Antimicrobial resistance in faecal samples from buffalo, wildebeest and zebra grazing together with and without cattle in Tanzania.

AIM: The aim of this study was to determine whether the practice of co-grazing with cattle and wild life constitutes a risk of transmission of antibiotic resistant bacteria to wild ungulates. METHODS AND RESULTS: Faecal samples were collected from buffalo (n = 35), wildebeest (n = 40), zebra (n = 40) and cattle (N = 20) from Mikumi National Park, Tanzania (MNP), where cattle is prohibited and from Ngorongoro Conservation Area (NCA) where co-grazing is practiced. The number of coliforms and enterococci resistant to selected antibiotics was determined. Wild life generally harboured higher number of resistant Escherichia coli and Enterococci than cattle, but with no general influence in wild life of co-grazing with cattle. Vancomycin-resistant Enterococci were detected in wild life samples, and E. coli resistant to cefotaxime and enrofloxacin were observed among isolates from all wild life, but not from cattle. Culture independent estimates of the number of sulII gene copies obtained by qPCR did not differ between wild life from the two sample sites, while tetW was significantly higher in samples from MPN than from NCA. CONCLUSIONS: Antibiotic resistant bacteria were not more frequently found in ungulates grazing together with cattle than ungulates without this interaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This study did not indicate that transmission of antibiotic resistant bacteria is a frequent event following co-grazing of wild life and cattle.

Prevalence and characterization of verotocytoxin producing Escherichia coli O157 from diarrhoea patients in Morogoro, Tanzania.

Escherichia coli O157:H7 is an important agent of haemorrhagic colitis and haemolytic uraemic syndrome in children less than five years old and elderly people. The objective of this study was to investigate the prevalence of verotocytoxin producing E. coli 0157 (VTEC O157) among human patients with diarrhoea in Morogoro, Tanzania. Faecal samples originating from 275 human patients with diarrhoea were screened for presence of E. coli O157:H7. A total of 96 E. coli isolate were identified. Of these, 10 isolates were grouped into sorbitol non-fermenting and glucuronide negative and 49 isolates were sorbitol positive and glucuronide positive. The remaining 37 were sorbitol negative and glucuronide positive. Using the polymerase chain reaction techniques, a total often verotocytocin producing E. coli isolated in this study were used. The overall two (15%) and one (7%) of the isolated of E. coli possessed both attaching and effacing (eae A) and enterohemolysin (ehly) A genes respectively. Other enterobacterial agents including Pseudomonas spp, Proteus spp and coliforms were also isolated. The VTEC O157 isolates were 100% resistant to oxytetracycline, chloramphenicol, streptomycin, and amoxyclav. In conclusion, the isolation of diarrhoeaogenic E. coli O157:H7 in this region suggests that the pathogen is an important aetiology of acute gastroenteritis in Tanzania. There is therefore, need to improve sewage and refuse disposal system, the provision of safe potable water, sanitation, personal hygiene and health education in order to reduce infection with this and other enteric pathogens.

Prevalence and Characterization of Verotocytoxin Producing Escherichia coli O157 from Diarrhoea Patients in Morogoro; Tanzania

Escherichia coli O157:H7 is an important agent of haemorrhagic colitis and haemolytic uraemic syndrome in children less than five years old and elderly people. The objective of this study was to investigate the prevalence of verotocytoxin producing E. coli 0157 (VTEC O157) among human patients with diarrhoea in Morogoro; Tanzania. Faecal samples originating from 275 human patients with diarrhoea were screened for presence of E. coli O157:H7. A total of 96 E.coli isolate were identified. Of these; 10 isolates were grouped into sorbitol non-fermenting and glucuronide negative and 49 isolates were sorbitol positive and glucuronide positive. The remaining 37 were sorbitol negative and glucuronide positive. Using the polymerase chain reaction techniques; a total of ten verotocytocin producing E. coli isolated in this study were used. The overall two (15) and one (7) of the isolated of E. coli possessed both attaching and effacing (eae A) and enterohemolysin (ehly) A genes respectively. Other enterobacterial agents including Pseudomonas spp; Proteus spp and coliforms were also isolated. The VTEC O157 isolates were 100resistant to oxytetracycline; chloramphenicol; streptomycin; and amoxyclav. In conclusion; the isolation of diarrhoeaogenic E. coli O157:H7 in this region suggests that the pathogen is an important aetiology of acute gastroenteritis in Tanzania. There is therefore; need to improve sewage and refuse disposal system; the provision of safe potable water; sanitation; personal hygiene and health education in order to reduce infection with this and other enteric pathogens

