Comparative use of faecal egg count reduction test,egg hatch assay and beta-tubulin codon 200 genotyping in small strongyles (cyathostominae) before and after benzimidazole treatment

A survey on benzimidazole (BZ) resistance in small strongyles was performed on three farms in the tenth region in Chile. Samples from a total of 100 horses were tested using the faecal egg count reduction test (FECRT), the egg hatch assay (EHA) and an allele-specific PCR for the detection of beta-tubulin isotype 1 genes coding for phenylalanine (phe) or tyrosine (tyr) at codon 200. In the past, BZ-type drugs have been used within anthelmintic campaigns on all the three farms. This has predictably led to a high degree of BZ resistance at the Valdivia and Ri?ihue farms and to a lesser degree at the Frutillar farm, as demonstrated by all the three tests. The FECRT indicated resistance in every farm by faecal egg count reductions (FECR) of 27% (S.D. +/- 33), 26.5% (S.D. +/- 26.9) and 83.9% (S.D. +/- 22.8) for the Valdivia, Ri?ihue and Frutillar farms, respectively. With the EHA, the following mean LD(50) values were found before and after treatment with fenbendazole (FBZ): 0.093, 0.141 and 0.066 microg TBZ/ml and 0.149, 0.158 and 0.091 microg TBZ/ml, respectively, for the Valdivia, Ri?ihue and Frutillar samples. The corresponding LD(96) values were 0.222, 0.263 and 0.188 microg TBZ/ml before treatment and 0.316, 0.322 and 0.221 microg TBZ/ml after treatment, indicating BZ resistance in all the cases. Genotyping was performed on more than 1700 single larvae, at least 10 per faecal sample, for 98 pre- and 66 post-treatment samples. Despite a general trend toward higher percentages of phe/tyr and tyr/tyr individuals following treatment, no statistically significant difference was found between these two and the phe/phe genotype percentages. However, a significantly negative correlation was detected between the LD(50) values and the phe/phe percentages and there was a positive correlation between the FECRT results and the phe/phe percentages. Thus, there seems to be a difference in the significance of the codon 200 polymorphism in the mechanisms of BZ resistance in small strongyles of the horse and sheep trichostrongyles.     

Emergence from inhibited development of cyathostome larvae in ponies following failure to remove them by repeated treatments with benzimidazole compounds

The effect of three albendazole treatments at 5-week intervals, beginning at turnout in April, on cyathostome infections in Shetland ponies was compared with the effect of sequential treatments with albendazole, oxfendazole and oxibendazole. The results showed a substantial reduction in faecal egg output after the first albendazole treatment. Since faecal egg counts remained very low, no estimation of the effect of the second treatment was possible. The third treatment with albendazole and oxibendazole was followed by an increase in faecal egg counts to values of greater than 100 eggs g-1 within 4 weeks. A final albendazole treatment in December, 1 week before necropsy, failed to reduce faecal egg counts. These results suggest resistance to albendazole and oxibendazole in the cyathostome populations of the ponies. The increase in faecal egg counts after the third anthelmintic treatment in July occurred, although overwintered pasture infectivity was very low. The most likely explanation for this increase is resumption of the development of worms which overwintered as inhibited larvae in the host.     

Effect of repeated oxfendazole treatments on small strongyle infections in Shetland ponies

Overwintering of horse cyathostomes as inhibited third stage larvae (L3) and the effect of repeated oxfendazole (OFZ) treatment on strongyle infections were studied in an experiment with two groups of three Shetland ponies. Both groups were grazed together from May 28 to November 11, 1986 and subsequently housed. Treatments with 10 mg OFZ kg-1 were given on May 26, July 1 and July 28 and again one week before each group was necropsied in December and April, respectively. Worm populations of both groups were dominated by inhibited early L3. The proportion of fourth stage larvae (L4) was significantly higher in April than in December. The faecal egg counts showed that the OFZ treatment in May was more effective than those in July. This resulted in high pasture larval counts from the end of August onwards. The final treatments revealed a low efficacy against L3 and L4 but not against adult worms.     

