18thKhonKaen Veterinary Annual International Conference 2017

March 30th-31st, 2017 KhonKaen (Thailand)

“Safe food & Good practice for One Health”

DEADLINE for Abstract Submissions: February 28th, 2017

All accepted papers of the conference will be published in the conference proceeding.

KEYNOTE TOPICS

  • Foot and Mouth disease: Trading in Thailand and ASIA
  • DOPP 2.0 : Early Detection & Rapid Response for One Health
  • Food borne zoonosis in Bangladesh
  • Small animals:
  • Canine Glaucoma: Diagnosis and Treatment
  • Management of Lower Urinary Tract Obstruction
  • Post-op Management in Small Animal Orthopedic Patients
  • TreatmentModalities for Improving Outcome in Neuro-orthopedic Patients
  • Contraception and Hormonal Deficiency after Spaying
  • Ruminant DiseasesProduction
  • Para TB in Goats
  • Bovine Viral Diarrhea
  • TB in Swamp Buffalos
  • a key success for Dairy production
  • Swine Group: Diseases & Farm Regulations
  • Poultry Group: Research for Poultry Business & Field Practice for Poultry Production
  • Cholangiocarcinoma ResearchCenter, KhonKaen University: Advanced Research topics

REGISTRATION FEE:

Participants / Until February 28th, 2017 / After February 28th, 2017
International delegate / 3,000 Baht (US$ 100 ) / 3,500 Baht (US$ 115 )
Thai delegate / 2,000 Baht / 2,500 Baht
Alumni (Vet. KKU.) / 1,000 Baht / 1,500 Baht
Student / 500 Baht / 1,000 Baht

THEME & TOPICS: “Safe food & Good practice for One Health”

  • Animal Production, Medicine, Surgery, and Welfare
  • Exotic Pet Medicine
  • Emerging and re-emerging diseases
  • Food safety
  • Zoonosis
  • Animal welfare

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Prevalence of Chytrid Fungus (Batrachochytriumdendrobatidis) in Wild Amphibians at Nam Nao National Park, Thailand

Nuntita Ruksachat1, Kanit Chukanhom1*, Fanan Suksawat1, Michael G. Levy2

1Faculty of Veterinary Medicine, KhonKaen University, Muang, KhonKaen. Thailand, 40002

2College of Veterinary Medicine, North Carolina State University, USA

*Corresponding author Email:

Abstract

Objective To study the prevalence of Batrachochytriumdendrobatidisin wild amphibians at Nam Nao National park, Thailand

Materials and Methods Theprevalenct study of chytrid fungus (Batrachochytrimdendrobatidis) in wild amphibians was employed in 2013. The sampling site was in the buffer zone used by humans, domestic animals and wildlife at Nam Nao National Park, Thailand. One hundred and sixty-one wild amphibians of 3 families; Megophryidae, Microhylidae and Ranidae were collected by swabbing from amphibian skin. Real time Taqman PCR was used to detect Chytrid fungal DNA.

Results The real time Taqman PCR results of one hundred sixty-one samples were all negative for chytrid fungus.

Conclusion The study is useful and necessary for surveillance of chytrid fungus, the emerging infectious disease in wild amphibians.

Keywords: Chytrid fungus, Batrachochytrimdendrobatidis, Amphibian, Nam Nao National Park

Introduction

The globalization has affected on the biological of amphibians; for example, biological condition, metabolism, fertilization, habitat, and food sources. In addition, the climate changes are likely to affect the transmission of the emerging infectious diseases causing the amphibian population declines for all regions of the world [1]. Chytridiomycosis is the important infectious disease in amphibian caused by Chytrid fungus (Batrachochytriumdendrobatidis) [2]. The fungus originated in Africa and then found to be causing problems in Australia, North America, South America, Europe and Asia. The African clawed frogs (Xenopuslaevis) have been exported from Africa since 1930, which could be the beginning and spreading of the chytrid fungi to the other regions of the world [3]. The first detection of Chytrid fungi in Asia was from the native and imported amphibians in Japan, 2006[4]. In Southeast Asia [5] reported the chytrid fungal detection from native amphibians in the Peninsula Malaysia including 6 families and 47 species, the results showed 7.87% (10/127) of native amphibians were infected. In China, [6] detected chytrid fungus from wild and market amphibian in 10 provinces of China, the prevalence of fungal detection were 13.5% (89/659) in wild amphibians and 7.6% (157/2,075) in market amphibians. In Thailand, there was preliminary study on Chytrid fungus infection in Thailand in 60 years kept specimens from amphibians kept in museums. Histopathologic diagnosis was employed for 123 samples from 23 species of amphibians. However, the result was all negative [7]. During July 2009 to August 2010 [8] had done the molecular studyforchytrid fungus in quarantine amphibians and in wild amphibian at KhaoKheow Open Zoo, the resulting prevalence were 11.9% (20/168) in quarantined amphibians, but 492 samples of wild amphibians were negative.

