Qualitative and quantitative detection of SARS-CoV-2 RNA in untreated wastewater in Western Cape Province, South Africa.

Recent studies have shown that the detection of SARS-CoV-2 genetic material in wastewater may provide the basis for a surveillance system to track the environmental dissemination of this virus in communities. An effective wastewater-based epidemiology (WBE) system may prove critical in South Africa (SA), where health systems infrastructure, testing capacity, personal protective equipment and human resource capacity are constrained. In this proof-of-concept study, we investigated the potential of SARS-CoV-2 RNA surveillance in untreated wastewater as the basis for a system to monitor COVID-19 prevalence in the population, an early warning system for increased transmission, and a monitoring system to assess the effectiveness of interventions. The laboratory confirmed the presence (qualitative analysis) and determined the RNA copy number of SARS-CoV-2 viral RNA by reverse transcription polymerase chain reaction (quantitative) analysis from 24-hour composite samples collected on 18 June 2020 from five wastewater treatment plants in Western Cape Province, SA. The study has shown that a WBE system for monitoring the status and trends of COVID-19 mass infection in SA is viable, and its development and implementation may facilitate the rapid identification of hotspots for evidence-informed interventions.

5-Substituted 2-benzylidene-1-tetralone analogues as A1 and/or A2A antagonists for the potential treatment of neurological conditions.

Adenosine A1 and A2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A1 and A2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A1 and A2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A1 receptor with Ki values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A2A receptor. These substitution patterns led to enhanced adenosine A1 and A2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A1 receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A1 receptors. In conclusion, compounds 3 and 12, showed the best adenosine A1 and A2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.

Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine.

Recent research exploring C8 substitution on the caffeine core identified 8-(2-phenylethyl)-1,3,7-trimethylxanthine as a non-selective adenosine receptor antagonist. To elaborate further, we included various C8 two-chain-length linkers to enhance adenosine receptor affinity. The results indicated that the unsubstituted benzyloxy linker (1e A1Ki = 1.52 µM) displayed the highest affinity for the A1 adenosine receptor and the para-chloro-substituted phenoxymethyl (1d A2AKi = 1.33 µM) linker the best A2A adenosine receptor affinity. The position of the oxygen revealed that the phenoxymethyl linker favoured A1 adenosine receptor selectivity over the benzyloxy linker and, by introducing a para-chloro substituent, A2A adenosine receptor selectivity was obtained. Selected compounds (1c, 1e) behaved as A1 adenosine receptor antagonists in GTP shift assays and therefore represent selective and non-selective A1 and A2A adenosine receptor antagonists that may have potential for treating neurological disorders.

Investigation of isoniazid and ethionamide cross-resistance by whole genome sequencing and association with poor treatment outcomes of multidrug-resistant tuberculosis patients in South Africa.

