Tuberculosis at Farmer-Cattle Interface in the Rural Villages of South Gondar Zone of Northwest Ethiopia

Background Tuberculosis (TB) has been an important public health concern in Ethiopia, particularly at areas of human-animal intersection. However, limited epidemiological information is available in this respect in the country. Therefore, the present study was conducted to investigate the transmission of TB at human-cattle interface, associated risk factors and public awareness about the disease at South Gondar Zone, northwest Ethiopia. Methods A cross sectional study was conducted between March 2015 and April 2018 on 186 farmers and 476 cattle in South Gondar Zone, northwest Ethiopia. Bacteriological examination, region of difference (RD) 9-based polymerase chain reaction (PCR), single intradermal comparative tuberculin test (SICTT), and questionnaire were used for undertaking this study. Results Culture positivity in farmers was 59.7% (111/186) and all the culture positive isolates were M. tuberculosis. About 68% (74/111) of culture positive respondents did not know about the transmission of TB from cattle to human or vice versa. The animal and herd prevalence of bovine TB were 1.5% (7/476) and 7.4% (7/95), respectively. Although the result was not statistically significant, the odds of bovine TB in cattle owned by TB positive households was slightly higher than those owned by TB free households (adjusted odds ratio, AOR = 1.39; 95% CI: 0.31–7.10; p = 0.76). Conclusion Although SIDCTT reactivity was slightly higher in cattle owned by TB positive households, all the human isolates were M. tuberculosis and no M. bovis was isolated from farmers, which could be due to the low prevalence of bovine TB in the area.


Introduction
Tuberculosis (TB) is an infectious disease of humans and animals.The most common cause of human TB is known to be Mycobacterium tuberculosis (M.tuberculosis), and the main cause of TB in animals is Mycobacterium bovis (M.bovis).Zoonotic TB is a form of TB that is caused by M. bovis and transmitted from animals to humans.While reverse zoonotic TB is the form of TB that is caused by M. tuberculosis and is transmitted from humans to animals.
M. bovis often causes extra pulmonary and can also cause pulmonary TB which is clinically indistinguishable from TB caused by M. tuberculosis.In 2016, an estimated 147, 000 new cases of zoonotic TB were reported globally, and 12, 500 deaths due zoonotic TB.The highest burden of zoonotic TB was reported from the African, followed by the South-East Asian region [1].
The transmission of TB from cattle to human is due to consumption of raw/undercooked infected animal products such as milk and meat or through inhalation due to close contact bet -ween the cattle and humans.It is estimated that in countries where pasteurization of milk is rare and bovine TB is common, 10 to 15% human cases of TB are caused by M. bovis [2].
According to some studies from Tanzania, Nigeria and Uganda, M. bovis accounted for 20% or more of the MTBC isolated from human TB cases [3][4][5].
Data on TB transmission at the human-animal interface are important in designing a 'one health' approach for the control of the disease, particularly, rural settings.In the Amhara Region, north-western Ethiopia, information on the public health risk of zoonotic TB is scarce.Few cross sectional studies conducted in cattle reported animal prevalence of 3.55% and 8.7% [7,8].In addition, M. bovis was isolated from humans in north-western Ethiopia [8,9].In South Gondar Zone, Amhara Region, north-western Ethiopia, the practices of inhabitants could promote the transmission of TB from cattle to humans or vice versa.However, there is scarcity of epidemiological data on public awareness, risk factors and transmission of TB between humans and their cattle in the Zone.Hence, the present study was conducted to investigate the public awareness, risk factors to BTB and the potential transmission of TB from cattle to humans or vice versa in South Gondar Zone of north-western Ethiopia.

