PAKET INFORMASI BIDANG PENYAKIT HEWAN

PERPUSTAKAAN BBLITVET
NO.1; 2016

DISUSUN OLEH :
Siti Kuraesin

BALAI BESAR PENELITIAN VETERINER

Jl. R.E. Martadinata No.30 Bogor
2016
 KATA PENGANTAR

Paket informasi perpustakaan BBLITVET adalah kumpulan data bibliografi dan

abstrak informasi bidang penyakit hewan, terdiri dari bidang bakteriologi, virologi,
toksikologi, parasitologi dan informasi lain-lain yang sedang menarik. Disusun berdasarkan
subyek dan disebarkan untuk meningkatkan daya guna serta memudahkan pemustaka dalam
memperoleh informasi yang dibutuhkan.
Jika diperlukan artikel lengkapnya, pemustaka dapat menghubungi perpustakaan
BBLITVET melalui email, telpon atau datang langsung.
Akhirnya, kepada semua pihak yang telah membantu disampaikan terimakasih.

Bogor, 30 Februari 2016

Penanggung Jawab Perpustakaan

Siti Kuraesin, S.IIP

NIP. 19741219 200112 2 002
 Paket Informasi Perpustakaan BBLITVET
No.1, 2016

Paket Informasi Bidang Bakteriologi

1. Usage of Leptospira spp. local strains as antigens increases the sensitivity of the serodiagnosis of
bovine leptospirosis. Priscila S. Pinto, Ana P. Loureiro, Bruno Penna, Walter Lilenbaum. Acta Tropica
149 (2015) 163–167

Abstract :
Poultry is the most frequent reservoir of non-typhoid Salmonella enterica for humans. Understanding the
interactions between chickens and S. enterica is therefore important for vaccine design and subsequent
decrease in the incidence of human salmonellosis. In this study we therefore characterized the
interactions between chickens and phoP, aroA, SPI1 and SPI2 mutants of S. Enteritidis. First we tested
the response of HD11 chicken macrophage-like cell line to S.Enteritidis infection monitoring the
transcription of 36 genes related to immune response. All the mutants and the wild type strain induced
inflammatory signaling in the HD11 cell line though the response to SPI1 mutant infection was different
from the rest of the mutants. When newly hatched chickens were inoculated, the phoP as well as the SPI1
mutant did not induce an expression of any of the tested genes in the cecum. Despite this, such chickens
were protected against challenge with wild-type S. Enteritidis. On the other hand, inoculation of chickens
with the aroA or SPI2 mutant induced expression of 27 and 18 genes, respectively, including genes
encoding immunoglobulins. Challenge of chickens inoculated with these two mutants resulted in repeated
induction of 11 and 13 tested genes, respectively, including the genes encoding immunoglobulins. In
conclusion, SPI1 and phoP mutants induced protective immunity without inducing an inflammatory
response and antibody production. Inoculation of chickens with the SPI2 and aroA mutants also led to
protective immunity but was associated with inflammation and antibody production. The differences in
interaction between the mutants and chicken host can be used for a more detailed understanding ofthe
chicken immune system.

2. Leptospirosis in Greece. Anna Papa, Tzimoula Kotrotsiou. Acta Tropica 149 (2015) 135–137

Abstract :
Leptospirosis is a zoonotic disease with increased public health concern worldwide. The disease occurs
more often in temperate and tropical regions. The present study analyzes the demographic,
epidemiological and clinical data of 168 leptospirosis cases laboratory diagnosed in northern Greece
during 1998–2014. Most patients were males, aged 50–69 years, working in animal husbandry or
farming. Caseswere observed more frequently in summer and autumn. Severity of the disease was
correlated with presence of pulmonary involvement and hemorrhagic manifestations.

3. Overexpression of the pleiotropic regulator CodY decreases sporulation, attachment and pellicle
formation in Bacillus anthracis. Monisha Gopalani , Alisha Dhiman , Amit Rahi , Rakesh Bhatnagar.
Biochemical and Biophysical Research Communications 469 (2016) 672e678
Abstract :
CodY, a global transcriptional regulator, primarily functions as a nutrient and energy sensor. It is
activated by metabolic effectors like BCAA and GTP. In low G þ C Gram positive bacteria, it facilitates
coupling of changes in the cellular metabolite pool with those required in the transcriptome of the cell.
This pleiotropic regulator controls the expression of a vast number of genes as the cell transits from
exponential to the stationary phase. Earlier studies have shown that CodY is required for the virulence of
Bacillus anthracis. We sought to investigate the effect of its overexpression on the physiology of B.
anthracis. In our study, we found that cellular CodY levels were unchanged during this phasetransition.
Expression of endogenous CodY remained the same in different nutrient limiting conditions.
Immunoblotting studies revealed CodY presence in the whole spore lysate of B. anthracis indicating it to
 be a component of the spore proteome. We could also detect CodY in the secretome of B. anthracis.
Further, CodY was overexpressed in B. anthracis Sterne strain and this led to a 100-fold decrease in the
sporulation titer and a 2.5-fold decrease in the in vitro attachment ability of the bacteria. We also
observed a decrease in the pellicle formation by CodY overexpressed strain when compared to wildtype
bacilli. The CodY overexpressed strain showed chaining phenotype during growth in liquid media and
pellicle.

4. Presentation of peptides from Bacillus anthracis protective antigenon Tobacco Mosaic Virus as an
epitope targeted anthrax vaccine. Ryan C. McComba, Chi-Lee Hob, Kenneth A. Bradleyb, Laurence K.
Grilla, Mikhail Martchenkoa, Vaccine 33 (2015) 6745–6751

Abstract :
The current anthrax vaccine requires improvements for rapidly invoking longer-lasting
neutralizingantibody responses with fewer doses from a well-defined formulation. Designing antigens
that targetneutralizing antibody epitopes of anthrax protective antigen, a component of anthrax toxin,
may offer asolution for achieving a vaccine that can induce strong and long lasting antibody responses
with fewerboosters. Here we report implementation of a strategy for developing epitope focused virus
nanoparticlevaccines against anthrax by using immunogenic virus particles to present peptides derived
from anthraxtoxin previously identified in (1) neutralizing antibody epitope mapping studies, (2) toxin
crystal struc-ture analyses to identify functional regions, and (3) toxin mutational analyses. We
successfully expressedtwo of three peptide epitopes from anthrax toxin that, in previous reports, bound
antibodies that werepartially neutralizing against toxin activity, discovered cross-reactivity between
vaccine constructs andtoxin specific antibodies raised in goats against native toxin and showed that
antibodies induced by ourvaccine constructs also cross-react with native toxin. While protection against
intoxication in cellular andanimal studies were not as effective as in previous studies, partial toxin
neutralization was observed inanimals, demonstrating the feasibility of using plant-virus nanoparticles as
a platform for epitope definedanthrax vaccines.

5. Neutralizing antibody and functional mapping of Bacillus anthracis protective antigen—The first
step toward a rationally designed anthrax vaccine. Ryan C. McComb, Mikhail Martchenko. Vaccine
34 (2016) 13–19

Abstract :
Anthrax is defined by the Centers for Disease Control and Prevention as a Category A pathogen for
its potential use as a bioweapon. Current prevention treatments include Anthrax Vaccine Adsorbed
(AVA). AVA is an undefined formulation of Bacillus anthracis culture supernatant adsorbed to aluminum
hydroxide. It has an onerous vaccination schedule, is slow and cumbersome to produce and is slightly
reactogenic. Next-generation vaccines are focused on producing recombinant forms of anthrax toxin in a
well-defined formulation but these vaccines have been shown to lose potency as they are stored. In
addition, studies have shown that a proportion of the antibody response against these vaccines is focused
on non-functional, non-neutralizing regions of the anthrax toxin while some essential functional regions
are shielded from eliciting an antibody response. Rational vaccinology is a developing field that focuses
on designing vaccine antigens based on structural information provided by neutralizing antibody epitope
mapping, crystal structure analysis, and functional mapping through amino acid mutations. This
information provides an opportunity to design antigens that target only functionally important and
conserved regions of a pathogen in order to make a more optimal vaccine product. This review provides
an overview of the literature related to functional and neutralizing antibody epitope mapping of the
Protective Antigen (PA) component of anthrax toxin.
6. Experimental Study of the Pathogenicity of Pasteurella multocida Capsular Type B in Rabbits.
S. Katoch, L. Verma, M. Sharma, R. K. Asrani†, S. Kumar, R. Chahota and S. Verma. J. Comp. Path.
2015, Vol. 153, 160e166

Summary :
The increased frequency of isolation of Pasteurella multocida capsular type B from rabbitries in north-
western India prompted this investigation into the role of this organism in inducing disease in rabbits. Ten
rabbits were divided into two groups of five animals. Group I rabbits were infected intranasally (IN) with
1 ml of inoculums containing 2_105 colony forming units/ml, while rabbits in group II were given 1ml
phosphate buffered saline IN. The rabbits in group I developed respiratory distress, increased rectal
temperature and severe dyspnoea, with death occurring 24e48 h post infection. The main pathological
findings were severe congestion and haemorrhage in the trachea, fibrinopurulent pneumonia, bacteraemia
and septicaemia. The nasal secretions of all group I animals contained P. multocida. These observations
indicate that in addition to P. multocida capsular types A and D, P. multocida capsular type B can also be
highly pathogenic for rabbits.

7. Recombinant expression of Bacillus anthracis lethal toxin components of Indian isolate in

Escherichia coli and determination of its acute toxicity level in mouse model. Suryanarayana
Nagendra, Vanlalhmuaka, Sarika Verma, Urmil Tuteja, Kulanthaivel Thavachelvam. Toxicon 108 (2015)
108e114

Abstract :
Bacillus anthracis lethal toxin (LeTx) is the principle factor responsible for toxaemia and anthrax related
death. Lethal toxin consist of two proteins viz protective antigen (PA) and lethal factor which combines
in a typical fashion similar to other toxins belonging to A-B toxin super family. The amount of LeTx
required to kill a particular organism generally differs among strains owing to their geographical
distributions and genetic variation. In the present study, we have cloned PA and LF genes from B.
anthracis clinical isolate of Indian origin and expressed them in soluble form employing Escherichia coli
expression system. Both the proteins were purified to near homogeneity level using Immobilized metal
ion affinity chromatography (IMAC). Further we have used equal ratio of both the proteins to form LeTx
and determined its acute toxicity level in Balb/c mice by graphical method of Miller and Tainter. The
LD50 value of LeTx by intravenous (i.v) route was found to be 0.97 ± 0.634 mg kg 1 Balb/c mice. This
study highlights the expression of recombinant LeTx from E. coli and assessing its acute toxicity level in
experimental mouse model.

8. Inhibition of Listeria monocytogenes in vitro and in goat milk by liposomal nanovesicles containing
bacteriocins produced by Lactobacillus sakei subsp. sakei 2a. Patrícia S. Malheiros, Iolanda M.
Cuccovia, Bernadette D.G.M. Franco. Food Control 63 (2016) 158e164

Abstract :
Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacteria isolated from a Brazilian
meat product, capable to inhibit Listeria monocytogenes in vitro and in foods. In this study, bacteriocin
produced by this strain were encapsulated in phosphatidylcholine (FC) and 1,2-dioleoyloxy-3-
trimethylammonium-propane (DOTAP) liposomes, separately and in combination, were characterized
and evaluated for activity against L. monocytogenes in vitro and in experimentally contaminated UHT
goat milk, during storage at 7 C for 14 days and 30 C for 24 h. The FC and DOTAP/FC (3:1)
nanovesicles containing bacteriocins (12.800 AU mL1) presented zeta potential of 1.54 and þ 38.13 mV,
Entrapment Efficiency of 80.0% and 94.1%, and diameter of 91.19 and 81.49 nm, respectively.
DOTAP/FC nanovesicle presented excellent stability, and maintained the same physicochemical
characteristics over 28 days. Both free and encapsulated bacteriocins controlled L. monocytogenes
growth in BHI medium and goat milk stored at 30 C for at least 8 h, in a similar pattern. After 24 h in
BHI medium, bacteriocins encapsulated in FC nanovesicles were more effective (p < 0.05) than free
bacteriocins. However, in goat milk, no significant differences (p 0.05) were observed for the two types
of nanovesicles. At 7 C, both free and encapsulated bacteriocins retarded the L. monocytogenes growth,
 and after 5 days, counts were 5 log lower than in the controls, both in BHI and in goat milk.
Encapsulation of bacteriocin in FC and DOTAP/FC nanovesicles did not affect the antimicrobial activity,
but the advantages of their application for control of L. monocytogenes in goat milk based dairy
products, when compared to free bacteriocins, remain unclear and more studies are needed.
9. Generation and characterization of recombinant bivalent fusionprotein r-Cpib for immunotherapy
against Clostridium perfringens betaand iota toxemia. Das Shreya, Saugata Majumder, Joseph J.
Kingston, Harsh V. Batra Molecular Immunology 70 (2016) 140–148

