**4. Pathogens in cattle, humans and the environment**

Sample size of 100 clusters was calculated as previously described [16]. Face-to-face interviews were conducted to cattle keeping household members about cattle and manure management practices. Interview was also conducted to cattle keeping household neighbors who do not keep cattle about possible contact with cattle and manure. Individual fecal samples from 446 cattle, 100 stool samples from individuals keep cattle and 100 who do not keep cattle, 200 soil and 200 water samples from sources within homesteads were collected for bacteria isolation.

Out of 119 respondents, 5% reported to have heard about manure-associated pathogens which can infect human. There were 125 responses to problems related to manure management which respondents encounter. Out of these 125 responses, 77 (61.6%) said they encounter no problem, while 15 (12%) responses reported that poor infrastructure impedes manure management practices. Lack of working facilities such as utensils and transport was reported in 13 (10.4%) responses as one of the problems cattle keepers face, whereas land scarcity appeared in 6 (4.8%) responses. Health problems related to respiratory tract, injuries and foot rot to manure handlers were mentioned in 5 (4%) responses, same as for the presence of poor cattle housing facilities. Odor and water scarcity were each mentioned in 2 (1.6%) responses as among problems of manure management practices in urban and peri-

During the interview, Livestock Officers presented documents such as "Environmental Sanitation By-Laws" and "Animals in Urban areas By-Laws" which give directives on animal keep in the area and how to deal with wastes including manure. From interviews and the documents, the guideline which allows maximum herd size of four cattle per herd in urban area does not give area requirement specification and is not observed, and cattle manure is regarded by the by-laws and treated like any solid household waste [15]. It was observed that cattle keeping households are randomly distributed among non-cattle keeping households and there are no preconditions for a household to start keeping cattle. Anybody can start a herd of

The current manure management practices differ from those methods used a few decades ago in both the actual practices and resource base available which is shared between humans, animals and manure. Increased manure production in populated urban and peri-urban areas has resulted into the problems mentioned by cattle keepers. Some of these problems such as land scarcity odor and increased flies population have been previously reported to be due to exclusion of livestock farming during urban and peri-urban land use planning [4]. Increased manure production in a shrinking space has forced cattle keepers to collect, convey, store and finally dispose manure. Diverse cattle and manure management practices are determined by customs, convenience and availability of resources including land and equipment. Some farmers said that they keep cattle and handled the manure by the same methods since childhood; others opted for a particular cattle and manure management practice because it was easy to execute. Generally, there was direct contact between humans, cattle and manure and there was environmental contamination by fresh manure. In this scenario, humans,

Sample size of 100 clusters was calculated as previously described [16]. Face-to-face interviews were conducted to cattle keeping household members about cattle and manure management practices. Interview was also conducted to cattle keeping household neighbors who do not keep cattle about possible contact with cattle and manure. Individual fecal sam-

cattle anywhere in urban and peri-urban areas of Morogoro at any time.

animals and environment are exposed to manure-related pathogens.

**4. Pathogens in cattle, humans and the environment**

urban areas of Morogoro.

78 Livestock Science

*Escherichia coli* was isolated and characterized as described earlier [16]. In summary, nonsorbitol fermenting (NSF) *E. coli* were isolated by using sorbitol MacConkey agar, and suspect colonies were characterized biochemically by use of MacConkey agar, Brilliance *E. coli* agar and indole test. Confirmed NSF *E. coli* isolates were assessed for the presence of virulence genes: intimin gene (*eae*), verocytotoxin 1 (*vtx1*), verocytotoxin 2 (*vtx2*), heat-stable enterotoxin, human variant (*estA*-human), heat-stable enterotoxin, porcine variant (*estA*-porcine), heatlabile enterotoxin (*eltA*) and invasive plasmid antigen (*ipaH*) by multiplex diarrheagenic *E. coli* (DEC) PCR. Dot-blot DNA hybridization was done by using *vtx1, vtx2, eae, ehxA, EAF, bfpA, saa, astA and vtx2f* DNA probes to confirm the presence of virulence genes in isolates positive by DEC PCR. The colonies were lysed, denatured and neutralized using standard conditions and then hybridized as formerly described [17].

Somatic antigen O and flagella antigen H on diarrheagenic *E. coli* were typed by using specific antisera at Statens Serum Institut, Denmark, using a standard protocol [18]. In summary, both somatic O and flagella H antigens were tested by agglutination method against both pooled and specific antisera. For somatic O antigen, a boiled culture of *E. coli* isolate was tested against a pooled O antisera and culture with positive agglutination test was further tested against single specific O antisera. Somatic O antigen was assigned a number according to positive agglutination on a specific single O antigen. For flagella H antigen, an *E. coli* culture was tested for motility in semi-solid medium and fixed with formaldehyde 0.5%. Fixed culture was tested against pooled H antisera, and positive culture was further tested against single specific H antisera. Fluffy reaction indicated positive result, and the isolates were assigned a number.

Phenotypic activity of virulence genes was assessed on Vero cell monolayers to test for cytopathic effects using protocol formerly described [17].

