Serological and PCR investigation of Yersinia pestis in potential reservoir hosts from a plague outbreak focus in Zambia.

BACKGROUND: Plague is a bacterial zoonotic disease, caused by Yersinia pestis. Rodents are the natural hosts with fleas as the vehicle of disease transmission. Domestic and wild dogs and cats have also been identified as possible disease hosts. In Zambia, plague outbreaks have been reported in the Southern and Eastern regions in the last 20 years. Based on these observations, Y. pestis could possibly be endemically present in the area. METHODS: To substantiate such possibility, sera samples were collected from rodents, shrews, dogs and cats for detection of antibodies against Fraction 1 gene (Fra1) of Y. pestis while organs from rodents and shrews, and fleas from both dogs and rodents were collected to investigate plasminogen activator gene (pla gene) of Y. pestis using ELISA and PCR respectively. RESULTS: A total of 369 blood samples were collected from domestic carnivores, shrews and domestic and peri-domestic rodents while 199 organs were collected from the rodents and shrews. Blood samples were tested for antibodies against Fra1 antigen using ELISA and 3% (5/165) (95% CI 0.99-6.93%) dogs were positive while all cats were negative. Of 199 sera from rodents and shrews, 12.6% (95% CI 8.30-17.98%) were positive for antibodies against Fra1 using anti-rat IgG secondary antibody while using anti-mouse IgG secondary antibody, 17.6% (95% CI 12.57-23.60%) were positive. PCR was run on the organs and 2.5% (95% CI 0.82-5.77%) were positive for plasminogen activator gene of Y. pestis and the amplicons were sequenced and showed 99% identity with Y. pestis reference sequences. All 82 fleas collected from animals subjected to PCR, were negative for pla gene. The specific rat-flea and dog-flea indices were 0.19 and 0.27 respectively, which were lower than the level required to enhance chances of the disease outbreak. CONCLUSIONS: We concluded that plague was still endemic in the area and the disease may infect human beings if contact is enhanced between reservoir hosts and flea vectors. The lower specific rodent-flea Indices and absence of Y. pestis in the potential vectors were considered to be partly responsible for the current absence of plague outbreaks despite its presence in the sylvatic cycle.

Observations on the endemicity of plague in Karatu and Ngorongoro, northern Tanzania.

Commensal and field rodents and wild small carnivores were live-trapped in five villages of Karatu district and one settlement in the Ngorongoro Conservation Area in Ngorongoro district in Tanzania. Blood samples were taken and serologically tested for plague, using the Blocking ELISA technique. Some domestic dogs and cats in the Karatu villages were aseptically bled and similarly tested for plague. Fleas were collected from the examined animals and from randomly selected residential houses. A total of 241 rodents, 1 Crocidura spp, 43 dogs, 12 cats and 4 slender mongooses were involved in the survey. Of the rodents, 14.5% were infested with fleas, which comprised of Xenopsylla brasiliensis (45.8%) and Dinopsyllus lypusus (54.2%), with an overall population density of 0.2 fleas/animal. Thirty one (72.1%) of the dogs were infested with fleas, all of which were Ctenocephalides spp. Thirty five (63.3%) houses were infested with fleas whose population was composed of Ctenocephalides spp, Pulex irritans, Tunga penetrans and Echinophaga gallinacea. Infected rodents were found in all the villages while the infected dog was found at Rhotia-Kati. Nineteen (11%) of the rodents and one (2%) dog harboured specific plague antibodies. It was broadly concluded that sylvatic plague was endemic in Karatu district and Ngorongoro Conservation Area and that outbreaks of the disease can occur in the area any time if and when relevant conditions become favourable. Prompt application of appropriate preventive and control measures and survey for substantiating the status in the Lake Manyara National Park, which is adjacent to some of the infected villages, are recommended.

Plague epidemiology and control in eastern and southern Africa during the period 1978 to 1997.

Human plague is one of the important re-emerging zoonotic diseases threatening public health in some African, Asian and South American countries. During the period 1978 to 1997, Africa reported the largest numbers of plague cases and deaths in the world. During the decade 1978 to 1987, 19 countries reported 10 537 cases. Ten of the countries and 41.9% of the reported cases were in Africa. At least 14.5% of the cases reported in Africa died. Similarly, during the decade 1988 to 1997, 22 countries, 12 of which were in Africa, reported a total of 17,674 cases and 1,465 deaths. Of these, 11,711 cases of which 9.6% died, were reported from Africa.

Preliminary observations on factors responsible for long persistence and continued outbreaks of plague in Lushoto district, Tanzania.

