The effects of hair density of beef cattle on Haematobia irritans horn fly populations

Abstract We show the relationships that exist between the amount of hair and quantity of sebum on cattle skin and the population density of the horn fly, Haematobia irritans. Brahman and Chianina steers had means of 2390 and 1587 hairs per cm², respectively, significantly more than the mean number of hairs on Angus, Brahman x Angus Crossbred, Charolais, and Red Poll steers. The Chianina steers had > 30% more sebum present on their skin and hair (0.58g/929cm²) than the Angus, Charolais, and Red Poll steers at the Beef Cattle Research Station Savoy, Arkansas. The Brahman steers had a significantly greater amount of sebum present on the skin (1.51 g/ 929 cm²) than the Crossbred and purebred Angus steers (0.55 and 0.25g/929cm², respectively) at the South Central Family Farms Research Centre Booneville, Arkansas. The Brahman and Chianina steers had means of 61.9 and 17.0 horn flies per steer, respectively, during the fly season, whereas the Angus, Crossbred, Charolais and Red Poll steers had fly season means that ranged from 76.9 to 265.8 flies per steer. Regression analysis showed that an increase of 100 hairs per cm², was associated with a reduction of 11 horn flies in the Angus II, 5 in Angus I, 20 in Charolais, 37 in Red Poll, and 0.4 in Chianina steers at the Savoy Station and a reduction of 6.6 horn flies for the Angus, Brahman, and Crossbred steers at the Booneville Centre. Regardless of cattle breed, an increase of 1.0 g of sebum per 929 cm² output by the steer was associated with 478.5 additional hairs per cm² on the animal. Each increase of 0.25 g of sebum per 929 cm² resulted in a decrease of 9.2 horn flies per steer. We conclude that some of the factors responsible for fly-resistance in cattle are hair density and the corresponding amount of sebum present on cattle skin and hair.     

Role of Rusa deer Cervus timorensis russa in the cycle of the cattle tick Boophilus microplus in New Caledonia

Two field experiments were conducted to evaluate the efficiency of Rusa deer in the development of the cattle tick Boophilus microplus in comparison with that of steers in the same pastures and under the same conditions of infestation. No difference was noted between a mixed steer/deer herd and a pure steer or pure deer herd in the infestation pattern of each host, suggesting that attachment to the alternative host is mechanical and not affected by the simultaneous presence of the primary host on the pasture. Deer are capable of producing engorged viable females, with weight and reproductive performances similar to or even better than females fed on steers. For moderate levels (1 million larvae per hectare) and high levels (32 million larvae per hectare) of pasture infestation, tick burdens on steers were not very different (e.g. average 1911 and 2681ticks per m² skin, respectively, on day 24). This may be because of saturation of steer skin sites at the moderate larval dose. Deer harboured 2.7–33 times fewer ticks than steers and produce no engorged females at the moderate larval level and 32 times fewer engorged females than steers at the high larval level. Infestation of deer was dosedependent with averages of 12 and 399ticks per m² skin on day 25 at the moderate and high larval levels, respectively. At a high infestation level of the environment, Rusa deer may contribute, but to a limited extent, to infestation of pastures and, consequently, of cattle. However, their role in sustaining a viable tick population requires further investigation.     

Estimation of the effects of buffalo fly (Haematobia irritans exigua) on the milk production of dairy cattle based on a meta-analysis of literature data

Published reports on the effect of buffalo fly Haematobia irritans exigua De Meijere (Diptera: Muscidae) and the closely related horn fly (H. irritans) were examined and analysed using non-linear weighted regression techniques in an attempt to establish the relationship between daily production loss (D), average number of parasites (n) and the average damage per parasite per day (d), and to provide estimates of expected losses in milk yield (MYD) and live-weight gain (LWG) in dairy cattle. A Mitscherlich three-parameter model was used to explain the relationship between the total loss of production attributable to buffalo flies and the average number of flies associated with cattle. This model was significant (P<0.01), with R2 = 20.2% and predicted a threshold number of flies (n = 30) below which no adverse effects would be noted. At a moderate level of infestation (n = 200) dMYD was 2.6 ml/fly/day and dLWG was 0.14 g/fly/day, resulting in estimated daily losses in milk yield (D(MYD)) and live-weight gain (D(LWG)) of 520 ml and 28 g, respectively.     

