Excess of seminomas observed in Vietnam service U.S. military working dogs.

During the Vietnam War, US military working dogs served with their companion dog handlers in close proximity, sharing common exposures to war-related activity, many zoonotic infectious agents, chemical pesticides, phenoxy herbicides, and extensive use of therapeutic drugs. To gain insight into the effects of the Vietnam experience, we investigated the occurrence of neoplasms in military working dogs based on standard necropsy examination by the Armed Forces Institute of Pathology. We observed that these dogs experienced significant elevated risks for testicular seminoma and, independently, testicular dysfunction. Experimental evidence shows testicular dysfunction and impaired spermatogenesis in laboratory animals exposed to phenoxy herbicides, dioxin, or tetracycline, and antibiotic used extensively in military working dogs in Vietnam. Because an unexplained significant decrease in sperm quality in Vietnam veterans has been observed by the Centers for Disease Control, further research is warranted if we are to clarify military service in Vietnam as a risk factor for testicular dysfunction. The testis should be made a priority site in the study of Vietnam experience-related cancers.

Narrowing the zone of uncertainty between research and development in biological warfare defense.

Although "research" is not prohibited by the Biological Weapons Convention, States Parties to the Convention have maintained the spirit of the Convention in actions relating to research. The confidence-building measures agreed to at RC2 refer to research facilities, publication of research results, and promotion of contacts between scientists engaged in research related to the Convention. However, assessment of basic research on biological agents is not a productive way to distinguish an offensive from a defensive program. Additionally, if a country were to initiate a biological weapons program, basic research on biological agents may not be necessary. For example, the extensive published research on Bacillus anthracis, both as a cause of anthrax in cattle and other species and as a biological-warfare agent, would enable any motivated group or nation to initiate a biological weapons program that could immediately advance to the development and scale-up stages. Research on biological agents for offensive purposes would be characterized by activities such as selection for growth, virulence, and toxin production; improving stability under varying environmental conditions; and selection of strains that might overcome existing means of prophylaxis and treatment. A biological program with an offensive intent would in most cases be characterized by evidence of development efforts in mass production and dissemination, which are often agent-specific. Thus, an assessment of development may distinguish offensive from defensive programs. If a country were to initiate a biological weapons research program, and were willing to risk worldwide condemnation should existence of such a program become known, it is likely that such a program would include development and production capabilities. If a country were not committed to production capability, there would be no rationale for an offensive biological research that would bring worldwide condemnation. Critics of the U.S. Biological Defense Research Program have suggested that the program could easily and quickly be turned into an offensive effort. To accomplish this, however, we have to assume that all military personnel, including the civilians employed by the Department of the Army, are unethical and willing to break the law and run the risk of placing the U.S. in a noncompliance status. The Army is under constant scrutiny by governmental agencies, by visiting scientists, by audiences at scientific meetings, by scientists who review manuscripts for publications, by news media, and by private citizens.(ABSTRACT TRUNCATED AT 400 WORDS)

Serological evidence for a high incidence of transmission of Rickettsia tsutsugamushi in two Orang Asli settlements in Peninsular Malaysia.

Two communities of Orang Asli (aborigines) in Peninsular Malaysia were observed for evidence of Rickettsia tsutsugamushi infection over periods of 1-8 mo. Sequential sera were examined for antibody by the indirect immunofluorescence test. The incidence of infection in the two self-selected populations in the two communities was calculated to be 3.9% per month and 3.2% per month.

Adaptation of a microimmunofluorescence test to the study of human Rickettsia tsutsugamuskh antibody.

A microimmunofluorescence (micro-IFA) test has been adapted to the study of naturally occurring antibody to Rickettsia tsutsugamushi in humans. The micro-IFA test was comparable to the present immlnofluorescent assay in sensitivity and reproducibility, but offered distinct advantages in the quantity of reagents necessary. Also the micro-IFA could be used to simultaneously titer a serum to as many as nine strains of the organism resulting in a great saving of time.

Evidence of a serologic relationship between Ehrlichia canis and Rickettsia sennetsu.

A serologic relationship between Rickettsia sennetsu, the etiologic agent of human sennetsu rickettsiosis in western Japan, and Ehrlichia canis, the agent of canine ehrlichiosis, has been demonstrated. Using the indirect fluorescent antibody test, convalescent sera from patients with sennetsu fever reacted with Ehrlichia canis antigen. In the direct fluorescent antibody test, immunoglobulins from four patients with sennetsu rickettsiosis stained E. canis morulae in canine monocytes. This finding is significant in view of the morphologic uniqueness of the two agents and a lack of serologic relatedness with other major rickettsial agents.

