Rickettsia,

Bartonellosis, Q Fever

Decha Pangjai D.V.M.

National Institute of Health,

Department of Medical Sciences, Thailand

What is a Rickettsial Disease?

Genus Bartonella

Bartonella spp are small, gram-negative aerobic bacilli that are difficult to grow in culture, mainly transmitted by vector. These microorganisms infect erythrocytes of their mammalian hosts, and some species cause a wide spectrum of human illness, such as chronic bacteremia, fever and endocarditis.

Bartonella

spp.

Reservoir Accidental

host

Vector or potential

vector*

Current geographic

distribution

Present

zoonotic

status Bartonella

bacilliformis Humans No Pheblotomines (sand

flies)

(Lutzomyia verrucarum)

Andes (Peru, Ecuador

,Colombia, Bolivia,Chile,

Guatemala)

No

B. quintana Humans No Human body lice

(Pediculus humanis

corporis)

Worldwide† No

B. henselae Cats (Felis catus) Humans,

dogs

Fleas

(Ctenocephalides felis)

Ticks?

Worldwide† Yes

B. clarridgeiae Cats (F. catus) Humans,

dogs

Fleas (C. felis) Cosmopolitan‡ Yes

B. koehlerae Cats (F. catus) Unknown Fleas (C. felis) California, France No

B. vinsonii

subsp

arupensis

White-footed mice

(Peromyscus

leucopus)

Humans Fleas? Ticks? United States (Midwest) Yes

B. vinsonii

subsp

berkhoffii

Coyotes (Canis

latrans)

Humans,

dogs

(Canis

familiaris)

Ticks? Cosmopolitan‡ Yes

B. vinsonii

subsp vinsonii Meadow voles

(Microtus

pennsylvanicus)

No Ear mites? (Trombicula

microti)

Canada No

B. talpae Moles (Talpa

europaea)

No Unknown United Kingdom No

B. peromysci Field mice

(Peromyscus spp)

Unknown Unknown United States No

Bartonella

spp.

Reservoir Accidental

host

Vector or potential

vector*

Current geographic

distribution

Present

zoonotic

status B. birtlesii Wood mice

(Apodemus spp)

Unknown Unknown France, United Kingdom No

B. grahamii Bank voles

(Clethrionomys

glareolus)

Humans Fleas? United Kingdom Yes

B. taylorii Wood mice

(Apodemus spp)

Unknown Fleas? United Kingdom No

B. doshiae Meadow voles

(M agrestis)

Unknown Unknown United Kingdom No

B. elizabethae Rats (Rattus

norvegicus)

Humans,

dogs

Fleas Worldwide Yes

B. tribocorum Rats (R norvegicus) Unknown Unknown Cosmopolitan‡ No

B. alsatica Rabbits(Oryctolagus

cuniculus)

Unknown Fleas? Ticks? France No

B. washoensis Ground squirrels

(Spermophilus

beecheyi)

Humans,

dogs

Fleas? Ticks? United States (Western) Yes

Bartonella sp

Unknown Prairie dogs

(Cynomys

ludovicianus)

Unknown Fleas? United States (Western) No

Bartonella

spp.

Reservoir Accidental

host

Vector or potential

vector*

Current geographic

distribution

Present

zoonotic

status B. bovis Domestic cattle

(Bos taurus)

Unknown Biting flies? Ticks? Cosmopolitan‡ No

B. capreoli Roe deer

(Capreolus

capreolus)

Unknown Biting flies? Ticks? Europe No

B. choenbuchensis Roe deer(C

capreolus)

Unknown Biting flies? Ticks? Europe No

B. chomelii Domestic cattle

(B. taurus)

Unknown Biting flies? Ticks? France No

*Potential vectors are organisms that have been suggested as vectors but for which there is no experimental proof. †Worldwide

distribution

There are only a few reports on detection of Bartonella in Thailand.

-Castle et al. (2004) reported a prevalence of 9% in

Bandicota indica and Rattus spp. in Chiangrai, Thailand

-Parola et al. (2003) identified Bartonella BNfRs in flea

(Nosopsyllus fasciatus) collected from Rattus surifer in

Kanchanaburi province, on the Thai-Myanmar border.

-The newly described B. tamiae pathogen was isolated fromhuman patients in Thailand (Kosoy et al., 2008)

-while B.henselae was recently isolated from biopsy skin lesions of bacillary angiomatosis (Paitoonpong et al., 2008).

