In vitro acaricidal activity of Piper longum L. against amitraz resistant Rhipicephalus microplus (Acari: Ixodidae).

The cattle tick, Rhipicephalus microplus Canestrini (Acari: Ixodidae) is one of the most important tick species severely affecting health and causes huge losses to dairy industry. Chemical acaricides are mainly applied for tick control but development of resistance, environmental pollution and contamination of milk and meat products with residues has led to exploration alternative eco-friendly tick control strategies. The dried fruits of Piper longum L. (Indian long pepper, Thippali or Pippali) generally used as flavoring agent have also been shown to have insecticidal property. Different concentrations (0.625%-10%) of alcoholic and aqueous extracts of Piper longum L. were prepared and evaluated for acaricidal activity against amitraz resistant R. microplus adult and larval stages. Against larval stages a dose-dependent mortality response was recorded for both extracts and higher acaricidal property was exhibited by the alcoholic extract with LC50 and LC95 (95% CL) values of 0.488% (0.48-0.49) and 1.39% (1.35-1.44), respectively. Similarly, against adult engorged females, ethanolic extract showed higher acaricidal property with LC50 and LC95 (95% CL) values of 4.67% (4.61-4.74) and 12.38% (12.05-12.73), respectively. Significant (p < 0.05) reduction was recorded in reproductive index of ticks treated and but no effect on hatchability of eggs was recorded in treated groups. The present study establishes acaricidal activity of P. longum fruit extracts against both larval and adult stages of amitraz resistant population of cattle tick.

Copro-prevalence and Risk Factor Assessment of Gastrointestinal Parasitism in Indian Domestic Pigs.

The aim of the present study was to determine copro-prevalence of gastrointestinal parasites and their associated potential risk factors in pigs of Punjab (India). A total of 839 faecal samples were collected from pigs of all age group and sex from different agro-climatic zones of Punjab covering all seasons and subjected to qualitative and quantitative examination. Among the samples examined, 28.4 % were positive for gastrointestinal parasites and their respective prevalences were Ascaris suum (11.1 %), coccidia (9.41 %), Trichuris suis (6.43 %), Balantidium coli (4.5 %), amphistome (3.33 %), strongyle (2.14 %) and Ascarops strongylina (1.78 %). Upon sporulation of coccidian positive samples, 8 species of Eimeria were recorded (Eimeria polita, E. spinosa, E. scabra, E. perminuta, E. suis, E. debliecki, E. neodebliecki and E. porci). Among the various risk factors analysed, season, agro-climatic zones and managemental practices had a signifi cant (p<0.05) effect on gastrointestinal parasitism of pigs. Quantification of the infection levels in various seasons and age groups revealed the highest mean egg per gram in rainy season (1966. 6± 1146.5) and grower pigs (1457.1 ± 500.4). Coproculture analysis revealed the presence of larvae of Hyostrongylus rubidus and Oesophagostomum species. The results of the current study would be of immense help in formulation and implementation of control strategies for effective control of gastrointestinal parasitism in pigs.

In vitro assessment of the acaricidal activity of Piper longum, Piper nigrum, and Zingiber officinale extracts against Hyalomma anatolicum ticks.

Ticks and tick-borne diseases are a major constraint for the sustainable cattle industry in the tropical and subtropical regions including the Indian subcontinent. The development of resistance to most of the commonly used acaricides leads to an attempt to screen plant extracts and their combinations for their possible acaricidal activity to develop an eco-friendly tick control alternative. An alcoholic and various aqueous extracts of Piper longum, Piper nigrum and Zingiber officinale and their combinations were evaluated for acaricidal activity against the three-host ixodid tick, Hyalomma anatolicum by larval immersion test using 14-21 days old unfed larvae. The efficacy was assessed by measuring larval mortality (%) and the lethal concentrations for 50% (LC50) and 95% (LC95) with their 95% confidence limits (CL) values were estimated by applying regression equation analysis to the probit transformed data of mortality. A concentration-dependent mortality response was recorded in all extracts prepared from seeds of P. longum and P. nigrum and their combinations. The highest acaricidal property was exhibited by the alcoholic extract of P. longum seeds with the minimum LC50 and LC95 (95% CL) values of 0.071% (0.07-0.072) and 0.135% (0.13-0.14), respectively, followed by alcoholic combinations. Interestingly, no acaricidal activity was recorded in extracts prepared from the rhizome of Z. officinale. The results indicated that the ethanolic extracts of P. longum and P. nigrum and their combinations can be used effectively for tick control in an integrated format.

