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Rickettsia,Bartonellosis, Q Fever

Rickettsia,Bartonellosis, Q Fever

Rickettsia, Bartonellosis, Q

Decha Pangjai D.V.M. National Institute of Health, Department of Medical Sciences, Thailand What is a Rickettsial Disease? Genus 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 .

Bartonella Reservoir Accidental Vector or potential Current geographic Present spp. host vector* distribution zoonotic status Bartonella Humans No Pheblotomines (sand Andes (, Ecuador No bacilliformis flies) ,Colombia, Bolivia,Chile, ( verrucarum) Guatemala) B. quintana Humans No Human body lice Worldwide† No (Pediculus humanis corporis) B. henselae Cats (Felis catus) Humans, Worldwide† Yes dogs (Ctenocephalides felis) Ticks? B. clarridgeiae Cats (F. catus) Humans, Fleas (C. felis) Cosmopolitan‡ Yes dogs B. koehlerae Cats (F. catus) Unknown Fleas (C. felis) California, France No

B. vinsonii White-footed mice Humans Fleas? Ticks? United States (Midwest) Yes subsp (Peromyscus arupensis leucopus) B. vinsonii Coyotes (Canis Humans, Ticks? Cosmopolitan‡ Yes subsp latrans) dogs berkhoffii (Canis familiaris) B. vinsonii Meadow voles No Ear mites? (Trombicula Canada No subsp vinsonii (Microtus microti) pennsylvanicus) B. talpae Moles (Talpa No Unknown United Kingdom No europaea) B. peromysci Field mice Unknown Unknown United States No (Peromyscus spp) Bartonella Reservoir Accidental Vector or potential Current geographic Present spp. host vector* distribution zoonotic status B. birtlesii Wood mice Unknown Unknown France, United Kingdom No (Apodemus spp) B. grahamii Bank voles Humans Fleas? United Kingdom Yes (Clethrionomys glareolus) B. taylorii Wood mice Unknown Fleas? United Kingdom No (Apodemus spp) B. doshiae Meadow voles Unknown Unknown United Kingdom No (M agrestis) B. elizabethae Rats (Rattus Humans, Fleas Worldwide Yes norvegicus) dogs B. tribocorum Rats (R norvegicus) Unknown Unknown Cosmopolitan‡ No

B. alsatica Rabbits(Oryctolagus Unknown Fleas? Ticks? France No cuniculus) B. washoensis Ground squirrels Humans, Fleas? Ticks? United States (Western) Yes (Spermophilus dogs beecheyi) Bartonella sp Prairie dogs Unknown Fleas? United States (Western) No Unknown (Cynomys ludovicianus) Bartonella Reservoir Accidental Vector or potential Current geographic Present spp. host vector* distribution zoonotic status B. bovis Domestic cattle Unknown Biting flies? Ticks? Cosmopolitan‡ No (Bos taurus) B. capreoli Roe deer Unknown Biting flies? Ticks? Europe No (Capreolus capreolus) B. choenbuchensis Roe deer(C Unknown Biting flies? Ticks? Europe No capreolus)

B. chomelii Domestic cattle Unknown Biting flies? Ticks? France No (B. taurus) *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 (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 (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 : ; 11: negative control; 12 : 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 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: [email protected] Prevalence of Bartonella species by region and province.

Bartonella species and No. of bacteremic animal Region Province No. examined No. (%) positive B. tribocorum B. rattimassiliensis B. elizabethae B. queenslandensis

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

Chiang Rai 41 11 (28.6) 6 1 2 2 Northern 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

Nakhon Si 18 3 (16.6) 0 3 0 0 Thammarat

Southern 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 • 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

(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.)

No. No. Number of animals infected with Bartonella species/(Type of animals infected with Bartonella species) Province (%) examined B. new B. species new B. species new B. species positive B. elizabethae B. henselae B. queenslandensis B. rattimassiliensis B. tribocorum coopersplainsensis A B 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: [email protected] 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 JQ765388 JQ765385 JQ765384 JQ765386 JQ765387 JX006076 JX006075 - sp. nov. 91.1 - 98.4 82.6 - 97.1 85.4 - 97.9 82.9 - 97.7 77.1 - 96.1 97.8 - 99.8 42.2 - 92.8 84.0-97.4 . is belonging to the order, Coxiellaceae family Introduction . 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 .