Blood was inoculated onto a blood agar plate

(Brain Heart Infusion agar base containing 5% whole defibrinated rabbit blood).

Plates were then incubated for up to 35 days at 35°C in a moist,

5% CO2 atmosphere. Plates were checked after 3 days’ incubation

(DNA extraction Boiled Method from Isolation)

DNA Extraction

Agarose gel electrophoresis of rpoB primer (893 bp)

Fig C. PCR amplification of Bartonella spp. DNA from rodents blood using gltA ; BhCS.781p and BhCS.1137n primers. M : DNA ladder (100 bp). ; 1 : Sample B1 ; 2 : Sample B2 ; 3 : Sample B3; 4 : Sample B4; 5 : Sample B5; 6 : Sample B6; 7: Sample B7; 8 : Sample B8; 9: Sample B9; 10 : Francisella tularensis ; 11: negative control; 12 : Bartonella henselae DNA (312 bp); Arrow 500 bp

Sequencing and phylogenetic analysis of rpoB, gltA, ftsZ, groEL, ribC, 16S rRNA and the 16S–23S rRNA intergenic spacer region (ITS)

The QIAquick PCR purification kit (Qiagen,Germantown,MD) was used to prepare amplicons for sequencing. The PCR products were sequenced by using specific primers for rpoB, gltA, ftsZ, groEL, ribC, 16S rRNA and the 16S–23S rRNA intergenic spacer region (ITS) and the BigDye Terminator Cycle sequencing Ready Reaction (ABI PRISM3130xl genetic analyzer, Applied Biosystems Foster City, CA)

The GeneDoc program was used for the alignment of the multisequences obtained in this study with those of known Bartonella species from the GenBank databases.

A phylogenetic tree was drawn based on the sequences of rpoB, gltA, ftsZ, groEL, ribC, 16 rRNA and the 16S–23S rRNA intergenic spacer region (ITS) using the neighbor-joining method with Kimura’s two-parameter distance method in Molecular Evolutionary Genetics Analysis version 5 (MEGA 5). Bootstrap analysis was carried out with 1,000 resamplings.

GenBank accession numbers of the sequences used to construct the tree are shown in the table A homology to rpoB, gltA, ftsZ, groEL, ribC, 16 rRNA and the 16S–23S rRNA intergenic spacer region (ITS) was used to define groups.

Neighbor-Joining tree based on concatenated 7 gene sequences .The phylogenetic tree was created using the Kimura 2-parameter model. The tree was rooted by using Brucella melitensis as the out group. Bootstrap values resulting from 1,000 bootstrap trials are indicated for each branch. Bar, 0.01 estimated nucleotide substitutions per site.

National Center for Biotechnology Information(NCBI)

DNA Data Bank of Japan (DDBJ)

European Molecular Biology Laboratory - EMBL

Serological test

Prevalence and Genetic Diversity of Bartonella Species among small mammals in 9 Provinces of Thailand Decha Pangjai1, Soichi Maruyama2*, Sumalee Boonmar3, Hidenori Kabeya2, Burin Nimsuphan4, Wimol Petkanchanapong1, Wattanapong Wootta1, Piyada Wangroongsarb1, Maskiet Boonyareth1, Poom Preedakoon1, Watcharee Saisongkorh1 and Pathom Sawanpanyalert1

1 Department of Medical Sciences, National Institute of Health, Ministry of Public Health, Nonthaburi, Thailand 11000 2 Laboratory of Veterinary Public Health, College of Bioresource Sciences, Nihon University Fujisawa, Kanagawa 252-0880, Japan 3 International Emerging Infections Program (IEIP) Thailand MOPH-U.S. CDC Collaboration (TUC) Dept. of Medical Sciences,

Ministry of Public Health, Muang, Nonthaburi, Thailand 11000 4 Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart Universit, Bangkok, Thailand 10900

*Corresponding author E-mail: maruyama.soichi@nihon-u.ac.jp

Prevalence of Bartonella species by region and province.

