Assessment of consistency of minimum post-mortem intervals estimated by thermal summation-based methods in medico-legal cases associated with blowflies.

BACKGROUND: This study is based on blow fly samples collected from 8 medico-legal cases in Tamil Nadu, India. The fly life stages were identified and the consistency of minimum post-mortem intervals (PMImin) estimated by different thermal summation-based methods was assessed. METHODS: PMImin of 8 medico-legal cases was estimated using six different thermal summation constants and lower developmental temperatures that are based on C. megacephala and C. rufifacies developmental data. Limits of agreement (LoA), intra class correlation coefficient (ICC) between PMImin values and margin of error of mean of difference between PMImin values were calculated. RESULTS: Intra-class correlation between the PMImin values estimated using different thermal summation constants based on C. megacephala ranged between 0.89 and 0.98 and coefficient of determination ranged between 0.93 and 0.98. Intra-class correlation between the PMImin values estimated using different thermal summation constants based on C. rufifacies ranged between 0.91 and 0.99 and coefficient of determination ranged between 0.95 and 0.99. The mean difference of PMImin values estimated using different thermal summation methods based on C. megacephala ranged between 1.8 hr and 6.6hr and margin of error ranged between 2.51 and 6.93hr. The mean difference of PMImin values estimated using different thermal summation methods based on C. rufifacies ranged between 3.33 and 31.33hr and margin of error ranged between 4.66 and 32hr. CONCLUSION: Consistency of PMImin values estimated by different thermal summation methods was good to excellent. Thermal summation constants useful in estimation of PMImin with lowest mean difference and margin of error were described.

Incidence of Onchocerca gibsoni in subcutaneous nodules of cross bred Jersey cows: case report.

Onchocerca gibsoni subcutaneous nodules in four cross bred Jersey cows aged 5-6 years brought for post mortem with calcified and caseated skin nodules in the lateral flank region is reported. Examination and dissection of these nodules revealed that they were "worm nests" of Onchocerca sp. of filarid nematodes. The worm nests were carefully dissected and filarid worms were extracted out. Multiple numbers of worms were recovered from each nodule ranging from 15 to 20. Female worms were found inside the worm nests and were found to be filled with microfilariae. The nodules weighed 2-3 g. Based on the morphological features the worms were identified as O. gibsoni.

Deltamethrin resistance in south Indian isolates of Rhipicephalus (Boophilus) microplus.

The present communication deals with the detection and characterization of deltamethrin resistance in tick populations using biological (larval packet test), biochemical (esterase enzyme assay) and molecular assays. Ticks were collected from cattle farms of Korutla, Telangana (KOR), Mehboob Nagar, Telangana (MBN), Nagpur, Maharashtra (NAG), Parbani, Maharashtra (PBN), Madhavaram, Tamil Nadu (MAD), Cuddalore, Tamil Nadu (CUD), Sakhleshpur, Karnataka (SAK) and Buvenduvella, Karnataka (BUV). Out of eight field isolates, seven were identified as Rhipicephalus (Boophilus) microplus while one isolate (CUD) was identified as R. (B.) annulatus. The LC50 values and resistance factors (RF) of field isolates were assessed by larval packet test (LPT). RF values of two isolates viz., Korutla and Parbhani (KOR, PAR) were close to that of reference susceptible isolate. R. (B.) microplus isolate from Nagpur (NAG) and Sakleshpur (SAK) revealed slightly higher RF values (6.42 and 4.51). They revealed slightly elevated esterase enzyme activity too. Other isolates did not reveal higher values for RF or esterase activity. Previously identified mutations conferring synthetic pyrethroid resistance in R. (B.) microplus populations were analysed by sequencing the mutation flanking regions of the carboxyl esterase and the sodium channel genes (domain III S6 and domain II S4-5 linker region). However, these point mutations were not detected in the field isolates. The results of the present study revealed that low levels of synthetic pyrethroid resistance had developed in field populations of ticks of southern India.

Dirofilaria repens in scrotum of dogs.

Out of 150 apparently healthy male dogs from Kerala, India examined, five revealed single worm in the scrotum. They were found protruding out from beneath the tunica vaginalis when it was incised during the castration operation. Genomic DNA isolated from these worms amplified D. repens specific 246 bp PCR products (KP050552, KP050553). Thickened and enlarged epididymis along with interductal fibrosis, congestion of veins and widening of the cavernous spaces of testes and epididymis were observed in the worm infected dogs. Semeniferous tubules were intact and showed various developmental stages of sperms. The infection resulted in less impact on spermatogenesis.The present communication forms the first report from India on the presence of D. repens in the peritoneal location, which is frequently observed in the subcutaneous locations.

