Abstract
Background
This study was conducted with the objectives of estimating the morbidity and mortality rates in layer chickens, identifying the risk factors associated with morbidity and mortality, and identifying the major bacterial pathogens affecting small-scale commercial layers in Hawassa. A longitudinal observational study design was employed from November 2019 to March 2020. The chickens on selected farms were checked for morbidity and mortality twice a week. During each visit, clinical examination of sick birds and pathological investigation of dead birds were conducted. Cloaca samples were collected for isolation and identification of Salmonella spp. and E. coli. A Cox proportional hazard model was used to quantify the effects of various risk factors on the morbidity and mortality rates observed.
Results
Of the 8976 chickens followed, 106 developed clinical disease, giving a morbidity of 1.18% (95% CI: 0.97, 1.43). The overall morbidity rate was 2.37 (95% CI: 1.94, 2.87) per 1000 chicken months. A total of 101 of the chickens under study were found dead, yielding a mortality of 1.13% (95% CI: 0.92, 1.37) and a mortality rate of 2.26 (95% CI: 1.84, 2.75). Multivariable Cox regression analysis showed that farm hygiene, the experience of farm manager, housing condition, housing systems, the availability of veterinary services and age of chicken were important risk factors for morbidity and mortality. Out of 58 cloacal samples collected from sick chickens, 7 (12.07%; 95% CI: 4.99, 23.29) yielded positive results for Salmonella spp., while 25 (43.10%; 95% CI: 30.16, 56.77) yielded positive results for E. coli. Out of swabs collected from 8 randomly selected sick chickens after necropsy, 3 (37.5%) were found to be positive for Salmonella spp. Four (50%) of them were positive for E. coli. Swabs were collected and cultured from 15 dead chickens, and of these, 2 (13.33%) and 7 (46.67%) were found to be positive for Salmonella spp. and E. coli, respectively. Farm hygiene, age of chickens, housing conditions and frequency antibiotics use were important risk factors for colibacillosis and salmonellosis.
Conclusions
Although the incidence of chicken morbidity and mortality was relatively low in the present study, important risk factors have been identified in the poultry farms of Hawassa City, southern Ethiopia. Therefore, comprehensive poultry farm management practices are needed to mitigate risk factors for morbidity and mortality as well as colibacillosis and salmonellosis. Identification of the serotypes of Salmonella spp. and E. coli should be carried out.
Keywords: Chicken, E. coli, Morbidity, Mortality, Salmonella
Chicken; E. coli; Morbidity; Mortality; Salmonella.
1. Introduction
Poultry production is an integral part of urban agriculture in Ethiopia. It is a viable income generation activity to support and improve the welfare of the producers and the community. Poultry products are a cheap source of protein that is vital for maintaining the health of children, mothers, and needy people. It seeks to create employment opportunities for the youth and women so that they cater for themselves and, to some extent, the household needs. Hawassa city is one of the densely populated cities in Ethiopia. There are a large number of needy mothers, children, and others who need sufficient protein. In addition, the city is one of the destinations for tourists. Basically, there is a huge demand for animal protein in Hawassa. Poultry production, thus, plays an important role in improving the national economy, household sustainability, and food and nutrition security in developing countries [1, 2]. Therefore, poultry production contributes to the reduction of the supply-demand gap in animal proteins.
Taking this into account, several government-led programs have attempted to improve chicken production as a strategy to reduce poverty [3]. Elsewhere in the country, especially in central Ethiopia, there is a gradual increase in commercial and small-scale chicken enterprises [4]. In contrast, in Hawassa, which is the capital of the Sidama Region, as well as a business and tourism center in the southern half of the country, commercial chicken production is a more recent enterprise. Several small-scale farms have been established and have started to produce and supply table eggs and meat to various supermarkets, kiosks, and roadside restaurants in Hawassa and beyond.
Chicken production in Hawassa is, however, affected by several technical and nontechnical factors. Among the known problems that negatively affect the productivity of chickens are the occurrences of infectious and noninfectious diseases [5]. Infectious diseases reduce egg production and retard growth (meat production), thereby significantly impairing the economics of the poultry industry [1, 6]. Optimum poultry production, therefore, requires an understanding of the epidemiology of infectious diseases, such as the incidence of morbidity and mortality, which are useful for quantifying the financial losses incurred. The magnitude of morbidity and mortality in small-scale poultry farms in Hawassa has not been studied. The differential diagnosis of the pathogens responsible for morbidity and mortality has also not been established. This study was carried out with the objectives of (1) estimating the morbidity and mortality rates in layer chickens, (2) identifying the risk factors associated with morbidity and mortality, and (3) identifying the major bacterial pathogens affecting small-scale commercial layers in Hawassa.