Identification and antigenic typing of Rickettsia tsutsugamushi in naturally infected chiggers (Acarina: Trombiculidae) by direct immunofluorescence.

Rickettsia tsutsugamushi organisms were detected and typed antigenically by direct immunofluorescence in mites from laboratory-maintained infected colonies of Leptotrombidium (Leptotrombidium) fletcheri and L. (L.) arenicola. Rickettsiae were identified most readily in unengorged larvae, but were also discernable in engorged larvae and all post-larval stages of the vectors.

Characterization of Rickettsia tsutsugamushi strains in two species of naturally infected, laboratory-reared chiggers.

The strains of Rickettsia tsutsugamushi found in naturally infected, laboratory-reared Leptotrombidium (Leptotrombidium) arenicola and L. (L.) fletcheri chiggers were characterized by direct immunofluorescence (FA) and by mouse and monkey virulence tests. The strains existing in the L. (L.) arenicola chiggers consisted of different combinations of TA716, TA763, TA686, Karp, and Kato. In addition to these five strains, Gilliam was found in the L. (L.) fletcheri chiggers. Results indicate that individual chiggers can be simultaneously infected with several antigenic strains of R. tsutsugamushi. Although these antigens appear to remain stable within familial lines when several generations were viewed, the antigenic patterns observed in two succeeding generations did not always correlate. This variable expression of antigens was considered to be due to a quantitative fluctuation from one generation to the next in the strains of rickettsiae combined with a lack of sensitivity of the direct FA test in detecting small numbers of antigenically different rickettsiae. Phenotypic variation was considered to be a less probable explanation. Morbidity and mortality were minimal in ICR mice fed upon by individual chiggers of either species, but infection rates were 85-99%. Tissue suspensions prepared from mice infected by L. (L.) arenicola produced higher mortality and longer duration of illness in mice than those prepared from L. (L.) fletcheri-infected mice. Silvered leaf and cynomolgus monkeys were fed upon by the two species of chiggers or inoculated with the mouse tissue suspensions. In both cases, minimal clinical responses were observed.

Febrile illness in Malaysia--an analysis of 1,629 hospitalized patients.

We studied 1,629 febrile patients from a rural area of Malaysia, and made a laboratory diagnosis in 1,025 (62.9%) cases. Scrub typhus was the most frequent diagnosis (19.3% of all illnesses) followed by typhoid and paratyphoid (7.4%); flavivirus infection (7.0%); leptospirosis (6.8%); and malaria (6.2%). The hospital mortality was very low (0.5% of all febrile patients). The high prevalence of scrub typhus in oil palm laborers (46.8% of all febrile illnesses in that group) was confirmed. In rural Malaysia, therapy with chloramphenicol or a tetracycline would be appropriate for undiagnosed patients in whom malaria has been excluded. Failure to respond to tetracycline within 48 hours would usually suggest a diagnosis of typhoid, and indicate the need for a change in therapy.

The longevity of antibody to Rickettsia tsutsugamushi in patients with confirmed scrub typhus.

Serological surveillance for up to two years of 114 patients with laboratory confirmed scrub typhus showed that antibody to Rickettsia tsutsugamushi as demonstrated by the indirect fluorescent antibody test is short-lived. The mean reversion time from mean peak titre (1:499) was 48.9 weeks and the calculated annual reversion rate to a titre less than 1:50 was 61%. This can be used to estimate attack rates based on point prevalence of antibody. The relationship between antibody prevalence and attack rates observed by other workers was confirmed using this model. The possible uses of the finding and its implications in Malaysia are briefly discussed.

Single dose doxycycline therapy for scrub typhus.

A single dose of 200 mg of doxycycline was shown to be as effective as a seven day course of tetracycline, in patients suspected of having scrub typhus. 65 (44%) of the 149 patients studied fulfilled the criteria for definite diagnosis of scrub typhus; 10 had an additional diagnosis. Rickettsia tsutsugamushi was isolated from 49 (75%) patients. There was no difference between the two treatment groups in time to defervescence, abolition of cough and headache, or in the time taken to recover well-being. There were no relapses in either group. Of the remaining 84 patients, a causal diagnosis was achieved in 52. Irrespective of a diagnosis there was no difference in apparent response to either doxycycline or tetracycline.

Scrub typhus: a common cause of illness in indigenous populations.