Chytrid fungal infection can be diagnosed by histopathological examination from the carcasses of amphibians but not from the living amphibians. Therefore, it is not applicable for surveillance and monitoring. In this study, real-Time Taqman PCR method was used since it can detect the Chytrid fungal DNA at the first phase of infection in the amphibians infected with only one zoospore [9]. In Thailand, many amphibianspecies have been increasingly imported as food and also raised as exotic pets. This may lead to the dissemination of chytrid fungal infection in native species either raised in farms or in wild amphibians. Suitable climate and efficient factors in national parks in Thailand may facilitate the chytrid fungal growth. Therefore, the survey of chytrid fungal prevalence of in natural areas is very important for disease surveillance in wild amphibians.

Materials and Methods

Sampling site: It was the Nam Nao National Park in Phetchabun Province located in the lower Northern part of Thailand in contact the Northeastern part. The Northern and Northeastern parts of Thailand in this area are connected by the route number 12. Before sampling, 8 areas of Nam Nao National Park had been observed including Haew-Sai Waterfalls, HuaiSanamsai Point, Sammakhao Unit, Wildlife Observation Tower, Phrom Song Unit, PhromLaeng Unit, Fai Nao Unit and Nam Nao National Park Office. Nam Nao National Park Office area was chosen as the sampling site. This area has been exploited by humans and wildlife. There are the natural trail, the stream flowing through and the campsite for tourists. In addition, the height of this area is of 845 meters above sea level and the transportation is convenient that it is only 500 meters away from the route number 12. So it is suitable for the sample collection.

The sample collection: One hundred and sixty-one wild amphibians from 4 families including Megophryidae, Microhylidae, RanidaeandRhacophoridae were collected at Nam Noa National Park (Table1). The sampling was variable, not limited by age, gender, or weight. Hand nets were used to capture the amphibians in the sampling site with gentle catching, and non-powder gloves were used and had been changed every time before the next capturing begun. In addition, the swab test was done Hyatt methods as previously described [10]. According to Hyatt methods, cotton swabs were applied to 7 areas of amphibian skin including stomach, abdomen, both sides of hind legs on and toes by smearing for 5 times on each area. The first point was two sides of stomach followed by abdomen, the inside area of the hind legs and toes, respectively. Each amphibian was applied with a cotton swab soaked in the tube which contained absolute ethyl alcohol, and the tube was closed firmly for sending to laboratory, to be detected by real time Taqman. Subsequently, the examination and the data were recorded in the amphibian sampling forms. The photos were also taken, and the identification of amphibian species was done by following the manual of amphibians in Thailand [11].

Real time Taqman PCR

DNA extraction: DNA was extracted from swabs by PrepMan Ultra. Fifty μl of PrepMan Ultra was added to each sample along with 30-40 mg of Ziriconium/silica beads 0.5 mm diameter. The sample was homogenized for 45 sec in a bead-beater. After brief centrifugation (30 sec at 13000×g in microfuge) to settle all materials to the bottom of the tube, homogenization and centrifugation was repeated. The homogenized sample was immersed in a boiling water bath for 10 min, cooled for 2 min, then centrifuged at 13000×g for 3 min in a microfuge tube; 20 μl of supernatant was recovered and stored -80 ◦C.

TaqMan assay: The mater mix is prepared with primer 1 (Forward primer), primer 2 (Reverse primer) and the probe. Add 5 μl DNA at 1/10 dilution was included in the master mix of 1 well of the 3 triplicates. Detection system was set as the following programme: 50 ◦C for 2 minutes 1 cycle; 95◦C for 10 minutes 1cycle; 95 ◦C for 15 seconds, 60 ◦C for 1 minute; 45 cycles.

Interpretation of data: Positive results are the presence of specific amplicons, indicated by a characteristic amplification curve similar to the positive control with a CT value less than or equal to 39 (in all three wells). Negative results are defined by the absence of specific amplicons, indicated by no characteristic amplification curve and having a CT value greater than 41(in all three wells). Inconclusive result was the presence of specific amplification curves similar to the positive control but with a CT value between 39 and 41 or a low number of zoospore equivalents in only one or two wells. Such results necessitate a repeat of the assay.

