Thesis-MS

Browse

Now showing 1 - 20 of 30

Histopathological examination and optimization of Multiplex PCR protocol for diagnosis of viral respiratory diseases in commercial poultry
(A thesis submitted in the partial fulfillment of the requirements for the degree of Master of Science (MS) in Pathology Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Khulshi, Chittagong-4225, Bangladesh, 2013-06) Pervin, Shahnaj
The present work was to optimize a multiplex PCR protocol for rapid and accurate diagnosis of avian respiratory viral diseases. Four sets of specific oligonucleotide primers were used in this study that amplified products of predicted sizes from each virus in the sRT-PCR as well as in the mPCR assays (1023, 320, 647, 149 bp for AIV, NDV, ILTV, and IBV respectively) with the use of 1% agarose gel electrophoresis for PCR amplified products. A total of 48 specimen samples included trachea, lungs, liver, spleen, proventriculus etc. were collected from different dead birds that were tentatively diagnosed as respiratory viral infection on the basis of anamnesis and postmortem lesions by the expert veterinary clinician. Samples were preserved in 10% buffered formalin for histopathological study and in 100% alcohol with -80oC refrizaration for molecular study like RNA/DNA extraction for PCR or RT-PCR. A questionnaire was used during the period from August 2013 to March 2014 to record additional information and history of the cases such as age and type of birds, litter type, vaccination history, farm category, sex, pathological lesions, histopathology etc. Out of 48 cases 35.42% was tentatively diagnosed as AI, 33.33% was ND and 12.25% was IB where visceral gout was most prominent lesion. No ILT was diagnosed tentatively through necropsy. Mixed infection is common in commercial chicken flock, among the cases 4.17% was diagnosed as AI+ND, 12.25% and 2.08% was diagnosed as AI/ ND and IB/ND respectively. Among 48 test samples both the PCR screening process a total of 33(68.75%) samples showed positive band in 1% agarose gel electrophoresis where 15(31.25%), 16(33.33%) and 2(4.16%) samples were found positive for AI, ND and IB respectively. No ILT was found in this screening. Among the diseases AI was diagnosed accurately in 70.58% cases through necropsy and 81.25% and 33.33% for ND and IB respectively. ILT was not diagnosed tentatively which was also proved by molecular diagnosis that indicates the 100% accuracy of the tentative diagnosis. Microscopically congestion and hemorrhage in trachea, lungs, liver and spleen were found commonly in most of the samples, in addition with exudates in trachea and hemorrhagic kidney with urate crystal in renal tubules in IB infected sample. Hemorrhage in proventriculus is also found in only AI and ND infected cases.
Molecular Investigation of Cryptosporidium Spp. in Stray and Pet dog at Chittagong Metropolitan Area
(A thesis submitted in the partial fulfillment of the requirements for the degree of Master of Science (MS) in Pathology Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Khulshi, Chittagong-4225, Bangladesh, 2014-06) Islam, Md. Shafiqul
Cryptosporidium is a zoonotic pathogen composed of genetically distinct but morphologically identical genotypes. Recent molecular study indicates that dogs may transmit the cattle genotype, which is known to be pathogenic to humans. The aim of the present study was to evaluate the present condition of Cryptosporidium infection in stray and pet dogs at Chittagong metropolitan area in Bangladesh. One hundred and thirty (130) non diarrheic fecal samples obtained from 98 stray dogs and 32 pet dogs were investigated during the period from January 2014 to May 2014. Initial identification of Cryptosporidium spp. was carried out on stool samples by Ziehl-Neelsen (Z-N) acid fast staining method where Cryptosporidium oocysts were revealed as pink to bright red spherical or slightly ovoid bodies. DNA was extracted from Z-N positive fecal samples and Cryptosporidium spp. were confirmed by amplifying 18srRNA gene. The prevalence was determined to be 8.46% in Z-N techniques and 3.85% by nested PCR amplification of 18srRNA gene. Five fecal samples were found positive in PCR where one sample was found positive from pet dog. Four sets of specific oligonucleotide primers were used in this study that amplified products of predicted sizes for each gene in PCR assays [581, 550, 800, 448 bp for 18srRNA(nested), COWP, Gp60(nested) and HSP70 gene respectively] with the use of 1% agarose gel electrophoresis for PCR amplified products. C. parvum was found to be present in one isolates from stray dog which showed successful amplification of a specific DNA fragment at 550 bp with C. parvum outer wall protein (COWP) gene amplicon which may contain cattle genotype of zoonotic significance but it’s unclear up to further sequence study. There was no significant (p>0.05) difference in the prevalence between male and female stray and pet dogs. There was significant (p<0.05) prevalence in young in compared to adult stray dogs. Results indicate that PCR-based diagnostic methods are useful tool for the accurate diagnosis of Cryptosporidium infection in dogs. Significant raise (p<0.05) in lymphocytic and monocyte counts was found in Cryptosporidium infected dogs. This is the first investigation on the presence of C. parvum among stray dogs in Bangladesh based on COWP gene amplification and it has pointed to existence of a genotype that may play an important role as a source of human and farm animal cryptosporidiosis, although gene sequence of the positive isolates is needed for clear identity of the genotypes. The results of this research give an idea that stray dogs may act as reservoirs for zoonotic intestinal protozoan parasites and should be considered important to public health. So that, it is imperative for human to avoid fecal contamination in streets, public gardens and parks. Also stray dog population should be controlled by euthanasia or by performing neutering of male stray dog, specially in urban areas.