2. Results
2.1. Morbidity and mortality rates in laying chickens in Hawassa city
Of the 8976 chickens followed for five months (November 2019 to March 2020), 106 developed clinical diseases. That is, the morbidity was 1.18% (95% CI: 0.97, 1.43). The study chickens developed the clinical disease within a total of 44,666 chicken months at risk given an overall morbidity rate of 2.37 (95% CI: 1.94, 2.87) per 1000 (Table 1). The morbidity rate was highest in Farm 1 and lowest in Farm 3. During the period under investigation, 101 of the chickens under study were found dead, yielding a mortality of 1.13% (95% CI: 0.92, 1.37). The overall chicken months elapsed for mortality was 44,674, giving a mortality rate of 2.26 (95% CI: 1.84, 2.75). Similar to the morbidity rate, the highest mortality rate was observed in chickens reared on Farm 1, whereas the lowest was observed on Farm 3 (Table 2). The case fatality observed in this study was 95.28% (95% CI: 89.33, 98.45).
Table 1.
The morbidity rates observed in layer chickens at different farms in Hawassa city, Ethiopia.
| Farm | Chicken at risk | No. of cases | Chicken months | Morbidity rate per 1000 |
|---|---|---|---|---|
| Farm 1 | 798 | 21 | 3940 | 5.32 (95% CI: 3.29, 8.12) |
| Farm 2 | 300 | 4 | 1492 | 2.68 (95% CI: 0.73, 6.85) |
| Farm 3 | 1000 | 6 | 4983 | 1.20 (95% CI: 0.44, 2.62) |
| Farm 4 | 6878 | 75 | 34,251 | 2.19 (95% CI: 1.72, 2.74) |
| Total | 8976 | 106 | 44,666 | 2.37 (95% CI: 0.97, 1.43) |
Table 2.
Mortality rates in layer chickens reared on different farms in Hawssa city, southern Ethiopia.
| Farm | Chicken at risk | Deaths | Chicken months | Incidence rate per 1000 |
|---|---|---|---|---|
| Farm 1 | 798 | 51 | 3868 | 13.19 (95% CI: 9.83, 7.29) |
| Farm 2 | 300 | 3 | 1496 | 2.00 (95% CI: 0.41, 5.85) |
| Farm 3 | 1000 | 0 | 5000 | 0 .00 (95% CI: 0.00, 5.97) |
| Farm 4 | 6878 | 47 | 34,310 | 1.37 (95% CI: 1.01, 1.82) |
| Total | 8976 | 101 | 44,674 | 2.26 (95% CI: 1.84, 2.75) |
2.2. Factors associated with morbidity in layer chickens in Hawassa city
The results of multivariable Cox regression analysis indicated that farm hygiene, the experience of the farm manager, housing condition, and age of chicken were significantly associated with morbidity (Table 3). Keeping layer chickens on farms with good hygiene significantly reduced morbidity (HR = 0.005, P < 0.001) compared to keeping them on farms with poor hygiene. In farms that were managed by more experienced managers, the hazard of morbidity was lower by nearly 44% (HR = 0.435, P = 0.002) compared to those farms managed by less experienced managers (Table 3).
Table 3.
Results of multivariable Cox regression analysis revealing the risk variables significantly associated with the morbidity of layer chickens in Hawassa city.
| Variable | Category | HR | P value | 95% CI |
|---|---|---|---|---|
| Farm hygiene | Good | 0.005 | 0.000 | 0.003, 0.009 |
| Poor | ||||
| Farm experience | ≥3 yrs. | 0.435 | 0.002 | 0.254, 0.746 |
| <3 yrs. | ||||
| Housing condition | Good | 0.190 | 0.000 | 0.112, 0.325 |
| Poor | ||||
| Age category | 4–5 month | 0.155 | 0.000 | 0.080, 0.302 |
| >5 month |
HR = hazard ratio; CI = confidence interval.
The experience of the farm managers, breed of chicken, availability of veterinary service, regular vaccination programs, type of farm ownership, housing system, and age of chicken were significantly associated with mortality. The results of multivariable Cox regression analysis showed that the relative hazard of mortality was significantly lower in chickens managed by more experienced farm managers than in chickens managed by less experienced farm managers. The relative hazard of mortality was significantly lower in Bovans brown than in Sasso. Interestingly, the hazard of mortality was nearly 22 times higher in farms that did not have adequate veterinary services than in those farms having adequate veterinary services. The occurrence of mortality was significantly higher in private farms (HR = 2.508) than in government-owned farms in chickens reared on deep litter (HR = 4.102) than in those reared in cage systems (Table 4).
Table 4.