Morphology of the Leather Defect Light Flecks and Spots

The skin histology and the scanning electron microscope morphology of the hide defect light flecks and spots after tanning were studied in 11 steers infested with biting lice (Damalinia bovis). Nine steers from herds free of lice were used as controls. Skin biopsies from 6 of the animals in the lice infested group showed mild to moderate hyperkeratosis and moderate perivascular to diffuse dermatitis with infiltration of mainly mononuclear cells and some eosinophilic granulocytes. The steers were slaughtered at an age of 18 to 23 months. Light flecks and spots occurred on all examined hides from the infested group after tanning. No examined hides from the control group demonstrated similar damage. Both light microscopic examination of sections of tanned hide with light flecks and spots and scanning electron microscopy of the same defects showed superficial grain loss and craters with a irregular fibre base encircled by smooth and intact grain.     

Differential survival of male and female partially resistant horn flies (Diptera: Muscidae) on steers treated with permethrin

Males and females from a heterozygous, resistant strain (SR) of the horn fly, Haematobia irritans (L.), were tested for survival until reproductive maturity on steers with ear tags in outdoor, screened enclosures and on steers sprayed on the neck in an indoor isolation room. After 6 d, female SR flies on outdoor steers with one tag had 10 times greater survival than males; almost no SR flies on steers with two ear tags survived. Survival of male and female SR flies on steers sprayed on the neck was reduced during the first 24 h, but not thereafter. Lower survival of males compared with females on treated steers reflected differential survival of the sexes during exposure to treated cloths in a laboratory bioassay. Hair samples from neck, back, rump, and lower legs of steers with ear tags in outdoor pens were tested for toxicity to the SR flies. These bioassays indicated high localization of insecticide on the neck of steers with ear tags.     

Cattle breed-variation in infestation by the horn fly Haematobia irritans

A study was carried out to assess the resistance of pure and cross-bred groups of cattle to the horn fly Haematobia irritans (Linnaeus) (Diptera: Muscidae) in northern Argentina. Pure-bred cattle were Criolla, Iberian Bos taurus Linnaeus (Artiodactyla: Bovidae) and Nellore, Bos indicus Linnaeus (Artiodactyla: Bovidae). Cross-bred cattle were Hereford, British B. taurus (34%) X Nellore (66%) and Hereford (66%) X Nellore (34%). All were heifers and animals were maintained in two groups, each containing a mixture of pure and cross-breeds. The lowest fly numbers were found on Criolla heifers and the highest on Hereford X Nellore cross-breeds. However, it could not be determined from this study whether this was a consequence of breed and/or size, as Criolla heifers were lighter than the corresponding Hereford X Nellore heifers. Fly numbers on the heifers followed an approximately negative binomial distribution. However, the ranking of individual animals in their level of infestation within subgroups was not consistent. Hence, culling the most infested heifers on any given date would at best give only a small improvement in H. irritans control.     

Roles of the mushroom bodies in olfactory learning and photoperiodism in the blow fly Protophormia terraenovae

Mushroom bodies (MBs) in Protophormia terraenovae were ablated by hydroxyurea (HU) treatment to larvae just after hatching in order to examine roles of the MBs in olfactory learning and photoperiodism. In all individuals the structures of the alpha, beta, and gamma lobes, the pedunculi, and the calyces of the MB were not found after HU treatment. The other structures of the brain were not obviously damaged. The volumes of both the antennal lobes and the central complex, however, were smaller in the HU-treated flies than those in the control flies. The HU-treated and non-treated flies were tested for their appetitive olfactory learning ability and photoperiodism. In the olfactory learning paradigm, an odor of methylsalicylate or coumarin was paired with a reward of sucrose. The non-treated flies learned to associate both odors with the reward, but the HU-treated flies did not. In the test for photoperiodism, both the HU-treated and non-treated flies responded to photoperiod. Both groups of flies developed ovaries under long-day conditions but entered diapause under short-day conditions. The results imply that the MBs are indispensable for olfactory learning but not for photoperiodism, and that storage of daily cycles of photoperiodic information occurs by a neural system other than the MBs.     