Abstract :
Clostridium perfringens beta (CPB) and iota (CPI) toxaemias result in some of the
most lethal forms ofhaemorrhagic and necrotic enteritis and sudden death
syndrome affecting especially neonates. WhileCPB enterotoxemia is one of the most
common forms of clostridial enterotoxemia, CPI enterotoxemiathough putatively
considered to be rare is an emerging cause of concern. The similarities in clinical
man-ifestation, gross and histopathology findings of both types of toxaemias
coupled to the infrequency of CPItoxaemia might lead to symptomatic
misidentification with Type C resulting in therapeutic failure due tohabitual
administration of CPB anti-toxin which is ineffective against CPI. Therefore in the
present study,to generate a composite anti-toxin capable of neutralizing both
toxaemias, a novel bivalent chimera r-Cpib was constructed by splicing the non-
toxic C terminal binding regions of CPB and CPI, via a flexibleglycine linker (G4S) by
overlap-extension PCR. The fusion protein was characterized for its
therapeuticabilities toward CPI and CPB toxin neutralizations. The r-Cpib was found
to be non-toxic and could com-petitively inhibit binding of CPB to host cell receptors
thereby reducing its cytotoxicity. Immunization ofmice with r-Cpib generated
specific antibodies capable of neutralizing the above toxaemias both in vitroand in
vivo. Caco-2 cells exposed to a mixture of anti-r-Cpib sera and native CPI or CPB,
displayed signif-icantly superior protection against the respective toxins while
passive challenge of mice with a similarmixture resulted in 83 and 91% protection
against CPI and CPB respectively. Alternatively, mice exposedto a mixture of sham
sera and native toxins died within 2–3 days. This work thus demonstrates r-Cpibas a
novel bivalent fusion protein capable of efficient immunotherapy against C.
perfringens CPI and CPBtoxaemia.
10. Markers of endothelial cell activation and immune activation are increased in patients with severe
leptospirosis and associated with disease severity. Marco Goeijenbier, M. Hussein Gasem, Joost C.M.
Meijers, Rudy A. Hartskeerl, Ahmed Ahmed, Marga G.A. Goris, Bambang Isbandrio, Simone S. Schuller,
Albert D.M.E. Osterhaus, Byron E.E. Martina, Eric C.M. van Gorp, Jarlath E. Nally, Jiri F.P. Wagenaar.
Journal of Infection (2015) 71, 437e446

Abstract :
Objectives: Previous studies concluded that haemorrhage is one of the most accurate prognostic factors
of mortality in leptospirosis. Therefore, endothelial cell activation was investigated in relation to disease
severity in severe leptospirosis. Methods: Prospective cohort study of severe leptospirosis patients.
Plasma levels of sEselectin and Von Willebrand factor (VWF) were determined. Consequently, an in
vitro.
11. Rapid detection of Bacillus anthracis by γ phage amplifcation and
lateralfow immunochromatography. Christopher R. Cox ,Kirk R. Jensen Roy R.
Mondesire , Kent J. Voorhees. Journal of Microbiological Methods 118 (2015) 51–56

Abstract :
New, rapid point-of-need diagnostic methods for Bacillus anthracis detection can enhance civil and
military responses to accidental or deliberate dispersal of anthrax as a biological weapon. Current
laboratory-based methods for clinical identification of B. anthracis require 12 to 120 h, and are confirmed
by plaque assay using thewell-characterized γ typing phage,which requires an additionalminimumof 24 h
for bacterial culture. To reduce testing time, the natural specificity of γ phage amplification was
investigated in combination with lateral flow immunochromatography (LFI) for rapid, point-of-need B.
anthracis detection. Phage-based LFI detection of B. anthracis Sterne was validated over a range of
bacterial and phage concentrations with optimal detection achieved in as little as 2 h from the onset of
amplification with a threshold sensitivity of 2.5 × 104 cfu/mL. The novel use of γ phage amplification
detected with a simple, inexpensive LFI assay provides a rapid, sensitive, highly accurate, and field-
deployable method for diagnostic ID of B. anthracis in a fraction of the time required by conventional
techniques, and without the need for extensive laboratory culture.

12. Animal models to study the pathogenesis of human and animal Clostridium perfringens infections
Francisco A Uzal, Bruce A McClane , Jackie K. Cheung, James Theoret, Jorge P. Garcia, Robert J.
Moore, Julian I. Rood. Veterinary Microbiology 179 (2015) 23–33

Abstract :
The most common animal models used to study Clostridium perfringens infections in humans and
animals are reviewed here. The classical C. perfringens-mediated histotoxic disease of humans is
clostridial myonecrosis or gas gangrene and the use of a mouse myonecrosis model coupled with genetic
studies has contributed greatly to our understanding of disease pathogenesis. Similarly, the use of a
chicken model has enhanced our understanding of type A-mediated necrotic enteritis in poultry and has
led to the identification of NetB as the primary toxin involved in disease. C. perfringens type A food
poisoning is a highly prevalent bacterial illness in the USA and elsewhere. Rabbits and mice are the
species most commonly used to study the action of enterotoxin, the causative toxin. Other animal models
used to study the effect of this toxin are rats, non-human primates, sheep and cattle. In rabbits and mice,
CPE produces severe necrosis of the small intestinal epithelium along with fluid accumulation. C.
perfringens type D infection has been studied by inoculating epsilon toxin (ETX) intravenously into
mice, rats, sheep, goats and cattle, and by intraduodenal inoculation of whole cultures of this
microorganism in mice, sheep, goats and cattle. Molecular Koch’s postulates have been fulfilled for
enterotoxigenic C. perfringens type A in rabbits and mice, for C. perfringens type A necrotic enteritis and
gas gangrene in chickens and mice, respectively, for C. perfringens type C in mice, rabbits and goats, and
for C. perfringens type D in mice, sheep and goats.

13. Binding of Clostridium perfringens to collagen correlates with the ability to cause necrotic enteritis
in chickens. B. Wadea, A.L. Keyburna, T. Seemann, J.I. Roodb, R.J. Moorea. Veterinary Microbiology
180 (2015) 299–303

Abstract :
This study investigated the ability of Clostridium perfringens isolates derived from chickens to bind to
collagen types I–V and gelatin. In total 21 strains from three distinct backgrounds were studied: (i)
virulent strains isolated from birds suffering from necrotic enteritis, (ii) avirulent strains isolated from
birds suffering from necrotic enteritis and (iii) strains isolated from healthy birds. All strains isolated
from diseased birds had been assessed for virulence in a disease induction model. The virulent isolates all
displayed collagen binding ability. However, most strains in the other two classes showed negligible
binding to collagen. The prevalence of a previously described C. perfringens putative collagen
dhesinencoding gene was investigated by PCR screening. It was found that
five of the strains carried the putative collagen adhesin-encoding gene and that all of these strains were
 virulent isolates. Based on these studies it is postulated that collagen adhesion may play a role in the
pathogenesis of necrotic enteritis. Crown

14. Technical Note: Simple, scalable, and sensitive protocol for retrieving Bacillus anthracis (and other
live bacteria) from heroin. Gregor Grass, Bjoern Ahrens , Uwe Schleenbecker, Linda Dobrzykowski ,
Matthias Wagner , Christian Kru¨ ger , Roman Wo¨lfel Forensic Science International 259 (2016) 32–35

Abstract :
We describe a culture-based method suitable for isolating Bacillus anthracis and other live bacteria from
heroin. This protocol was developed as a consequence of the bioforensic need to retrieve bacteria from
batches of the drug associated with cases of injectional anthrax among heroin-consumers in Europe. This
uncommon manifestation of infection with the notorious pathogen B. anthracis has resulted in 26 deaths
between the years 2000 to 2013. Thus far, no life disease agent has been isolated from heroin
duringforensic investigations surrounding these incidences. Because of the conjectured very small
number of disease-causing endospores in the contaminated drug it is likely that too few target sequences
are available for molecular genetic analysis. Therefore, a direct culture-based approach was chosen here.
Endospores of attenuated B. anthracis artificially spiked into heroin were successfully retrieved at 84-
98% recovery rates using a wash solution consisting of 0.5% Tween 20 in water. Using this approach, 82
samples of un-cut heroin originating from the German Federal Criminal Police Office’s heroin analysis
program seized during the period between 2000 and 2014 were tested and found to be surprisingly poor
in retrievable bacteria. Notably, while no B. anthracis was isolated from the drug batches, other bacteria
were successfully cultured. The resulting methodical protocol is therefore suitable for analyzing un-cut
heroin which can be anticipated to comprise the original microbiota from the drug’s original source
without interference from contaminations introduced by cutting.

15. Egg white versus Salmonella Enteritidis! A harsh medium meets a resilient
pathogen. Florence Baron , Françoise Nau , Catherine Gu_erin-Dubiard , Sylvie
Bonnassie , Michel Gautier , Simon C. Andrews, Sophie Jan. Food Microbiology 53
(2016) 82e93

Abstract :
Salmonella enterica serovar Enteritidis is the prevalent egg-product-related food-borne pathogen. The
egg-contamination capacity of S. Enteritidis includes its exceptional survival capability within the harsh
conditions provided by egg white. Egg white proteins, such as lysozyme and ovotransferrin, are well
known to play important roles in defence against bacterial invaders. Indeed, several additional minor
proteins and peptides have recently been found to play known or potential roles in protection against
bacterial contamination. However, although such antibacterial proteins are well studied, little is known
about their efficacy under the environmental conditions prevalent in egg white. Thus, the influence of
factors such as temperature, alkalinity, nutrient restriction, viscosity and cooperative interactions on the
activities of antibacterial proteins in egg white remains unclear. This review critically assesses the
available evidence on the antimicrobial components of egg white. In addition, mechanisms employed by
S. Enteritidis to resist egg white exposure are also considered along with various genetic studies that have
shed light upon egg white resistance systems.We also consider how multiple, antibacterial proteins
operate in association with specific environmental factors within egg white to generate a lethal protective
cocktail that reserves sterility.
16. Biofilm formation of Salmonella Enteritidis under food-related environmental stress conditions and
its subsequent resistance to chlorine treatment. Yishan Yang , Marta Mik_s-Krajnik , Qianwang Zheng
, Sang-Bong Lee , Seung-Cheol Lee , Hyun-Gyun Yuk Food Microbiology 54 (2016) 98e105

Abstract :
This study determined the effects of temperature (4 and 25 C), pH (5.3, 7.3, and 8.3), and nutrient
availability (TSB and 20 times diluted TSB (1/20 TSB)) on Salmonella Enteritidis biofilm formation and
its resistance to chlorine treatment (pH 6.8, 50 ppm for 1 min). The results showed that biofilm density
was significantly higher (P < 0.05) at 25 C or in 1/20 TSB, regardless of pH and bacterial strains.
Moreover, 1/ 20 TSB significantly enhanced the chlorine resistance of biofilms formed at 25 C,
especially for S. Enteritidis with rdar morphotype, with an average reduction of 1.52 log CFU/cm2
compared to that of biofilm in TSB with 4.07 log reduction. All biofilms formed at 4 C were very
sensitive to chlorine treatment. In most cases, acidic pH sensitized biofilms to chlorine treatment
compared with neutral and alkaline pHs. The further analysis of cellulose production of biofilms
indicated that it had a positive impact on biofilm resistance to chlorine treatment. This study suggests that
environmental stress conditions encountered in food processing plant might alter S. Enteritidis biofilm
resistance to sanitizertreatment possibly by acting on the cellulose production

17. Physiology, pathogenicity and immunogenicity of live, attenuated Salmonella enterica serovar
Enteritidis mutants in chicks. Wei Si, Xiumei Wang, Huifang Liu, Shenye Yu, Zhaoli Li, Liping Chen,
Wanjiang Zhang, Siguo Liu. Microbial Pathogenesis 83-84 (2015) 6e11

Abstract :
To construct a novel live, attenuated Salmonella vaccine, the lon, cpxR and cpdB genes were deleted
from a wild-type Salmonella enterica serovar Enteritidis-6 (SM-6) strain using the phage l Red
homologous recombination system, resulting in SM-△CpxR, SM-△C/Lon and SM-△C/L/CpdB. The
growth curves of strain SM-△C/Lon grew more rapidly than the other strains and had OD 600 values
higher than the other strains starting at the 4 h time point. The growth curves of strain SM-△C/L/CpdB
were relatively flat. The colonization time of SM-△C/L/CpdB is about 8e10 days. Deleting the
lon/cpxR/cpdB (SM-6) genes resulted in an approximate 103-fold attenuation in virulence assessed by
the analysis of the LD50 of specific pathogen-free (SPF) chicks. This result indicated that the deletion of
the lon, cpxR and cpdB genes induced significant virulence attenuation. The protective effects of SM-
△C/L/CpdB vaccination in SPF chicks against 5 109 colony forming units (CFU) of S. Enteritidis were
resulted from the induction of an effective immune response. These findings demonstrate the potential of
mutant SM-△C/L/CpdB to be used as an effective vaccine.

