Babesia spp. infection in Boophilus microplus engorged females and eggs in Sao Paulo State, Brazil

Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01).     

Comparison of duplex PCR and microscopic techniques for the identification of Babesia bigemina and Babesia bovis in engorged female ticks of Boophilus microplus

A single-step duplex polymerase chain reaction (PCR) technique and traditional microscopic examination of haemolymph smears were used to detect Babesia bigemina and/or Babesia bovis infection in engorged female ticks of Boophilus microplus recovered from calves raised in an endemic area of the State of Minas Gerais, Brazil. In the PCR amplification of tick-derived DNA, pairs of oligonucleotide primers specific for a 278-bp sequence from B. bigemina and for a 350-bp sequence from B. bovis were used conjointly. The microscopic examination of haemolymph revealed that 16.7% of the engorged ticks were infected with Babesia spp., although no significant differences (rho > 0.05) were found in the infection rate of ticks collected from calves of different age groups. PCR analysis showed that 77.8% of the engorged ticks whose haemolymph contained sporokinetes were infected with B. bigemina, 7.8% with B. bovis and 14.4% with both protozoan species. However, the PCR assay further revealed that, amongst the engorged female ticks whose haemolymph was apparently negative for the presence of sporokinetes, 15.6% were infected with B. bigemina, 2.2% with B. bovis and 10.0% with both species. The duplex PCR method is thus more efficient and sensitive than the microscopic assay and also permits facile identification of the protozoa species present in engorged female ticks.     

Studies on ticks of veterinary importance in Nigeria. VI. Comparisons of oviposition and the hatching of eggs of Hyalomma species

Fully engorged Hyalomma spp. in Nigeria oviposited greater numbers of eggs than those partially engorged. Hyalomma impressum was a more prolific egg layer than H. rufipes, H. impeltatum and H. truncatum. The variations in the egg output as well as the recognizable peaks in the number of eggs during oviposition were described for each species. No species of Hyalomma below the engorged weight of 0.2 g oviposited; oviposition started with ticks of weight 0.3 g. Eggs produced by ticks weighing below 0.3 g did not hatch; the highest percent egg eclosion occurred with ticks of weight 0.6 g (H. rufipes) and 0.7 g (other Hyalomma species). The pre-oviposition, oviposition and eclosion periods were shortened when eggs were laid and incubated at high temperatures, although the number of oviposited eggs did not increase significantly. At the standard temperature of 24 degrees C, the longest eclosion period was seen in the eggs of H. rufipes (41 days) while those of H. truncatum, H. impressum and H. impeltatum were similar to each other (29 days). Only eggs of H. rufipes hatched at an incubation temperature of 15 degrees C. Eggs of Hyalomma species laid at the same time hatched over a 2--4 day period, except at 15 degrees C when the hatching period of H. rufipes lasted 10 days. The eclosion period was longest in the earlier ovipositions and shorter in the later ones. It is suggested that some intrauterine larval development might have started in the eggs before they were released at a later oviposition period. The percentage mortality of eggs at various temperatures showed that eggs of H. rufipes were more tolerant of low temperatures than those of H. impressum, H. truncatum and H. impeltatum, while the eggs of the latter 3 species were more tolerant of high temperatures than those of H. rufipes. The relevance of these results of the distribution and abundance of the Hyalomma species in Nigeria was discussed.     

Detection of Babesia bigemina in cattle of different genetic groups and in Rhipicephalus (Boophilus) microplus tick

Babesia bigemina infections were investigated in four genetic groups of beef cattle and in Rhipicephalus (Boophilus) microplus engorged female ticks. Blood samples and engorged female ticks were collected from 15 cows and 15 calves from each of the following genetic groups: Nelore, Angus x Nelore, Canchim x Nelore, and Simmental x Nelore. Microscopic examination of blood smears and tick hemolymph revealed that merozoites of B. bigemina (6/60) as well as kinetes of Babesia spp. (9/549) were only detected in samples (blood and ticks, respectively) originated from calves. PCR-based methods using primers for specific detection of B. bigemina revealed 100% infection in both calves and cows, regardless the genetic group. Tick infection was detected by nested-PCR amplifications showing that the frequency of B. bigemina was higher (P<0.01) in female ticks collected from calves (134/549) than in those collected from cows (52/553). The frequency of B. bigemina was similar in ticks collected from animals, either cows or calves, of the four genetic groups (P>0.05).     

