Morphological and molecular evidence of sandfly (Diptera: Psychodidae: Phlebotomine) and its relevance to recent cases of leishmaniasis from Jammu region of North India.

Present study was conducted to carry out morphological and molecular confirmation of sandflies collected at the Department of Parasitology, Faculty of Veterinary and Animal Sciences, R.S. Pura, Jammu, India. Larva was maggot like with large head, thorax and abdomen with typical black head, 12 abdominal segments and last abdominal segment carried two pairs of caudal bristles with matchstick hairs on each segment. The adult fly possessed head, abdomen and thorax. Head consisted of pair of long, hairy and beaded antenna, proboscis and one pair of prominent black eyes. Thorax possessed a pair of wings and three pairs of legs, wings were hairy and pointed with 2nd longitudinal vein branched twice. The abdominal segments were covered with small hairs and last abdominal segment was having a pair of anal recti. These identification characteristics confirmed the fly under study as Phlebotomus argentipes that confirms its occurance in this region. Molecular characterization of identified flies was carried out on positive morophological flies. Confirmation of Phlebotomus species was ascertained by amplifying the 18S ribosomal RNA gene sequence using PCR. Clear amplification was observed for Phlebotomus argentipes (538 bp). After sequencing/genotyping, Phlebotomus argentipes (OP646634) isolate of present study was clustering in same clade with Phlebotomus argentipes sequences obtained from GeneBank from other locations across globe, irrespective of their geographical location, thus providing the molecular evidence of this species present in Jammu region of North India.

Comparative haemato-biochemical profile of induced anchylostomiasis in healthy and diabetic murine model.

A study aims to determine comparative pathology of induced anchylostomiasis was conducted in murine model. L3 larvae of Ancylostoma caninum were given at the rate of 200 larvae per rat orally and 1000 per rat through per cutaneous route in both healthy and diabetic groups as per the design of the experiment. Blood samples were collected on day 15, 30 and 45 for haemato-biochemical investigations. Decrease in Hb, PCV, TEC was observed in all groups as compared to healthy control. Leukocytosis with neutrophilia and eosinophilia was also recorded. An increase in glucose, cholesterol, ALT and AST was recorded in diabetic groups as compared to their counterparts. BUN and creatinine was elevated in all infected group. Similarly total protein and albumin gradually declines (after initial increase) in all the groups. Most of the parameters remain unaffected in group V as compared to control. It was concluded that the pathology of induced anchylostomiasis in murine model is more sever in diabetic group as evident by the significantly altered haemato-biochemical parameters when compare to their counterparts. This may be due to damage caused by non treated diabetes to different body system in addition to the anchylostomiasis.

Acceleration of cutaneous wound healing by Lucilia sericata maggots in diabetic Wistar rats.

The study was aimed to evaluate the effectiveness of maggot therapy in healing of cutaneous infected wound in streptozotocin (STZ) induced diabetic Wistar rat. For live maggots, the sterilized eggs of Lucilia sericata were obtained from colonies established in laboratory. Diabetes model was established in 48 male Wister rat by intra-peritoneal injection of STZ at the dose of 60 mg/kg body-weight. Cutaneous wounds exposed with mixed colonies of bacteria like Staphylococcus aureus, E. coli and Pseudomonas aeruginosa were prepared in all rat. The animals equally divided in 4 groups with 12 rats each being presented as treatment group of control, antibiotic, maggot and maggot with antibiotic in combination. All treatments were done once and hold for 24 hours. Wound kinetics and bacterial bio burden were measured at weekly interval to till complete healing. Significant reduction in wound area with maximum contraction was found (>95%) in maggot treated group when compared to antibiotic treated (79%) and control (72%). In maggot as well as maggot and antibiotic in combination group showed early elimination of bacterial bio-burden 7.88±0.03log CFU/ml to 1.12±0.65log CFU/ml and 7.86±0.04) log CFU/ml to 1.54±0.52log CFU/ml respectively in three weeks of time. Early healing indication was also experienced on histomorphological examination of wounded tissue of maggot treated groups by early and better epithelialization, collagenation and neovascularization with complete healing of wound in three weeks in comparison to antibiotic and control respectively. However, the present study did not show any difference in healing of wound with use of maggot alone or in antibiotic combination. Live maggot of Lucilia sericata effectively lower bacterial bioburden and and accelerate healing of infected cutaneous wound in diabetic conditions.

