Humoral immune response and hematologic evaluation of pregnant Jersey cows after vaccination with Anaplasma centrale

The main objective of this work was to evaluate the safety of an Anaplasma centrale vaccine in pregnant pure bred Jersey cows selected from a herd located at Miranda State, Venezuela. Ten cows of 3-5 months of gestation were chosen and previous vaccination all cows were tested for Anaplasma antibodies by the indirect immunofluorescence assay (IFA), so only seronegative cows were included in the group, and for blood parameters, rectal temperature, and pregnancy. Selected cows were vaccinated intramuscularly with 1ml of an A. centrale live vaccine which had 10(8) A. centrale per ml. Over the next 2 months cows were checked weekly for hematological parameters and Anaplasma antibodies, and then for the next 2 months these evaluations were performed monthly. Among the values monitored were: A. centrale parasitemia, hematocrit, hemoglobin, and white blood cells (WBCs) (neutrophil, lymphocyte and eosinophil counts). Levels of Anaplasma antibodies were measured by IFA. Anaplasma were observed for the first time in blood films of two vaccinated cows at 14 days post-vaccination (PV), 6 out of 10 cows were A. centrale positive at 30 days PV, and all cows were A. centrale positive at 42 days PV. A. centrale often showed low parasitemia, 1-3%. Anaplasma antibodies were detected at day 14 PV in all vaccinated cows with a mean group titre of 360 (range: 80-1280). All vaccinated cows showed few changes in their hematologic parameters or in rectal temperature, and all gave birth to healthy calves. In conclusion, adult pregnant cows were safely vaccinated with this live A. centrale vaccine, which may help to develop a cross-protective immunity against field strains of A. marginale.     

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.     

Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection

The immunity induced by frozen and fresh Anaplasma centrale vaccines against anaplasmosis caused by A. marginale was tested in 12-month old Friesian steers. A. centrale parasitaemia occurred in all cattle inoculated with both types of vaccine. The average maximal decrease in PCV for the frozen and fresh vaccines was 41.0 and 40.3% respectively. All cattle recovered spontaneously. Vaccinated and control steers of the same age were challenged six months later with doses of 10(6), 10(7) or 10(8) A. marginale organisms. Vaccinated cattle showed average maximal A. marginale parasitemia of 1.2-4.0 versus 10.3-12.0% in control cattle. The average maximal decrease in packed cell volume (PCV) was 33.1 and 30.0% for steers vaccinated with frozen or fresh vaccine, respectively, and 57.4% for the non-vaccinated steers. All vaccinated cattle recovered spontaneously from the A. marginale infection while 7 out of 8 control steers required specific treatment. It thus appears that both frozen and fresh A. centrale vaccines are equally capable of inducing partial protection against infection with A. marginale and of preventing severe red blood cell destruction.     

Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.     

Efficacy of attenuated Anaplasma marginale vaccine under laboratory and field conditions in Colombia

Four-month-old Holstein-Friesian calves were inoculated with 3 different doses (1, 2, and 3 ml) of attenuated Anaplasma marginale vaccine. Vaccinated calves showed mild anaplasma parasitemia, slight decrease in packed cell volume, low serologic conversion, and no clinical illness. An artificial challenge exposure of vaccinated and unvaccinated calves with virulent Colombian A marginale showed that the vaccine provided protection against clinical signs of the disease, including parasitemia and anemia. The volume of the vaccinal dose did not alter the degree of protection provided. A 2nd group of 8- to 9-month-old Holstein-Friesian calves was then inoculated with 3 ml of anaplasma vaccine and premunized with both Babesia bigemina and Babesia argentina while being housed in an area free of these diseases. Calves were moved to an enzootic region heavily infested with various arthropods, including ticks, for natural field challenge exposure. Control calves, which were not given anaplasma vaccine, suffered clinical illness manifested by severe anemia and an average weight loss of 50.6 kg due to anaplasma field challenge exposure. In contrast, vaccinated calves did not show anemia and their weight loss was 3.9 kg.     

