Type of the Paper (Article
LMRJ Volume 3 Issue 3 70 | P a g e
Research article
RICKETTSIAL AGENTS DETECTION FROM BLOOD OF TICK-INFESTED ANIMALS IN
LOWER SINDH- COMPARISON OF CONVENTIONAL AND MOLECULAR APPROACH
Iram Shaikh1,*, Naheed Baloch1, Abdullah G. Arijo2, Riffat Sultana1 and Saleem Raza Solangi1
1Department of Zoology, University of Sindh, Jamshoro-Pakistan. 2Department of Veterinary Parasitology,
Sindh Agriculture University, Tando Jam-Pakistan.
ABSTRACT
Ticks are important vectors of human and animal pathogens. They are consid-
ered as main vectors for transmission of rickettsial agents affecting animal and
human health. The study was designed to investigate district wise pattern and
detection of rickettsial agents by using molecular and conventional techniques
in blood samples of infected cattles, buffalos, sheep and goats. A survey study
was carried out in lower Sindh (Tharparkar, Badin, Hyderabad, Karachi, Tando
Muhammad khan, Thatta and Mirpurkhas). Blood samples were collected ran-
domly from infected Cattles, buffalos, sheep and goats and transported to the
Molecular Parasitology laboratory, Sindh Agriculture University, Tandojam,
followed by examinations under stereomicroscope and Polymerase Chain Re-
action (PCR). The study showed that overall infection of Rickettsial agents
among infected animals was recorded following Microscopy/ Blood smear test
in cattles, buffalos, sheep and goats was 41.79, 49.09, 46 and 41.66% respectively,
whereas overall infection through PCR in cattle, buffalo, sheep and goat was
39.55, 43.55, 46 and 55.55% respectively. Whereas animal-wise data through
PCR indicates that in case of Goats (55.55%) were more susceptible to rickettsial
infection as compared to sheep (46%), buffaloes (43.55%) and cattle (39.55%).
The highest rate of rickettsial agents was found in district Tharparkar and low-
est rate was found in district Karachi. Microscopy/Blood smear method indi-
cates that Buffaloes were more susceptible for infection. Whereas PCR indicates
Goats were more susceptible for infection.
Key Words: Ticks (Vectors), Rickettsial agents (Pathogens), Molecular and Conventional techniques, Lower Sindh
INTRODUCTION
Ticks are potential vectors and reservoirs of many infectious agents such as. Pasteurella multocida, brucella
abortus and salmonella typhimurium in both humans as well as animals (1). Ticks have enormous capability
to adapt to changing geoclimatic conditions and can therefore expand their distribution range (2). They are
known as main vectors for transmission of many pathogens such as viral, bacterial, rickettsial and parasitic
infestations (3). Till date 899 species of ticks are known which belong to three families, namely Ixodidae,
Argasidae and Nuttalliellidae (represented by a mono- typic species restricted to South Africa (4). After suck-
ing blood, the outer surface of a tick grows to 200-600 times as compared to its unfed body weight (5). Prev-
alence of tick-borne pathogens (TBPs) and their occurrence in bovines have been found all over the Pakistan
(6). Hyalomma anatolicum transmitted some of tick-borne pathogens (TBP) which have zoonotic importance
(e.g. Crimean Congo haemorrhagic fever) (6). Generally, rickettsioses is the term used for those diseases
which have continuous spectrum of severity of illness and overlapping clinical manifestations. R. rickettsii,
R. prowazekii, R. conorii, and R. typhi are rickettsial agents with a potential to cause life-threatening diseases
(7). The main cause of granulocytic anaplasmosis is Anaplasma phagocytophilum which is considered as one
of the most important species from humans’ point of view because of its zoonotic potential.