Study area
The study was conducted in South Gondar Zone, north-western Ethiopia.The Zone is located in the Amhara Region, 660 km north east of Addis Ababa, the Capital of Ethiopia.The Zone is known for its diverse topography ranging from flat and low grazing land to high cold mountains.The altitude of the Zone ranges from1500 to 3,600 meters above sea level.The average yearly rainfall of the Zone ranged from 700-mm to 1300 mm in while the average daily temperature was 17 o C in 2017.South Gondar Zone consisted of 10 districts and covers a total area of 14 320 square km.According to the Central Statistical Agency of Ethiopia, the Zone has a total population of 2,051,738 and a population density of 145.56 [10].Majority of the population (90.47%) of the Zone were rural inhabitants.A total of 468,238 households were counted in this Zone, which resulted in an average of 4.38 persons to a household [10].
The majority of the population has depended on subsistence farming and dairy cattle rearing [11].The Zone has been known for its indigenous milk cattle such as the 'Fogera' and 'Dera' cattle.Dairying is commonly practiced using small herd size.

Study population and sampling
Human TB cases were recruited from districts health centres, peripheral health posts, Zonal and regional government as well as private referal hospitals (Debretabor, Felegehiwot and Gambi hosptals).The human cases included those active TB patients who visited the health facilities seeking for TB-treatment.The control human subjects were households who did not have TB cases for the last five years and residing in the same villages with the households which have TB cases in their family members.Cattle owned by both TB positive households and TB negative households were tested for bovine TB.

General inclusion and exclusion criteria
Small scale dairy farmers who were permanently living in the zone for at least two months prior to the study, aged five years and above at the time of the study as well as who were consented and willing to participate in the study were included.Whereas, individuals who had no association with cattle, those who were not willing to participate, aged below five years and seriously ill ones were excluded from participating in the study.Both local and cross breed cattle older than six months and owned by households with active TB cases and TB free households were included.Young cattle with age less than six months, clinically sick ones, and cows one month pre-and post partum were excluded from the study.

Study design and sample size determination
A comparative cross sectional study was conducted between December 2015 to February 2018 The sample size for this study was calculated with the assumption of a 15% bovine TB prevalence in TB positive households and a 5% bovine TB prevalence among TB free households, a 95% confidence interval (CI) with a power of 80%, a ratio of cases to comparison groups of 3:1 (based on findings of 15.3% bovine TB among TB positive households and a 5.9% BTB among TB free households [8].Adding a 10% non-response rate, the required sample size was 105 for TB positive households and 35 TB for free households.However, in the present study, due to several limitations we have only managed to reach out only 63 TB positive and 32 TB negative households.Based on this, a total of 476 cattle (315 from TB positive households and 161 from TB free households) were tested for bovine TB.

Questioner survey
All human study participants were interviewed with questionnaire in order to assess their awareness and practices about the zoonotic transmission of TB, and the possible risk factors for the disease.Their socio-demographic characteristics, knowledge, attitudes and practices towards TB including their consumption habits of raw milk/meat as well as husbandry practices were asked.Moreover, all herd owners of tuberculin tested cattle were interviewed for possible risk factors of TB positive cattle.Data on age and sex of individual animal, herd size, origin, breed type, recent introduction of new animals into the herd, and keeping of different livestock together were collected.

Laboratory methods
After informed consent was obtained, two sputum samples (on-spot and morning) from suspected pulmonary TB (PTB) patients and fine needle aspirate (FNA) samples from suspect -ed extrapulmonary TB (EPTB) patients were collected by trained laboratory technicians and pathologists, respectively.
The samples were first digested and concentrated/decontaminated by the N-acetyl-Lcysteine-Sodium hydroxide (NALC-NaOH) method.Smears of the final deposits from the various specimens were stained by the Ziehl-Neelsen (ZN) method and examined under oil immersion using a binocular light microscope.All smear positive TB samples were stored at +4°C at the study site and then transported to Regional Health Research Llaboratory Center (Bahirdar) and kept at +4 °C until bacteriological culture was performed.
Similarly, FNA specimens were collected by pathologists and stored in cryo-tubes in phosphate buffer saline (PBS) with pH 7.2.ZN staining was performed.AFB-positive specimens were stored at -20 °C until mycobacterial culture was performed.
The samples were processed for culturing according to the standard methods described earlier [12,13].Both sputum and FNA samples were cultured at the Bahir Dar Regional Health Research Laboratory Centre.
To differentiate M. tuberculosis from other members of the M. tuberculosis complex (MTBC) species, RD9 based PCR was performed according to protocols previously described [14].
M. tuberculosis H37Rv, M. bovis bacille Calmette-Guérin (BCG) were included as positive controls and water was used as a negative control.Interpretation of the result was based on bands of different sizes (396 base pairs (bp) for M. tuberculosis and 375bp for M. bovis) as previously described [15].