Results of multivariable Cox regression analysis revealing risk factors significantly associated with mortality of layer chickens.
| Variable | Category | HR | P value | 95% CI |
|---|---|---|---|---|
| Farm experience | ≥3 yrs. | 0.007 | 0.000 | 0.003, 0.012 |
| <3 yrs. | ||||
| Breed | Bovans brown | 0.279 | 0.000 | 0.161, 0.483 |
| Sasso | ||||
| Availability of veterinary services | In adequate | 21.667 | 0.000 | 5.635, 83.306 |
| Adequate | ||||
| Regular vaccination | Yes | 0.016 | 0.000 | 0.004, 0 .085 |
| No | ||||
| Farm ownership type | Private | 2.508 | 0.002 | 1.403, 4.482 |
| Government | ||||
| Housing system | deep litter | 4.102 | 0.000 | 2.300, 7.315 |
| cage system | ||||
| Age category | 4–5 month | 0.269 | 0.010 | 0.099, 0.732 |
| >5 month |
2.3. Results of clinical and necropsy examination
The prominent clinical findings observed in chickens suspected of infection with E. coli include depression, reduced appetite, weight loss, diarrhea with pasting of feathers around the vent, sitting on hocks, arching back, drooping of the head, neck, and wings, and reduced egg production. The common signs recorded in chickens affected by salmonellosis comprise depression, pale comb, ruffled feathers, chalky white diarrhea stained with green bile, vent soiled with feces, sulfur (yellow)-colored diarrhea, and reduced egg production. The results of a necropsy revealed a congested trachea, swollen and discolored liver, congested spleen, hemorrhagic lung, presence of yolk material in the peritoneal cavity, hemorrhagic enteritis and distended intestinal loops with fluid, hemorrhagic oviduct, congested ovarian follicles, congested and swollen kidney and salpingitis in the case of colibacillosis. In the case of salmonellosis, the major gross lesions observed were swollen and fragile liver with a distinctive copper-bronze sheen on its surface, congested and swollen spleen, irregularly shaped ova with prominently thick stalks, caseous exudate in the oviduct, and salpingitis.
2.4. Results of the bacteriological investigation
A total of 58 cloacal samples were collected from 58 sick chickens and subjected to bacteriological analysis, of which 7 (12.07%; 95% CI: 4.99, 23.29) gave positive results for Salmonella spp. Similarly, 25 (43.10%; 95% CI: 30.16, 56.77) of them yielded positive results for E. coli. Swabs were also collected from the organs of 8 randomly selected sick chickens after necropsy, of which 3 (37.50%) were found to be positive for Salmonella spp. Four (50.00%) of them were positive for E. coli. Swabs were collected and cultured from 15 dead chickens, and of these, 2 (13.33%) and 7 (46.67%) were found to be positive for Salmonella spp. and E. coli, respectively.
2.5. Risk factors associated with colibacillosis and salmonellosis
Farm hygiene, age of study chicken, and housing condition were significantly associated (P < 0.05) with the occurrence of colibacillosis. The hazard of colibacillosis was significantly lower (HR = 0.006) in chickens raised on farms with good hygiene than in those reared on farms with poor hygiene, in young chickens (HR = 0.079) than in older ones and in chickens reared on well-constructed houses (0.074) than in those reared on poorly constructed houses (Table 5). Farm hygiene, use of antibiotics, and age of chicken were found to be significantly associated with salmonellosis. The hazard of salmonellosis was significantly lower (HR = 0.011) in chickens kept on farms with good hygiene than those kept on farms with poor hygiene; in flocks given antibiotics (HR = 0.044) regularly than those that did not receive antibiotics (Table 6).
Table 5.
Results of Cox regression analysis revealing risk factors for colibacillosis in layer chickens in Hawassa.
| Variable | Category | HR | P value | 95% CI |
|---|---|---|---|---|
| Farm hygiene | Good | 0.006 | 0.000 | 0.003, 0.015 |
| Poor | ||||
| Age of chicken | 4–5 month | 0.079 | 0.000 | 0.027, 0.232 |
| >5 month | ||||
| Housing condition | Good | 0.074 | 0.000 | 0.035, 0.154 |
| Poor |
Table 6.
Results of Cox regression analysis revealing risk factors for salmonellosis in layer chickens in Hawassa.
| Variables | Categories | HR | P value | 95% CI |
|---|---|---|---|---|
| Farm hygiene | Good | 0.011 | 0.000 | 0.002, 0.046 |
| Poor | ||||
| Use of antibiotics | Yes | 0.044 | 0.000 | 0.011,0.169 |
| No | ||||
| Age of chicken | 4–5 month | 0.069 | 0.003 | 0.012,0.398 |
| >5 month |
3. Discussion
3.1. Morbidity and mortality in layer chickens
In Ethiopia, poultry production is an important economic activity providing cash, nutritional and sociocultural services. The Ethiopian government has made poultry production a priority sector for achieving food and nutrition security. The goal is to raise the number of small-scale and commercial poultry farms with greater emphasis on small-scale farms. Poultry enterprises are particularly interesting in highly growing urban centers such as Hawassa. To achieve this goal, the identification of standing problems that can counteract the productivity of chickens is of vital importance. This study provided preliminary results on the morbidity and mortality of layer chickens in Hawassa city. Factors that have the potential to set back the productivity of layers in the area have also been identified. Therefore, veterinary and livestock authorities should take this into account when planning poultry development activities and setting up systems of livestock production and health monitoring.
