Spatial clustering of livestock Anthrax events associated with agro-ecological zones in Kenya, 1957-2017.

BACKGROUND: Developing disease risk maps for priority endemic and episodic diseases is becoming increasingly important for more effective disease management, particularly in resource limited countries. For endemic and easily diagnosed diseases such as anthrax, using historical data to identify hotspots and start to define ecological risk factors of its occurrence is a plausible approach. Using 666 livestock anthrax events reported in Kenya over 60 years (1957-2017), we determined the temporal and spatial patterns of the disease as a step towards identifying and characterizing anthrax hotspots in the region. METHODS: Data were initially aggregated by administrative unit and later analyzed by agro-ecological zones (AEZ) to reveal anthrax spatio-temporal trends and patterns. Variations in the occurrence of anthrax events were estimated by fitting Poisson generalized linear mixed-effects models to the data with AEZs and calendar months as fixed effects and sub-counties as random effects. RESULTS: The country reported approximately 10 anthrax events annually, with the number increasing to as many as 50 annually by the year 2005. Spatial classification of the events in eight counties that reported the highest numbers revealed spatial clustering in certain administrative sub-counties, with 12% of the sub-counties responsible for over 30% of anthrax events, whereas 36% did not report any anthrax disease over the 60-year period. When segregated by AEZs, there was significantly greater risk of anthrax disease occurring in agro-alpine, high, and medium potential AEZs when compared to the agriculturally low potential arid and semi-arid AEZs of the country (p < 0.05). Interestingly, cattle were > 10 times more likely to be infected by B. anthracis than sheep, goats, or camels. There was lower risk of anthrax events in August (P = 0.034) and December (P = 0.061), months that follow long and short rain periods, respectively. CONCLUSION: Taken together, these findings suggest existence of certain geographic, ecological, and demographic risk factors that promote B. anthracis persistence and trasmission in the disease hotspots.

Co-creation and priority setting for applied and implementation research in One Health: Improving capacities in public and animal health systems in Kenya.

Background: The Kenyan government has successfully been implementing sector specific and multisectoral projects aligned to the Global Health Security Agenda (GHSA). For operational readiness and to enhance the effective planning and implementation of Global Health Security Programs (GHSP) at national and subnational level, there is an urgent need for stakeholders' engagement process to seek input in identifying challenges, prioritise activities for field implementation, and identify applied research and development questions, that should be addressed in the next five years. Methods: The modified Child Health and Nutrition Research Initiative (CHNRI) method was used to identify global health security related priorities for multisectoral implementation in Kenya. Subject matter experts from human, animal and environmental health sectors at national and subnational level contributed to predefined research questions from a number of sources and activities for consideration for implementation using a One Health approach. Sixty-two experts scored the 193 questions based on five pre-defined criteria: 1) feasibility and answerability; 2) potential for burden reduction; 3) potential for a paradigm shift; 4) potential for translation and implementation; and 5) impact on equity. Data resulting from this process was then analysed in a Microsoft Excel spreadsheet to determine the research priorities and experts' agreements. Results: Among the priority activities identified for implementation research were; strengthening One Health governance and legal frameworks; integration of ecosystem health into One Health programming; strengthening disease reporting, integrated data collection, information sharing and joint outbreak response; socio-anthropological and gender-based approaches in improving risk and behavioural change communication and community engagement; and one health workforce development. In addition, the potentials to invest in collaborative predictive risk modelling to enhance epidemic intelligence systems, while strengthening the One Health approach in the food safety incident and emergency response plans are feasible. Interpretation: Successful multisectoral implementation of global health security program in Kenya calls for a whole of society approach that will harness community and private sector knowledge to build preparedness and response capacities while targeting neglected and marginalised populations. This research provides a framework that is worth emulating for cost-effective planning and implementation of overarching One Health programs.

Korean hemorrhagic fever: propagation of the etiologic agent in a cell line of human origin.

The etiologic agent of Korean hemorrhagic fever has been propagated in a human cultured cell line derived from a carcinoma of the lung. The cells, described as type II, alveolar epithelial, support replication of the agent and successive passages. Antigen of the Korean hemorrhagic fever agent is readily detected in infected cells by means of direct or indirect fluorescent antibody techniques. Previous attempts to propagate this agent in vitro had been unsuccessful.

Spontaneous ileostomy closure.