A study on prevalence and molecular identification of haemoprotozoan diseases in cattle at Chittagong district
(A thesis submitted in the partial fulfillment of the requirements for the degree of Master of Science in Parasitology Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Chittagong-4225, Bangladesh, 2014-12) Bary, Md. Akramul
This present survey was conducted to reveal out the prevalence through microscopic examination and molecular identification of haemoprotozoan parasitic diseases in cattle at Chittagong district, Bangladesh. A total 300 blood samples were randomly collected (150 crossbred and 150 local cattle) in three consecutive seasons (summer, rainy and winter) from four selected areas, namely Nasirabad, Patia, Bayezid and Jointika. The effects of topography, season, age and gender were tested in both crossbred and local cattle. Molecular detection technique (PCR) was performed after consequence screening by light microcopy, which exhibited that 22 samples (14 for Anaplasma sp, 6 for Babesia sp and 2 for mixed infections were positive.The overall prevalence of haemoprotozoan diseases were 9.33% in crossbred and 5.33% in local cattle, among these babesiosis, anaplasmosis were recorded 2.66% and 6.00 % in crossbred cattle and 1.33% and 3.33 % in local cattle, respectively. Anaplasmosis was common in all areas.The highest prevalence of anaplasmosis was found in Patia (9.33%) followed by Bayezid (4.00%), Nasirabad (2.67%) and Jointika (2.66%) and babesiosis was recorded in Bayezid (4.00%) followed by Jointika (2.66%) and Patia (1.33%). Moreover, haemoprotozoan diseases occurred more frequently in summer followed by rainy and winter season. Among these seasons the highest prevalence of anaplasmosis was recorded 12.00% in crossbred cattle followed by 6.00% in local cattle in summer whereas frequency of babesiosis was highest in summer which was 4.00% in crossbred cattle followed by 2.00% in local cattle. Prevalence of anaplasmosis increased significantly (P<0.05) with the increase of age in crossbred cattle. The highest prevalence of anaplasmosis was 13.72% and 6.94% in adult crossbred and local cattle, respectively. Occurrence of babesiosis was the highest in adult (5.88%) in crossbred than young (2.78%) in local cattle, respectively. It was observed that, the breed and age were important predictor of haemoprotozoan diseases. It was revealed that haemoprotozoan diseases were more common in female cattle, among these highest prevalence of anaplasmosis was recorded (6.11%) in female crossbred cattle and (4.00%) in local cattle, respectively. Positive samples were analyzed by using RNA gene based PCR technique, where 9 samples were amplified among these 4 samples (1.33%) of Babesia sp and 5 samples (1.67%) of Anaplasma sp. As this study is a new invention under these areas, further investigation is recommended with special emphasis on the identification of available vectors for better understanding of such diseases which will help to take necessary preventive measures.
Molecular identification of babesiosis in stray dog at Chittagong Metropolitan Area
(A thesis submitted in the partial fulfillment of the requirements for the degree of Master of Science in Parasitology Department of Pathology and Parasitological Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Chittagong-4225, Bangladesh, 2014-12) E-Azam, Md. Nur
An epidemiological study of babesiosis in stray dogs was conducted at Chittagong Metropolitan area, Bangladesh, for six months. Blood samples of 130 stray dogs were collected from nine randomly selected locations along with the information of age and sex. Blood samples were initially examined by Giemsa's stained blood smear method. DNA was extracted from microscopy positive blood samples and Babesia spp were confirmed by amplifying 18S rRNA gene. The prevalence was determined 6.92% in microscopic technique and the percentages of PCR positive samples from the microscopy positive samples were 66.67% (6 out of 9). The highest prevalence of babesiosis was found in Alankar (16.67%) compared to other areas. Significantly higher frequency of babesiosis was found in adult dog than younger (p<0.05). Again, Babesia infection was common in male (11.94%) than female (1.59%) and were statistically significant (p<0.05). Furthermore, we sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asia genotype), B. canis subsp. vogeli, B. canis subsp. canis and B. canis subsp. rossi DNA in canine blood samples. An outer primer pair was designed to amplify a 340 bp fragment of the 18S rRNA genes in the seminested primary reaction. Then specific internal primers were designed for B. gibsoni (Asian type) (BgibAsia-F) and B. canis subsp. canis (BCC-F) that were paired with the outer reverse primer in the seminested secondary reaction to amplify 185 bp and 198 bp respectively. Based on seminested PCR and sequencing of the PCR products, all parasitic isolates were identified as Babesia gibsoni (Asian type) and B. canis subsp. canis. These results recommended that Babesia gibsoni (Asian type) and B. canis subsp. canis parasites are widespread in stray dog in Chittagong Metropolitan area, Bangladesh. Further investigations are required to clarify the origin, distribution, vector and pathogenesis of these parasites tracking in dogs in Bangladesh.
Isolation and molecular detection of enteric bacteria from neonatal calf in Chittagong
(A thesis submitted in the partial fulfillment of the requirements for the degree of Master of Science in Pathology Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Chittagong-4225, Bangladesh, 2016-12) Tofazzal, Md. Rakib
Calf mortality due to neonatal calf diarrhea is a major problem in cattle farming in Bangladesh where certain groups of bacteria play crucial role. The aim of the present study was to carry out pathological and molecular investigation on enterotoxigenic Escherichia coli (ETEC) and Salmonella typhimurium in neonatal calves. During the study, fecal samples were collected from 200 calves (less than 40 days of age) from a total of 64 dairy and beef farms located in Chittagong Metropolitan Area and Patiya Upazila of Chittagong. The study was conducted during June, 2015 to March, 2016 when 5 dead calves with a history of diarrheal illness were examined through necropsy for gross and histopathological screening. Alongside classical culture technique molecular diagnostic tools like Polymerase chain reaction (PCR) was employed to identify the pathogens including E. coli and Salmonella spp. Two separate primer pairs namely ECO and K99 were used during this study where ECO is indicative of E. coli, and K99 is indicative for Enterotoxigenic E. coli (ETEC) K99. Besides, Salmonella spp. was confirmed by InvA gene in PCR. Typh and 104 primers were also used to identify S. typhimurium and multidrug resistant S. typhimurium DT104, respectively. Results indicated 19 (9.5%) cases of ETEC K99 out of 200 isolates, whereas S. typhimurium was recovered from only 8 calves (4%) and S. typhimurium DT104 was recorded from 2 calves. None of the dead animals was found positive for S. typhimurium through PCR. Gross pathological changes observed during necropsy include watery intestinal contents with bad odor, mucus and blood (in two cases) with dilatation of intestinal lumen. Histopathological investigation of intestine showed villous atrophy in all five cases of dead calves. Thickening of intestinal epithelium, sloughing off epithelium, congestion in lamina propria, infiltration of reactive cells were the findings of diarrhea caused by ETEC K99. Two out of five dead calves were found positive for ETEC K99 and one was isolated for mixed infection with E. coli and rotavirus in confirmatory molecular identification. Statistically significant difference (p<0.05) were observed in the prevalence of E. coli and S. typhimurium collected from different locations and herd size. Highest prevalence of ETEC K99 and S. typhimurium were found in Bakalia (30.43%) and Chawkbazar (27.27%), respectively. Compared to female animals, male calves were mostly affected by both ETEC K99 (13.27%) and S. typhimurium (27.27%). Small farms having <10heads had highest prevalence of ETEC K99 (42.86%). But S. typhimurium occurred highest in medium sized farms (5.81%) having 21-50 heads. Multilevel univariable and multivariable logistic regression was used to evaluate the risk factors associated with both infections. E. coli was significantly affected by farm size (OR = 3.54, 95% CI: 1.02, 12.78), population density (OR = 1.02, 95% CI: 1.00, 1.05), age (OR = 5.51, 95% CI: 1.59, 19.04), coexistence of other animal species in same pen (OR = 3.24, 95% CI: 1.12, 9.34), suckling as feeding regime (OR = 5.07, 95% CI: 1.43, 17.16). Interestingly, level of dehydration showed inverse association with prevalence of ETEC K99. Infection with Salmonella spp. was found to be significantly affected by the source of drinking water supply (OR = 32.28, 95% CI: 5.76-180.94), status of antibiotic therapy (OR = 7.03, 95% CI: 1.30, 37.81) and recent introduction of new calf in the farm (OR = 0.09, 95% CI: 0.01, 0.90). The results of the present study indicate the importance of PCR as rapid, effective and reliable tool alongside conventional culture techniques for screening of ETEC and Salmonella spp. when confronted with cases of undifferentiated calf diarrhea. Moreover, identification of the risk factors associated with the spreading of bacteria causing diarrheal illness may be helpful towards developing suitable prevention and control strategies.