Single intradermal comparative tuberculin test
Single intradermal comparative tuberculin test (SIDCTT) was performed for detecting TB in cattle.SIDCTT was done using both bovine and avian purified protein derivatives (PPDs) (Prionics Lelystad B. V., The Netherlands).The tuberculin test measures the hypersensitivity reaction on the skin due to the administered antigens (PPDs).
Two sites were shaved in the middle of the side of the neck, one above the other, separated by about 12 cm for injection of the two PPDs.The thickness of the skin fold at both injection sites were measured using a calliper and recorded before injection.The sites were injected with 0.1ml (2000IU) aliquot of bovine PPD and 0.1ml (2500IU) of avian PPD interdermally.
After 72 hours the two sites were measured for change in skin thickness, and the result was interpreted according international [16] and local [17] criteria.

Data Analysis
Data were entered in to Excele file format and transferred in to SPSS software version 25 for statistical analysis.Descriptive statistics were used to depict the demographic variables.Chisquare ( 2 ) test was used to test differences in proportions and the association between categorical variables with raw milk consumption habit.Bivariate and multivariate logistic regressions were used to determine the association between background variables with awareness of zoonotic TB and tuberculin reactivity in cattle.Results were considered statistically significant whenever p-value was less than 5%.

Ethics approval and consent to participate
Ethical clearance for the study was obtained from the Ethical Committee of Addis Ababa University, Department of Microbial, Cellular and Molecular Biology (Ref.CNSDO/491/07/ 15).In addition, written permission was sought from the Amhara Regional Health Bureau Research Ethical Committee (Ref.HRTT/1/271/07).Each study participant was consented with a written form and agreed to participate in the study after a clear explanation of the study objectives and patient data confidentiality (S1 Appendix).In case of participants under the age of 18 years, consent was obtained from their parents/guardians.The animals used in this study were privately owned by the study participants and a written consent was sought from the owners to take samples from the animals.A high standard veterinary care was taken to minimize cattle suffering during tuberculin test.9

Tuberculosis in farmers
Culture positivity was obtained in 59.7% (111/186) of the active TB cases.Of which, 59.5% (66/111) was isolated from extra pulmonary TB (EPTB) patients.The molecular typing of culture positive isolates using RD9-based PCR revealed that all isolates had intact RD9 locus and were subsequently classified as M. tuberculosis.No M. bovis was detected (Fig 1).

Awarness on zoonotic transmission of TB and food consumption habit
About 68% (74/111) of the respondents did not know about the transmission of TB from cattle to human or vice versa.About 69% (77/111) of the respondents had habit of consuming raw milk and other uncooked dairy products (Table 1).The logistic regression, taking log-odds of awarness about zoonotic transmission as an outcome variable, resulted patient origin and educational status were observed to be significantly associated (p<0.05)(Table 1).The study participants in Libo kemkem and Simada were 11 and 10 times more aware about the zoonotic tramnsmission of TB as compared to those of Dera District (Libo kemkem vs. Dera AOR= 11.84; 95% CI: 1.67-73.2;p = 0.003) and (Simada vs. Dera AOR= 10.47; 95% CI: 0.72-116.3;p = 0.038).The odds of having awareness on zoonotic transmission of TB was higher among individuals with secondary school educational level (AOR=4.16;95% CI: 1.05-15.57;p = 0.029) compared to those of illiterates (Table 1).
However, other patient characteristics such as age groups, sex, TB history in the family, raw milk consumption habit, and patient category (new or retreatment cases) were not significantly associated with particpants' over all awarness about zoonotic transmission of TB (Table 1).