Acquired resistance of horses to Amblyomma cajennense (Fabricius, 1787) ticks

Acquired immunity of horses to larvae, nymphs and adults of the Amblyomma cajennense tick was evaluated through three consecutive experimental infestations of tick-bite na?ve hosts. Data from these infestations were compared to those from field-sensitized horses and donkeys. It was observed that tick-bite na?ve horses developed a low level of resistance after two infestations as shown by a significant decrease in larval yield and a tendency for lower engorged weight of nymphs during third infestation. Ticks fed on field-sensitized horses had a similar biological performance to that observed on the third infestation of tick-bite na?ve horses but the mean engorged nymph weight was significantly lower than that of the first infestation from tick-bite na?ve horses. Donkeys presented the strongest resistance with significantly lower engorged weights of all instars and of the egg mass compared to the first infestation of tick-bite na?ve horses. Donkeys also displayed a significantly higher resistance than field-sensitized horses as demonstrated by significantly lower egg mass weights. Overall these results indicate that donkeys but not horses maintain a strong resistance to A. cajennense ticks. The importance of these findings in relation to vectoring of tick-borne diseases is discussed.     

Virulence of Beauveria bassiana, Metarhizium anisopliae and Paecilomyces lilacinus to the engorged female Hyalomma anatolicum anatolicum tick (Acari: Ixodidae)

The tick is a common ectoparasite of livestock and humans, and is responsible for the transmission of pathogens among hosts. Direct and indirect impacts of ticks include limiting the sustainable development of the animal husbandry industry and detrimental effects on human health. Despite these negative effects, the main method of controlling ticks remains the application of chemical acaricides, which can lead to ambient pollution and the development of tick resistance to them. The biocontrol of ticks is one of the alternative control methods that has received recent research attention. The present study used Tenebrio moliter bait methods to collect 13 species of entomopathogenic fungi from different areas in China that were then tested to observe their effects on engorged female Hyalomma anatolicum anatolicum ticks. The results showed that more than half of the isolates had some pathogenic effects on the ticks; in particular, two Beauveria bassiana strains (B.bAT01, B.bAT17) and one Metarhizium anisopliae strain (M.aAT26) were highly virulent, causing up to 90% mortality. In addition, H. anatolicum anatolicum females were treated with B. bassiana B.bAT17 using different concentrations of the fungus. Results revealed that B. bassiana B.bAT17 is highly pathogenic against engorged H. anatolicum anatolicum females. This is the first report of the pathogenic effect of entomopathogenic fungi on engorged H. anatolicum anatolicum females. However, studies of the efficiency of this fungus against ticks in the field are required before it can be used for tick management in practice.     

吸血行为对草原革蜱和森林革蜱中肠菌群结构的影响

本研究旨在探明随吸血时间的延长,草原革蜱和森林革蜱中肠菌群结构的特征及变化。从内蒙古呼伦贝尔和宁夏固原绵羊体表分别采集半饱血、饱血草原革蜱和半饱血、饱血森林革蜱,无菌条件下收集蜱中肠内容物,提取细菌总DNA,扩增细菌16S rDNA V3-V4区,IonS5^TMXL高通量测序,对比分析各样本菌群结构的特征。结果显示,森林革蜱半饱血中肠内的细菌多样性最高,草原革蜱饱血和半饱血中肠内的细菌多样性次之,森林革蜱饱血中肠内的细菌多样性最低;变形菌门为4个样品的优势菌门;无形体属、立克次体属、寡养单胞菌属和柯克斯体属为4个样品的优势菌属,其中无形体属在草原革蜱和森林革蜱饱血中肠内的含量高于2种蜱半饱血中肠,立克次体属和柯克斯体属在2种蜱半饱血中肠内的含量明显大于饱血中肠;边缘无形体、弯曲假单胞菌和柯克斯体科RFE02菌为4个样品的优势菌种,其中边缘无形体的分布特点与无形体属在2种蜱中肠内的分布特点相一致。结果表明,草原革蜱和森林革蜱中肠菌群结构易受到吸血行为的影响,共有菌属和菌种在不同蜱种和不同饱血状态下的相对丰度变化较大。     

Culicoides bottimeri as a vector of Haemoproteus lophortyx to quail in California, USA

