Molecular characterization and prevalence of bovine hemoprotozoan and rickettsial organism from Bhubaneswar, Eastern India.

The blood parasites adversely affect the bovine population and are one of the major hindrances to productivity and health of livestock, globally. This study aimed to study the prevalence of some bovine hemoparasites, their molecular characterization, and associated risk factors in and around Bhubaneswar, a place in Eastern India. Microscopy (Giemsa stained thin blood smear) and polymerase chain reaction (PCR)-based prevalence studies were undertaken for a total of 106bovine blood samples belonging to different breed, age, and sex presented to Veterinary Clinical Complex. DNA was extracted by conventional method and primer targeting msp5 gene (382 bp) for Anaplasma marginale, Tams1 gene (572 bp) for Theileria annulata and VSG RoTat 1.2 gene (110 bp) for Trypanosoma evansi, were used. Sequencing and phylogenetic analysis were also carried out for representative PCR products. The risk factors were correlated with prevalence by logistic regression analysis. Blood smear examination revealed a prevalence an overall of 48.11% (51/106) out of which the prevalence of T. annulata, A. marginale and T. evansi, and concurrent infection were reported to be 26.42%, 10.38%, 3.77%, and 7.55% respectively. All the samples found positive in microscopy were also found positive in PCR. PCR assay revealed an overall prevalence of 73.58% (T. annulata: 37.74%, A. marginale:17.92%, T. evansi: 5.66% and concurrent infection:12.26%). A higher prevalence was observed in the age group of 2-4 years (37.74%), females (61.32%) and cross-bred Jersey (50%). Representative PCR products were sequenced and assigned by GenBank (OL550058: A. marginale; OL604429: T. annulata and OL550059: T. evansi). There is a paucity of disease mapping of these parasites in most parts of the country. The study is probably the first report of molecular characterization of T. evansi and A. marginale from the region which gives some information of possible genetic diversity of the isolates circulating in the region. Future research should include large-scale epidemiological surveys as well as concurrent diagnosis of these hemoparasites for better treatment and control strategies.

Determining Theileria annulata parasitemia through qPCR in lactating cows of Odisha, - a theileriosis-endemic region of India.

Study was undertaken in a theileriosis-endemic region of India during May 2018 to April 2019 among milch cows. Blood samples collected from apparently healthy (n = 65) and Theileria-suspect cows (n = 65) were screened against T. annulata and T. orientalis infection by SYBR Green‒based real time PCR using primers designed from the isolates of study area. Cows having single infection with T. annulata with/without clinical signs of inappetence, low milk yield, pale mucous membranes, fever, enlarged prescapular lymph node, soil licking, panting, coughing, salivation and lachrymation were subjected to further investigation where parasitaemia and piroplasms per 1000 erythrocytes ranged from 1.6 × 107 to 1.2 × 108 parasites/mL of blood and 3-24 piroplasms in moderate group (16/65), 4.4 × 108 to 6.9 × 109 parasites/mL of blood and >88 piroplasms in severe group (30/65) and 1.6 × 104 to 5.5 × 106 parasites/mL of blood and 0-1 piroplasms in asymptomatic or carriers (17/65), respectively. Study unfolded significant difference in T. annulata parasitaemia among apparently healthy and ill cows. Phylogenetic analysis of our T. annulata isolates (NCBI accession numbers MN098316, MN098317 and MN098318) exhibited maximum similarity with the isolates detected in other parts of India.

Nested PCR assay for detection of Theileria annulata in Hyalomma anatolicum infesting cattle from coastal Odisha, India.

Ticks are economically important obligatory blood feeding arthropods that have a pivotal role in transmission of infection. The present study was conducted in ixodid ticks collected from four districts of coastal Odisha, India to investigate the prevalence of Theileria annulata. Adult semi engorged Hyalomma anatolicum ticks (n = 178) were dissected, the salivary gland was isolated and DNA was extracted. A nested PCR targeting the Tams1 gene of T. annulata, utilizing two sets of primers (N516F, N517R, and Ta14136iF, Ta249R) was utilized for detection of the parasite. The PCR products were then sequenced and subjected to BLAST analysis, alignment, and phylogenetic study. Two sequences deposited in GenBank were assigned Accession No MH477290.1 and Accession No MH477291.1. The molecular investigation of T. annulata revealed an overall prevalence of 14.6% in tick vectors, and nested PCR was found to have significant (p < 0.05) higher results than primary PCR. A significant higher presence (p < 0.05) was recorded in female ticks compared with male ticks. This is the first report of detection of the parasite in tick vectors in the state of Odisha.

Transplacental transmission of Theileria annulata in cattle confirmed by molecular techniques.

Theileria annulata (T. annulata) is a tick-borne apicomplexan parasite affecting ruminants and causes severe economic loss. The present paper reports the vertical transmission of T. annulata in a three day old crossbred Jersey calf. Molecular diagnosis of tropical theileriosis was done by PCR, cloning, sequencing and phylogenetic analysis of isolated parasites. Calf showed very low hematocrit value (HCT) (17.82%), red blood cells count (RBC) count (6.9 × 106/µL), and hemoglobin concentration (Hb) (6.6 g/dL). Phylogenetic analysis of Tams 1 gene showed that T. annulata Khorda isolate (MW123091) shared 99.23%, 99.23% and 99.11% nucleotide homology with Puri, India (MN818858), Bahrain (AF214797) and Hyderabad, India (MK034702), respectively. This is the first study in Odisha, India to give an insight into the molecular detection, phylogeny and hematological analysis of T. annulata infected crossbred Jersey calf which got the infection through transplacental transmission.

Effects of commonly used chemical fertilizers on development of free-living stages of Haemonchus contortus in experimentally infected pasture.

AIM: The effects of N-P-K fertilizers in the form of urea, single super phosphate and muriate of potash on development of free-living stages of Haemonchus contortus were studied. MATERIALS AND METHODS: Five parasite free experimental plots of 1 m×1 m area, each of paddy leaves (15-day-old) and an equal number of Cynodon dactylon grass were infested with about 10×104 eggs/ml phosphate buffer saline along with the application of the calculated amount of fertilizers solution. On the 10th day of posttreatment, the pasture was cut, processed, larvae recovered by Baermann method and counted, which was expressed as number of L3 per kg dry matter (DM) of pasture. RESULTS: The average recovered population of L3 of H. contortus per kg DM varied significantly (p<0.05) between the paddy leaves (5933.57±22.718) and Cynodon grass (4861.00±22.718). When different doses of chemical fertilizer and their impact on different pasture were analyzed for control (T-1, 0-0-0 kg/ha N-P-K), the mean L3 recovery per kg DM of paddy (19512.7±50.80) was more than that of Cynodon grass (16540.9±50.80). Larvae recovery per kg DM for different pastures under treatment were in decreasing order as follows: T-2 of paddy (6981.33±50.80, 35.77%), T-2 of Cynodon (5545.38±50.80, 33.52%), T-3 of paddy (317378±50.80, 16.26%), and T-3 of Cynodon (2218.72±50.80, 13.41%) which showed significant difference (p<0.05) among the treatments. In T-4 (paddy) and T-5 (Cynodon), the average number of recovery of larvae was nil implying no significant variation (p>0.05). CONCLUSION: This study shown that when N-P-K fertilizers administered at recommended level, significantly reduced larval translation of H. contortus minimizing pasture infectivity for the free range grazing animals.