Transmission of Anaplasma marginale by adult Dermacentor andersoni during feeding on calves

Laboratory-reared Dermacentor andersoni ticks experimentally infected as nymphs with Anaplasma marginale were allowed to feed as adults from 1 to 9 days on susceptible, splenectomized calves to determine when, during feeding, the hematozoan was transmitted from ticks to cattle. In experiment 1, ticks were allowed to feed on calves for 1, 2, 3, 4, 5, or 6 days and anaplasmosis did not result. The same calves were used for experiment 2, and ticks were allowed to feed for 1, 3, 6, 7, 8, or 9 days and anaplasmosis occurred in all calves on which ticks fed for greater than or equal to 6 days. In 2 trials in experiment 3, ticks were allowed to feed on calves for 1 to 9 days. Anaplasmosis developed only in calves on which ticks fed for 7, 8, or 9 days. The prepatent periods shortened with longer tick feeding, and linear regression analysis of combined prepatent periods of both trials of experiment 3 indicated a significant (P = 0.05) slope with an estimated daily decrease of 7.75 days from day 7 to 9 of feeding. There was no apparent correlation between length of tick feeding and severity of clinical signs in those calves that developed anaplasmosis. Seemingly, A marginale can be transmitted to cattle by adult D andersoni ticks no earlier than the 6th or 7th day of feeding.     

Pen studies on the control of cattle tick (Rhipicephalus (Boophilus) microplus) with Metarhizium anisopliae (Sorokin)

Three field trials were conducted over 12 months to assess the pathogenicity of Metarhizium anisopliae to parasitic stages of Rhipicephalus (Boophilus) microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30 degrees C), good spore production characteristics and ability to quickly kill adult engorged ticks in the laboratory. Spores were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated in the laboratory to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. In each trial the formulation rapidly caused 100% mortality in unengorged ticks that were removed from cattle and cultured in the laboratory. A significant reduction in egg production was recorded for engorged ticks collected in the 3 days post-treatment. However, there was little effect of the formulation on the survival of ticks on cattle, indicating that there is an interaction between the environment of the ticks on the cattle and the biopesticide, which reduces its efficacy against ticks.     

Transstadial and attempted transovarial transmission of Anaplasma marginale by Dermacentor variabilis

Transstadial and transovarial transmission of Anaplasma marginale by Dermacentor variabilis were attempted with with ticks exposed to the organism once by feeding as larvae or nymphs, and twice by feeding as larvae and nymphs. Typical colonies of A marginale were in gut tissues of adults that were infected as larvae, larvae and nymphs, and as nymphs; repeated exposure of ticks did not appear to result in an increase in the number of colonies in the gut of subsequently molted adults nor did it affect severity of the clinical disease that developed in cattle they fed on. In contrast, colonies of A marginale were not found in the midgut epithelium of unfed nymphs exposed as larvae, even though companion nymphs transmitted the parasite, causing severe clinical anaplasmosis in susceptible calves. The organism was not transmitted transovarially by F1 larvae or nymphs from the groups exposed as parent larvae, nymphs, larvae and nymphs, and as adults. Some of the calves fed on by F1 progeny had a few erythrocytic marginale bodies that looked suspiciously like A marginale, as well as postchallenge exposure prepatent periods that were longer than other calves in the transovarial transmission study. Sera from these calves were tested for antibody to A marginale, using a highly sensitive immunoblot technique. Antibodies were not detected in any of the sera.     

Attempted transmission to cattle of Anaplasma marginale from overwintered Dermacentor andersoni ticks.

Since the 1983 summer outbreak of anaplasmosis in southern Saskatchewan, the role of the tick, Dermacentor andersoni as an overwintering reservoir for Anaplasma marginale has been questioned. The purpose of this study was to determine if spring-collected ticks carried virulent A. marginale. Sixteen splenectomized calves were assigned randomly to two groups of 14 principals and two controls. Adult D. andersoni, collected in April from areas having high transmission rates of A. marginale, were confined to the ears of the principals by special bags and allowed to feed for eight days. The two control calves were subsequently challenged intravenously with blood from a calf infected with the Virginia strain of A. marginale. Principals and controls were monitored for 60 and 50 days postexposure respectively for signs of infection by clinical, hematological and serological procedures. None of the principals developed anaplasmosis but both control calves developed signs of disease.     