Results

All samples were negative for chytrid fungus by real time Taqman PCR (Table 1 and Table 2).

Discussion

Chytridiomycosis has affected the amphibian population declines around the world due to the difficulty to determine and to cure. The diagnosis can be done mostly in late-stages of the diseases. The molecular biology method (real time Taqman PCR method) has been developed to diagnose the beginning stage of infection by only one zoospore that the examination could be done effectively and expeditiously. For this reason, the disease treatment, prevention and surveillance in amphibians were implemented effectively [12]. The diagnostic method which suggested by OIE applied in this research study was real-time Taqman PCR method in order to diagnose chytrid fungal infection, effectively. The growth of Chytrid fungus is well developed in low temperature about 17-25 degree Celsius and at pH 6-7 [12] which is closely to the amphibian study in the natural areas of Nam Nao National Park, Petchabun Province. The height above sea level of the National Park office area is 845 meters, and the average annual temperature is 25 degree Celsius. According to all negative results from 161 samples by real time TaqmanPCR,this is correlated with [7] studied 123 samples of amphibians which have been kept in museums in Thailand for more than 60 years. There was no chytrid fungal infection in all the samples either. [8] was no chytrid fungal infection in wild amphibians. The result also suggested that there was no chytrid fungal infection in natural areas. In addition, from the result in Chiang Dao wildlife reservation area, it was also found that there was no chytrid fungus infection in 661 samples of wild amphibians. Furthermore, the Department of national parks, wildlife and plant conservation also survey chytrids in PhuLuang wildlife reservation area and the area of Halabala wildlife reservation area. From the results, chythid fungus in wild amphibians was not found. Moreover, according to PimchanokSuwanthada et al. (the data has not been published yet) detected chytrid fungus in captive frog which raised in farms in KhonKaen province by real time Taqman PCR, and the result also showed that there was no chytrid fungal infection. According to the information discussed above, it can be concluded that there is no incidence of chytrid fungus infection in both wild amphibians and captive amphibians. Thus, it could be assumed that climate change is not the factor affecting the incidence of the disease in amphibians. However, [8] found 11.9% (20/ 168) of imported living amphibians kept in the quarantine area were positive by PCR method, and 2.0% (6/ 50) of dead amphibians were positive to chytrid fungal infection by histopathological examination. According to [12], it was discovered that the chytrid fungus could be examined in some amphibian species which lived in forests without clinical signs or death. However, it cannot be obviously explained how they become disease carrier in long period of time. This was correlated with [5] found chytrid fungus in native amphibians without clinical sign. It could be suggested that chytrid fungus was endemic pathogen in this region. The surveillance of chytrid fungus in wild amphibians should be implemented especially during breeding season which chytrid fungus infection causing the highest death rate of crawfish frog were found. However, the death rate was gradually decline in summer [14] .

Acknowledgements

We would like to thank Department of national parks, wildlife and plant conservation, all staffs of Nam Nao National Park for help us provide facilitation, assist and sampling collection of wild amphibians in this research.

References

1. Daszak P, Berger L, Cunningham A A, Hyatt AD, Green GE, Spease R.. Emerging infectious disease and amphibian population decline.Emerg Infect Dis.1999; 5:735-748.

2. Gervasi SS, Gondhalekar C, Blaustein AR. Physiological responses of amphibians after exposure to the fungal pathogen, Batrachochytriumdendrobatidis. Integr Comp Biol. 2012; 52: E64.

3. Weldon C, du Preez LH, Hyatt AD, Muller R, Spears R. Origin of the amphibian chytrid fungus. Emerg. Infect. Dis. 2004: 10:2100-2105.

4. Goka K, Yokoyama JUN, Une Y, Kuroki T, Suzuki K, Nakahara M, Kobayashi A, Inaba S, Mizutani T, Hyatt AD. Amphibian chytridiomycosis in Japan: distribution, haplotypes and possible route of entry into Japan. Mol Ecol. 2009; 18:4757-4774.

5. Savage AE, Grismer LL, Anuar S, Onn CK, Grismer JL, Quah E, Muin MA, Ahmad N, Lenker M, Zamudio KR. First record of Batrachochytriumdendrobatidisinfecting four frog families from Peninsular Malaysia. Eco Health. 2011; 8:121-128.

6. Bai CM, Liu X, Fisher MC, Garner TWJ, Li YM. Global and endemic Asian lineages of the emerging pathogenic fungus Batrachochytriumdendrobatidis widely infect amphibians in China. Divers Distrib. 2012;18:307-318.