Molecular prevalence of Babesia bigemina in Rhipicephalus microplus ticks infesting cross-bred cattle of Punjab, India.

Babesiosis is an economically important tick-borne apicomplexan protozoan disease of cattle in tropical and subtropical regions. In the present study, Rhipicephalus microplus engorged female ticks were collected from 135 apparently healthy cattle from different agro-climatic zones of Punjab, India, to investigate the carrier status of Babesia bigemina infection in vector tick by using microscopy and PCR based assays. PCR when applied on DNA extracted from the egg masses harvested from ticks showed 1.48% (2/135) samples as positive, whereas 4.44% (6/135) samples were positive when product of primary PCR was used as template in nPCR. Further, among the DNA samples isolated from the unfed larval stages that emerged from egg masses laid by ticks, only 1.48% (2/135) samples were detected as positive for B. bigemina in PCR, while 7.41% (10/135) samples were detected positive in nPCR assay. Statistically, non-significant (p > 0.05) difference in prevalence rates was observed across different agro-climatic zones and between different age groups of cattle from which engorged ticks were collected. It can, thus, be concluded that prevalence of B. bigemina in the vector tick, R. microplus in Punjab state of India indicates an endemic status of the organism and a further study is needed for the management and control of the bovine babesiosis.

Assessment of risk factors associated with prevalence of coccidiosis in dairy animals of Punjab.

A total of 2089 faecal samples (956 buffaloes and 1133 cattle) were collected from 21 districts of five major agro-climatic zones of Punjab during April, 2013-May, 2014. An overall prevalence rate of 32.17 % (672/2089) was recorded for coccidiosis in dairy animals with a significantly higher (P < 0.01) infection rate in buffaloes (35.46 %) in comparison to cattle (29.39 %). Quantitative analysis revealed oocyst per gram of faeces ranging from 200 to 10,000 (1083.72 ± 227.20) and 100 to 2400 (748.72 ± 81.38), in cattle and buffalo populations, respectively. The results of multivariate analysis showed that prevalence of coccidiosis in cattle population was associated with various risk factors viz. districts (P = 0.000; OR 2.749; 95 % CI 1.238-6.107), agro-climatic zones (P = 0.004; OR 2.707; 95 % CI 1.707-4.293) and seasons (P = 0.000; OR 1.106; 95 % CI 0.811-1.509). Similarly, in buffalo population an association with districts (P = 0.000; OR 2.163; 95 % CI 0.702-6.668), agro-climatic zones (P = 0.000; OR 1.237; 95 % CI 0.673-2.277) and seasons (P = 0.100; OR 1.382; 95 % CI 1.003-1.903) was recorded. The findings of the current study would provide a basis for evolving effective control strategy for the management of coccidiosis in dairy animals of the region.

Assessment of risk factors associated with prevalence of strongyle infection in equines from Central Plain Zone, Punjab.

A total of 311 equine faecal samples (190 horses and 121 mules) collected from six districts of Central Plain Zone, Punjab were examined using standard coprological methods. The results showed an overall prevalence of 27.33 % for strongyles with rare to mild type of infection as evident from egg per gram of faeces. In particular, amongst the examined samples, 17.90 % of horses and 42.14 % of mules were infected and the difference was statistically significant (P < 0.01). Identification of the faecal culture harvested larval stages, showed 56 % and 46 % of horses and mules, respectively, positive for small strongyles (Cyathostomes). Amongst the large strongyles, highest proportion was recorded for Strongylus vulgaris whereas, S. equinus was found in least proportion. Results indicated that the various risk factors viz. area, season, age and sex of the host had no significant effect on prevalence of strongyle infection in equines.

Fenvalerate resistance status in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from Punjab, India.