Region Province No. examined No. (%) positive Bartonella species and No. of bacteremic animal

B. tribocorum B. rattimassiliensis B. elizabethae B. queenslandensis

Northern

Mae Hong Son 44 4 (9.1) 3 1 0 0

Chiang Rai 41 11 (28.6) 6 1 2 2

Nan 10 0 ND ND ND ND

subtotal 95 15 (15.8) 9 2 2 2

North-Eastern Loei 55 5 (9.1) 5 0 0 0

Eastern Sa Kaeo 113 23 (20.4) 13 0 8 2

Central Nonthaburi 48 0 ND ND ND ND

Southern

Nakhon Si Thammarat

18 3 (16.6) 0 3 0 0

Surat Thani 25 3 (12.0) 0 3 0 0

Phang Nga 21 9 (42.9) 0 9 0 0

subtotal 112 15 (13.4) 0 15 0 0

Total 375 58 (15.5) 27 17 10 4

ND: not detected

(Kosoy M, Bai Y, Sheff K, Morway C, Baggett H, Maloney SA, et al.Identification of Bartonella infections in febrile human patients fromThailand and their potential animal reservoirs. Am J Trop Med Hyg2010;82(6):1140–5.)

• B. elizabethae DNA has been detected in the blood of febrile patients in Chiang Rai and Khon Kaen provinces inThailand

• the DNA related to B. tri-bocorum and B. rattimassiliensis has been detected in febrile human patients in Thailand

Province No.

examined

No.

(%)

positive

Number of animals infected with Bartonella species/(Type of animals infected with Bartonella species)

B.

coopersplainsensis B. elizabethae B. henselae B. queenslandensis B. rattimassiliensis B. tribocorum

new B. species

A

new B. species

B

new B. species

C

Khon Kaen 369 8(2.2) 0 0 0 3/(Rr=1,Bi=2) 2/(Bi=1,Rr=1) 3/(Mm=3) 0 0 0 Nakhon Phanom 45 14(35.0) 0 0 0 0 3/(Rr=3) 11/(Re=8,Rn=1,Rr=2) 0 0 0

Tak 41 1(2.5) 0 0 0 0 0 1/(Bi=1) 0 0 0

Chon Buri 40 5(12.5) 2/(Bi=1,Rr=1) 0 0 0 2/(Rr=2) 0 1/(Rr=1) 0 0

Chanthaburi 148 25(16.9) 4/(Rr=4) 0 0 2/(Rr=2) 8/(Rr=8) 2/(Rr=2) 5/(Rr=5) 4/(Rr=4) 0

Ranong 114 40(35.1) 0 10/(Rn=8,Rr=2) 2/(Rn=1,Rr=1) 10/(Rn=8,Rr=2) 3/(Rn=2,Rr=1) 10/(Rn=8,Rr=2) 1/(Rr=1) 0 4/(Sm=4)

Phuket 39 3(7.7) 0 0 0 0 2/(Rr=2) 0 0 1/(Rr=1) 0

Songkhla 37 2(5.4) 0 0 0 0 0 0 1/(Rt=1) 0 1/(Sm=1) Satun 27 1(3.7) 0 0 0 0 0 0 0 1/(Rt=1) 0

Total 860 99(11.5) 6(6.1) 10(10.1) 2(2.0) 15(15.2) 20(20.2) 27(27.3) 8(8.1) 6(6.1) 5(5.1)

Prevalence of “Bartonella siamensis sp. novel ” isolates from deer

(Rusa timorensis) in Thailand

Decha Pangjai1, Santaya Intachinda2, Soichi Maruyama3*,Sumalee Boonmar4, Hidenori Kabeya3, Wimol Petkanchanapong1,

Wattanapong Wootta1, Piyada Wangroongsarb1,

Maskiet Boonyareth1, Poom Preedakoon1,

Watcharee Saisongkorh1 and Pathom Sawanpanyalert1

1 National Institute of Health ,Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand 11000 2 Nongkwang Livestock Breeding and Research Center, 128, Moo 10, Tambol Khaochangum, Photharam District, Ratchaburi,

Thailand 70120 3 Laboratory of Veterinary Public Health, College of Bioresource Sciences, Nihon University Fujisawa, Kanagawa 252-0880, Japan 4 International Emerging Infections Program (IEIP) Thailand MOPH-U.S. CDC Collaboration (TUC) Dept. of Medical Sciences, Ministry of

Public Health, Muang, Nonthaburi, Thailand 11000

*Corresponding author E-mail: maruyama.soichi@nihon-u.ac.jp

Neighbor-Joining tree based on concatenated 7 gene sequences .The phylogenetic tree was created using the Kimura 2-parameter model. The tree was rooted by using Brucella melitensis as the out group. Bootstrap values resulting from 1,000 bootstrap trials are indicated for each branch. Bar, 0.01 estimated nucleotide substitutions per site.

