Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle

Resistance to the drugs used to control African animal trypanosomosis is increasingly recognised as a constraint to livestock production in sub-Saharan Africa. The most commonly used tests for detection of trypanocidal drug resistance are tests using mice or ruminants, but these suffer from lack of standardisation and hence it may be difficult to compare the results of different investigators. Tests in mice are less expensive than tests in ruminants, but while tests in mice they may be useful as a general guide to resistance in a geographic area they should not be extrapolated to cattle on an individual trypanosome level. Moreover, the commonly used protocols are too laborious for their application to large number of trypanosome isolates on an area-wide basis. This paper presents guidelines for standardised testing of trypanocidal drugs in vivo, and introduces a simplified single-dose test for use in mice, which is convenient for use in areas with limited laboratory facilities. The single-dose test is appropriate for characterisation of geographic areas in terms of trypanocidal drug resistance using large numbers of trypanosome isolates, for making comparisons between areas, and for monitoring changes in trypanocidal drug resistance over time. Multiple-dose tests may be used to determine the degree of resistance of individual stabilates to be determined precisely in mice are also described, but for logistical reasons these will rarely be conducted on more than a few stabilates, and testing of a larger number of stabilates in the single-dose test will generally provide more useful information. Finally, we describe tests in cattle that may be used to determine the efficacy of recommended curative doses of trypanocidal drugs for the treatment of infection with individual trypanosome isolates, including Trypanosoma vivax, which is rarely infective for mice.     

Pathogenesis of experimental bovine spongiform encephalopathy (BSE): estimation of tissue infectivity according to incubation period

This paper reports the results of tissue infectivity assays of bovine spongiform encephalopathy (BSE) agent in orally exposed cattle at stages during the incubation period. Estimations of the titre of infectivity in central nervous system (CNS), certain peripheral nerve ganglia and distal ileum tissue were made according to time post exposure from the relationship between incubation period and dose for RIII mice and C57bl mice using data from titrations of brain material from cases of BSE. The rate of increase of infectivity in the bovine CNS was then estimated, taking into account these tissue infectivity titres, the variability of the brain titre of clinical field cases of BSE, and the probability density of the expected number of months before clinical onset of each infected bovine. The doubling time for CNS was shown to equal 1.2 months. The titre in the thoracic dorsal root ganglia (DRG) was, on average, approximately 1 log units less than CNS, and cervical DRG approximately 0.5 log less than thoracic DRG. The pattern of increase of infectivity in the distal ileum is that of an initial increase up to 14-18 months post exposure, followed by a decrease, which is likely to be highly variable between animals. These results will be informative for future risk assessments of BSE, especially in relation to reviewing current control measures.     

Delayed-type hypersensitivity test for assessing tick-immune status of cattle in Zambia

Delayed-type hypersensitivity skin reactions were used to assess the tick resistance status of Tonga calves in Zambia. The antigen used in the tests was a homogenate of unfed nymphal Rhipicephalus appendiculatus which had been shown to give protective immunity in guinea pigs to adult female R appendiculatus. There was a significant negative correlation between the intensity of the reactions and the total number of ticks (Amblyomma variegatum, R appendiculatus, Hyalomma truncatum, Boophilus decoloratus and Rhipicephalus species) on the animals.     

Conformational change in hamster scrapie prion protein (PrP27-30) associated with proteinase K resistance and prion infectivity

The scrapie prion protein (PrP27-30) is a crucial component of the prion and is responsible for its transmissibility. Structural information on this protein is limited because it is insoluble and shows aggregated properties. In this study, PrP27-30 was effectively dispersed using sonication under the weak alkaline condition. Subsequently, the small PrP27-30 aggregates were subjected to different pH, heat, and denaturing conditions. The loss of proteinase K (PK) resistance of PrP27-30 and prion infectivity were monitored along with spectroscopic changes. Prion inactivation could not be achieved by the loss of PK resistance alone; a significant loss of the PrP27-30 amyloid structure, which was represented by a decrease in thioflavin T fluorescence, was required for the loss of transmissibility.     

Logarithmic normal distribution of incubation times for canine diseases (Sartwell's model)

The distribution of incubation periods for most infectious diseases follows a logarithmic normal distribution (Sartwell's model). The same is true for human neoplastic diseases of known cause and for genetic diseases, when the age at onset of the disease was used as a surrogate measure of the incubation period. In this study, a similar distribution was observed when Sartwell's model was applied to canine diseases of known genetic cause, but not to diseases of an environmental cause. The model was also applied to canine diseases of suspected multifactorial causes for the purpose of distinguishing between the relative importance of genetic and environmental factors that act before or after birth. Limitations of this method were discussed.     