Characteristics of the study cattle
The majority of the cattle were females accounting for 54.2% (258/476) of the study cattle.
Cattle within the age range of 5-10 years had the greatest share (47%) from the total cattle tested with a mean age of 5.5 years.Many of the cattle were Zebu breed while only 6.1% (29/476) of them were cross breed (Table 2).

Prevalence of TB in cattle
Animal prevalence was 1.6% (5/315) and 1.2% (2/161) at ≥2mm cut-off value in TB positive and TB free households, respectively.Using the same cut-off value, 7.9% (5/63) and 6.3% (2/32) herd prevalence was recorded in cattle owned by TB positive and TB free households, .CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which was this version posted April 10, 2019.; https://doi.org/10.1101/605154doi: bioRxiv preprint respectively.The overall animal and herd prevalence were 1.5% (7/476 and 7.4% (7/95), respectively.However, non of the tested cattle were positive for bovine TB at the international cut-off value of >4mm.

Risk factors for bovine TB
Risk factor analysis to the occurrence of bovine TB in cattle revealed that age groups between 5-10 years were more reactive to tuberculin test than younger age groups (AOR=3.1;95% CI: 0.35-35.69;p=0.16).Cattle with apparently good (AOR= 8.53; 95% CI: 0.85-83.34;p =0.02) and medium (AOR= 3.00; 95% CI: 0.27-28.38;p = 0.33) body conditions were more likely to be reactive to the tuberculin test as compared to those with apparently poor body condition, and the difference was statistically significant (p<0.05)(Table 2).Although the difference was not statistically significant (p>0.05, the odd of bovine TB that cattle owned by TB positive cases was slightly higher than those owned by TB free households (AOR=1.39;95% CI: 0.31-7.10;p = 0.76).Despite the observed differences, sex, breed type, source of cattle and households TB status were not significantly associated with the occurrence of BTB in the present study (Table 2).

Zoonotic transmission of TB
In the present study, molecular typing of culture positive isolates using RD9-based PCR confirmed that all the human isolates were M. tuberculosis (Fig 1).Furthermore, no M. bovis was detected even from those TB patients who owned tuberculin reactor cattle.Hence, this study did not revealed evidence of direct transmission of tuberculosis from cattle to their closely associated owners.cattle owned by households with active TB cases than TB free households [8,35,39,40].Such observation could suggest the existence of TB transmission between cattle and their owners.The transmission could be zoonotic (transmission of M. bovis from cattle to humans) or reverse zoonotic (transmission of M. tuberculosis from humans to cattle).In the present study, isolation of mycobacteria was not done from cattle and diagnosis of bovine TB was made by SIDCTT.On the other hand, all the human isolates were M. tuberculosis.This could imply that M. tuberculosis might have been transmitted to cattle from their owners and positivity to SIDCTT was due to sensitization to infection with M. tuberculosis as it was observed earlier by other authors (14,39,41).
The present study has some limitation in conducting pathological examination and there by strain identification of mycobacteria isolates from tuberculin reactor cattle.

Conclusion
All the human isolates recovered from farmers with active TB cases were M. tuberculosis and no M. bovis was isolated from farmers.Moreover, the overall prevalence of bovine TB in the area was low; but it was slightly higher in cattle owned by households with active TB cases than in cattle owned with active TB free households; which could suggest the presence of zoonotic and or reverse zoonotic transmission of TB between cattle and their owners.This could also be exacerbated by the low level of awareness of the farmers on the transmission of mycobacterial species between cattle and their owners.

Fig 1 .
Fig 1.Electrophoretic separation of PCR products by RD9 deletion typing.The figure repre peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made

Table 1 . Association of demographic factors with awarness to zoonotic transmission of TB among AFB culture positive TB patients (N=111), South Gondar Zone, northwest Ethiopia (2015-2017). Aware of zoonotic TB Demographic factors
COR: crude odds ratio, AOR: adjusted odds ratio, CI: confidence interval.