Iatrogenic ileostomies are routinely placed during colorectal surgery for the diversion of intestinal contents to permit healing of the distal anastomosis prior to elective reversal. We present an interesting case of spontaneous closure of a diverting ileostomy without any adverse effects to the patient. A 65-year-old woman, positive for hereditary non-polyposis colorectal cancer type-I, with locally invasive cancer of the distal colon underwent en-bloc total colectomy, hysterectomy, and bilateral salpingoophorectomy with creation of a proximal loop ileostomy. The ostomy temporarily closed without reoperation at 10 weeks, after spontaneously reopening, it definitively closed, again without surgical intervention at 18 weeks following the original surgery. This rare phenomenon has occurred following variable colorectal pathology and is poorly understood, particularly in patients with aggressive disease and adjunct perioperative interventions.

Phylogenetic analysis of gag genes from 70 international HIV-1 isolates provides evidence for multiple genotypes.

OBJECTIVE: To determine the extent of genetic variation among internationally collected HIV-1 isolates, to analyse phylogenetic relationships and the geographic distribution of different variants. DESIGN: Phylogenetic comparison of 70 HIV-1 isolates collected in 15 countries on four continents. METHODS: To sequence the complete gag genome of HIV-1 isolates, build multiple sequence alignments and construct phylogenetic trees using distance matrix methods and maximum parsimony algorithms. RESULTS: Phylogenetic tree analysis identified seven distinct genotypes. The seven genotypes were evident by both distance matrix methods and maximum parsimony analysis, and were strongly supported by bootstrap resampling of the data. The intra-genotypic gag distances averaged 7%, whereas the inter-genotypic distances averaged 14%. The geographic distribution of variants was complex. Some genotypes have apparently migrated to several continents and many areas harbor a mixture of genotypes. Related variants may cluster in certain areas, particularly isolates from a single city collected over a short time. CONCLUSIONS: The genetic variation among HIV-1 isolates is more extensive than previously appreciated. At least seven distinct HIV-1 genotypes can be identified. Diversification, migration and establishment of local, temporal 'blooms' of particular variants may all occur concomitantly.

Neoadjuvant chemotherapy before surgery in cervical cancer.

Eighteen phase II clinical trials have demonstrated the relative ease of administering intravenous neoadjuvant chemotherapy before radical surgery in patients with cervical cancer. Toxicity has been modest, with the exception of occasional severe bleomycin-induced pulmonary toxic effects. All regimens tested have been cisplatin based with no clearly superior combination. Overall objective response rates were 85% for patients with International Federation of Obstetrics and Gynecology stage IB or IIA disease, 88% for those with stage IIB disease, 74% for those with stage III disease, and 47% for those with stage IV disease. There was a corresponding decrease in clinical complete response rates with increasing stage from 28% for those with stage IB or IIA disease to 7% for those with stage IV disease. Operability was also stage dependent: 94% for patients with stage IB and IIA disease, 74% for patients with stage IIB disease, and 58% for patients with stage III or IV disease. All inoperable patients received primary radiation therapy. Fifty-five percent of operable patients received postoperative radiation therapy. Most phase II trials using historical controls demonstrated comparable survival. Only a few trials showed improved survival with the use of neoadjuvant chemotherapy before radical surgery. To date, there has been only one reported prospective trial of neoadjuvant chemotherapy before surgery. Patients with stage IB disease were randomly assigned to receive radical hysterectomy and pelvic lymphadenectomy with or without neoadjuvant chemotherapy. A survival advantage was present only in patients with a tumor volume of greater than 60 cm3. Randomized, prospective trials of neoadjuvant chemotherapy followed by radiation therapy have shown either no advantage or a statistically significant reduction in survival when neoadjuvant chemotherapy is administered before radiation therapy. In light of this finding and the operability of only 58% of the patients with stage III or IV disease, it would be prudent to limit neoadjuvant trials to patients with stage I or II disease with an initial tumor volume of greater than 60 cm3. Four such trials either are under way or are approved an awaiting activation.

Genetic analysis of HIV-1 isolates from Brazil reveals presence of two distinct genetic subtypes.