动物源粪肠球菌对7种抗生素耐药表型及耐药基因检测

为查明动物源(牛、羊、猪、鸡)粪肠球菌分离株对7种常见抗生素的耐药情况(包括耐药表型及相关的耐药基因),采用药敏纸片法、浓度稀释法和VITEK-AMS全自动药敏法3种不同的方法,根据CLSI(2007)判定标准,检测40株分离株的耐药表型;采用聚合酶链反应(PCR)方法,检测分离株中β-内酰胺类抗生素耐药相关基因(TEM)、氨基糖苷类抗生素耐药相关基因(aac(6’)/aph2’’,aph(3’)-Ⅲ,ant(6)-I)、四环素耐药相关基因(tetM)、红霉素耐药相关基因(ermB,mefA)和万古霉素耐药相关基因(vanA,vanB,vanC)。结果表明,分离株对庆大霉素、链霉素、四环素、红霉素、青霉素、阿莫西林表型耐药率分别为:60.0%(24/40),57.5%(23/40),52.5%(21/40),67.5%(27/40),60.0%(24/40),55.0%(22/40),本试验中未发现耐万古霉素粪肠球菌。耐药基因aac(6’)/aph2’’,ant(6)-Ⅰ,aph(3’)-Ⅲ,tetM,ermB,TEM的检出率分别为:55%(22/40),55%(22/40),25.0%(10/40),42.5%(17/40),50.0%(20/40),45.0%(18/40);未检测到mefA,vanA,vanB,vanC基因的菌株。动物源性粪肠球菌多重耐药现象严重,携带抗生素相关耐药基因是导致分离株对抗生素产生耐药的主要原因,从耐药表型和基因型的角度均可证实分离株粪肠球菌具有多重耐药性。     

Effects of the repeated distribution of sainfoin hay on the resistance and the resilience of goats naturally infected with gastrointestinal nematodes

Due to the high prevalence of anthelmintic resistance in goats, the need to explore novel approaches to control nematodes and to reduce the exclusive reliance on chemotherapy is strongly demanded in this host species. In sheep, several studies have shown that the consumption of tannin-rich legume forages was associated with positive effects on host resilience and resistance to parasite infection. In goats, studies on such interactions between tanniferous plants and nematode infections remain few. The objectives of the current study were to examine under natural conditions the effects of consumption of sainfoin hay by goats on the parasite populations and on host resilience. Eighteen adult cull goats naturally infected with Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis were used in the study. At the start of the assay, the goats were allocated into two groups, balanced according to weight and the levels of egg excretion. The two groups grazed separate pastures for 3 months with similar stocking rates. Goats from group S received each month indoors, for 7 days, sainfoin hay and control goats (group C) received hay of ryegrass. The diets in both groups were made isoenergetic and isoproteic and the refusals measured. Individual parasitological and pathophysiological measurements were performed fortnightly in order to compare host resistance and resilience. At the end of the study, five goats per group were necropsied. The distribution of sainfoin was associated with: (1) a higher consumption of hay; (2) significant, lower levels of nematode egg excretion which was associated with a decrease in worm fertility but no change in worm population; however, the number of intestinal worms was reduced by 50% in group S; (3) a better host resilience. In particular, after 2 months of grazing, two control goats died and half of the remaining animals needed to be treated whereas this was not the case in group S. These differences were related to significant changes in pepsinogen and phosphate values (PCV) but not in pepsinogen and phosphate concentrations. These results demonstrate that a repeated distribution of sainfoin hay to grazing goats might be beneficial in regard of pasture contamination and host resilience. They suggest that administration of sainfoin hay might represent a valuable alternative and adjunct to reduce nematode infections in dairy goat flock.     