Human plague has been an important public health problem in Tanzania for over a century. Recorded outbreaks of the disease have been reported from various parts of Tanzania, including Iringa, Kagera, Singida, Mbulu, Arusha and Kilimanjaro since 1886. Since 1980 however, only Lushoto, Singida and Karatu districts have experienced outbreaks of the disease. Of these areas, Lushoto has disproportionately high incidences of the disease and this has persisted for nearly 17 years. Efforts to curb the disease through conventional methods, including control of vectors and reservoirs, chemotherapy and chemoprophylaxis, enforcement of sanitation improvement as well as health education, have been applied every year, but plague cases and deaths continue to occur in the area to date. During the period April 1980 to December 1996, a total of 6599 cases with 580 (8.8%) deaths were recorded. Biological factors, such as the presence of suitable rodent reservoirs, efficient flea vectors and plague bacillus, could be partly responsible for the long persistence and recurrence of the disease. Since such factors are also common in other plague foci where the disease has never been persistently recurrent, and where indigenous people are culturally different from those in Lushoto, it is assumed that socio-cultural factors play an important role as determinants of the disease in the latter district. This paper reports preliminary observations on socio-cultural, biological and environmental factors which are thought to be, at least partly, responsible for the long persistence and repeated outbreaks of plague in the district. These include traditional beliefs on the cause and health seeking behaviour for treatment of plague, sleeping and food storage habits, large populations of rodents and fleas, and status of the immediate environment.

PIP: Human plague has been an important public health problem in Tanzania for more than a century, with outbreaks of the disease reported from various parts of the country, including Iringa, Kagera, Singida, Mbulu, Arusha, and Kilimanjaro since 1886. However, since 1980, only Lushoto, Singida, and Karatu districts have experienced outbreaks of plague. Of those areas, Lushoto has had disproportionately high incidences of the disease for the past 17 years. Plague cases and deaths continue to occur despite the annual application of conventional measures to check the disease. 6599 cases with 580 (8.8%) deaths were recorded between April 1980 and December 1996. Biological factors such as the presence of suitable rodent reservoirs, efficient flea vectors, and plague bacillus could be partly responsible for the long persistence and recurrence of the disease. However, sociocultural factors also help to propagate and perpetuate plague in Lushoto. Preliminary observations are reported upon sociocultural, biological, and environmental factors thought to be partly responsible for the persistence and repeated outbreaks of plague in the district. Facilitating factors include traditional beliefs upon the cause and health-seeking behavior for treatment of plague, sleeping and food storage habits, large populations of rodents and fleas, and the status of the immediate environment.

Bait bases for Mastomys natalensis Smith 1834: a murid of public health interest in Africa.

Mastomys natalensis is widely distributed in the whole of Africa and is a reservoir of important zoonoses including plague. In order to control this species efficiently using rodenticides, tests were carried out to find the most preferred cereal and bait additives to form a suitable bait base. Investigations were based on food choice tests in caged animals. Among the cereals tested, rice was significantly preferred (P=0.01) followed by wheat, sorghum and maize. Of the additives tested, both sexes of M. natalensis significantly preferred baits containing either 1,3 and 5% sucrose (p=0.05), or 3 and 5% glycerine, or 3% fish, or 1% sucrose. Also both sexes rejected bait containing 1 and 3% molasses. Additives such as 1% glycerine, 1 and 3% groundnut did not significantly influence bait intake. Sex preference differential was observed on baits containing 1 and 5% fish, 1 and 5% groundnut, 5% cotton oil, 5% cod liver oil and 0.5% salt. It was concluded that bait additives which scored 70% or more should be considered suitable for M. natalensis bait. However further studies are recommended on this subject.

Importance of intersectoral co-ordination in the control of communicable diseases, with special reference to plague in Tanzania.

Human health, agriculture, including livestock, energy, education, wildlife, construction, forestry and trade sectors are inter-related and their co-ordination is an important pre-requisite for successful control of most communicable diseases including plague. Similar linkage between research, policy, training and extension activities in each sector are essential for any successful control strategy. Inadequate agricultural produce, inaccessibility of people to the available food and ignorance on proper preparation and usage of available food materials are responsible for malnutrition, and malnourished people are very vulnerable to disease. Irrigation schemes facilitate breeding of various disease vectors and transmission of some communicable diseases. Forests are ecologically favourable for some disease vectors and reservoirs for tsetse flies and rodents, while deforestation leads to soil erosion, lack of rainfall and consequently reduced productivity in agriculture which may result in poor nutrition of the population. Wildlife and livestock serve as reservoirs and/or carriers of various zoonoses including plague, trypanosomiasis and rabies. Lack of proper co-ordination of these sectors in communicable disease control programmes can result in serious and undesirable consequences. Indiscriminate killing of rodents in order to minimize food damage by these vermin forces their flea ectoparasites to seek alternative hosts, including man, a development which may result in transmission of plague from rodents to man. Similarly, avoidance of proper quarantine during plague epidemics, an undertaking which is usually aimed at maintaining economic and social links with places outside the affected focus, can result in the disease becoming widespread and consequently make any control strategies more difficult and expensive.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of dogs in plague epidemiology in Tanzania. Serological observations in domestic animals in Lushoto District.