18. Comparative clinicopathological changes in buffalo and cattle following infection by Pasteurella
multocida B:2. S. Annas, M. Zamri-Saad , F.F.A. Jesse , Z. Zunita. Microbial Pathogenesis 88 (2015)
94e102

Abstract :
Haemorrhagic septicaemia (HS) is an acute, septicaemic disease of cattle and buffalo of Asia and Africa
caused by Pasteurella multocida B:2 or E:2. Buffaloes are believed to be more susceptible than cattle. In
this study, 9 buffaloes of 8 months old were divided equally into 3 groups (Groups 1, 3, 5). Similarly, 9
cattle of 8 months old were equally divided into 3 groups (Groups 2, 4, 6). Animals of Groups 1 and 2
were inoculated with PBS while Groups 3 and 4 were inoculated subcutaneously with 105 cfu/ml of
P. multocida B:2. Animals of Groups 5 and 6 were inoculated intranasally with the same inoculum. Both
buffaloes and cattle that were inoculated subcutaneously succumbed to the infection at 16 h and 18 h,
respectively. Two buffaloes that were inoculated intranasally (Group 5) succumbed at 68 h while the
remaining cattle and buffaloes survived the 72-h study period. Endotoxin was detected in the blood of
infected cattle (Group 4) and buffaloes (Groups 3 and 5) prior to the detection of P. multocida B:2 in the
blood. The endotoxin was detected in the blood of buffaloes of Group 3 and cattle of Group 4 at 0.5 h
post-inoculation while buffaloes of Group 5 and cattle of Group 6 at 1.5 h. On the other hand,
bacteraemia was detected at 2.5 h in buffaloes of Group 3 and cattle of Group 4 and at 12 h in buffaloes
 of Group 5 and cattle of Group 6. Affected cattle and buffaloes showed lesions typical of haemorrhagic
septicaemia. These included congestion and haemorrhages in the organs of respiratory, gastrointestinal
and urinary tracts with evidence of acute inflammatory reactions. The severity of gross and
histopathology lesions in cattle and buffalo calves that succumbed to the infection showed insignificant
(p > 0.05) difference. However, inoculated buffalo and cattle that survived the infection showed
significantly (p < 0.05) less severe gross and histopathological changes than those that succumbed. In
general, cattle are more resistant to intranasal infection by P. multocida B:2 than buffaloes
19. Clinico-pathology, hematology and biochemistry responses in buffaloes towards Pasteurella
multocida type B: 2 immunogen lypopolysaccharide via oral and intravenous routes of infection
Eric Lim Teik Chung, Faez Firdaus Jesse Abdullah, Hayder Hamzah Ibrahim, Ali Dhiaa Marza, Mohd
Zamri-Saad, Abdul Wahid Haron, Mohd Azmi Mohd Lila, Mohd Jefri Norsidin. Microbial Pathogenesis
91 (2016) 141e154

Abstract :
Haemorrhagic septicaemia is a disease caused by Pasteurella multocida serotype B: 2 and E: 2. The
organism causes acute, highly fatal septicaemic disease with high morbidity and mortality in cattle and
more susceptible in buffaloes. Lipopolysaccharide can be found on the outer cell wall of the organism.
Lipopolysaccharide is released during multiplication which leads to inflammatory reaction. It represents
the endotoxin of P. multocida type B: 2 and responsible for toxicity in haemorrhagic septicaemia which
plays an important role in the pathogenesis of the disease. Therefore, the aim of this study was to
investigate the clinical signs, blood parameters, gross post mortem lesions and histopathology changes
caused by P. multocida type B:2 immunogen lipopolysaccharide infections initiated through intravenous
and oral routes of infection. 9 buffalo heifers were divided equally into 3 treatment groups. Group 1 was
inoculated orally with 10 ml of phosphate buffer saline (PBS); Group 2 and 3 were inoculated with 10 ml
of lipopolysaccharide broth intravenously and orally respectively. For the clinical signs, there were
significant differences (p < 0.05) in temperature between the control, intravenous and oral group. In
hematology and biochemistry findings, there were significant differences (p < 0.05) in erythrocytes,
haemoglobin, PCV, MCV, lymphocytes, monocytes, eosinophils, GGT and albumin between the control,
intravenous and oral group. However, there were no significant differences (p > 0.05) in the MCHC,
leukocytes, band neutrophils, basophils, thrombocytes, plasma protein, icterus index, total protein,
globulin and A:G ratio between intravenous and oral group. For Group 2 buffaloes, there were gross
lesions in the lung, trachea, heart, liver, spleen, and kidney. In contrast, lesions were only observed in the
lung, trachea and liver of Group 3 buffaloes. There were significant differences (p < 0.05) in hemorrhage
and congestion; necrosis and degeneration; and inflammatory cells infiltration between experimental
groups and control group. However, there were no significant differences (p > 0.05) in edema lesion
between groups. In conclusion, this study is a proof that oral route infection of P. multocida type B:2
immunogen lipopolysaccharide can be used to stimulate host cell responses where oral vaccine through
feed could be developed in the near future.

20. Direct construction of predictive models for describing growth of Salmonella Enteritidis in liquid
eggs e A one-step approach. Lihan Huang. Food Control 57 (2015) 76e81

Abstract :
The objective of this study was to develop a new approach using a one-step approach to directly
construct predictive models for describing the growth of Salmonella Enteritidis (SE) in liquid egg white
(LEW) and egg yolk (LEY). A five-strain cocktail of SE, induced to resist rifampicin at 100 mg/L, was
used to inoculate LEW and LEY. Kinetic studies were conducted isothermally at different temperatures
between 8 and 43 C to generate growth curves at each temperature. This study first solved an inverse
problem globally, using the growth curves to estimate the temperature-dependent kinetic parameters, and
then applied the parameters to predict growth (a forward problem). Once the growth curves were
generated, they were assembled and analyzed using nonlinear regression to determine kinetic parameters
of both primary and secondary models in one step, with an objective to minimize the global residual sum
of squares (RSS) for the entire data set. For growth in LEW, a three-parameter logistic model was used.
For growth in LEY, the Huang model was used as the primary model. The Ratkowsky square-root model
was used to evaluate the growth rates. The results showed that the one-step approach resulted in accurate
 estimation of the kinetic parameters that were used later to successfully predict the growth of SE in LEY
and LEW. The estimated nominal minimum growth temperatures of SE were 7.4 C and 9.9 C, while the
estimated maximum growth temperatures were 45.2 C and 46.8 C, respectively, in LEW and LEY. As a
validation, the predictive models were tested with independent growth curves of SE in LEY and LEW at
37 C. The root mean square error (RMSE) was only 0.36 and 0.28 log CFU/ml over a total scale of 8.4
and 7.8 log CFU/ml, respectively, for the growth models of SE in LEY and LEW, suggesting that the one-
step approach can generate accurate models for predicting the growth of SE in LEY and LEW. The
results from this study can be used to predict the growth of SE and evaluate the safety of LEY and LEW.

21. Technological properties and bacteriocins production by Lactobacillus curvatus 54M16 and its use
as starter culture for fermented sausage manufacture. Annalisa Casaburi, Veronica Di Martino,
Pasquale Ferranti, Luca Picariello, Francesco Villani. Food Control 59 (2016) 31e45

Abstract :
Lactobacillus curvatus 54M16 was isolated from traditional fermented sausages of Campania region
(Italy) and identified by 16S rRNA and hsp60 gene sequencing and by SDS-PAGE of whole cell proteins.
The strain produced more than one bacteriocin, carrying the genes for sakacin X, T and P as
demonstrated by PCR studies. The ability of L. curvatus 54M16 to produce multiple bacteriocins is
confirmed by molecular mass spectrometry analysis and N-terminal amino acid sequencing. Among the
bacteria tested, the pathogens Listeria monocytogenes and Bacillus cereus and the meat spoilage
Brochotrix thermosphacta were sensitive to the bacteriocins. No inhibition of tested Gram-negative
bacteria, some strains of Lactobacillus and of strains of Staphylococcus aureus was observed. All the in
vitro conditions tested for the production of the bacteriocins (temperature, pH and NaCl), indicate that the
strain of L. curvatus 54M16 is able to grow and to produce the antagonistic substances at pH 4.5 and in
the presence of 4% NaCl. The strain has good acidifier capability and it is able to hydrolyse sarcoplasmic
but not myofibrillar proteins, lipids and to reduce nitrates. Moreover it shown high values
of SOD, while the aminopeptidase activity is restricted to whole cells that hydrolyse at high rates the
amino acids L-arginine, L-valine, L-phenylalanine and L-lysine. The suitability of L. curvatus 54M16 as
starter culture was assayed during sausage fermentation. Its use can improve the quality and safety of the
traditional fermented sausages prepared without antimicrobial additives.

22. Prevalence, acquired antibiotic resistance and bacteriocin production of Enterococcus spp. isolated
from tunisian fermented food products. Amel Rehaiem, Imene Fhoula, Amine Faouzi Slim, Ilhem
Boutiba Ben Boubaker, Abdellatif Boudabous Chihi, Hadda-Im ene Ouzari. Food Control 63 (2016)
259e266

Abstract :
In this study, a total of 100 fermented food products including dairy (Lben, Rayeb, Rigouta, and Jben)
olive and vegetable products, harvested in Northwestern Tunisia, were investigated for the presence of
Enterococcus spp. Our results showed high levels of contamination with Enterococcus spp., identified
according to standard bacteriological, biochemical and phenotypic criteria. 143 isolates were recovered;
Enterococcus faecium (46.15%) was the predominant species, followed by Enterococcus faecalis
(27.27%), Enterococcus casseliflavus (12.58%), E. durans (8.39%) and E. mundtii (5.59%). None of the
isolates showed acquired resistance againts clinically relevant drugs used for enterococcal infections
treatment in human medicins, and no haemolytic activity was demonstrated. Furthermore, over 50% of
the isolates within each species exhibited antilisterial bacteriocin production. Further data are needed to
enhance understanding of bacteriocin production of enterococci in fermented food products as well as the
potential risks to quality and safety, including possible transmission of antibiotic resistant organisms to
human consumers.
23. Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative
plasmids. Miseon Park, Joanna Deck, Steven L. Foley, Rajesh Nayak, J. Glenn Songer, Janice R. Seibel,
Saeed A. Khan, Alejandro P. Rooney, David W. Hecht, Fatemeh Rafii. Anaerobe 38 (2016) 25e35

Abstract :
Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe
infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which
may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C.
perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals
(porcine, bovine and canine), from different geographic areas in the United States between 1994 and
2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel
electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using
SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could
not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of
the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the
same domestic animal species were clustered more closely with each other than with other strains.
However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the
clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers
for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified
identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized
plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed
diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C.
perfringens extended to the plasmids carrying conjugative genes.

24. Multidrug resistance in Clostridium perfringens isolated from diarrheal neonatal piglets in
Thailand. Bhinyada Ngamwongsatit, Wimonrat Tanomsridachchai, Orasa Suthienkul, Supanee Urairong,
Wichian Navasakuljinda, Tavan Janvilisri. Anaerobe 38 (2016) 88e93

Abstract :
Clostridium perfringens causes diarrhea in neonatal piglets, thereby affecting commercial swine farming.
The objective of this study was to determine the prevalence and characterize antimicrobial resistance in
C. perfringens isolated from diarrheal neonatal piglets in Thailand. A total of 260 rectal swab samples
were collected from 13 farms and were subjected to C. perfringens isolation. A total of 148 samples were
PCR-positive for C. perfringens toxin genes, from which 122 were recovered. All isolates were cpb2-
encoding C. perfringens type A and enterotoxin gene negative. Most of the isolates were susceptible to
ampicillin, bacitracin, chlorotetracycline, doxycycline, and oxytetracycline with MIC50 values ranging
from 0.32 to 8 mg/ml. The high resistance rates were observed for ceftiofur, enrofloxacin, erythromycin,
lincomycin, and tylosin. Among resistant isolates, 82% were resistant to more than one type of
antibiotics. The distinct pattern of multiple drug resistance in C. perfringens was observed in different
regions, potentially reflecting the farm specific usage of these agents.
25. Isolation and biochemical characterisation of a bacteriocin-like substance produced by Bacillus
amyloliquefaciens An6. Hanen Ben Ayed, Hana Maalej, Noomen Hmidet, Moncef Nasri. Journal of
Global Antimicrobial Resistance 3 (2015) 255–261

Abstract :
This study focuses on the isolation and characterisation of a peptide with bacteriocin-like properties from
Bacillus amyloliquefaciens An6. Incubation conditions were optimised, and the effects of the incubation
period and of carbon and nitrogen sources were investigated. The produced bacteriocin was partially
purified with ammonium sulphate precipitation, dialysis and ultrafiltration and was then biochemically
characterised. Maximum bacteriocin production was achieved after 48 h of incubation in a culture
medium containing 20 g/L starch and 10 g/L yeast extract, with an initial pH 8.0 at 30 8C under
continuous agitation at 200 rpm. The bacteriocin was sequentially purified and its molecular weight was
determined to be 11 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE).
The bacteriocin was relatively heat-resistant and was not sensitive to acid and alkaline conditions (pH
4.0–10.0). Its inhibitory activity was sensitive to proteinase K but was resistant to the proteolytic action
of alcalase, trypsin, chymotrypsin and pepsin. In conclusion, bacteriocin An6, owing its wide spectrum of
activity as well as its high tolerance to acidic and alkaline pH values, temperature and proteases shows
great potential for use as a food biopreservative.

