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Prevalence and control of babesiosis in the Americas.

This review presents up-to-date information on the distribution and control measures of babesiosis in Latin America. Bovine babesiosis caused by Babesia bovis and B. bigemia will be emphasized. The disease is endemic in most countries and poses a serious economic burden on livestock production in the region (U.S. $1365 million/year, FAO, 1989). Of the estimated 250 million cattle in Central and South America, approximately 175 million (70%) are in tick-infested regions. Humid, tropical and subtropical areas favor development of the main vector, the one-host tick Boophilus microplus. In many regions bovine babesiosis is enzootically stable as a consequence of a balanced host-parasite relationship. However, Latin America offers a wide range of epidemiologic conditions that are influenced by variations from tropical to cool climates and by susceptible purebred cattle that are regularly imported to upgrade local stocks. The control measures employed in most countries for babesiosis essentially rely on chemotherapy, use of acaricides for B. microplus, and to a lesser degree, on immunization methods. In general, these measures are expensive, time consuming, and in many cases, provide limited success. Finally, the zoonotic potential of babesiosis will be addressed, with special emphasis on the situation in the United States. Even though bovine babesiosis has long been eradicated from the U.S.A., human babesiosis is endemic in the northeastern region of the country.

Anaplasmosis↗

Antigenic characterization of Brazilian isolates of Anaplasma marginale.

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

Anaplasma↗

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays.

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis--63.7 and 56.4% and B. bigemina--53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.

Anaplasma marginale↗

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Anaplasma marginale↗

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Anaplasma marginale↗

Variation among geographically separated populations of Dermacentor andersoni (Acari: Ixodidae) in midgut susceptibility to Anaplasma marginale (Rickettsiales: Anaplasmataceae).

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle that is endemic throughout large areas of the United States. Cattle that survive acute infection become life-long persistently infected carriers. In the intermountain west the Rocky Mountain wood tick, Dermacentor andersoni Stiles, is the most common vector of A. marginale. Male D. andersoni acquire A. marginale when feeding on persistently infected cattle and biologically transmit it when they transfer from infected to susceptible hosts. Host-seeking adult D. andersoni were collected from four widely separated natural populations and tested for susceptibility to midgut colonization with A. marginale. Male ticks were fed on calves persistently infected with a strain of A. marginale naturally transmitted by D. andersoni. Gut infection rates ranged from 12.5% of ticks collected from a mountain site near Hamilton, MT, to 62.5% of ticks from a rangeland site near Riley, OR. Sites near Miles City, MT, and Kamloops, British Columbia, Canada, had intermediate levels of susceptibility. The infection rates differed significantly among populations, and the same populations sampled in two consecutive years were not significantly different from one year to the next. Although there was variation among the populations in the size of ticks, size was unrelated to acquisition of gut infection. Quantitative polymerase chain reaction (PCR) demonstrated that there was no significant difference between populations in the mean number of genome copies in the guts of infected ticks. A. marginale from infected ticks was genotyped to confirm that they were all infected with the laboratory strain, and a sample of 682 field-collected D. andersoni was surveyed for A. marginale by nested PCR; none were found to be naturally infected. Infection of the gut is an essential constituent of vector competence for A. marginale; in this study, we have demonstrated significant variation among populations in this key component of vector competence.

Anaplasma marginale↗

Relative efficiency of biological transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni (Acari: Ixodidae) compared with mechanical transmission by Stomoxys calcitrans (Diptera: Muscidae).

Anaplasma marginale Theiler is a tick-borne intraerythrocytic rickettsial pathogen of cattle that also can be mechanically transmitted by biting flies. Rickettsemia during the acute phase of infection may reach as high as 10(9) infected erythrocytes (IEs) per milliliter of blood. Animals that survive acute infection develop a life-long persistent infection that cycles between 10(2.5) and 10(7) IE/ ml of blood. We compared stable fly Stomoxys calcitrans (L.) -borne mechanical transmission during acute infection with Rocky Mountain wood tick, Dermacentor andersoni Stiles-borne biological transmission in the persistent phase of infection to demonstrate quantitatively that biological transmission by ticks is considerably more efficient than mechanical transmission by biting flies. Stable flies that partially fed on an acutely infected calf and were immediately transferred to susceptible calves to complete their bloodmeals failed to transmit A. marginale. Ticks that fed on the original acquisition host after it reached the persistent phase of infection (>300-fold lower rickettsemia) successfully transmitted A. marginale after transfer to the same calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >300-fold higher than that from which ticks transmit with 100% efficiency demonstrates that tick-borne biological transmission is at least 2 orders of magnitude more efficient than direct stable fly-borne mechanical transmission.

Anaplasma marginale↗

A Canadian bison isolate of Anaplasma marginale (Rickettsiales: Anaplasmataceae) is not transmissible by Dermacentor andersoni (Acari: Ixodidae), whereas ticks from two Canadian D. andersoni populations are competent vectors of a U.S. strain.