7. McLeod DS, Sheridan JA, Jiraungkoorskul W, Khonsue W. A survey for chytrid fungus in Thai amphibians. Raffles Bull Zool. 2008; 56:199-204.

8. Sommanustweechai A, Pirarat N, Wongsakorn S, Sailasutra A, Siriaroonrat B, Kamolnorranart S. Risk of Thai frog to new emerging ‘chytridiomycosis’disease. 1st conference on Thai elephant and wildlife. 2010. p37. [In Thai]

9. Boyle DG. Boyle DB, Olsen V, Morgan JA, Hyatt AD. Rapid quantitative detection of chytridiomycosis (Batrachochytriumdendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis Aquat Organ. 2004; 60:141-148.

10. Hyatt AD, Boyle DG, Olsen V, Boyle DB, Berger L, Obendorf D, Dalton A, Kriger K, Hero M, Hines H, Phillott R, Campbell R, Marantelli G. Diagnotic assays and sampling protocols for the detection of Batrachochytriumdendrobatidis. Dis Aquat Org. 2007; 73:175-192

11. Chan-ard T. Handbook of Amphibians in Thailand. 2003. Dansutha press. Bangkok. 175 pp [In Thai]

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13. Piotrowski JS, Annis SL, Longcore JE.Physiology of Batrachochytriumdendrobatidis, a chytrid pathogen of amphibians.Mycologia.2004; 96:9-15.

14. Kinney VC, Heelmeyer JL, Pessier AP, LannooMj. Seasonal Pattern of BatrachochytriumdendrobatidisInfection and Motiality in Lithobatesareolatus: Afirrmation of Vredenburg’s “10,000 Zoospore Rule”. PLos One. 2011; 6(3): e16708.

Radiographic Finding and Surgical Removal of Partial Intestinal Obstruction Causing by a Large Plastic Bottle Cap in a Dog

Chalermkwan Nonthakotr1*, Piyasak Wipoosak1, Naruepon Kampa2

1Veterinary Teaching Hospital, Faculty of Veterinary Medicine, KhonKaen University.

2Department of Surgery any Gynecology, Faculty of Veterinary Medicine, KhonKaen University, KhonKaen 40002, Thailand

*Corresponding author Email:

Abstract

Case Description An eight-year-old male American Pit Bull Terrier weighting 19.3 kg was presented at Veterinary Teaching Hospital, KhonKaen University with a three-week history of periodic vomiting, lethargy, emaciation and non responsive to medical interventions.

Clinical Findings Radiographic finding: Lateral and ventrodorsal plain abdominal radiographs were obtained. The radiographs showed markedly dilated loops of small intestine with gas and heterogeneous soft tissue and small mineral opaque materials in the lumen, the largest diameter measuring was 0.8 cm. This finding likely indicated the obstruction of small intestine. However, no evidence of radiopaque foreign (FB) body was seen. The barium contrast study and ultrasonography were performed. The delay transit time of contrast study was noticed and there was suspicious of non-radiopaque material from the filling defect pattern. The FB cannot be found by ultrasonography. The blood samples were collected for hematology and clinical biochemical parameters at Veterinary Diagnostic Laboratory prior to surgery. All blood results were within normal range. Intestinal partial obstruction was suspected based on clinical parameters.

Treatment and Outcome Exploratory laparotomy was performed in order to assess the abnormality of gastrointestinal (GI) tract. Severe inflammation with thickening of jejunum and a foreign body were noted. A large plastic cap was removed surgically. Antimicrobials and supportive treatment were given postoperatively for two weeks. The dog recovered with no sign of vomiting and abdominal cramp, postoperatively.

Clinical Relevant GI obstruction is commonly found in dog which can be classified as partial or complete. Plain radiograph can be used as a useful tool to help the diagnosis of GI obstruction. The ileus sign (dilated of small intestine) is recognized as one of criteria of GI obstruction. The foreign body may not be found by ultrasonography if there is gas filled in GI tract. The exploration is recommended to confirm the finding in the suspected cases. In conclusion, suspected partial gastrointestinal tract obstruction, proper medical intervention prior to surgery is necessary. Surgical intervention should be considered very soon after successfully medical stabilization to attain an excellent outcome.

Keywords: Foreign bodies, Gastrointestinal tract, Periodic vomiting, Dog

SPEAKER NOTE Submission Guideline

A speaker note in English should be submitted only via official email to the 18th KVAC chairperson: . Any note sent by facsimile or air-mail will not be accepted. Please send the manuscript before 28th February, 2017