The prevalence of morbidity and mortality reported in this study was lower than those reported elsewhere in the world, Bangladesh [14], Nigeria [15], Europe [16] and [17], which does not rule out the negative effects it has on the sustainability of the farms. The lower morbidity and mortality observed in the present study could be ascribed to the fact that commercial poultry production is a recent activity in the area where there is limited contact among farms, thereby limiting the spread of the pathogens. In addition, there are only few commercial poultry farms holding less than 20,000 chickens, which contributed to a lowered chance of accumulation of the disease agents in the area. The morbidity and mortality rates observed in this study are, however, comparable to previously published reports from Bangladesh [18, 19], who studied morbidity and mortality rates in the Fayoumi and Rhode Island Red breeds, respectively. This suggests that although the proportion of chickens that succumb to illness and die is low, the speed with which it occurs in the flocks in the study area is relatively high, implying the need to take action before it becomes very high.
3.2. Factors affecting morbidity and mortality
In this study, the age of chickens was significantly associated with morbidity and mortality. It was shown that both morbidity and mortality rates were higher in adult chickens than younger ones. This could be due to the increased susceptibility of adult layers as a result of the effects of egg production, which stresses the chickens and suppresses their immunity [20]. This could also happen due to the increased chance of exposure to disease agents or predisposing factors compared with the newly arrived younger ones. That is, once the chickens start laying eggs, they need close observation and necessary health provision. Consistent with our observation [14, 21, 22, 23], similar effects of age on morbidity and mortality rates have been published elsewhere in the world. However, our results disagree with the reports of [18, 24, 25], who observed higher morbidity and mortality rates in younger chickens than adults. This difference could be due to differences in the level of management and biosecurity measures.
Higher morbidity and mortality rates were observed in layer farms with poor hygiene than in those with good hygiene. This underlines the importance of maintaining farm hygiene to sustain the health of chickens. Unhygienic farms are favorable for the survival and multiplication of pathogens, especially bacteria. This is further supported by the fact that the occurrence of both Salmonella and E. coli infections was significantly associated with poor farm hygiene. The effect of farm hygiene on the occurrence of diseases and mortality in poultry farms as a source of contamination and spread of infectious pathogens from bird to bird and farm to farm has been well documented [21, 26, 27]. The higher morbidity and mortality rates in chickens reared on deep litter systems than those reared on cage systems could also be associated with farm hygiene. In deep litter systems, there is an accumulation of waste materials, especially feces, and contaminated water for long periods of time, especially on farms with poor hygienic practices. This allows the survival and multiplication of bacterial pathogens such as Salmonella spp. and E. coli since both are environmental pathogens. The chickens can ingest the pathogens contaminating the feed and water while feeding and watering and spreading to others. In contrast, in cage systems, excreta and other wastes drop to the ground, and the probability of chickens acquiring pathogens is lower. This observation is supported by the reports of [28, 29], who observed higher morbidity and mortality in chickens reared on deep litter systems than those kept on cage systems.
The higher morbidity and mortality in chickens kept in poorly maintained buildings could be due to the access of rodents to the poultry house. Rodents can transmit pathogens to chickens and contaminate feed and water. In addition, poorly constructed buildings also stress chickens due to the direct effects of temperature and sun light, which increase the susceptibility of chickens to diseases. This is in agreement with the findings of [25, 30, 31], which showed flock-to-flock spread of infectious agents in chickens reared on poorly constructed houses.
The effects of other factors, such as the availability of adequate veterinary services in the vicinity of the farms, regular administration of vaccination, and the experience of farm managers in reducing the magnitude of morbidity and mortality, are directly associated with the implementation of biosecurity measures. Experienced managers understand the importance of vaccines and veterinary services in the prevention of diseases and implement the necessary measures to keep chickens healthy. This justifies the significantly lower morbidity and mortality in chickens having more than 3 years of farm experience. A similar observation has been published by [32], in which poor veterinary services and poor understanding of biological chicken behavior were associated with increased morbidity and mortality.
The higher mortality reported in the Sasso breed than in Bovans brown in this study reveals the genetic liability between the two breeds to diseases or climatic and other predisposing factors. In agreement with our observation, these studies [14, 16, 19, 20, 21, 29, 33] reported significant differences in mortality among various breeds of layers.