Larval packet test was used for evaluating the resistance levels in Rhipicephalus (Boophilus) microplus collected from different districts of central plain zone of Punjab state, India against fenvalerate. The regression graphs of probit mortality of larvae plotted against log values of progressively increasing concentrations of fenvalerate were utilized for the estimation of lethal concentration for 50 % (LC50) and 95 % (LC95) values against various field isolates of R. (B.) microplus. The slope of mortality (95 % confidence levels) varied from 0.730 ± 0.097 (0.419-1.043) to 1.455 ± 0.281 (0.558-2.352) and the value of R(2) varied from 0.881 to 0.997. From the regression equation the values of LC50 and LC95 were recorded in range of 184.39-1,338.01 and 3,253.33-112,706.26 ppm, respectively. Among the various tick isolates resistance factors in range of 1.56-54.34 were determined and all field isolates studied were found resistant against fenvalerate. Two field isolates (Jalandhar and Ludhiana) showed level I resistance; three (Patiala, Fatehgarh Sahib and Amritsar) showed level II and Kapurthala isolate showed level IV resistance. The data generated on fenvalerate resistant status will help in judicious use of the drug and formulation of effective tick control strategy for the region.

Detection of goldfish haematopoietic necrosis herpes virus (Cyprinid herpesvirus-2) with multi-drug resistant Aeromonas hydrophila infection in goldfish: First evidence of any viral disease outbreak in ornamental freshwater aquaculture farms in India.

This outbreak report details of a mortality event where Cyprinid herpes virus-2 (CyHV-2) was detected in association with multidrug-resistant Aeromonas hydrophila infection in goldfish, Carassius auratus, from commercial farms. The goldfish exhibited large scale haemorrhages on the body, fins and gills, lepidorthosis, necrosed gills, protruded anus and shrunken eyes. White nodular necrotic foci in spleen and kidneys were noticed, along with necrosis and fusion of gill lamellae. Transmission electron microscopy of affected tissues revealed the presence of mature virus particles. Involvement of CyHV-2 was confirmed by PCR, sequencing and observed cytopathic effect in koi carp fin cell line along with experimental infection study. A bacterium isolated from the internal organs of affected fish was found to be pathogenic Aeromonas hydrophila having resistance to more than 10 classes of antibiotics. We postulate that CyHV-2 was the primary etiological agent responsible for this outbreak with secondary infection by A. hydrophila. The experimental infection trials in Labeo rohita and koi carp by intraperitoneal challenge with CyHV-2 tissue homogenates failed to reproduce the disease in those co-cultured fish species. This is the first report of a viral disease outbreak in organised earthen ornamental fish farms in India and bears further investigation.

Molecular detection of Babesia bigemina infection in apparently healthy cattle of central plain zone of Punjab.

Bovine babesiosis is an important tick-borne disease caused by the parasites belonging to the genus Babesia, distributed worldwide and infecting a wide range of domesticated and wild cattle, occasionally man. The present study was conducted to determine the prevalence of B. bigemina infection in apparently healthy cattle from central plain zone of Punjab, India. Examination of Giemsa-stained peripheral thin blood smears revealed 2.45 % (5/204) animals to be positive for piroplasms of B. bigemina. However, genomic DNA isolated from these blood samples when subjected to primary PCR revealed a positivity of 7.35 % (15/204) as detected by the amplification of a 278-bp product in the agarose gel. PCR products obtained from the primary PCR of B. bigemina, when employed as template in nested PCR produced the amplicons of desired size (170 bp) was detected in 30.39 % (62/204) of the samples. It can thus be concluded that B. bigemina infection is prevalent in apparently healthy cattle population of this region and PCR assays can serve as a valuable tool for epidemiological studies in endemic areas.

Effect of Malathion on Reproductive Parameters of Engorged Female Rhipicephalus (Boophilus) microplus Ticks of Punjab Districts, India.