Arthropod sampling via periodic direct bird examination and regular light trapping was conducted between June 2000 and October 2002 to survey for potential vectors of Haemoproteus lophortyx to nonnative, captive-raised bobwhite quail (Colinus virginianus) in northern California, USA. Examination of individual bobwhite quail (from 5 weeks of age through adult, total n=76) was conducted on several dates during the transmission period (June-October). No ectoparasites, including hippoboscid flies (reported as Haemoproteus vectors to wild quail in early literature), were collected from the birds. Trapping with ultraviolet light suction traps near the quail revealed nine species of biting midges (Culicoides spp.). Of these, 94% were C. bottimeri, which was abundant near the birds, and 65% of collected C. bottimeri were engorged with blood. C. bottimeri adult activity began in late-April, slightly before the onset of disease in the quail. Activity peaked between July and late-September, coincident with maximum reported transmission, and adult activity ceased by early-November. Nonengorged C. bottimeri had a parity rate of 43.6% overall, suggesting excellent survival for biological transmission of a pathogen like H. lophortyx. A controlled study was done injecting a macerated slurry of pooled, nonengorged, wild-caught C. bottimeri into the peritoneum of 1-day-old bobwhite quail hatchlings held in insect-proof containers. Blood smears 13-19 days later confirmed H. lophortyx infection in zero controls but all insect-injected quail. Biting midges, especially C. bottimeri, transmit H. lophortyx to captive quail and probably are the dominant vector to native California quail (Callipepla californica) as well.     

Factors affecting the detachment rhythm of engorged Boophilus microplus female ticks (Acari: Ixodidae) from Charolais steers in New Caledonia

As in most parts of the world where the cattle tick Boophilus microplus is established, resistance of ticks to acaricides occurs in New Caledonia. In order to implement laboratory resistance tests on larvae, engorged females collected in suspected farms are necessary. Investigations on the detachment schedule of the engorged females were conducted to explain certain field situations such as the lack or scarcity of engorged females on highly infested cattle driven from the pasture to the pen in the morning. Three experiments on Charolais steers naturally infested on pastures showed that: (1) engorged female burdens at sunrise are similar whether the steers spend the night in pasture or in a pen; (2) compared with steers maintained in a pen, morning detachment of females increases when the steers stay on the pasture or move from the pasture to the pen; (3) detachment rhythm of engorged females on steers staying the morning in a pen, is not influenced by feeding activity or exposition of steers to sun; (4) detachment occurs earlier for females attached on anatomical sites exposed to sun, and earlier from these sites for the steers in pasture or walking than for steers in a pen.     

盾糙璃眼蜱传播的牛泰勒虫分离鉴定及生物学特性

自新疆伊宁地区黄牛体表采集的盾糙璃眼蜱饥饿或半饱血成虫,带回实验室感染除脾牛.经血涂片检查发现,于感染后第21天,病牛血液中出现一种以圆环形与卵圆形为主的泰勒虫.经形态学观察和18 S rRNA基因测序和进化关系分析证明,它们为环形泰勒虫.将此饱血成蜱经实验室饲喂条件下,孵化出的幼虫分别饲喂于牛与家兔两种不同的动物,结果在饱血若虫阶段所有的蜱均从两种动物体表脱落,不再叮咬,饥饿成蜱又更换另一宿主,证明其是二宿主蜱.     

Acaricidal activity of thymol on larvae and nymphs of Amblyomma cajennense (Acari: Ixodidae)

The aim of the present study was to evaluate the acaricidal efficacy of thymol on unengorged and engorged larvae and engorged nymphs of Amblyomma cajennense. To perform the test for unengorged larvae, the larval packet technique was employed and mortality was evaluated 24h after the test, while for engorged larvae and engorged nymphs the immersion technique was employed and mortality was evaluated after 15 days. In all the experiments, the following concentrations of thymol were tested: 2.5, 5.0, 10.0, 15.0 and 20.0mg the thymol/ml. The control group was exposed to water and 1% DMSO and there were 10 repetitions for each treatment. The values found for mortality of unengorged larvae were 18.2%, 51.8%, 97.6%, 93.5% and 94.5%, and for engorged nymphs the values were 26.0%, 92.2%, 100.0%, 100.0%, 100.0%, in concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0mg of thymol/ml, respectively. In the test with engorged larvae, mortality was 100.0% in all treatments. Based on the results, it is possible to conclude that thymol has acaricidal activity against immature stages of A. cajennense.     

Investigations into the volatility of female pheromones and the aggregation-inducing property of guanine in Argas (Persicargas) walkerae

During investigations into the volatility of female pheromones of A. walkerae in a Y-shaped olfactometer, engorged conspecific male ticks responded weakly both to female-contacted filter paper discs and to intact females enclosed in nylon bags. In tests in which female-contacted filter paper discs were separated from engorged male ticks by means of a screen, no aggregation was observed. However, replete male ticks showed a vigorous response to liquefied air and hexane-trapped volatiles collected from intact females. Guanine showed a potent aggregation attraction for engorged and unfed larvae and 1st and 2nd stage nymphal ticks as well as for male and female imagines of A. walkerae over a wide concentration range. It is concluded that at least 2 pheromones, 1 volatile and 1 non-volatile, are operative in the communication system of this tick species.     