3.3. Occurrence of infection with Salmonella spp. and E. coli
The occurrence of infection with E. coli observed in this study is slightly higher than that in previous reports [18, 34] from Bangladesh. In contrast, it is lower than the reports of [23, 35, 36] from Bangladesh. The difference observed in the magnitude of infection with E. coli might result from the difference in the study design, isolation techniques used, the difference in sample type and size, the difference in geographical location, breeds of chickens, management system, and difference in the standard of laboratory facilities available. The incidence rate of colibacillosis recorded in this study is also comparable to the reports of [18] from Bangladesh. Apart from its negative effects on the productivity of chickens, the occurrence of E. coli bears public health implications. Therefore, veterinary and public health authorities should take this into account and monitor its spread to consumers.
The occurrence of infection with Salmonella spp. observed in this study is lower than the results reported previously [18, 23, 34, 35] elsewhere in the world. The incidence rate of infection with Salmonella spp. is also in agreement with the reports of [19] in Fayoumi and RIR chickens. The lower prevalence of infection with Salmonella spp. could be due to the relatively recent establishment of commercial small-scale poultry farms in Hawassa. Since the environment is an important source of Salmonella spp., a sufficient amount of the bacteria has not been accumulated on the firms. However, the low prevalence does not rule out its importance, as Salmonella infection causes a prolonged reduction in egg production, and some of the serotypes also cause human infection.
The occurrence of infection with Salmonella spp. was significantly lower in chickens reared on farms that used antibiotics than in those that did not use or less frequently used antibiotics. On the one hand, this shows the importance of veterinary services, including the application of antibiotics for treatment and prophylaxis, and implies a lower prevalence of Salmonella infection reported in this study since antibiotic administration augments the immunity of chickens and ultimately reduces the incidence of infection. On the other hand, the inadvertent use of antibiotics can lead to the development of resistance and the outbreak of disease that cannot be treated with available antibiotics. The lower prevalence of Salmonella spp. in poultry farms that frequently use antibiotics has also been reported previously [27].
4. Conclusions
The present study revealed the occurrence of relatively lower morbidity and mortality in chickens in Hawassa city. Farm hygiene, housing systems, housing conditions, age of chickens, and the experience of farm managers are important factors that affect the morbidity and mortality of chickens in the area. Limited number of farms willing to be involved in the follow-up and non-identification of bacterial serotypes using further laboratory methods was the limitation of this study. The occurrence of infection with Salmonella spp. and E. coli is identified, suggesting their role in affecting productivity and public health. The veterinary and public health authorities should take this into account. Identification of the serotypes of Salmonella spp. and E. coli should be carried out.
5. Methods
5.1. The study area
The study was conducted from November 2019 to March 2020 in commercial poultry farms in Hawassa city. Hawassa is the capital of Sidama Regional State, covering an area of 50 square km. It is located between 7°3′N latitude and 38°28′E longitude at an elevation of 1,708 m above sea level. It is located 273 km south of Addis Ababa in the Ethiopian Great Rift Valley. The average monthly minimum and maximum temperatures and mean annual rainfall of the area are 13 °C, 27 °C, and 1,091 mm, respectively. According to the Hawassa city office of Agriculture, there are 13 small-scale and 5 medium-scale registered layer farms holding a total number of 17,914 chickens. Hawassa city has a total of eight subcities and 32 Kebeles (lower administration units) (Figure 1).
Figure 1.
Map of Ethiopia and Hawassa city, Sidama Regional State showing the selected study areas.
5.2. Study population
The study populations were layer chickens reared on poultry farms in Hawassa city. The chickens included in the study were chickens belonging to Bovans brown and Sasso between 4-12 months of age. The farms were owned by the government (Hawassa University Enterprise Poultry Farm) and private farms (all commercial small- and medium-scale farms). Data obtained from the Hawassa city livestock and fishery department revealed that there were 13 small-scale and 5 medium-scale layer farms holding a total of 17,914 chickens. Four farms willed to participate in the follow-up study from each of the four subcities designated Mehal Ketema, Tula, Tabor, and Misrak were selected. To maintain the confidentiality of the data generated from farms, their names were not mentioned. The numbers of chickens recruited for the study were 798 from Farm 1, 300 from Farm 2, 1000 from Farm 3, and 6878 from Farm 4, with a total of 8976.
5.3. Study design
A longitudinal prospective study design was employed for five months from November 2019 to March 2020. The poultry farms included in the study were selected based on willingness to participate in the follow-up study, and all chickens reared on the selected farms were followed for five months. During the study period, chickens reared on the selected farms were monitored for morbidity and mortality twice per week. However, the farm attendants were instructed to record the time at which incidents occurred whether it was signs of disease or death. During each visit, clinical examination of sick birds and pathological investigation of dead birds were conducted. Samples of cloaca were collected for isolation and identification of Salmonella spp. and E. coli as described below. In addition, a questionnaire survey was administered to farm owners or managers to gather information on the potential risk factors for morbidity and mortality.