Venous blood was collected aseptically from clinically healthy domestic dogs, goats, sheep, cats and fowl in various plague-infected villages of Lushoto District, Tanzania, at the time when the disease was actively prevalent in the area. Flea ectoparasites were collected from the animals, processed, identified and counted. Serum samples were tested for specific plague antibodies, using the passive haemagglutination technique and checked by passive haemagglutination inhibition tests. Altogether 389 animals, of which 201 were domestic dogs, were involved. 11 (5.5%) dogs had significantly elevated specific plague antibodies at titres ranging from 20 to 1280. All the dogs were also heavily infested with fleas at a mean index of 7.7 fleas per animal. Of 1,871 fleas collected from the dogs, 93.8% were Ctenocephalides felis and 6.2% were C. canis. All the other animals examined were negative for plague. It was concluded that domestic dogs could play an important role as plague carriers in the area and that the animals could serve as sentinel animals for the detection of plague in villages where human plague outbreaks have not previously occurred.

The current epidemiology and control of trypanosomiasis and other zoonoses in Tanzania.

The epidemiology and control strategies of African trypanosomiasis, plague, rabies, brucellosis, anthrax and hydatidosis, the most important and well documented zoonotic diseases in Tanzania, have been described. Bovine tuberculosis, tetanus, taeniosis, trichinosis and tungosis are also endemic in some parts of the country but records of their incidences are not available. Initial outbreaks of trypanosomiasis in Tanzania were caused by Trypanosoma gambiense which originated from West Africa and reached Tanzania via Zaire around 1902. T. rhodesiense which is currently responsible for human trypanosomiasis in Tanzania was introduced from Mozambique around 1910 and quickly spread to many parts of the country. The disease is currently prevalent in the western, north and northwestern parts, the southern highlands and southern regions. Over 6000 cases have reported since 1979. Control strategies against sleeping sickness in Tanzania include chemical control of vectors, treatment of patients with trypanocides and avoidance of humantsetse contact. Plague is mostly endemic in central, northern and north-eastern Tanzania. A total of 8161 cases with 1885 deaths have been recorded since 1890. The disease is currently prevalent in Lushoto district where outbreaks have been experienced since 1980, and in Singida district where it has been endemic since 1918. Integrated control measures are currently applied and were possibly responsible for the 1989 decline of outbreaks in the area. Financial constraints which led to deterioration of control activities from July 1989 probably accounted for the severe outbreaks in 1990/91 which spread to other parts of the country. Rabies is endemic country-wide except in Mtwara, Lindi and Zanzibar. Domestic dogs are the principal transmitters and prompt vaccination and destruction of unvaccinated stray dogs are the main control measures. Brucellosis is widely endemic in livestock and potentially so in humans. Destruction of infected animals, immunisation of susceptible ones, proper boiling of milk and its products and chemotherapy are the currently applied control measures against the disease. Anthrax and hydatidosis are sparsely endemic in the country, and they are mostly controlled by appropriate meat inspection and consequent condemnation and proper disposal of the affected meat. Vaccination and treatment of animals are also effective against anthrax.

Observations on the epidemiology of plague in Tanzania during the period 1974-1988.

Field and commensal rodents and shrews were live-trapped from selected areas in each of the six zones of the Republic, namely North-eastern, Eastern, Central-western, South-western, Southern and Seaports. The captured animals, small ruminants, humans and small carnivores were serologically tested for specific plague antibodies, using the Passive haemagglutination (PHA) and Passive haemagglutination Inhibition (PHAI) techniques. Taxonomic studies and population densities of rodents and their ectoparasasites were carried out. A total of 5638 animals were captured and 79.9% of them were tested for plague. Of those tested, 2.4% contained agglutinating plague antibodies at serum dilutions of 1:20 and above. The positive rodents were found in Lushoto, Mbulu, Chunya and Monduli districts, as well as at Tanga seaport. These include R. rattus, M. natalensis, A. niloticus and Otomys spp. A total of 7480 human, 293 carnivorous and 425 small ruminant sera were also tested for plague. Seven (2.4%) of the carnivores were positive at serum titres of 1:20-1:80. All the small ruminants were negative. Likewise, 1.4% of the tested human sera contained specific plague antibodies at titres ranging from 1:20-1:60. Out of 6480 fleas collected from the captured small mammals, 5476 were identified. Of these, 34.5%, 20.4%, and 17.8% were X. brasiliensis, D. lypusus and X. cheopis respectively. It was generally concluded that plague was still endemic in several parts of the country, that some foci were active, and that there was a potential risk of accidental introduction and/or transfer of the disease into or out of the country. Establishment and maintenance of surveillance services and facilitating research and control programmes of the disease were recommended.