Correspondence:
Iram Shaikh,
Department of Zoology,
University of Sindh,
Jamshoro, Sindh, Paki-
stan
Email: shaikh-
iram37@gmail.com
DOI:
10.38106/LMRJ.2021.3.03-
05
Received: 03.09.2021
Accepted: 15. 09.2021
Published: 30. 09.2021
LMRJ Volume 3 Issue 3 71 | P a g e
Ticks are known as etiological agents of tick-borne fever in ruminants and equine, canine and human gran-
ulocytic anaplasmosis (EGA, CGA and HGA, respectively) (8). Genetic diversity has been recognized among
various European strains of A. phagocytophilum shown through phylogenetically analysis of genes such as
groEL (chaperone protein encoding gene) (8) Rickettsiae are commonly defined as genetically related, oblig-
atory intracellular bacteria that reside in an arthropod host during a part of their zoonotic cycle. (9) Ticks as
parasites are vectors of many important human and animal pathogens such as Q fever Babesiosis, tick paral-
ysis, haemorrhagic fever, Lyme disease (LD), tick-borne encephalitis and tick-borne muscular fever. Rocky
mountain spotted fever, which is caused by Rickettsia rickettsii, is a life-threatening, tick-borne disease that
occurs throughout much of the United States (11). It has been estimated that 10% of the known tick species
act as vectors of the pathogens of above mentioned diseases (12). They also pose a great threat to global
animal production in terms of economic expenditure incurred through treatment of various inflammatory
and hematologic conditions that occurred in humans and animals through these tick-borne diseases. It has
further been suggested that around 80% of cattle production worldwide is at increased risk of tick-borne
infections. (6) Rickettsia have a comparatively small genome developed though reductive evolution because
of their dependence on the host for survival and to carry out essential functions. (18) The genomes of various
species of rickettsia have been sequenced such as Rickettsia prowazekii and Rickettsia conorii (19). There was
limited literature available looking at the presence of rickettsial organisms in ticks in lower Sindh. Therefore,
this study was aimed to investigate district wise pattern and compare detection of rickettsial agents by using
molecular and conventional techniques in blood samples of infected cattles, buffalos, sheep and goats.
METHODOLOGY
A survey was conducted in lower Sindh including Tharparkar, Badin, Hyderabad, Karachi, Tando Muham-
mad khan, Thatta and Mirpurkhas districts. Blood samples were collected randomly from infected Cattle,
buffalos, sheep and goats and transported to the Molecular Parasitology laboratory, Sindh Agriculture Uni-
versity, Tandojam, followed by examinations under stereomicroscope and Polymerase Chain Reaction
(PCR)(24).
Blood Collection
Host that carried ticks were selected for blood sampling, 5 ml blood from each infested host was collected
from jugular vein or ear vein from large and small animals respectively. The blood transferred to Ethylene
diamine tetra acetic acid (EDTA) containing tubes and stored until further diagnosis of pathogen (Viz. Blood
Filming and DNA Extraction) was carried out.
Blood Sampling Procedure
Hairs from collection site were removed using automatic hair shaver. Cotton swab soaked in the antiseptic
(alcohol) was applied for disinfection to avoid any secondary contamination in the sample. Ear vein was
gently punctured with sterilized needle and blood was allowed to ooze out. A thin and thick blood smear
was prepared fixed on spot in 70% alcohol to avoid rupturing of erythrocytes. In case of collection from
jugular vein, syringe was gently used and 5ml of blood was drawn and preserved in EDTA tubes. The blood
vials were soaked by rotating between palms of two hands for proper mixing of the anti-coagulant. The
collection tubes were labelled with the name of owner, type of host and refrigerated at -20˚ C. Relevant in-
formation on host, sex, age and date of collection was obtained and recorded on a Proformma specifically
designed for this project.
Blood Smear Method
Two methods were applied for blood examination viz. thin and thick blood smear(s).
Thin Smear method
For making thin blood smear, a glass slide was dipped in 95% alcohol. About 2ul of blood was placed on one
end of the slide (called microscopic slide). Another slide (called spreader slide) was placed on microscopic
LMRJ Volume 3 Issue 3 72 | P a g e
slide containing the droplet of blood, positioning it about an inch in front of the droplet. The spreader slide
was quickly run on the surface of microscopic slide at angle of 45 degrees. In a smooth motion, the spreader
slide was pushed forward to spread the blood in a layer. Prepared blood slide was allowed to air dry for one
minute and fixed in absolute alcohol for 5 minutes. Slides were removed from alcohol jars and air dried.