5.4. Study techniques used
5.4.1. Questionnaire survey and farm visits
During the study, the attendants or managers of each farm selected for this study were interviewed using a semistructured questionnaire. The information gathered during an interview included biosecurity measures, flock management practices, waste handling activities, general farm history, disease management information, housing conditions, flock number, source of drinking water, and hygiene status of the farms. Emphasis was given to critical management practices and risk factors that might have a potential association with morbidity and mortality. Each farm was regularly visited twice weekly. In addition to the questionnaire, data were recorded on husbandry practices related to the health of the chicken, clinical signs, duration of illness, and associated mortality. In addition, for any mortalities that occurred on other days (other than the days of visits), data enumerators were assigned to record the incidents.
5.4.2. Sample collection and processing
Whenever chickens were affected clinically with diseases suspected to be salmonellosis or colibacillosis, samples were collected for isolation and identification of the causative bacteria following the recommendations of OIE [7]. The samples were labeled individually with specific identification numbers, dates of sampling, and sample type. Fifty-eight (58) cloacal samples were collected from clinically sick chickens using sterile cotton-tipped swabs moistened with buffered peptone water. Each swab sample was placed in separate screw-capped sterile test tubes containing buffered peptone water, kept in an icebox containing ice packs and transported to Hawassa University's Microbiology laboratory. In addition, 23 clinically sick and dead chickens were autopsied in the farms following standard postmortem procedures [8, 9, 10]. At necropsy, each organ and system of the body of each chicken was examined for gross lesions, and the changes observed were recorded. When gross lesions suggesting the occurrence of salmonellosis and/or colibacillosis were encountered, tissue samples were collected from vital organs such as the liver, spleen, ovary, and kidney using sterile forceps and scalpel blade following aseptic conditions. Before sampling, the surface of each organ was decontaminated using a hot sterile spatula. After making an incision on each organ using a sterile scalpel blade, sterile cotton-tipped swabs were inserted. The swabs were kept in sterile test tubes containing buffered peptone water and transported to the laboratory.
5.4.3. Isolation and identification of Salmonella and E. coli
For isolation and identification of E. coli, a loop full of inoculum from swab samples pre-enriched in buffered peptone water was streaked onto MacConkey agar and incubated at 37 °C for 24 h. Then, the suspected colonies of E. coli were inoculated onto EMB agar. Finally, the pure colonies of E. coli with characteristic red color on MAC agar and green metallic shine appearance on EMB agar were further analyzed by biochemical tests such as indole production tests, triple sugar iron (TSI) tests, and citrate utilization tests [11, 12].
For isolation and identification of Salmonella spp., the pre-enriched samples from buffered peptone water were transferred onto selective enrichment media (Rappaport Vassiliadis) and incubated at 41.5 °C for 24–48 h. Then, the loop full of the samples was inoculated onto xylose lysine deoxycholate (XLD) and incubated at 37 °C for 48 h. The pure colonies that resemble Salmonella spp. with characteristic red color with black center on XLD agar were subjected to biochemical tests such as triple sugar iron (TSI) test, citrate utilization test, and indole production test following the procedures described by [11, 12].
5.5. Data management and analysis
Epidemiological data obtained from the questionnaires, follow-up of the farms and laboratory tests were summarized and analyzed using STATA version 14 (StataCorp, 4905 Lakeway Drive, College Station, Texas 77845, USA). Data were gathered on thirty-two hypothesized explanatory variables to test their association with morbidity and mortality in layer chickens in the study area. The overall cumulative incidence, morbidity, and mortality rates as well as the cause-specific rates (CSR) were calculated using the formula described by Thrusfield [13]. During the analysis, all chickens developed an outcome variable (became sick, died, infected with E. coli or Salmonella spp.) constitute the numerator, and the time in months that the chickens were at risk was used as a denominator for computation of the morbidity and mortality rates. The rates computed were expressed as the number of cases per 1000 chicken months at risk. Cox's proportional hazard model was used to quantify the effects of various risk factors on the morbidity and mortality rates observed. Initially, the association of individual risk factors with morbidity and mortality was screened by univariable Cox regression. The explanatory variables (factors) significantly associated with morbidity at a 5% significance level in the univariable analysis were recruited for multivariable analysis using multiple Cox regression. In the multivariable Cox regression analysis, the final model was fitted for morbidity by stepwise backward elimination of those variables that had no significant effect (P > 0.05).
Declarations
Author contribution statement
Gizachew Hailegebreal: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Kabech Gedeno: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Bereket Molla Tanga; Mishamao Sulayeman: Contributed reagents, materials, analysis tools or data; Wrote the paper.
Teshale Sori: Analyzed and interpreted the data; Wrote the paper.
Funding statement
This study was supported by Hawassa University, Vice President for Research and Technology Transfer.
Data availability statement
Data will be made available on request.
Declaration of interest's statement
The authors declare no competing interests.
Additional information
No additional information is available for this paper.
