Livestock Research for Rural Development 25 (7) 2013 Guide for preparation of papers LRRD Newsletter

Citation of this paper

Hatchery hygiene evaluation by questionnaire and microbiological screening of hatchery samples

E S Swai, P N Sanka* and C J Daborn**

Ministry of Livestock Development and Fisheries, Box 9152, Dar-es-Salaam
ESswai@gmail.com
* Veterinary Investigation Centre, PO Box 1068 Arusha, Tanzania
** Tropical Veterinary Services, PO Box 266, Karatu, Tanzania

Abstract

Questionnaire and microbiological-based surveys were conducted, during the period of March to August 2011, in 5 commercial broiler and layer hatcheries located in northern Tanzania to evaluate hatchery hygiene. Information on farm/hatchery history, hygienic practices and bio-security measures employed was collected. Hatchery samples (n=59), comprising day old dead chicks (DOC), dead-in-shell embryos (DES) and walls/ premises swabs (SWB), were aseptically collected for detailed bacteriological screening. Both non-selective (nutrient plate agar), selective and differential media (MacConkey agar plate) prepared according to the manufacturer’s instructions, were employed to differentiate between Gram-positive, Gram-negative and lactose fermenting organisms. Culture positive colonies were characterized further using Triple Sugar Iron (TSI) in order to establish the presence of enteric based pathogens.    

Overall, 3 units in Kilimanjaro and 2 units in Arusha were visited and bio-security measures reported to be used in order to avoid disease occurrence was routine usage of disinfectants (n=4; 80%), strict entry prohibition of non-authorized personnel (n=4; 80%), avoidance of mixing birds of different production purposes together (i.e. layers viz broilers, n=1; 20%) and change of clothes after each unit operation (n=1; 20%). The most cited targeted areas for disinfection were unit floor (n=5; 100%) and poultry and hatchery walls (n=4; 80%). The colonies that grow on the agar plates were about 2-4mm in size, irregular and whitish in colour. Stained colonies revealed that Gram negative bacteria were the commonest microorganisms, comprising 92% of all culture positives. Pathogen isolation rate was highest in hatchery D followed by hatcheries A, C and B with negative results in hatchery E. The isolation rate was highest in the DOC and DES derived samples and lowest in the SWB samples. Characterisation of positive culture samples, using the TSI biochemical test, revealed the following proportions of bacterial agents: Proteus vulgaris (60.7%), Enterobacter aerogenes (42.8%), Enterobacter cloacae (17.8%), Salmonella vulgaris (10.7%) and Salmonella typhi (7.1%). The high isolation rate and wide range of microbial pathogens found by this survey, reveals a serious problem of inadequate biosecurity practice indicating that there is considerable room for improvement in hatchery operations particularly with regards to hygiene and sanitation. Adequate training of hatchery operators is needed to raise awareness of the crucial role hygiene and biosecurity plays in ensuring high chick quality and their consequent survivability rates.

Key words: commercial hatchery, microbiological screening, poultry, Tanzania


Introduction

Worldwide, hatchery hygiene is recognized as an important factor in healthy poultry production (Chen et al 2002; Rodgers et al 2003). Poor standards of hatchery hygiene may lead ultimately to an explosion of pathogenic organisms resulting in severe losses to a country’s poultry industry (Harry and Gordon 1966). Despite an increasing demand for quality day-old chicks, the hatchery environs and operations in Tanzania are not well organized. Major constraints to this include, irregular or intermittent electric supply, frequent power break-down, lack of adequate training of personnel, weak regulatory services, under capacities of existing units and  inadequate supply of day-old chicks from the hatcheries due to hatchery and none-hatchery- borne disease mortalities. The environment of a poultry hatchery is very susceptible to contamination by vermin, rodents and microorganisms which can adversely affect hatchability of the eggs and can result in embryonic deaths. Important microorganism that may adversely affect hatchery hygiene and performance include contagious and non-contagious microbial pathogens such as Staphylococci species, Streptococci species, E. coli, Salmonella species and fungal infections such as Aspergillus fumigatus (Shane, 1999; Saif et al 2003; Ayachi et al 2010). There is an inadequate amount of credible information concerning hatchery hygiene in Tanzania. Understanding the prevailing disease status in hatcheries would contribute to a wider knowledge of the magnitude, impact and factors contributing to the establishment and persistence of identified pathogens. 