Demonstration of the inclusion appendage of Anaplasma marginale in nymphal Dermacentor andersoni

Dermacentor andersoni nymphs were placed in stockinettes and allowed to feed on a splenectomized calf with experimentally induced anaplasmosis when the parasitemia was 3%-5%. Nymphs were selected on each of the 6 days of feeding and every 5 days from repletion through molting to the adult stage (25 days postrepletion); they were killed and midgut tissues were processed and examined by light and electron microscopies. No stages of A marginale were seen in tissues of feeding ticks. Visualization of individual components of gut contents was difficult owing to presence of the concentrated, electrondense blood meal containing hemoglobin. Inclusion appendages were observed in midgut tissues of nymphs at 5 and 10 days postrepletion, but not at 20 or 25 days. The morphology of the appendages was similar to that described for inclusion appendages commonly associated with anaplasmal inclusions in bovine erythrocytes. Some appendages were free in the lumen of the midgut and occurred either alone or with clusters of small vesicular particles. Occasionally, initial bodies like those generally found in bovine erythrocytes were seen with the appendage, but most of them were swollen and appeared to be degenerating. Frequently, inclusion appendages were observed attached to the luminal surface of the midgut cell membrane by a blunt, electron-dense attachment complex. The attachment of the appendage appeared to be extracellular, with the pointed end extending into the lumen. Often, small particles were observed immediately across the cell membrane from where the appendages were attached; the small particles appeared to be generated from the appendage itself and to have passed through the membrane of the midgut cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

INFECTIVITY FOR CATTLE OF ANAPLASMA MARGINALE EXTRACTED FROM BOOPHILUS MICROPLUS TICKS EXPOSED TO CERTAIN TEMPERATURES

SUMMARY: Boophilus microplus ticks collected from calves with patent Anaplasma marginale infections were incubated at either 4 to 5°C, 14°C, 22°C, 27°C or 37°C for up to 14 days. Extracts prepared either from larvae, nymphs, immature females, adult males or mixtures of both sexes were infective for 14 of the 16 splenectomised calves inoculated. Extracts either from nymphs or from adult ticks deriving from nymphs moulted in vitro were infective for 11 of 12 nonsplenectomised calves. Possible application of the findings to producing a vaccine strain of A. marginale is discussed.     

The sex ratio of Amblyomma cajennense (Acari: Ixodidae) with notes on the male feeding period in the laboratory

To evaluate the sex ratio of field collected nymphal Amblyomma cajennense ticks, we collected 5326 engorged nymphs from naturally infested horses in Pirassununga county and allowed them to molt to adults in the laboratory. They yielded a sex ratio of 1:1.83 (M:F). Three and two engorged females were collected from horses pastured at Pirassununga county and from tapirs pastured in Sorocaba county, respectively. These females were allowed to oviposit and their progeny were reared until the adult stage in the laboratory. Engorged females collected from Pirassununga yielded a sex ratio of 1:1.57 (M:F) and a sex ratio of 1.14:1 (M:F) were obtained for those ticks collected from tapirs. In addition, unfed tick larvae were collected from Pedreira county and reared in the laboratory until the adult stage. This collection yielded a sex ratio of 1.11:1 (M:F). These results showed significantly different (P<0.05) sex ratio constitutions among different tick populations. Laboratory rabbits were infested once with A. cajennense male ticks, which showed feeding periods varying from 7 to 86 days. During this period, the rabbits were re-infested regularly with A. cajennense female ticks. A total of 179 engorged females were collected from the rabbits and their engorged weight, feeding, preovioposition and egg incubation periods, weight of deposited eggs, percent of hatched eggs and egg production efficiency were compared to the male feeding period and to the number of live males present on the host. None of the female variables were affected by the male feeding period. Male ticks remained fertile for the whole feeding period. Percent of hatched eggs was the only female variable that significantly decreased as the number of live males decreased on the host. The results showed that although some A. cajennense populations are composed of more females than males after molting, this female predominance is compensated by a long male feeding period and maintenance of its reproductive performance.     