Acceleration of cutaneous wound healing by Lucilia sericata maggots in diabetic Wistar rats

@#The study was aimed to evaluate the effectiveness of maggot therapy in healing of cutaneous infected wound in streptozotocin (STZ) induced diabetic Wistar rat. For live maggots, the sterilized eggs of Lucilia sericata were obtained from colonies established in laboratory. Diabetes model was established in 48 male Wister rat by intra-peritoneal injection of STZ at the dose of 60 mg/kg body-weight. Cutaneous wounds exposed with mixed colonies of bacteria like Staphylococcus aureus, E. coli and Pseudomonas aeruginosa were prepared in all rat. The animals equally divided in 4 groups with 12 rats each being presented as treatment group of control, antibiotic, maggot and maggot with antibiotic in combination. All treatments were done once and hold for 24 hours. Wound kinetics and bacterial bio burden were measured at weekly interval to till complete healing. Significant reduction in wound area with maximum contraction was found (>95%) in maggot treated group when compared to antibiotic treated (79%) and control (72%). In maggot as well as maggot and antibiotic in combination group showed early elimination of bacterial bio-burden 7.88±0.03log CFU/ml to 1.12±0.65log CFU/ml and 7.86±0.04) log CFU/ml to 1.54±0.52log CFU/ml respectively in three weeks of time. Early healing indication was also experienced on histomorphological examination of wounded tissue of maggot treated groups by early and better epithelialization, collagenation and neovascularization with complete healing of wound in three weeks in comparison to antibiotic and control respectively. However, the present study did not show any difference in healing of wound with use of maggot alone or in antibiotic combination. Live maggot of Lucilia sericata effectively lower bacterial bioburden and and accelerate healing of infected cutaneous wound in diabetic conditions.

Synthetic pyrethroid resistance in Rhipicephalus (Boophilus) microplus ticks from north-western Himalayas, India.

In the present study, adult immersion test (AIT) was used for evaluation of resistance against synthetic pyrethroids (deltamethrin and cypermethrin) in Rhipicephalus (Boophilus) microplus ticks collected from nine districts of three agro-climatic zones of north-western Himalayan region of India. Resistance factors (RFs) were calculated between 0.94 to 50.71 for deltamethrin and 0.32 to 13.18 for cypermethrin. Resistance to deltamethrin was detected at level I in two, level II in four, level III and level IV in one isolate each while one isolate was susceptible. Against cypermethrin, resistance at levels I and II was detected in three isolates each while three isolates were found susceptible. The low altitude sub-tropical zone revealed higher density of resistant ticks where intensive animal husbandry practices were followed and the synthetic pyrethroid usage was common. Data generated on pyrethroid resistance status of ticks in north-western Himalayan region will provide new insights in acaricidal resistance particularly from remote areas of this region and will help in formulating suitable control measures.

In vitro acaricidal activity of Piper nigrum and Piper longum fruit extracts and their active components against Rhipicephalus (Boophilus) microplus ticks.