Plague in Lushoto district, Tanzania, 1980-1988.

Rodents were live-trapped in selected plague-inflicted villages from June 1980 to March 1988. Flea infestation rates were determined and the animals were serologically tested for plague. Clinically suspected and clinically healthy people in the affected areas were similarly tested for plague antibodies. Of 1596 rodent sera tested, 91 (5.7%) were positive for plague. These were mostly from Rattus rattus, Mastomys natalensis, Otomys spp. and Pelomys fallax. A total of 1772 fleas, of which Dinopsyllus lypusus, Xenopsylla brasiliensis and Ctenophthalmus calceatus comprised the largest proportion, was collected from the captured rodents. Total flea indices ranged from 0.67 to 1.12 fleas per rodent. A total of 2809 human cases and a mortality rate of 10.2% were recorded in 1980-1988. It was concluded that most rodent species in the area were suitable reservoirs of plague and that D. lypusus, X. brasiliensis and C. calceatus were probably responsible for transmitting the pathogen. Lack of effective quarantine measures during outbreaks was partly responsible for the spread of the disease to many villages, while inadequate rodent and flea control and poor sanitary measures could be responsible for continued outbreaks of plague in the area.

A decade of plague epidemiology and control in the western Usambara mountains, north-east Tanzania.

Outbreaks of human plague have been occurring in the Western Usambara mountains since 1980, involving many cases and deaths. Epidemiological surveys and control activities were carried out from June 1980 to May 1990. Rodents were trapped live, identified and serologically tested for plague, using the passive haemagglutination and passive haemagglutination inhibition tests. Rodent fleas were collected, processed, identified and counted. House fleas were caught with light traps and similarly treated. People and domestic carnivores were serologically tested for the disease. Various plague control measures were undertaken. A total of 2433 animals, 2254 rodent fleas and 1366 house fleas were collected. Average indices of rodent and house fleas were 0.93 and 5.38 respectively. Rattus rattus and Mastomys natalensis were the most abundant rodent species. Xenopsylla brasiliensis and Dinopsyllus lypusus were their commonest flea ectoparasites while Pulex irritans was the major house flea. Of 2044 rodent, 1880 human and 176 dog sera tested, 5.5%, 0.5% and 6.3% respectively were positive. It was concluded that plague was active in the focus despite the control measures and that common reservoirs and efficient vectors were present. It was suggested that dogs were probably involved in the epidemiology of the disease, that P. irritans was not susceptible to the insecticide used and that the flea was probably involved in murine plague transmission. Bacteriological research on the causative agent to establish the nature of its long persistence in the area and maintenance of a surveillance service are recommended.

Seasonal patterns of rodents, fleas and plague status in the Western Usambara Mountains, Tanzania.

Field and commensal rodents were live-trapped at three villages in an active focus of plague (Yersinia pseudotuberculosis pestis) in Lushoto District, Western Usambara Mountains, Tanga Region, Tanzania, from December 1983 to November 1984. Their flea ectoparasites were collected, identified and counted. The rodent carcasses were serologically examined for specific plague antibodies and antigens, and bacteriologically examined for bipolar staining bacilli. A total of 1758 traps were set during the 12-month period and 924 animals were caught. From these, 1037 fleas were collected. Rattus rattus (L.), Praomys natalensis (Smith) and Lophuromys flavopunctatus Thomas comprised the largest proportions of the rodent population, while Dinopsyllus lypusus Jordan & Rothschild, Ctenophthalmus calceatus Waterston and Xenopsylla brasiliensis (Baker) were the dominant flea species. Rodents were most abundantly trapped during December and January. Flea indices were highest from December to May. Human plague was most active from November to March. Rodents contained plague antibodies every month except May and July, with a peak in September. Plague antigens and bipolar bacilli were detected in rodent organs during January-April. From the product of abundance and infection rate, the most prevalent rodent hosts of plague appeared to be R. rattus, Otomys angoniensis Wroughton, P. natalensis and Pelomys fallax (Peters). Continuous integrated control of rodents and fleas was recommended, reinforced by quarantine and maintenance of a surveillance service for clinical detection, diagnosis and treatment of patients in the plague endemic area.