GenBank accession numbers / The percentage genetic similarity of the concatenated sequences from Bartonella siamensis sp. nov. compared with other reference Bartonella spp.

GenBank accession numbers/Percent genetic similarity to reference Bartonella spp.

rpoB gltA ftsZ groEL ribC 16S rRNA ITS Concatenated

B. siamensis

sp. nov.

JQ765388

91.1 - 98.4

JQ765385

82.6 - 97.1

JQ765384

85.4 - 97.9

JQ765386

82.9 - 97.7

JQ765387

77.1 - 96.1

JX006076

97.8 - 99.8

JX006075

42.2 - 92.8

-

84.0-97.4

Q fever is belonging to

the Legionellales order, Coxiellaceae family

Q fever has been in a wide variety of other animals, including other species of livestock and in domesticated pets.

Humans are often very susceptible to the disease

very few organisms may be required to cause infection.

Introduction

World Health Organization (WHO)

Food and Agriculture Organization of the United Nations(FAO)

World Organisation for Animal Health (OIE)

European Centre for Disease Prevention and Control (ECDC)

include Q fever among

‘emerging infectious disease’

Transmission model for Q fever

Hendrik-Jan Roest, 2013

Introduction

Goat

Q fever natural history in the absence of treatment

C. burnetii is considered a biological-threat agent by its very low infectious dose and high transmissibility. It is listed as a group B bioterrorism agent by the Centres of Disease Control and Prevention in the USA .

For this reason, C. burnetii is considered a containment level 3

Most of these techniques are limited in their sensitivity and rely on enrichment of the bacterium by culture before it can be genotyped.

C. burnetii culture is mostly performed by the use of tissue culture under biosafety level 3 conditions,

In addition, culturing of C. burnetii is bound by strict government regulations due to its infectious potential and status as a category B biowarfare agent

Rationale / Background / Review literature

Results of several seroprevalence and molecular studies in Thailand.

Since 1966. C. burnetii infection has been widespread in Thailand both in human and animals (goats, sheep, and cattle).

The prevalence in asymptomatic persons varies from 0.4% to 2.6% , and studies indomestic animals show that the highest prevalence of this infection occurs in dogs (28.1%).

The prevalence in goats, sheep, and cattle varies from 2.3% to 6.1% using the complement fixation test.

(Sangkasuwan et al., 1966)

Epidemiological aspect:

Study of Q fever in Thailand

In 2003 .A case series of acute Q fever was diagnosed and reported in a prospective study in patients with acute febrile illness

A total of 1,171 serum specimens from 678 patients were tested for Q fever by the microimmunofluorescent antibody.

Nine patients (1.3%) fulfilled the diagnosis of acute Q fever.

These data confirmed that Q fever is widespread in

this country. (Suputtamongkol et al., 2003) Yupin

Epidemiological aspect: Study of Q fever in Thailand

• In 2012.Two cases of fatal endocarditis in Khon Kaen Province in northeastern Thailand were found to be caused by C. burnetii confirmed by Serology, PCR and immunohistochemistry diagnostic.

(Pachirat et al., 2012)

• In 2012. report a Q fever surveillance of normal placentas (Beef cattle, Dairy cattle, Goat, Buffalo ) indicate a high frequency of C. burnetii infections which match locations where fatal human cases of endocarditis have occurred in Thailand analyzed by PCR for IS1111

(Yingst et al., 2013)

Seroprevalence of Coxiella burnetii on Goat Farms of

Chiang Rai, Kanchanaburi and Nakorn Pathom Province,

Thailand, 2014-2015

Decha Pangjai1,5, Leenawat Sukkasem2, Panupong Mahaprom3, Gilbert J. Kersh4, Maskiet Boonyareth1, Piyada Wangroongsarb1 and

Theera Rukkwamsuk5*

*Corresponding author

Objective

•To determine seroprevalence of Coxiella burnetii Infection in Goats Raised in Chiang Rai , Kanchanaburi and Nakornpathom Province by the indirect immunofluorescent antibody test (IFA)

Coxiella burnetii Phase 1 and Phase 2

Nine Mile

Phase 1

Nine Mile

Phase 2

Growth, pathology

Limited

growth and pathology

Q fever IFA

Q Fever-IFA

Drop Goat IgG-FITC ( 5 l )

Mount slide wit glycerol buffer

FA Microscope

Incubate 37 0 C 30 min Wash slide with PBS 3x5 min

Drop diluted serum (1: 16 -1:2,048) ( 5 l )