Immunity following use of Australian tick fever vaccine: a review of the evidence

OBJECTIVE: To review the evidence available on the degree and duration of immunity provided by Australian tick fever vaccines against Babesia bovis, B. bigemina and Anaplasma marginale infections in Australia and overseas. BACKGROUND: Vaccines containing attenuated strains of B bovis and B bigemina as well as A. centrale grown in splenectomised calves have been used in Australia since 1964 to immunise cattle against tick fever. About 800,000 doses of vaccine are supplied annually and much of the evidence for protection is field evidence rather than conventional immunological measures or pen trials. CONCLUSIONS: Immunity to Babesia bovis and B. bigemina--A single inoculation generally provides sound, long-lasting protection both in Australia and overseas. No evidence was found of a loss of immunity with time. Vaccine failures to B. bovis do occur, but are uncommon and evidently caused by a number of factors, including immune responsiveness of the vaccinated animals, and immunogenicity of the vaccine strain. Immunity to Anaplasma marginale--The vaccine containing A. centrale provides partial, variable protection against A. marginale. Protection against challenge in Australia is adequate in most cases to prevent disease and use of the vaccine in this country appears to be justified. Protection against antigenically diverse, highly virulent stocks of A. marginale in other countries is, at times, clearly inadequate and better vaccines are required in situations where the challenge is severe.     

The persistence of colostral Anaplasma antibodies and incidence of in utero transmission of Anaplasma infections in calves under laboratory conditions

Twenty-six calves, born from 25 Anaplasma-infected, intact and splenectomized cows, from a herd kept under strict tick-free laboratory conditions, were monitored for the presence of Anaplasma antibodies, using the rapid card agglutination test. Serum was collected at birth, weekly for 12 weeks, and then monthly for approximately 6 months. Specific antibodies passively acquired could be detected in calf sera for an average period of 8 weeks after birth. Calves that remained positive for longer than 12 weeks were suspected of having contracted in utero infections. Infection of the calves was confirmed by splenectomy. It was concluded that 4 calves in Group I contracted in utero infections. Two of the dams were chronically infected, whilst the other 2 underwent acute primary reactions during the 1st and 2nd trimesters of gestation, respectively. Subsequently all calves born from infected cows in this tick-free herd were serologically screened before being splenectomized at an average age of 8 months. Out of 50 cows, 8 in utero infected calves were identified serologically and this finding was confirmed through splenectomy or subinoculation of blood. Both Anaplasma centrale and Anaplasma marginale were carried transplacentally. Splenectomized and intact cows, chronically infected or undergoing primary reactions during the 1st, 2nd or 3rd trimester of gestation, produced infected calves. A 15,6% incidence of in utero transmitted infections were observed amongst 77 calves under these conditions. None of the 13 splenectomized cows, undergoing primary A. centrale infections during gestation, aborted. Clinical signs of disease were not observed in any of the 12 in utero infected calves prior to splenectomy. The implications of these findings are discussed.     

The effect of oxytetracycline treatment on immunity induced by Anaplasma centrale.

Calves vaccinated with Anaplasma centrale were treated with 20 mg/kg of long-acting oxytetracycline (OTC/LA) before or simultaneously with vaccination or up to seven months later. Of 40 animals given one or two of OTC/LA from 3 to 13 days before vaccination, 23 become patent after vaccination, with an average prepatent period almost twice as long as that in non-treated vaccinated controls. Upon challenge with 2 x 10(8) A. centrale per dose all 17 previously non-patent calves showed average maximum parasitemias of 2 to 3.8%. Out of 30 calves treated with two to four doses of OTC/LA from one to four weeks after vaccination, 29 remained negative for A. centrale and reacted to challenge infection with average maximum parasitemias of 6.9-7.8%. Five out of 10 calves receiving OTC/LA simultaneously with the vaccination, and all of a separate group of 10 calves treated with a single dose seven days after vaccination, become patent an average of 51.6 and 63.5 d, respectively, after vaccination. Upon challenge, the five previously non-patent calves showed an average of 5.2% maximum parasitemia. In all groups, only rare parasites were seen in previously patent calves after challenge. Thirty calves treated with 2-4 doses of OTC/LA about six months after vaccination showed no or only a few parasites upon challenge. The above results show that treatment with single or multiple doses of OTC/LA a few weeks before or after administration of live A. centrale vaccine can interfere with elaboration of immunity.     

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.     

Heterologous antibody responses of calves to Anaplasma centrale and A. marginale

Antigens of Anaplasma centrale, Onderstepoort isolate, and A. marginale, Wacol isolate, were analysed by a Western blotting technique. Sera from A. centrale-infected calves reacted to 41- and 38-kDa antigens in A. centrale and a 41-kDa antigen in A. marginale. Serum collected during the primary reaction from an A. marginale-infected calf reacted only to the 41-kDa antigen of A. marginale; heterologous antibody response to the 41-kDa antigen of A. centrale did occur later during the infection, but remained markedly weaker than the homologous response. The serologic cross-reactivity to this 41-kDa Anaplasma antigen confirms that it is common to the genus and also that it is a heterogeneous complex.     