The present study was aimed at evaluating effects of malathion on the various reproductive parameters, namely, egg mass weight (EMW), reproductive index (RI), percentage inhibition of oviposition (%IO), and hatchability percentage of eggs of Rhipicephalus (Boophilus) microplus (Canestrini 1887) females from 19 districts of Punjab, India. The effect on various parameters was found to be dose dependent and more discernible upon exposure to higher concentrations. Complete cessation of egg laying was recorded in tick isolates on exposure to 5000 ppm and above. The values of %IO ranged in 4.4-68.6, 25.2-76.2, 35.6-100.0, 45.7-100.0, and 71.4-100.0 in groups treated with 1250, 2500, 5000, 10000, and 20000 ppm of malathion, respectively. A low hatching % was recorded in eggs of all treated female ticks in comparison to control treated with distilled water and complete inhibition of hatching was recorded at 10000 ppm and above. However, the survival of the hatched larvae was not affected and was similar to control group. The results of the current study can be of immense help in formulation and implementation of effective tick control measures.

Occurrence of Setaria labiatopapillosa in peritoneal cavity of a crossbred cattle.

The current study reports the occurrence of Setaria labiatopapillosa, a filarid worm, during post mortem examination in the peritoneal cavity of an adult female cross-bred cattle died due to foreign body syndrome from Ludhiana district, Punjab. The worms were cleared in lactophenol after collection and mounted in glycerol for identification. The worms were 90-95 mm in length, milky white, tapering, long with spirally coiled hind ends. The anterior end of the mounted parasite revealed peribuccal chitinous rings with lateral epaulette like structures in the head region. The posterior end of the female revealed thin, cone shaped, two lateral appendages and terminating into a prominent rosette like structure carrying a number of differently sized spikes.

Detection of amitraz resistance in Rhipicephalus (Boophilus) microplus from North Gujarat, India.

Amitraz has become one of the most extensively used chemical acaricide for control of cattle tick due to development of resistance against most of the organophosphates and synthetic pyrethroid acaricides. The resistance status of amitraz was evaluated against Rhipicephalus (Boophilus) microplus collected from Banaskantha district, Gujarat, India by adult immersion test (AIT). The different concentrations of amitraz utilized in the AIT were 125, 250, 500, 750 and 1,000 ppm. The adult female ticks showed an upward trend in the mortality percentage with increase in drug concentration. The regression graph of probit mortality of ticks plotted against log values of progressively increasing concentrations of amitraz was utilized for the determination of slope of mortality which was 1.868 ± 0.2068. The lethal concentration (LC95) was calculated as 3098.2 ppm and the RF was 24.78 which indicated level II resistance status. The dose response curves for egg masses, reproductive index and inhibition of oviposition of R. (B.) microplus were also validated and the slope was -0.5165 ± 0.08287, -0.1328 ± 0.04472 and 24.22 ± 8.160, respectively. The current study appears to be the pioneer report of amitraz resistance in R. (B.) microplus from India and the data generated could be of immense help to develop effective control strategies against ticks.

Prevalence of Theileria annulata infection in Hyalomma anatolicum anatolicum collected from crossbred cattle of Ludhiana, Punjab.

The prevalence of Theileria infection in tick vector Hyalomma anatolicum anatolicum collected from healthy animals of Ludhiana district, Punjab was recorded to assess the natural infection level of theilerial parasite in the field condition. A total of 60 semi-engorged H. a. anatolicum were collected from cattle and their salivary glands were dissected out. One half of the salivary gland was stained with methyl green pyronin (MGP) and the other half was utilized for DNA isolation for molecular detection of Theileria infection. A PCR and nested PCR assays were standardized for the detection of T. annulata infection in salivary gland of H. a. anatolicum. The prevalence of T. annulata infection was recorded as 8.3, 20.0 and 60.0 % by MGP staining, primary PCR and nested PCR, respectively. Further, the prevalence was higher in female ticks (8.8 %) than male ticks (6.6 %). The results demonstrated that both primary and nested PCR assays are a valuable technique for detection of T. annulata infection in vector tick under field conditions.

Detection of Malathion Resistance in Hyalomma anatolicum anatolicum from Bathinda District, Punjab.

