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Outline:

Brucellosis
is the second most important zoonotic disease in the world. Different Brucella spp. and their different
biovars have been reported all over the world in both animals and humans. In
Pakistan most of the work is done on animals. Most prevalent biovar of B. abortus in animals, in Pakistan is biovar
1and regarding humans, it is still unknown. The main focus of this study is to
identify, isolate and molecularly characterize the different biovars of Br.
abortus, which are prevalent in humans of Pakistan. This study will increase
the understanding regarding B. abortus
in humans in Pakistan.

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Introduction

Brucellae
are Gram-negative, nonmotile, aerobic, non-spore forming, facultative
intracellular bacilli. They have the ability to invade macrophages, epithelial
cells, dendritic cells, and placental trophoblasts (Asif et al., 2014). It has the ability to escape from the
immune system and thus can live intracellularly (Al Dahouk et al.,
2013).
On the bases of difference in host specificity the genus is divided into 10
nomo-species. Among six classical species, two major species are; B. melitensis biovar 1–3; B. abortus bv 1– 9 (Din et al., 2013). 

In Pakistan, a serious threat for expecting women
and their unborn children is Brucellosis. The seroprevalence of Brucellosis in
pregnant women with history of an abortion, having contacts to aborted women or
contact to animals that aborted were 14.6 %, 15.8 %, and 12.5 % respectively (Ali et al., 2016). In humans, it causes undulant fever, and male
sterility in humans and in animals, B.
abortus causes abortion (Din et al., 2013). Primarily the disease
is presented as fever (of unknown origin) with various clinical signs and
symptoms. These include serious focal problems such as neurobrucellosis or
Brucella endocarditis and spondylitis. Besides that failure of treatment by primary
antibiotic, relapses, and chronic disease occur frequently (Baba et al., 2001;
Grillo et al., 2006). 

Brucellosis
is primarily a contagious disease of domestic animals – goats, sheep, cattle,
swine, camels, and dogs. People are infected through ingestion of fresh milk,
cheese, and cream; through direct contact with infected animals (e.g., among
shepherds, farmers, and veterinarians); and through inhalation of infectious
aerosols (e.g., by workers in abattoirs and microbiology laboratories).
Brucellosis is a       global zoonotic
disease associated with significant morbidity that can lead to increased   rates of spontaneous abortions in livestock    and also in humans. The disease            is widely distributed   throughout the            developing world, considered to be serious problem in at
least 86 countries (Din et al., 2013).

Annually,
>500,000 cases had been reported globally. But it is still believed that
ratio of undetected cases is higher than reported cases (Geresu et al., 2016). This
disturbing circumstance can be recognized by non-speci?c clinical image of
human Brucellosis, low awareness of the disease in non-endemic areas and
shortcomings in laboratory diagnosis. The number of human cases is directly associated
with the number of infected animals within a marked area. Effective counter measures
to reduce the chance of human brucellosis which are based on surveillance and
control of livestock and pasteurization or cooking of contaminated food
products. Once the disease has been transferred from primary host to reservoir
to humans, only early diagnosis and adequate antibiotic therapy can prevent
serious sequelae in patients (Al Dahouk et al., 2013).

Pakistan shares its
boundaries with India, Iran, Afghanistan & China. Many outbreaks of
Brucellosis have been reported form these countries. WHO (World Health
Organization) declared Brucellosis as neglected disease mainly in Pakistan
(WHO, 2012). As poverty ratio is high in Pakistan (about 23.9% population is
under poverty line). So, mainly poor farmers kept ruminants for their basic
needss (Ali et al., 2014). A
study (Buchanan et al. 1974) revealed that brucellosis is an abattoir associated
disease and slaughter house workers have the greatest risk of contracting the
disease.
In a study, the
prevalence of Brucellosis among humans, based on RBPT, was 14%.  The prevalence rates of Brucellosis in cattle
and buffalo at slaughterhouses were 10% and 9.5%, respectively, with RBPT, and
8.33% and 7%, respectively, with ELISA (Hussain
et al., 2008).