OBJECTIVE/BACKGROUND: Ethionamide (ETH) and isoniazid (INH) are part of the backbone regimen used for the treatment of multidrug-resistant tuberculosis (MDR-TB). Both ETH and INH are structurally similar and are activated by ethA and katG gene products. Resistance to INH among MDR-TB patients may cause ETH to be ineffective, as both target nicotinamide adenine dinucleotide-dependent enoyl-acyl carrier protein reductase inhA protein and mutations within inhA gene may lead to their cross-resistance. Furthermore, ETH resistance is caused by mutations within ethA and ethR genes forming part of the ETH drug activation pathway. Nicotinamide adenine dinucleotide is coded by the ndh gene, and its overexpresion may lead cross-resistance between INH and ETH drugs. Phenotypic drug susceptibility testing of ETH is difficult and often unreliable. We used whole genome sequencing to compare inhA, inhA promoter, ethA, ethR ndh, and katG genetic regions in serial isolates (baseline and follow-up) with treatment outcomes. METHODS: MDR-TB strains were collected from 46 patients before and during second-line drug treatment in KwaZulu-Natal and Eastern Cape between 2005 and 2009. All patients had phenotypically determined MDR-TB at baseline and had treatment outcomes documented. Unfavorable treatment outcomes were defined as death, default, and failure, while favorable outcomes were cure and treatment completion. Each strain had baseline and at least one strain collected on follow-up. From each strain, DNA was extracted from colonies grown on Löwenstein-Jensen slants, and fragment and jumping paired-end Illumina DNA libraries were constructed and sequenced on the Illumina HiSeq 2000 (Broad Institute, Cambridge, MA, USA). Sequences were aligned to H37Rv genome and Pilon was run to generate a list of SNPs. In silico spoligotyping was performed to a database 43 unique spacer sequences. Cross-resistance was defined as the presence of both inhA and either ethA or ethR mutations in clinical isolates. RESULTS: A total of 92 sequences from 46 serial isolates of MDR-TB patients from KwaZulu-Natal (29 isolates) and Eastern Cape (17 isolates) were analyzed. Most patients (29/46; 63.0%) had unfavorable outcomes, 13 (28.3%) had favorable outcomes, while four (8.7%) had unknown outcomes. Phylogenetic reconstruction revealed that primary genotype differed by province. The Beijing genotype was predominant in Eastern Cape, while EuroAmerican lineage (S, T, LAM, X) was found in KwaZulu-Natal. Whole genome analysis revealed nonsynonymous insertions and deletions within katG, ethA, ethR, ndh, and inhA and its promoter region. Among patients with treatment outcome data, mutations were detected in 92.8% in katG, 50% in inhA, 53.6% in ethA, 2.4% in ethR, and 19% in ndh. The majority of mutations causing ETH (20/29; 68.9%) and INH (18/29; 62.1%) resistance occurred among patients with unfavorable outcomes. Both inhA and either ethA or ethR mutations were detected in 16/29 (55.2%) patients with unfavorable outcomes. Cross-resistance of both INH and ETH drugs was associated with unfavorable treatment outcomes (p=0.021) in 16/29 (55.2%) patients compared with favorable treatment outcomes in 2/13 (15.4%) patients. CONCLUSION: Baseline ETH molecular resistance before second-line treatment is a concern. Unfavorable treatment outcomes of patients with ethA, ethR, and inhA mutations highlight the importance of genotypic testing before initiation of treatment containing ETH. The clinical significance of whole genome analysis for early detection of mutations predictive of treatment failure needs further investigation.

Integration of TB-HIV services at an ANC facility in Frances Baard District, Northern Cape, South Africa.

BACKGROUND: Integrated tuberculosis-human immunodeficiency virus (TB-HIV) service delivery as part of maternal health services, including antenatal care (ANC), is widely recommended. This study assessed the implementation of collaborative TB-HIV service delivery at a hospital-based ANC service unit. METHODS: A record review of a random sample of 308 pregnant women attending the ANC service between April 2011 and February 2012 was conducted. Data were extracted from registers and patient case notes. Outcomes included the proportion of women who underwent HIV counselling and testing (HCT), CD4 count testing, antiretroviral treatment (ART), cotrimoxazole preventive treatment (CPT), TB screening and isoniazid preventive treatment (IPT). Analysis measured variations in patient characteristics associated with service delivery. RESULTS: All women underwent HCT; 80% of those who tested HIV-positive were screened for TB. Most (85.9%) of the HIV-positive women received a CD4 count. However, only 12.9% of eligible women received ART prophylaxis onsite, only 35.7% were referred for initiation of ART, only 42.3% commenced IPT and none received CPT or further investigations for TB. HIV-negative women had 2.6 higher odds (95%CI 1.3-5.3) of receiving TB screening than their HIV-positive counterparts. CONCLUSIONS: Although the identification of HIV-positive women and TB suspects was adequate, implementation of other TB-HIV collaborative activities was sub-optimal.