26. Rapid diagnosis of virulent Pasteurella multocida isolated from farm animals with clinical
manifestation of pneumonia respiratory infection using 16S rDNA and KMT1 gene. Gamal
Mohamedin Hassan, Zaki Ahmed El-Feky, Eissa Ahmed Eissa, Ayaat Ahmed Teleb. Asian Pac J Trop Dis
2016; 6(1): 21-26

Abstract :
Objective: To characterize intra-isolates variation between clinical isolates of Pasteurella multocida (P.
multocida) isolated from sheep, cattle and buffalo at molecular level to check the distribution of
pneumonia and hemorrhagic septicemia in some regions of Fayoum, Egypt. Methods: These isolates
were obtained from various locations in the Fayoum Governorate, Egypt and they were identified by
amplifying 16S rDNA and KMT1 genes using their DNA as a template in PCR reaction. Results: The
results demonstrated that the five selective isolates of P. multocida had similar size of PCR products that
generated one band of 16S rDNA having 1 471 bp and KMT1 gene having 460 bp. The phylogenetic tree
and similarity of the five selective isolates of P. multocida which were collected from GenBank database
were calculated and analyzed for the nucleotide sequence of 16S rDNA and KMT1 genes. The
sequencing result of 16S rRNA gene product (1 471 bp) for the five selective isolates of P. multocida
showed that the isolates of sheep (FUP2) shared 94.08%, 88.10% homology with the buffalo isolate
(FUP8) and cattle isolate (FUP9) respectively, whereas, the buffalo isolate (FUP5) shared 98.18% and
94.40% homology with the cattle isolates (FUP12 and FUP9). Conclusions: The results indicated the
relationships of P. multocida isolated from buffalo and cattle rather than the close relationships between
P. multocida isolated from cattle and sheep. Diagnosis of P. multocida by 16S rDNA and KMT1 gene
sequences was important to determine the antigen that is responsible for protective cover within the same
group of animals and to help for the production of new vaccines for the control of microbial infection for
domestic animals.
27. Prevalence, characterization and antibiotic resistance of Pasteurella multocida isolated from bovine
respiratory infection. Hossein Jamali, Mojtaba Rezagholipour, Sepideh Fallah, Arezoo Dadrasnia,
Shamini Chelliah, Rita Devi Velappan, Kelvin Swee ChuanWei, Salmah Ismail. The Veterinary Journal
202 (2014) 381–383

Abstract :
The objectives of this study were to determine the prevalence, characterization and antibiotic resistance
of Pasteurella multocida isolated from calves with respiratory infection in Iran. P. multocidawas detected
in 141/169 bovine respiratory infection cases on Iranian dairy and beef farms. P. multocida were grouped
into serogroups A (126/141), D (12/141), and B (3/141). Of the P. multocida isolates, all harboured the
psl, ompH, oma87, fimA, ptfA, nanB, and nanH genes, 139/141 had hsf-2, and 115/141 pfhA, and tadD.
The isolates were most frequently resistant to penicillin G (43/141 resistant isolates; 30.5%) and
streptomycin (31/141; 22%).

Paket Informasi Bidang Lain-lain

1. Paradigms of climate change impacts on some major food sources of the world: A review on
current knowledge and future prospects. Ashutosh Tripathia, Durgesh Kumar Tripathib, D.K.
Chauhana, Niraj Kumarc, G.S. Singhd. Agriculture, Ecosystems and Environment 216 (2016) 356–373

Abstract :
Due to the adverse impacts of climate change on earth systems the research in this field has been
profoundly taken a part in all scientific arenas since last few decades. The deleterious impacts of climate
change on agricultural production are challenging the food security of the world in terms of quantity and
quality both. Wheat, rice, maize, vegetables, fruits and fish-food provide food security for more than half
of the world and are under immense pressure of changing climate. This review is an overview of the
significant impacts associated with climate change on these food sources. In present synthesis, various
phenological, physiological, biochemical and reproductive responses in major food crops have been
summarized emphasizing the vulnerable growth and development stages. Winter and summer sensitivity
responses, and morpho-biochemical acclimation patterns have also been summarized. Sustenance in
wheat and rice production is evident but impacts of increasing temperatures are negating this on bio-
physiological level impacts. Maize crops are experiencing more impacts on yield as compared to wheat
and rice. Fruits and vegetable production is highly vulnerable to climate change at their reproductive
stages and also due to more disease prevalence. Fisheries as a critical animal food source; is in extreme
danger as apparent changes in their habitat and unmanageable environmental conditions are producing
extreme losses. This review also provides an account of stress responses and useful adaptive measures.
This synthesis may be helpful in understanding manifold dimensions and interactions of climate change
impacts on selected major food sources of the world.
2. A study of medicinal plant susedasethno veterinary:Harnessing Potential lphytotherapy in Bheri,
DistrictMuzaffarabad(Pakistan). Muhammad JamilAhmed, GhulamMurtaza. Journal
ofEthnopharmacology159(2015)209–214

Abstract :
Ethnopharmacological relevance : Medicinal plants are utilized for handling health care system and in
preventing a variety of diseases. A survey was conducted to document the rapidly disappearing
traditional knowledge of medicinal plants in union council Bheri, District Muzaffarabad, Azad Kashmir,
Pakistan. Materials and methods : Questionnaire format was used to collect the medicinal uses of
plants. The 180 informants were interviewed from six villages in total, 30 from each village (20 male and
10 female) regarding the ethnoveterinary uses of plants in several ailments. For the reliability of
ethnoveterinary knowledge, the informant consensus factor (FIC), and fidelity level (FL) were calculated
and the literature cited was surveyed. The medicinal information was gathered from local inhabitants,
healers, shepherds and old men and women of different age groups. Results : A total of 24 medicinal
plant species used as ethnoveterinary were found belonging to 22 genera and 19 families. The most
dominant family was Polygonaceae (3 species) followed by Araceae, Asteraceae, Lamiaceae each with 2
species and remaining families having one species. The important medicinal plant species showed the
highest fidelity level (FL) such as: Rumex nepalensis, Primula denticulata, (100%) used for dysuria, red
urination, Skimmia laureola (100%), Swertia paniculata (99%), and Angelica glauca (97%), used for
ague, cold, shivering, gastric ailments, Melia azedarach (100%), used to reduce intestinal worm load in
cattle showing the conformity of knowledge on these species. Highest FIC was recorded for foot and
mouth diseases and ectoparasite (1) followed by ague (0.98) and dysuria (0.99) depicting that a few
species were used to cure various animals‫ ׳‬ailments. Conclusions : The findings of the research revealed
that merely a few species are used as ethnoveterinary medicine supported by pharmacology study. Due to
anthropogenic pressure the extinction of each species from the areas could result in disappearing
knowledge regarding century‫׳‬s old traditional methods of curing diseases from these plant species.

3. The relationship between climate, diseases of domestic animals and human-carnivore conflicts.
Igor Khorozyana, Mahmood Soofib, Arash Ghoddousia, Amirhossein Khaleghi Hamidib. Basic and
Applied Ecology 16 (2015) 703–713

Abstract :
Human-carnivore conflicts over livestock predation threaten biodiversity conservation and rural
development, but the impact of climate and its change on such conflicts is insufficiently studied. The
effect of climatic factors on diseases of predation-prone domestic animals and then on conflicts is
unstudied, but potentially significant. This empirical case study addressed the conflict between people
and leopards (Panthera pardus) in the Hyrcanian humid temperate forest (Iran). We analyzed our
questionnaire and other data from all 34 villages around Golestan National Park in terms of probabilities
of human-leopard conflicts over livestock predation, diseases of domestic animals and WorldClim
bioclimatic variables. Using multiple predictive modeling approaches (generalized linear modeling
GLM, Multivariate Adaptive Regression Splines MARS, Bayesian Belief Network BBN, BIOCLIM and
DOMAIN), we show that climate continentality and precipitation patterns affect diseases, and more
diseases lead to more conflicts. The Community Climate System Model (CCSM4) scenarios forecast
aridization of the study area in 2041–2080 and a resultant decline of disease and conflict probabilities by
18.4–21.4% and 10.4–11.9%, respectively. We conclude that diseases can drive human-carnivore
conflicts which may become less intense with projected aridization of the studied humid environment.
4. Helminthiasis and medicinal plants: a review. Mahesh Bandappa Manke, Shashikant Chaburao
Dhawale, Prasad Govindrao Jamkhande. Asian Pac J Trop Dis 2015; 5(3): 175-180

Abstract :
Helminthiasis is the most common infection caused by worms that is contaminant parts. Normally, the
worms live in the gastrointestinal tract, liver and other organs t. oT hhue mcuanrr ebnotdlyy
davieatiylalcbaler baanmthaezlimnein, tiivce drmruegcst,i nin, cpluradziinqgu aalnbteenl,d aarzeo lew,i
dmeelyb eunsdeadz otloe ,c tohnitarboel nhdealzmolien,t hniiarsidisa.z oBluet, vthoemsiet idnrgu, gasb
dhoamvei nsaelr ipoauisn ,d hreaawdbaacchkes asnudc hd iaasr rhheeap.a Ttohtouxs,i ciitt yis, nloescse
sosfa aryp ptoe tlioteo,k d fiozrz imneosres , enffaeucstievae, oann thtrealdmitiinotnica ld mruegdsi
cwinitehs tahned m pinlainmtu emxt rsaidcets eafnfedc ttsh.e E aigchtitvye p ceorncsetnittu oefn tthse a
wreo rulsde’sd ptoo pmuelaetti opne orpelliee’ss ipnr itmhea rtyr ehaetmalethn tc oafr eh enlemedinst.h
Tiahsisis r.

Paket Informasi Bidang Parasitology

1. Anthelmintic activity of Ocimum sanctum leaf extract against ovine gastrointestinal nematodes in
India. Dharmendra Kanojiya, Daya Shanker, Vikrant Sudan, Amit Kumar Jaiswal, Rahul Parashar.
Research in Veterinary Science 99 (2015) 165–170

Abstract :
Leaves of Ocimum sanctum have been traditionally used for various ethno-veterinary practices as well
as medicinal purpose. In vitro ovicidal and larvicidal potential of crude aqueous and hydro-alcoholic
extracts of the bulb of O. sanctum was investigated. Alkaloids, carbohydrates, steroids and tannins were
identified in phytochemical analyses. The various blood parameters coupled marker enzymes and
antioxidant status were also evaluated during in vivo trial. Aqueous extract showed better EC50 and
EC99 values in comparison with methanolic extract in egg hatch assay and larval development test,
respectively. However, in the larval paralysis test, both aqueous and methanolic extracts showed almost
similar efficacy. A 77.64% reduction in fecal egg output was observed on day 14. No deleterious ill effect
was found in any of the hematological and biochemical parameters suggesting that the plant could be
safer for use in sheep.
2. In vitro screening of forty medicinal plant extracts from the United States Northern Great Plains
for anthelmintic activity against Haemonchus contortus. Jyotsna Acharya, Michael B. Hildreth, R.
Neil Reese. Veterinary Parasitology 201 (2014) 75–81

Abstract :
An egg hatch assay (EHA) and a larval migration assay (LMA) involving Haemonchus contortus was
used to evaluate the anthelmintic activity of methanol extracts from 40 plants that are native or
naturalized within the U.S.A. Northern Great Plains. Only one of these 40 plants (i.e. Lotus corniculatus)
had been previously evaluated for activity against any gastrointestinal nematode. The various extracts
were initially screened at 50 mg/ml diluted either in 0.5% dimethyl sulphoxide (DMSO) or 3-(N-
morpholino) propanesulfonic acid (MOPS buffer), and plants showing 100% inhibition at 50 mg/ml,
were further evaluated at 8 other concentrations(25–0.19 mg/ml). Extracts with 100% activity with the
EHA were again screened with the LMA (50 mg/ml). Two extracts with the highest LMA inhibition were
also evaluated at lower concentrations (25–3.1 mg/ml). Of the 40 methanolic extracts screened, 7
(Chrysothamnus viscidiflorus, Ericameria nauseosa, Liatris punctata, Melilotus alba, Melilotus
officinalis, Perideridia gairdneri, and Sanguinaria canadensis) showed significant egg-hatch inhibition in
DMSO and MOPS buffer. Three extracts (Geranium viscosissimum, L. corniculatus, and Rhus
aromatica) only showed significant inhibition in DMSO. The 8 extracts showing 100% efficacy at 50
mg/ml exhibited dose-dependent effects at the 8 lower concentrations, and R. aromatica and E. nauseosa
extracts had the lowest ED50 values. Similarly, when these 8 plant extracts were further evaluated with
the LMA, the extracts of E. nauseosa and R. aromatic again exhibited the highest activity (p < 0.001),
 with ED50 values of 4.0 mg/ml and 10.43 mg/ml respectively. Three other extracts (C. viscidiflorus, M.
alba and M. officinalis) also showed inhibitory activity in the LMA. These results support the need for
additional evaluations of the nematocidal properties for at least these 5 plants.

