称号及び氏名

博士(獣医学) Randy Rhon Simoun P. Lapuz

学位授与の日付

平成 1９年９月３０日

論「Rodents:Major risk factor in the transmission and persistence of文

名Salmonella contamination in layer farms(養鶏場のサルモネラ汚染伝搬と維持における主たるリスクファクターとしてのネズミの重要性）」
論文審査委員

主査

馬場栄一郎

副査

小崎 俊司

副査

稲葉 俊夫

副査

笹井 和美

論文要旨
Introduction
Microbial pathogens of the genus Salmonella are among the leading
causes of food-borne illness in the world. Reports of almost 380,000 human and66,000 nonhuman Salmonella isolations worldwide were entered into the WHO
global surveillance report database during 2000-2002. In Japan, most cases ofbacterial food poisoning cases have generally been attributed to Salmonella in
the past 20 years. From 2004 to 2006, the top two Salmonella serovars
associated with human salmonellosis in Japan were Salmonella enterica subsp.
enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica subsp.
enterica serovar Infantis (S. Infantis).
Most of the outbreaks of S. Enteritidis infection are often associated withcontaminated eggs and egg products. In addition, S. Enteritidis is the only
human pathogen that routinely contaminates the egg. S. Enteritidis seems to beable to colonize the ovary and oviduct of laying hens and this route ofcontamination has been identified as major risk factor in the emergence ofhuman illness. Since the primary contamination of S. Enteritidis occurs at thefarm level, on-farm prevention and control measures and quality assurance
programs were initiated in the 1990s.
The concept of Hazard Analysis and Critical Control Point (HACCP) has
been generally known to Japanese farmers. The Japanese layer industry hasbeen carrying out numerous measures to prevent egg contamination in layerhouses. These measures include,
stocking layer houses with
Salmonella-negative birds from uninfected breeders, regular monitoring of feed
samples and sometimes vaccination against S. Enteritidis. However, inclusion ofrodents as part of intervention strategies to prevent Salmonella contamination
has not been fully realized by the Japanese layer industry probably due to limited
documentations of significant public health threat that rodents pose in livestock
operations. In Japan, roof rats (Rattus rattus), are the dominant rodent species
in poultry premises. Investigations of the prevalence of Salmonella infection
among rodents in layer farms are limited and its epidemiological role in thespread of Salmonella contamination is not yet fully understood.
In the present study, the prevalence of Salmonella spp. in rats found inlayer farms was investigated. Additionally, isolation of Salmonellae from eggs,
environment samples, layer hens, liquid egg samples were also conducted.
Pulsed-field gel electrophoresis (PFGE) analyses of Salmonella isolates fromrats, eggs, environment samples, layer hens, and liquid egg samples were also
performed to elucidate the epidemiological role of rats in transmission ofSalmonella contamination in layer farms. Furthermore, a field experiment wasinitiated on one of the confirmed Salmonella-contaminated farm to determine theeffect of rodent control program in reducing Salmonella contamination levels inthe layer houses.
Chapter I. The role of roof rats (Rattus rattus) in the spread of Salmonella
contamination in layer farms
The prevalence of Salmonellae in rodents, eggs, and environment
samples of 7 commercial layer farms in eastern Japan was investigated between
2004 and 2006 to determine the role of roof rats (Rattus rattus) in theepizootology of S. Enteritidis contamination in layer farms. Also, the rodent
density of each farm was estimated using visual inspection and Rodent Indexing(RI) developed by Henzler. Several Salmonella serovars such as S. Enteritidis, S.
Infantis, S. Thompson, S. Potsdam, S. Mabandaka, and S. Virchow weredetected from environment samples of different layer farms. S. Enteritidis and S.
Infantis strains were the only serovars isolated from pooled egg samples.
Persistent S. Enteritidis contamination of environment and pooled egg sampleswere observed in 3 (Farms A, B, and C) of 7 layer farms. These 3 persistently S.
Enteritidis-contaminated farms have high rodent populations. A total of 113(12.6 %) and 158 (17.6%) out of 900 roof rats examined were positive for S.
Enteritidis and S. Infantis, respectively. In addition, 13 (1.4 %) rats from Farm E
were infected with S. Thompson. There were no rodents caught in Farms D, F,and G.