Incubate 37 0 C 30 min Wash slide with PBS 3x5 min

Negative : no bright green coccobacilli

Positive :

bright green coccobacilli

+ positive negative

Diagnosis

Province District Positive Total %

Kanchanaburi Bo Phloi 25 198 12.6

Dan Makham Tia 3 68 4.4

Mueang 37 210 17.6

Phanom Thuan 28 110 25.5

Tha Muang 30 140 21.4

Tha Maka 5 50 10.0

Sai Yok 1 48 2.1

Nakornpathom Kamphaeng Saen 23 190 12.1

Chiang Rai Mueang 1 23 4.13

Mae Fa Luang 5 199 2.5

Total 158 1236 12.8

Table 2 Seroprevalence against C. burnetii in 3 provinces of Thailand Result

Province Positive Total %

Kanchanaburi 129 824 16.7

Nakornpathom 23 190 12.1

Chiang Rai 6 222 2.7

Total 158 1236 12.8

Result

Kanchanaburi>Chiang Rai Nakornpathom> Chiang Rai (0.000) nominal significance level:P<0.05;

Bonferroni correction for significance was calculated as P<0. 016.

Province Positive Herd %

Kanchanaburi 48 76 63.2

Nakornpathom 12 22 54.5

Chiang Rai 2 2 100

Total 62 100 62.0

Result

Discussion •High seropositivities of infection would imply that meat goats were an effective source of C. burnetii.

• Intensive surveillance of Q fever in meat goats should be rapidly implemented. Comparison with caution

• Study population

• Diagnostic tests [type and performance]

• Study period

Discussion

• This study showed the seropositive of C. burnetii infection would imply that goats were an effective source of C. burnetii in Thailand.

• Further studies focusing on the burden and risk factors of C.burnetii infection among animals and humans should be implemented to prevent the occurrence of the disease in Thailand.

Q fever Serology test in serum from Brucelosis suspect samples (Rachaburi province 55 samples)

- Screening test by ELISA - Antibody titer by IFA found 9 samples were positive

Lab No.

IFA Phase I IFA Phase II

IgM IgG IgM IgG

13-56-10227 <1:16 1:512 <1:16 1:64

13-56-10229 <1:16 1:64 <1:16 <1:16

13-56-10237 <1:16 1:32 <1:16 1:16

13-56-10240 <1:16 1:512 <1:16 1:512

13-56-10242 <1:16 1:2048 <1:16 1:1024

13-56-10262 <1:16 1:128 <1:16 <1:16

13-56-10263 <1:16 1:2048 <1:16 <1:16

13-56-10267 <1:16 1:512 <1:16 > 1:2048

13-56-10276 <1:16 1:1024 <1:16 1:128

Q fever Serology - Antibody titer by IFA in Dog, Cat, Rodent from

Phuket, Ranong, Songkla, Satune ,

Nakhon Phanom Province

all samples were negative

• In phylogenetic analyses, which are important for

epidemiological investigations, 2 methods based on PCR

and sequencing are used:

Multilocus Variable Number Tandem Repeats Analysis and Multi Spacer Typing. MLVA is an improved Variable Number Tandem Repeats (VNTR) method, in which series of tandem repeats are flanked by designed primers and multiplied by PCR . Tandem repeats can be located in different parts of the genome, and also occur in many copies .

MLVA has been successfully used for the molecular typing of C. burnetii, which is especially important in epidemiological investigations.

Molecular diagnostics – PCR method

• Another molecular method allowing determination of similarities between C. burnetii strains is MST, which is based on the sequencing of intergenic spacers, since in the spacer regions a single nucleotide polymorphism or insertion/deletion mutations can occur randomly.

• The profiles obtained by this method can be easily compared using web sites, where 34 different MST profiles have been already described (see: http://ifr48.timone.univ-mrs.fr/

MST_Coxiella/mst).

Molecular diagnostics – PCR method

Glazunova O, Roux V, Freylikman O, Sekeyova Z, Fournous G, Tyczka J, Tokarevich N, Kovacova E, Marrie TJ, Raoult D: Coxiella burnetii genotyping. Emerg Infect Dis 2005, 11:1211–1217.

Glazunova O, Roux V, Freylikman O, Sekeyova Z, Fournous G, Tyczka J, Tokarevich N, Kovacova E, Marrie TJ, Raoult D: Coxiella burnetii genotyping. Emerg Infect Dis 2005, 11:1211–1217.