3. Anthelmintic resistance in gastrointestinal nematodes ingoats and evaluation of FAMACHA

diagnostic marker in Uganda. Immaculate Nabukenyaa, Chris Rubaire-Akiikia, Deogracious
Olilaa,Denis Muhangia, Johan Höglundb. Veterinary Parasitology 205 (2014) 666–675

Abstract :
Gastrointestinal nematodes (GIN) are a challenge to goat production globally causingreduced growth,
morbidity and mortality. We report here results of the first nation-wideanthelmintic resistance (AR) study
and validation of assessment of clinical anaemia withFAMACHA eye scores in goats in Uganda. From
August to December 2012 the efficacyof albendazole (7.5 mg/kg), levamisole (10.5 mg/kg) and
ivermectin (0.3 mg/kg) againststrongyle nematodes was tested on 33 goat farms in Soroti, Gulu, Mpigi,
Mbarara andSsembabule districts of Uganda. Altogether 497 goats were subjected to a total of 45 dif-
ferent faecal egg count reduction tests (FECRT), each involving 5–20 goats. On one farmall substances
were tested. Faecal and blood samples were collected and FAMACHA eyescores evaluated on the day of
treatment and 15 days later. A questionnaire survey wasconducted on frequency, type and dose of
anthelmintics used, farm size and grazing man-agement system. Examination of infective third stage
larvae (L3) from pooled faecal culturesdemonstrated Haemonchus to be the predominant genus (>75%).
Resistance to at least oneanthelmintic group was detected on 61% of the 33 farms and in 49% of the 45
test groups.Prevalence of resistance to ivermectin, levamisole and albendazole was respectively
58%,52% and 38%. Correlation between pre-treatment packed cell volume determinations
andFAMACHA scores (r498= −0.89) was significant. Paddock grazing system (Odds ratio 4.9, 95%CI
1.4–17.3) and large farm size of >40 goats (odds ratio 4.4, 95% CI 1.2–16.1) were significantpredictors
of AR. In all districts, resistance to all three anthelmintics was higher on large-scale goat farms practising
mostly paddock grazing. Interestingly, resistance to albendazole,the most commonly used anthelmintic in
Uganda, was lower than that to ivermectin andlevamisole. We recommend adaptation of FAMACHA to
goats to help restrict anthelmintictreatment to heavily infected individuals. This will limit selection
pressure and hence delaydevelopment of anthelmintic resistance.

4. Anthelmintic resistance in nematodes of beef cattle insouth-west Western AustraliaJ.L. Cotter, A.

Van Burgel, R.B. Besier. Veterinary Parasitology 207 (2015) 276–284

Abstract :
Anthelminthic resistance in nematodes of beef cattle is an emerging issue globally withimplications for
effective parasite control. The prevalence of resistance in beef cattle in theMediterranean-style climatic
zone of south-west Western Australia was assessed on 19farms, using faecal egg count reduction tests.
Pre-treatment faecal worm egg counts werecompared with counts at 14 days after treatments with
ivermectin (injectable), fenbenda-zole (oral), or levamisole (oral). A separately grazed group treated with
topical ivermectin(pour-on) and sampled at 28 days was included as a comparison against injectable iver-
mectin. The results demonstrate that resistance is common, with failure of at least oneanthelmintic
(<95% reduction for each species, by arithmetic means) for either of the majorspecies Cooperia
oncophora or Ostertagia ostertagi on 17 of the 19 properties. Resistance toivermectin (injectable) was
demonstrated in C. oncophora in 59% of tests, but ivermectinwas fully effective against O. ostertagi by
this route. Conversely, O. ostertagi resistant to fen-bendazole and levamisole were present on 50% and
67% of farms respectively, with bothfully effective against C. oncophora. The finding of Haemonchus
placei on several proper-ties was unexpected but the egg counts were low and there is no suggestion of
pathogeniceffects. An indication of reduced efficacy of the pour-on ivermectin formulation comparedto
the injectable was apparent against both C. oncophora and O. ostertagi, and this may haveimplications
for resistance development, given the widespread use of topical treatmentsreported in this region. This
survey confirms that anthelminthic resistance in nematodesof beef cattle is common in Western Australia
and the pattern of occurrence is in generalagreement with surveys elsewhere in Australia and in other
countries.
5. Resistance of gastrointestinal nematodes to the most commonly used anthelmintics in sheep, cattle
and horses in Spain. M. Martínez-Valladares, T. Geurden, D.J. Bartram, J.M. Martínez-Pérez, D.
Robles-Pérez, A. Bohórquez, E. Florez, A. Meana, F.A. Rojo-Vázquez. Veterinary Parasitology 211
(2015) 228–233

Abstract :
The objective of this study was to evaluate the status of anthelmintic resistance (AR) in ruminants and
horses in Spain. The efficacy of commonly used macrocyclic lactones (MLs) – ivermectin (IVM) and
moxidectin (MOX) – was measured in sheep, cattle and horses. In addition, albendazole (ABZ) and
levamisole (LEV) were evaluated in sheep and oxibendazole (OXI) and pyrantel (PYR) in horses.
Efficacy was evaluated based on the difference between the arithmetic mean pre- and post-treatment
faecal egg count (in cattle and horses), or compared to an untreated control group (in sheep). AR was
present when the percentage reduction in egg count was <95% and the lower 95% confidence interval
(CI) was <90%; if only one of these two criteria was met, the finding was recorded as suspected AR
(SAR). In horses, AR–PYR and OXI was considered when the percentage reduction in egg count was
≤90% and the lower 95% CI was ≤80%. For each animal species, at least 10 study sites were selected.
AR to at least one of the drugs was detected in all 10 sheep flocks; the main parasite identified after
treatment was Teladorsagia circumcincta. Moreover, in 5 flocks multidrug resistance was identified, on
4 farms to drugs from different families, on one farm to both MOX and IVM and on another farm to all
drugs tested. In cattle, the efficacy of both MOX and IVM was 100% on 4 and 3 farms, respectively, and
therefore 60% of these farms were considered to have AR or SAR to both MLs. The most frequent
parasite identified after treatment was Trichostrongylus spp., although Ostertagia ostertagi was also
identified after treatment on one farm. In contrast to ruminants, the 4 drugs evaluated in horses were
highly efficacious against strongyles, with efficacies for the MLs and OXI between 95 and 100% and
between 94 and 100% for PYR, although 3 herds were SAR against PYR. In conclusion, AR to at least
one of the commonly used drugs was identified on all sheep flocks investigated in the northwest of
Spain. The occurrence of AR to MLs in cattle was higher than expected but consistent with what was
observed in sheep. In horses, all currently used drugs were confirmed as effective against strongyles.

6. Anthelmintic efficacy and pharmacokinetics of pour-oneprinomectin (1 mg/kg bodyweight) against

gastrointestinaland pulmonary nematode infections in goats. Dietmar Hamel, Martin Visser, Michael
Kellermann, Valerie Kvaternick, Steffen Rehbein. Small Ruminant Research 127 (2015) 74–79

Abstract :
Two controlled studies were performed to assess the efficacy and pharmacokinetics oftopical 0.5% w/v
eprinomectin (EPRINEX®Pour-on, Merial) against nematode infectionsof goats. Each study included 16
male castrated goats, less than one year old, harboringinduced infections of adult gastrointestinal and
pulmonary nematodes. Following blockingon pre-treatment bodyweight and random allocation to one of
two groups, half the goatsin each study were treated once with eprinomectin (1 mg/kg bodyweight,
administeredtopically), and half remained untreated and served as controls. Plasma concentrations
ofeprinomectin were determined in blood samples collected prior to and at multiple timepoints up to
necropsy. Efficacy was determined based on the nematode counts of theanimals following necropsy 14
days after treatment.Efficacy of treatment was 100% against adult Haemonchus contortus,
Nematodirusbattus, Nematodirus spathiger, Oesophagostomum venulosum, Teladorsagia
circumcinctaand Dictyocaulus filaria was >94% against adult Cooperia curticei, Trichostrongylus
axeiand Trichostrongylus colubriformis, and was 89.4% against adult Strongyloides papillo-sus (p <
0.01). Basic pharmacokinetic parameters of eprinomectin (B1a component) forthe goats in the two
studies were: AUClast, 23.5 ± 5.19/36.0 ± 8.74 day*ng/mL; and Cmax,3.65 ± 1.12/5.25 ± 1.39 ng/mL,
respectively; individual maximum plasma concentrationswere observed 1 or 2 days after treatment.
Results of the studies consistently demonstrateda high therapeutic efficacy of topical eprinomectin at 1
mg/kg bodyweight against a broadrange of gastrointestinal and pulmonary nematode parasites of goats.
7. Climate change, biodiversity, ticks and tick-borne diseases: The butterfly effect. Filipe Dantas-
Torres. International Journal for Parasitology: Parasites and Wildlife 4 (2015) 452e461

Abstract :
We have killed wild animals for obtaining food and decimated forests for many reasons. Nowadays, we
are burning fossil fuels as never before and even exploring petroleum in deep waters. The impact of these
activities on our planet is now visible to the naked eye and the debate on climate change is warming up
in scientific meetings and becoming a priority on the agenda of both scientists and policy decision
makers. On the occasion of the Impact of Environmental Changes on Infectious Diseases (IECID)
meeting, held in the 2015 in Sitges, Spain, I was invited to give a keynote talk on climate change,
biodiversity, ticks and tick-borne diseases. The aim of the present article is to logically extend my
rationale presented on the occasion of the IECID meeting. This article is not intended to be an exhaustive
review, but an essay on climate change, biodiversity, ticks and tick-borne diseases. It may be anticipated
that warmer winters and extended autumn and spring seasons will continue to drive the expansion of the
distribution of some tick species (e.g., Ixodes ricinus) to northern latitudes and to higher altitudes.
Nonetheless, further studies are advocated to improve our understanding of the complex interactions
between landscape, climate, host communities (biodiversity), tick demography, pathogen diversity,
human demography, human behaviour, economics, and politics, also considering all ecological processes
(e.g., trophic cascades) and other possible interacting effects (e.g., mutual effects of increased
greenhouse gas emissions and increased deforestation rates). The multitude of variables and interacting
factors involved, and their complexity and dynamism, make tick-borne transmission systems beyond
(current) human comprehension. That is, perhaps, the main reason for our inability to precisely predict
new epidemics of vectorborne diseases in general.
8. Anthelmintic resistance to ivermectin and moxidectin in gastrointestinal nematodes of cattle in
Europe. Thomas Geurden , Christophe Chartier, Jane Fanke, Antonio Frangipane di Regalbono, Donato
Traversa, Georg von Samson-Himmelstjerna, Janina Demeler, Hima Bindu Vanimisetti, David J.
Bartram, Matthew J. Denwood. International Journal for Parasitology: Drugs and Drug Resistance 5
(2015) 163e171

Abstract :
Anthelmintic resistance has been increasingly reported in cattle worldwide over the last decade, although
reports from Europe are more limited. The objective of the present study was to evaluate the efficacy of
injectable formulations of ivermectin and moxidectin at 0.2 mg per kg bodyweight against naturally
acquired gastro-intestinal nematodes in cattle. A total of 753 animals on 40 farms were enrolled in
Germany (12 farms), the UK (10 farms), Italy (10 farms), and France (8 farms). Animals were selected
based on pre-treatment faecal egg counts and were allocated to one of the two treatment groups. Each
treatment group consisted of between 7 and 10 animals. A post-treatment faecal egg count was performed
14 days (±2 days) after treatment. The observed percentage reduction was calculated for each
treatment group based on the arithmetic mean faecal egg count before and after treatment. The resistance
status was evaluated based on the reduction in arithmetic mean faecal egg count and both the lower and
upper 95% confidence limits. A decreased efficacy was observed in half or more of the farms in
Germany, France and the UK. For moxidectin, resistance was confirmed on 3 farms in France, and on 1
farm in Germany and the UK. For ivermectin, resistance was confirmed on 3 farms in the UK, and on 1
farm in Germany and France. The remaining farms with decreased efficacy were classified as having an
inconclusive resistance status based on the available data. After treatment Cooperia spp. larvae were
most frequently identified, though Ostertagia ostertagi was also found, in particular within the UK and
Germany. The present study reports lower than expected efficacy for ivermectin and moxidectin (based
on the reduction in egg excretion after treatment) on European cattle farms, with confirmed anthelmintic
resistance on 12.5% of the farms.
Paket Informasi Bidang Toksikology

1. Fast gas chromatographic residue analysis in animal feed using splitinjection and atmospheric
pressure chemical ionisation tandemmass spectrometry. M. Tienstra, T. Portolésa, F. Hernández,
J.G.J. Mol. Journal of Chromatography A, 1422 (2015) 289–298