By PFGE analysis, a variety of Pulsed-field patterns were generated byS. Enteritidis isolates from rats, eggs, and environment samples of one
contaminated farm (Farm A), however, there were also very closely related S.
Enteritidis and S. Infantis strains observed. In addition, PFGE analysis of S.
Enteritidis strains from rats, eggs and environment samples of Farms B (Fvalue=0.92) and C (F value=1.0) yielded genetically related clones. Theseresults suggest that roof rats are persistent carriers of S. Enteritidis and S.
Infantis and may have an important role in the transmission and persistence of
these pathogens inside the layer premises.
Chapter II. Comparison of the prevalence of Salmonella infection in layerhens among layer farms with low and high rodent density
There are evidences that S. Enteritidis gains access to egg contents by
migrating from the cloaca to the reproductive organs, or via trans-ovariantransmission following systemic infection and localization in the ovaries, oviducts
or peritoneum of laying hens. Consequently, infected flocks are difficult toidentify.
There are limited studies that have documented the prevalence of
Salmonella in naturally infected layer hens in field conditions. This study wasconducted to compare the prevalence of Salmonella infection in layer hens
housed in layer farms with high rodent populations and with low rodent
populations. In addition, PFGE analysis of Salmonella isolates from layer hens,
rats, eggs and environment samples were conducted to determine theepidemiological link between rats and layer hens in the transmission ofSalmonella contamination in layer farms. A total of 380 layer hens from 7
commercial layer farms were examined for Salmonella infection from 2004 to2006. Portions of the liver, spleen, kidney, intestine, ovary, oviduct, and cloacal
swabs were cultured for Salmonella isolation. S. Enteritidis strains were onlyisolated from farms with high rodent density (Farms A, B, and C). Salmonella
infection rate in layer hens ranged from 5.7 % to 8.8 %. Oviduct (48.0 %) was theorgan with the highest isolation rate for S. Enteritidis followed by liver. Results ofPFGE analysis revealed that S. Enteritidis strains from layer chickens had thesame Pulsed-field patterns generated by Salmonella isolates originating from theresident rat population. These findings suggest that high rodent population is amajor risk factor in the transmission and persistence of S. Enteritidis infection inlayer hens.
Chapter III. Epidemiological analysis of S. Enteritidis and S. Infantis
isolates from liquid eggs by Pulsed-field gel electrophoresis
In order to determine the epidemiology of Salmonella contamination inliquid eggs, Salmonella isolates from environment, rodent, layer chicken, andegg samples were compared with isolates from liquid eggs from a liquid egg
factory, in which some of the eggs supplied originated from a rodent-infested
layer farm. The prevalence of Salmonella contamination in unpasteurized liquid
eggs was investigated. A total of 1,585 batches of unpasteurized liquid eggs
were examined for Salmonella contamination. The contamination rates of S.
Enteritidis and S. Infantis strains in liquid egg samples were 1.2 % and 0.2 %,respectively. By PFGE analysis, S. Enteritidis and S. Infantis isolates from liquid
eggs are very closely related to Salmonella isolates from rodents, layer chickens,
eggs and environment samples of the layer farm. Salmonella contamination inliquid eggs can be traced back to the rodent-infested farm.
Chapter IV. Effect of reducing rodent population on Salmonella
contamination levels in layer houses
The high rodent population inside the layer farms might be an importantfactor in the transmission of Salmonella. To determine the effect of rodent control
program in reducing and ultimately eliminating Salmonella contamination in layerhouses, a field experiment involving initiation of rodent control measures on
confirmed highly rodent-infested S. Enteritidis-contaminated layer houses wasundertaken for six months. Three groups of 2 layer houses each were examinedfor Salmonella contamination on eggs and environmental samples. Two types ofrodent control programs were implemented on groups 1 and 2, while, in group 3(control group), no rodent control measures were applied. Rodent control
measures include repair of rodent-damaged walls and attic of layer houses,
application of adhesive traps and rodenticide baiting. Rodent density was also
estimated monthly using Rodent Index developed by Henzler. The
implementation of rodent control measures resulted in gradual reduction ofrodent population inside the layer houses and in turn, Salmonella contamination
rates of eggs and environmental samples were also significantly reduced.
Conclusions
1. Roof rats are persistent carriers of S. Enteritidis and S. Infantis and may havean important role in the transmission and persistence of these pathogens
inside the layer premises.
2. High rodent population is a major risk factor in the transmission andpersistence of S. Enteritidis infection in layer hens.
3. Salmonella contamination in liquid eggs can be traced back to therodent-infested farm.
4. The implementation of rodent control measures resulted in the reduction ofrodent population inside the layer houses and Salmonella contamination ratesof eggs and environmental samples were also significantly reduced.