Dried slides were stained in freshly prepared Romanowisky stain (commonly called Giemsa’s stain) for 5
minutes.
Thick Smear method
Procedure for making thick blood smear was same except that the spreader slide was moved slowly to make
a thick film on microscopic slide.
Nucleic Acid Extraction from Blood
DNA was extracted and obtained from collected blood by commercial kit (GeneJET Genomic DNA purifica-
tion Kit #K0722, Thermo Scientific, USA) as per manufacturer’s instructions. 20 µl of Proteinase-K solution
and 400 µl of lysis solution were added to 200 µl of whole blood. The mixture was mixed by vortexing in
order to obtain a uniform suspension. It was then kept in incubation at 56˚ C for around 10 minutes or till the
cells were completely hemolysed. Afterwards, ethanol in a quantity of 200 µl was added and vortexed. The
solution obtained was then transferred to GeneJET genomic purification column and was centrifuged at
6000xg for up to one minute. The flow through solution in the collection tube was discarded whereas purifi-
cation column was transferred in a new collection tube. Wash buffer (500 µl) was added to this collection
tube which was then centrifuged at 8000xg for one minute. The flow through solution was again discarded
while transferring the purification column to a new collection tube to which 500 µl of wash buffer 2 (with
ethanol already added) was added and further centrifuged at 12000xg for three minutes. The purification
column was transferred into 1.5ml micro tube whereas collection tube containing flow through solution was
again discarded. 200 µl of elution buffer was added to 1.5 ml micro tube containing purification column and
it was then incubated at room temperature for two minutes and then centrifuged at 8000xg for one minute.
The DNA thus extracted is obtained by discarding the supernatant and its concentration was evaluated by
spectrophotometer (Thermo scientific Nano drop 1000).
PCR Process
Table-1 shows components and volume used in PCR process, the sample tubes were loaded in Thermal cycles
(Applied Bio- system, USA). The cycles were already set. The lid of machine was closed to start the operation.
DNA was denatured at 94oC for 5 min. Annealing process took place at 550C for 1 min. Two complementary
copies of DNA were obtained from one DNA at 72⁰C for I min, the cycle again started from 940C. The PCR
product was subjected to electrophoresis.
Table -1 Components used in PCR process.
Components Volume
Master mix 25 µl
Piro Primer(F) 8 µl
Piro Primer(R) 8 µl
DNA extract 2 µl
Distilled water 7 µl
Total 50 µl
Method
All primers were diluted with 20ul of TE Buffer. Piro primer (F) =8µl was added in 25 µl of master mix in a
small tube (Neptune company). Piro primer (R) =8µl was added in 25 µl of master mix in a small tube (Nep-
tune company). 2µl of DNA extract were added in Piro (F) and (R) primers respectively.
Agarose Gel (1%)
LMRJ Volume 3 Issue 3 73 | P a g e
Agarose gel powder was taken in a quantity of 0.5 grams in a conical flask. 50 ml of 0.5 TAE buffer was then
added to the agarose powder and microwaved for about one minute in order to dissolve the powder. It was
then allowed to cool down to 60˚ C. afterwards, 2 µl of ethidium bromide was added to gel solution and it
was then poured down slowly into the tank. Comb was correctly positioned in the tank and it was then left
for at least 30 minutes to solidify. Before using the gel, it was submerged in 0.5 TAE buffer in the tank.
Table-2 Primers used
Primers Nucleotides Species References
PIRO-F AATACCCAATCCTGACACAGGG
All Piroplasms Karimi et al.,2012
PIRO-R TTAAATACGAATGCCCCCAAC
All Piroplasms Karimi et al.,2012
Bi-F AATAACAATACAGGGCTTTCGTCT
Babesia
bigemina
Kim et al., 2007
Bi-R ACGCGAGGCTGAAATACAAC
Babesia
bigemina
Kim et al., 2007
T. annulata-F CACCTTCGACAAGAAAGAAGTCGG Theileria
Designed in
Mather lab, USA
T.annulata-R TGAGAAGACGATGAGTACTGAGGC
Theileria
Designed in
Mather lab, USA
Sample loading
Before loading the samples in the gel, DNA ladder (Fermantas EU) was loaded down in the very first well of
agarose gel in order to quantify the size of the samples. Afterwards, 4 µl of each sample was loaded in the
subsequent wells. After all samples have been added, electrophoresis unit was allowed to run with 80 volts
and 100 amperes for 30-45 minutes allowing samples to travel a sufficient distance.