Duplex real-time polymerase chain reaction for simultaneous detection and quantification of Anaplasma marginale and Anaplasma centrale

Anaplasma marginale and Anaplasma centrale are rickettsial pathogens responsible for acute disease and mild infections, respectively, in cattle herds. A duplex real-time polymerase chain reaction (PCR) assay with probes labeled with different fluorophores was developed for simultaneous detection and quantification of A. marginale and A. centrale DNA in bovine blood samples. The assay was able to detect as few as 10(1) and 10(2) DNA copies for A. marginale and A. centrale, respectively, with optimal specificity and reproducibility. Analysis by real-time and nested PCR carried out on 54 samples previously tested by reverse line blot hybridization showed that the established duplex real-time PCR assay can detect and quantify the 2 Anaplasma spp., even if present simultaneously in the same blood samples. Such an assay could be used in pathogenesis studies on bovine acute anaplasmosis.     

Post-thawing viability of vaccines for bovine babesiosis and anaplasmosis cryopreserved with glycerol

Live frozen vaccines containing Babesia bovis, Babesia bigemina or Anaplasma centrale were prepared using glycerol as cryoprotectant and stored in liquid nitrogen. The viability of the vaccines was tested inoculating calves 1 h (n = 12), 2 h (n = 12), 12 h (n = 6) and 24 h (n = 6) after thawing. Babesia bovis and A. centrale were detected in thin and/or thick blood smears in all vaccinated calves; however, 1 of 12 calves inoculated 1 h after thawing and 3 of 6 calves inoculated 24 h after thawing did not develop a B. bigemina parasitaemia. The longer post-thawing durability of frozen vaccines cryoprotected with glycerol compared with those cryoprotected with dimethyl sulfoxide, presented by other authors, will extend their use under field conditions.     

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.     

DNA probes for the detection of Anaplasma centrale and Anaplasma marginale

Anaplasmosis can be diagnosed either by immunological techniques or by direct microscopic examination of blood smears. Both methods are time-consuming and labour intensive. The use of DNA probes in an hybridization assay may simplify the diagnosis of anaplasmosis in cattle and sheep. A genomic DNA library of Anaplasma centrale was constructed in an expression vector and screened to detect clones containing A. centrale DNA. Four probes which hybridized to A. centrale and Anaplasma marginale DNA were isolated. One of these (AC-1) hybridized only to A. centrale DNA, whereas AC-2, AC-3 and AC-4 could detect DNA from both A. centrale and A. marginale. Probes AC-1 and AC-2 could detect 127 ng and 8 ng DNA respectively, while AC-3 and AC-4 detected 64 ng A. centrale DNA.     

牛传染性鼻气管炎弱毒与牛病毒性腹泻弱毒抗原免疫干扰的研究

本试验使用3~6月龄健康易感牛9头(牛传染性鼻气管炎病毒(IBRV)和牛病毒性腹泻病毒(BVDV)抗原、抗体均阴性),共分3组,每组3头犊牛。第1组首免肌肉注射IBRV-LNM弱毒疫苗株种毒,接种1周后,每头牛接种BVDV-SM弱毒疫苗株;第2组只接种BVDV-SM弱毒疫苗株种毒,接种时间同第1组;第3组为对照组,接种MDBK细胞培养液。接种BVDV-SM疫苗毒后每周采血至疫苗毒接种后28 d,测定接种后BVDV抗体效价,并采用BVDV-JL检验用强毒进行攻毒试验。结果表明,第1组与第2组试验动物血清中牛病毒性腹泻病毒抗体水平无明显差异,能够抵抗BVDV-JL强毒攻击达到免疫保护的效果,说明牛传染性鼻气管炎病毒IBRV-LNM弱毒疫苗株接种后在牛体内对牛病毒性腹泻病毒BVDV-SM疫苗毒不产生免疫干扰作用。     

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.     