Abstract :
Significant speed improvement for instrumental runtime would make GC–MS much more attractive
fordetermination of pesticides and contaminants and as complementary technique to LC–MS. This was
thetrigger to develop a fast method (time between injections less than 10 min) for the determination of
pes-ticides and PCBs that are not (or less) amenable to LC–MS. A key factor in achieving shorter
analysis timewas the use of split injection (1:10) which allowed the use of a much higher initial GC oven
temperature. Ashorter column (15 m), higher temperature ramp, and higher carrier gas flow rate (6
mL/min) further con-tributed to analysis-time reduction. Chromatographic resolution was slightly
compromised but still wellfit-for-purpose. Due to the high sensitivity of the technique used (GC–APCI-
triple quadrupole MS/MS),quantification and identification were still possible down to the 10 g/kg level,
which was demonstratedby successful validation of the method for complex feed matrices according to
EU guidelines. Other advan-tages of the method included a better compatibility of acetonitrile extracts
(e.g. QuEChERS) with GC, anda reduced transfer of co-extractants into the GC column and mass
spectrometer.
2. Carry-over of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and
polychlorinated biphenyls (PCBs) in dairy cows fed smoke contaminated maize silage or sugar beet
pulp. Ron L.A.P. Hoogenboom, Arie Klop, Rik Herbes, Jan C.H. van Eijkeren, Marco J. Zeilmaker, Ad
M. van Vuuren, Wim A. Traag. Chemosphere 137 (2015) 214–220

Abstract :
Fires and improper drying may result in contamination of feed with PCDD/Fs and PCBs. To predict the
impact of elevated feed levels, it is important to understand the carry-over to edible products from food
producing animals. Therefore, a carry-over study was performed with maize silage contaminated by a
fire with PVC materials, and with sugar beet pulp contaminated by drying with coal, containing particles
from a plastic roof. Levels of PCDD/Fs and dl-PCBs in the maize silage were 0.93 and 0.25 ng TEQ kg1,
those in beet pulp 1.90 and 0.15 ng TEQ kg1 (both on 88% dry matter (DM)). Dairy cows (3 per
treatment) received either 16.8 kg DM per day of maize silage or 5.6 kg DM per day of sugar beet pellets
for a 33-d period, followed by clean feed for 33 days. This resulted in a rapid increase of PCDD/F levels
in milk within the first 10 days with levels at day 33 of respectively 2.6 and 1.7 pg TEQ g1 fat for maize
silage and beet pulp. Levels of dl-PCBs at day 33 were lower, 1.0 and 0.5 pg TEQ g1 fat. In the case of
the maize silage, the carry-over rates (CORs) at the end of the exposure were calculated to be 25% and
32% for the PCDD/F- and dl-PCB-TEQ, respectively. For the dried beet pulp the CORs were 18% and
35%. This study shows that the carry-over of PCDD/Fs and dl-PCBs formed during drying processes or
fires can be substantial.
2. Dioxins and PCBs in feed and food-Review fromEuropean perspective. Rainer Malisch, Alexander
Kotz. Science of the Total Environment 491–492 (2014) 2–10

Abstract :
During the 1990s, a number of adverse contamination incidents focussed the attention of the media and
the general public on food safety. This led to the evaluation of safety measures with regard to dioxin
intake from food. Important aspects regarding dioxins and PCBs in the food chain are reviewed here,
allowing a contextual understanding of the present situation through its chronological developments.
About 90–98% of the average exposure of humans to dioxins and PCBs results from dietary intake, with
food of animal origin being the predominant source. Therefore, animal feed contributes considerably to
the presence of these compounds in food. The detection of the “real” source of a contamination event in
the food chain is a complex scientific problem and requires specific knowledge on production processes
and changes of patterns during bioaccumulation. This is demonstrated by complex investigations
performed in three studies on two continents to identify the source (e.g. from contamination of cow's
milk in Germany, to citrus pulp pellets from Brazil as an ingredient in feed, then to contaminated limefor
neutralization and finally to a landfillwith residues of vinyl chloridemonomer production). This example
shows also the substantial economic losses resulting fromincidents in the food chain and the
consequences to global trade. In 2001, the EU Scientific Committee on Food established a group
tolerable weekly intake (TWI) of 14 pgWHOTEQ/kg bodyweight and concluded that a considerable
proportion of the European populationwould exceed this TWI. On the global level, the Joint FAO/WHO
Expert Committee on Food Additives (JECFA) provides scientific advice to the Codex Alimentarius
Commission and therefore contributes to harmonized international food standards. In its evaluation of
2001, JECFA derived a provisional tolerable monthly intake (PTMI) of 70 pg TEQ/kg body weight. The
sum of the median intake of PCDD/F-TEQ and PCB-TEQ exceeded the PTMI in Western European
countries, was in the PTMI range in North America, but lower in Japan and NewZealand. The 90th
percentile of PCDD/F-TEQ exceeded the PTMI inWestern European countries and North America, the
90th percentile of coplanar PCBs in Western European countries. Therefore, in 2001 the EU Commission
developed a strategy to reduce the presence of dioxins and PCBs in the environment and in the food
chain. The legislative measures comprised maximum levels and action levels for feed and food, and a
Rapid Alert Systemfor detected incidents was introduced. The network of the EU Reference Laboratory
and National Reference Laboratories contributes to harmonization within the EUMember States and
developed analytical criteria for screening and confirmatory methods for control of feed and food. After
all these efforts it is of general interest to seewhether these measures had an effect. The 2012 evaluation
of the European Food Safety Authority (EFSA) based on comprehensivemonitoring data of 26 European
countries shows a general decrease in dietary exposure of dioxins and DL-PCBs between 2002–2004 and
2008–2010, estimated to be between 16.6% and 79.3% for the different population groups. A smaller
decrease was observed for NDL-PCBs. The percentage of individuals exposed above the TWI of 14 pg
TEQ/kg b.w. was estimated to be between 1.0 and 52.9%. Toddlers and other children were the most
exposed groups (being at the upper end of these ranges). Fish, meat and dairy products appeared to be the
highest contributing food groups to dietary exposure.
3. Valorisation of food waste to produce new raw materials for animal feed. D. San Martin, S. Ramos,
J. Zufía. Food Chemistry 198 (2016) 68–74

Abstract :
This study assesses the suitability of vegetable waste produced by food industry for use as a raw material
for animal feed. It includes safety and nutritional viability, technical feasibility and environmental
evaluation. Vegetable by-products were found to be nutritionally and sanitarily appropriate for use in
animal feed. The drying technologies tested for making vegetable waste suitable for use in the animal
feed market were pulse combustion drying, oven and microwave. The different meal prototypes obtained
were found to comply with all the requirements of the animal feed market. An action plan that takes into
account all the stages of the valorisation process was subsequently defined in agreement with local
stakeholders. This plan was validated in a pilot-scale demonstration trial. Finally, the technical feasibility
was studied and environmental improvement was performed. This project was funded by the European
LIFE+ program (LIFE09 ENV/ES/000473).
4. European developments following incidents with dioxins and PCBs in the food and feed chain.
Ron Hoogenboom, Wim Traag, Alwyn Fernandes, Martin Rose. Food Control 50 (2015) 670e683

Abstract :
Incidents with dioxins and PCBs have resulted in a strategy within the EU to reduce the exposure of the
population to these compounds. Maximum levels were set for food and feed products and criteria were
developed for the analytical methods (both confirmatory and screening) used for official control
measurements. Ideally, any analysis performed with the aim of comparing the result with the legal limits
should be performed according to these criteria. It should also apply to monitoring, performed to
estimate human exposure and trend analysis rather than compliance with limits, since risk assessments
and EU-policies rely heavily on these data. In recent years, analytical capacity has largely increased to
complement the additional testing. In line with the responsibility of producers for the safety of their
products, self-control has strongly increased and has played an important role in the discovery of several
of the incidents. However, the increased monitoring seems not to have resulted in a clear further decrease
in the levels reported for food and feed in the last decade. This may in part be due to a lack of follow up
when elevated levels (above action levels) are found, which would lead to a reduction of output from
remaining sources. It may also be related to the sensitivity of applied methods and the data collected in
databases. This paper reviews the incidents and developments that have taken place within the EU over
the last 15 years in the area of dioxins and PCBs, including the role of applying screening and
confirmatory methods for achieving the desired further reduction in the levels.

Paket Informasi Bidang Virologi

1. Successful cross-protective efficacy induced by heat-adapted liveattenuated nephropathogenic

infectious bronchitis virus derivedfrom a natural recombinant strain. Tae-Hyun Lim, Ha-Na Youn,
Seong-Su Yuk, Jung-Hoon Kwon, Woo-Tack Hongb,Gyeong-Bin Gwon, Jung-Ah Lee, Joong-Bok Lee,
Sang-Won Lee, Chang-Seon Song. Vaccine 33 (2015) 7370–7374

Abstract :
A natural recombinant nephropathogenic K40/09 strain of infectious bronchitis virus (IBV) was heat-
adapted for possible future use as live attenuated vaccine. The K40/09 strain was selected
duringsuccessive serial passages in specific-pathogen free (SPF) embryonated eggs at sub-optimal higher
tem-perature (56◦C). Unlike the parental strain, the attenuated strain, designated K40/09 HP50, was
foundto be safe in 1-day-old SPF chicks, which showed neither mortality nor signs of morbidity, and
rarelyinduced ciliostasis or histological changes in the trachea and kidney after intraocular and fine-
sprayadministration. K40/09 HP50 provided almost complete protection against two distinct subgroups
of anephropathogenic strain (KM91-like and QX-like subgroup) and elicited the production of high titers
ofneutralizing antibody (neutralization index of 3.6). We conclude that the K40/09 HP50 vaccine virus
israpidly attenuated by heat adaptation and exhibits the desired level of attenuation, immunogenicity,
andprotective efficacy required for a live attenuated vaccine. These results indicate that the K40/09
vaccinecould be helpful for the reduction of economic losses caused by recently emergent
nephropathogenic IBVinfection in many countries.
2. Impact of climate change in the epidemiology of vector-borne diseases in domestic carnivores. F.
Beugnet, K. Chalvet-Monfray. Comparative Immunology, Microbiology and Infectious Diseases 36
(2013) 559– 566

Abstract :
Vector-borne diseases are medically important in humans and animals but were long considered tropical
and known to first affect production animals. This is no longer true and we can see today that they are
common in domestic animals and that they are also present in temperate countries, especially in Europe.
In recent years, an increase in the diagnosis of vector borne diseases among humans and animals has
been observed, which may partly due to the development of diagnostic tools. Their study requires
exchanges and collaborations between the many actors involved, especially since the epidemiology
seems to be constantly evolving. The veterinary practitioner is the first one to notice the emergence of
cases and to implement prevention measures. He also acts as a sentinel to alert epidemiologists. Many
factors can explain the epidemiological changes, i.e. all human factors, such as the increase in
commercial transportation, but also owners traveling with their pet during the holidays, the development
of “outdoor” activities, the increase of individual housings with gardens; to these human factors must be
added the ignorance of the risks, linked to animals in general and to wildlife in particular; then the
environmental changes: forest fragmentation, establishment of parks; the increase of wild mammal
populations (deer, carnivores, rodents, etc.); finally, climate changes. Climate change is a reality which
may explain the increase of density of arthropod vectors, but also of their hosts, changes in periods of
activity and variations in geographical distribution. The authors show the proof of the climate
modifications and then explain how it has an impact in Europe on ticks, mosquitoes, sandflies and even
fleas. They conclude on the practical consequences for veterinary practitioners, especially with the
diagnosis of parasitic diseases or diseases in areas where they usually do not occur. However, not any
epidemiological modification should be linked to climate change, since many other factors are involved
and often even overriding.