Dynamics and mechanisms of permethrin resistance in a field population of the horn fly, Haematobia irritans irritans

A study was conducted at the Pressler ranch, near Kerrville, Texas, USA between 2002 and 2006 to determine the dynamics and mechanisms of resistance to permethrin in a field population of the horn fly, Haematobia irritans irritans (L.). Changes of resistance to pyrethroid insecticide associated with use of a pour-on formulation of cyfluthrin in 2002 and use of diazinon ear tags in subsequent years were studied using a filter paper bioassay technique and a polymerase chain reaction assay that detects two sodium channel mutations, kdr and super-kdr resistance alleles. A maximum of 294-fold resistance to permethrin was observed in the summer of 2002. A significant decrease in the resistance level was observed in spring 2003, and resistance continued to decline after animals were treated with diazinon ear tags. In response to pyrethroid treatments, the allelic kdr and super-kdr frequency increased from 56.3% to 93.8% and from 7.5% to 43.8%, respectively in 2002, and decreased significantly in 2003 when the pyrethroid insecticide was no longer used to treat animals. Females were found to have a higher allelic super-kdr frequency than males in 2002, while no difference was detected between males and females in the allelic kdr frequency. There was a significant positive correlation between frequencies of the sodium channel mutations and levels of permethrin resistance, suggesting that the sodium channel mutations, kdr and super-kdr , are the major mechanisms of resistance to pyrethroids in this horn fly population. Results of synergist bioassays also indicated possible contributions of two metabolic detoxification mechanisms, the mixed function oxidases (MFO) and glutathione S-transferases (GST). Compared to a horn fly infestation of an untreated herd, treatments with the pyrethroid pour-on formulation failed to control horn flies at the Pressler ranch in 2002. Sustained control of horn flies was achieved with the use of diazinon ear tags in 2003 and subsequent years.     

Long-term study of Haematobia irritans (Diptera: Muscidae) seasonal distribution in central Argentina with focus on winter fly abundance.

The seasonal distribution of Haematobia irritans (Linnaeus, 1758) was evaluated at 31 degrees 12' S-61 degrees 29' W, Santa Fe, Argentina from November 1992 to August 2000 by weekly fly counts on 20 Holstein cows not treated against ectoparasites. The analysis was mainly focused on winter fly abundance. Two peaks of abundance were found from spring to autumn. Adults of H. irritans were consistently found on cattle during winter, with increasing numbers from the end of July to late August. The only climatic parameter soundly correlated with the four week period, before each winter fly count, was the mean air temperature with the exception of year 1998. No significant relationship was found between level of cow infestation and relative humidity, saturation deficit and rainfall. Horn fly infestation on cows was lower than 20% from end of June to end of August only in 1995, when mean air temperature was consistently lower than 11 degrees C during the four week period previous to most fly counts. Conversely, the mean temperature was higher than 12 degrees C previous to fly counts in 1998, when most cows remained infested. The results indicate that a proportion (unknown) of immature stages of H. irritans were insensitive to diapause inducing factors and developed through winter.     

Microbial community on industrial salty bovine hides: From the slaughterhouse to the salting

The leather-making industry is an age-old industry and desiccation with salt has been one of the most used methodologies for obtaining valuable skins. However, halophiles may proliferate and affect the integrity of the hide-collagen structure, as well as leading to undesirable red colorations or less-frequent purple stains. To understand the basis of these industrial hide contaminations, the microbial community from raw hide samples, salt-cured samples and four different industrial salts, was analyzed by 16S rRNA gene metabarcoding together with standard cultivation methods.Comparison of raw hides and correctly cured hides revealed a core microbiome that was absent from contaminated hides. In addition, archaea were missing from well-cured hides, whereas Psychrobacter and Acinetobacter were highly represented (23 % and 17.4 %, respectively). In damaged hides, only a few operational taxonomic units (OTUs), from among the hundreds detected, were able to proliferate and, remarkably, a single Halomonas OTU represented 57.66 % of the reads. Halobacteria, mainly Halovenus, Halorubrum and Halovivax, increased by up to 36.24–39.5 % in the red- and purple-affected hides. The major contaminants were isolated and hide infections, together with collagenase activity, were evaluated. The results showed that hides enriched with the non-pigmented isolate Halomonas utahensis COIN160 damaged the collagen fibers similarly to Halorubrum, and together they were considered to be one of the major causes. Putative degrading inhibitors were also identified from among the Alkalibacillus isolates.It was concluded that hide contaminations were driven by clonal outbreaks of a few specific microbes, which may have been non-pigmented collagen degraders. Acinetobacter and Alkalibacillus, members of the core microbiome of raw and well-cured salted hides, are suggested as hide contaminant inhibitors that need further analysis.     