Gel Documentation
After electrophoresing the samples, the gel was removed and put in gel documentation system (Cleaver
Scientific, Ltd, UK) in order to visualized the bands of samples and to determine their size by comparing
them with the ladder.
Statistical methods
Statistical package for Social Sciences (SPSS Version 21) was used for results analysis. Frequencies and per-
centages were analysed and presented.
RESULTS
Blood samples were collected from different districts of lower Sindh, in order to compare sensitivity of pol-
ymerase chain reaction with that of blood smear method, Table-3 and 4 reveals the detection of rickettsial
agents through blood smear method then blood samples were subjected to PCR for detection of rickettsial
infection. Table-5,6 and 7 reveals that PCR is more sensitive diagnostic method as samples that were negative
via blood smear method were found positive when diagnosed through PCR. Presence of rickettsial agents
through blood smear test was confirmed under high power magnification, whereas through PCR the detec-
LMRJ Volume 3 Issue 3 74 | P a g e
tion was confirmed by looking at the bands that appeared at 405, 150, 170 and 290 base pairs on gel docu-
mentation. (Figure-1-3), Table- 3 to 6 shows data on Cattle, Buffalo, Sheep and Goat that were diagnosed
positive for rickettsial agents via blood smear test and PCR, the highest ratio of differences in districts was
found in District Tharparkar 83:83, 81:87, 66:66, 57:71 in cattle, buffalo, sheep and goat, whereas lowest ratio
of differences is found in district Karachi was 36:30, 45:41, 25:25, 28:42 in cattle, buffalo, sheep and goat re-
spectively.
Table 3. Detection of rickettsial agents in Cattle and Buffalo through blood smear method in lower Sindh
Districts
CATTLE
BUFFALO
Observed Infested Random
blood
samples
Infected
samples
(%)
Observed Infested Random
blood
samples
Infected
samples%
Karachi 96 33 12 36.36 117 24 11 45.83
Hyderabad 27 20 8 40 67 33 14 42.42
Badin 27 17 6 35.29 26 15 7 46.66
Tharparkar 10 6 5 83.33 20 16 13 81.25
T.M Khan 59 24 10 41.66 119 35 16 45.71
Mirpurkhas 37 24 11 45.83 73 29 14 48.27
Thatta 16 10 4 40.00 44 11 5 45.45
Total 272 134 56 41.79 466 163 80 49.07
Table 4. Detection of rickettsial agents in Sheep and Goat through blood smear method in lower Sindh
Districts
SHEEP GOAT
Observed Infested Random
blood
samples
Infected
samples
%
Observed Infested Random
blood
samples
Infected samples %
Karachi 11 4 1 25 32 7 2 28.57
Hyderabad 15 6 2 33.33 10 3 1 33.33
Badin 18 14 5 35.71 16 5 2 40
Tharparkar 12 3 2 66.66 13 7 4 57.14
T.M Khan 15 5 2 40.00 16 5 2 40
Mirpurkhas 9 3 1 33.33 12 4 2 50.00
Thatta 14 4 2 50.00 10 5 2 40.00
Total 94 39 15 38.46 109 36 15 41.66
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Table 5. Detection of rickettsial agents in Cattle and Buffalo through PCR in lower Sindh
Districts
CATTLE BUFFALO
Observed Infested At Ran-
dom
blood
samples
PCR detec-
tion(%)
Observed Infested At Ran-
dom
blood
samples
PCR detec-
tion%
Karachi 27 20 6 30 67 33 9 27.27
Hyderabad 96 33 10 30.30 117 24 10 41.66
Badin 27 17 7 41.17 26 15 6 40