A study on prevalence, molecular identification and characterization of blood and tissue protozoa of domestic ruminants in Chittagong, Bangladesh
(A thesis submitted in the total fulfillment of the requirements for the degree of Doctor of Philosophy from Department of Pathology and Parasitology Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Chittagong-4225, Bangladesh, 2017-01) Mannan, Abdul
Blood and tissue parasites infect both the small and large ruminants causing significant economic losses all over the world including Bangladesh. The actual extent of these parasitic diseases and their epidemiologic features is not yet comprehensively studied in Bangladesh. Accurate identification and characterization are essential for any epidemiologic investigation. With the availability of modern DNA based tools and resources, the capacity for accurate and reliable detection of any disease agents are now possible which was not available before. The present study was therefore designed to investigate the prevalence followed by molecular identification of selected hemoparasites (Babesia, Anaplasma, Trypanosoma) of cattle in different geographic areas of the country. In addition, an attempt was made towards the molecular characterization of tissue protozoan (Toxoplasma gondii) in small ruminants in the Chittagong Metropolitan area located in southern part of Bangladesh. Further analyses of the associated risk factors were also done to help to develop necessary prevention and control options against these important protozoa. Blood samples collected from cattle were preserved in the laboratory for further examination with conventional microscopy and modern molecular tools like PCR and DNA sequencing. Sample size varied among different protozoa species and initial screening was aided by Giemsa stained thin smear examinations. A variable numbers of samples were assessed to detect hemoprotozoa including Anaplasma (n=1680), Babesia (n=1680) and Trypanosoma (n=480) where 8.21%, 1.43% and 0.63% prevalence was recorded respectively from different study areas inside Bangladesh. Bovine anaplasmosis was found as an important rickettsial disease with limited reports related to its prevalence inside Bangladesh. Comparative prevalence of this hemoparasitewas recorded as 9.33% in the hilly areas, 9.00% in coastal areas and 5.83% in plain lands inside the country. Further analyses indicated a higher prevalence (P>0.05) in crossbred cattle compared to local or indigenous breeds. Variations in seasonal incidence were also observed with high case numbers recorded in winter season of the year. Age of the animals was also found an important epidemiologic factor where animals aged 18-30 months were more vulnerable to anaplasmosis than other age groups. Housing and floor types also found to be associated with variable prevalence and animals living in non-cemented or soil-type floors were more prone to suffer from anaplasmosis. Molecular characterization through PCR amplification of 16S rRNA gene was achieved where an 80% cases (40 out of 50 samples) were found positive for A. marginale infections. Further amplification of MSP4 gene fragments successfully identified all forty positive cases in 16S rRNA screening. Further DNA sequencing and phylogenetic studies revealed that all the isolates of this present study were closely related to the isolates already reported from countries like Argentina, Australia and China. Bovine babesiosis is an economic important protozoan disease with sporadic reports on its prevalence and incidence in Bangladesh. During this study, prevalence of babesiosis was found as 1.17%, 1.67% and 1.46% in the selected hilly, coastal and xviii plain areas respectively in the study areas. Further data analysis showed crossbred and female animals more vulnerable than local or indigenous cattle and their male counterparts. High number of babesiosis was recorded in summer season compared to other time of the year. Animals aged 6-18 months were more susceptible to babesiosis than other age groups. Molecular identification of the organism were achieved through PCR amplification of 18SrRNA gene fragments where all positive cases (n=24) screened by conventional microscopy were successfully amplified. Further DNA sequencing and phylogenetic analysis revealed that two distinct isolates of Babesia, Babesia bigemina and Babesia ovata were present in the country. Trypanosomiasis is one of the least studied blood parasites of cattle in Bangladesh and thought to be distributed in different parts where relevant vectors are available. During this study out of 480 blood samples, only three were found positive in a single dairy farm located in Chittagong Metropolitan area. All positive cases were recorded in female and Holstein Frisian crossbred cattle and in one farm where previous incidence and disease record was not available. The time of the year when these cases were found was in rainy season and all the animals were aged 6 years or more and none of the younger animals were found positive. Molecular identification was achieved through PCR amplification of 18SrRNA gene fragment following previously published protocols. Sequencing of amplified DNA and further phylogenetic analysis revealed that Trypanosoma evansi were circulating in Bangladesh which is closely related to the isolates reported from countries like Taiwan, Thailand and Japan. Toxoplasmosis is an important zoonotic tissue protozoan disease of small ruminants in Bangladesh and has been documented by a number of publications. Several studies reported variable prevalence rate of toxoplasmosis in different parts of the country. The present study revealed an overall prevalence of toxoplasmosis in sheep and goats as 30.43% and 41.30% respectively. Further statistical analysis of the data indicated that animals aged two years or more were highly seropositive to Toxoplasma gondii. History of abortion and sources of drinking water were found to be significantly associated with toxoplasmosis. Animals with previous history of abortion were highly seropositive compared to the other groups. High seroprevalence was recorded in farms where ponds and municipality supply water are used as water sources for animals. Apart from serodiagnosis, acute and chronic cases of toxoplasmosis are always troublesome to detect hindering their prevention and control efforts. Combined histopathological and molecular approaches were applied during this study to detect the Toxoplasma gondii from several aborted tissues of sheep and goats. Prevalence of T. gondii was detected as high as 25% in sheep (n=4) and 36.84% (n=19) in goats. All the positive cases were detected in animals that aborted at 3.5 months of gestation. Further sequencing of amplified DNA and phylogenetic analysis of the sequences confirms the presence of T. gondii in a single cluster with minimum genetic variations among the four isolates which are closely related to the isolates recorded from countries like India and Iran.