广西不同养殖模式猪源大肠杆菌耐药性和耐药基因检测分析

本研究目的是了解广西不同养殖模式猪源大肠杆菌的耐药情况和耐药基因流行情况,为临床合理用药治疗猪大肠杆菌病以及减缓细菌耐药性产生提供参考。采用K-B纸片法测定了180株分离自广西3种养殖模式猪源大肠杆菌的耐药情况,并通过PCR检测其携带EABLs(bla_(TEM)、bla_(CTX))、PMQR因子(qnrA、oqxA、oqxB、aac(6′)-Ib-cr)、大环内酯类耐药基因(ermB)、磺胺类耐药基因(suL)和氟苯尼考耐药基因(floR)的情况。结果显示,大规模、中小规模和散养户养猪场的大肠杆菌对头孢西丁、头孢他啶和阿米卡星的敏感率高于70%,而对青霉素G、氨苄西林、阿莫西林、利福平、林可霉素和复方新诺明的耐药率均高于90%。大规模养猪场大肠杆菌对恩诺沙星的耐药率极显著低于散养户(P<0.01),对氧氟沙星、庆大霉素和氟苯尼考的耐药率显著低于散养户(P<0.05);中小规模养猪场大肠杆菌对恩诺沙星、强力霉素和氟苯尼考的耐药性显著低于散养户(P<0.05);散养户大肠杆菌对环丙沙星、卡那霉素和壮观霉素的耐药率极显著高于大规模养猪场和中小规模养猪场(P<0.01),而对头孢噻肟的耐药率极显著低于大规模养猪场和中小规模养猪场(P<0.01)。大规模养殖场、中小规模养殖场和散养户大肠杆菌的多重耐药指数(MARI)分别为0.62、0.63和0.68,分离自散养猪场的83.3%菌株(50株)表现为对13-23种药物的多药耐药,极显著高于大规模养猪场(50%,30株,P<0.01),显著高于中小规模养猪场(61.7%,37株,P<0.05)。在3种模式养猪场的大肠杆菌中均检测到bla_(TEM)、bla_(CTX、)qnrA、oqxA、oqxB、aac(6′)-Ib-cr、ermB、suL和floR 9种耐药基因,其中bla_(TEM)和suL的阳性率高达100%,bla_(CTX)的阳性率最低,各模式猪场均低于57%。3种养殖模式之间的耐药基因阳性率相比差异显著,但与耐药表型不具有显著相关性。结果表明,3种养殖模式的猪场分离的大肠杆菌均具有严重的耐药问题,且菌株可携带多种耐药基因,表现为多重耐药,而不同养殖模式猪场分离的菌株耐药性存在显著性差异。     

The prevalence of pyrethroid resistance phenotype and genotype in Rhipicephalus (Boophilus) microplus in Yucatan, Mexico

A field survey was conducted to evaluate susceptibility of Rhipicephalus (Boophilus) microplus to cypermethrin on 49 farms in three areas of Yucatan, Mexico. The modified larval packet test was used to evaluate larval mortality at different cypermethrin concentrations. Dose-mortality regressions, lethal concentrations (LC(50)-LC(99)), confidence intervals and slope were estimated by probit analysis. Phenotype was defined as susceptible, tolerant or resistant when the resistance factor (RF) derived from both LC(50) and LC(99) determinations were <3, 3-5 or >5, respectively. An allele specific PCR (AS-PCR) was used to determine the frequency of a sodium channel mutation (F1550I, Phe→Ile) associated with pyrethroid resistance. Overall, 26.5%, 40.8% and 32.6% of tick populations were susceptible, tolerant and resistant to cypermethrin, respectively. A substantial inter-population variation in the level of cypermethrin response was evident (resistance factors ranged from 0.3 to 2599 and from 0.7 to >5000 when were indicated by the LC(50) and LC(99), respectively). The F1550I mutation (R allele) in R. microplus was present in all studied areas. The increasing presence of the R allele correlated well with increased levels of response indicated by both the LC(50) (r(2)=0.659, p=0.001) and LC(99) (r(2)=0.688, p=0.001) to cypermethrin. These results indicated that the F1550I mutation is a major common mechanism responsible for pyrethroid resistance in field populations of R. microplus ticks in the Mexican tropics. Both bioassay and AS-PCR showed that the prevalence of cypermethrin-resistant/tolerant R. microplus is high in Yucatan, Mexico and the relationship between the RF and the frequency of the R allele supports the role of F1550I as one of the most important mechanisms conferring pyrethroid resistance in these R. microplus populations.     