The spread of the human immunodeficiency virus type 1 (HIV-1) is by now virtually worldwide. An understanding of the genetic, biological, and immunological differences among isolates collected in different geographic locales is crucial for the development of globally effective vaccines. Here we report the genetic characteristics of 21 HIV-1 isolates from Brazil. The isolates were initially characterized using a heteroduplex mobility assay. The majority (17 of 21) were related to North American/European reference isolates of genetic subtype B. Four isolates belonged to a more recently identified genotype, termed subtype F. The subtype F sequences from Brazil are distinguishable in both gag and env from five other genetic subtypes of HIV-1 currently recognized. Like many locales, Brazil harbors more than one HIV-1 subtype.

PIP: The spread of the human immunodeficiency virus type 1 (HIV-1) is by now virtually worldwide. An understanding of the genetic, biological, and immunological differences among isolates collected in different geographic locales is crucial for the development of globally effective vaccines. The genetic characteristics of HIV-1 isolates from whole blood samples of 21 HIV-1-seropositive Brazilian patients collected during 1989 and 1990 are reported. Virus was isolated by cocultivation of patient peripheral blood mononuclear cells (PBMCs) with phytohemagglutinin (PHA)-stimulated donor PBMCs. The isolates were initially characterized using a heteroduplex mobility assay, which estimates the DNA sequence homology of a selected genomic region from different HIV-1 isolated from the migration of heteroduplexes in polyacrylamide gels. Five distinct HIV-1 envelope subtypes were identified, and a sixth subtype, termed F, was identified using isolates from Brazil and Romania. One Brazilian isolate was identified as subtype B by the more rapid relative migration of heteroduplexes. The majority (17 of 21) were related to North American/European reference isolates of genetic subtype B, whereas 4 isolates belonged to subtype F, a more recently identified genotype. The gag and env genes of several Brazilian isolates belonging to subtypes B and F were cloned and sequenced to allow a detailed analysis of their phylogenic relationships. This further established the existence of two distinct and well-separated genetic subtypes among Brazilian HIV-1 isolates. The subtype F sequences from Brazil are distinguishable in both gag and env from 5 other genetic subtypes of HIV-1 currently recognized. Like many locales, Brazil harbors more than one HIV-1 subtype.

Genetic comparison of HIV-1 isolates from Africa, Europe, and North America.

PIP: Laboratory scientists used anchored polymerase chain reaction (PCR) and DNA sequencing to compare HIV-1 isolates from countries in Africa (Ivory Coast, Gabon, Zaire, Kenya, and others), Europe (Belgium and other countries), and the US. The US isolates had the most homogenous PCR profile followed by the European pattern. There was considerable PCR primer mispairing for the African isolates, especially those from Kenya, indicating that the range of HIV-1 variation could have been rather extensive. This virus diversity could greatly affect therapy or intervention in sites in Africa with such a complex mix of variants. Nevertheless, the genetic information of these diverse isolates could bring about research leading to an anti-HIV-1 vaccine. For example, the expanded DNA sequence data base could record phylogenetic relationships, thereby, helping researchers choose prototypic variants for vaccine development. More information would allow researchers to generate new PCR primers for better discrimination of variants. They could apply PCR typing to huge sample sizes to adequately document HIV-1 variation in Africa. It could also prove invaluable as a means to determine incidence and prevalence of local variants during vaccine field trials. It can also discern the limiting criteria for HIV-1 genetic variation.^ieng

Viral DNA burden and decline in percentage of CD4-positive cells in the lymphoid compartment of SIV-infected macaques.

The decline in CD4+ cells and increased viral DNA and RNA burden in the blood of human immunodeficiency virus (HIV)-infected individuals have been used as closely related correlates of disease progression. However, little is known about levels of total or unintegrated viral DNA in lymphoid tissue of HIV-infected patients and how they relate to CD4+ cell decline or disease progression. Exploiting the similarities between HIV- and simian immunodeficiency virus (SIV)-induced disease, we examined lymphoid organs and peripheral blood from SIV-infected macaques for total (pol) and unintegrated 2-LTR circular viral DNA by polymerase chain reaction (PCR). Two SIV isolates (SIVmac/251 and SIVmne/E11S) that differ markedly in their biological and clinical properties were studied. The results indicate that total viral DNA burdens vary considerably between isolates. There was no strong association between total viral DNA levels and CD4% in lymphoid tissues when isolates were compared and death was not associated with any particular level of viral pol DNA. In contrast, accumulation of unintegrated viral DNA was closely associated with decline in CD4/CD8 ratios in lymphoid organs and AIDS. The appearance of both pol and unintegrated viral DNA in thymus of infected macaques also emerged as one of the single best correlates or possible predictors of advanced disease yet studied. Their roles in pathogenesis are discussed.