Thailand Cow’s Placenta Found Type 20 and 18/25 Using SNIP genotyping (Department of Livestock Development)

However, thorough genotyping data to provide epidemiological insight Thailand are still missing. A new typing techniques is single-nucleotide-polymorphism (SNP)-based typing. SNP methods are rapid, sensitive, easy to perform, and unambiguous in result interpretation and might be well-suited for genotyping of C. burnetii in an outbreak setting.

Therefore, we try to set up and develop an SNP genotyping assay for C. burnetii directly applicable to human and animal samples.

Thank you for your attention

โรคบารโตเนลโลสส (Bartonellosis) • Bartonella henselae ท ำใหเกดโรคแมวขวน (Cat-scratch disease), Bacillary

angiomatosis และ Peliosis hepatitis

• Bartonella quintana ท ำใหเกดไขเทรนช (Trench fever) และ Bacillary angiomatosis

• Bartonella bacilliformis ท ำใหเกดโรคคำรเรยน (Carrión's disease)

คนตดเชอบารโตเนลลาโดยผานการกดของแมลงพาหะ ไดแกพวกเหบ ไร หมด และแมว อาการสวนใหญไมรนแรง บอยครงทหายเองได

(Cat-scratch disease) ตมนนแดงหรอตมหนอง ขนาด 3-5 มม. จะเกดตรงต าแหนงทถกแมวทมเชอขวนหรอขบหลงจากนนประมาณ 3-10 วน แลวจะคอย ๆ หายไปเองใน 1-3 สปดาหตอมา แตจะมตอมน าเหลองบรเวณนนโตและกดเจบเกดขนแทน ผปวยสวนใหญไมคอยรสกวามไขหรอมอาการรวมอยางอน ตอมน าเหลองทโตและกดเจบนจะอยนาน 2-4 เดอน แลวจะคอย ๆ ยบไปเองอกเชนกน นอยรายมากทจะเกดภาวะแทรกซอนของระบบอน เชน เยอบหวใจอกเสบ สมองอกเสบ หนวยไตอกเสบ กระดกตดเชอ เมดเลอดแดงแตก ปอดบวม หรอตาแดงขางหนงรวมกบตอมน าเหลองหนาหขางนนโต (Parinaud’s oculo-glandular syndrome)

โรค Bacillary angiomatosis และ Peliosis hepatitis

ทงสองโรคนมกเกดรวมกนในคนไขเอดส หลงถกแมลงทมเชอกดจะมรอยโรคทผวหนงรวมกบอำกำรของตบอกเสบ รอยโรคทผวหนงมลกษณะคลำย Kaposi sarcoma คอเปนตมนน สแดงเขม ภำยในมกลมของหลอดเลอดฝอย ผ ปวยจะมไขหนำวสน ปวดกระดก ปวดทอง อำเจยน ตวเหลองตำเหลอง ตอมน ำเหลองและตบมำมโต ตรวจกำรท ำงำนของตบพบวำเอนไซม transaminases สงไมมำก แต Alkaline phosphatase จะสงมำก (5-10 เทำของคำปกต) อำกำรไขและปวดทองจะเปนอยประมำณ 1-8 สปดำห แตรอยโรคทผวหนงยงคงอยตอไปอกหลำยเดอน

Bartonella henselae is a proteobacterium that can cause bacteremia, endocaditis, bacillary angiomatosis, and peliosis hepatis. It is also the causative agent of cat-scratch disease (Bartonellosis) which, as the name suggests, occurs after a cat bite or scratch. The disease is characterized by lymphadenopathy(swelling of the lymph lymph nodes) and ferver.

• Papulopustular lesions of a primary inoculation site on the hand of a 16-year-old patient. These lesions had been present for approximately 3 weeks. A cat scratch antigen skin test was positive with 15-mm induration. No treatment was administered, and her condition resolved spontaneously in 2.5 months. Courtesy of Andrew Margileth, MD.

• A crusted primary inoculation papule on the neck of a 4-year-old child. Note the adjacent lymphadenitis. This patient had contact with cats and had multiple scratches. Courtesy of Andrew Margileth, MD.

• This 13-year-old girl developed fatigue and malaise after being licked and scratched by a cat. The typical conjunctival granuloma was accompanied by a parotid mass and intraparotid adenitis. No treatment was administered, and all her signs and symptoms resolved in 3 months. Courtesy of Andrew Margileth, MD.