Evaluation of a larval development assay (DrenchRite) for the detection of anthelmintic resistance in cyathostomin nematodes of horses

A larval development assay (LDA, DrenchRite) was evaluated to determine the effectiveness of this method in detecting anthelmintic resistance in cyathostomin nematodes of horses. A total of 15 horse farms from Georgia and South Carolina (USA) and Population S ponies from the University of Kentucky (USA) were included in this study. Nematode eggs were extracted from pooled fecal samples and placed into the wells of a DrenchRite plate for testing against thiabendazole (TBZ), levamisole (LEV) and 2 ivermectin (IVM) analogs (IVM-1, IVM-2). After a 7-day incubation larvae in each well were counted and data were analyzed by logistic regression. Resistance status of each farm for different drugs was determined in a separate study using a fecal egg count reduction test. LDA were performed on the 15 farms once, however, the Population S cyathostomins were assayed on 3 separate occasions to estimate the consistency of results between assays. Mean TBZ LC50 for oxibendazole resistant, suspected resistant and sensitive farms were 0.2015, 0.1625, and 0.1355 microM, respectively. For LEV, mean LC50 for PYR resistant, suspected resistant and sensitive farms were 1.590, 1.8018 and 1.4219 microM, respectively. All 15 farms had worms susceptible to IVM; mean LC50 for IVM-1 and for IVM-2 were 7.5727 and 87.9718 nM, respectively. A linear mixed model was fitted to the data to determine the relationship between LC50 and LC95 and resistance status for each farm. No meaningful relations were found. Consistency of assays varied between drugs, being best for TBZ and worst for LEV and IVM-1. All farms in this study had benzimidazole-resistant nematodes; therefore usefulness of DrenchRite for discriminating susceptibility versus resistance to this drug class could not be accurately assessed. Moreover, since all farms tested were sensitive to IVM and resistance to this drug class has not yet been reported in cyathostomins, it is not possible to assess accurately the usefulness of DrenchRite LDA for detecting IVM resistance at this time. Assay results for LEV suggest that LEV in a LDA does not yield data that is useful in estimating PYR efficacy in vivo. Based on results for PYR/LEV, the current high prevalence of benzimidazole resistance, no known cases of IVM resistance, and the sometimes extreme variation in results seen in many of the assays, DrenchRite LDA cannot be considered a useful tool for the diagnosis of resistance in cyathostomins of horses at present.     

Effect of hay dust extract and cyathostomin antigen stimulation on cytokine expression by PBMC in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is an inflammatory, obstructive airway disease induced by exposure of susceptible horses to inhaled organic dust particles. The immunological process underlying RAO is still unclear. Previous studies have shown that RAO is linked to the Interleukin-4 receptor (IL-4R) gene in one Warmblood family (F1), but not in another (F2). It has also been shown that in F1, but not in F2, RAO is associated with resistance against parasites, suggesting that this association may have an immuno-genetic basis. Therefore, we hypothesized that the T helper (h)1/Th2/regulatory (Treg) cytokine profiles of RAO-associated antigen- and parasite-antigen-stimulated peripheral blood mononuclear cells (PBMC) differ between RAO-affected and healthy horses depending on their genetic background. In our study, PBMC from 17 RAO-affected and 14 healthy control horses of F1 and F2 were stimulated for 24 h with antigens relevant to RAO [hay dust extract (HDE), Aspergillus fumigatus extract (AFE) and lipopolysaccharids (LPS)]; cyathostomin extract (CE) and recombinant cyathostomin antigen (RCA) or with concanavalin A (ConA). Total mRNA levels of IL-4, IL-4R, IL-13, interferon (INF)-γ and IL-10 were examined by qRT-PCR. Stimulation with either HDE or RCA resulted in significant differences in IL-4R mRNA levels between RAO-affected and control horses in F1, but not in F2. For IL-10 mRNA expression, a significant difference between RAO-affected and control horses in F1 but not in F2 was observed only following stimulation with HDE. In contrast to HDE, stimulation with CE resulted in a significant difference of IL-10 mRNA expression level between RAO-affected horses of F2 and healthy horses of F1. No significant differences were detected upon stimulation with any of the other challenge agents. These findings indicate that the immunological response, specifically IL-4R expression, in response to hay dust and cyathostomin antigens, differs between RAO-affected and healthy horses depending on their genetic background. This study shows that analysis of PBMC reveals systemic changes associated with RAO and helps to elucidate immunological pathways involved in this disease.     