Dog feeding test for assessing the nutritional adequacy of practical diets

The nutritive value of dog foods declared by the manufacturer as nutritionally complete and balanced can be best assessed by feeding trials with dogs. A protocol of a feeding trial has been developed and tested with working dogs fed two different commercial complete and balanced diets for 8 weeks. The parameters used for evaluating the effect of diets were general health status, body and hair coat condition, change of body weight, haematological parameters (white blood cell (WBC) count, red blood cell (RBC) count, haemoglobin, packed cell volume), and biochemical parameters in blood serum (alanine aminotransferase, urea, albumin). The trial protocol proved to be appropriate to monitor the dogs' nutritional status and to reveal differences between diets. This method of evaluation is recommended for use in supporting the nutritional claims (labelling) of dog foods.     

A larval development test for the detection of anthelmintic resistance in nematodes of sheep

First stage larvae of a number of species of parasitic nematodes of sheep have been shown to develop to third stage larvae in the presence of a defined medium consisting of Earle's balanced salt solution and yeast extract. A larval development test, based on this culture technique, was used as a screen for detecting the presence of anthelmintic resistance in nematodes of sheep. It was found to be sensitive and simple to use and also appeared capable of detecting resistance to any of the main anthelmintic groups. Available anthelmintic sensitive and resistant strains of Haemonchus contortus and Ostertagia circumcincta showed differences in development when incubated in the presence of either thiabendazole, levamisole and ivermectin. These differences were expressed as the minimum inhibitory concentration required to prevent larval development over the incubation period.     

Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola hepatica

A sheep trial was performed to standardise a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole (TCBZ) in Fasciola hepatica). The CRT employs the BIO K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) to test for the presence of F. hepatica coproantigens in a faecal sample. If it is coproantigen-positive, the CRT protocol recommends that faecal samples are re-tested for coproantigens at 14 days post-treatment (dpt), with negative testing at this point indicating TCBZ success. Initial work aimed to confirm the sensitivity of the BIO K201 ELISA for Fasciola infection and investigate whether coproantigens represent a robust reduction marker of TCBZ efficacy. Thirty-eight, indoor-reared sheep were artificially infected with F. hepatica isolates known to be susceptible (Cullompton) and resistant (Sligo) to TCBZ action, respectively. Treatment was administered at 12 weeks post-infection (wpi), with 2 sheep groups, infected with each isolate, culled at 2 and 4 weeks post-treatment (wpt), respectively. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period. Additional work focused on the effect of temperature on faecal sample collection and storage. Faecal samples collected from sheep positive for F. hepatica infection were sub-sampled and left at room temperature. Individual sub-samples were tested by ELISA on consecutive days and these readings compared to the original test result on the day of collection. In addition, ELISA values were compared between faecal sub-samples prepared on the day of sampling and post storage at -20°C. Also, an immunocytochemical study was performed to determine the tissue site of origin of the coproantigen protein in the fluke. Results showed that the BIO K201 ELISA was sensitive for Fasciola coproantigens, with coproantigens detectable from 5 wpi onwards. The suitability of coproantigens as a diagnostic marker of TCBZ efficacy was supported by the absence and presence of coproantigens in TCBZ-treated Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) F. hepatica infections at 2 and 4 wpt, respectively. Study results suggest that low to moderate temperature has little, if any, impact on coproantigen stability in faecal samples, but that higher temperatures may have. Immunolabelling for the coproantigen showed that it was specific to the gastrodermal cells of both adult and juvenile flukes.     

Comparative infectivity of oocysts and bradyzoites of Toxoplasma gondii for intermediate (mice) and definitive (cats) hosts

Tachyzoites, bradyzoites (in tissue cysts), and sporozoites (in oocysts) are the three infectious stages of Toxoplasma gondii. The prepatent period (time to shedding of oocysts after primary infection) varies with the stage of T. gondii ingested by the cat. The prepatent period (pp) after ingesting bradyzoites is short (3-10 days) while it is long (18 days or longer) after ingesting oocysts or tachyzoites. The conversion of bradyzoites to tachyzoites and tachyzoites to bradyzoites is biologically important in the life cycle of T. gondii and it has been proposed that the pp can be used to study stage conversion. In the present study, infectivity of oocysts and bradyzoites released from tissue cysts of a recent isolate of T. gondii, TgCkAr23, to cats and mice was compared. Ten-fold dilutions of oocysts or bradyzoites were administered orally to cats, and orally and subcutaneously to mice. Of the 29 cats each fed 1-10 million oocysts only one cat shed oocysts and the pp was 23 days; all cats remained asymptomatic. In contrast, all mice administered the same 10-fold dilutions of oocysts either orally or subcutaneously died of toxoplasmosis. The results confirm that infectivity of the oocysts to cats is lower than for mice and that oocysts are non-pathogenic for cats. Of the 41 cats each fed 1-1,000 free bradyzoites, 15 shed oocysts with a short pp of 4-9 days, and all remained asymptomatic. The infectivity of bradyzoites to mice by the oral route was approximately 100 times lower than that by the subcutaneous route. The results confirm the hypothesis that the pp in cats is stage and not dose dependent, and that transmission of T. gondii is most efficient when cats consume tissue cysts (carnivory) or when intermediate hosts consume oocysts (fecal-oral transmission).     

