Wood Vinegar inhibit Salmonella Enteritidis in Piglets
Wood Vinegar inhibit Salmonella Enteritidis in Piglets
1. Background and rationale
Salmonella is one of the most important pathogens that cause foodborne illness in developing countries. It has a wide range of animal reservoirs and can cause salmonellosis in both humans and animals. Salmonellosis is a zoonotic disease which means that it can be transmitted between animals and humans. The majority of cases of human salmonellosis are due to consumption of contaminated eggs, poultry, pork, beef and milk products (del Cerro et al., 2003). Symptoms of human salmonellosis can be divided into four syndromes: enteric fever (typhoid-like disease), gastroenteritis (food poisoning), bacteremia with or without gastroenteritis, and the asymptomatic carrier state (Nair et al., 2002). Salmonellosis is primarily a disease confined to the gastrointestinal tract, but may cause serious extraintestinal tract disease especially in the infant, old aged who are the immunologically compromised host (Levine et al., 1991). The incidence of nontyphoidal salmonella infections has increased considerably in many countries (Makanera et al., 2003). The most common clinical sign caused by Salmonella species in animals is diarrhea. Some animals may be infected and shed the agent in their feces or milk but show no signs of the disease (Fraser et al., 1991). Subclinical salmonellosis infection in animals is common and may become asymptomatic carriers that intermittently shed the bacteria into the environment for variable periods of time (Guerin et al., 2005).
Antimicrobial-resistant strains have been isolated with increasing frequency from human infections, farm animals, and foodstuffs and may cause a prolonged or more severe illness than antimicrobial-susceptible strains (Rabsch et al., 2001). This multi-resistance is of considerable importance in both human and veterinary medicine (Glynn et al., 1998). There has been concern about antimicrobial resistance in Salmonella, which has leaded to failure of treatment for Salmonella and other bacterial pathogens (Travers and Barza, 2002; Butt et al., 2003). Since foods of animal origin are a major source of human salmonellosis, it has been suggested that antimicrobial use in food animal production may contribute to the presence of antimicrobial resistance in Salmonella species that infect humans (Iovine and Blaser, 2004). Of particular concern is the increasing association of human infections with multidrug-resistant Salmonella strains.
Wood vinegar is a byproduct from charcoal production. It is a liquid generated from the gas and combustion of fresh wood burning in airless condition. When the gas is cooled, it condenses into liquid. Raw wood vinegar has more than 200 chemicals, such as acetic acid, formaldehyde, ethyl-valerate, methanol, tar, etc. Wood vinegar improves soil quality, eliminates pests and controls plant growth, but is slightly toxic to fish and very toxic to plants if too much is applied. It accelerates the growth of roots, stems, tubers, leaves, flowers, and fruit. In certain cases, it may hold back plant growth if the wood vinegar is applied at different volumes. A study shows that after applying wood vinegar in an orchard, fruit trees produce increased amounts of fruit. Wood vinegar is safe to living matters in the food chain, especially, insects that help pollinate plants. Wood vinegar is made from burning fresh wood in a charcoal kiln, made from a 200-liter oil drum and 120-cm-tall concrete chimney with a 4-inch diameter. The kiln contains 63-83 kg of fresh wood. Wood good for vinegar must have a heart-wood. (http://www.agnet.org/library/pt/2005025/; 23 September 2008)
There was only one study to elucidate the effect of wood vinegar on suppression of Salmonella Enteritidis in chickens. The result was decrease bacteria compare with control group (Watarai and Tana, 2004). Mekbungwan et al., 2003 report to 3% of wood vinegar was improved feed efficiency after feeding. In previous study, wood vinegar was effective to inhibit Salmonella in vitro. However, there was no study to use for prevention of diarrhea pigs. Although, there was introduce to use wood vinegar 2 liters mix with charcoal 8 kilograms in feed 990 kilograms but it is not improve by scientific study. The purpose of this study was to know effect of wood vinegar inhibit Salmonella in piglets.