The effects of stage-specific selection on the development of benzimidazole resistance in Haemonchus contortus in sheep

Resistance to the benzimidazole (BDZ) class of anthelmintics in nematodes of sheep has become a common and global phenomenon. The rate at which the selection process and development of resistance occurs is influenced by a number of factors. Of these, the effects of stage-specific exposures to anthelmintic were investigated with a BDZ-resistant strain of Haemonchus contortus (HCR) over five parasite generations. Sheep were infected at each generation with the HCR strain and were treated with thiabendazole (TBZ), either 5 days post-infection (p.i.) (larval line), 21 days p.i. (adult line), or left untreated (no selection line). Additionally eggs from each generation were exposed to TBZ (egg line). Geometric worm burdens were calculated from post-mortem worm counts, both at the start of the study, and after the final selection studies for each of the selection lines. Egg hatch assays (EHAs) were also conducted throughout the study. All data relating to worm burdens and EHAs for each generation were analysed by linear regression to produce dose titration curves and lethal dose(50) (LD(50)) values for each of the selection lines. Over the five generations, LD(50) values on dose-response were increased and worm survival occurred at higher dose rates of TBZ irrespective of the parasite stage exposed to treatment. A similar picture was seen with ED(50) values, which showed a fluctuating but generally upward trend for each of the three selection lines. In contrast, LD(50) and ED(50) values were decreased in the no selection line, indicating some degree of reversion albeit to levels still considered to be BDZ-resistant.     

Sequential mesenteric arteriography in pony foals during repeated inoculations of Strongylus vulgaris and treatments with ivermectin

Semiselective mesenteric arteriography was performed at regular intervals (inoculation weeks [IW] 0, 11, 18, and 24) in 9 of 10 pony foals raised to be free of parasites. Fifty infective larvae (L3) of Strongylus vulgaris were administered weekly for 4 weeks, then every 2 weeks through the 20th week. Three ponies were given ivermectin (oral paste, 0.2 mg/kg of body weight) treatment at IW 8, 16 and 24. Four ponies were inoculated, but did not receive ivermectin, and a third group of 2 ponies acted as uninoculated controls. Control ponies did not have gross or arteriographic lesions, whereas the inoculated untreated ponies had gross and progressive arteriographic lesions typical of verminous arteritis. Arteriographic lesions in the ivermectin-treated inoculated ponies were not as severe those in the untreated inoculated group, and there was either a partial resolution or a lack of progression of arteriographic lesions in all treated ponies. One untreated inoculated pony did not have progressive arterial lesions as did the 3 others in the group, and may develop resistance to the parasite.     

Synergistic action of mebendazole and levamisole in the treatment of a benzimidazole-resistant Haemonchus contortus in sheep

One-year old worm free, merino wethers, were each infected with 5000 H. contortus larvae of a strain resistant to mebendazole at a rate of 52 mg/kg body weight of sheep. After 21 days, they were assigned to two trials. The preliminary trial showed that mebendazole and levamisole acted synergistically on the H. contortus infection. In the second trial, sheep were treated with 0.35 mg/kg levamisole (one seventh the minimum effective dose against susceptible worms) or 40 mg/kg mebendazole (40 times the minimum effective dose against susceptible worms). In each case the anthelmintics did not reduce worm burdens, although mebendazole depressed egg production. However, when mebendazole and levamisole, at the above dose rates, were administered simultaneously, total worm counts in sheep were reduced by almost 60%. Similar results were obtained when the levamisole was administered 8 h or 14 h after mebendazole treatment. The implications of these observations for the treatment of benzimidazole-resistant haemonchiasis in sheep are discussed.     