An explanation was sought for the disparity between the low reported incidence of scrub typhus and the high prevalence of antibody to Rickettsia tsutsugamushi in the rural population of Malaysia. A combination of isolation of the organism, titration of antibody by indirect immunofluorescence, and the Weil-Felix test was used to confirm infections. Scrub typhus was found to be very common, causing 23% of all febrile illnesses at one hospital. The infection was particularly prevalent in oil-palm workers, causing an estimated 400 cases annually in a population of 10,000 people living on one plantation. The clinical syndrome, whether mild or severe, was difficult to distinguish from that due to other infections. Eschars, rashes and adenopathy were uncommon. When used to examine early sera, the Weil-Felix test failed to confirm the diagnosis in most infections.20

Kinetics of antibody response to Ehrlichia canis assayed by the indirect fluorescent antibody method.

The kinetics of antibody production response to experimentally induced infection of dogs with Ehrlichia canis was determined by ion-exchange and molecularsieve chromatography and by indirect fluorescent antibody (IFA) test. The first IFA antibody at 7 days after inoculation resided in immunoglobulin M (IgM) and immunoglobulin A (IgA) classes. At approximately 21 days after inoculation, the antibody was in IgM, IgA, and immunoglobulin G (IgG) classes. Thereafter, antibody concentrations continued to increase in the IgG class; those in the other 2 immunoglobulin classes had a variable pattern. In 2 dogs which died 60 and 114 days after inoculation, a decrease of antibody concentration in the 3 immunoglobulin classes was evident at the time of death. In the carrier dog, however, which was killed 147 days after inoculation, antibody concentrations sustained increasing titers in the 3 immunoglobulin classes.

Experimentally induced infection of dogs, cats, and nonhuman primates with Ehrlichia equi, etiologic agent of equine ehrlichiosis.

Dogs (German Shepherd Dogs and Beagles), cates, rhesus macaques (Macaca mulatta), and baboons (Papio anubis) were inoculated with Whrlichia equi, the etiologic agent of equine ehrlichiosis. Within 3 to 7 days after inoculation, morulae were observed in the eosinophils of cats, neurtrophils of macaques and baboons, and in both neutrophils and eosinophils of dogs. The severe disease produced in horses by this agent was not a feature of E equi infection in dogs, cats, macaques, and baboons. However, a susceptible horse, inoculated with the pooled blood of 2 infected macaques, developed severe clinical signs of equine ehrlichiosis. Infection with E equi did not alter the susceptibiltiy of dogs to infection with Ehrlichia canis and did not prevent development of signs of disease resulting from this infection. The broad experimental host range from this infection. The broad experimental host range of E equi distinguishes it from other ehrlichial agents which are characterized by a rather narrow host range. The susceptibiltiy of nonhuman primates to infection with E equi provides a basis for consideration of the potential transmission of ehrlichial agents to man.

Immune response of ponies to experimental infection with Ehrlichia equi.

Four ponies experimentally infected with Ehrlichia equi developed substantial cell-mediated immune responses, as measured by the leukocyte migration-inhibition test. Serum anti-E equi antibodies up to 1:1,280 were demonstrated by the indirect fluorescent antibody test. Cell-mediated immune responses returned to a base-line value by day 200 after primary inoculation, but serum antibody titers persisted for at least 300 days after inoculation. Two additional susceptible ponies, which were inoculated with convalescent blood or organ homogenates from ponies recovered from acute equine ehrlichiosis, treated with tetracycline, and subsequently challenge exposed with E equi-infective blood, did not develop clinical disease. This study suggested that ponies are resistant to reinfection with E equi following clinical ehrlichiosis.

Transmission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus).

Two strains of Rhipicephalus sanguineus acquired Ehrlichia canis by feeding as either larvae or nymphs on acutely infected dogs and, in subsequent instars, transmitted the agent to normal dogs. Three strains of R sanguineus transmitted E canis as adults after their larval and nymphal stages fed on infected dogs. More than 400 adult female ticks were fed on infected dogs as larvae or nymphs or both, but none transmitted E canis transovarially.

Development of Ehrlichia canis, causative agent of canine ehrlichiosis, in the tick Rhipicephalus sanguineus and its differentiation from a symbiotic Rickettsia.