Histoarchitecture of ovary of Haemaphysalis bispinosa during engorgement period.

The ovary of Haemaphysalis bispinosa was of panoistic type with asynchronous development of oocytes. The wall of the ovary was composed of a layer of epithelial cells to which the oocytes were attached by means of pedicel cells with elongated nucleus. The oocytes were classified into stages I to V based on morphologic characteristics like size and shape, presence / absence of germ vesicle, cytoplasmic appearance, presence or absence of yolk granules and presence of chorion. Day wise changes were in the form of occurrence of oogonia from partially fed upto day zero of engorgement, presence of all stages of oocytes on day one and two after engorgement and onset of degenerative changes in oocytes from day three onwards. Degeneration was complete on day eight with the appearance of polymorphism, vacuolation, cytoplasmic blebbing and autophagic activity in oocytes.

Histoarchitecture of ovary of Haemaphysalis bispinosa during engorgement period

The ovary of Haemaphysalis bispinosa was of panoistic type with asynchronous development of oocytes. The wall of the ovary was composed of a layer of epithelial cells to which the oocytes were attached by means of pedicel cells with elongated nucleus. The oocytes were classified into stages I to V based on morphologic characteristics like size and shape, presence / absence of germ vesicle, cytoplasmic appearance, presence or absence of yolk granules and presence of chorion. Day wise changes were in the form of occurrence of oogonia from partially fed upto day zero of engorgement, presence of all stages of oocytes on day one and two after engorgement and onset of degenerative changes in oocytes from day three onwards. Degeneration was complete on day eight with the appearance of polymorphism, vacuolation, cytoplasmic blebbing and autophagic activity in oocytes.

Acaricidal effects of fenvalerate and cypermethrin against Rhipicephalus (Boophilus) annulatus.

The acaricidal effects of two most commonly used acaricides viz., fenvalerate and cypermethrin against Rhipicephalus (Boophilus) annulatus were studied using Adult Immersion Test (AIT). The LC50 values observed for fenvalerate and cypermethrin were 1570 ppm and 184 ppm respectively. The death of ticks was not an immediate process. Fenvalerate caused death only after 7 days while cypermethrin after 5 days of treatment. The eggs laid by treated ticks did not hatch at all concentrations tested.

Low level deltamethrin resistance in ticks from cattle of Kerala, a south Indian state.

The deltamethrin resistance status in Rhipicephalus (Boophilus) annulatus and R. (B.) microplus ticks collected from cattle of five organized farms of Kerala, south India was evaluated. Resistance was characterized using biological (larval packet test), biochemical (esterase enzyme activity assay) and molecular tools (PCR amplification and sequencing of deltamethrin resistance-associated genes). Characterization of field isolates revealed level I resistance in ticks collected from four out of five farms. Elevated level of α/ß esterase activity was not recorded in isolates showing level I resistance. Previously reported point mutations in the carboxyl esterase (G1120A) and sodium channel (T2134A and C190A) genes were not observed in any of the field isolates. The present study showed a low level (level I) resistance is developed in the most economically important ticks infesting cattle of this state and it cautions the development of large scale resistance in future.

Phylogenetic analysis of bovine Theileria spp. isolated in south India.

The objective of the present study is to determine the phylogenetic position of the Theileria organisms in blood of cattle of southern India using molecular tools. Theileria annulata (Namakkal isolate, Tamil Nadu) and three Theileria field isolates (free of T. annulata) from Wayanad, Kerala (Wayanad 1, 2, 3) were used. The small subunit ribosomal RNA (SSU rRNA) and major piroplasm surface protein (MPSP) gene products were cloned, sequenced and the phylogenetic tree constructed. SSU rRNA gene of Wayanad 1 isolate (JQ706077) revealed maximum identity with Theileria velifera or Theileria cervi. The phylogenetic tree constructed based on SSU rRNA genes revealed that Wayanad 1 isolate belonged to a new type which share common ancestor with all the other theilerial species while Wayanad 2 and 3 isolates (JX294459, JX294460) were close to types A and C respectively. Based on MPSP gene sequences, Wayanad 2 and 3 (JQ706078, JX648208) isolates belonged to Type 1 and 3 (Chitose) respectively. When, the previously reported MPSP type 7 is also considered from the same study area, Theileria orientalis types 1, 3 and 7 are observed in south India. SSU rRNA sequence of South Indian T. annulata (JX294461) showed a maximum identity with Asian isolates while the Tams1 merozoite surface antigen (MSA) gene (JX648210) showed maximum identity with north Indian isolate.