The brown dog tick Rhipicephalus sanguineus and the dog as experimental hosts of Ehrlichia canis

Adult Rhipicephalus sanguineus ticks efficiently transmitted Ehrlichia canis to susceptible dogs for 155 days after detachment as engorged nymphs from a dog in the acute phase of ehrlichiosis. Adult ticks that had similarly engorged on a dog in the chronic phase of ehrlichiosis failed to transmit E canis to susceptible dogs. Infected but unfed adult ticks may be of greater importance than the chronically infected carrier dog as a natural reservoir of E canis.     

Preparation of suspensions of Hyalomma excavatum ticks infected with Theileria annulata

Partially engorged adult Hyalomma excavatum ticks infected with Theileria annulata were reared for the preparation of an infective suspension. About 6000 partially engorged infected adults were obtained from 420 non-infective adult ticks. The entire cycle required about 4 months. Six rabbits were used for the production of the fully engorged females. The larvae were fed on 180 gerbils, the nymphs on 17 infected calves and the partially fed adults on 22 goats, 7 sheep and 7 rabbits The adult ticks did not show any host preference.     

Hosts,seasonal occurrence and life cycle of Rhipicentor nuttalli (Acari: Ixodidae)

There are only two species worldwide within the genus Rhipicentor, namely Rhipicentor bicornis and Rhipicentor nuttalli and both occur only in Africa. Rhipicentor nuttali has a widespread distribution in South Africa and the present investigation was initiated to elucidate its host preference, seasonality and life cycle. Rock elephant shrews, Elephantulus myurus were examined for ticks at four localities in the Free State Province, one in Gauteng Province and two in Limpopo Province, South Africa. Cape elephant shrews, Elephantulus edwardii were examined at two places in the Western Cape Province, and a single specimen of the bushveld elephant shrew, Elephantulus intufi was examined in central Namibia. Small mammals of other species were also examined at two of these localities. The majority of E. myurus at two sites in the Free State, at the locality in Gauteng and both sites in Limpopo Province were infested with larvae and/or nymphs of R. nuttalli, while the single E. edwardii examined at one site in the Western Cape Province and the single E. intufi examined in Namibia were infested with nymphs of this tick. Not one of the other small animals was infested. Although larvae and nymphs of R. nuttalli were present on E. myurus throughout the year, the former were generally most numerous during the period March to September, and the latter during May to October. The preferred hosts of the adults are domestic dogs, leopards, Panthera pardus and South African hedgehogs, Atelerix frontalis. Adult females engorged on Atelerix frontalis in 16-32 days and, after a preoviposition period of 2-4 days, produced approximately 170,00 eggs during the following 60-70 days. The average incubation period of the eggs was 59 days. Larvae engorged on E. myurus in 4-10 days and moulted to nymphs 12-20 days later. Nymphs required 11-15 days to engorge on E. myurus and moulted to adults 32-47 days later. Allowing 14 days for the exoskeletons and mouthparts of each of the three parasitic stages to harden before they can attach to a host, the life cycle took approximately 214 days to complete in the laboratory. The length of this period, considered in conjunction with the times of maximum seasonal occurrence of the immature stages, indicates that the life cycle probably takes a year to complete in the field.     

Immunization of cattle with nymphal Hyalomma anatolicum anatolicum extracts: effects on tick biology

Antigens derived from partially engorged nymphs of Hyalomma anatolicum anatolicum were used in immunizing crossbred (Bos indicus×Bos taurus) cattle against larval, nymphal and adult H. a. anatolicum and H. dromedarii. The cattle were either infected with Theileria annulata at low parasitaemia or were uninfected. Whole nymphal extract (WNE), nymphal membrane antigens (NMA) and nymphal soluble antigens (NSA) were used for immunization. The group immunized with WNE showed significant and better rejection of H. a. anatolicum ticks as compared to calves immunized with either NMA or NSA. The moulting rates of both engorged larvae and nymphs remained unaffected. Nymphs which engorged on the immunized calves were fully susceptible to infection by T. annulata as indicated by the intensity and abundance of Theileria infections in the resulting adult ticks from immunized and unimmunized Theileria infected cattle. These ticks also transmitted fatal theileriosis to susceptible calves.     