2. Literature review
Human salmonellosis is an important public health problem in Thailand. Common symptoms can range from bacterial diarrhea to septicemia. Human infections are often related directly or indirectly to infection in animals. Transmission can occur via a variety of specific routes including food-borne, water-borne, animal to person contact, person to person contact (either direct or indirect), and transfusion. The sources of food-borne salmonellosis outbreaks indicate that foods of animal origin are the most common source. Most of human salmonellosis cases are traced to Salmonella infected farm animals (Hoszowski and Wasyl, 2001). Salmonella can be introduced and perpetuated on a farm through contaminated feeds, the introduction of infected stock, or by wild animals. The incidence of salmonellosis is higher in children than in adult and the majority of cases are sporadic (Grisaru-Soen et al., 2004). Human salmonellosis can also be contracted through contact with infected animals. Food containing products from farm animals, especially from pigs, are an important source of human Salmonella infections (van Duijkeren et al., 2002). Because Salmonella species can survive and proliferate outside their host, the “Salmonella problem” is basically acontinuous faecal-oral cycle and thus also a “hygiene problem”. Many Salmonella species can survive for several months in soil and for several weeks on stems and leaves of feed crops.
Salmonella is an enteric pathogen that colonizes the intestinal tracts of a variety of animal; foods that have contributed most to the incidence of salmonellosis are pork and products 10% and 16% (White et al., 2001). Contamination of pork products by Salmonella is often the result of feces being spread onto carcasses during the slaughter and processing of swine (Wonderling et al., 2003). National Animal Health Monitoring System was reported 38% Salmonella positive isolated from swine herd. Infected swine are frequently non-symptomatic, it is critical to prevent or minimize the spread of fecal matter onto carcasses (Duffy et al., 2001). In Thailand, investigation epidemiology of Salmonella species reported by The National Salmonella and Shigella Center, 2006
Infections in healthy animals may be asymptomatic. Symptomatic infections are often precipitated by stressors, such as transport, drought, malnutrition or food deprivation, crowding and some drugs. Symptomatic infections may result in several syndromes: acute septicemia, acute enteritis, subacute or chronic slmonellosis, and abortion (Berends et al., 1998).
In Thailand, Salmonella is widespread in pigs and pork was 25% and 65% in Khon Kaen province (Angkititrakul et al., 2005). Consequently, in pre-slaughter pigs with percentage between 6 to 69.5 % and pork at the Chieng Mai province market was 29% (Padungtod and Kaneene, 2006).The reduction of the prevalence of Salmonella in pigs at the herd level should reduce the contamination in pork products. Concerning the epidemiology of Salmonella from farm to table in the pig production line, need exists to establish the status of Salmonella with the objective of set up food quality standards and risk assessment in Thailand.
Wood vinegar or pyroligneous acid is a byproduct from condensation of charcoal production while the temperature in kiln is between 300-400 degrees Celsius. It is a light brown liquid and smoke wood smell, generated from the gas and combustion of fresh wood burning in airless condition. When the gas is cooled, it condenses into liquid. Raw wood vinegar has 8% (volume/weight of wood) and remain to 5% if purification. It has more than 200 chemicals, such as acetic acid, formaldehyde, ethyl-valerate, methanol, tar, etc. Composition of wood vinegar is water (85%), organic acid (3%) and other (12%). pH of wood vinegar is 3 and specific gravity is 1.012-1.024 upon type of wood. Most of ingredients are acetic acid, phenol, formaldehyde and methanol. Purification of wood vinegar has 3 methods as (1) Sedimentation: Leave the raw wood vinegar for 3 months to become silted. The vinegar will turn yellow like vegetable oil. After which, it will turn light brown and the tar will become silted. The top layer will be light, clear oil, middle layer is wood vinegar and bottom layer is tar. Remove the tar and light oil, as well as the dark brown translucent oil and the remainder will be wood vinegar. (2) Filtration: use of 5% charcoal; by weight (3) Distillation: process after complete precipitation.
Wood vinegar has also traditionally been used as improves soil quality, help plant grow better, stronger, eliminates pets, insect repellant, deodorizer, antibacterial agent, sterilizer, alkaline bath and wash, and food additive. Wood vinegar is known to be able to improve our blood circulation. This improved circulation can lead to relief from fatigue, headache, double vision, blood pressure, arthritis, and many other similar symptoms. It is safe to human, animals, plants and environment. Farmers can produce wood vinegar from branches trimmed from trees which saving cost of chemicals and low cost of production.
3. Justification to do the study:
The number of human salmonellosis cases reported in Thailand has been increasing since 1993 and the most infection occurs in children (< 5 years), elderly (> 60 years) and immunosuppression patients. During 2004 – 2006, prevalence of human salmonellosis was raise from 62% to 78%.