Molecular and Histopathological detection of Cryptosporidiosis in calves in Chittagong
(Chittagong Veterinary and Animal Sciences University, Khulshi-Chittagong-4225, Bangladesh, 2017-06) Parvez, Md. Anwar
Cryptosporidium is an intestinal protozoan parasite of livestock and human and cause cryptosporidiosis which is also a common disease of cattle especially neonatal calves. Cryptosporidiosis is commonly asymptomatic but sometimes associated with persistent diarrhea in calves. The aim of the present study was to carry out pathological and molecular investigation of cryptosporidium infection in calves. The study was conducted during June, 2015 to January, 2016. During the study period fecal sample were collected from 160 calves with less than 45 days age from a total of 50 dairy and beef farms located in Chittagong Metropolitan Area and Patiya Upazilla of Chittagong when 5 dead calves with a history of diarrheal illness were also examined for gross and histopathological examination. The fecal samples were first screened by modified ZiehlNeelsen (Z-N) staining technique for the detection of cryptosporidium oocysts under light microscope which is characterized by pink to bright red spherical bodies and later confirmed by polymerase chain reaction (PCR) using 18s rRNA gene. Z-N positive fecal samples were used for DNA extraction Cryptosporidium spp. was confirmed by amplifying 18srRNA gene primer. The positive samples yielded 580bp band with the use of 1% agarose gel electrophoresis for PCR amplified products. The overall prevalence of Cryptosporidiosis in crossbred calves was 9.37% (15/160) by Z-N techniques and 1.88% (3/160) by nested PCR amplification which is statistically significant (p<0.05). The prevalence of Cryptosporidium was also significantly higher in diarrhoeic calves (42.85%) than non diarrhoeic (7.9%) calves. During necropsy examination bad odors, bloody mucous membrane with fluid filled watery fecal contents were found in two dead calves. For histopathological examination tissue samples were collected from dead calves and preserved in 10% buffered formalin. After tissue processing and routine H&E staining of intestinal tissue section revealed atrophied villi and sloughing of mucosa with reactive cellular infiltration. It is evident that the prevalence of cryptosporidiosis in bovine calves in these areas is under diagnosed and the clinical status of infection was not potentially high. It can be concluded that Cryptosporidium spp. infection is prevalent in Bangladesh and adult cattle could serve as reservoirs for the zoonotic infection in humans.
Occurrence of Eimeria spp. in neonatal calves in Chittagong metropolitan area
(Chittagong Veterinary and Animal Sciences University, Khulshi-Chittagong-4225, Bangladesh, 2017-06) Rafiul Hoque, Mohammad
Calf diarrhea is a major problem in cattle farming in Bangladesh where coccidiosis is common phenomenon. The specific objective of the present study was to determine the prevalence of coccidia infection in neonatal calves as well as to assess the risk factors associated with the infection. During the study, fecal samples were collected from 130 calves (less than 45 days of age) from a total of 39 dairy and beef farms located in Chittagong Metropolitan Area and Patiya Upazila of Chittagong. The study was conducted during June, 2015 to December, 2015. The prevalence of Eimeria spp. was found 20.77% in diarrheaic calves in the study area. 26% and 27% prevalence of Eimeria spp. was found considering age in calves between 2-4 weeks and > 4 weeks, respectively. Univariable and multivariable logistic regression models were used to evaluate the risk factors associated with Eimeria spp. infection. In univariable logistic regression analysis, 5 variables (age, flooring of calving pen, source of drinking water, fecal consistency and dehydration test) showed significant (P-value <0.2) association with the prevalence of Eimeria spp. In multivariable analysis, age and fecal consistency were found significantly (P-value <0.05) associated with presence of Eimeria spp. Calves with more than 4 weeks of age and calves with 2 to 4 weeks of age had odds ratio of 9.03 and 6.58, respectively compared to calves with less than 2 weeks of age. Calves with liquid (OR=4.5) and semi-solid (OR=1.4) fecal consistency had higher chance to get the infection compared to calves with solid feces. The results of the present study identified the risk factors associated with occurrence of Eimeria spp. causing diarrhea in neonatal calves will be helpful for developing suitable targeted control program in the study area.
HISTOPATHOLOGICAL AND MOLECULAR DETECTION OF SALMONELLOSIS IN POULTRY IN CHITTAGONG
(Chittagong Veterinary and Animal Sciences University, Khulshi-Chittagong-4225, Bangladesh, 2017-06) Mohajan, Prima
Salmonellosis is one of the most important bacterial diseases of poultry because it causes high economic loss due to mortality and reduced egg production in layer birds. Salmonella infection in poultry can occur in either acute or chronic form by the member of genus Salmonella under Enterobacteriaceae family. There are mainly two biovars of avian species (host) specific Salmonella enterica named Salmonella Gallinarum and Salmonella Pullorum which are responsible for fowl typhoid and pullorum disease respectively. The present study was undertaken with the aim to carry out pathological and molecular identification of Salmonella isolated from commercial chickens in Chittagong. For this purpose a cross sectional survey was done with the collection of 90 different samples from dead chickens (38 Layers, 37 Broilers and 15 Sonali) brought to the department of Pathology and Parasitology, Chittagong Veterinary and Animal Sciences University (CVASU) for postmortem examination. A preformed questionnaire was also used to record additional information such as production type, farm size, vaccination etc. All the postmortem samples were primarily inoculated into selenite cystine broth followed by streaking into XLD agar to determine typical colony characteristics of Salmonella. Out of 90 samples 16 (17.78%, 95% CI 10.5-27.3%) were found primarily positive on XLD agar based on colony morphology which was also tested by Polymerase Chain Reaction (PCR) using previously published primer to detect invA gene specific for Salmonella. After PCR, 13(14.44%) samples were found positive (95% CI 7.9-23.4%) with the amplicon size of 284bp in agarose gel electrophoresis. Additionally liver, lung, spleen, kidney and intestine were also collected and preserved in 10% buffered formalin for histopathological examination. Gross pathological changes observed during necropsy included misshapened ova, unabsorbed yolk sac in chicks, bronze colored liver, haemorrhages and necrotic foci in spleen and intestine. Histopathological investigation showed congestion and hemorrhages in lungs; coagulation necrosis and fatty change in liver; congestion and haemorrhage in kidney which was also associated with coagulation necrosis and fatty change in renal tubule in some cases. Marked destruction of intestinal epithelia with caseous mass in intestinal lumen was found along with haemorrhage and sloughed off villi. Depletion of lymphocytic nodule and lymphocytes along with vascular thickening in spleen was markedly observed in most of the cases. Necrosis, proliferation of Reticuloendothelial (RE) cells and presence of secondary follicles were also noticeable lesions in spleen. Univariable logistic regression was used to identify the effect of different variables on the estimated prevalence (14.44%) of Salmonella. Statistically significant difference (p<0.05) was observed in the prevalence of salmonellosis in different production type of chickens. Prevalence in layer (26%, OR=4) was significantly higher compared to broiler (8%, OR=1). The result of the present study indicates the importance of PCR as a rapid and reliable tool alongside conventional cultural techniques for screening Salmonella. The data of risk factor analysis in this study suggest the prevalence of Salmonella enterica is very high in commercial layers in Chittagong.