Contexte : Une offre de services intégrée de tuberculose et du virus de l'immunodéficience humaine (TB-VIH)­en tant qu'élément des services de santé maternelle, notamment des consultations prénatales (ANC)­est largement recommandée. Cette étude a évalué la mise en œuvre d'une offre de services intégrée TB-VIH dans une unité hospitalière de CPN.Méthodes : Les dossiers d'un échantillon aléatoire de 308 femmes enceintes qui ont fréquenté le service ANC entre avril 2011 et février 2012 ont été revus. Les données ont été extraites à partir des registres ainsi que des dossiers des patients. Les résultats attendus comprenaient la proportion de femmes bénéficiant d'un conseil et test VIH (HCT), d'un comptage des CD4, d'un traitement antirétroviral (ART), d'un traitement préventif par cotrimoxazole (CPT), d'un dépistage de TB et d'un traitement préventif par isoniazide (IPT). L'analyse a mesuré les variations des caractéristiques des patients associées à l'offre de services.Résultats : Toutes les femmes ont bénéficié du HCT et 80% de celles ayant eu un test VIH positif ont eu un dépistage de TB. La majorité (85,9%) des femmes VIH-positives ont eu un comptage des CD4. Cependant, seulement 12,9% des femmes éligibles ont reçu une prophylaxie ART sur place ; seulement 35,7% ont été référées pour une mise en route de l'ART ; seulement 42.3% ont commencé l'IPT ; et aucune n'a reçu de CPT ni d'autres investigations relatives à la TB. Les femmes VIH négatives avaient 2,6 fois (IC95% 1,3­5,3) plus de chances de bénéficier d'un dépistage de TB que leurs homologues VIH positives.Conclusions: L'identification des femmes VIH positives et de celles suspectes de TB a été satisfaisante, mais la mise en œuvre des autres activités de collaboration TB-VIH a été insuffisante.

Marco de referencia: La prestación integrada de servicios de atención de la tuberculosis (TB) y la infección por el virus de la inmunodeficiencia humana (VIH) se recomienda ampliamente como parte de los servicios que se ofrecen a las madres durante la atención prenatal (ANC). En el presente estudio se evaluó la introducción de los servicios integrados de TB y VIH en una unidad hospitalaria de ANC.Método: Se examinaron las historias clínicas de una muestra aleatoria de 308 embarazadas que acudieron al servicio de ANC entre abril del 2011 y febrero del 2012. Se extrajeron datos de los registros y las historias clínicas de las pacientes. Los criterios de evaluación fueron la proporción de mujeres en quienes se practicó la orientación y las pruebas diagnósticas del VIH (HCT), el recuento de linfocitos CD4, el tratamiento antirretrovírico (ART), el tratamiento preventivo con cotrimoxazol (CPT), la detección sistemática de la TB y el tratamiento preventivo con isoniazida (IPT). En el análisis se midieron las variaciones en las características de las pacientes asociadas con la prestación de los servicios.Resultados: Todas las mujeres recibieron HCT y en 80% que obtuvieron un resultado positivo, se practicó la detección sistemática de la TB. En la mayoría de las pacientes positivas frente al VIH se practicó el recuento de linfocitos CD4 (85,9%). Sin embargo, solo 12,9% de las mujeres aptas recibieron la profilaxis ART en el lugar de la consulta; solo 35,7% se remitieron con el fin de comenzar el ART; apenas 42,3% de las pacientes comenzaron el IPT; y ninguna recibió CPT ni tuvo investigaciones complementarias por TB. Las mujeres con resultados negativos frente al VIH exhibieron un cociente de posibilidades 2,6 veces inferior de beneficiar de la detección sistemática de la TB, en comparación con las mujeres VIH positivas.Conclusión: Se constató una detección adecuada de las mujeres positivas frente al VIH y de casos con presunción clínica de TB, pero una prestación deficiente de las demás actividades de los servicios integrados del VIH y la TB.

Epidemiology of drug-resistant tuberculosis among children and adolescents in South Africa, 2005-2010.