Tharparkar 10 6 5 83.33 20 16 14 87.5
T.M Khan 59 24 9 37.5 119 35 13 37.14
Mirpurkhas 37 24 10 41.66 73 29 12 41.37
Thatta 16 10 6 60.00 44 11 7 63.63
Total 272 134 53 39.55 466 163 71 43.55
Table 6. Detection of rickettsial agents in Sheep and Goat through PCR in lower Sindh
Districts SHEEP GOAT
Observed In-
fested
At Ran-
dom
blood
samples
PCR de-
tection%
Observed Infested At Ran-
dom blood
samples
PCR detec-
tion%
Karachi 11 4 1 25 10 3 1 33.33
Hyderabad 15 6 2 33.33 32 7 3 42.85
Badin 18 14 5 35.71 16 5 3 60
Tharparkar 12 3 2 66.66 13 7 5 71.42
T.M Khan 15 5 2 40.00 16 5 3 60
Mirpurkhas 9 3 1 33.33 12 4 2 50.00
Thatta 14 4 2 50.00 10 5 3 60.00
Total 94 39 15 38.46 109 36 20 55.55
Graph-1 showing the comparison between Microscopy
and PCR
Figure-1 Gel electrophoresis of amplified PCR
product of rickettsial agents of buffalo blood DNA
LMRJ Volume 3 Issue 3 76 | P a g e
Table-7 shows the pooled data of all districts indicates that through blood smear test Buffaloes were (49.09%)
more susceptible to rickettsial infection as compared to sheep (46%), cattle (41.79%) and goats (41.66%),
Whereas through PCR, the data indicates that in case of goats 55.55% were more susceptible to rickettsial
infection as compared to sheep (46%), Buffaloes (43.55) and cattle (39.55%).
Table 7. Pure and mixed infection of rickettsial agents in cattle, buffalo, sheep and goat
Figure-2 Gel electrophoresis of amplified PCR
product of rickettsial agents of Cattle blood DNA
Figure-3 Gel electrophoresis of amplified
PCR product of rickettsia agents of Buffalo
blood DNA
DISCUSSION
The blood samples of tick-carrying cattle, buffalo, sheep and goat conventionally confirmed blood samples
were subjected for PCR detection of Piroplasms, for this purpose, DNA was extracted from positive blood
samples and quantified on Nano-drop spectrophotometer.
Primers used for PCR reaction are described in Table 2. Different concentrations of MgCl2 were used for PCR
reaction i.e. 5ul & 6 ul for T. annulata whereas for B. bovis and B. bigemina concentration of MgCl2 was 3ul.
PCR was done for 30 cycles with following conditions: Denaturation at 94˚ C for 5 min, 94˚ C for 30 sec. Tem-
perature was lowered for several minutes to allow both forward and backward (right or left) primers to
anneal with the complementary sequences. At this stage three conditions 50˚ C, 55˚ C and 60˚ C for 30 secs
S.
No.
Name
Of
Animal
Microscopy PCR
Total
no. of
Ani-
mals
ob-
served
Total
no. of
animals
in-
fested
At Ran-
dom
blood
samples
% of in-
fected
samples
through
Smear
method
Total no.
of Ani-
mals ob-
served
Total
no. of
animals
in-
fested
At Random
blood sam-
ples
% of infected
samples
through
PCR
01 Cattle 272 134 56 41.79% 272 134 53 39.55%
02 Buffalo 466 163 80 49.09% 466 163 71 43.55%
03 Sheep 99 50 23 46% 99 50 23 46%
04 Goat 109 36 15 41.66% 109 36 20 55.55%
Total 946 383 174 45.43% 946 383 167 43.60%
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were checked for each primer set. Finally, extension was carried out 72˚C for 45 secs. Analysis of amplified
product by electrophoresis was done with a 1% agarose gel.