Investigations of the occurrence of Avibacterium paragallinarum infections in Uganda.

Investigations were conducted to determine the occurrence of Avibacterium paragallinarum in poultry in Uganda. A total of 710 each of bacteriologic and serum samples were taken from chickens and turkeys for demonstration of A. paragallinarum and antibodies. Samples for isolation of A. paragallinarum were also subjected to direct polymerase chain reaction (PCR) for demonstration of the organism's presence. Antibodies to A. paragallinarum were demonstrated in the sera using the hemagglutination inhibition test. A total of five isolates were recovered from two out of five commercial layer chicken farms investigated where suspected cases of infectious coryza were reported, and all of them belonged to Page's serovar C. PCR detected more positive samples (11/68) than did culture (5/68). Isolates were not recovered from free-range poultry nor were there any positive samples by PCR. The overall seroprevalence was 40.5% and the seroprevalence to serovars A, B, and C were 18%, 0.5%, and 22%, respectively. Antibodies to all Page's serovars A, B, and C were demonstrated in free-range chickens but only serovar C antibodies were demonstrated in commercial chickens. No antibodies were demonstrated in turkeys. This is the first time infectious coryza has been confirmed in Uganda and the causative agent, A. paragallinarum, isolated. A high seroprevalence observed in free-range chickens seems to indicate a subclinical infection under extensive village management conditions.

Virulence characterization of Avibacterium paragallinarum isolates from Uganda.

Avibacterium paragallinarum isolates from Uganda were characterized for their virulence by comparison of their pathogenicity and their resistance to serum. Pathogenicity was evaluated using commercial Hisex Brown layer chickens, local indigenous chickens, local turkeys and local guineafowls inoculated with 108 colony-forming units of Av. paragallinarum and comparing their overall mean disease scores over a period of 20 days. Persistence of the bacteria in the host and water was also investigated for a 60-day period by culture and polymerase chain reaction as well as use of sentinel chickens. Serum resistance was measured by comparison of the growth kinetics and survival indices at 3 and 6 h. There was no difference in the virulence of the isolates. Commercial layer chickens and local indigenous chickens were equally susceptible to challenge, while turkeys and guineafowls only showed transient mild signs and did not transmit infection. Turkeys and guineafowls did not acquire the infection when placed in contact with infected chickens. The isolates were resistant in normal chicken serum at both 3 and 6 h of incubation but were resistant at 3 h and sensitive at 6 h in turkey and guineafowl sera. The resistance of the isolates to serum correlated with their pathogenicity in the different hosts. No carrier status was demonstrated in this study using polymerase chain reaction and culture. The present study demonstrates that Ugandan Av. paragallinarum isolates are pathogenic to chickens with only transient signs in turkeys and guineafowls, and that serum resistance could be a subject for further investigation as a predictor of virulence of these bacteria. The role of turkeys and guineafowls in transmission of Av. paragallinarum was not demonstrated in the present investigation.

Differences in resistance to Salmonella enterica serovar Gallinarum infection among indigenous local chicken ecotypes in Tanzania.