Acknowledgements
We are grateful to the University of Hawassa, Office of Vice President for Research and Technology Transfer for Funding the Study under the Thematic Research Program. The authors express special thanks to owners or managers of the poultry farms included in the study for their collaboration during the study period. Hawassa University, Faculty of Veterinary Medicine Laboratory Staffs and Members of the veterinary departments in the surveyed subcities are also very much appreciated for their cooperation in the laboratory and fieldwork.
References
- 1.Habte T., Amare A., Bettridge J., Collins M., Christley R., Wigley P. ILRI Manual 25. International Livestock Research Institute (ILRI); Nairobi, Kenya: 2017. Guide to chicken health and management in Ethiopia. [Google Scholar]
- 2.Mottet A., Tempio G. Global poultry production: current state and future outlook and challenges. World Poultry Sci. J. 2017;73 Animal Production and Health Division, Food and Agriculture Organization of the United Nations, Rome, Italy. [Google Scholar]
- 3.Sebho H.K. Exotic chicken status, production performance and constraints in Ethiopia: a review. Asian J. Poultry Sci. 2016;10(1):30–39. [Google Scholar]
- 4.Yemane N., Tamir B., Mengistu A. Constraints, opportunities and socio-economic factors affecting flock size holding in small scale intensive urban poultry production in Addis Ababa, Ethiopia. Agric. Biol. J. N. Am. 2016;7(3):2151–7525. [Google Scholar]
- 5.Sambo E., Bettridge J., Dessie T., Amare A., Habte T., Wigley P., Christley R.M. Participatory evaluation of chicken health and production constraints in Ethiopia. Prev. Vet. Med. 2015;118:117–127. doi: 10.1016/j.prevetmed.2014.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Nour H.S.H. Department of Tropical Veterinary Medicine, College of Veterinary Medicine, Mekelle University; Tigray Region: 2019. Review on Major Bacterial Disease of Poultry that Cause Economic Loss on Ethiopia. [Google Scholar]
- 7.OIE . In: Terrestrial Animal Health Code. OIE, editor. World Organization for Animal Health (OIE); Paris, France: 2017. Chapter 10.7. Fowl typhoid and Pullorum disease; pp. 1–2. [Google Scholar]
- 8.Kidsadagon P. Faculty of Veterinary medicine, Chiang Mai University manual; 2012. Field Necropsy Techniques in Mammal and Poultry; pp. 41–50. [Google Scholar]
- 9.Bello A., Umaru M.A., Baraya Y.S., Adamu Y.A., Jibir M., Garba S., Hena S.A., Raji A.A., Saidu B., Mahmuda A., Abubakar A.A. Postmortem procedure and diagnostic avian pathology. Sci. J. Zool. 2012;1(3):37–41. [Google Scholar]
- 10.Morishita T.Y. The Ohio State University; Columbus, Ohio: 2019. Poultry Necropsy Basics. Extension Fact Sheet. PHR Factsheet 2019-04. [Google Scholar]
- 11.Leboffe M.J., Pierce B.E. fourth ed. 2011. A Photographic Atlas for the Microbiology Laboratory. San Diego City College, Morton. [Google Scholar]
- 12.Quinn P.J., Markey B.K., Leonard F.C., Hartigan P., Fanning S., FitzPatrick E.S. second ed. Wily Blackwell; Oxford: 2011. Veterinary Microbiology and Microbial Disease; pp. 263–286. [Google Scholar]
- 13.Thrusfield M. third ed. Blackwell Science Ltd.; London: 2018. Veterinary Epidemiology; pp. 182–198. [Google Scholar]
- 14.Uddin M.Z., Samad M.A., Kabir S.M.L. Mortality and disease status in hy-line and isa-brown strains of layer chickens reared in cage system in Bangladesh. Bangladesh J. Vet. Med. 2011;9(1):1–16. [Google Scholar]
- 15.Muhammad M., Muhammad L.U., Ambali A.G., Mani A.U. A survey of early chick mortality on small-scale poultry farms in jos, Central Nigeria. Int. J. Poultry Sci. 2010;9(5):446–449. [Google Scholar]
- 16.Appiah M.O. An investigation of factors that may influence the occurrence of early chick mortality on some farms in Ghana. Int. J. Anim. Sci. 2018;2(5):1–8. [Google Scholar]
- 17.Jasper L.T., Delezie H.E., Kempen I., Zoons J., Ampe B., Rodenburg T.B., Frank A.M. Specific characteristics of the aviary housing system affect plumage condition, mortality and production in laying hens. Poultry Sci. 2015;94:2008–2017. doi: 10.3382/ps/pev187. [DOI] [PubMed] [Google Scholar]