In vitro acaricidal activity of Piper nigrum and P. longum fruit extracts and their active components (piperine for P. nigrum and piperine and piperlonguminine for P. longum) was evaluated against adults engorged females of Rhipicephalus (Boophilus) microplus using adult immersion test. Three concentrations of each extract with four replications were used in the bioassay. Extracts significantly affected mortality rates of ticks in dose-dependent manner ranged 12.5-95.8% for P. nigrum and 29.2-87.5% for P. longum, with an additional effect on the reproductive physiology of ticks by inhibiting oviposition (28.1-96.9% by P. nigrum and 36.1-89.3% by P. longum). However, the acaricidal and oviposition limiting properties were decreased significantly when the active component(s) of each extract was tested separately. However, the combination of piperine and piperlonguminine (obtained from P. longum extract) caused 79.2% mortality of ticks which is equivalent to the corresponding concentration (~ 5%) of the extract. It can be concluded that the fruit extracts of P. nigrum and P. longum had both acaricidal and oviposition limiting actions against the adults of R. (B.) microplus which could make it a valuable component of developing sustainable strategy for integrated tick management.

Therapeutic use of Lucilia sericata maggot in controlling bacterial bio-burden in Rat wound model.

Delayed wound healing due to extraneous bacterial contamination, antibacterial resistance and other associated factors are of great concern in dealing patients having chronically infected wound. Medicinal properties of certain maggots of Calliphoridae family are known for its effective wound debridement therapy. The objective of the study was to evaluate the wound healing potential of maggots of Lucilia sericata in an experimentally infected cutaneous wound model in Wistar rat. The study was carried out by using male Wistar rats (n=48) by creating excisional wounds and later contaminated with mixed population of gram positive and gram-negative bacteria. Animals were divided randomly in to four groups with 12 individuals each, being denominated as control, antibiotic treated, maggot treated, and antibiotic plus maggot combination treated group. Ten pre-sterilized maggots were applied per centimetre square wound bed for 24 hours. Different wound kinetics in L. sericata maggot treated wounds revealed significant reduction in wound area with maximum contraction, early elimination of bacterial bioburden as compared to group of infected control and group of rats receiving only antibiotic treatment. The histopathological examination of wounded tissue of maggot treated groups showed early and better epithelialization, collagenation and neovascularization with complete healing of wound in two weeks. The maggot effects on healing when used singly or in combination with antibiotic were recorded to be similar. The results of the present study clearly demonstrate that the maggots of L. sericata possesses a definite antibacterial action along with removal of dead tissues and effectively reduced the bacterial bio-burden in infected wound and induced wound healing quickly.

Therapeutic use of Lucilia sericata maggot in controlling bacterial bio-burden in Rat wound model

@#Delayed wound healing due to extraneous bacterial contamination, antibacterial resistance and other associated factors are of great concern in dealing patients having chronically infected wound. Medicinal properties of certain maggots of Calliphoridae family are known for its effective wound debridement therapy. The objective of the study was to evaluate the wound healing potential of maggots of Lucilia sericata in an experimentally infected cutaneous wound model in Wistar rat. The study was carried out by using male Wistar rats (n=48) by creating excisional wounds and later contaminated with mixed population of gram positive and gram-negative bacteria. Animals were divided randomly in to four groups with 12 individuals each, being denominated as control, antibiotic treated, maggot treated, and antibiotic plus maggot combination treated group. Ten pre-sterilized maggots were applied per centimetre square wound bed for 24 hours. Different wound kinetics in L. sericata maggot treated wounds revealed significant reduction in wound area with maximum contraction, early elimination of bacterial bioburden as compared to group of infected control and group of rats receiving only antibiotic treatment. The histopathological examination of wounded tissue of maggot treated groups showed early and better epithelialization, collagenation and neovascularization with complete healing of wound in two weeks. The maggot effects on healing when used singly or in combination with antibiotic were recorded to be similar. The results of the present study clearly demonstrate that the maggots of L. sericata possesses a definite antibacterial action along with removal of dead tissues and effectively reduced the bacterial bio-burden in infected wound and induced wound healing quickly.

Detection of amitraz and malathion resistance in field populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in Jammu region of India.