A study was conducted to evaluate the disease resistance potential in 105 chickens of six indigenous local chicken ecotypes in Tanzania by orally challenging 1-week-old chicks with 2.5 x 10(8) colony-forming units of virulent S. Gallinarum. For 14 days post infection, clinical signs, necropsy findings, antibody titres, packed cell volume, leukocyte population count, and viable bacterial cell counts in the liver and spleen were recorded. Clinical signs were recorded daily but other parameters were recorded on the day of infection, then on days 3, 6, 10 and 14 after infection. Clinical signs of fowl typhoid were evident in chickens from day 3 post infection and disappeared by day 9 post infection. Pathological lesions on sacrificed birds included enlargement of the liver and spleen with foci of necrosis on the liver, spleen and myocardium. The mean viable bacterial cell counts in the liver and spleen varied between ecotypes, although the differences were not statistically significant. There were significant differences in the leukocyte population in the peripheral blood, with one ecotype (Morogoro-medium) showing a consistent and significantly higher heterophil count compared with other ecotypes. It was concluded that there is a selectable resistance potential to S. Gallinarum among the local chicken ecotypes in Tanzania that may be attributable to non-specific host immune responses. Further studies are suggested.

Productivity of local chickens under village management conditions.

The productivity of local chickens under village management conditions was studied in six villages situated in three climatic zones within Morogoro District in Tanzania. Two villages were picked in each climatic zone (warm and wet, warm and dry, cool and wet) for the study. The data were obtained by actual measurement, qualitative observations and interview of members of the households directly responsible for the care of chickens. In addition, data sheets were given to selected farmers to record the performance of their chickens. The mean flock size for the three zones was 16.2, with a range of 2 to 58. The overall mean clutch size, egg weight and hatchability were 11.8, 44.1 g and 83.6%, respectively. The overall mean chick survival rate to 10 weeks of age was 59.7%. The mean live weights for cocks and hens were 1948 g and 1348 g, respectively. The mean growth rates to the age of 10 weeks were 4.6 g/day and 5.4 g/day, while those from 10 to 14 weeks of age were 8.4 g/day and 10.2 g/day for female and male birds, respectively. The age at first lay ranged between 6 and 8 months, and the average hen had three laying cycles per year. Most of the chickens were left to scavenge during the day and were provided with simple housing at night (95.2% of the owners). Only small amounts of supplementary feeds were occasionally given and minimal health care was provided. It was concluded that the low productivity of chickens was partly due to the prevailing poor management practices, in particular the lack of proper health care, poor nutrition and housing.

Comparative pathogenesis of experimental infections with Salmonella gallinarum in local and commercial chickens.

The pathogenicity of a virulent strain of Salmonella gallinarum was studied in local chickens and commercial layers. Sixty 4-month-old chickens were used. Of these, 50 chickens were orally infected with S. gallinarum, comprising 25 commercial layers and 25 local chickens. Five chickens in each group were used as uninfected negative controls. The clinical signs and pathological features of acute, subacute and chronic fowl typhoid were observed in both groups. Chickens in both groups seroconverted, but the antibody titre was significantly higher (p < 0.001) in the commercial layers. The antibody titre remained high to the end of the experiment in all the surviving chickens. Only one commercial layer chicken died during the course of the experiment. The PCV decreased significantly (p < 0.001) in the infected chickens of both groups as compared to the controls. The viable cell count of S. gallinarum in the liver and spleen reached a maximum on day 9 after infection in both groups. However, there was a significantly higher cell count (p < 0.05) in the commercial layers. The severity of the disease appeared to be slightly greater in the commercial layers, although the susceptibility was similar in both groups. It was concluded that, under experimental conditions, local chickens are just as susceptible to S. gallinarum infection as are commercial layers.

Nutrient status of crop contents of rural scavenging local chickens in Tanzania.