In order to optimize day old chick production from the existing hatcheries, it’s imperative that a number of routine management interventions are made. A critically important intervention is the development and maintenance of an effective hatchery sanitation program, which is essential for the successful operation of a poultry hatchery. 

This study was initiated with the objective of determining the levels of hygiene at commercial poultry hatcheries using microbiological examination of hatchery derived samples as a screening tool to estimate pathogen contamination levels. The purpose was to use the findings to inform poultry diseases policy reform and control strategies.


Materials and Methods

Study area

This study covered five commercial broiler and layers hatcheries located in two regions namely Arusha and Kilimanjaro, northern Tanzania. All five hatcheries are located at distances not >40 km from the centre of the regional city of each study regions. Broadly the location of both hatcheries were on latitude 3 16’ 21.61” S and longitude 37 34’ 32.19” E at an elevation of 1200-1900 m above sea level. Two of the hatcheries designated as unit D, and E are located in Arusha region and the third, fourth and fifth designated as units, A, B, and C in Kilimanjaro region. With reference to Tanzania day-old chick production capacities (< 5000 breeder stock), units A, B, and D were designated as large and units C and E as small operations, respectively. 

Data collection 

A questionnaire-based survey and microbiological sampling were conducted in all selected hatcheries. The study was conducted from March to August 2011. The questionnaire incorporated open and closed ended questions designed to gather hatchery level information (data) related to unit operations and hygiene. Questionnaires were extensively piloted and modified to ensure that data quality is maximized. Questionnaire administrations were undertaken jointly between researchers/research assistants and local area based livestock personnel. Questions in the survey covered farm/hatchery history, management practices employed with keen attention to disease bio-security and hygiene. Data collection techniques also included direct questioning and discussions with hatchery operators including a review of hatchery records where possible. Observation was also used to verify data. 

Biological sampling and handling 

To maximize sample collection and quality, a farm unit/hatchery visit was planned in such a way that it coincided with day of day-old-chick production. Sources of the samples were from hatchery debris/wall swabs (SWB), dead-in shell embryos (DES) and fresh dead day-old chicks (DOC). Collection of hatchery wall samples was made using the following protocol. Sterile moistened swabs with sterile normal saline were used to swab walls and floors of each sampling site. Swabs were placed in sterile test tubes containing sterile normal saline and transferred to the laboratory in an ice box. Collected samples were carefully labelled, packed and kept cool until they could be refrigerated (approx 2-4 hrs). Samples were stored at -200 C upon arrival from the field. Three to four days later, all samples were thawed and processed. 

Microbial assessment 

Non-selective (nutrient agar) and selective or differential (MacConkey agar) plate (Oxoid) were used to establish growth of both Gram-positive and Gram-negative group of bacterial pathogens from the different DES, DOC and SWB samples collected. For samples of necropsied day old chicks, the surface of the collected organs was seared with hot spatula and a sample was obtained by inserting a sterile wire loop through the heat sterilized surface and were streaked on to the plates of nutrient agar and MacConkey agar and incubated at 37o C for 24-48 hrs and observed for bacteria growth (Plate 1a). MacConkey agar was used to check for the presence of Gram negative lactose fermentors coliform bacteria (Plate 1b). All media used were prepared according to manufacturer’s instruction. Gram staining was done to identify the Gram-positive and Gram-negative bacteria from the bacteria isolated on different culture media. The slides were viewed under Microscope using x 100 magnification or oil immersion (Plate 1c). Sub-cultured using different media were made where necessary for distinct colony types and for confirmation of organisms earlier identified.