Intermediate site of development of Anaplasma marginale in feeding adult Dermacentor andersoni ticks that were infected as nymphs

The development of Anaplasma marginale in midgut epithelial cells was studied in feeding, transmitting adult Dermacentor andersoni ticks. Laboratory-reared ticks experimentally infected as nymphs were allowed to feed from 1 to 9 days on susceptible calves. Gut tissues from ticks were collected on each day they fed (total, 9 days) and were processed for light and transmission electron microscopy. Colonies of A marginale were abundant during the first 6 days of feeding, after which numbers decreased. Colonies were adherent to the basement membrane of gut cells early during feeding, with resultant flattening of the colonies. Colonies also were seen in muscle cells on the hemocoel side of the basement membrane. Morphologic features of A marginale within muscle cells varied and were similar to those observed in gut cells. In addition, however, a large reticulated form in the colonies was observed in muscle cells and appeared to give rise to small particles by budding. Development of A marginale in muscle cells appears to represent an intermediate site of development between those in gut and in salivary glands.     

Infectivity and antigenicity of Anaplasma marginale from tick cell culture

The infectivity and immunogenicity of Anaplasma marginale grown in a tick cell culture from embryonic Dermacentor variabilis ticks were assessed in splenectomized and intact calves, respectively. Culture 1 consisted of the cell line inoculated with midguts of adult ticks infected with the Mississippi isolate of A marginale and dissected 5 to 10 days after repletion and detachment from an experimentally infected calf. Cultures 2 and 3 consisted of the cell line inoculated with midguts of ticks infected with the Virginia isolate of the organism. Inoculum for culture 2 was derived from nymphal ticks dissected 5 to 10 days after repletion and detachment from the infected calf; inoculum for culture 3 was midguts from adult ticks that were fed as nymphs, allowed to molt in the laboratory and dissected 21 to 24 days after molting. In trial 1, cultures 1, 2, and 3 were maintained at pH 6.9 and incubated at 28 C; in trial 2, cultures 1 and 3 were maintained at pH 7.4 and incubated at either 28 C or 37 C. Cultures 1, 2, and 3 failed to induce infection when injected IV and SC into 6 calves in 2 separate trials. Pre-challenge sera from these calves reacted with 2 purified Anaplasma antigens in the ELISA, but failed to react in the complement-fixation test. Results of a trial to use cultures 1 and 3 in combination with an oil-in-water adjuvant to immunize intact calves against A marginale were inconclusive. However, pre-challenge sera from immunized calves reacted with the 2 purified Anaplasma initial body antigens in the ELISA but failed to react in the complement-fixation text.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and infectivity of Anaplasma marginale in Dermacentor andersoni nymphs

The development of Anaplasma marginale was studied in Dermacentor andersoni nymphs after they had fed on a calf with ascending Anaplasma infection. Gut tissues were collected on day 4 of tick feeding, from newly replete (fed) nymphs and on postfeeding days (PFD) 5, 10, 15, 20, and were processed for light and electron microscopy to determine density of A marginale colonies. Homogenates of gut tissues were prepared from nymphs collected on the same days and inoculated into susceptible, splenectomized calves to test for infectivity. Anaplasma colonies were detected in gut cells on PFD 5, 10, 15, and 20. Although colony density appeared to be higher on PFD 10 and 15, differences were not significant. Nymphal type-1 colonies were detected in highest numbers on PFD 5 and 10, transitional colonies were seen in highest numbers at PFD 10 and 15, and nymphal type-2 colonies were observed only on PFD 20. Gut homogenates that were collected from ticks at 4 days of feeding, when newly replete, and on PFD 20 caused anaplasmosis when injected into susceptible calves, but homogenates made from ticks collected on PFD 5, 10, and 15 were not infective. The data indicate that of the colony types of A marginale that develop in replete nymphs, nymphal type-1 and transitional colonies may contain organisms that are not infective for cattle.     

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).     

The maturation of Theileria annulata in Hyalomma anatolicum anatolicum stimulated by incubation or feeding to produce sporozoites

Adult Hyalomma anatolicum anatolicum ticks infected with Theileria annulata (Hissar strain) were incubated at 36 degrees C or fed on rabbits. Tick salivary glands were stained whole with methyl green pyronin or ground up and deposited on microscope slides and stained with Giemsa's solution. Separate batches of ticks from both treatments were ground up, centrifuged and filtered to produce sporozoite suspensions. The suspensions were examined as deposits on microscope slides stained with Giemsa's solution. The Theileria in the salivary glands of the fed ticks matured more completely and rapidly than in the incubated ticks. The peak numbers of sporozoites from the fed ticks was greater by at least tenfold than the peak from the incubated ticks. This peak was on the third day of feeding or on the fourth day of incubation. It was confirmed that fed ticks will be more suitable for sporozoite production for infection of cattle and production of stabilates.     