Titration and characterization of two rhesus-derived SIVmac challenge stocks.

Simian immunodeficiency virus infection of macaques is a model for human immunodeficiency virus infection of humans. In vivo-titrated stocks of SIV are essential for the utilization of this model for vaccine development. The elicitation of anti-human cell antibodies by some vaccines prepared in human cells and the related protective effects of the vaccine produced in human cells suggest a need for new macaque-derived SIV stocks. Here we describe the titration and characterization of two stocks of SIVmac that were produced in primary rhesus macaque cells. The first virus is SIVmac251, isolated from tissues of macaque 251, and the second is a molecular clone designated as SIVmac239. A 50% rhesus monkey infectious dose (MID50) was titrated for each virus stock by intravenous inoculation. An additional five macaques were inoculated with 10 MID50 of the SIVmac251 stock and were followed for disease outcome. All five monkeys developed antigenemia by 14 days postchallenge. Two of the five monkeys developed strong anti-SIV humoral immunity, whereas three developed little or no humoral immunity. As has been observed previously, the rapidity of disease progression correlated with the lack of a strong antibody response. The three animals with low humoral immunity died within 7 months of challenge, with antigenemia, cachexia, hypoproteinemia, hypoalbuminemia, weight loss, and intractable diarrhea, while maintaining their circulating CD4 numbers. One animal died at 1.5 years of more typical simian AIDS.

Decline in the CD4+ lymphocyte population in the blood of SIV-infected macaques is not reflected in lymph nodes.

Although loss of CD4+ lymphocytes in peripheral blood is a standard criterion for evaluating the course of HIV disease, little is known about changes within lymphoid organs, which contain the bulk (> 50%) of the body's lymphocytes. Because such studies are feasible only by using non-human primates, we have examined lymph nodes (LNs), spleen, and blood from monkeys infected with two isolates of simian immunodeficiency virus (SIV). During both the acute and chronic phases of these infections, characteristic reductions in the blood CD4+ cell levels are not reflected in LN, where the CD4+ pool remains within normal levels. However, when circulating CD4/CD8 ratios have consistently fallen to approximately 0.5, striking decreases in the percentage of CD4 cells (CD4%) and CD4/CD8 ratios in LN occur concomitantly with dramatic increases in viral antigen expression on follicular dendritic cells within LN germinal centers (GCs). The data suggest that loss from the total T cell pool in minimal until the final stages of SIV and HIV disease and that the immunological deterioration of LN is the event that precipitates the increased susceptibility to infections and progression to AIDS.

HIV-1 infection in pigtailed macaques.

Four pigtailed macaques were inoculated with autologous cells expressing low levels of human immunodeficiency virus type 1 (HIV-1). During the first 10 weeks, infectious virus was recovered from peripheral blood mononuclear cells (PBMCs) and lymph nodes from three of the animals. Subsequently, HIV-1 DNA was frequently detected in uncultured PBMCs from all three animals, and virus was isolated from one of them at weeks 38 and 61. The fourth animal, which was rechallenged at week 10 with cell-free virus isolated from one of the others, never became virus isolation positive, but harbored HIV-1 proviral genomes. These virus infections were accompanied by the development of varied HIV-1-specific humoral immune responses. Antibodies to gp160 were first apparent at week 8 in the three initially infected animals and persisted. The animal from whom virus was isolated at late times also developed persisting antibodies to HIV-1 p24 and gp120. Antibodies to gp120 and gp160 became apparent in the rechallenged animal at 1 week following reinoculation, but they waned with time. In vivo passage of the virus was attempted at week 6. One recipient pigtailed macaque and one recipient cynomolgus monkey failed to become detectably infected following transfusion of virus-positive blood and lymph node cells. The long-term presence of HIV-1-specific antibodies and proviral genomes in these animals, and the recovery of infectious virus more than 1 year following inoculation, are indicative of persistent infection, and confirm previous reports that pigtailed macaques are susceptible to HIV-1.

Immunological and virological changes associated with decline in CD4/CD8 ratios in lymphoid organs of SIV-infected macaques.

