**5. Transmission of bacteria between cattle, humans and environment**

The study on transmission of bacteria involved 100 clusters, and each cluster was formed by a pair of a cattle keeping household and a neighboring non-cattle keeping household. Each cluster contributed two stool samples, two water samples and two soil samples, one of the samples from cattle keeping household and another from a non-cattle keeping household. Isolation, characterization and quantification of the risk of transfer of *E. coli* were done as earlier reported [19]. In summary, isolation of *E. coli* was carried out by inoculating a loopful suspension of cattle feces and stool from cattle keepers and non-cattle keepers, soil and water on MacConkey agar followed by 24-h incubation at 37°C. *E. coli* suspected isolates were confirmed and screened for double antimicrobial resistance to ampicillin and tetracycline on antimicrobial embedded Petrifilm™ *Select E. coli* count (SEC) plate. Preparation of antimicrobial stock solution and screening procedure was done according to Ref. [20]. Ampicillin-tetracyclineresistant *E. coli* isolates were genetically assessed by pulsed-field gel electrophoresis (PFGE) according to Ref. [21]. Analysis and comparison of PFGE gel pictures were done by using GelCompar II software (Applied Maths, St-Martens-Latem, Belgium) as previously reported [18]. Isolates from cattle, humans, soil and water with 100% band pattern homology were considered genetically identical. A face-to-face interview was conducted to each household in the cluster. Semi-structured questionnaire which aimed at gathering information related to cattle and manure management (for cattle keeping households) and events or scenario leading to contact with cattle and manure (for non-cattle keeping households) was administered.

Logistic regression was run to quantify risk factors for the presence of isolates from cattle, humans, water or soil which are genetically identical to at least one other isolate from same or different clusters by using PROCGENMOD in SAS as earlier described [19]. The response variable was the occurrence of identical PFGE band pattern of *E. coli* isolates (yes or no), while the independent variables comprised of factors focusing on cattle herd characteristics and management (the presence of species other than cattle and labor division), cattle housing infrastructure (roof, floor and beddings), feeding and water system and manure management issues (collection and disposal). Univariable analysis was performed to all explanatory variables and those with an arbitrary *p*-value of equal or less to 0.25 were included in a multivariable model. A final model was obtained by a backward stepwise strategy. Chi-square test was used to check for association between different cattle and manure management factors at 5% significance level.

From 1046 samples, 118 (11.28%) samples produced ampicillin-tetracycline-resistant *E. coli*. Forty samples with resistant *E. coli* isolates (34%) were human stool, 50 (42%) were cattle feces, 21 (18%) were soil and 7 (6%) were water. One ampicillin-tetracycline-resistant *E. coli* isolate per sample was taken for further analyses. The 118 ampicillin-tetracycline-resistant *E. coli* isolates came from 44 out of the total 100 clusters. Twenty-three out of 44 clusters showing ampicillin-tetracycline-resistant isolates (52.3%) yielded at least one isolate with identical PFGE band pattern to another isolate from another source, suggesting that transfer of *E. coli* was a common event. Eight distinct PFGE band patterns designated arbitrary letters A, B, D, E, F, G, H and I for distinguishing purposes were identified. Inclusion of *Salmonella enterica* serovar *Braenderup* in all the gels showed a band pattern reproducibility of 100% (type C) (**Figure 2**) [19]. These PFGE band patterns cut across different clusters and were from cattle, humans, soil and water. Sixteen clusters out of 44 (36%) yielded at least one *E. coli* isolate which was identical to another isolate from another source by 100%. Seven clusters (16%) had isolate with similarity between 95 and 99.1% (**Figure 2**). PFGE band pattern A was comprised of five clusters, pattern B had two clusters, pattern D had three clusters, pattern E had six clusters, pattern F had two clusters, pattern G had one cluster, pattern H had two clusters and pattern I had also two clusters. Twelve isolates from cattle, human and soil constituted PFGE band pattern A, while pattern E was made up of eight isolates from cattle, soil and water (**Table 4**). Public Health Aspect of Manure Management in Urban and Peri-Urban Livestock Farming in Developing Countries http://dx.doi.org/10.5772/65346 83

MacConkey agar followed by 24-h incubation at 37°C. *E. coli* suspected isolates were confirmed and screened for double antimicrobial resistance to ampicillin and tetracycline on antimicrobial embedded Petrifilm™ *Select E. coli* count (SEC) plate. Preparation of antimicrobial stock solution and screening procedure was done according to Ref. [20]. Ampicillin-tetracyclineresistant *E. coli* isolates were genetically assessed by pulsed-field gel electrophoresis (PFGE) according to Ref. [21]. Analysis and comparison of PFGE gel pictures were done by using GelCompar II software (Applied Maths, St-Martens-Latem, Belgium) as previously reported [18]. Isolates from cattle, humans, soil and water with 100% band pattern homology were considered genetically identical. A face-to-face interview was conducted to each household in the cluster. Semi-structured questionnaire which aimed at gathering information related to cattle and manure management (for cattle keeping households) and events or scenario leading to contact with cattle and manure (for non-cattle keeping households) was administered.