Prevalence of gastrointestinal parasitic infections in sheep of Subarnachar upazilla in Noakhali district of Bangladesh
(A thesis submitted in the partial fulfillment of the requirements for the degree of Master of Science in Parasitology. Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Chittagong-4225, Bangladesh, 2017-12) Hossain, Md. Meraj
Gastrointestinal parasitic infection is a major cause of failures in sheep production in Bangladesh. A study was conducted to determine the prevalence of gastrointestinal parasitic infections of sheep in Subarnachar upazilla of Noakhali district in Bangladesh during January 2017 to December 2017. In total, 200 rectal fecal samples of non-descriptive indigenous sheep were randomly collected and were examined by routine coproscopical methods and modified McMaster technique. The effects of topography, age, sex, rearing system, health status and management system on gastrointestinal parasitic infections were evaluated by chi-square test. The overall prevalence of gastrointestinal parasitic infections in sheep was 79.50%. The highest prevalence of gastrointestinal parasitic infections was found in Char Clark union (83.33%) than the others, although their relationship were statistically insignificant (P>0.05). Species wise prevalence of gastrointestinal parasitic infections was Fasciola sp. (37.5%), Paramphistomam sp. (41.0%), Strongyloides sp. (46.0%), Strongyle-type (39.5%), Trichuris sp. (28.0%) and Moniezia sp. (34.0%). The occurrence of gastrointestinal parasitic infections was more in young (90.0%) than the adult sheep (70.90%). Female sheep displayed a higher infection (85.0%) as compared to male (71.25%) and free range grazing sheep (82.66%) in bathan area was more infected as compared to semi-intensive system (77.66%). Good healthy animals had infection with EPG level of 380-1000 as compared to moderate healthy (>1000-2000) and poor healthy (>2000) animals. The animals those were reared under proper management system including regular deworemed by anthelmentics, proper housing and feeding with nutritional supplement were less infected by gastrointestinal parasites. As the present study was fresh of its type in the study areas, further investigation is recommended to evaluate the region specific risk factors.
DNA barcoding and morphotaxonomic analyses of mosquitoes collected from Chittagong Metropolitan area
(A thesis submitted for partial fulfillment of the requirements for the degree of Master of Science in Parasitology Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Chittagong-4225, Bangladesh, 2018-01) Sarkar, Md. Suruzzaman
Mosquitoes are important vectors for a wide variety of pathogens and parasites. Identification of different mosquito species are crucial for their eventual control. We have used the classical morphometry and modern molecular methods like DNA barcoding approach for reliable identification of mosquitoes that were collected from different locations of Chittagong Metropolitan area of Bangladesh. A total of 20 randomly collected specimens were included in this study. Different morphological features of head, clypeus, vertex, antenna, maxillary palp, thorax, wings, legs and abdomen of the mosquitoes were examined using three-dimensional microscopy. The morphological characteristics of the mosquitoes observed under stereo binocular microscope in the laboratory shown highly similarity with the Aedes aegypti species. Genomic DNA was extracted from all of the 20 samples using commercial kits and specific primers were used for amplification of partial cytochrome oxidase genes for molecular characterization. PCR products were then sequenced and further bioinformatics analyses was accomplished. Sequence similarity based BLASTn and phylogenetic analyses (MEGA 6) of the sequence indicated similarity with three different species namely Aedes aegypti, Culex pipiens and Aedes albopictus. Among these 5 samples, 3 samples were Aedes aegypti, 1 sample was Aedes albopictus and 1 sample was Culex pipiens. The findings were compatible with the morphological data and reliably characterized the mosquito species. The study was first DNA barcoding analyses on mosquito in the country and showed the feasibility of using molecular tools for their characterization. Further study about the origin and vector potential of these mosquitoes will enhance our capacity to improve existing vector surveillance and control program.
Occurrence of enteric parasites in household and free range ducks at Hakaluki and Tanguar haor of Sylhet division, Bangladesh
(A thesis submitted for partial fulfillment of the requirements for the degree of Master of Science in Parasitology Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Chittagong-4225, Bangladesh, 2018-06) Uddin, Md. Sarwar
Bangladesh has the third largest duck population in the world with the stocks of 52.24 million. Duck rearing has potential to give maximum return with minimum investments. This study of enteric parasites of ducks was undertaken to estimate the prevalence and relationships between prevalence and the age, breed of ducks at Hakaluki and Tanguar haor of Sylhet division, Bangladesh. A total of 600 fecal samples of ducks of different ages and breeds were collected and examined using standard examination techniques includes flotation, sedimentation and direct smear under microscopy. The prevalence of helminths was found to be 48.33% (290) from which the prevalence of nematode, cestode, and trematode infections were 39.67% (238), 3.33% (20) and 9.33% (56) respectively. The nematodes identified include Capillaria spp., Ascaridia galli, and Amidostomum anseris and Tetrameres spp. The reported trematode and cestode were Prosthogonimus spp. and Hymenolepis spp. respectively. The helminths infections were recorded in Muscovy 77.78% (7), Deshi Pati Duck 52.15% (182) and Khaki Campbell 41.74% (101) were more in the age group of more than 6 to 24 months. The seasonal variation of helminths infection were observed and recorded in three different seasons. The highest rate of infection was observed in monsoon season 52.5% (105) followed by summer 50% (100) and winter season 42.5% (85). The present study suggests that age, breed of ducks and seasons of the year influence the enteric parasitic infection to a greater extend in the duck in Hakaluki and Tanguar haor of Sylhet division, Bangladesh.