The effectiveness of bird hides in mitigating recreational disturbances of birdwatchers

Bird hides are often built to ameliorate the disturbance of birdwatchers at birdwatching sites. However, the effectiveness of such shelters is rarely evaluated. This study investigated the recreational disturbances induced by birdwatchers at Mai Po Nature Reserve in Hong Kong and examined the effectiveness of bird hides in mitigating birdwatcher impacts. A bird disturbance survey was conducted to record the disturbances caused by birdwatchers and responses shown by birds at three sampling sites in Mai Po Nature Reserve. A total of 469 observations were recorded in 25 field days from Oct 2019 to April 2020. The results indicated that bird hides significantly mitigated the frequency of disturbance and level of response of birds. With a bird hide, the frequency of disturbance was lower than the unsheltered site given that both sites have a similar level of visitation, yet, disturbance frequency was not significantly lower if the bird hide was highly utilized by birdwatchers. Birds had shown less vigorous and slower responses to disturbances at sites with bird hides than that without. On the other hand, at those sites with bird hides, birds responded at a farther distance than birds at the unsheltered site, which a longer distance may represent a greater level of disturbance. Upon disturbances, birds at the unsheltered site tended to stay in the original area, while birds around bird hides tend to settle further from the original habitat. Visual disturbances were well avoided at bird hides when compared to an unsheltered environment, however, disturbance from noise was found to be more prevalent at bird hides. Implications of these findings were discussed, and recommendations regarding the use, setting, and design of bird hides, and visitor management were given.     

Isolated fibrillar damage in tendons stimulates local collagenase mRNA expression and protein synthesis

The etiology of repetitive stress injuries in tendons has not been clearly identified. While minor trauma has been implicated as an inciting factor, the precise magnitude and structural level of tissue injury that initiates this degenerative cascade has not been determined. The purpose of this study was to determine if isolated tendon fibril damage could initiate an upregulation of interstitial collagenase (MMP13) mRNA and protein in tendon cells associated with the injured fibril(s). Rat tail tendon fascicles were subjected to in vitro tensile loading until isolated fibrillar damage was documented. Once fibrillar damage occurred, the tendons were immediately unloaded to 100g and maintained at that displacement for 24h under tissue culture conditions. In addition, non-injured tendon fascicles were maintained under unloaded (stress-deprived) conditions in culture for 24h to act as positive controls. In situ hybridization or immunohistochemistry was then performed to localize collagenase mRNA expression or protein synthesis, respectively. Fibrillar damage occurred at a similar stress (41.13+/-5.94MPa) and strain (13.24+/-1.94%) in the experimental tendons. In situ hybridization and immunohistochemistry demonstrated an upregulation of interstitial collagenase mRNA and protein, respectively, in only those cells associated with the damaged fibril(s). In the control (stress-deprived) specimens, collagenase mRNA expression and protein synthesis were observed throughout the fascicle. The results suggest that isolated fibrillar damage and the resultant upregulation of collagenase mRNA and protein in this damaged area occurs through a mechanobiological understimulation of tendon cells. This collagenase production may weaken the tendon and put more of the extracellular matrix at risk for further damage during subsequent loading.     

Summer mastitis experimentally induced by Hydrotaea irritans exposed to bacteria

Abstract. Summer mastitis is an acute suppurative bacterial infection of the udder in heifers and dry cows. To ascertain the possible role of flies in the transmission of the disease, experimental exposures of recipient heifers to Hydrotaea irritans previously exposed to bacteria were carried out. Flies were allowed to feed on secretions from clinical cases of summer mastitis. The pathogens present were the bacteria Staphylococcus aureus, Streptococcus dysgalactiae, Actinomyces pyogenes , Stuart-Schwan cocci, Peptococcus indolicus, Fusobacterium necrophorum and Bacterioides species. The teats of eight heifers were exposed to flies with verified pathogen content. Two teats of each animal were deliberately damaged before fly exposure. One teat was cut, another pricked with insect needles to mimic insect bites. Two of the heifers developed summer mastitis in the quarters where teats had been cut. The bacterial species isolated from these quarters corresponded to those that had previously been fed to the flies. For the first time, it is now demonstrated that H.irritans is capable of transmitting summer mastitis pathogens and so causing summer mastitis in recipient heifers. Lesions on the teat orifice may be a predisposing factor in the development of the disease.     