Pork and pork products are now recognized as one of the most important sources of human salmonellosis (Wegener and Baggesen, 1996). Mostly pig farms were overuse or misuse of antimicrobial agents. They were not only for therapy and prevention, but may also be added continuously to animal feeds to promote growth and increase feed efficacy.
Wood vinegar is natural substance and safe to humans, animals and environment. In previous study, 7% of wood vinegar can inhibit Salmonella Enteritidis in vitro. There was only one study used wood vinegar decreased Salmonella in chickens and one study used improved to feed efficiency in piglets. However, no scientific study use wood vinegar inhibits Salmonella in pig.
If this study can prove to inhibit Salmonella Enteritidis in piglets that farmer should be use for control Salmonella in pig farms. Reduce of use antimicrobial agents in pig farms and decrease antimicrobial residue in pork which important problem in public health. The advantage of wood vinegar, cost is lower than antimicrobial agents.
4. Research question
Primary research question: Can wood vinegar inhibit colonization of Salmonella Enteritidis in piglets?
Secondary research question: What is dilution of wood vinegar inhibit colonization of Salmonella Enteritidis in piglets?
Third research question: Was improved feed efficiency in piglets?
5. Conceptual framework
Primary objective: To know the effect of wood vinegar to inhibit Salmonella Enteritidis in piglets
Secondary objective: To determine dilution of wood vinegar to inhibit Salmonella Enteritidis in piglets
Tertiary objective: To improve feed efficiency in piglets
7. Key words & Operational definitions
Salmonella Enteritidis, wood vinegar, piglets
8. Research design
Experimental study (control and treatment group)
9. Research methodology
Sixteen piglets are select using the follow inclusion criteria: (1) Piglets are healthy and Salmonella free, (2) hybrid of piglets, (3) 15 kilograms body weight. Each experiment piglets is allotted in 1.5×2.0 m2 pens and feed and water were purchase ad libitum.
A control group contains 4 piglets providing feed without wood vinegar. In 3 of treatment groups are providing feed contain 3%, 5%, and 7% of wood vinegar, respectively. Three days later, all piglets are orally challenged with Salmonella Enteritidis suspension (1x 107 cfu). Fresh fecal samples (approximately 10 grams) are collect from each piglet on day 1 to 7 after challenge.
All fresh fecal samples are weight and suspend in sterile PBS at 2X dilution. They are then serially dilute 10 fold in PBS, and 100 µl of each dilution is spread onto XLD agar plates. After cultivation at 37 oC for 24 hours, dark colonies of Salmonella Enteritidis on the plates are count.
Group 1: control and feed without wood vinegar
Group 2: feed with 3% wood vinegar
Group 3: feed with 5% wood vinegar
Group 4: feed with 7% wood vinegar
Target population: all piglets in farm
Sample population: Piglet which healthy, Salmonella free, hybrid, 15 kilograms body weight
Sampling technique: randomization technique
11. Sample size calculation
The sample size calculation bases on the objective study to determine dilution of wood vinegar to inhibit Salmonella Enteritidis in piglets
There is no previous report of dilution of wood vinegar to inhibit Salmonella Enteritidis in piglets.
The sample size will be calculated to detect the difference dilution of wood vinegar between both groups use hypothesis testing for two population mean at a significant level of 95% and power of 80% (two-sided test). The number of sample size is 4 piglets per group.
n = number of participants
= population standard deviation
α = level of significant
1-β = power of test
µ = value of the population mean
Dilution of wood vinegar 3%, 5% and 7%
13. Outcome variables
– Primary outcome: Presence or absence of colony of Salmonella Enteritidis (dichotomous data: yes/no)
– Secondary outcome: The number of colony of Salmonella Enteritidis (continuous data)
– Tertiary outcome: Body weight of piglets (continuous data)
14. Measurement of the outcome(Data collection)
– Measure the outcome: Colony count of Salmonella Enteritidis on XLD agar
– Tool will be used: Standard Conventional Method (ISO: 6579). Culture Salmonella growth in Incubation 37oC 24 hours
– Do this job: Farm owner was managing piglets such as feeding and sanitation, technician was culture Salmonella in laboratory.
– Collect the data: January 2009
This study use statistical analysis by one way analysis of variance (one way ANOVA). Because I would like to know some group of treatment may be different from other group compare with control group. There is the same condition of each group such as environment, feed, water, and management.