Prevalence of Blood Protozoa inDuck at Hakaluki and Tanguar Haor of Sylhet Division in Bangladesh
(A thesis submitted in the partial fulfillment of the requirements for the degree of Master of Science in Parasitology. Department of Pathology and Parasitology Faculty of Veterinary Medicine Chittagong Veterinary and Animal Sciences University Chittagong-4225, Bangladesh., 2018-06) Hoque, Azizul
The blood protozoa of avian are under the genera Haemoproteus, Plasmodium and Leucocytozoon which causes high mortality of birds and render huge economic losses of the farmers. The present study was undertaken to find out the prevalence of haematozoan parasites in ducks at Hakaluki and Tanguar haor of Sylhet division in Bangladesh. A total of 400 blood samples in summer (n=200) and rainy (n=200) seasons were examined under light microscopy and data on the breed, age, sex, deworming, types of scavanging, types of housing were recorded during the study period. Among the microscopically examined samples, 22% (44/200) duck were found to be infected with different blood protozoa during summer season and 15% (30/200) during rainy season. Of those, 14% (28/200) of birds were infected with Haemoproteus spp., 7% (14/200) with Plasmodium spp. and 10.5% (21/200) with Leucocytozoon spp. during summer season. On the other hand, 8.5% (17/200) of birds were infected with Haemoproteus spp., 11% (22/200) with Plasmodium spp. and 3% (6/ 200) with Leucocytozoon spp. during rainy season. Among the breeds, highest infection was 13.8% (12/87) of ducks (KK) for Haemoproteus spp. during summer season and lowest infection was 0.0% (0/3) of ducks (Muscovy) for Leucocytozoon spp. during both summer and rainy season. Highest infection was 40.0% (6/15) in the birds whose age was less than six months or equal in case of Haemoproteus during summer season and lowest infection was found 0.00% (0/12) in these birds whose age was less than six months or equal in case of Leucocytozoon during rainy season. Non vaccinated duck became more infected than vaccinated. In summer season, highest infection was 14.3% (17/119) in case of Haemoproteus spp. and lowest infection was 1.27 % (1/79) in case of Leucocytozoon spp. during rainy season. Non-dewormed duck became more infected than dewormed duck for both during rainy and summer season. Highest infection was 15.25% (9/59) for Haemoproteus spp. during summer season but lowest was 1.75% (1/57) for Leucocytozoon spp. in rainy season Sick ducks became more infected than healthy duck. Highest infection was 26.32% (5/19) during summer season in case of Haemoproteus and lowest infection was 0.00% (0/15) in case of Leucocytozoon spp. during rainy season. Highest infection was 33.3% (4/12) during summer season in case of household Page | xi scavenging for Haemoproteus and lowest infection was 0.00% (0/4%) in case of Ponds cum wetland for Haemoproteus, Leucocytozoon and Plasmodium during both summer and rainy season. During rainy season, highest infection was 18.97% (11/58) in case of within house for Plasmodium and lowest infection was 0.00% (0/1) in case of yard cum house for Haemoproteus, Leucocytozoon and Plasmodium during both summer and rainy season. There was no statistical significant in the infection rates among the study sites. Two birds were positive for both Haemoproteus spp. and Plasmodium spp. Prevalence of infection varied among different breeds. My research work will help to control of blood protozoa and flock management easily of ducks at haor areas.
Prevalence of gastrointestinal parasitic infections, haemoprotozoan diseases and their associated risk factors in sheep and goat in selected hilly areas of Bangladesh
(Chattogram Veterinary and Animal Sciences University Chattogram-4225, Bangladesh, 2020-06) Kamal, Towhida
Gastrointestinal parasitism and haemoparasitism in small ruminants are known to impose substantial economic burdens on owners. A cross sectional study was conducted to investigate the prevalence of gastrointestinal parasites and haemoparasites of small ruminants from three unions of Khagrachari district, namely Panchori, Golabari and Sadar. The fecal samples, blood samples and tick samples were collected from a total of 279 goats (97) and sheep (182) from study areas. All the specimens were subjected to microscopic examination technique first then blood samples (118) and tick samples (12) were examined by polymerase chain reaction for the detection of haemoparasites. Selected positive samples (9) were sent for sequencing and then phylogenetic analysis was done using sequenced data. The overall prevalence of gastrointestinal parasitic infection was found 55.67% (54) and 63.19% (115) in goats and sheep, respectively. The prevalence of parasites was variable with Fasciola sp. (27.84%), Strongyloides sp. (15.38%), Trichostrongylus sp. (14.84%), Paramphistomum sp. (9.89%), Trichuris sp. (8.24%) and Oesophagostomum sp. (6.04%). Overall haemoprotozoan infections were 42.27% and 40.11% in goats and sheep, respectively. The prevalence of Babesia sp., Anaplasma sp. and Theileria sp. were detected (38.64%, 29.73%), (38.64%, 28.38%) and (25.00%, 17.57%) in goats and sheep respectively by polymerase chain reaction. Parasitic ova and haemoparasitic infection rate in male and female exhibited no significant variations (p>0.05) between them. The infection was significantly higher in adult than young. Seasonal variation was found between winter and summer season. Haemoparasites are more prevalent in summer but opposite observation was found in case of gastrointestinal parasites. Two types of ticks were identified where Boophilus sp. is more prevalent than Haemophysalis sp. in both goats and sheep. Babesia sp. is identified from extracted DNA of Haemophysalis sp. tick. Finally, the random sequencing of isolates from Babesia sp., Anaplasma sp. and Theileria sp. revealed Babesia ovis, Anaplasma bovis, Anaplasma phagocytophilum and Theileria lewenshuni from goat and sheep and phylogenetic analysis prove the transmission of Babesia ovis though Haemophysalis sp. tick. Further investigation is necessary for a structured surveillance to investigate more variances of them to formulate effective control measures.
PREVALENCE OF ASCARIDIA GALLI AND IT’S ASSOCIATED RISK FACTORS CAUSING ASCARIDIASIS OF HILLY CHICKENS AT RANGAMATI, KHAGRACHARI AND BANDARBAN IN CHATTAGRAM
(Chattogram Veterinary and Animal Sciences University Chattogram-4225, Bangladesh, 2020-06) Chakma, Kalpana
Ascariasis caused by Ascaridia galli is a common parasitic infection in chicken throughout the world.This infection is prevalent in all kind of production systems and cause substantial economic problem in terms of lower feed consumption, reduced weight gain, lower egg production and even death in case of heavy infections. Information about phenotypic variation is ultimate understanding the host parasite relationship as well as to design an effective control measures against this economically important parasite. Therefore, this study was conducted to get updated information on the prevalence of Ascaridia galli infection in chickens. For this purpose, a total of 200 chickens‘ feces was collected from hilly area (Rangamati, Khagrachari and Bandarban). 21 chickens out of 200 were found positive to the infection giving the prevalence as 10.5% of A. galli infection in chickens of the study area, of which males were 12 (57.14%) whereas females were 9(42.85%). The result of this observation indicates that there is a marked pattern of Ascaridia galli infection in both sexes. From this result without significant test it is difficult to prove it proved that males are more susceptible than females. Relatively young animals were more susceptible than adult. Age group wise prevalence was as follows 57.14 % in 14 -52 wks, 33.33 % in 1.5 -2 yrs and 9.52% in 2.5-3yrs respectively. It was documented that the rate of occurrence of A. galli in Summer 41.15%, Rainy 44.67% and winter 14.18%. Free range housing system chickens were more infected than semi intensives housing system. Anthelmintic were used more in semi intensives than free range housing system.