OBJECTIVE: The resistance status against malathion in Hyalomma anatolicum anatolicum ticks collected from Bathinda district, Punjab, was evaluated by adult immersion test (AIT). MATERIALS AND METHODS: Technical grade malathion was used for conduction of AIT with 2 min immersion time protocol. The regression graph of probit mortality of ticks was plotted against log values of increasing concentrations of malathion and was utilized for the determination of slope of mortality, LC50, LC95 (95% confidence interval [CI]) and resistance factor (RF). The reproductive parameters of treated ticks viz. egg mass weight, reproductive index (RI), and percentage inhibition of oviposition (% IO) were also studied. RESULTS: The increasing concentration of malathion showed an upward trend in per cent tick mortality. The slope of mortality (95% CI) was 2.489 ± 0.719 (2.489 ± 0.719) and value of goodness of fit (R(2)) was 0.799. The LC50 (95% CI) and LC95 (95% CI) values were recorded as 9099.2 (8378.6-9881.7) and 41,511.3 (35,060.2-49,149.4) ppm, respectively, with RF as 16.60 indicating level II resistance status. A negative dose-dependent slope of egg mass weight (-91.79 ± 25.15 [-171.8 to -11.76]) was recorded as the survived ticks laid significantly (P = 0.0355) fewer eggs. The mean RI of treated ticks decreased with increasing concentrations of drug and the slope (95% CI) was -0.293 ± 0.059 (-0.482 to -0.105). Further, a dose-dependent significant increase (P = 0.0157) in the mean % IO was recorded in treated ticks. CONCLUSIONS: The results of the current study would be useful in the formulation and implementation of effective tick strategies in the region.

Malathion resistance in Rhipicephalus (Boophilus) microplus from Ludhiana district, Punjab.

The resistance status of Rhipicephalus (Boophilus) microplus collected from Noorpur Bet village of Ludhiana district, Punjab was evaluated against malathion by Adult Immersion Test. The adult female ticks showed an upward trend in the mortality percentage with increase in drug concentration. The regression graph of probit mortality of ticks plotted against log values of progressively increasing concentrations of malathion was utilized for the determination of slope of mortality (95 % confidence intervals) which was 2.469 ± 0.5744 (0.6413-4.297) whereas, the value of goodness of fit (R(2)) was 0.8603. The LC50 (95 % CI) and LC95 (95 % CI) were recorded as 1875.05 (1725.14-2,038) and 8,654 (7296.8-10263.8) ppm, respectively and the resistance factor was 3.46 (Level I). The slope of egg mass (95 % CI) was -0.1500 ± 0.04071 (-0.2795 to -0.02045) and was negative because with the increasing concentrations of acaricide the ticks died. The reproductive index when plotted against increasing log concentrations of malathion revealed a slope value of -0.414 ± 0.055. Further, a significant variation (p = 0.0049) was recorded in the inhibition of oviposition among the various groups treated with increasing concentrations of malathion.

Effect of various acaricides on hatchability of eggs of Rhipicephalus (Boophilus) microplus.

The effect of commonly used acaricides (amitraz, cypermethrin, deltamethrin, fenvalerate, and flumethrin) on the eggs of Rhipicephalus (Boophilus) microplus collected from Ludhiana, Punjab, was evaluated by egg hatch assay (EHA). The regression graph of probit hatchability and per cent inhibition of hatching (IH%) of eggs was plotted against log values of concentration of various acaricides. All concentrations of flumethrin and amitraz caused complete inhibition of hatching, whereas a hatchability of 31.0 ± 6.1, 40.0 ± 5.2 and 19.3 ± 1.7% was only recorded at the highest concentration of cypermethrin, deltamethrin, and fenvalerate, respectively. An increase in the concentration of the acaricide showed a significant effect on the IH% of eggs for cypermethrin (P < 0.01) and deltamethrin (P < 0.05) but was nonsignificant for fenvalerate. The slope of the regression curve of IH% was utilized for the calculation of the dose of various acaricides causing inhibition of hatching for 95% eggs (LC95) and the discriminating dose (DD). Results indicated that maximum DD was recorded for fenvalerate (2.136%), followed by cypermethrin (0.214%) and deltamethrin (0.118%). The results of the current study will be helpful in formulating effective control strategies against ticks.

Canine babesiosis in northwestern India: molecular detection and assessment of risk factors.