1. A total of 144 chickens purchased from peasants in Morogoro, Tanzania were slaughtered, their crops dissected and the contents analysed. The birds consisted of 48 chickens from each of three climatic zones (warm and wet, warm and dry, cool and wet). Seventy-two chickens were slaughtered in each season, that is short rainy and long rainy season. 2. Cereal grains, bran, green forages, insects/worms and kitchen food wastes were the main crop contents and their composition varied significantly with season and climate. 3. The overall mean chemical compositions (g/kg) of the crop contents were: 430.8 +/- 107.78 dry matter (DM), 104.4 +/- 43.47 crude protein (CP), 61.2 +/- 36.48 ether extract (EE), 58.2 +/- 26.29 crude fibre (CF), 125.4 +/- 58-27 ash, 6.3 +/- 5.19 calcium (Ca) and 3.6 +/- 2.41 phosphorus (P). 4. CP, EE, ash and P contents varied significantly (P<0.05) with season but contents of DM, CF and Ca were not significantly different. 5. CP content was significantly higher (P<0.05) in growers than in adults while the reverse was true for Ca content. Other variables did not differ significantly. 6. Significant differences (P<0.05) in chemical composition were observed among the zones in all parameters except for CP content. 7. The study showed that the chemical composition of feeds eaten by rural scavenging chickens was below the nutritional requirements and varied with season, climate and age of birds.

Efficacy of crude extract of Aloe secundiflora against Salmonella gallinarum in experimentally infected free-range chickens in Tanzania.

The ethnoveterinary use of Aloe species extract in free-range local chickens against fowl typhoid was investigated. Five-months-old local chickens, free of antibodies against fowl typhoid were used. The chickens were randomly assorted into five groups including pretreated and infected (G1, n=21), infected and untreated (G2, n=21), infected and treated (G3, n=21), untreated and uninfected (G4, n=10) and treated uninfected (G5, n=10). Groups 1, 2 and 3 were inoculated with 5.0 x 10(8) c.f.u/ml of Salmonella gallinarum, following which the chickens were monitored for 15 days. There was a delay on the occurrence of the clinical signs and reduced severity of the disease in the Aloe treated chickens (G1 and G3). The mortality rates were 23.8% (5/21) in G1 (pretreated and infected), 42.8% (9/21) in G2 (infected and untreated) and 14.2% (3/21) in G3 (infected and treated). Antibody levels were raised among the infected and untreated group (G2) while they remained relatively low in the Aloe treated groups (G1 and G3). However, there was a sharp increase in the levels of interleukin 6 (IL-6) in the Aloe treated groups (G1 and G3) as compared to the untreated group (G2) until day 9 post-infection. The results of this work indicate that the extract of Aloe secundiflora may be used in the control of fowl typhoid in chickens. Studies to determine the active ingredients of the plant extract are in progress.

Phenotypes including immunocompetence in scavenging local chicken ecotypes in Tanzania.

A study was conducted to determine the variations in physical characters and immunocompetence among scavenging local chicken ecotypes in Tanzania. Eighty-four adult scavenging local chickens from four eco-climatic regions of Tanzania were studied. Measurements of adult body weight, body length, shank length and egg weight and observations of plumage colour and pattern, earlobe colour, skin colour and the shape of the comb were conducted. The antibody response to sheep red blood cells, serum haemolytic complement and the cutaneous response to phytohaemagglutinin-P were assessed. Five ecotypes were identified and named Mbeya, Morogoro-medium, Ching'wekwe, Kuchi and Singamagazi. Singamagazi and Kuchi were significantly heavier, with longer shanks and heavier eggs than the other ecotypes. The average adult body weight for males ranged from 1621 g (Mbeya) to 2915 g (Singamagazi). Average female weights ranged from 1108 g (Morogoro-medium) to 2020 g (Singamagazi). Mean egg weights ranged from 37.65 g (Ching'wekwe) to 45.61 (Singamagazi). The Kuchi had mostly rose and walnut combs, while the other ecotypes were mostly single combed. In each ecotype there were chickens with a high or low antibody response to red blood cells, but there was a significant difference between the ecotypes.