審査結果の要旨
サルモネラは食材媒介性疾病の主要な病原菌の一つであり、これらの汚染防止は世界的な課題である。日本でも、最近 20 年間の細菌性食中毒事例の多くがサルモネラによると報告されており、中でも重要な血清型は Salmonella enterica subsp. enterica serovar Enteritidis（S.
Enteritidis）と同 serovar Infantis（S. Infantis）であり、ここ数年間に日本で分離された血清型の上位を占めている。多くの S. Enteritidis 感染事例が汚染された卵や卵製品と関連し、 S.
Enteritidis の主要な汚染現場は産卵養鶏場であるといわれている。そのため養鶏場での汚染防止対策がとられ、導入ヒナ、餌、鶏舎などの衛生管理をはじめワクチンなどを用いたあらゆる方法が講じられた結果、食中毒の発生率は減少傾向にあるが、未だ、十分とはいえない。
申請者は、これまであまり積極的に捉えられてなかった養鶏場におけるネズミのサルモネラ汚染に関わる役割に着目した。日本ではクマネズミ（Rattus rattus）が優勢なげっ歯類であるが、被害の実態についての報告は少ない。そこで、養鶏場におけるネズミとサルモネラ汚染について現場に密着した調査を実施し、さらに、ネズミの被害を抑えることによってサルモネラ汚染を抑制できるかどうかを実際の養鶏場を用いて試験した。成績は以下のように要約される。
第１章では、サルモネラ汚染が確認されていた施設を含む７ヵ所の採卵養鶏場について、３年に亘って詳細な検査を実施し、以下の結果を得た。各養鶏場では調査期間中に少なくとも１度は鶏群を入れ換えている。７養鶏場のうち３ヵ所では、卵や環境材料に持続的な S.
Enteritidis あるいは S. Infantis 汚染がみられ、同時にこれらの養鶏場ではヘンツラーの RodentIndex（RI）で比較したネズミの棲息密度が高かった。サルモネラの分離頻度を見ると、環境材料よりもネズミの方が高かった。また、分離菌株のパルスフィールド電気泳動（PFGE）パターンの比較から、それぞれの養鶏場内での分離株は互いに密接に関連していることが判った。他の４養鶏場では環境材料から S. Enteritidis や S. Infantis 以外のサルモネラ血清型が分離されたが、卵からは分離されず、ネズミの棲息密度も低かった。これらの結果から、ネズミは S.
Enteritidis と S. Infantis の持続的なキャリアであって、養鶏場内の伝播と病原体の持続的な生息に重要な役割を持つことが示唆された。
第２章では、鶏卵を汚染するサルモネラの血清型が S. Enteritidis や S. Infantis に集中する機序を解明する一助として、養鶏場の産卵鶏についてサルモネラの臓器分布を調べ、分離株についてネズミ由来株間での PFGE パターンを比較した。７養鶏場のうち汚染のみられた３養鶏場の産卵鶏の臓器から S. Enteritidis あるいは S. Infantis が検出され、特に卵管からの分離率（48.0%）が他の臓器よりも高かった。PFGE の結果では、産卵鶏から分離した全ての S.
Enteritidis 株のパターンはネズミ由来株と一致した。したがって、ネズミに由来する S.
Enteritidis あるいは S. Infantis が産卵鶏を汚染し、卵管に到達して卵を汚染した可能性が示唆された。
第３章では、液卵加工された製品から検出された汚染サルモネラと、養鶏場のネズミが保有するサルモネラとの関係を調べるために、それぞれの分離株について PFGE パターンとファージ型を比較した。液卵 1,585 サンプルから S. Enteritidis が 1.2%、S. Infantis が 0.2%の割合で分離された。これらの菌株と、液卵の由来養鶏場のネズミ分離菌株とを比較したところ、両者は非常に密接に関連することが明らかになり、液卵のサルモネラ汚染は、ネズミに侵害された養鶏場にまで遡ることができることが示唆された。
第４章では、S. Enteritidis に汚染された鶏舎を用いてネズミを制御した場合にサルモネラ汚染が軽減されるかどうかを試験した。６鶏舎を３群に分け、金属板でネズミの出入りを遮断し殺鼠剤を用いた群、殺鼠剤のみを用いた群、および無処置群を設けた。6 ヵ月間に亘り、ヘンツ
ラーの RI、および卵と環境材料のサルモネラ分離によって評価した。ネズミの棲息を制御した
２つの群では S. Enteritidis 汚染の有意な低下がみられ、養鶏場でのネズミの棲息がサルモネラ汚染とその持続に関わっていることが証明された。
以上の結果は、鶏卵のサルモネラ汚染に養鶏場のネズミが深く関与していることを証明したものである。これらの結果は、畜産現場におけるサルモネラ汚染対策上重要な示唆を与えるものであり、獣医衛生学、獣医公衆衛生学に寄与するところが大きい。したがって、本論文の審査および最終試験の結果とあわせて博士（獣医学）の学位を授与することを適当と認める。