• This 9-year-old boy developed cat scratch disease (CSD) encephalitis and a papular pruritic dermatitis after sustaining cat scratches and developing regional lymphadenitis. He was in a coma for 4 days but experienced a complete and rapid recovery within 3 weeks. Biopsy of the skin rash revealed nonspecific changes. The CSD antigen skin test result was positive. Courtesy of Andrew Margileth, MD.

• This 2.5-year-old boy was recovering from cat scratch disease acquired 10 months before when he developed this neck abscess over a period of 3 weeks. Biopsy revealed caseating granulomas; acid-fast bacillus and Warthin-Starry stain results were negative. Courtesy of Andrew Margileth, MD.

Trench fever

โรคนจะคลำยโรคมำลำเรยและไขกลบซ ำ แตไมมกำรแตกของเมดเลอดแดง อำกำรไขจะเรมหลงถกหมดทมเชอ Bartonella quintana กดประมำณ 2 สปดำห โดยจะมไขสงเกดขนทนททนใดในวนแรก ปวดศรษะมำก ตำแดง ปวดเนอตว ปวดตำมขอ และปวดขำ จำกนนในวนทสองและสำมอำจมไขขนบำงไมสงมำก เวนไปอกสองวนกจะกลบมำมไขสงฉบพลนเชนเดยวกบวนแรก ไขจะมำเปนชด ๆ แบบน ชดละ 5 วน ชำวบำนจงเรยกโรคนวำ "five-day fever" หรอ "quintan fever" ชวงทมไขกจะปวดศรษะ ตำแดง ปวดเนอตวมำกดงกลำว วนเวยนอยอยำงนประมำณ 2-6 สปดำหกจะหำยไปเอง

Carrión's disease อำกำรจะม 2 ระยะคอ

1.ระยะเฉยบพลน (Oroya fever) หลงถกแมลงประเภท sand fly ทมเชอ Bartonella bacilliformis กดประมำณ 3-12 สปดำห ผ ปวยจะมไขสงฉบพลน ซด ตำเหลอง ตอมน ำเหลองโต ตบมำมโต และทรดลงอยำงรวดเรวจำกภำวะเมดเลอดแดงแตก (severe hemolytic anemia) บำงรำยจะมอำกำรทำงสมองดวย โดยจะปวดศรษะ หำยใจเรว ซมลง และชก กวำรอยละ 40 จะเสยชวต จดเปนภำวะทรำยแรงทสดของโรคกลมบำรโตเนลโลสส

Carrión's disease • 2. ระยะเรอรง (Verruga Peruana) จะพบเฉพำะในผทรอดตำยโดยไมไดรบกำรรกษำ หลงจำกฟนไขหลำยสปดำหจะมตมเกดขนทผวหนง ขนำดตำง ๆ กน ภำยในเปนกลมของหลอดเลอดฝอยทรวมตวกน เมอโตขนจะแตกออก มเลอดไหล แลวแผลจะหำยเอง วนเวยนกนอยำงน ผ ปวยสวนใหญยงคงมภำวะซดและตบมำมโตตอมำจำกระยะเฉยบพลน และอำจมไขกลบมำใหมได

M. Maurin and D. Raoult

Clin. Microbiol. Rev. 1999, 12(4):518.

Q fever

is a zoonotic disease

the microorganism was given the name C burnetii in 1948 in honor of Dr Cox and Dr Burnet,

A small obligate intracellular gram-negative bacterium that is distributed globally.

Cattle, sheep, and goats are the primary reservoirs of C. burnetii.

C. burnetii is a member of the family of the Coxiellaceae bacteria and replicates intracellularly in cells of different species.

Phylogenetically related bacteria include Legionellaceae, Francisellaceae, Pseudomonaceae, and other Gammaproteobacteria.

Coxiella are small Gram-negative, pleomorphic, coccoidbacteria with a size of 0.2–1.0 µm.

They occur in 3 different forms:

-small cells (small cell variant, SCV) which are highly infectious

- large cells (large cell variant, LCV) which develop also in cell culture

- spore-like particles (SLP) which are infectious and very robust to environmental conditions.

Dependent on the host system, Coxiella undergoes a phase variation during growth .

In mammalian cells, bacteria grow as LCV, and form spore-like particles and 2 different antigenic forms described as Phase I and II.