Detection of resistance levels against amitraz and malathion in Rhipicephalus (Boophilus) microplus collected from four districts of Jammu region (India) was carried out using the adult immersion test. The regression graphs of probit mortality of ticks plotted against log values of concentration of drugs were utilised for the determination of slope of mortality, lethal concentration for 50% (LC50), 95% (LC95) and resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index and percentage inhibition of oviposition) the resistance level was categorised as I, II, III and IV. Out of these four districts, resistance to amitraz was detected at level I in Udhampur (RF = 2.81), Jammu (RF = 2.53) and Samba isolates (RF = 2.24) whereas Rajouri isolate was found susceptible (RF = 1.0). Resistance to malathion was detected at level I in Udhampur (RF = 4.01) and Jammu isolates (RF = 1.76) whereas Rajouri (RF = 0.472) and Samba (RF = 0.199) isolates were found susceptible. The data generated on amitraz and malathion resistance status will help in formulating a tick control strategy in the region.

Comparative efficacy of pour-on eprinomectin and ivermectin against Sarcoptes scabiei in buffaloes.

The efficacy of eprinomectin and ivermectin pour-on was evaluated against Sarcoptes scabiei in naturally infested buffaloes. Eighteen animals were allocated into three groups (A, B and C) of six animals each. Animals in group A were treated with eprinomectin (pour-on) @ 0.5 mg/kg; group B with ivermectin (pour-on) @ 0.2 mg/kg while group C served as untreated controls. Skin scrapings were examined on days 0, 7, 14, 21, 28 and 42 post-treatment (PT). Skin scrapings were found free for mites on day 21 PT, with marked clinical improvement in the lesions after day 28 of treatment. Untreated control group continued to show severe progressive lesions throughout the study period. No clinical side-effects of the tested drugs were observed after treatment. This preliminary report on the efficacy of eprinomectin (pour-on) against S. scabiei in buffaloes is encouraging and results could be of considerable importance.

Prevalence of ixodid ticks in dairy animals of Jammu region.

To determine the prevalence of ticks, 960 bovines (cattle 480, buffaloes 480) were examined from organised and unorganised dairy units of Jammu district during March 2012 to February 2013. The overall infestation rate was found to be 42.18 %. The infestation rate in cattle was found to be 47.08 % while 37.29 % of examined buffaloes were infested with ixodid ticks. Among the ixodid ticks, only one species i.e. Rhipicephalus (Boophilus) microplus was recorded. A significantly (p < 0.05) higher prevalence was recorded in monsoon season as compared to other seasons. Agewise, the animals aged <6 months showed the highest prevalence and the lowest was found in animals >1 year of age. Sexwise, the males had higher infestation rate than the females.

Prevalence and analysis of associated risk factors for Cryptosporidium infection in lambs in Jammu district.

An epidemiologic study was carried out to investigate the prevalence and analysis of risk of Cryptosporidium infection in lambs in Jammu district. Faecal samples of 120 lambs of different age groups viz., <1 month, 1-3 months and 3-6 months were assessed. Cryptosporidium oocysts were identified by using modified Zeihl Neelsen technique. Statistical analysis showed that infection rates were significantly higher in lambs of <1 month age group (65 %) than other two age groups (p < 0.05). Similarly, cryptosporidial infection rates were significantly higher in diarrhoeic (54.41 %) than in non diarrhoeic lambs (34.61 %). Winter records highest prevalence (73.33 %) which varied significantly. Sex wise higher prevalence was observed in females (51.56 %) as compared to males (39.28 %). The relationship between intensity of infection and various epidemiological factors showed that highest intensity was observed in lambs of 0-1 month age group, having diarrhoea, in winter season.

Incidence of Haemoproteus columbae in pigeons of Jammu district.