Certain aspects of the development of Ehrlichia canis, causative agent of canine ehrlichiosis (tropical canine pancytopenia) in Rhipicephalus sanguineus ticks were studied. It was found that partial feeding of nymphs infected as larvae with E canis was a desirable, if not necessary, preliminary treatment for successful infection of dogs with ground-up ticks. It remains unclear whether feeding increased the number or altered the virulence of ehrlichiae within tick tissues. Ehrlichia canis organisms were detected by immunofluorescent microscopy in the midgut and hemocytes and by electron microscopy in the midgut and salivary glands of partially engorged adult ticks which had been infected as larvae and nymphs. Organisms were not observed in the ovary. Intracytoplasmic inclusions contained 1 to 80 elementary bodies, each provided with 2 distinct membranes. Infection of the midgut and salivary gland was confirmed by injecting homogenates of these tissues into susceptible dogs. Staining of gut smears of partially engorged adult ticks by fluorescein-conjugated anti-E canis antibody was found to be a reliable indicator of the infection.

Studies on the pathogenesis of Rickettsia rickettsii in the dog: clinical and clinicopathologic changes of experimental infection.

Beagle dogs inoculated with the agent of Rocky Mountain spotted fever, Rickettsia rickettsii, developed a clinical syndrome that extended from febrile exanthema to death and appeared to be dose related. Infected dogs were anorectic and lethargic and developed cutaneous lesions characteristic of Rocky Mountain spotted fever, including petechia, ecchymosis, edema, and necrosis. Hematologic changes after inoculation included anemia, leukopenia proceeding to leukocytosis, and thrombocytopenia. Changes in blood chemistry values included increases in serum alkaline phosphatase and cholesterol, and hyponatremia and hypochloremia. The prominent histopathologic change was necrotizing vasculitis. The canine disease is comparable with human Rocky Mountain spotted fever on a clinical, hematologic, biochemical, and pathologic basis, and may provide a model system for this disease in man. The results suggest the dog may be involved in the epidemiology of R rickettsii infections.

Autologous lymphocyte-mediated cytotoxicity against monocytes in canine ehrlichiosis.

The 51Cr-release technique for detecting cytotoxicity had been adapted to immunologic studies of canine ehrlichiosis. Lymphocytes from dogs infected with Ehrlichia canis, cause of canine ehrlichiosis, were shown to be cytotoxic for autologous monocytes. The effect was dependent on the concentraton of lymphocytes, being optimal at 100:1 lymphocyte:monocyte ratio. Neither immune serum and complement, nor anti-canine globulin had any observable effect on cytotoxicity. The monocytotoxicity bore a temporal relationship to the thrombocytopenia. It is suggested that T lymphocyte activation accompanying ehrlichiosis contributes to pathogenesis of the disease and that the specific immune elimination of parasitized monocytes is antibody independent.

Cell-mediated and humoral immune responses of German Shepherd Dogs and Beagles to experimental infection with Ehrlichia canis.

The cell-mediated and the humoral immune responses of 12 German Shepherd Dogs and 5 Beagles inoculated with Ehrlichia canis were evaluated. Results indicated that specific and nonspecific immunosuppression due to E canis occurred in the German Shepherd Dogs. Canine leukocyte migration-inhibition factor was successfully isolated and shown to be physically and functionally similar to human and guinea pig migration inhibition factor. Of the German Shepherd Dogs, 58% developed positive cell-mediated responses; 80% of the Beagles became positive. German Shepherd Dogs that developed severe chronic ehrlichiosis did not respond to as great a degree as did the German Shepherd Dogs and Beagles with mild chronic disease. The cell-mediated responses decreased with time and disappeared by 147 days after inoculation. Humoral antibody titers in all inoculated dogs increased with time and remained at increased concentrations. Treatment of four inoculated dogs with antilymphocyte serum did not modify the course of the disease. The findings indicated that cell-mediated immunity may have a significant role in determining the course of disease in dogs infected with E canis.

Epizootiology of tropical canine pancytopenia.

Tropical canine pancytopenia (TCP) is a newly recognized infectious disease of dogs in diverse tropical and subtropical areas. The disease is characterized by hemorrhage, pancytopenia, severe emaciation and persistent infection. Dogs with TCP are often presented with epistaxis, which is the most dramatic sign of the disease; however, a large number of affected dogs develop severe pancytopenia and die without manifesting clinical signs of hemorrhage. The disease has been reported most frequently in the German Shepherd. Pathological findings consist of petechial and ecchymotic hemorrhages on serosal and mucosal surfaces of numerous organs. The most prominent histological finding is a perivascular plasma cell infiltrate in most organs. Disease, indistinguishable from the natural disease, has been produced in laboratory dogs inoculated with whole blood from affected dogs. Ehrlichia canis has been consistently recovered from all experimentally infected dogs. Attempts to transmit the disease to other laboratory animals and to propagate the agent in cell cultures and embryonating eggs have been unsuccessful. The tick is the probable vector of the disease.