The decline in CD4/CD8 ratios in lymph nodes (LNs) of SIV macaques and HIV-infected individuals occurs later than that in blood. In a previous study, long-term SIV-infected macaques were delineated into two groups: (1) those whose LNs had normal CD4/CD8 ratios and (2) those whose LNs had low CD4/CD8 ratios. In the present investigation, LNs, spleens, and blood from these groups have been further analyzed to ascertain the cellular and virological events, particularly those involving CD8+ cells, that occur concomitantly with LN CD4% decline. An increase in the percent of CD69-, IL-2R(p75)-, CD45RA1o CD8+ cells was the most constant event observed in lymphoid tissue from mid- to late-stage SIV-infected monkeys. Such cells were sometimes observed in LNs prior to any other immunological or morphological changes. However, decline in LN CD4/CD8 ratios and the associated degeneration of follicular dendritic cells (FDCs) in the germinal centers (GCs) of these nodes were observed only when both CD8+ cell infiltration of GCs and accumulation of viral antigens within the FDC network could be demonstrated. These dramatic changes were also associated with significantly reduced responsiveness to mitogens throughout the lymphoid compartment. In terms of viral burden, immunological and structural collapse of LNs was not always associated with increased viral DNA levels. Despite the CD4+ cell decline in blood during HIV and SIV infections, the immunological and architectural collapse of the lymphoid compartment, which comprises the bulk of the lymphocytes in the body, appears to be a critical event leading to the onset of AIDS. The present findings suggest that increased CD8+ cell activity as well as decrease in CD4+ cell function both contribute to this process.

Novel adjuvant strategies for experimental malaria and AIDS vaccines.

Adjuvant research has improved the ability of biotechnology to generate novel vaccines. Numerous strategies for enhancing the immunogenicity of synthetic peptides and proteins have been identified. This overview focuses on adjuvant development and vaccine delivery systems that provide new tools for amplifying the effectiveness of ongoing malaria and AIDS vaccine development programs. In addition, some of the complex challenges and issues that have become associated with the delivery of modern vaccines in man are outlined. As adjuvant research continues to open new opportunities in vaccine development, there is renewed expectation that further generations of safe and potent vaccines will be possible against a broad spectrum of infectious agents and cancer.

Papanicolaou smears by the Bethesda system in endometrial malignancy: utility and prognostic importance.

OBJECTIVE: To evaluate the prognostic significance of the Bethesda system's cytologic categories in patients with endometrial malignancy. METHODS: Patients with biopsy or hysterectomy-proven endometrial malignancy and a Papanicolaou smear result reported using the Bethesda system within 1 year of diagnosis were identified through retrospective review of our computerized database. RESULTS: After introduction of the Bethesda system in our laboratory on November 1, 1992, until January 1, 1997, 112 eligible patients were identified (108 with carcinomas and four with carcinosarcomas). Patients with cytologic diagnoses of malignancy (n = 17) were significantly more likely to have International Federation of Gynecology and Obstetrics (FIGO) grade 3 tumors and high-risk histology (serous, clear cell, and adenosquamous carcinoma and carcinosarcoma) than those with atypical glandular cells of uncertain significance (n = 33) or those with cytology not suspicious for malignancy (n = 63). Patients with malignant smears were also significantly more likely to have cervical extension, malignant peritoneal cytology, and FIGO stage II, III, or IV than those with atypical glandular cells of uncertain significance or those with cytology not suspicious for malignancy. CONCLUSION: Papanicolaou smears obtained within 1 year of histologic diagnosis of endometrial malignancy and interpreted using the Bethesda system were suspicious for (atypical glandular cells of uncertain significance) or diagnostic of malignancy in nearly half of all cases (29 and 15%, respectively). Patients having malignant glandular cells were more likely to have poor prognostic pathologic findings.

Fallopian tube carcinoma.

Seventy-one cases of primary adenocarcinoma of the fallopian tube treated at The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston were reviewed. The most common presenting symptoms were abdominal pain, abnormal uterine bleeding, and vaginal discharge. The most common physical finding was a palpable abdominal or pelvic mass. The preoperative diagnosis was correct for two patients. Initial therapy consisted of surgery alone, surgery plus radiation therapy, surgery plus chemotherapy, and a combination of surgery, chemotherapy, and radiation therapy in 10, 32, 21, and eight cases, respectively. The median survival for patients in these treatment groups was 33, 22, 27, and 22 months, respectively; the median survival for all patients was 23 months. No statistically significant differences emerged among the survival curves of patients treated with each of the above regimens.