The results were photographed with Gel Documentation System (Gel Doc USA). In order to compare sensi-
tivity of polymerase chain reaction, with that of blood smear method, blood samples were subjected to PCR
detection of rickettsial infection. (2) gave findings take out at Maharashtra (India) by (3) Boophilus, Haema-
physalis, Hyalomma, Amblyomma, Nosoma and Rhipicephalus were found tick infesting in subfamily Bovinae
animals, which includes cattle, buffalo, and kudus at 40, 16.96, 20.14, 10.22, 4.56, and 1.96 percent attentive-
ness, correspondingly. He discovered 8 different tick genera of ticks to be precise as Boophilus, Rhipicephalus,
Hyalomma, Amblyomma, Dermacentor, Haemaphysalis, Ixodes, and Aponoma from many segments of Pakistan.
The lessen quantity of the genera perceived possibly would be the reason of looked-for the partial region
stipulated for the present investigation, also in a partial investigation takeout by (4) and (5) stated 4 genera,
even if dissimilar from every one, for the tick troublesome invasion resident of a tract of land on which crops
and often livestock are raised for livelihood in their particular investigation.
CONCLUSION
Information regarding to cattle, buffalo, sheep and goat farms (946 observed animals out of which 383 were
the infested animals and 45.43% were infected animals through Microscopy and 43.60% were infected
through PCR) According to Microscopy Buffaloes were more susceptible to rickettsial infection as compared
to cattle, sheep and goat. According to PCR Goats were more susceptible to rickettsial infection as compared
to cattle, buffalo and sheep, Highest rate of rickettsial infection is found in district Tharparkar. Lowest rate
of rickettsial infection is found in district Karachi.
Ethical Consideration: The study was approved by the Ethical committee of xxxx
Conflict of Interest: There is no conflict of interest.
Funding: This study was not funded by any agency
REFERENCES
1. Jongejan F, Uilenberg G. The global importance of ticks. Parasitology. 2004;129(SUPPL.):129.
2. Buczek A, Bartosik K, Buczek AM, Buczek W, Stanko M. Conspecific hyperparasitism in the hyalomma
excavatum tickand considerations on the biological and epidemiological implications of this phenomenon.
Ann Agric Environ Med. 2019 Aug 6;26(4):548–54.
3. Ganjali M, Dabirzadeh M, Sargolzaie M. Species diversity and distribution of ticks (Acari: Ixodidae) in
Zabol County, Eastern Iran. J Arthropod Borne Dis [Internet]. 2014 Dec [cited 2020 Jan 8];8(2):219–23. Avail-
able from: http://www.ncbi.nlm.nih.gov/pubmed/26114136
4. Yu Z, Wang H, Wang T, Sun W, Yang X, Liu J. Tick-borne pathogens and the vector potential of ticks in
China. Vol. 8, Parasites and Vectors. BioMed Central Ltd.; 2015.
5. D.E. S. Biology of ticks. New York. Oxford Univ Press. 1991; 1:1–449.
6. Ghafar A, Cabezas-Cruz A, Galon C, Obregon D, Gasser RB, Moutailler S, et al. Bovine ticks harbour a
diverse array of microorganisms in Pakistan. Parasit Vectors [Internet]. 2020 Jan 3 [cited 2020 Jan 7];13(1):1.
Available from: http://www.ncbi.nlm.nih.gov/pubmed/31900233
7. Dabaja MF, Tempesta M, Bayan A, Vesco G, Greco G, Torina A, et al. Varietà e distribuzione di zecche
in ruminanti domestici in Libano. Vet Ital. 2017 Apr 1;53(2):147–55.