Haemoproteus columbae is the major protozoan infection reported in pigeon and appeared in the erythrocyte of the peripheral circulation. Incidence and parasitaemia of H. columbae in pigeon was studied in different localities of Jammu, India for a period from April to September 2010 using thin blood smear examination. Of the 150 pigeons (wild: 70, domestic: 80), 92 (61.33 %) were found to be infected with H. columbae. Domestic pigeon showed higher incidence rate (74.28 %) than the wild (50 %). Mature and immature gametocyte encircled the erythrocyte nucleus to form a halter shaped appearance were characteristic feature of the parasite. Pseudolynchia canariensis, the vector for H. columbae was also recovered from beneath the feathers of pigeons. No other haemoprotozoan parasite was observed in the blood smear of examined pigeon. Counting of infected erythrocyte revealed higher affection of 1-2 erythrocytes indicative of milder infection in the birds. A long term study within bird population is essential in order to disclose seasonal variation in parasite, vector density and age of infection such as nesting area.

Incidence of Buxtonella sulcata in bovines in R.S. Pura, Jammu.

Incidence of Buxtonella sulcata was recorded as 20.9 % in bovines (n = 374) of R.S. Pura, Jammu with cattle showing higher infection rate (23.6 %) than the buffaloes (18.5 %). The infection was significantly (p < 0.0001) higher in young animals (33.1 %) than the adults (13.9 %). Animals with diarrhoeic faeces had a significantly higher (p < 0.0001) infection rate (38.5 %) than the animals with normal faeces (9.9 %), suggesting the influence of protozoan on the incidence of diarrhoeal symptoms in bovines.

Occurrence of Setaria digitata in a cow.

The present investigation reports the occurrence of filarial worm, Setaria digitata, recovered during the surgery of a cow suffering from intussusception. The worms were thread like, milky white, long with tapering ends especially towards the hind end with an average size of 62.8 ± 9.89 mm in length. On examination of anterior end, the cuticular rings surrounding the mouth region with dorsal, ventral and lateral prominences were observed. The posterior end had few spines along with a pair of appendages near the tip of the tail. On the basis of morphological characteristics the worms were identified as female S. digitata.

Deltamethrin resistance in field populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in Jammu and Kashmir, India.

Detection of resistance levels against deltamethrin in Rhipicephalus (Boophilus) microplus collected from six districts of Jammu and Kashmir (India) was carried out using the adult immersion test. The regression graphs of probit mortality of ticks plotted against log values of concentration of drug were utilised for the determination of slope of mortality, lethal concentration for 50% (LC50), 95% (LC95) and resistance factor (RF). On the basis of the data generated on mortality, egg mass weight, reproductive index and percentage inhibition of oviposition, the resistance level was categorised as I, II, III and IV. Out of these six districts, resistance to deltamethrin at level I was detected in one district (RF = 1.9), at level II in two districts (RF = 7.08-10.07) and at level IV in three districts (RF = 96.08-288.72). The data generated on deltamethrin resistance status will help in formulating tick control strategy in the region.

In vitro acaricidal activity of ethanolic and aqueous floral extracts of Calendula officinalis against synthetic pyrethroid resistant Rhipicephalus (Boophilus) microplus.

Detection of resistance levels against deltamethrin and cypermethrin in Rhipicephalus (Boophilus) microplus collected from Jammu (India) was carried out using larval packet test (LPT). The results showed the presence of resistance level II and I against deltamethrin and cypermethrin, respectively. Adult immersion test (AIT) and LPT were used to evaluate the in vitro efficacy of ethanolic and aqueous floral extracts of Calendula officinalis against synthetic pyrethroid resistant adults and larvae of R. (B.) microplus. Four concentrations (1.25, 2.5, 5 and 10 %) of each extract with four replications for each concentration were used in both the bioassays. A concentration dependent mortality was observed and it was more marked with ethanolic extract. In AIT, the LC50 values for ethanolic and aqueous extracts were calculated as 9.9 and 12.9 %, respectively. The egg weight of the live ticks treated with different concentrations of the ethanolic and aqueous extracts was significantly lower than that of control ticks; consequently, the reproductive index and the percent inhibition of oviposition values of the treated ticks were reduced. The complete inhibition of hatching was recorded at 10 % of ethanolic extract. The 10 % extracts caused 100 % mortality of larvae after 24 h. In LPT, the LC50 values for ethanolic and aqueous extracts were determined to be 2.6 and 3.2 %, respectively. It can be concluded that the ethanolic extract of C. officinalis had better acaricidal properties against adults and larvae of R. (B.) microplus than the aqueous extract.