Isolation & molecular characterization of Campylobacter jejuni and virulent gene associated avian fecal Escherichia coli in broiler, Bangladesh Md. Sirazul
(Chattogram Veterinary and Animal Sciences University Chattogram-4225, Bangladesh, 2020-06) Md. Sirazul Islam, Md. Sirazul
The intestinal environment of broilers is a potential Campylobacter jejuni and avian fecal Escherichia coli (AFEC) reservoir. Understanding the intestinal microbiota in broilers and their subsequent human transmission is a key public health concern here, where C. jejuni and intestinal virulent associated gene (VAG) carriage E. coli are simultaneously studied in apparently healthy birds from broiler farms, live bird markets (LBMs) and supershops. In the period from June 2019 to February 2020, pooled cloacal samples were collected from broiler farms located in 6 districts of Bangladesh and pooled meat samples were collected from LBMs and super shops in Chattogram. Data on farm management, biosecurity, and hygiene practices were collected through a face-to-face interview during sampling using a structured questionnaire. To examine the occurrence of C. jejuni and VAG AFEC in broiler chickens, microbial culture and PCR-based methods were applied. Positive PCR amplicon was confirmed by gene sequencing and phylogenetic analysis was subsequently performed. To evaluate the risk factors, epidemiological data were analyzed using univariable logistic regression models followed by multivariable logistic regression. Among the 216 farms, 27 were positive to C. jejuni; thus, the farm level prevalence was estimated to be 12.5% (95% CI: 8.5%–17.7%). Remarkably, at LBMs and super shops, a higher prevalence of 27.08% (95% CI: 15.28%–41.84%; N=48) of C. jejuni was recorded in broiler meat. In risk factor analysis, downtime of less than 14 days, no separate footwear for shed entry, increased number of flock rotations per shed per year, and entry of more than 1 person to sheds were found to be significantly associated with C. jejuni infection. Phylogenetic analysis showed a close connection between C. jejuni strains isolated from Bangladesh and other strains isolated from humans, pigs and bats of India, South Africa and Grenada. VAGs AFEC was isolated from the apparently healthy chickens with a prevalence of 55.6% (95% CI: 48.7%–62.3%). Virulence Associated Genes; astA, iucD, iss, irp2 and cva/cvi were detected in a rate of 46.3%, 25.5%, 17.6%, 12.9% and 2.8%, respectively. Geographical locations and flock age of less than 21 days were significantly associated with the VAGs AFEC positive status in broilers. The results of this study showed a high level of microbial contamination of zoonotic importance and the presence of pathogenic bacteria in the intestine and meat of Bangladeshi broilers. Potential sources of contamination and anthropogenic factors associated with the alarming occurrence of C. jejuni and VAGs AFEC noted in this study would assist in developing interventions under the 'One Health' banner that includes chickens, humans and environmental perspectives to minimize the increasing risks of broiler-associated pathogens.
Lumpy skin disease outbreak in commercial cattle farms of Chattogram, Bangladesh
(Chattogram Veterinary and Animal Sciences University Chattogram-4225, Bangladesh, 2020-06) Hasib, F. M. Yasir
Lumpy Skin Disease (LSD) is an important viral disease capable of incurring significant economic loss in commercial livestock production. The disease is of major economic importance due to production losses resulting from severe emaciation, lowered milk production, abortion, secondary mastitis, loss of fertility, extensive damage to hides and a loss of draft from lameness. Moreover, death or culling of sick cattle impose indirect losses due to the cattle movement and trade restriction. In mid-2019, an outbreak of LSD was reported in cattle population from different parts of Bangladesh including Chattogram Division. During this study a cross-sectional survey was undertaken from August to December 2019 to investigate the prevalence and associated risk factors of LSD in cattle in Chattogram district. A total of 3327 cattle from 19 commercial farms were investigated for the LSD associated skin lesions and possible risk factors. A total of 120 skin biopsies were collected from the suspected animal for histopathological examination followed by molecular detection through PCR and DNA sequencing. Partial genome sequencing and phylogenetic analyses were also explored on selected specimens. It was revealed that demonstrated that the overall prevalence of LSD in the study population was 10% (95% confidence interval: 9.4 to 11%) where the highest farm level outbreak frequency was 63.33% and lowest 4.22%. Crossbred (OR=3.56) and female (OR=3.56) cattle showed significantly higher susceptibility to the disease compared to their counterparts. Introduction of new animals in any farms was found to be one of the most significant risk factors (OR=2.34) associated with the transmission of the disease. Notably all suspected skin biopsies were positive for LSD virus (LSDV) infection with granulomatous and pyogranulomatous dermatitis as revealed through histopathology. Phylogenetic analysis based on the inverted terminal repeat region of the LSDV genome suggested that the locally circulating strain was closely related to the strains isolated from Middle East and North African countries.
Endoparasitic Infection and Antibiotic Resistance Patterns of Escherichia coli and Salmonella Isolated from Pet Birds in Chattogram
(Chattogram Veterinary and Animal Sciences University Chattogram-4225, Bangladesh, 2020-06) Md. Bayzid, Md.