Postmolar trophoblastic disease in women using hormonal contraception with and without estrogen.

One hundred thirteen patients with a molar pregnancy evacuated from January 5, 1976, through February 15, 1980, had close follow-up at the Los Angeles County-University of Southern California Women's Hospital. Twenty-seven patients (23.9%) developed postmolar trophoblastic disease, all of whom achieved remission with treatment. Of the 113 patients, 71 (62.8%) used only oral contraceptives, 32 patients (28.3%) used only intramuscular medroxyprogesterone acetate, seven patients (6.2%) used both oral contraceptives and medroxyprogesterone acetate, and three patients (2.7%) used nonhormonal contraception after evacuation of their molar pregnancy and before titer remission. Analysis of significant clinical and laboratory correlates of postmolar trophoblastic disease for the study group revealed no substantial bias in assignment of contraceptive method. There was no significant difference between the oral contraceptive and the medroxyprogesterone acetate groups with respect to the development of postmolar trophoblastic disease or the time to spontaneous titer remission. The study shows no apparent adverse effect of the estrogen (50 mg mestranol) component in oral contraceptives on the frequency of postmolar trophoblastic disease.

The African green monkey as an alternate primate host for studying Machupo virus infection.

African green monkeys (Cercopithecus aethiops) are highly susceptible to Bolivian hemorrhagic fever (BHF). Six monkeys were inoculated with 1,000 plague-forming units of Machupo virus, the etiologic agent of BHF. They were observed and monitored for clinical signs, body temperature, viremia, hematologic changes, and virus neutralizing antibody. Onset of fever, anorexia, and depression was noted on days 3 to 6 postinoculation. These and other signs increased in severity and all monkeys died: 5 of 6 died by day 13 and one survived until day 24. The median time to death for the group was 12.5 days. The mean value for hematocrit determinations gradually decreased to 30 on day 10 but subsequently increased. Mean neutrophil and lymphocyte values increased slightly until day 3, and then decreased to minimal values of 3,000 and 2,000, respectively, on day 10. Four monkeys were viremic by day 7 and all were viremic on day 10. The monkey that survived until day 24 had a neutralizing antibody titer of 1:32 on day 14 and appeared to recover from the initial acute illness by day 16. It died following onset of severe neurologic signs on day 23. BHF in the African green monkey is similar to the disease described in two species of macaques.

Pathology of Bolivian Hemorrhagic fever in the African green monkey.

Gross and microscopic pathological findings are presented for an African green monkey model of fatal Bolivian hemorrhagic fever. Six animals were inoculated with 1,000 plaque-forming units of Machupo virus, the etiological agent of Bolivian hemorrhagic fever. Five of the monkeys died within 13 days with signs of fever, anorexia, shock, and hemorrhage. The sixth monkey survived until the 24th day and died with signs of central nervous system disease. Gross lesions in the five monkeys that die in the acute stage included hepatic necrosis, necrotic enteritis, bronchopneumonia, and hemorrhages in the subcutis, lungs, intestine, liver, and lymph nodes. Microscopically, necrosis was consistently seen in liver, intestine, skin, oral cavity, and adrenal cortex. Acute thrombosis was observed in four monkeys, in blood vessels of the intestine, lung and choroid of the brain. Gram-negative bacteria were seen in many tissues, suggesting terminal bacteremia. The sixth monkey was emaciated and had bronchopneumonia, but did not have the necrotic hepatic and enteric lesions observed in the other five monkeys. The significant microscopic lesions in this monkey included encephalomyelitis, ganglionitis, and bronchopneumonia.

A microprecipitation test for rapid detection and identification of Venezuelan, eastern and western equine encephalomyelitis viruses.

The development of a new diagnostic procedure for the identification of Venezvelan, eastern and western equine encephalomyelitis (VEE, EEE, WEE) viruses is described. The procedure utilizes virus precipitation with reference fluorescein-conjugated gamma globulin, followed by cellulose acetate electrophoresis. Clinical specimens containing varying concentrations of virus yielded, in primary duck embryo cell culture, sufficient virus for detection within 22 to 44 hours. Identification of VEE, EEE and WEE virus in specimens was accomplished by microprecipitation within this time. In contrast to conventional identification methods, our procedure eliminates the cost of utilizing laboratory animals and considerably reduces the time required for virus identification.