A new in vitro test to evaluate the resistance level against acaricides of the cattle tick, Rhipicephalus (Boophilus) microplus

In this article we present a new bioassay to assess the resistance status of ticks to acaricides. The Larval Tarsal Test (LTT) is a sensitive, highly time-effective in vitro test. It allows the investigation of a large number of compounds and doses on the cattle tick Rhipicephalus (Boophilus) microplus in a short period of time. The ability of the LTT to assess the lethal concentration at 50% mortality (LC(50)) and resistance ratios (RRs) of a susceptible and a resistant R. microplus strain was compared with the FAO-recommended Larval Packet Test (LPT). Representative compounds of the carbamate, organophosphate (OP), synthetic pyrethroid (SP), formamidine (FOR), macrocyclic lactone and pyrazole classes were used for this comparison. The resistance status against OP, SP and FOR of the resistant R. microplus strain was confirmed in vivo. The LTT resulted in resistance ratios comparable to those obtained with the LPT. However, the lethal concentrations were up to 150-fold lower in the LTT than in the LPT. The advantage of the LTT is to simplify the methodology by avoiding the handling of larvae and using multi-well plates. The LTT is therefore a suitable test for the assessment of the level of resistance of R. microplus and is very promising to evaluate the resistance profile of field strains. Additionally, the LTT is also suitable to test other ixodid species.     

poly(A)尾对猪繁殖与呼吸综合征病毒基因组感染性的影响

大部分单股正链RNA病毒的基因组末端含有poly（A）尾,这对病毒基因组RNA的感染性起着非常重要的作用。本研究以猪繁殖与呼吸综合征病毒（PRRSV）感染性克隆pCBC2为平台进行反向遗传操作,通过突变PCR获得poly（A）尾长度分别为0、5、8、9、10、22、35的全长突变体克隆,经体外转录转染MARC-145细胞,观察细胞病变（CPE）,对于能产生CPE的突变克隆,抽提细胞上清中的病毒RNA,通过G-Tailing法鉴定子代病毒基因组的poly（A）尾长度。结果表明：1、poly（A）尾影响PRRSV基因组的感染性,且最小长度为10;2、PRRSV基因组poly（A）尾在感染细胞过程中能被修复。     

Evaluation of a portable test system for assessing endotoxin activity in raw milk

The aim of the present study was to compare endotoxin activities detected in raw milk samples obtained from cattle by a commercially available portable test system (PTS) and traditional microplate limulus amebocyte lysate (LAL)-based assay, which determined activities using a kinetic turbidimetric (KT) assay. Raw milk samples were obtained from 53 and 12 dairy cattle without and with clinical mastitis, respectively. Comparison between the KT and PTS was performed by the Friedman test. The Pearson product moment correlation coefficients were calculated to evaluate associations between any two continuous variables. Linear regression model analysis was also performed to obtain the equation describing the relationship between PTS and KT assay. The endotoxin activities detected in 200- or 400-fold diluted milk samples were similar between PTS and KT assay, whereas a significant difference was observed in 100-fold diluted milk (P<0.001). The results obtained from 200- (r²=0.778, P<0.001) and 400-fold diluted milk samples (r²=0.945, P<0.001) using PTS correlated with those using KT assay. The median milk endotoxin activities in Gram-positive and Gram-negative clinical mastitis cows were 0.655 and 11,523.5 EU/ml, respectively. The results of the present study suggest that PTS as a simple and easy test to assess endotoxin activity in raw milk is efficient, simple and reproducible.     