Managing the horn fly (Diptera: Muscidae) using an electric walk-through fly trap

An electric walk-through fly trap was evaluated for the management of the horn fly, Hematobia irritans (L.), on dairy cattle in North Carolina over 2 yr. The trap relies on black lights and electrocution grids to attract and kill flies that are brushed from the cattle passing through. During the first season, horn fly densities were reduced from >1,400 to <200 flies per animal. Horn fly density averaged 269.2 +/- 25.8 on cattle using the walk-through fly trap twice daily, and 400.2 +/- 43.5 on the control group during the first year. The second year, seasonal mean horn fly density was 177.3 +/- 10.8 on cattle using the walk-through fly trap compared with 321.1 +/- 15.8 on the control group. No insecticides were used to control horn flies during this 2-yr study.     

Chronology of permethrin resistance in a southern Illinois population of the horn fly (Diptera: Muscidae) during and after selection by pyrethroid use

From 1985 through 1988, horn flies (Haematobia irritans (L)) collected at the Dixon Springs Agricultural Center (DSAC) in southern Illinois were tested in 22 h bioassays for permethrin resistance with residues on cotton cloths. The LC90 for a susceptible field population collected in June 1985 was 0.19 micrograms/cm2. In comparison, flies collected from pyrethroid-tagged cattle in 1985 and 1986 exhibited 25- to 116-fold resistance to permethrin. A 25-fold level of resistance allowed survival on treated cattle 8 wk after pyrethroid tag application. Flies representing the local background population were collected periodically from an untreated herd 2.4 km from the nearest cattle treated with a pyrethroid; these flies exhibited up to 18-fold resistance. Although pyrethroids were not used on DSAC animals after October 1986, all bioassays done in 1987 and 1988 indicated resistance levels of greater than or equal to 7-fold. The 95% confidence intervals for LC90s from all 1987 bioassays overlapped the confidence interval from the corresponding July 1986 estimate for resistant flies collected from pyrethroid-tagged cattle. Although some decline in resistance was evident in 1988, bioassays done at the end of the season produced resistance ratios of 7.4 and 15.3. Survivorship at a diagnostic dose indicated that resistance frequencies remained at 4-8% throughout 1988. Two years' abstinence from pyrethroid use was insufficient to allow an adequate decline in resistance levels.     

Effects of horn fly and house fly (Diptera: Muscidae) larvae on the development of parasitic nematodes in bovine dung

Laboratory studies were conducted to determine the effects of horn fly, Haematobia irritans (L.), and house fly, Musca domestica L., larvae on the development of a mixed population of parasitic nematodes in compressed and crumbled bovine dung. Fresh dung (100 g per sample) from a single calf passing trichostrongyle type eggs was infested with 150 horn fly or 150 house fly eggs. After 14-15 d, more horn flies and house flies had emerged from the compressed dung than from the crumbled dung, but more third stage parasitic nematode larvae were recovered from the crumbled dung containing either fly species than from dung containing no flies.     

Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay

The correlation of oxidative stress on development and DNA damage in bovine embryos was investigated by the comet assay (single-cell microgel electrophoresis), an effective technique for detecting single-strand DNA breakage. After in vitro maturation and fertilization, one-cell stage embryos without cumulus cells were cultured for 8 days in SOF medium containing amino acids plus 5% FCS under low (5%) and atmospheric (20% ) oxygen concentration. After 8 days of culture, the extent of blastocyst formation was significantly decreased (P<0.001) when embryos were cultured under 20% oxygen concentration (5.8 +/- 2.4%) when compared to embryos cultured under 5% oxygen concentration (35.1 +/- 6.7%). At the day 3 of development, DNA damage of individual embryos cultured under 5% or 20% oxygen concentration was measured by the comet assay, which entails microgel electrophoresis that can readily detect damaged DNA. After measuring the DNA damage in individual embryos by the comet assay, the length (149.9 +/- 15.3 microm) of the migrating DNA fragment that is indicative of damaged DNA was significantly increased (P<0.001) in the embryos cultured under 20% oxygen concentration when compared to embryos cultured in 5% oxygen concentration (42.3 +/- 7 microm). The length of damaged DNA in more than 50% of embryos was less than 50 microm. when embryos were cultured under 5% oxygen concentration. In contrast, the distribution of damaged DNA shifted to the more damaged extent when embryos were cultured under 20% oxygen concentration. These results demonstrate that the retardation in bovine embryo development than in likely due oxidative stress as a consequence of the higher atmospheric oxygen concentration is positively correlated with an increase in the extent of DNA damage. Moreover, these results demonstrate that the comet assay is a useful method to evaluate embryo culture conditions.     