Logistic regression was run to quantify risk factors for the presence of isolates from cattle, humans, water or soil which are genetically identical to at least one other isolate from same or different clusters by using PROCGENMOD in SAS as earlier described [19]. The response variable was the occurrence of identical PFGE band pattern of *E. coli* isolates (yes or no), while the independent variables comprised of factors focusing on cattle herd characteristics and management (the presence of species other than cattle and labor division), cattle housing infrastructure (roof, floor and beddings), feeding and water system and manure management issues (collection and disposal). Univariable analysis was performed to all explanatory variables and those with an arbitrary *p*-value of equal or less to 0.25 were included in a multivariable model. A final model was obtained by a backward stepwise strategy. Chi-square test was used to check for association between different cattle and manure management factors

From 1046 samples, 118 (11.28%) samples produced ampicillin-tetracycline-resistant *E. coli*. Forty samples with resistant *E. coli* isolates (34%) were human stool, 50 (42%) were cattle feces, 21 (18%) were soil and 7 (6%) were water. One ampicillin-tetracycline-resistant *E. coli* isolate per sample was taken for further analyses. The 118 ampicillin-tetracycline-resistant *E. coli* isolates came from 44 out of the total 100 clusters. Twenty-three out of 44 clusters showing ampicillin-tetracycline-resistant isolates (52.3%) yielded at least one isolate with identical PFGE band pattern to another isolate from another source, suggesting that transfer of *E. coli* was a common event. Eight distinct PFGE band patterns designated arbitrary letters A, B, D, E, F, G, H and I for distinguishing purposes were identified. Inclusion of *Salmonella enterica* serovar *Braenderup* in all the gels showed a band pattern reproducibility of 100% (type C) (**Figure 2**) [19]. These PFGE band patterns cut across different clusters and were from cattle, humans, soil and water. Sixteen clusters out of 44 (36%) yielded at least one *E. coli* isolate which was identical to another isolate from another source by 100%. Seven clusters (16%) had isolate with similarity between 95 and 99.1% (**Figure 2**). PFGE band pattern A was comprised of five clusters, pattern B had two clusters, pattern D had three clusters, pattern E had six clusters, pattern F had two clusters, pattern G had one cluster, pattern H had two clusters and pattern I had also two clusters. Twelve isolates from cattle, human and soil constituted PFGE band pattern A, while pattern E was made up of eight isolates from cattle, soil and water (**Table 4**).

at 5% significance level.

82 Livestock Science

**Figure 2.** PFGE band pattern for ampicillin- and tetracycline-resistant *E. coli* isolates from humans, cattle, soil and water.

This shows that there was sharing of genetic characteristics between bacteria isolates from different sources. There was also genetic relatedness in cluster seven between isolates from cattle keeping human (7H1), cattle (7B2) and non-cattle keeping human (7H2). This scenario suggests that sharing of bacteria go beyond cattle keeping households to their non-cattle keeping neighbors. In some instance, like in cluster six, isolates from cattle (6B2, 6B4 and 6B6) did not resemble humans in the same household, but had PFGE band pattern identical to neighboring non-cattle keeping human (6H2). Sharing of genetic features was also observed in isolates from cattle, humans and the environment. For instance, isolate from cattle in cluster eight (8B1) was identical to isolate from non-cattle keeping human (8H2) and isolate from soil collected from cattle keeping household (8S1) in the same cluster eight. In PFGE band pattern E, isolates from water sources of non-cattle keeping households (40W2 and 44W2) had identical PFGE patterns to isolates from cattle (11B1, 17B2, 20B2 and 20B3) and soil (17S1 and 18S1) from cattle keeping households (**Table 4**). Some isolates with identical PFGE band patterns from cattle, e.g., in PFGE band pattern A, came from different households/herds, signifying the role of communal grazing in sharing of bacteria between cattle.



a *E. coli* isolates from humans (H), water (W) and soil (S) with odd last digit originated from cattle keeping households while those with even last digit were obtained from non-cattle keeping neighbors.