The
brucellosis is diagnosed on the basis of serological, bacteriological, allergic
skin reaction and molecular methods. The most important confirmatory method of Brucella infection is bacteriological
diagnosis since its specificity is much higher than that of other diagnostic
methods and it is used as a gold standard diagnostic method (Sathyanarayanan et al., 2011).  Also currently Brucella specie is detected by molecular diagnostic methods in
various samples. The existence of different Brucella
biotypes among the Brucella spp.
and their identification in human is important to confirm the infection and trace
the source of the infection.

 

Sero-prevalence
studies showed that Brucellosis is regional in animals in Pakistan. However, the
biovars of Brucella species, which
are endemic in Pakistan are still unknown. on the basis of species-specific PCR
and biochemical tests, the evidence have been collected in previous studies that
biovar 1 of B. abortus is present in
cattle and buffaloes (Ali et al., 2014). Hence, there
is no up to date knowledge of any Pakistani investigation of Brucella in humans. Available pieces of
information related to Brucellosis especially in human beings are scarce. B. abortus biovar 1 is the cause of most
infections all over the world primarily, but distribution of biotypes also
depends on geographic differences. B.
abortus biovars 1, 2, and 4 are found in cattle in India
and confirmed by the characterization of Brucella
abortus. (Kanani 2007)

 

Aims
& Objectives

 

In
Pakistan most studies on Brucellosis were conducted on two platforms 1. organized
government livestock farms and 2. private livestock farms, and, to some extent,
in humans. There is very little knowledge is gathered about the prevalence of B. abortus in humans in Pakistan. The current study aims to isolate and
characterize the Brucella abortus and
also to investigate the biovars of B. abortus (bv)
1–6 and 9 in the human population.

 

·        
Sampling of Brucella positive human
blood from hospitals

·        
Isolation of B. abortus by using culture media (Modified Farrell’s Serum
dextrose Agar)

·        
Characterization of B. abortus through AMOS PCR

·        
Identification of different species of
Brucella and its biovars through bio-chemical tests

·        
Comparison
of molecular characteristics of the obtained isolates with other strains in the
region and outside.

 

Material
& Methods

Outline:

Brucella positive human blood samples will be collected from
hospitals of Rawalpindi and Islamabad. First, we will isolate Brucella spp. on selective media and
different biochemicals will confirm our organism. Secondly, DNA will be
extracted using kit method from human blood sample. This DNA will be amplified
in AMOS PCR and will confirm different species of Brucella. Finally, different set of tests proposed by Alton and
Jones (1967), will be performed for biovar typing of Br. abortus.

Methodology
Flow Chart

 

1.    Sample Collection

A
total of 300 Brucella positive blood
sample will be randomly collected from different hospitals of Rawalpindi and
Islamabad.

2.    Isolation

Bacteriological analysis will be performed in a
safety level-3 bio-containment facility. According to
standard procedures (Farrell and Robinson 1972; Alton et al. 1988) isolation of Brucella spp.
will be done by inoculating the samples on modified Farrell’s serum
dextrose agar. The components of modified Farrell’s serum dextrose agar are 5 %
horse serum, 1 % dextrose. In the presence of 5–10 % carbon dioxide at 37 °C,
we will inoculate the plates with samples and incubate it aerobically

Biochemical tests

Catalase,
oxidase and urease tests will confirm the Brucella isolates after isolation on
selective media.

3.    Characterization

DNA extraction from
blood samples 

We
will use DNA purification kit (MBI Fermentas, Graiciunau 8, Vilmius 2028, and
Lithuania) to extract DNA from all the blood samples, as per the directions of
manufacturer. Purified DNA pellet will be dissolved in 100 µl of double
distilled deionized water and will store at -20o C.

DNA Amplification by
PCR

Positive
samples will be subjected to AMOS PCR for species identification. The extracted
DNA samples will be amplified by PCR using specific set of primers for B. abortus.
After that amplified DNA, PCR products will be examined by using Gel Electrophoresis.

Biovar Typing of B.
abortus

Brucella isolates, confirmed by results of
catalase, oxidase, and urease tests, will be examined for biovar typing
according to the standard methods described by Alton et al. Briefly, biovars will
be identified based on agglutination with A and M monospecific antisera, CO2 requirement for growth, H2S production, and growth on media containing
20??g/mL basic fuchsin and thionin dyes.