Acaricidal activity of ethanolic extract of Artemisia absinthium against Hyalomma anatolicum ticks.

The aim of the study was to evaluate the in vitro efficacy of different concentrations of ethanolic extract obtained from the aerial parts of Artemisia absinthium in comparison to amitraz on adults, eggs and larvae of Hyalomma anatolicum using the adult immersion test (AIT), egg hatchability test and larval packet test (LPT), respectively. Four concentrations of the extract (2.5, 5, 10 and 20%) with three replications for each concentration were used in all the bioassays. In AIT, the mortality rates at 2.5, 5 and 10% were significantly different (p < 0.05) in comparison to the control group; however, at 20%, it was similar to the positive control group. Maximum mortality of 86.7% was recorded at 20%. The LC50 and LC95 values were calculated as 6.51 and 55.43%, respectively. The oviposition was reduced significantly by 36.8 and 59.1% at concentrations of 10 and 20%, respectively. Egg hatchability was reduced significantly at all concentrations (2.5-20%) in comparison to the control. In LPT, the extract caused 100% mortality of larvae at all the concentrations after 24 h. The results show that ethanolic extract obtained from the aerial parts of A. absinthium has acaricidal properties and could be useful in controlling H. anatolicum.

In vitro evaluation of ethanolic extracts of Ageratum conyzoides and Artemisia absinthium against cattle tick, Rhipicephalus microplus.

In vitro efficacy of ethanolic extracts obtained from the aerial parts of Ageratum conyzoides and Artemisia absinthium was assessed on Rhipicephalus microplus using adult immersion test (AIT). Five concentrations of the extract (1.25%, 2.5%, 5%, 10%, and 20%) with three replications for each concentration were used in the bioassay. In AIT, the maximum mortality was recorded as 40% and 66.7% at 20% concentration for A. conyzoides and A. absinthium, respectively. Acaricidal activity was found to be higher in the extract of A. absinthium with LC50 and LC95 values of 11.2% and 61.7%, respectively. Egg mass weight of the live ticks treated with different concentrations of the extracts was significantly (P<0.05) lower than that of control ticks; consequently, the reproductive index and oviposition values of the treated ticks were reduced significantly (P<0.05). The A. conyzoides inhibited 90% hatching of eggs at the 20% concentration, whereas A. absinthium showed 100% inhibition at 5%, 10%, and 20% concentrations. The results show that A. absinthium has better acaricidal properties than A. conyzoides and could be useful in controlling R. microplus.

In vitro acaricidal activity of Atropa belladonna and its components, scopolamine and atropine, against Rhipicephalus (Boophilus) microplus.

In vitro efficacy of methanolic extract of Atropa belladonna and its components scopolamine and atropine was assessed against Rhipicephalus (Boophilus) microplus. Five concentrations of the extract (1.25%, 2.5%, 5%, 10%, and 20%) were used whereas scopolamine and atropine were each tested at 0.1%. In adult immersion test, the extract was lethal to ticks at 20% concentration. The LC50 and LC95 values were determined as 6.875% and 17.306%, respectively. The extract caused a significant reduction (P < 0.05) in egg mass production at 10% concentration. In larval packet test, the extract was lethal to larvae in the concentrations of 10% and 20% after 24 h, with LC50 and LC95 values of 1.321% and 4.935%, respectively. Scopolamine and atropine showed 93.3% and 60.0% mortality of adult ticks, respectively, but they caused complete (100%) blocking of hatching as well as 100% larval mortality. Scopolamine and atropine were observed to be more potent than the crude extract at an equivalent concentration in both the bioassays.