3. Genotype-specific variation in West Nile virus dispersal in California. Nisha K.Duggal,

WilliamK.Reisen, YingFang, RuchiM.Newman, XiaoYang, GregoryD.Ebel, AaronC.Brault.
Virology485(2015)79–85

Abstract :
West Nile virus (WNV) is an arbovirus that was first reported in North America in New York in 1999
and, by 2003, had spread more than 4000 km to California. However, variation in viral genetics
associated with spread is not well understood. Herein, we report sequences for more than 100 WNV
isolates made from mosquito pools that were collected from 2003 to 2011 as part of routine surveillance
by the California Mosquito-borne Virus Surveillance System. We performed phylogeographic analyses
and demonstrated that 5 independent introductions of WNV (1 WN02 genotype strain and 4 SW03
genotype strains) occurred in California. The SW03 genotype of WNV was constrained to the
southwestern U.S. and had a more rapid rate of spread. In addition, geographic constraint of WNV strains
within a single region for up to 6 years suggest viral maintenance has been driven by resident, rather than
migratory, birds and overwintering in mosquitoes.
4. Genotypes of infectious bronchitis viruses circulating in the MiddleEast between 2009 and 2014.
Kannan Ganapathy, Christopher Ball, Anne Forrester. Virus Research 210 (2015) 198–204

Abstract :
We are reporting on the infectious bronchitis virus (IBV) genotypes circulating within seven MiddleEast
countries and the alterations in genotype distributions between 2009 and 2014. Tissue samples onFTA
cards were received over the six-year period. Viral RNA was extracted using phenol chloroform
andsubjected to nested RT-PCR targeting a 393 bp region of the S1 gene before being followed by
sequencing.From the 461 submitted samples, 363 were IBV positive by RT-PCR (77.01%). Of these, 355
(97.80%) gavesequences that can be genotyped. They belonged to six genotypes; 793B (43.66%),
IS/1494/06 (18.31%),Massachusetts (Mass) (12.96%), IS/885/00 (11.27%), Q1 (11.27%) and D274
(2.25%). The prominence of793B is not surprising, given that 793B vaccine strains are widely used in
the Middle East. Sequenceanalysis demonstrated that the majority of 793B (67.13%) and Mass (81.13%)
strains were closely relatedto vaccine strains based on 99–100% homology with the partial-S1 gene.
Vaccinal strains belonging tothe D274 genotype were present but only at a low level. Variable
proportions of 793B, Mass, D274,IS/1494/06, IS/885/00 and Q1 field strains were identified in different
countries. After 2012, the 793Bfield strain showed distinct clustering compared to strains from earlier
years. Translated amino acidalterations were minimal but still may have played an important role in the
persistence of this virusdespite the use of live 793B vaccines. Huge challenges for an efficient protection
against virulent IBVsand chicken production are posed by co-circulating793B, Mass and D274 viruses
with less than 99%homology to the respective vaccine strains, along with the recently emerged variant
IBVs, despite activeIBV vaccination strategies in the Middle East, continuous surveillance of IBV
genotypes is essential informulating optimal control strategies, including the choice and development of
new vaccine strains andformulation of vaccination programmes.
5. Identification of two amino acids within E2 important for thepathogenicity of chimeric classical
swine fever virus. Rui Wu, Ling Li, Yu Zhao, Jun Tu, Zishu Pan. Virus Research 211 (2016) 79–85

Abstract :
Our previous study demonstrated that a chimeric classical swine fever virus (CSFV) vSM/CE2
containingthe E2 gene of the vaccine C-strain on the genetic background of the virulent CSFV strain
Shimen (vSM)was attenuated in swine but reversed to virulence after serial passages in PK15 cells. To
investigatethe molecular basis of the pathogenicity, the genome of the 11th passage vSM/CE2 variant
(vSM/CE2-p11) was sequenced, and two amino acid mutations, T745I and M979K, within E2 of
vSM/CE2-p11 wereobserved. Based on reverse genetic manipulation of the chimeric cDNA clone
pSM/CE2, the mutatedviruses vSM/CE2/T745I, vSMCE2/M979K and vSM/CE2/T745I;M979K were
rescued. The data from infec-tion of pigs demonstrated that the M979K amino acid substitution was
responsible for pathogenicity.Studies in vitro indicated that T745I and M979K increased infectious virus
production and replication. Ourresults indicated that two residues located at sites 745 and 979 within E2
play a key role in determiningthe replication in vitro and pathogenicity in vivo of chimeric CSFV
vSM/CE2.
6. Pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis
and innate immune responses. Rajesh Chhabra, Suresh V Kuchipudi, Julian Chantrey, Kannan
Ganapathy. Virology 488 (2016) 232–241

Abstract :
To establish a characteristic host response to predict the pathogenicity and tissue tropism of infectious
bronchitis viruses (IBV), we investigated innate immune responses (IIR) and apoptosis in chicken
embryo kidney cells (CEKC) and tracheal organ cultures (TOC) infected with three IBV strains. Results
showed nephropathogenic IBV strains 885 and QX induced greater apoptosis in CEKC than M41, which
induced greater apoptosis in TOCs compared to 885 and QX. Elevated IIR is associated with tissue
tropism of different IBV strains. Compared to M41, 885 and QX caused greater induction of toll like
receptor 3 (TLR3), melanoma differentiation associated protein 5 (MDA5) and interferon beta (IFN-β) in
CEKC. In contrast, M41 infection caused greater expression of these genes than 885 or QX in TOCs. In
summary, greater levels of apoptosis and elevated levels of TLR3, MDA5 and IFN-β expression are
associated with increased pathogenicity of IBV strains in renal and tracheal tissues.
7. Enhanced expression of the Ernsprotein of classical swine fever virusin yeast and its application in
an indirect enzyme-linkedimmunosorbent assay for antibody differentiation of infected
fromvaccinated animals. Yuzi Luo, Lin Li, Sophia Austermann-Busch, Mei Dong, Jingjing Xu, Lina
Shao, Jianlin Lei, Na Li, Wen-Rui He, Bibo Zhao, Su Li, Yongfeng Li, Lihong Liu, Paul Becher, Yuan
Sun, Hua-Ji Qiu. Journal of Virological Methods 222 (2015) 22–27

Abstract :
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating disease of
swineworldwide. Although a mandatory vaccination with the modified live vaccine C-strain has been
imple-mented in China for decades, CSF remains a serious threat to the swine industry. To facilitate the
controland eradication of CSF in China, the E2-based marker vaccine rAdV-SFV-E2, an adenovirus-
delivered,alphavirus replicon-vectored vaccine, has been developed. Accordingly, an accompanying
discriminatorytest that allows differentiating infected from vaccinated animals (DIVA) is required. Here,
the enhancedexpression of Ernsprotein of CSFV was achieved in the methyltropic yeast Pichia pastoris
by codon-optimization of the Ernsgene, and an indirect enzyme-linked immunosorbent assay (iELISA)
based on theyeast-expressed Erns(yErns) was developed and evaluated. The optimized iELISA was able
to detect CSFV-specific antibodies in the serum samples from the CSFV-infected pigs as early as 6 days
post-infection,and discriminate the CSFV-infected pigs from those vaccinated with rAdV-SFV-E2. The
iELISA was evalu-ated using a panel of swine sera, and showed comparable sensitivity (94.6%) and
specificity (97.1%), andthe consistence rates with the virus neutralization test were 96.8% for CSFV-
infected swine sera, 83.3%for C-strain-vaccinated swine sera, and 95.0% for field swine sera. In
addition, the iELISA showed highersensitivity (90.4%) compared with PrioCHECK CSFV Erns(59.6%).
Taken together, the yErns-based iELISA isspecific and sensitive, representing a promising DIVA test for
E2-based marker vaccines against CSF.

8. Genotyping of classical swine fever virus using high-resolution melt analysis. Ilya Titov, Sodnom
Tsybanov, Alexander Malogolovkin. Journal of Virological Methods 224 (2015) 53–57

Abstract :
Discrimination between different field and vaccine strains of classical swine fever virus (CSFV) is
crucial for meaningful disease diagnosis and epidemiological investigation. In this study, a rapid method
for differentiating vaccine strains and outbreak CSFV isolates by combined RT-PCR and high-resolution
melt (HRM) analysis has been developed. The assay is based on PCR amplification of short fragments
from the most variable region of CSFVgene E2, followed by HRM analysis of amplicons. Real-Time
PCR/HRM for CSFV detection and differentiation analysis has sensitivity comparable to RT-qPCR and
genotyping resolution comparable to E2 nucleotide sequencing. This assay in one step enables rapid and
sensitive identification and genotype discrimination of CSFV in field samples, and thus will be valuable
for CSF outbreak response and disease control.
9. Construction of infectious cDNA clone derived from a classical swinefever virus field isolate in
BAC vector using in vitro overlap extensionPCR and recombination. Aman Kamboj, Mohini Saini,
Lekshmi S. Rajan, Chhabi Lal Patel, V.K. Chaturvedic,Praveen K. Gupta. Journal of Virological Methods
226 (2015) 60–66

Abstract :
To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required
which is often challenging due to many steps involved. In this study, we report cloning of full-length
cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in
vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps.
The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension
PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method
to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli
Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and
transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV
was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of
CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-
infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to
construct stable full-length cDNA clone of RNA virus in BAC vector.
10. The N-terminus of classical swine fever virus (CSFV) nonstructural protein 2 modulates viral
genome RNA replication. Ling Li, Rui Wu, Fengwei Zheng, Cheng Zhao, Zishu Pan. Virus Research
210 (2015) 90–99

Abstract :
Pestivirus nonstructural protein 2 (NS2) is a multifunctional, hydrophobic protein with an important but
poorly understood role in viral RNA replication and infectious virus production. In the present study,
based on sequence analysis, we mutated several representative conserved residues within the N-terminus
of NS2 of classical swine fever virus (CSFV) and investigated how these mutations affected viral RNA
replication and infectious virus production. Our results demonstrated that the mutation of two aspartic
acids, NS2/D60A or NS2/D60K and NS2/D78K, in the N-terminus of NS2 abolished infectious virus
production and that the substitution of arginine for alanine at position 100 (NS2/R100A) resulted in
significantly decreased viral titer. The serial passage of cells containing viral genomic RNA molecules
generated the revertants NS2/A60D, NS2/K60D and NS2/K78D, leading to the recovery of infectious
virus. In the context of the NS2/R100A mutant, the NS2/I90L mutation compensated for infectious virus
production. The regulatory roles of the indicated amino acid residues were identified to occur at the viral
RNA replication level. These results revealed a novel function for the NS2 N-terminus of CSFV in
modulating viral RNA replication.
11. Generation of a recombinant West Nile virus stably expressing theGaussia luciferase for
neutralization assay. Pan-Tao Zhang, Chao Shan, Xiao-Dan Li, Si-Qing Liu, Cheng-Lin Deng, Han-
Qing Ye, Bao-Di Shanga, Pei-Yong Shi, Ming Lv, Bei-Fen Shen, Cheng-Feng Qin, Bo Zhanga. Virus
Research 211 (2016) 17–24

Abstract :
West Nile virus (WNV) is a neurotropic human pathogen that has caused increasing infected cases
overrecent years. There is currently no licensed vaccine or effective drug for prevention and treatment
ofWNV infection in humans. To facilitate antiviral drug discovery and neutralizing antibody detection,
aWNV cDNA clone containing a luciferase reporter gene was constructed through incorporating
Gaussialuciferase (Gluc) gene within the capsid-coding region of WNV genome. Transfection of BHK-
21 cells withthe cDNA clone-derived RNA generated luciferase reporter WNV (WNV-Gluc) and the
stable WNV-Glucwith high titers (>107PFU/ml) was obtained through plaque purification. Luciferase
activity was used toeffectively quantify the viral production of WNV-Gluc. Using the reporter virus
WNV-Gluc, we developeda luciferase based assay in a 12-well format for evaluating neutralizing
antibodies. The reporter virus couldbe a powerful tool for epidemiological investigation of WNV,
vaccine evaluation, antiviral drug screening,and the study of WNV replication and pathogenesis.
12. Altered pathogenicity of a tl/CH/LDT3/03 genotype infectiousbronchitis coronavirus due to natural
recombination in the 5- 17 kbregion of the genome. Zongxi Han, Tingting Zhang, Qianqian Xu,
Mengying Gao, Yuqiu Chen, Qiuling Wang,Yan Zhao, Yuhao Shao, Huixin Li, Xiangang Kong,
Shengwang Liu. Virus Research 213 (2016) 140–148

Abstract :
An infectious bronchitis coronavirus, designated as ck/CH/LGX/130530, was isolated from an IBV
strainH120-vaccinated chicken in this study. Analysis of the S1 gene showed that isolate
ck/CH/LGX/130530 wasa tl/CH/LDT3/03-like virus, with a nucleotide sequence similarity of 99%.
However, a complete genomicsequence analysis showed that ck/CH/LGX/130530 was more closely
related to a Massachusetts typestrain (95% similarity to strain H120) than to the tl/CH/LDT3/03 strain
(86%), suggesting that recombina-tion might have occurred during the origin of the virus. A SimPlot
analysis of the complete genomicsequence confirmed this hypothesis, and it showed that isolate
ck/CH/LGX/130530 emerged from arecombination event between parental IBV H120 strain and
pathogenic tl/CH/LDT3/03-like virus. Theresults obtained from the pairwise comparison and nucleotide
similarity showed that the recombinationbreakpoint was located in the nsp14 gene at nucleotides 17055–
17083. In line with the high S1 genesequence similarity, the ck/CH/LGX/130530 isolate was
serotypically close to that of the tl/CH/LDT3/03strain (73% antigenic relatedness). Furthermore,
vaccination with the LDT3-A vaccine, which was derivedfrom the tl/CH/LDT3/03 strain by serial
passaging in chicken eggs, provided good protection againstchallenge with the tl/CH/LDT3/03 strain, in
contrast to the poor protection offered with the H120 vac-cine. Interestingly, isolate ck/CH/LGX/130530
exhibited low pathogenicity toward specific-pathogen-freechickens compared with the nephropathogenic
tl/CH/LDT3/03 strain, which was likely due to naturalrecombination in the 517-kb region of the genome.
Our results also indicate that the replicase gene ofIBV isolate ck/CH/LGX/130530 is associated with
viral pathogenicity.