OBJECTIVE: To describe the demographic and clinical characteristics of children and adolescents diagnosed with resistance to any anti-tuberculosis drug (drug-resistant tuberculosis; DR-TB) in South Africa. DESIGN: We retrospectively reviewed medical records of all children (<13 years) and adolescents (13 to <18 years) with DR-TB at specialty hospitals in four South African provinces from 2005 to 2010. RESULTS: During the review period, 774 children and adolescents (median age 11.3 years) were diagnosed with DR-TB at selected facilities. A high proportion of patients had a history of previous TB treatment (285/631; 45.2%), human immunodeficiency virus (HIV) infection (375/685; 54.7%), contact with a TB case (347/454; 76.4%), and smear-positive (443/729; 60.8%), cavitary (253/680, 38.7%) disease. Eighty-two per cent of patients with HIV infection received antiretroviral therapy. Of 626 patients diagnosed with multidrug-resistant TB (MDR-TB), 561 (89.6%) received a regimen consistent with national guidelines; the median length of treatment was 22 months (IQR 16-25). Among 400 patients with any DR-TB and a known outcome, 20.3% died during treatment. CONCLUSION: Pediatric DR-TB in these provinces is characterized by complex clinical features at diagnosis, with one in five children dying during treatment. History of previous treatment and contact with a TB patient indicate opportunities for earlier diagnosis and treatment to improve outcomes.

Rapid impact of effective treatment on transmission of multidrug-resistant tuberculosis.

BACKGROUND: Effective treatment for drug-susceptible tuberculosis (TB) rapidly renders patients non-infectious, long before conversion of sputum acid-fast smear or culture to negative. Multidrug-resistant TB (MDR-TB) patients on treatment are currently assumed to remain infectious for months. While the resources required for prolonged hospitalization are a barrier to the scale-up of MDR-TB treatment, the safety of community treatment is clear. OBJECTIVES: To estimate the impact of treatment on infectiousness among MDR-TB patients. METHODS: A series of five human-to-guinea pig TB transmission studies was conducted to test various interventions for infection control. Guinea pigs in adjacent chambers were exposed to exhaust air from a hospital ward occupied by mostly sputum smear- and culture-positive MDR-TB patients. The guinea pigs then underwent tuberculin skin testing for infection. Only the control groups of guinea pigs from each study (no interventions used) provide the data for this analysis. The number of guinea pigs infected in each study is reported and correlated with Mycobacterium tuberculosis drug susceptibility relative to treatment. RESULTS: Despite exposure to presumably infectious MDR-TB patients, infection percentages among guinea pigs ranged from 1% to 77% in the five experiments conducted. In one experiment in which guinea pigs were exposed to 27 MDR-TB patients newly started on effective treatment for 3 months, there was minimal transmission. In four other experiments with greater transmission, guinea pigs had been exposed to patients with unsuspected extensively drug-resistant tuberculosis who were not on effective treatment. CONCLUSIONS: In this model, effective treatment appears to render MDR-TB patients rapidly non-infectious. Further prospective studies on this subject are needed.

Health care workers' fears associated with working in multidrug- and or extensively-resistant tuberculosis wards in South Africa.

SETTING: Twenty-four drug-resistant tuberculosis (TB) hospitals and wards across all nine provinces of South Africa. OBJECTIVE: To assess health care workers' (HCWs') fears of working in multidrug-resistant TB (MDR-TB) or extensively drug-resistant TB (XDR-TB) wards. DESIGN: A cross-sectional descriptive study was conducted from June to September 2009 in 24 drug-resistant TB hospitals across South Africa. HCWs completed a self-administered questionnaire, including one open-ended question regarding personal concerns about their fear of contracting MDR- or XDR-TB. Responses were analysed by content analysis. RESULTS: Among the 24 hospitals, 499 HCWs were surveyed, of whom 363 (73%) responded to the open-ended question: 286 (86%) were nurses, 38 (11%) medical officers and 10 (3%) others. Six major themes regarding fears associated with the personal risk of acquiring drug-resistant TB emerged. These included the fear of 1) developing MDR- and XDR-TB, 2) the treatment course, 3) the financial implications, 4) family concerns, 5) working environment and 6) psychosocial issues. CONCLUSIONS: These data suggest that the greatest fear of HCWs working in drug-resistant TB wards is contracting MDR- or XDR-TB and infecting others. This fear may negatively impact the provision of quality patient-centred care, and highlights the need for training of HCWs in infection control measures, and specifically on how HCWs can protect themselves and others from developing TB.

Occupational health policies and practices related to tuberculosis in health care workers in KwaZulu-Natal, South Africa.