**Table 4.** Identical PFGE patterns of ampicillin- and tetracycline-resistant *E. coli* isolated from cattle keeping and non-cattle keeping neighbor households in peri-urban areas of Morogoro, Tanzania.

*Escherichia coli* isolates from cattle were found in all clusters with identical PFGE bands patterns (**Figure 2**), proposing that cattle are the focal point of bacteria sharing and manure is the center of contact between cattle, humans and the environment. These roles of cattle and manure in bacteria sharing between cattle, humans and the environment lead to a hypothetical bacteria transmission pathways presented in **Figure 3**. The bacteria sharing pathways can be used to set up strategies to break the contact and transmission pathways. However, there is a need to develop procedures which can be used to determine the donor-recipient bacteria transmission relationship, something that was not done in the current study.

Isolates with distinct PFGE band patterns within clusters had a good temporal relationship in terms of sampling and isolation. Most of them came from samples collected on one day or

> 7B2 7H1 7H2 16 July 2011 8B1 8H2 8S1 20 July 2011 9B3 20 July 2011 30B1 20 July 2011

38 38H2 21 September 2011

4 4B1 15 September 2011

3 3S1 22 July 2011

 17B2 17S1 15 January 2012 18S1 15 January 2012 40W2 15 January 2012 20B2 20B3 18 January 2012 44W2 11 January 2012

9 9S1 20 July 2011

*E. coli* isolates from humans (H), water (W) and soil (S) with odd last digit originated from cattle keeping households

*Escherichia coli* isolates from cattle were found in all clusters with identical PFGE bands patterns (**Figure 2**), proposing that cattle are the focal point of bacteria sharing and manure is the center of contact between cattle, humans and the environment. These roles of cattle and manure in bacteria sharing between cattle, humans and the environment lead to a hypothetical bacteria transmission pathways presented in **Figure 3**. The bacteria sharing pathways can be used to set up strategies to break the contact and transmission pathways. However, there is a need to

**Table 4.** Identical PFGE patterns of ampicillin- and tetracycline-resistant *E. coli* isolated from cattle keeping and

**Clonal group Cluster Isolate IDa Sample date** A 6 6B2 6B4 6B6 6H2 16 July 2011

B 36 36B1 21 September 2011

D 28 28H2 28S1 15 September 2011

E 11 11B1 24 September 2011

F 33 33H1 20 July 2011

while those with even last digit were obtained from non-cattle keeping neighbors.

non-cattle keeping neighbor households in peri-urban areas of Morogoro, Tanzania.

within a week (**Table 4**) [19].

84 Livestock Science

a

**Figure 3.** Hypothetical transmission pathways of enteric bacteria in urban and peri-urban livestock farming systems in Morogoro, Tanzania.

From univariable analysis, five explanatory variables, namely manure responsible personnel (family member or hired laborer), cattle house roof (present or absent), cattle house floor (concrete or earth), use of bedding (yes or no) and animal water source (tap or surface water) qualified and progressed to multivariable logistic regression analysis. There were no detected confounders during the model building process, and the final logistic regression model was made up of a single explanatory variable, the type of cattle house roof. The cattle house with a roof was at 11 times odds of having isolates with identical PFGE band pattern to another isolate from another source (OR = 11.2, 95% CI 1.1–119.3). Generally, isolates with PFGE band pattern identical to at least one isolate from another source were 33, 86.8% of which were isolated from cattle houses with a roof. The model goodness-of-fit test, expressed as the ratio of deviance to degree of freedom, was 1.2, while the correlation of 0.1344 existed between sample sources from different clusters. This shows that the variables were well explained by the model.

From this study, it seems that there was transmission of bacteria in roofed cattle houses than in cattle houses without roof. This could be due to the effect of direct sun rays in open cattle houses killing the bacteria before the transmission.

Cattle feeding system was statistically associated with cattle water sources (X2 = 28.5, df = 1, *p* ≤ 0.0001), whereby free range cattle used surface water and cattle under zero grazing used tap water which was also used by humans. On the other hand, distance from residence to manure disposal site was statistically associated with the way manure was handled (X2 = 8, df = 1, *p* = 0.005). That is, cattle keeping households which stored manure in heaps disposed manure within residential areas, whereas households which opted to spread fresh manure on land did it outside residential area [19].