13. Pathogenicity and tissue tropism of currently circulating highly pathogenic avian influenza A virus
(H5N1; clade 2.3.2) in tufted ducks (Aythya fuligula). Caroline Bröjer, Geert van Amerongen, Marco
van de Bildt, Peter van Run, Albert Osterhaus, Dolores Gavier-Widén, Thijs Kuiken. Veterinary
Microbiology 180 (2015) 273–280

Abstract :
Reports describing the isolation of highly pathogenic avian influenza (HPAI) virus (H5N1) clade 2.3.2 in
feces from apparently healthy wild birds and the seemingly lower pathogenicity of this clade compared
to clade 2.2 in several experimentally infected species, caused concern that the new clade might be
maintained in the wild bird population. To investigate whether the pathogenicity of a clade 2.3.2 virus
was lower than that of clades previously occurring in free-living wild birds in Europe, four tufted ducks
were inoculated with influenza A/duck/HongKong/1091/2011 (H5N1) clade 2.3.2 virus. The ducks were
monitored and sampled for virus excretion daily during 4 days, followed by pathologic,
immunohistochemical, and virological investigations. The virus produced severe disease as evidenced by
clinical signs, presence of marked lesions and abundant viral antigen in several tissues, especially the
central nervous system. The study shows that HPAI-H5N1 virus clade 2.3.2 is highly pathogenic for
tufted ducks and thus, they are unlikely to maintain this clade in the free-living population or serve as
long-distance vectors.
14. Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis
coronavirus in China. Lingfeng Chen, Tingting Zhang, Zongxi Han, Shuling Liang, Yang Xu, Qianqian
Xu, Yuqiu Chen, Yan Zhao, Yuhao Shao, Huixin Li, Kexiong Wang, Xiangang Kong, Shengwang Liu.
Veterinary Microbiology 181 (2015) 241–251

Abstract :
In this study, 418 IBVs were isolated in samples from 1717 chicken flocks. Twenty-nine of the isolates
were classified as the Massachusetts genotype. These 29 isolates, as well as two previously isolated
Massachusetts genotype IBV strains, were studied further. Of the 31 strains, 24 were H120-like and two
were M41-like isolates as determined by complete genomic sequence analysis, indicating that most of
the IBV isolates were likely the reisolated vaccine virus. The remaining five IBV isolates,
ck/CH/LHB/111172, ck/CH/LSD/111219, ck/CH/LHB/130598, ck/CH/LDL/110931, and
ck/CH/LHB/130573, were shown to have originated from natural recombination events between an
H120-like vaccine strain and other types of viruses. The virus cross-neutralization test found that the
antigenicity of ck/CH/LHB/111172, ck/CH/LSD/ 111219, and ck/CH/LHB/130598 was similar to that of
H120. Vaccination with the H120 vaccine offered complete protection against challenge with these
isolates. However, isolates ck/CH/LDL/110931 and ck/CH/LHB/130573 were serotypically different
from their parental viruses and from other serotypes in this study. Furthermore, vaccination with the
H120 vaccine did not provide protection against challenge with these two isolates. The results of this
study demonstrated that recombination is the mechanism that is responsible for the emergence of new
serotype strains, and it has the ability to alter virus serotypes. Therefore, IBV surveillance of chicken
flocks vaccinated with IBV live vaccines, as well as the consideration of new strategies to effectively
control IBV infection using inactivated or/and genetically engineered vaccines, is of great importance.

15. Different counteracting host immune responses to clade 2.2.1.1 and 2.2.1.2 Egyptian H5N1 highly
pathogenic avian influenza viruses in naïve and vaccinated chickens. Ahmed A. Samy, Mona I. El-
Enbaawy, Ahmed A. El-Sanousi, Soad A. Nasef, Mahmoud M. Naguib, E.M. Abdelwhab, Hirokazu
Hikono, Takehiko Saito. Veterinary Microbiology 183 (2016) 103–109

Abstract :
In Egypt, two distinct lineages of H5N1 highly pathogenic avian influenza (HPAI) viruses, “classic
2.2.1.2” and “variant 2.2.1.1” strains, have evolved. The underlying host immune responses
counteracting these viruses in chickens remain not well understood. In the present study, the cytokine
responses to a classic strain (C121) and those to a variant strain (V1063) were compared in naïve and
vaccinated chickens. In naïve chickens, the C121 replicated more efficiently than the V1063. Both the
C121 and the V1063 increased interferon (IFN)-g and interleukin (IL)-10 gene expression at 48 h post
inoculation (hpi) in the lung and spleen but the levels of these cytokines were lower in chickens infected
with the C121 than those infected with the V1063. In contrast, in chickens vaccinated with inactivated
C121-based vaccine, the C121 replicated less than the V1063. Both challenge with the C121 and that
with the V1063 did not increase IFN-g gene expression at 48 hpi; rather, the C121 increased IL-4 gene
expression in the lung accompanied with lower viral titer and higher HI titers. These results suggested
that the pathogenicity of HPAI viruses correlated with IFN-g-producing helper and/or cytotoxic T cell
responses in naïve chickens, whereas vaccine efficacy to HPAI viruses correlated with IL-4 producing
helper T cell responses in the lung in vaccinated chickens. It implies that IL-4 in the lung, in addition to
the traditional serum HI titers, could be used to screen novel vaccine strategies, such as strains, adjuvant,
prime/boost protocols, against HPAI in chickens.
16. Chronic West Nile virus infection in kea (Nestor notabilis). Tamás Bakonyia,b,1, Gyula K. Gajdonc,1,
Raoul Schwingc, Wolfgang Vogld, Annett-Carolin Häbiche, Denise Thallerf, Herbert Weissenböckf, Ivo
Rudolfg, Zdenek Hubálekg, Norbert Nowotnya. Veterinary Microbiology 183 (2016) 135–139

Abstract :
Six kea (Nestor notabilis) in human care, naturally infected with West Nile virus (WNV) lineage 2 in
Vienna, Austria, in 2008, developed mild to fatal neurological signs. WNV RNA persisted and the virus
evolved in the birds’ brains, as demonstrated by (phylo)genetic analyses of the complete viral genomes
detected in kea euthanized between 2009 and 2014. WNV antibodies persisted in the birds, too. Chronic
WNV infection in the brain might contribute to the circulation of the virus through oral transmission to
predatory birds.

17. Association between high expression macrophage migration inhibitory factor (MIF) alleles and
West Nile virus encephalitis. Rituparna Das, Kerry Loughran, Charles Murchison, Feng Qian, Lin
Leng, Yan Song, Ruth R. Montgomery, Mark Loeb, Richard Bucala. Cytokine 78 (2016) 51–54

Abstract :
Infection with mosquito-borne West Nile virus (WNV) is usually asymptomatic but can lead to severe
WNV encephalitis. The innate cytokine, macrophage migration inhibitory factor (MIF), is elevated in
patients with WNV encephalitis and promotes viral neuroinvasion and mortality in animal models. In
a case-control study, we examined functional polymorphisms in the MIF locus in a cohort of 454 North
American patients with neuroinvasive WNV disease and found patients homozygous for high-expression
MIF alleles to be >20-fold (p = 0.008) more likely to have WNV encephalitis. These data indicate that
MIF is an important determinant of severity of WNV neuropathogenesis and may be a therapeutic target.

18. Phylogenetic and pathogenic analyses of three H5N1 avian influenza viruses (clade 2.3.2.1) isolated
from wild birds in Northeast China. Zhaobin Fan, Yanpeng Ci, Liling Liu, Yixin Ma, Ying Jia, Deli
Wang, Yuntao Guan, Guobin Tian, Jianzhang Ma, Yanbing Li, Hualan Chen. Infection, Genetics and
Evolution 29 (2015) 138–145

Abstract :
From April to September 2012, periodic surveillance of avian influenza H5N1 viruses from different ild
bird species was conducted in Northeast China. Three highly pathogenic avian influenza (HPAI) H5N1
viruses were isolated from a yellow-browed warbler, common shoveler, and mallard. To trace the genetic
lineage of the isolates, nucleotide sequences of all eight gene segments were determined and
phylogenetically analyzed. The data indicated that three viruses belonged to the same antigenic virus
group: clade 2.3.2.1. To investigate the pathogenicity of these three viruses in different hosts, chickens,
ducks, and mice were inoculated. The results showed that chickens were susceptible to each of the three
HPAI H5N1 viruses, resulting in 100% mortality within 2–6 days after infection, whereas the three
solates exhibited distinctly different virulence in ducks and mice. The results of this study demonstrated
that HPAI H5N1 viruses of clade 2.3.2.1 are still circulating in wild birds through overlapping migratory
flyways. Therefore, continuous monitoring of H5N1 in both domestic and wild birds is necessary to
prevent a potentially wider outbreak.
19. Purpura fulminans associated with acute West Nile virus encephalitis. Sheevam Shah, Laura Paul
Fite, Natalie Lane, Palak Parekh. Journal of Clinical Virology 75 (2016) 1–4

Abstract :
Purpura fulminans is a progressive thrombotic disorder that presents with widespread purpura due to
deficiency or dysfunction of protein C or protein S. Lesions present as well-demarcated erythematous
macules that progress to irregular areas of hemorrhagic necrosis.West Nile virus is a member of the
Flaviviridae family transmitted to humans through the bite of various mosquito species. It manifests as
West Nile fever in 25% of those infected and less commonly as neuroinvasive disease. An African
American man in his fortiespresented with altered mental status and was noted to have evidence of
disseminated intravascular coagulation according to his lab data. He then developed dusky skin
discoloration and systemic flaccid bullae with desquamation. Biopsy was consistent with purpura
fulminans and the patient eventually developed symmetric peripheral gangrene, requiring amputations of
all four extremities. Infectious work up revealed positive testing for IgM and IgG antibodies in serum
and cerebrospinal fluid leading to the diagnosis of acute West Nile Virus encephalitis. We present this
case to describe the rarely reported association of purpura fulminans with West Nile Virus infection.

20. Emergence of novel clade 2.3.4 influenza A (H5N1) virus subgroups in Yunnan Province, China.
Tingsong Hua, Jianling Song, Wendong Zhang, Huanyun Zhao, Bofang Duan, Qingliang Liu, Wei Zeng,
Wei Qiu, Gang Chen, Yingguo Zhang, Quanshui Fan, Fuqiang Zhang. Infection, Genetics and Evolution
33 (2015) 95–100

Abstract :
From December 2013 to March 2014, a major wave of highly pathogenic avian influenza outbreak
occurred in poultry in Yunnan Province, China. We isolated and characterized eight highly pathogenic
avian influenza A (H5N1) viruses from poultry. Full genome influenza sequences and analyses have been
performed. Sequence analyses revealed that they belonged to clade 2.3.4 but did not fit within the three
defined subclades. The isolated viruses were provisional subclade 2.3.4.4e. The provisional subclade
2.3.4.4e viruses with six internal genes from avian influenza A (H5N2) viruses in 2013 were the novel
reassortant influenza A (H5N1) viruses which were associated with the outbreak of H5N1 occurred in
egg chicken farms in Yunnan Province. The HA genes were similar to subtype H5 viruses isolated from
January to March of 2014 in Asia including H5N6 and H5N8. The NA genes were most closely related to
A/chicken/Vietnam/NCVD-KA423/2013 (H5N1) from the subclade 2.3.2. The HI assay demonstrated a
lack of antigenic relatedness between clades 2.3.4.4e and 2.3.4.1 (RE-5 vaccine strain) or 2.3.2.2 (RE-6
vaccine strain). lack of antigenic relatedness between clades 2.3.4.4e and 2.3.4.1 (RE-5 vaccine strain) or
2.3.2.2 (RE-6 vaccine strain).

21. A new subgenotype 2.1d isolates of classical swine fever virus in China, 2014. Hongliang Zhang,
Chaoliang Leng, Liping Feng, Hongyue Zhai, Jiazeng Chen, Chunxiao Liu, Yun Bai, Chao Ye, Jinmei
Peng, Tongqing An, Yunchao Kan, Xuehui Cai, Zhijun Tian, Guangzhi Tong. Infection, Genetics and
Evolution 34 (2015) 94–105

Abstract :
The lapinized attenuated vaccine against classical swine fever (CSF) has been used in China for over half
a century and has generally prevented large-scale outbreaks in recent years. However, since late 2014, a
large number of new cases of CSF were detected in many immunized pig farms in China. Several of
these CSV viruses were isolated and characterized. Phylogenetic and genomic sequence analyses indicate
that these new isolates, as well as some reference isolates, form a new subgenotype named 2.1d, and
share several consistent molecular characteristics. Since these new isolates emerged in disparate
geographic regions within 5 months, this suggests that these isolates may be widespread. Given that
current vaccines do not appear to provide effective protection against this new subgenotype, further
investigation of these strains is urgently needed.