Comparison of four methods of extracting DNA from D. gallinae (Acari: Dermanyssidae)

Dermanyssus gallinae is one of the most serious ectoparasites of poultry and it has been implicated as a vector of several major pathogenic diseases. Molecular detection of such pathogens in mites is crucial and therefore, an important step is the extraction of their DNA from mites. So, we compared four DNA extraction protocols from engorged and unfed individual mites: a conventional method using a Cethyl Trimethyl Ammonium Bromide buffer (CTAB), a Chelex resin, a Qiamp DNA extraction kit and a more recent one filter-based technology (FTA). The DNA samples have been tested for their ability to be amplified by an amplification of a D. gallinae 16S rRNA gene region. The best results were obtained using CTAB and Qiagen methods at the same time with unfed and engorged mites (96% and 100% of amplified samples). FTA produced similar results when using unfed mites but not when processing engorged ones (96% and 70%). Finally, the Chelex method was the least efficient in terms of DNA amplification, especially when applied on engorged individuals (50%). The possible inhibitor role of these Chelex extracted DNA was demonstrated by the means of a PCR control on PUC plasmid. No difference was observed with CTAB, Qiamp DNA extraction kit or FTA methods using DNA extracted one year before.     

Bacterial cholangiohepatitis in a dog

A 9-year-old female Yorkshire terrier was presented for vomiting and diarrhea. Blood chemistry tests revealed hepatic dysfunction, cholestasis, and inflammation. Liver ultrasonography and liver biopsy were consistent with cholangiohepatitis. Fine-needle aspiration of the gallbladder revealed the presence of bacteria later identified as Clostridium spp. The cholangiohepatitis was successfully treated.     

Transmission of a Trypanosoma sp. to cattle by the tick Hyalomma anatolicum anatolicum

Hyalomma anatolicum anatolicum engorged nymphs and flat adults were collected from two areas in northern Sudan. Various developing stages of Trypanosoma theileri-like flagellates were observed in the engorged nymphs, freshly moulted adults and mature adults partially engorged on rabbits. When these ticks were applied to two calves, one calf became infected with the trypanosome. The parasites were observed for one day in the enlarged lymph node nearest to the tick-feeding site 5 days after the tick application. Subsequently the trypanosomes were re-isolated in vitro from the infected calf. Inoculation of a ground-up tick supernatant suspension from the infected batch of ticks containing 10(4) trypanosomes into a calf did not produce a patent infection.     

Amblyomma variegatum in cattle in Marie Galante, French Antilles: prevalence, control measures, and infection by Ehrlichia ruminantium

We report Marie Galante as one of the Caribbean islands most heavily infested by the tropical bont tick (TBT) Amblyomma variegatum which is associated with two major diseases of ruminants: heartwater and dermatophilosis. In 2005, a survey was undertaken to assess the prevalence of TBT infestation in cattle, the prevalence of Ehrlichia ruminantium infection in TBTs, and the tick control measures implemented by livestock owners. A random sample of 195 cattle herds out of 1885 recorded on the island was investigated by thoroughly counting adult ticks on each animal and filling a questionnaire. A randomly collected sample of 136 TBTs was tested for infection by E. ruminantium by pCS20 nested PCR. Cattle herd prevalence (hp) was 73.8% for infestation by at least one TBT, 17.9% for infestation by at least one engorged female TBT, and 8.2% for clinical dermatophilosis. Cattle individual prevalence was 42.3% for infestation by at least one TBT, 6.6% for infestation by at least one engorged female TBT, and 2.2% for clinical dermatophilosis. The minimum, maximum and average numbers of TBTs per infested animal were, respectively 1, 108 and 11.5. Prevalence of TBT infection by E. ruminantium was 19.1%. No significant difference in herd prevalence was found among parishes or among ecological zones. For cattle owners treating against ticks (97.9% of all owners), all used aspersion of amitraz and herd prevalence was significantly different among those treating every 1-2-week (hp=69.6%, n=148), and less often than every 2-week (hp=88.6%, n=35) (P=0.031). Of the 42 herd subunits treated less than 4 days before the survey, 27 (64%) were infested with at least one TBT, and 6 (14%) with at least one engorged female TBT. These results indicate a high level of TBT infestation in Marie Galante, the inefficacy of tick treatments currently performed, and the need for an improved tick control strategy. Persisting high levels of infestation in Marie Galante threaten the success of on-going TBT eradication programs in the Caribbean because TBT can spread through migrating birds and trade of animals or of animal hides to other islands and potentially the American continent.