8. Matei IA, Estrada-Peña A, Cutler SJ, Vayssier-Taussat M, Varela-Castro L, Potkonjak A, et al. A review
on the eco-epidemiology and clinical management of human granulocytic anaplasmosis and its agent in
Europe. Parasites and Vectors [Internet]. 2019 Dec 21 [cited 2020 Jan 7];12(1):599. Available from:
http://www.ncbi.nlm.nih.gov/pubmed/31864403
LMRJ Volume 3 Issue 3 78 | P a g e
9. Sahni A, Fang R, Sahni SK, Walker DH. Pathogenesis of Rickettsial Diseases: Pathogenic and Immune
Mechanisms of an Endotheliotropic Infection. Annu Rev Pathol Mech Dis [Internet]. 2019 Jan 24 [cited 2020
Jan 22];14(1):127–52. Available from: https://www.annualreviews.org/doi/10.1146/annurev-pathmechdis-
012418-012800
10. Díaz-Sánchez AA, Meli ML, Obregón Álvarez D, Fonseca-Rodríguez O, Cabezas-Cruz A, Hofmann-
Lehmann R, et al. Development and application of a multiplex TaqMan® real-time qPCR assay for the sim-
ultaneous detection of Anaplasma marginale and Theileria annulata and molecular characterization of An-
aplasma marginale from cattle in Western Cuba. Ticks Tick Borne Dis. 2020;11(2).
11. Demma LJ, Traeger MS, Nicholson WL, Paddock CD, Blau DM, Eremeeva ME, et al. Rocky Mountain
spotted fever from an unexpected tick vector in Arizona. N Engl J Med. 2005 Aug 11;353(6):587–94.
12. Zhang G, Zheng D, Tian Y, Li S. A dataset of distribution and diversity of ticks in China. Sci data. 2019
Jul 1;6(1):105.
13. Richards AL. Worldwide detection and identification of new and old rickettsiae and rickettsial diseases.
FEMS Immunol Med Microbiol. 2012 Feb;64(1):107–10.
14. Labruna MB, Whitworth T, Bouyer DH, McBride J, Camargo LMA, Camargo EP, et al. Rickettsia
bellii and Rickettsia amblyommii in Amblyomma Ticks from the State of Rondônia, West-
ern Amazon, Brazil. J Med Entomol. 2009 Oct 29;41(6):1073–81.
15. La Scola B, Raoult D. Laboratory diagnosis of Rickettsioses: Current approaches to diagnosis of old and
new Rickettsial diseases. Vol. 35, Journal of Clinical Microbiology. 1997. p. 2715–27.
16. Petri A.W. Overview of Rickettsial Infections - Infections - MSD Manual Consumer Version [Internet].
2018 [cited 2020 Feb 11]. Available from: https://www.msdmanuals.com/home/infections/rickettsial-and-re-
lated-infections/overview-of-rickettsial-infections
17. Venzal JM, Portillo A, Estrada-Peña A, Castro O, Cabrera PA, Oteo JA. Rickettsia parkeri in Am-
blyomma triste from Uruguay. Emerg Infect Dis. 2004;10(8):1493–5.
18. McLeod MP, Qin X, Karpathy SE, Gioia J, Highlander SK, Fox GE, et al. Complete genome sequence of
Rickettsia typhi and comparison with sequences of other rickettsiae. J Bacteriol. 2004 Sep;186(17):5842–55.
19. Martinez JJ, Seveau S, Veiga E, Matsuyama S, Cossart P. Ku70, a component of DNA-dependent protein
kinase, is a mammalian receptor for Rickettsia conorii. Cell. 2005 Dec 16;123(6):1013–23.
20. Mc Carthy VG. Ixodid ticks (Acarina: Ixodidae) of West Pakistan. PhD Thesis Univ Maryl. 1967;1–533.
21. Hines, S.A., G.H. Palmer, D.P., Jasmer WLG and TFM. Immunization of cattle with recombinant Babesia
bovis merozoite surface antigen-1 Infection and Immunity. 1998; 63:349-52.
22. Hussain S. Studies on ectoparasites of livestock of Sindh. Final Tech Report, Univ Sindh, Pakistan.
23. Khan MI. Taxonomical study of ticks of genus Rhipicephalus and their relation to the incidence of
haemoparasites and comparative efficacy of different acaricides on ticks in sheep and goats in Kaghan val-
ley. MSc Thesis, Coll Vet Sci Lahore, Pakistan. 1993;
24. Shaikh. I, A. G Arijo, N. Akhter, Saud Farooque, Comparing Blood Smear Test and PCR on Detection of Babesia
in Tick-infested Cattle and Buffalo. Proceedings of Parasitology, Dec.2012;54; 83-91.