. 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’

Introduction Transmission model for Q fever

Goat

Hendrik-Jan Roest, 2013 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 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 . . 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 , 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 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)

Q fever IFA

Coxiella burnetii Phase 1 and Phase 2

Nine Mile Growth, pathology Phase 1

Limited Nine Mile growth and pathology Phase 2 Q Fever-IFA

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

Incubate 37 0 C 30 min Wash slide with PBS 3x5 min Drop Goat IgG-FITC ( 5 l )

Incubate 37 0 C 30 min Wash slide with PBS 3x5 min Mount slide wit glycerol buffer + FA Microscope positive negative

Positive : Negative : bright green coccobacilli no bright green coccobacilli Diagnosis Result Table 2 Seroprevalence against C. burnetii in 3 provinces of Thailand

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 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 Kanchanaburi>Chiang Rai Nakornpathom> Chiang Rai (0.000) nominal significance level:P<0.05; Bonferroni correction for significance was calculated as P<0. 016. Result

Province Positive Herd % Kanchanaburi 48 76 63.2

Nakornpathom 12 22 54.5

Chiang Rai 2 2 100 Total 62 100 62.0

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

IFA Phase I IFA Phase II

Lab No. 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

Molecular diagnostics – PCR method

• 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). 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 โรคบาร์โตเนลโลสิส (Bartonellosis)

• Bartonella henselae ท ำให้เกิดโรคแมวข่วน (Cat-scratch disease), Bacillary angiomatosis และ Peliosis hepatitis • ท ำให้เกิดไข้เทรนช์ () และ Bacillary angiomatosis • ท ำให้เกิดโรคคำร์เรียน (Carrión's disease)

คนติดเช้ือบาร์โตเนลลาโดยผา่ นการกดั ของแมลงพาหะ ไดแ้ ก่พวกเห็บ ไร หมดั และแมว อาการส่วนใหญ่ไม่รุนแรง บ่อยคร้ังที่หายเองได ้

(Cat-scratch disease) ตุ่มนูนแดงหรือตุ่มหนอง ขนาด 3-5 มม. จะเกิดตรง ตา แหน่งที่ถูกแมวที่มีเช้ือข่วนหรือขบหลงั จากน้นั ประมาณ 3-10 วนั แลว้ จะค่อย ๆ หายไปเองใน 1-3 สปั ดาห์ต่อมา แต่จะมีต่อมน้า เหลืองบริเวณน้นั โตและกดเจบ็ เกิดข้ึน แทน ผปู้ ่วยส่วนใหญ่ไม่ค่อยรู้สึกวา่ มีไขห้ รือมีอาการร่วมอยา่ งอื่น ต่อมน้า เหลืองที่โต และกดเจบ็ น้ีจะอยนู่ าน 2-4 เดือน แลว้ จะค่อย ๆ ยบุ ไปเองอีกเช่นกนั นอ้ ยรายมากที่จะ เกิดภาวะแทรกซอ้ นของระบบอื่น เช่น เยอื่ บุหวั ใจอกั เสบ สมองอกั เสบ หน่วยไต อกั เสบ กระดูกติดเช้ือ เมด็ เลือดแดงแตก ปอดบวม หรือตาแดงขา้ งหน่ึงร่วมกบั ต่อม น้า เหลืองหนา้ หูขา้ งน้นั โต (Parinaud’s oculo-glandular syndrome) โรค Bacillary angiomatosis และ Peliosis hepatitis ทั้งสองโรคนี้มักเกิดร่วมกันในคนไข้เอดส์ หลังถูกแมลงที่มีเชื้อกัดจะมีรอยโรคที่ผิวหนังร่วมกับอำกำรของตับ อักเสบ รอยโรคที่ผิวหนังมีลักษณะคล้ำย Kaposi sarcoma คือเป็นตุ่มนูน สีแดงเข้ม ภำยในมีกลุ่มของ หลอดเลือดฝอย ผู้ป่วยจะมีไข้หนำวสั่น ปวดกระดูก ปวดท้อง อำเจียน ตัวเหลืองตำเหลือง ต่อมน ้ำเหลืองและ ตับม้ำมโต ตรวจกำรท ำงำนของตับพบว่ำเอ็นไซม์ transaminases สูงไม่มำก แต่ Alkaline phosphatase จะสูงมำก (5-10 เท่ำของค่ำปกติ) อำกำรไข้และปวดท้องจะเป็นอยู่ประมำณ 1-8 สัปดำห์ แต่รอยโรคที่ผิวหนังยังคงอยู่ต่อไปอีกหลำยเดือน

