titer usually was higher than in urine and sometimes higher than in blood samples taken simultaneously. Five of 16 heifers artificially inseminated with semen from infected bulls and 5 of 10 heifers inseminated with FMDV in various diluents developed FMD. It was concluded that semen of bulls could contain FMDV prior to signs of illness and that the disease could be transmitted by artificial insemination. The dilution and freezing techniques commonly used to preserve semen also favor survival of FMDV. The virus in semen is primarily in the fluid portion, but antiserum added to semen could not be relied upon to eliminate it. The semen of some FMD convalescent bulls apparently contains antibodies.

Relationship of foot-and-mouth disease virus plaque size on cell cultures to infectivity for cattle by intramuscular inoculation

With sixteen virus strains of cattle origin representing all seven types of foot-and-mouth disease virus (FMDV), infectivity for cattle by intramuscular (i.m.) inoculation was significantly related to plaque size on primary calf kidney cell cultures; the larger plaque viruses were more infectious. This relationship was not observed when these virus strains were inoculated into cattle tongues, mice or cell cultures (by CPE technique). For the 16 strains, the approximate minimum infective doses, expressed in bovine ID50 units, varied from 10 to 10,000 units for i.m. inoculation and from 0.01 to 2.0 units for tongue inoculation of cattle. The tongue route of inoculation for FMDV in cattle was from 100 to 200,000 times more sensitive for producing infection than i.m. inoculation. Of 195 cattle given various doses of FMDV by i.m. inoculation, 154 were infected, 13 were only antigenically stimulated or immunized and 28 were not clinically or serologically affected; with some strains, virus doses of as much as 15,000 bovine ID50 units failed to elicit a detectable response. Five of the FMD viruses that had 20 or more serial passages by cattle tongue inoculation produced smaller plaques in calf kidney cell cultures and were less infectious for cattle by i.m. inoculation than four field strains that had two to three laboratory passages and probably had natural passage primarily in cattle. Thus, serial passage of FMD viruses by cattle tongue inoculation may not be the ideal method for retaining field strain characteristics. Also, the hosts of natural passage may influence results.

Foot-and-mouth disease virus carrier state in cells cultured from tissues of convalescent cattle

Cultures were made from mucosal tissues of the pharynx, esophagus, rumen and tongue of cattle convalescent (7 days) from foot-and-mouth disease (FMD) infection. The persistence of FMD virus in the cell cultures was demonstrated by fluorescent antibody technique and by subcultures in primary swine kidney cells using cytopathic effect and plaque assay techniques. Virus persisted in the cell cultures and was found in the supernatant fluids, in washed and lysed cells, and in cells by fluorescent antibody reaction of the various samples for the number of weeks indicated, respectively: tongue — 3, 3, 5; rumen — 5, 5, 7; pharynx — 25, 25, 24; and esophagus — 25, 16 (intermittently), 24. Interferon was not detected in the supernatant fluids of any of the cultures. The virus-laden cultures did not show gross signs of infection, whereas mucosal cultures from similar areas of normal cattle were destroyed in about 18 hours after inoculation with stock virus. Both the infected cultures and noninfected normal cultures deteriorated after 25 to 26 weeks. The carrier virus isolated at 22 weeks from the esophageal and pharyngeal cultures showed decreased pathogenicity to different primary and cell line kidney cultures and to mice, as compared to the parent virus. The carrier viral isolates were infective for cattle. However, esophageal-pharyngeal (EP) fluids from steers inoculated with the carrier virus isolated from the esophageal culture at 22 weeks contained more virus at 7 and 14 days than the EP fluids from a steer inoculated with the carrier virus isolated from the pharyngeal cell cultures.