 

 

 

 

 

 

Gantt
chart

Activity

1st Month

2nd Month

3rd Month

4th Month

5th Month

6th Month

7th Month

8th Month

9th Month

10th Month

Human Blood Sampling

 

 

 

 

 

 

 

 

 

 

Isolation via Selective media

 

 

 

 

 

 

 

 

 

 

Bio-Chemical Tests for B. abortus

 

 

 

 

 

 

 

 

 

 

Mol. Characterization by AMOS PCR & Biovar Typing

 

 

 

 

 

 

 

 

 

 

 

References

AL DAHOUK, S., SPRAGUE, L. &
NEUBAUER, H. 2013. New developments in the diagnostic procedures for zoonotic
brucellosis in humans. Rev Sci Tech,
32, 177-88.

 

ALI, S., AKHTER,
S., NEUBAUER, H., SCHERAG, A., KESSELMEIER, M., MELZER, F., KHAN, I., EL-ADAWY,
H., AZAM, A. & QADEER, S. 2016. Brucellosis in pregnant women from
Pakistan: an observational study. BMC
infectious diseases, 16, 468.

 

ALI, S., ALI, Q.,
MELZER, F., KHAN, I., AKHTER, S., NEUBAUER, H. & JAMAL, S. M. 2014.
Isolation and identification of bovine Brucella isolates from Pakistan by
biochemical tests and PCR. Tropical
animal health and production, 46,
73-78.

 

Alton, G.G.,
Jones, L.M., Angus, R.D., Verger, J.M., 1988. Bacteriological methods. In: G.G.
Alton, L.M. Jones, R.D. Angus and J.M. Verger (eds), Techniques for the
Brucellosis Laboratory. Paris: Institut National De La Recherche Agronomique,
pp 13–61.

 

ASIF, M., WAHEED,
U., FAROOQ, M., ALI, T. & KHAN, Q. M. 2014. Frequency of Brucellosis in
High Risk Human Groups in Pakistan Detected through Polymerase Chain Reaction
and its Comparison with Conventional Slide Agglutination Test. International Journal of Agriculture &
Biology, 16.

 

Baba
MM, SE Sarkindared and F Brisibe, 2001. Serological evidence of brucellosis
among predisposed patients with pyrexia of unknown origin in the north eastern
Nigeria. Cent Eur J Pub Health, 9: 158– 161.

Buchanan,
T.M., Sulzer, C.R., Frix, M.K., Feldman, R.A.: Brucellosis in the United
States, 1960-1972. An abattoir associated disease, Part II. Diagnostic aspects.
Medicine, 1974: 53; 415-425.

 

DIN,
A., KHAN, S., AHMAD, I., RIND, R., HUSSAIN, T., SHAHID, M. & AHMED, S.
2013. A study on the seroprevalence of brucellosis in human and goat
populations of district Bhimber, Azad Jammu and Kashmir. J. Anim. Plant Sci, 23,
113-118

 

Farrell,
I.D., Robinson, L., 1972. A comparison of various selective media, including a
new selective medium for the isolation of brucellae from milk. Journal of
Applied Bacteriology, 35, 625–630.

 

GERESU,
M. A., AMENI, G., WUBETE, A., ARENAS-GAMBOA, A. M. & KASSA, G. M. 2016.
Isolation and Identification of Brucella Species from Dairy Cattle by
Biochemical Tests: The First Report from Ethiopia. World, 6, 80-88.

 Grillo MJ, MJ DeMiguel, PM Munoz, CM Marin, J
Ariza and JM Blasco, 2006. Efficacy of several antiniotic combinations against
Brucella melitensis Rev1 experimental infection in BALB/c mice. J Antimicrob
Chemother, 58: 622–626.

 

HUSSAIN,
I., ARSHAD, M. I., MAHMOOD, M. S. & AKHTAR, M. 2008. Seroprevalence of
brucellosis in human, cattle, and buffalo populations in Pakistan. Turkish Journal of Veterinary and Animal
Sciences, 32, 315-318.

Kanani,
A.N., 2007. Serological, cultural and molecular detection of Brucella infection
in breeding bulls. PhD thesis, Anand Agricultural University, India.

SATHYANARAYANAN,
V., RAZAK, A., SARAVU, K., ANANTHAKRISHNA, S.B., MUKHYPRANA, P.M. AND VANDANA,
K.E., 2011. Clinical profile of brucellosis from a tertiary care center in
southern India. Asian Pac. J. trop. Med., 397-400.

 

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