Effects of chemical treatments on pH and bacterial population in poultry litter: a laboratory experiment

1. The goal of this study was to evaluate the effects of three chemical treatments on pH and bacterial populations (total aerobic bacteria and gram-negative bacteria) in poultry litter under laboratory conditions. 2. Litter obtained from poultry houses was treated with three chemical treatments (alum, AlCl(3) and FeSO(4)) at the same concentration (8 g/100 g litter), while untreated litter served as a control. The study was conducted for 3 weeks. 3. All of the chemical treatments reduced total aerobic bacteria (22 to 87% of the untreated control) and gram-negative bacteria (63 to 99% of the untreated controls) populations and lowered litter pH values (5.95 to 6.64). However, a significant difference in gram-negative bacteria did not exist among chemical treatments at 0, 1 and 2 weeks. 4. These results suggest that the reduction in total aerobic bacteria and gram-negative bacteria populations is highly related to a decrease in litter pH, and acidifying treatment (alum, AlCl(3) and FeSO(4)) of poultry litter may serve as a means to help the reduction in pathogen populations and to improve economical benefits under commercial production conditions.     

荧光定量PCR检测小鹅瘟病毒VP3基因疫苗在小鼠体内的动态分布

小鹅瘟是由小鹅瘟病毒(GPV)引起的一种主要侵害30日龄以内雏鹅和雏番鸭的急性、高度接触性传染病,是目前危害养鹅业的主要传染病之一,传统的小鹅瘟弱毒苗在免疫预防小鹅瘟中发挥了重要作用,但仍存在潜在毒力易返强、不易区分自然感染与疫苗抗体等不足[1].     

Effect of anticonvulsant dosages of potassium bromide on thyroid function and morphology in dogs

A placebo-controlled experiment was performed to evaluate the effect of potassium bromide on the canine thyroid gland. Basal total thyroxine, free thyroxine, and basal thyrotropin serum concentrations were evaluated over a 6-month period in potassium bromide-treated and control dogs. A thyrotropin-releasing hormone stimulation test was also performed in all dogs at the beginning and conclusion of the study. Thyroid histopathology was compared between treated and control dogs at the end of the study. No difference was detected in any parameter between the two groups at the end of the study. A decline in thyroid hormone concentrations over the course of the study did occur in both groups of dogs. Potassium bromide does not appear to have a significant effect on canine thyroid function or morphology.     

Wide geographic distribution of Cryptosporidium bovis and the deer-like genotype in bovines

Recent studies in the United States reported that approximately 85% of pre-weaned dairy calves were infected with zoonotic Cryptosporidium parvum, whereas only 1-2% of post-weaned calves and 1-2-year-old heifers were infected with this species. Cryptosporidium bovis and Cryptosporidium deer-like genotype were much more prevalent in the post-weaned animals. It is not clear whether the same infection pattern also occurs in other geographic areas. In this study, to determine whether the same Cryptosporidium infection pattern was present in other geographic areas, we genotyped Cryptosporidium specimens collected from two farms in China and India, using specimens from farms in Georgia, USA for comparison. C. bovis was the most common species found in pre- and post-weaned calves in all three areas. In Georgia, the deer-like genotype was found frequently in pre- and post-weaned calves and Cryptosporidium andersoni was found in one post-weaned calf. Both C. bovis and the deer-like genotype were found in the few milking cows examined in Georgia. There were no differences in the small subunit rRNA gene sequences obtained from C. bovis or deer-like genotype among the three areas. One adult yak in China, however, was infected with a species similar to C. bovis, with only three nucleotide mutations in the target gene. All four common bovine Cryptosporidium spp. were differentiated from each other by restriction fragment length polymorphism analysis of PCR products with enzymes SspI and MboII. Thus, both C. bovis and the deer-like genotype are found in all age groups of cattle in diverse geographic areas and host adaptation of C. bovis might have occurred in yaks.     