The transovarial transmission of Babesia caballi by Hyalomma truncatum

Babesia caballi, isolated from a horse that originated from South West Africa/Namibia, was transmitted transovarially by adult Hyalomma truncatum. B. caballi proved to be highly infective for adult H. truncatum. Forty-five per cent of ticks feeding on a reacting animal with an extremely low parasitaemia became infected. In spite of a low parasitaemia, the ticks were severely affected by the parasite. Seventy per cent of the infected ticks either died during oviposition or after laying only a few eggs. The features of the infection in horses were: a prepatent period of 10 days, very low parasitaemias with low pathogenicity and spontaneous recovery of the infected animals.     

Experimental infection by capillary tube feeding of Rhipicephalus sanguineus with Bartonella vinsonii subspecies berkhoffii

It has been speculated that ticks may serve as vectors of Bartonella species. Circumstantial, clinical, epidemiological and serological evidence suggest that B. vinsonii subspecies berkhoffii (B. v. berkhoffii) might be transmitted by Rhipicephalus sanguineus. The purpose of the present study was to determine whether adult R. sanguineus ticks can be infected with a B. v. berkhoffii genotype II isolate via capillary tube feeding and whether the infection can then be transmitted from adult females to their eggs via trans-ovarial transmission. Furthermore, tick fecal material was also collected and screened as a possible source of infectious inoculum for canine infections. B. v. berkhoffii DNA was detected in 50% (7 of 14) of females that did not oviposit and in 14.3% (2 of 14) of female ticks that laid eggs, but not detected in egg clutches (100 eggs/female). DNA was also detected in tick feces collected on days 2 through 6 post-capillary tube feeding, however, dogs (n=3) did not become bacteremic or seroconvert when inoculated with tick fecal material. Therefore, trans-ovarial transmission of B. v. berkhoffii by R. sanguineus is unlikely, but further studies are needed to determine if tick fecal material can serve as a source of infection to canines.     

Fatal bovine anaplasmosis in a herd with new genotypes of Anaplasma marginale, Anaplasma ovis and concurrent haemoplasmosis

Haematological and molecular analysis of blood samples was carried out during an outbreak of bovine anaplasmosis in Hungary. Acute disease was observed in five animals, two of which died. Anaplasma-carrier state was diagnosed in 69 (92%) of cattle. Further evaluation of 24 blood samples revealed concurrent infections with Mycoplasma wenyonii and 'CandidatusM. haemobos' in 22 and 21 animals, respectively. In addition, two cows were identified with rickettsaemia. Regarding molecular investigation of potential hard tick vectors, Haemaphysalis inermis and Dermacentor marginatus males collected from the animals were PCR-negative. However, in one pool (out of 18) of Ixodesricinus males, and in six pools (out of 18) of D. reticulatus males the msp4 gene of Anaplasma marginale was detected. In the same I. ricinus pool Anaplasma ovis was also identified. All ticks were negative for haemoplasmas. Anaplasma sequences yielded 97-99% homology to sequences deposited in the Genbank. This is the first report of fatal bovine anaplasmosis associated with divergent A. marginale genotypes and concurrent 'CandidatusM. haemobos' infection, as well as of an A. ovis strain in ticks collected from cattle.     

Experimental transmission of bovine anaplasmosis (caused by Anaplasma marginale) by means of Dermacentor variabilis and D. andersoni (Ixodidae) collected in western Canada

Canadian cattle are free of bovine anaplasmosis, with the exception of 4 isolated incursions since 1968, which were eradicated. It is not known why the disease has not become established in regions of Canada adjacent to the United States where it is endemic. To assess the vector competence of wild-caught ticks in cattle-rearing regions, Dermacentor variabilis and D. andersoni were collected in western Canada and fed on calves experimentally infected with Anaplasma marginale (St. Maries strain). The 2 tick species were equally competent in transmitting A. marginale to splenectomized calves, all 15 tick-exposed calves becoming infected. The prepatent periods in 13 calves ranged from 18 to 26 d and did not vary in relation to the numbers of ticks fed or the duration of transmission feedings. The unusually long prepatent periods in 2 calves (45 and 55 d) were probably due to concomitant Eperythrozoon infection. This study clearly demonstrated that tick species present in western Canada are competent vectors of bovine anaplasmosis. Potential barriers, including climate, must be considered in developing strategies to prevent A. marginale from becoming established in anaplasmosis-free regions.     