- 18.Rashid M.H., Xue C., Islam M.R., Islam M.T., Cao Y. A longitudinal study on the incidence of mortality of infectious diseases of commercial layer birds in Bangladesh. Prev. Vet. Med. 2013;109:354–358. doi: 10.1016/j.prevetmed.2012.10.012. [DOI] [PubMed] [Google Scholar]
- 19.Gupta S.D., Biswas P.K., Anwar M.N., Dey B.C., Debnath N.C. A longitudinal study to identify the causes of mortality in Fayoumi and Rhode Island red chickens and the immune status of the birds against newcastle disease. Bangladesh J. Microbial. 2007;24(1):47–51. [Google Scholar]
- 20.Aerni V., Brinkhof M.W.G., Wechsler B., Oester H., Frohlich E. Productivity and mortality of laying hens in aviaries: a systematic review. World’s Poult. Sci. J. 2005;61(1) 15-12. [Google Scholar]
- 21.Farooq M., Mian M.A., Durrani F.R., Syed M. Prevalent diseases and mortality in egg type layers under subtropical environment. Livest. Res. Rural Dev. 2002;14:144–151. [Google Scholar]
- 22.Samad M.A. 1st Pub.; LEP No. 10, BAU Campus, Mymensingh, Bangladesh: 2005. Poultry Science and Medicine. [Google Scholar]
- 23.Hossain M.B., Chakma S., Noman A.A. Prevalence of infectious and non-infectious diseases in different age groups of commercial layer chicken in Feni. District, Bangladesh. Vet. J. 2015;26(1):35–38. [Google Scholar]
- 24.Carrique-Mas J.J., Davies R.H. Salmonella Enteritidis in commercial layer flocks in Europe: legislative background, on farm sampling and main challenges. Braz. J. Poultry Sci. 2008;10(1):1–9. [Google Scholar]
- 25.Shittu A., Raji A.A., Madugu S.A., Hassan A.W., Fasina F.O. Predictors of death and production Performance of layer chickens in opened and sealed pens in a tropical savannah environment. BMC Vet. Res. 2014;10:214. doi: 10.1186/s12917-014-0214-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Vandekerchove D., Herdt P.D., Laevens H., Pasmans F. Colibacillosis in caged layer hens: characteristics of the disease and the etiological agent. Avian Pathol. 2004;33:117–125. doi: 10.1080/03079450310001642149. [DOI] [PubMed] [Google Scholar]
- 27.Agada G.O.A., Abdullahi I.O., Aminu M., Odugbo M., Chollom S.C., Okeke L.A., Okwori A.E.J. Risk factors associated with salmonella species contamination of commercial poultry farms in jos, plateau state, Nigeria. Int. J. Curr. Res. 2014;6(4):6292–6301. [Google Scholar]
- 28.Roll V.F.B., Levrino G.A.M., Briz R.C. Rearing system and behavioral adaptation of laying hens to furnished cages. Ciência Rural. 2008;38:1997–2003. [Google Scholar]
- 29.Singh R., Cheng K.M., Silversides F.G. Production performance and egg quality of four strains of laying hens kept in conventional cages and floor pens. Poultry Sci. 2009;88:256–264. doi: 10.3382/ps.2008-00237. [DOI] [PubMed] [Google Scholar]
- 30.Davies R., Breslin M. Observations on Salmonella contamination of eggs from infected commercial laying flocks where vaccination for Salmonella enterica serovar Enteritidis had been used. Avian Pathol. 2004;33:133–144. doi: 10.1080/03079450310001652040. [DOI] [PubMed] [Google Scholar]
- 31.Pereira D.F., do Vale M.M., Zevolli B.R., Salgado D.D. Estimating mortality in laying hens as the environmental temperature increases. Braz. J. Poultry Sci. 2010;12(4):265–271. [Google Scholar]
- 32.Getu A., Birhan M. Chicken production systems, performance and associated constraints in north Gondar zone, Ethiopia. World J. Agric. Sci. 2014;10:25–33. [Google Scholar]
- 33.Barrow P.A., Neto F.O.C. Pullorum disease and fowl typhoid - new thoughts on old diseases: a review. Avian Pathol. 2011;40:1–13. doi: 10.1080/03079457.2010.542575. [DOI] [PubMed] [Google Scholar]
- 34.Islam M.R., Das B.C., Hossain K., Lucky N.S., Mostafa M.G. A study on the occurrence of poultry diseases in Sylhet region of Bangladesh. Int. J. Poultry Sci. 2003;2(5):354–356. [Google Scholar]
- 35.Badruzzaman A.T.M., Noor M., Mamun M.A., et al. Prevalence of diseases in commercial chickens at Sylhet division of Bangladesh. Int. Clin. Pathol. J. 2015;1(5):104–108. [Google Scholar]
- 36.Mamun M.,A., Islam K.M., Rahman M.M. Occurrence of poultry diseases at Kishoregonj district of Bangladesh. MOJ Proteomics Bioinf. 2019;8(1):7‒12. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data will be made available on request.