Molecular epidemiology of Salmonella gallinarum in chickens in Tanzania.

A molecular epidemiological investigation of Salmonella gallinarum infection in scavenging local chickens and commercial layers in Tanzania was conducted between August 1997 and April 1998. A total of 1152 chickens were randomly selected from 10 villages and seven commercial farms. For serological and cultural prevalence studies, 1152 blood samples and 912 cloacal swabs were collected. In scavenging local chickens, the individual serological and cultural prevalences were 6.3 and 0%, while the prevalences were significantly higher in commercial layers at 18.4 and 2.6% (P < 0.001), respectively. The risk of infection in flocks of scavenging local chickens that had contact with commercial chickens was six times greater than the risk of infection in flocks of scavenging local chickens that had no contact with commercial chickens. Thirty-four S. gallinarum isolated from commercial chickens in this study, together with 29 Tanzanian historical isolates, were characterized using plasmid profiling and ribotyping. Fifty-one isolates contained both 85 and 2.5kb plasmids, five isolates contained only one plasmid of 85kb, and seven isolates had no plasmids. Ribotyping using HindIII restriction endonuclease demonstrated seven different ribotypes. Forty-seven isolates had similar results in both typing systems, suggesting they belonged to one clone. It is concluded that S. gallinarum infection in chickens in Tanzania is more prevalent in commercial layers than in scavenging local chickens. One strain of S. gallinarum from chickens first isolated in a Dar es Salaam hatchery was found to be common throughout the country.

A disc ELISA for the detection of Salmonella group D antibodies in poultry.

An ELISA using lipopolysaccharide antigens prepared from Salmonella gallinarum and S enteritidis was developed for the serological diagnosis of fowl typhoid and S enteritidis infection in poultry. There was good agreement between the results of the ELISA and conventional serological tests when samples from naturally infected birds and S enteritidis immunised birds were tested. Some cross reactions were observed when serum samples from S typhimurium infected birds were tested by ELISA. Subsequently a disc ELISA, using filter paper discs, was developed to facilitate sampling and testing of poultry. There was good correlation between the results of the disc and serum ELISAs and the test is recommended for the field testing of birds.

Humoral factors of natural resistance of Bos indicus cattle selected for antibody titre to Brucella abortus.

The concentration of lysozyme, total immunoglobulin and bactericidal activity were measured in sera of Bos indicus cattle, retrospectively screened for specific antibodies to Brucella abortus and classified as being positive reactors or negative reactors. In addition, the effect of complement in the sera was studied to demonstrate complement dependence of antibody-mediated bacterial killing. It was observed that, under the test conditions, serum bactericidal activity and concentration of total immunoglobulin were associated with high specific antibody levels (P less than 0.001). Furthermore, there was a slight decrease in the lytic activity of lysozyme in the sera of animals with high antibody titres.

Serum, disc and egg ELISA for the serodiagnosis of Salmonella gallinarum and S. enteritidis infections in chickens.

An ELISA was evaluated for the serodiagnosis of fowl typhoid and paratyphoid due to Salmonella enteritidis in chickens. The hot phenol: water lipopolysaccharide (LPS) extract of Salmonella was used as the antigen. Chicken serum, eggs and discs impregnated with chicken blood were tested for the presence of antibodies against Salmonella factor 'O' 9 antigen. The substrate and chromogen used were hydrogen peroxide and orthophenylenediamine respectively. Serological results from the experimentally and naturally infected chickens showed close agreement between the conventional Serum Tube Agglutination Test (SAT) and serum ELISA while serum ELISA results were in close agreement with the egg and disc ELISA results. It was noted that ELISA was highly sensitive, convenient and versatile. It is concluded that ELISA, especially disc ELISA, ought to replace SAT for seroscreening chickens against S. gallinarum and other Salmonella Group D infections.