Infectivity of three Anaplasma marginale isolates for Dermacentor andersoni

Three isolates of Anaplasma marginale--Virginia (VAM), Illinois (IAM), and Florida (FAM)--were compared for infectivity for Dermacentor andersoni. The isolates were selected, in part, because of a tail-like appendage that has been demonstrated in the VAM and IAM, but not in the FAM. Ticks were exposed to the isolates as nymphs either naturally by feeding on a calf with anaplasmosis or artificially by percutaneous inoculation with infected bovine erythrocytes. They were examined for infectivity after molting to the adult stage by determining their capability to transmit the disease to susceptible calves and by demonstrating colonies in tick gut sections. Only those ticks exposed to the VAM proved to be infected with A marginale; ticks naturally exposed and those artificially infected with this isolate transmitted the disease to susceptible calves. Colonies of A marginale were observed only in gut tissues of ticks naturally infected with VAM. The IAM (appendage present) and FAM (appendage absent) could not be found in ticks exposed by either method, indicating that factors other than the presence of inclusion appendages may be involved in infection of ticks by A marginale.     

Preliminary studies of the development of Anaplasma marginale in salivary glands of adult, feeding Dermacentor andersoni ticks

On each day of feeding on susceptible calves, salivary glands obtained from groups of adult ticks that transmitted Anaplasma marginale were examined for A marginale colonies by use of light microscopy and transmission electron microscopy. On day 8 of feeding, salivary glands were examined, using fluorescein-labeled antibody and methyl green-pyronine stain. Use of fluorescein-labeled antibody consistently revealed small numbers of fluorescent foci in salivary gland acinar cells obtained from ticks that had fed for 8 days. Colonies of A marginale were seen by transmission electron microscopy only in salivary gland acini of male ticks; these colonies could not be identified, using light microscopy, in companion 1-micron plastic sections stained with Mallory stain. Methyl green-pyronine stain, used commonly to detect theilerial parasites in tick salivary glands, did not differentiate A marginale from cytoplasmic inclusions normally found in salivary gland acinar cells.     

Comparison of an amitraz-impregnated collar with topical administration of fipronil for prevention of experimental and natural infestations by the brown dog tick (Rhipicephalus sanguineus).

OBJECTIVE: To compare the efficacies of amitraz and fipronil for prevention of experimental and natural infestations of Rhipicephalus sanguineus. DESIGN: Prospective study. ANIMALS: 30 dogs. PROCEDURE: In 3 trials, dogs were allocated to 3 groups of 10 each. In trial 1, dogs were experimentally infested on day--1, and on day 0 were fitted with an amitraz-impregnated collar, treated topically with fipronil, or not treated. Ticks were counted daily until day 7, when viability of ticks and their progeny was determined. In trial 2, dogs were treated on day 0 and experimentally infested on days 7, 8, 10, and 13. Ticks were counted on days 8, 10, 13, and 18, and viability of ticks and their progeny was determined on day 18. In trial 3, dogs were exposed weekly to a tick-infested environment from day--3 to day 70. Dogs were treated on day 0, and ticks were counted and removed weekly from day 3 to day 77. RESULTS: Fipronil and amitraz were acaricidal and inhibited attachment and feeding. Amitraz had a significantly greater effect than fipronil on numbers of live, feeding ticks, egg hatchability, and larval viability, indicating partial ability to interrupt the tick life cycle. In field conditions, amitraz remained effective over the entire observation period. CLINICAL IMPLICATIONS: Amitraz had stronger and more sustained effects against tick infestation than fipronil.     

Isolate of Anaplasma marginale not transmitted by ticks

The tick-borne transmissibility of 2 isolates of Anaplasma marginale was compared. Dermacentor variabilis were exposed to A marginale as nymphs by feeding on 1 of 4 splenectomized calves during periods of ascending parasitemia (maximum 49% to 81% parasitized erythrocytes) induced by injection of a stabilate. Tick-borne transmission was attempted, using 26 to 224 adult ticks within 30 to 220 days after molting. Adult D variabilis did not transmit an Illinois isolate of A marginale in 7 tick-borne transmission experiments (P = 0.0047), including 2 experiments in which calves were inoculated IV with homogenates of adult ticks. In contrast, a Virginia isolate of A marginale was readily transmitted by the same tick colony. Thus, previously reported morphologic and immunologic differences among A marginale isolates may extend to tick-borne transmissibility. The Virginia and Illinois A marginale isolates had an inclusion appendage that was not a marker for tick transmissibility.