Photo 1a: Sample inoculation for bacteria culture Photo 1b: Plate culture growth


Photo 1c: Laboratory examination of samples Photo 1d: TSI sugar fermentors
Characterisation of positive cultures using triple sugar iron (TSI) 

Suspected Enteric based bacteria and Salmonella colonies on agar plates were inoculated on triple sugar iron agar (TSI) and incubated for 24 hrs at 37C(Plate 1d). TSI is a differential medium that contains lactose, sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the PH indicator phenol red. It’s used to differentiate enteric organisms based on the ability to reduce sulfur and ferment carbohydrates. If an organism can only ferment dextrose, the small amount of dextrose in the medium is used by the organism within the first ten hours of incubation. After that time, the reaction that produced acid reverts in the aerobic areas of the slant, and the medium in those areas turns red, indicating alkaline conditions. The anaerobic areas of the slant, such as the butt, will not revert to an alkaline state, they will remain yellow. This is seen with Salmonella and Shigella

Data management 

Data collected were entered and managed in MS Excel. Descriptive statistics to generate frequency distributions of isolates and graph were also performed in MS Excel. 


Results

Participating unit characteristics  

Overall, 5 units (3 in Kilimanjaro and 2 in Arusha) were visited, the owner or representative interviewed and sampling, as described above, conducted. Three units were classified as large scale operators, run and managed by registered institution/companies and the remaining 2 as small scale operators managed by individual local entrepreneurs. The education level of the respective units managers were 3 University graduate, 1 diploma graduate and 1 primary school leaver. The average age of the interviewed manager was 49.6 years (range 27-71). Most of the hatcheries (60%; 3/5) started during 80’s and only one started recently (2008). 

Bio-security measures employed in order to prevent microbial pathogens  

Hatchery owners were asked to describe the different bio-security measures used in order to avoid disease occurrence in their units. These were reported as: routine usage of disinfectants (n=4; 80%), strict entry prohibition of non-authorized personnel (n=4; 80%), avoidance of mixing birds of different production purposes together (i.e. layers viz broilers, n=1; 20%) and change of clothes after each unit operation (n=1; 20%). The most cited targeted areas for disinfection was unit floor (n=5; 100%) and poultry and hatchery walls (n=4; 80%). 

Culture results                                                                                                   

Microbial plate growth assessment revealed that colonies were about 2-4mm in size, irregular and whitish in colour. Based on morphological type and supported by Gram stain, the bacterial pathogens were mostly Gram-negative motile rods indicative of E. coli with a few Gram-positives mainly Staphylococcus spp (Fig 1). 


Figure 1: Types and proportion of microbial pathogens detected -
based on a Gram stain of plate growth colonies (n=59)

Microbial plate growth rate by units/farm and type of samples are shown in Fig 2-3. High growth rate was detected in unit/farm D (100%), closely followed by unit A and C. No growth was observed in unit E. With respect to sample type, over 95% of the plate growths were seen in day-old dead chicks followed by dead-in shell-embryo and least in hatchery wall swabs (Fig 3).


Figure 2: Microbial plate growth isolation rate by farm/units (n=59).

Figure 3: Microbial plate growth isolation rate by sample type (n=59).

A representative number of samples (n=59) culture positive were plated out on TSI medium, a differential medium that can distinguish between Gram-negative enteric bacteria based on their physiological ability (or lack thereof) to: metabolize lactose and/or sucrose; conduct fermentation to produce acid; produce gas during fermentation, and generate hydrogen gas (H2S). Salmonella organisms and other enteric bacteria pathogens were recovered from 14 out of 17(82.3%), 12 out of 22(54.5%) and 2 out of 20(10%) samples of dead–in-shell embryo, day-old chick and hatchery wall/surfaces, respectively. Detection rate of enteric microbial pathogens (TSI results) by farm/hatchery and sample types are given under table 1 and Figure 4. Overall TSI detection rate of pathogen by farm/units was 47.4%. Detection rate was 100% in hatchery D followed by hatchery C (55%), B (39%) and A (33%) with no isolates from hatchery E (Fig4). The most significant isolate was Proteus vulgaris and Enterobacter aerogenes (Fig 5).