Depletion of gentamicin in the milk of Holstein cows after single and repeated intramammary and parenteral treatments

Twenty-four healthy Holstein cows, 2.72 ± 0.64 (mean ± SD) years old, weighing 603.96 ± 73.22 kg (mean ± SD), and representing various levels of milk production, were used to determine the depletion of gentamicin (GT) in milk. The cows had not received antibiotics or other drugs that could interfere with study for at least 60 days before the beginning of the investigation. The cows were divided into six groups (n = 4) and treated with single (treatments A, B and C) or repeated (treatments D, E and F) doses of GT. Cows were acclimated for 7 days before administration of GT and milked twice a day at 12-h intervals (06.00 hours, 18.00 hours) throughout the duration of the study. Control milk samples were obtained after the arrival of the cows and assayed to establish their GT free status. On day 1 of each treatment, a baseline milk sample was collected from the milk produced (06.00 hours) by each cow. A single dose of GT was administered intramammarlly (A, i.m.m. left front quarter, 500 mg), intravenously (B, i.v., 5 mg/kg body weight) or intramuscularly (C, i.m., 5 mg/kg body weight). Cows in treatments D (i.m.m., 500 mg), E (i.v., 5 mg/kg body weight) and F (simultaneous i.m.m. 500 mg plus i.v. 5 mg/kg body weight) were treated twice a day for 5 consecutive days just after the morning and evening milkings. Milk samples from individual cows were collected every day after each milking during and after dosing until GT concentration in the milk was below the safe level of ± 30 ng/mL. The concentration of GT in milk was determined by a high-performance liquid chromatographic procedure. Depletion of GT to a concentration ± 30 ng/mL occurred at the seventh (84 h), third (36 h), third (36 h), eleventh (132 h), third (36 h) and nineteenth (228 h) post-dosing milking, for cows in treatments A, B, C., D, E and F respectively. The highest mean ± SEM) concentrations of GT were 14 710 ± 1213.89, 167.87 ± 46.94 and 91.62 ± 14.55 ng/mL measured in the first milking post dosing (12 h) for cows in treatment A, B and C respectively; for cows in treatments D, E and F, during the dosing period, they were 14067.50 ± 2989.09, 446.07 ± 100.92, and 22900 ± 2843.66 ng/mL and occurred at the seventh, third and eighth milking respectively. Because GT is not approved for use in dairy cattle and because of the long depletion time associated with some possible treatments, illegal and extra-label use is likely to cause residues in milk.     

Immunologic response and resistance to experimentally induced pneumonic pasteurellosis in cattle vaccinated with various dosages of lyophilized Pasteurella haemolytica

Pasteurella haemolytica was lyophilized in an enriched soybean polypeptone broth. Lyophilization in this medium resulted in a mean 10-fold loss in P haemolytica viability, as opposed to up to a 10(4)-fold loss in viability when other media were used. Lyophilized P haemolytica was reconstituted and used as a live vaccine in 3 experiments. Calves were challenge exposed by transthoracic injection with virulent P haemolytica. In experiment 1, 2 subcutaneous injections (7-day interval between injections) with 5 ml of recently harvested (1 X 10(9) colony-forming units [CFU]/ml) or lyophilized (1 X 10(8) CFU/ml) P haemolytica significantly (P less than 0.001) enhanced resistance against challenge exposure, compared with resistance in calves given saline solution or sterile medium (control calves) or calves vaccinated with lyophilized organisms at a concentration of 1 X 10(6) CFU/ml. In experiment two, 1, 2, or 5 ml of lyophilized P haemolytica (1 X 10(8) CFU/ml) significantly (P less than 0.05) enhanced resistance, compared with resistance in calves given saline solution (control calves). In experiment three, 1 or 2 injections of lyophilized P haemolytica significantly (P less than 0.01) enhanced resistance against challenge exposure, compared with that of calves given saline solution. The mean lesion score for calves given 1 injection was not significantly higher than the mean lesion score for the group given 2 injections. Vaccination with lyophilized P haemolytica vaccine caused significant (P less than 0.05) increases in serum antibody to P haemolytica somatic antigens, to a carbohydrate-protein subunit of the organism, and to leukotoxin.