Resistance to the Mexican rice borer (Lepidoptera: Crambidae) among Louisiana and Texas sugarcane cultivars

A 2-yr study to evaluate Louisiana and Texas sugarcane, Saccharum spp., cultivars for resistance to the Mexican rice borer, Eoreuma loftini (Dyar) was conducted in two locations in Texas, chosen for having different infestation levels. Criteria for assessment of resistance included percentage of bored internodes and adult emergence holes, the latter used to determine the relative impact of each cultivar on the potential areawide buildup or reduction of adult E. loftini populations. A recently released cultivar, HoCP 85-845, seemed to lose a portion of its resistance under heavy E. loftini infestation pressure, suggesting its value only in moderate-to-low infestation conditions. Cultivar CP 70-321 was the most resistant. Results indicated that cultivar LCP 85-384 was significantly (P < 0.05) more susceptible than NCo 310, traditionally the most susceptible cultivar commercially produced in Texas. In 2001, LCP 85-384, which now represents 85% of the production area in Louisiana, had the greatest moth production per hectare (17,052 +/- 3,956) under the lower infestation pressure, significantly (P < 0.05) higher than HoCP 85-845 (3,038 +/- 2,353). In a portion of the test at the high-infestation location, high levels of sodium and magnesium salt stress (15-30-cm soil depth) were associated with higher E. loftini damage in all cultivars except HoCP 91-555 and CP 70-321.     

Interactive response of the horn fly (Diptera: Muscidae) and selected breeds of beef cattle

Statistically significant differences were observed in the population density of the horn fly, Haematobia irritans irritans (L.), on different breeds of beef cattle. The European breed Chianina had a population density of horn flies generally less than or equal to 50% than that of the British cattle breeds (Angus, Hereford, Polled Hereford, and Red Poll) and another European breed (Charolais). Generally, no significant difference existed among numbers of horn flies on Hereford, Polled Hereford, and Red Poll cows in 1988 or among Angus, Hereford, Polled Hereford, and Red Poll cows in 1989. Factors other than color appeared to be involved in the selective process between the horn fly and its host. Population densities on two white European breeds (Charolais and Chianina) were significantly different on all weekly intervals except for 4 wk in both 1988 and 1989. No significant difference existed among Charolais and British breeds except during 4 wk in 1988 and 3 wk in 1989. When weaning weights of all calves were adjusted for the effects of age to 205 d, sex of calf, and age of dam, the indirect effect of the horn fly on weaning weight showed a significant linear regression. Each 100 flies per cow caused a reduction of 8.1 kg in calf weaning weight. Cows within each breed with low numbers of horn flies weaned significantly heavier calves than cows with higher numbers of horn flies.     

Trypanosoma congolense: drug resistance during cyclical transmissions in tsetse flies and syringe passages in mice

A drug-resistant Trypanosoma congolense strain with predetermined curative doses (CD50 and CD90) of samorin at 13.9 +/- 1.02 and 20.3 +/- 1.13 mg/kg body weight, respectively, was cyclically transmitted through tsetse flies and by syringe passages in mice in the absence of drug pressure. The changing levels of drug sensitivity were determined after every 3rd cyclic and 5th syringe passage intervals. It was noted that when the strain was maintained in tsetse flies through 12 cyclical transmissions, the CD50 and CD90 dropped slightly from 13.9 to 11.9 +/- 1.06 and from 20.3 to 18.0 +/- 1.08 mg/kg body weight, respectively. This decrease in the level of resistance was not significant (P greater than 0.05). However, when the trypanosomes were maintained by syringe passages in mice, there was a significant reduction (P less than 0.05) in the degree of resistance (CD50 from 13.9 to 11.4 +/- 1.07 and CD90 from 20.3 to 16.7 +/- 1.16 mg/kg), by the 15th syringe passage.