SETTING: Three district hospitals in KwaZulu-Natal, South Africa, with specialized drug-resistant tuberculosis (TB) wards. OBJECTIVE: To increase understanding of the implementation of occupational health (OH) and infection control (IC) guidelines for the prevention and control of TB among health care workers (HCWs). DESIGN: An operational cross-sectional study conducted between July and September 2011, consisting of interviews with OH and IC nurses and chart review of OH medical records. RESULTS: Although general national and provincial OH policies are in place, no specific OH policies exist for hospital settings. Two of three hospitals had a full-time OH nurse and all had a full-time IC nurse. All hospitals offered TB symptom screening; however, only 19% of HCWs were screened in 2010. TB incidence among HCWs was 1958 per 100 000 population in 2010. All hospitals offered HIV counseling and testing; however, only 22% of staff were tested across sites. Two hospitals offered isoniazid preventive therapy to HIV-positive staff and reassigned these staff to low TB risk areas. CONCLUSIONS: While OH policies and procedures are in place, implementation of these policies and procedures is inconsistent. This potentially places HCWs at risk of acquiring TB. These findings support the need for strengthening OH and IC services to prevent TB.

Missed opportunities for accessing HIV care among Tshwane tuberculosis patients under different models of care.

OBJECTIVE: To compare access to human immunodeficiency virus (HIV) care for tuberculosis (TB) patients in settings with antiretroviral treatment (ART) and TB care under one roof ('semi-integrated sites') and in settings with geographically separately rendered care in Tshwane, South Africa. METHODS: Historical cohort study of patients registered with TB at 46 TB treatment points, with follow-up until the end of anti-tuberculosis treatment. ART initiation for HIV-positive TB patients was established through linkage of TB register patient identifiers to the electronic ART register. Data analysis entailed univariate and multivariate competing risk analysis. RESULTS: The records of 636 and 1297 patients for semi-integrated and separate facilities, respectively, were reviewed. Cotrimoxazole prophylactic therapy and recording of CD4 count were lower in semi-integrated than in separate facilities, but the reverse was true for referral to HIV-related care. A higher percentage of patients started ART in the semi-integrated than in the separate facilities (70.5% vs. 44.6%, P < 0.001). In competing risk analysis (with death and lost to follow-up as competing risks), attending a semi-integrated facility (sub-hazard ratio [SHR] 2.49, 95%CI 1.06-5.88) and TB case load > 401 (SHR 1.45, 95%CI 1.04-2.03) were associated with increased ART initiation. CONCLUSIONS: ART and TB treatment under one roof appears to facilitate ART initiation for HIV-positive TB patients.

A national infection control evaluation of drug-resistant tuberculosis hospitals in South Africa.

BACKGROUND: The importance of infection control (IC) in health care settings with tuberculosis (TB) patients has been highlighted by recent health care-associated outbreaks in South Africa. OBJECTIVE: To conduct operational evaluations of IC in drug-resistant TB settings at a national level. METHODS: A cross-sectional descriptive study was conducted from June to September 2009 in all multidrug-resistant (MDR-TB) and extensively drug-resistant TB (XDR-TB) facilities in South Africa. Structured interviews with key informants were completed, along with observation of IC practices. Health care workers (HCWs) were asked to complete an anonymous knowledge, attitudes and practices (KAP) questionnaire. Multilevel modeling was used to take into consideration the relationship between center and HCW level variables. RESULTS: Twenty-four M(X)DR-TB facilities (100%) were enrolled. Facility infrastructure and staff adherence to IC recommendations were highly varied between facilities. Key informant interviews were incongruent with direct observation of practices in all settings. A total of 499 HCWs were enrolled in the KAP evaluation. Higher level of clinical training was associated with greater IC knowledge (P < 0.001), more appropriate attitudes (P < 0.001) and less time spent with coughing patients (P < 0.001). IC practices were poor across all disciplines. CONCLUSION: These findings demonstrate a clear need to improve and standardize IC infrastructure in drug-resistant TB settings in South Africa.

Auxotrophic, plasmid-cured Salmonella enterica serovar typhimurium for use as a live vaccine in calves.

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.

Characterization of south african isolates of Salmonella enteritidis by phage typing, numerical analysis of RAPD-PCR banding patterns and plasmid profiles.