Table 1: Enteric pathogens detection rate (TSI) by sample type (n=59)

Sample

No of samples

Detection rate

No. Positive

%

DES

17

14

82.3

DOC

22

12

54.5

SWB

20

2

10

Overall

59

38

64.4


Figure 4: Enteric pathogens detection rate (TSI) by farm/units (n=59)

Figure 5: Types and frequencies of enteric pathogens detection (TSI) (n=59)


Discussion

Results of the present study indicate that there was a wide range of bacteria pathogens prevailing in the five hatcheries surveyed. Identification of Salmonella spp by biochemical test strongly supports the results of other workers (Gosh and Panda 1998; Mdegella et al 2000). The incidence and extent of Salmonella and other enterobacteriaceae group of bacteria revealed by this study from the different samples taken is in agreement with findings of Gosh and Panda, (1998). There was greater rate of bacterial recovery from day old dead chick compared to dead in-shell embryo and least from the hatchery wall. Evidence of Salmonella as detected by this study indicates that this pathogen is circulating in some breeding units which, most logically, would be through trans-ovarian transmission. Bacterial pathogen recovery from dead DOC was noted to be high. The number of bacteria contaminated chicks in hatchery might be reduced if bacterial is totally eliminated from eggs. There are evidence that some chicks acquire Salmonella and other bacterial pathogens as they hatch from their own contaminated shells and shell membranes (William and Dillard 1968).The reason for lower incidence of bacterial pathogen positive samples between studied units could be due to the size of each respective units and the frequency and number of visitors/operators. For instance, the hatching eggs are gathered frequently because of the size of the flocks and the value of the eggs. Comparatively units C and E were identified to be the smallest units whereas units A and D were considered to be larger, both having more than 5,000 birds of different breeds, purposes and age.           

Based on the findings of this study it may be concluded that, some commercial poultry hatcheries in Tanzania are operating in a diseased environment. There is a need to undertake frequent monitoring of all commercial hatcheries for contamination by specific pathogens. Monitoring should include routine screening of live birds against Salmonella, collection of raw materials, faeces, swabs and from other critical control points for routine laboratory screening. At farm level, dirty or cracked eggs should be discarded, infected birds culled and strict bio-security and levels of hygiene observed at all steps/stages of the production cycle. A bio-security and sanitation plan should be developed and target specific areas (rooms, floors, foot baths), equipment (watering, feeders, egg collecting/storage), personnel, vehicles at each operation point and disinfectants  from quality controlled sources and with proven bactericidal qualities such as peroxides, chlorine, phenolics or formaldehyde/glutaraldehyde/quaternary ammonium compounds as recommended by OIE (Deeba et al 2003; Haynes and Smith 2003; Ledoux and Lines 2003; OIE 2008).           

Due to the limited laboratory capacity, a detailed molecular characterization of the isolates could not be made. Further studies should be directed at molecular typing and drug sensitivity tests of the isolates.


Conflict of interest statement


Acknowledgments

The authors wish to acknowledge the financial support from Northern Zone Agriculture and Research Development Fund, Project No NZARDEF/LP/11/04 without which this research would not have been possible. The authors are very grateful to the hatchery owners who gave their time to enable this research to be conducted. In addition, our appreciation is extended to the laboratory and veterinary field staff of Moshi, Kilimanjaro and Arusha for their cooperation in the study. This paper is published with the permission from the Director of veterinary services in Tanzania.  


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Received 23 May 2013; Accepted 12 June 2013; Published 1 July 2013

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