LCV (Large Cell Variant) and SCV (Small Cell Variant) -Small Cell Variant is characterized by exceptional resistance to physical (e.g. ultraviolet radiation) and chemical factors. In dried milk - 30 days In urine 49 days In the dust about 120 days In the Dermacentor andersoni faeces for up to 580

days. In wool stored at 4–6 oC12–16 months, In soil up to 5 months

Q fever in humans and animals, caused by C. burnetii, is found worldwide.

In humans, it causes a variety of diseases such as acute flulike illness, pneumonia, hepatitis, and chronic endocarditis.

In animals, C. burnetii is found in the reproductive system, both uterus and mammary glands and may cause abortion or infertility

Cat scratch disease (CSD), Bartonella henselae •Low-grade fever may be present •Enlarged, tender lymph nodes that develop 1–3 weeks after exposure •A papule or pustule at the inoculation site Rarely, unusual manifestations such as eye infections, severe muscle pain, or encephalitis may occur.

Introduction

• Sheep and goats are identified as the source in majority of outbreaks

• Recently the largest reported Q fever outbreak in Netherlands. 2007-2010

• This outbreak was linked to dairy goats

Centers for disease control and prevention, CDC, 2013

Woldehiwet, 2004

Hendrik-Jan Roest, 2013

Introduction • The most important risk factor for human Q fever is living

close (<5 km) to an infected dairy goat farm.

• In pregnant goats: target cells are trophoblasts of allantochorion Van der Hoek et al., 2012

• Infected goat: abortion, weak kids

• The bacteria have been detected in feces, vaginal mucus and milk. Abortion/ non-abortion goat CDC, 2013

The infection may occur as a result of direct animal-animal,animal-human or human-animal contact

Horizontal human-human infections occur very rarely.

Isolation of bacteria

-The frozen blood samples were thawed at room

temperature and centrifuged at 1,800 g for 60 min. After centrifugation, an aliquot of 100 µl sample of each was plated on brain heart infusion agar (BHIA: Difco, MI) plates containing 5% defibrinated rabbit blood (www.nlac.mahidol.ac.th).

-The plates were incubated at 35°C under 5% CO2 for 2-4 weeks. Small, rough gray colonies that required long culture periods (1 week or more) were selected.

- For pure culture, two or three selected colonies were picked from each plate, streaked out on new plates, and further cultured under the same conditions. (Inoue et al., 2008)

Titer Kanchanaburi Nakornpathom Chiang Rai Total %

<1:16 695 167 216 1078 87.2

1:16 7 0 0 7 0.6

1:32 7 3 0 10 0.8

1:64 19 1 2 22 1.8

1:128 20 4 1 25 2.0

1:256 21 3 0 24 1.9

1:512 19 2 0 21 1.7

1:1024 6 2 1 9 0.7

> 1:2048 30 8 2 40 3.2

Total 1824 190 222 1236

Table 1 Antibody titers against C. burnetii in 3

provinces of Thailand

Result

Sex Positive Total %

Female 136 1052 12.9

Male 22 184 11.9

Total 158 1236 12.8

Result

No significance between Female and Male

(0.716) nominal significance level:P<0.05;

Bonferroni correction for significance was

calculated as P<0. 05

Type Positive Total %

Diary 0 16 0.0

Meat 156 1144 13.6

Meat/Dairy 2 76 2.6

Total 158 1236 12.8

Result

Meat>Meat/Dairy (0.006) nominal significance level:P<0.05;

Bonferroni correction for significance was

calculated as P<0. 016

Fig. The 6 genogroups of C. burnetii and the MST genotypes assigned to these groups. Genogroup VI contains the so-called Dugway strain. The phylogenetic tree shows the close relationship of individual MST genotypes which have partly developed by convergent evolution. In this analysis, MST 14 and 15 are identical in Group II. Graphic modelled on Hornstra et al., 2011 (L. Guertler).

• C. burnetii strains isolated from humans with ‘acute’ symptoms, in most cases belong to I, II and III genomic group, while plasmids from IV and V genomic group were found in patients with ‘chronic’ symptoms.

• This correlation led to the hypothesis that plasmids encode specific virulence factors which can determine the different virulence levels of C. burnetii strains

Bartonella

• Human : B. bacilliformis, B. elizabethae, B. henselae, and Bartonella quintana

• Cat : B. henselae, B. clarridgeiae and B. kochleae

• Dog : B. henselae, B. vinsonii subsp. berkhoffii (Bvb) and B. henselae