For non-typhoidal Salmonella spp. isolation, 1 ml of the sample suspension was enriched by overnight incubation in selenite fecal broth at 37°C. The bacteria growth was subcultured on Salmonella-Shigella agar at 37°C for 24 h. Colorless colonies with a black center were biochemically tested by urease and lysine carboxylase tests. Urease-negative and lysine carboxylase-positive colonies were tested against Salmonella polyvalent agglutinating sera (REMEL30858201 ZC02—LOT 820883) and serotyped by Kauffmann-White M03-03-001 method at Danish Institute for Technology (DTU).

Vero cytotoxin-producing *E. coli* (VTEC) from cattle, enteropathogenic *E. coli* (EPEC) from cattle and water and attaching and effacing *E. coli* (A/EEC) from cattle were isolated (**Figure 1**). Overall prevalence of diarrheagenic *E. coli* in cattle (n = 446) was 2.2% (95% CI 0.99–3.67) and in water (n = 200) was 0.5% (95% CI 0.025–2.44). The prevalence of VTEC in cattle was 1.6% (95% CI 0.69–3.08), (**Table 2**) [16].

**Figure 1.** Multiplex DEC PCR for NSF *E. coli* isolates: lanes M: molecular weight size marker (100-bp plus DNA ladder); lane 1: *vtx2* and *eae*; lane 2: *vtx2* and *eae*; lane 3: *vtx2* and *eae*; lane 4: *vtx2* and *eae*; lane 5: *eae*; lane 6: *eae*; lane 7: *vtx1* and *vtx2*; lane 8: *vtx1* and *vtx2*; lane 9: *vtx1* and *vtx2*; lane 10: *eae*; lane 11: *eae*; lane P1: positive control for *vtx2*, *eae* and *vtx1*; lane P2: positive control for *ipaH, eltA* and *estA*; lane N: negative control.


**Table 2.** Zoonotic bacteria isolated from cattle, humans and environment in urban and peri-urban areas of Morogoro, Tanzania.

The prevalence of *Salmonella kentucky* in cattle was 0.45% (95% CI 0.001–0.016), while one *Salmonella weltevreden* and one *Salmonella amager* were isolated from different apparent healthy humans (**Table 2**).

The VTEC strains contained *vtx1a*, *vtx2b*, *vtx2c* and *vtx2d* subtypes either singly or in combinations, and phenotypic expression of virulence was confirmed by the cytopathic effect they caused to Vero cell monolayers (**Table 3**) [16].


**Table 3.** Vero cell assay (VCA) and *vtx* subtyping for non-sorbitol fermenting diarrheagenic *E. coli* isolates.

**Figure 1.** Multiplex DEC PCR for NSF *E. coli* isolates: lanes M: molecular weight size marker (100-bp plus DNA ladder); lane 1: *vtx2* and *eae*; lane 2: *vtx2* and *eae*; lane 3: *vtx2* and *eae*; lane 4: *vtx2* and *eae*; lane 5: *eae*; lane 6: *eae*; lane 7: *vtx1* and *vtx2*; lane 8: *vtx1* and *vtx2*; lane 9: *vtx1* and *vtx2*; lane 10: *eae*; lane 11: *eae*; lane P1: positive control for *vtx2*, *eae* and

Cattle O113:H2 VTEC vtx2

**Table 2.** Zoonotic bacteria isolated from cattle, humans and environment in urban and peri-urban areas of Morogoro,

Cattle O+:H16 VTEC vtx1 and vtx2 Cattle O113:H21 VTEC vtx1 and vtx2 Cattle O142:H34 EPEC eae, EAF and bfpA Water O142:H34 EPEC eae, EAF and bfpA Cattle O+:H- A/EEC eae ehxA and astA

Cattle O157:H7 VTEC vtx2, eae, ehxA and astA Cattle O157:H7 VTEC vtx2, eae, ehxA and astA Cattle O157:H7 VTEC vtx2, eae, ehxA and astA

*vtx1*; lane P2: positive control for *ipaH, eltA* and *estA*; lane N: negative control.

*Salmonella kentucky* Cattle *Salmonella kentucky* Cattle *Salmonella weltevreden* Human *Salmonella amager* Human

Tanzania.

80 Livestock Science

**Bacteria species Source Serotype Pathotype Virulence genes**

*Escherichia coli* Cattle O157:H7 VTEC vtx2, eae, ehxA and astA

Isolation of diarrheagenic *E. coli* and Salmonella species from cattle feces is an evidence of risk of infection to humans and environmental contamination. There was also isolation of diarrheagenic *E. coli* from water in the study area. The risk in this scenario is due to direct contact between cattle, humans and manure as well as direct spread of fresh manure onto land within residence. This risk can cross between cattle keeping households because different cattle herds come into contact during grazing, and the spread can reach the non-cattle keeping neighbors. Sharing of water sources between humans and cattle, at some instances during dry season, poses another threat to public health. It is fortunate that these highly pathogenic and fatal diarrheagenic *E. coli* were not detected in humans because only apparent healthy subjects were sampled. Isolation of *Salmonella amager* and *S. weltevreden* in human stool calls for an attention on pathogen transmission route because humans can also act as a source of pathogens to livestock and the environment.