In the current study, a total of 214 blood samples from dogs in and around Ludhiana, Punjab (India), suspected for canine babesiosis were examined with conventional and molecular assays. Examination of Giemsa-stained peripheral thin blood smears revealed an overall prevalence of 7.47% (16/214) for canine babesiosis encompassing 0.93% (2/214) of large Babesia and 6.54% (14/214) of Babesia gibsoni. However, molecular diagnosis revealed 15.42% (33/214) samples positive for B. gibsoni infection as evident by the presence of 671 bp amplicon. The results of multivariate analysis showed that the prevalence of B. gibsoni was associated with various risk factors, namely, age (P<0.001; OR: 0.398; CI 95%: 0.080-1.799), sex (P=0.022; OR: 0.849; CI 95%: 0.403-1.791), breed of host (P=0.371; OR: 3.345; CI 95%: 1.045-10.710), and season (P=0.230; OR: 2.143; CI 95%: 0.788-5.830). The prevalence of B. gibsoni was higher in summer as compared to winter season and in younger dogs, while breed and sex of the host were not significantly associated with the occurrence of the disease.

Detection of amitraz resistance in Rhipicephalus (Boophilus) microplus from SBS Nagar, Punjab, India.

The resistance status of Rhipicephalus (Boophilus) microplus collected from SBS Nagar, Punjab, was evaluated against amitraz by Adult Immersion Test (AIT). The regression graph of probit mortality of ticks plotted against log values of progressively increasing concentrations of amitraz revealed the slope of mortality (95% CI) as 2.36 ± 0.61 (0.38 to 4.33). The LC50 (95% CI) and LC95 (95% CI) values were recorded as 332.52 ppm (305.06-362.44) and 1646.93 ppm (1383.97-1959.84), respectively, and the resistance factor (RF) was 13.17 which indicated level II resistance status. The dose response curves for egg mass weight, reproductive index, and percentage inhibition of oviposition were also validated by AIT and the slopes (95% CI) were -7.17 ± 2.41 (-14.85 to 0.50), -0.009 ± 0.02 (-0.16 to -0.031), and 19.99 ± 4.77 (4.81 to 35.17), respectively. The current study reports the development of resistance in R. (B.) microplus to amitraz from Punjab state and the data generated would be useful in formulation of effective control strategies against ticks of this region.

Prevalence of parasitic infections in cattle of Ludhiana district, Punjab.

A total of 862 cattle were sampled for both haematological (703) and coprological (159) investigations at the Large Animal Clinics, GADVASU, Ludhiana, Punjab, India. Examination of Giemsa-stained peripheral blood smears exhibited that 22.9 % (161/703) of cattle were infected with haematozoa comprising Theileria annulata (14.65 %), Trypanosoma evansi (0.28 %), Babesia bigemina (1.56 %) and Anaplasma marginale (8.53 %) while mixed infection appeared in 2.13 % (15/703) animals. The prevalence of total haemoparasites and A. marginale infections were significantly higher (p < 0.01) in younger animals <1 year of age whereas, T. evansi and A. marginale infections were significantly higher (p < 0.05) in males. Coprological examination revealed that the overall prevalence of gastrointestinal (GI) parasitic infection was 16.98 % (27/159). Among the revealed parasites, amphistomes, Fasciola spp., Eimeria spp., Balantidium coli, strongyles and Trichuris spp. were detected from 3.77, 1.88, 3.77, 2.52, 10.69 and 1.26 % of examined animals. Except coccidiosis, there was no significant variation of GI parasitic infections in relation to sex as Eimeria spp. were found higher (p < 0.01) in males. The prevalence of Trichuris spp. was significantly higher (p < 0.05) in younger animals <6 months of age. The present work emphasized that strongyles and T. annulata were the most prevalent GI and haemoparasites, respectively.

First report of Ctenocephalides felis felis infestation of buffalo calves in Punjab, India.

Two female buffalo calves of approximately 3 months of age from Hoshiarpur and Gurdaspur districts of Punjab state, India were found to be moderately infested with the cat flea Ctenocephalides felis felis. The calves were apparently healthy with no clinical signs and skin lesions. The calves were also found to be simultaneously infested with ixodid tick (Rhipicephalus (Boophilus) microplus). Infestations of buffalo calves by C. felis felis is reported for the first time.