Observations on the susceptibility of Ctenocephalides felis (Siphonaptera: Pulicidae) to malathion and permethrin in Tanzania.

Laboratory-reared Ctenocephalides felis (Bouche) adults were tested with 0.5% malathion and 0.5% permethrin, using the standard WHO methods. After 24 h exposure to malathion (3.6 mg/cm2), 92% of the fleas died. The LT50 for malathion was approximately 8 h. Permethrin (0.45 mg/cm2) produced 100% mortality of exposed insects after 24 h while with a higher dose (0.9 mg/cm2) all fleas died after 8 h exposure. LT50 for the two doses of permethrin were 7.7 and 1.05 h, respectively. The failure of the diagnostic dose of malathion to kill 100% of the population was attributed to resistance. Permethrin is a suitable pesticide for controlling fleas of domestic animals in Tanzania.

The baseline susceptibility levels of Chunya Praomys natalensis, the commonest plague reservoir and crop pest in Tanzania, to warfarin.

P. natalensis were live-trapped at Kibwawa (Chunya district) in November 1977 and November 1979. An F3 generation was raised from animals which tolerated 0.025% a.i. warfarin. All three populations were tested using the WHO standard method (WHO/VBC/75.595), with 0.025% warfarin. The populations tested were fully susceptible to the rodenticide, and the latter was well acceptable to the rodents. In this area, warfarin can be successfully used for controlling P. natalensis. Failure to obtain 100% mortality with the 1977-caught wild population was probably due to vigour tolerance. Regular seminars are recommended to train staff in the rodenticide application.

Observations on the current status of plague endemicity in the western Usambara mountains, north-east Tanzania.

Epidemiological investigations were executed at 6 locations in the western Usambara mountains in north-east Tanzania from November 1980 to May 1982. Rodent, human and dog sera were checked for agglutinating plague antibodies, using the passive haemagglutination test. Fraction I plague antigen was similarly tested for in rodent organ macerates. Bubo aspirates and rodent organ-smears were microscopically examined for plague bacilli. A total of 257 rodents and 191 fleas were collected. Rattus rattus, Praomys natalensis and Arvicanthis niloticus were the commonest rodent species. Flea ectoparasites mostly comprised of Xenopsylla brasiliensis and Dinopsyllus lypusus. Specific plague antibodies were detected in 2.8%, 10% and 2.9% of the rodents examined in November 1980, November 1981 and May 1982 respectively. Similarly, plague antibodies were found in 0.6%, 1.2% and 0.5% of the human sera tested in September 1981, November 1981 and May 1982 respectively. All the dog sera were negative. The results suggested past contact with plague and existence of a persistent plague focus in the area. Health education for villagers, maintenance of a plague surveillance programme and regular seminars for medical and health personnel in the area were recommended in order to prompt early detection, reporting and control of an outbreak.

The first outbreak of human plague in Lushoto district, north-east Tanzania.

A one-week investigation was performed at Mkunki and Mavumo (ward of Shume, Lushoto district, Tanzania) at the time of outbreak (June, 1980). Rodents, people, domestic dogs and fleas were examined, 36 rats, mostly Rattus rattus, were caught and used in serological and bacteriological analysis for plague. 48 fleas, mostly Xenopsylla brasiliensis, were collected from the captured rodents. 413 people and 11 dogs were examined. All rodent, carnivorous and human sera were tested for plague antibodies, using the Passive Haemagglutination (PHA) test. Similarly protein extracts from rodent organs were tested for Yersinia pestis fraction I. Impression smears prepared from these organs were stained with methylene blue and examined for presence of bipolar bacteria. Bacteriological isolation of Y. pestis was performed on bubo aspirates and/or blood samples of ten patients. Over-all total and infested flea indices of rodents were 1.3 and 3.7, respectively. Bipolar organisms were demonstrated in 13.3% of the rodent smears while specific agglutinating plague antibodies were detected in 3.3% of the tested rodent sera. Y. pestis fraction I antigen was detected in 8.3% of the rodents. Of all the human sera tested, 1.4% contained specific plague antibodies. Y. Pestis was bacteriologically isolated and biochemically confirmed in 16.7% of the cultured specimens. All the dog sera were negative for plague. It was generally concluded that the causative agent of the outbreak was Yersinia pestis and that the disease probably spread from Rongai (Rombo district).