Eleven of the 33 strains of Salmonella enteritidis (S.E.) included in this study belonged to phage type 34. Six strains belonged to phage type 14, six strains to phage type 4 and four strains to phage type 7. The remaining six strains belonged to phage types 35, 1, 24var (a variation of phage type 24), 9a, 1b and an unknown phage type. The majority of S.E. phage type 34 strains (eight of the 11) grouped at R2 > or =0.45 into one RAPD-PCR cluster with two strains of phage types 4, a strain of phage type 24var and a strain of phage type 9a, indicating that they consist of a genetically heterogeneous collection of strains. Two of the remaining three phage type 34 strains grouped into two different clusters, well separated from the other phage type 34 strains. One strain of phage type 34 was genetically diverse and did not cluster with any of the strains included in this study. Three of the phage type 14 strains grouped into cluster 11 at R2 > or =0.72, suggesting that they are genetically closely related. However, the remaining three strains of phage type 14 grouped into two separate clusters. Strains of phage types 7, 35, and 1 grouped in one cluster at R2 > or = 0.55. Our results clearly indicated that S.E. strains of the same phage type are not always genetically related. On the other hand, strains of a high genetic relatedness classified as different phage types. No specific plasmid profile could be linked to any of the phage types. Based on results obtained by LD50 virulence tests, strains containing the 38 MDa plasmid are more virulent compared to strains which do not contain the plasmid.

Phage types of Salmonella enterica serovar Enteritidis isolated in South Africa from 1991-1995.

A total of 615 strains of Salmonella enterica serovar Enteritidis (SE), received from 1991-1995 at the Onderstepoort Veterinary Institute (OVI), were phage typed. Most SE isolates (54,7%) originated from poultry followed by humans (28,5 %) and poultry eggs (9,6 %). Phage type 34 was the most prevalent (40,5 %) of all isolates, followed by phage type 4 (33,8 %). Other phage types identified were 1, lb, 4a, 7, 7a, 9a, 14, 24, 24var and 35 (in total 2,4% of isolates). Most isolates of SE were received from the Western Cape Province (47,4%) and Gauteng (22,3%). In poultry phage type 4 was dominant, but in humans, eggs, goats, ducks, sheep, pigs and rabbits, phage type 34 was the dominant type. It appeared as if the poultry-associated epidemic of SE in South Africa that occurred from 1991-1995 originated in the Western Cape Province during 1991 amongst poultry and then spread from there to humans and eggs and then to the rest of the country, where it emerged during 1993. Results indicate that phage type 34 was the dominant phage type from 1991-1993, but during 1994-1995 its presence declined. During this latter period the presence of phage type 4 increased. This may suggest that two smaller epidemics consisting of the two different phage types might have been responsible for the epidemic that occurred from 1991-1995.

The production of an auxotrophic marked, plasmid-cured Salmonella ser. Typhimurium as a live attenuated vaccine.

A number of amino acid requiring auxotrophic strains of Salmonella Typhimurium were produced by chemical mutagenesis. One of them, strain 81, was cured of the virulence plasmid and attenuated for mice. This strain had an auxotrophic requirement for serine, which could be used as a marker for the differentiation of the vaccine strain from other isolates in the field. The strain still contained the smooth form of the O-antigen, was resistant to Complement-mediated killing of serum and produced type 1 fimbriae. Of the six auxotrophic mutants only this mutant differed in its outer membrane protein profile from that of the parent strain in that an outer membrane protein of about 30 kDa was absent. With the use of the polymerase chain reaction, using total DNA of the cell as template, and with primers targeted to the virulence plasmid, it was shown that the virulence plasmid of Salmonella Typhimurium was completely cured from this strain. This strain also had a LD50-value of 4 log units lower for mice than the parent strain. The plasmid-cured strain gave a very high degree of protection to mice after systemic immunization, but not after oral vaccination. Compared to the parent, strain 81 also had a lower multiplication rate in the liver and spleen after intraperitoneal inoculation, characteristics that could be attributed to plasmid-loss, and it could also not be recovered from the spleen and liver of orally inoculated mice.