Bartonella henselae is a proteobacterium that can cause bacteremia, endocaditis, bacillary angiomatosis, and . 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 (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 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 ; acid-fast bacillus and Warthin-Starry stain results were negative. Courtesy of Andrew Margileth, MD. Trench fever

โรคนี้จะคล้ำยโรคมำลำเรียและไข้กลับซ ้ำ แต่ไม่มีกำรแตกของเม็ดเลือดแดง อำกำรไข้จะเริ่มหลังถูก หมัดที่มีเชื้อ Bartonella quintana กัดประมำณ 2 สัปดำห์ โดยจะมีไข้สูงเกิดขึ้นทันทีทันใดในวันแรก ปวดศีรษะมำก ตำแดง ปวดเนื้อตัว ปวดตำมข้อ และปวดขำ จำกนั้นในวันที่สองและสำมอำจมีไข้ขึ้นบ้ำงไม่สูง มำก เว้นไปอีกสองวันก็จะกลับมำมีไข้สูงฉับพลันเช่นเดียวกับวันแรก ไข้จะมำเป็นชุด ๆ แบบนี้ ชุดละ 5 วัน ชำวบ้ำนจึงเรียกโรคนี้ว่ำ "five-day fever" หรือ "quintan fever" ช่วงที่มีไข้ก็จะปวดศีรษะ ตำแดง ปวดเนื้อตัวมำกดังกล่ำว วนเวียนอยู่อย่ำงนี้ประมำณ 2-6 สัปดำห์ก็จะหำยไปเอง

Carrión's disease

อำกำรจะมี 2 ระยะคือ 1.ระยะเฉียบพลัน (Oroya fever) หลังถูกแมลงประเภท sand fly ที่มีเชื้อ Bartonella bacilliformis กัด ประมำณ 3-12 สัปดำห์ ผู้ป่วยจะมีไข้สูงฉับพลัน ซีด ตำเหลือง ต่อมน ้ำเหลืองโต ตับม้ำมโต และทรุดลงอย่ำงรวดเร็วจำกภำวะ เม็ดเลือดแดงแตก (severe ) บำง รำยจะมีอำกำรทำงสมองด้วย โดยจะปวดศีรษะ หำยใจเร็ว ซึมลง และชัก กว่ำร้อยละ 40 จะเสียชีวิต จัดเป็นภำวะที่ร้ำยแรงที่สุดของ โรคกลุ่มบำร์โตเนลโลสิส

Carrión's disease

• 2. ระยะเรื้อรัง (Verruga Peruana) จะพบเฉพำะในผู้ที่รอดตำยโดยไม่ได้รับกำรรักษำ หลังจำกฟื้นไข้ หลำยสัปดำห์จะมีตุ่มเกิดขึ้นที่ผิวหนัง ขนำดต่ำง ๆ กัน ภำยในเป็นกลุ่มของหลอดเลือดฝอยที่รวมตัวกัน เมื่อ โตขึ้นจะแตกออก มีเลือดไหล แล้วแผลจะหำยเอง วนเวียนกันอย่ำงนี้ ผู้ป่วยส่วนใหญ่ยังคงมีภำวะซีดและ ตับม้ำมโตต่อมำจำกระยะเฉียบพลัน และอำจมีไข้กลับมำใหม่ได้

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 and replicates intracellularly in cells of different species.

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

. 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, , 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)

Result Table 1 Antibody titers against C. burnetii in 3 provinces of Thailand

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

Result

Sex Positive Total % Female 136 1052 12.9 Male 22 184 11.9 Total 158 1236 12.8

No significance between Female and Male

(0.716) nominal significance level:P<0.05; Bonferroni correction for significance was calculated as P<0. 05 Result

Type Positive Total %

Diary 0 16 0.0

Meat 156 1144 13.6 Meat/Dairy 2 76 2.6

Total 158 1236 12.8

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

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