Different parasitic and bacterial diseases are the crucial impediment in the rearing of pet birds. Gastrointestinal tract and blood are the major predilection sites for parasitic infection. The aim of this study was to identify the gastrointestinal (GI), as well as blood parasitic infection and the occurrence of Escherichia coli and Salmonella spp. with antimicrobial resistance (AMR) patterns from pet birds in Bangladesh. Between June, 2019 and March, 2020 a total of 549 (for GI parasite), 150 (for AMR of E. coli and Salmonella spp.) freshly voided faecal samples and 311 blood samples were collected from Chattogram metropolitan area, Bangladesh. For the detection of GI parasites coproscopy were performed and for isolation of E. coli and Salmonella spp. different microbiological tests were conducted followed by antibiotic sensitivity test (AST) and antibiotic resistant genes were identified by molecular technique. Blood samples tested through multiplex PCR method for Haemoproteus, Plasmodium and Leucocytozoon spp. detection. The occurrence of GI parasite was 18.9% (CI=15.7– 22.5%). Among them the protozoal cyst was 10.9% (CI=8.40–13.8%) containing Eimeria 7.7% (CI=5.6-10.2%), Isospora 2.9% (CI=1.7-4.7%), Caryospora 0.4% (CI=0-1.3%). Among nematode, Ascaridia infection rate was 3.5% (CI=2.1-5.4%), Capillaria 1.7% (CI=1-3%), Heterakis 0.91% (CI=0-2%). Within cestode, the Coanotaenia was found 0.36% (CI=0-1%). But no trematode egg was detected. The occurrence of blood parasitic disease was 2.25% (CI=0.91-4.58%). Among them 0.32% (CI=0.4-1.8%) was Haemoproteus, 1.3% (CI=0.35-3.26%) Plasmodium and 0.64% (CI=0.4-2.3%) Leucocytozoon. The overall prevalence of E. coli in pet birds was 48.7% (95% CI=40.4-57). The AST revealed that, the 91.78% isolates were resistant to ceftriaxone, 86.3% to tetracycline and 75.34% to sulfomethoxazole-trimethoprim. To the contrary, 98.63% isolates were sensitive to colistin sulfate followed by 97.26% to gentamicin. Majority of E. coli isolates were multidrug resistant and carried blaTEM, tetA, tetB, sul1, sul2, gyrA, gyrB, parC and CTX-M-2 genes. The overall infection rate of Salmonella spp. in pet birds were 2.7 % (95% CI=0.7-6.7). The AST evolved that the isolates were 100% resistant to ceftriaxone, 75% to azithromycin, 50% to amoxiciline and tetracycline followed by gentamicin 25%. To the contrary, 100% isolates were sensitive to ciprofloxacin, enrofloxacin and colistin sulfate. Different pet bird’s spp. was infected with various types of gastrointestinal and blood parasites. Due to cherished in intensive care with regular anthelmintic therapy, less vector availability, the occurrence of gastrointestinal as well as blood parasitic infections were less. In order to haphazard used of antibiotics, AMR were developed in E. coli and Salmonella spp.
Prevalence of Ectoparasites, Gastrointestinal Parasites and Blood Protozoan Infection of Pigeon in Chattogram Metropoliton Area, Bangladesh
(Chattogram Veterinary & Animal Sciences University, Khulshi-Chattogram, 2022-12) HASAN MAMUN, MD MOAZZEM
A cross sectional study was done with the aim to estimate prevalence of ectoparasite, gastrointestinal parasite and blood parasite infections in pigeons (Columba livia) in different parts the Chattogram metropolitan area in Bangladesh. A total of 130 pigeons were checked for ectoparasites, gastrointestinal parasites and blood protozoa. The analysis was conducted between the dates of 1 July 2022 to 31 December 2022 at the Department of Pathology and Parasitology, Chattogram Veterinary and Animal Sciences Univesity, Chattogram. For identification of gasteointestinal parasite we performed direct smear, sedimentation technique and simple floatation method and for morphological identification of ectoparasite I used microscope and giemsa stain for blood protozoa. All acquired information was entered into a Microsoft Excel spreadsheet and analysed using SPSS version 13. The study found that 59.2% of pigeon had ectoparasites on their skin. Out of 4 different ectoparasites identified, Lipeurus caponis (38.5%) caused the most infections. Columbicola columbae (31.5%) and Menopon gallinae (23.1%), which were more common than Menacanthus straminius (15.4%). Four species of gastro-intestinal (GI) parasites were identified, including Ascaridia sp., Capillaria sp., Heterakis sp., and Syngamus trachae. Ascaridia sp., Capillaria sp., Heterakis sp., and syngamus trachea had respective prevalences of 37%, 33.8%, 17%, and 5.4%. The prevalence of GI parasites was 62.31% and 45 (34.6%) pigeons had blood protozoa. Two species of protozoa, including Haemoproteus sp. (21.5%) and Leucocytozoon sp. (13.10%) were identified. The epidemiological study showed that the number of pigeon with parasites in their GI tract, ecoparasites, and blood protozoa was pretty high during the study period. Housing, sex, and deworming all might have a big effect on the prevalence rate. Thus, more epidemiological studies are needed to determine Chattogram district pigeon parasitic infection.
DEVELOPMENT OF SIMPLEX AND MULTIPLEX PCR ASSAY FOR RELIABLE IDENTIFICATION OF HAEMOPARASITIC DISEASES OF CATTLE, BANGLADESH
(Chattogram Veterinary & Animal Sciences University, Khulshi-Chattogram, 2022-12) Sultana, Ayesha
Bangladesh is a tropical, agro-based developing country. Anaplasmosis, Babesiosis, and Theileriosis are the most common vector-borne haemoparasitic diseases in cattle. This study not only describes the development and evaluation of a multiplex PCR assay for simultaneous detection of haemoparasitic diseases (Theileria annulata, Babesia bigemina, and Anaplasma marginale), but also their prevalence ratio. In the multiplex PCR assay, three sets of pre-designed primers were used that targeted the genes Tams1 for T. annulata, 18S rRNA for B. bigemina, and 16S rRNA for A. marginale, with desired amplicons sizes of 751 bp, 504 bp, and 270 bp, respectively, with the use of 2% agarose gel for electrophoresis of amplified PCR products. A total of 350 blood specimens were collected that were tentatively diagnosed as haemoparastic diseases on the basis of clinical signs from three districts of Bangladesh: Chattogram, Rangpur, and Sylhet. Blood samples were stained and preserved at -20°C for further molecular study. In this study, 52% (out of 350) cases were tentatively diagnosed as positive by microscopic examination, among the isolates A. marginale (30.3%), Babesia sp. (12.9%), and Theileria sp. (9%). (Whereas, 21.7% in Chattogram, 17.1% in Sylhet, and 13.1% in Rangpur.) However, in the simplex PCR assay, 35.7% of the cases (14.9% in Chattogram, 12% in Sylhet, and 8.9% in Rangpur) showed a positive band in electrophoresis. Mixed haemoparasitic infection is very common in cattle. Overall, 13.1% of infections were detected positive by multiplex PCR; among them, 2% were diagnosed as positive for all three haemoparasites and 3.71% were diagnosed as anaplasmosis+Babesiosis and so. The partial gene sequencing and phylogenetic analysis of the nucleotide sequences expressed the fidelity of the primer pairs that were used in multiplex PCR, which was found to be 100% sensitive and 85% specific for the detection of infections. In multiplex PCR, amplification of multiple target sequences in one assay is helpful for diagnosis of multiple organisms at a time. This is also time-saving and costeffective compared to other two methods, but requires several trials for optimization of annealing temperature and shows false negative results in minor errors. Beyond all limitations, multiplex PCR assay is precise and could be used as a robust tool for easy, sensitive, specific, and simultaneous diagnosis of haemoparasitic diseases in cattle.