毛皮动物源肺炎克雷伯菌部分毒力基因、耐药基因检测及药敏试验

为了了解河北秦皇岛毛皮动物(狐、貉、貂)源肺炎克雷伯菌毒力基因、耐药基因分布及药物敏感性等情况,采用常规分离纯化、生化鉴定及分子生物学的方法对病原菌进行鉴定,动物致病性试验检测其致病性,PCR方法检测其主要K血清型、毒力基因和耐药基因,K-B法对常用西药进行药物敏感性检测,琼脂平板打孔法对中药药物敏感性进行检测,试管二倍稀释法检测中药最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。结果显示:此次共分离到48株肺炎克雷伯菌;48株肺炎克雷伯菌均能够使小鼠发病死亡;48株肺炎克雷伯菌均检测到fimH、wabG毒力基因,检出率为100%;mrkD毒力基因检出率为87.5%;blaTEM、aadA1、gyrB、gyrA、blaSHV-1、sul1、sul2、sul3、blaROB-1、tetA和tetD耐药基因的检出率分别为100.0%、100.0%、89.6%、4.2%、39.6%、18.8%、27.1%、16.7%、12.5%、60.4%和37.5%,未检出ermF耐药基因。西药药敏试验结果显示,48株肺炎克雷伯菌临床分离菌株均对头孢拉定、复方新诺明、林可霉素、替米考星、磺胺间甲氧嘧啶耐药。中药药敏试验结果显示,48株肺炎克雷伯菌临床分离菌株对乌梅、五味子极度敏感,抑菌圈直径在20～25 mm之间,MIC和MBC为15.63～62.50 g/L。本试验调查了河北秦皇岛地区毛皮动物源肺炎克雷伯菌的流行情况,对毛皮动物源出血性肺炎的防治具有重要的指导意义,更具公共卫生学意义。     

寄生虫的抗药性管理

抗药性在畜禽寄生虫中普遍存在，且具有一些共同的特征。了解这些共同特征将会为寄生虫的抗药性管理提供一些新观点、新思路。本文着重讨论了寄生虫的抗药性问题，并对其共同特征做了描述；同时也论述了寄生虫综合管理方案，旨在通过总结一种寄生虫的综合管理方案，为其他寄生虫病的控制开辟一条新途径。     

Antimicrobial drug use and resistance in dogs

Fifteen years (1984-1998) of records from a Veterinary Teaching Hospital were analyzed to determine whether antimicrobial drug resistance in coagulase-positive Staphylococcus spp. (S. aureus, S. intermedius) isolated from clinical infections in dogs has increased, and whether there has been a change in the species of bacteria isolated from urinary tract infections in dogs. In coagulase-positive Staphylococcus spp., a complex pattern showing both increases and decreases of resistance to different classes of antimicrobial drugs was observed, reflecting the changing use of different antimicrobial drug classes in the hospital over a similar period (1990-1999). In canine urinary tract infections identified from 1984 to 1998, an increase in the incidence of multiresistant Enterococcus spp. was apparent, with marginal increases also in incidence in Enterobacter spp. and in Pseudomonas aeruginosa, both of which, like Enterococcus spp., are innately antimicrobial-resistant bacteria. A survey of directors of veterinary teaching hospitals in Canada and the United States identified only 3 hospitals that had any policy on use of "last resort" antimicrobial drugs (amikacin, imipenem, vancomycin). Evidence is briefly reviewed that owners may be at risk when dogs are treated with antimicrobial drugs, as well as evidence that some resistant bacteria may be acquired by dogs as a result of antimicrobial drug use in agriculture. Based in part on gaps in our knowledge, recommendations are made on prudent use of antimicrobial drugs in companion animals, as well as on the need to develop science-based infection control programs in veterinary hospitals.     

Comparison of two assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance to triclabendazole in Fasciola hepatica in sheep

A sheep trial was performed to evaluate two diagnostic assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance of Fasciola hepatica to triclabendazole (TCBZ). The FECRT defines successful TCBZ treatment as a 95% or greater reduction in fluke faecal egg counts (FECs) at 14 days post-treatment (dpt). The CRT defines effective TCBZ treatment as faeces negative for Fasciola coproantigens at 14dpt, as measured by the commercial BIO K201 coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium). Forty-nine indoor-reared sheep were split into four trial groups and each sheep was infected with 200 metacercariae of 1 of 4 F. hepatica isolates, previously described as susceptible (Cullompton and Fairhurst) and resistant (Leon and Oberon) to TCBZ action, respectively. TCBZ treatment was administered at 12 weeks post-infection (wpi) to one sub-group in each infected sheep group, and these sheep were culled at 4 weeks post-treatment (wpt). Untreated sheep sub-groups, were culled at a parallel time-point, that is, at 16wpi. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period, sub-sampled and examined by a standardised egg sedimentation protocol and by the BIO K201 ELISA. Results supported the use of both the FECRT and the CRT for the diagnosis of resistance of F. hepatica to TCBZ. In addition, the study confirmed the TCBZ susceptibility of the Cullompton and Fairhurst F. hepatica isolates and the TCBZ resistance of the Oberon F. hepatica isolate. However, the Leon F. hepatica isolate was found to be susceptible, rather than resistant, to TCBZ action.