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)     

Serologic and clinical responses of premunized, vaccinated, and previously infected cattle to challenge exposure by two different Anaplasma marginale isolates

Two Anaplasma marginale isolates, one originating in Florida (FAM) and the other from Virginia (VAM), were compared immunologically by cross-challenge exposure of 14 Anaplasma carrier cattle, 8 previously infected cattle, and 6 splenectomized carrier calves. In addition, 28 cattle vaccinated with a commercially available adjuvant killed vaccine and 22 nonvaccinated cattle were challenge exposed with either FAM or VAM. A detectable clinical response was not produced by either FAM or VAM challenge exposure in carrier and previously infected cattle; however, evidence of A marginale growth as characterized by low percentages of parasitemia and increased serum complement-fixation titers was seen in carrier cattle given a heterologous challenge organism and in previously infected cattle inoculated with either homologous or heterologous organisms. Among splenectomized calves, there was virtually no cross protection to the heterologous challenge exposure, whereas a homologous challenge failed to elicit any detectable response. Vaccinated cattle were resistant to VAM exposure, but the clinical response to FAM exposure was severe with a 47% mortality. Most of these cattle displayed typical acute anaplasmosis that was only marginally less severe than that encountered in nonvaccinated cattle.     

Phylogenetic analysis of the erythrocytic Anaplasma species based on 16S rDNA and GroEL (HSP60) sequences of A. marginale,A. centrale,and A. ovis and the specific detection of A. centrale vaccine strain

Phenotypic criteria for the identification of erythrocytic ruminant Anaplasma species has relied on subjective identification methods such as host pathogenicity (virulence for cattle or sheep) and/or the location of Anaplasma inclusion bodies within the host's red cells. Sequence comparisons of new and available GenBank Accessions were investigated to elucidate the relationships among these closely related Anaplasma species. Twenty-one 16S rDNA and GroEL (HSP60) sequences from 13 Anaplasma marginale (South Africa, Namibia, Zimbabwe, Israel, USA, Australia and Uruguay), three A. centrale (South Africa and Japan), two A. ovis (USA and South Africa), and two unknown Anaplasma species isolated from wild ruminants (South Africa), were compared. 16S rDNA maximum-likelihood and distance trees separated all A. marginale (and the two wild ruminant isolates) from the two South African A. centrale (including original vaccine strain, Theiler, 1911). The Japanese A. centrale (Aomori) demonstrated the lowest sequence identity to the remaining erythrocytic Anaplasma species. A. ovis inter-species relationships could not be resolved through the 16S rDNA analyses, whereas strong bootstrap branch support is demonstrated in the GroEL distance tree using A. ovis OVI strain. All erythrocytic Anaplasma species and isolates were confirmed to belong to the same cluster showing strong branch support to Anaplasma (Ehrlichia) phagocytophilum with Ehrlichia (Cowdria) ruminantium and Rickettsia rickettsii serving as appropriate out-groups. Based on groEL sequences, a specific PCR method was developed which amplified A. centrale vaccine (Theiler, 1911) specifically. This study confirms the suitability of 16S rDNA sequences to define genera and demonstrates the usefulness of GroEL sequences for defining species of erythrocytic Anaplasma.     

Study of an attenuated Anaplasma marginale vaccine in Mexico natural challenge of immunity in an enzootic area.

An evaluation was made of the protection induced by an attenuated Anaplasma marginale vaccine in young purebred cattle against the challenge exposure of naturally transmitted anaplasmosis in enzootic areas of Mexico. The cattle, which were raised in isolation units free of arthropods, consisted of 10 Brown Swiss calves (1 to 13 months of age) and 8 Holstein calves (5 to 7 months of age). They were paired by breed, age, and body weight, and allotted to 2 equal groups. Calves in 1 group were vaccinated, and at 6 weeks after vaccinations were done, calves in both groups were placed in the field where they were raised for approximately 1 year. Two Holstein and 3 Brown Swiss calves of the nonvaccinated group (group 2) developed clinical anaplasmosis, and the remaining calves of this group had hematologic evidence of the disease during the 2 to 4 months after introduction to the field. The vaccinated group, which remained free of anaplasmosis, showed consistently greater weight gain than did the controls. Among the Holstein calves, the maximum weight difference in favor of the vaccinated group was 50 kg/head at 5.5 months after field exposure, and among the Brown Swiss calves, the differences in weight gain in favor of vaccinated calves at the end of the 12-month period was between 11 and 30 percent. It is concluded that the vaccine provided a means for safe adaptation of high-quality young cattle to the tropics by protecting them against what appears to be the major obstacle to this practice, anaplasmosis.