Anaplasma marginale Theiler is a tick-borne rickettsial pathogen of cattle with a global distribution in both temperate and tropical regions. The pathogen is endemic in regions within the United States, whereas the Canadian cattle population is considered to be free ofA. marginale. Farmed bison, Bison bison L., in central Saskatchewan have been found to be infected with A. marginale; however, there is no evidence of transmission from bison to cattle. We tested a Saskatchewan bison isolate of A. marginale (SB1) to determine whether it is transmissible by the Rocky Mountain wood tick, Dermacentor andersoni Stiles. Colonized D. andersoni from the United States and Canada failed to transmit SB1. A separate transmission trial using D. andersoni adults reared from ticks collected in Alberta and British Columbia showed that ticks from these populations could successfully transmit the St. Maries, Idaho, strain of A. marginale. Although the Saskatchewan bison isolate of A. marginale seems not to be transmissible by D. andersoni, in the event of the introduction of a tick-transmissible strain, Canadian D. andersoni are likely to be competent vectors.

Anaplasma marginale↗

Effects of Anaplasma phagocytophilum infection on the molting success of Ixodes scapularis (Acari: Ixodidae) larvae.

We assessed the effects of sympatric (occupying the same or overlapping geographic areas) and allopatric (occurring in separate geographic areas) isolates of Anaplasma phagocytophilum on the survival of Ixodes scapularis Say larvae that were derived from ticks collected in Bridgeport, CT. Seven isolates of A. phagocytophilum, originating from different geographic regions of the United States, were tested: four isolates from the northeast (Bridgeport, Dawson, Gaillard, and NY-8), two from the Midwest (Webster and Sp-Is), and one from California (MRK). BALB/c mice were infected with each of the seven isolates via exposure to infected I. scapularis nymphs, whereas uninfected nymphs fed upon control mice. Both infected and control mice were infested with uninfected larvae at 1, 2, 3, 4, 6, and 9 wk after nymphal infestation. The molting success in cohorts of infected and uninfected ticks was calculated as the percentage of larvae successfully molting into nymphal stage, and the prevalence of infection in molted nymphs was determined by polymerase chain reaction. In ticks that became infected with the Bridgeport or Sp-Is isolates, the molting success decreased with an increase in the prevalence of infection. Ticks that fed upon mice infected with six allopatric isolates (Dawson, Gaillard, NY-8, Sp-Is, Webster, and MRK) showed significantly lower levels of survival than those fed upon control mice, regardless of the prevalence of infection, whereas in ticks fed upon mice infected with a sympatric isolate (Bridgeport), the overall molting success was similar to the control. Thus, some but not all of the A. phagocytophilum isolates have adverse effects on ticks. Ticks exposed to harmful isolates may experience higher levels of bacterial metabolism, and/or reduced quality of their blood meal, thereby reducing their survival. Noted differences between isolates may be due to the origin of a particular isolate and/or the degree of coadaptation between the pathogen and its vector on the population level.

Anaplasma↗

Comparative study of Anaplasma parasites in tick carrying buffaloes and cattle.

A comparative study on the prevalence of Anaplasma parasite was conducted on ticks carrying buffaloes and cattle. Five hundred blood samples of both animals (250 of each) were collected during February, March and April. Thin blood smears on glass slides were made, fixed in 100% methyl alcohol and examined. Microscopic examination revealed that 205 (41%) animals had Anaplasma parasites, out of which 89, 44 and 72 animals had Anaplasma marginale, Anaplasma centrale and mixed infection respectively. Infected buffaloes and cattle were 75 and 130 respectively. The infection in female was 53 and 92 in buffaloes and cattle respectively. Twenty-two and 92 blood samples of male were found positive in buffaloes and cattle respectively. Comparative study revealed that the cattle were 26.82% more susceptible than buffaloes. The parasite prevailing percentage in female of both animals was slightly higher than that of the male. This investigation was aimed at studying the comparative prevalence of Anaplasma parasite in tick carrying buffaloes and cattle.

Anaplasma↗

Advances in the diagnosis of some parasitic diseases by monoclonal antibody-based enzyme-linked immunosorbent assays.

Advances in diagnostic assays for parasitic diseases include the use of monoclonal antibodies (MAbs) in antigen capture and competitive inhibition enzyme-linked immunosorbent assays (C-ELISA). Antigen capture ELISAs for Anaplasma marginale and Cryptosporidium parvum provide direct detection of these parasites during clinical disease, and the C-ELISA format has been adapted for detection of anti-Babesia equi, anti-A. marginale and anti-bluetongue virus antibodies. False-positive results may occur when antigen preparations in other ELISA formats are contaminated with Escherichia coli, erythrocyte or cell-culture antigens. The C-ELISA format overcomes problems of antigen purity, since the specificity of the C-ELISA depends solely on the MAb used. For this reason, the C-ELISA format is highly suited for use with recombinant antigens. Also, the use of recombinant protein in diagnostic assays precludes the need to infect animals for antigen production when the antigen cannot be produced in cell culture.

Anaplasmosis↗

Immunodiagnosis of and immunoprophylaxis against the haemoparasites Babesia sp. and Anaplasma sp. in domestic animals.

The haemoparasites Babesia bovis and Anaplasma marginale are extremely important pathogens of cattle, affecting more than 300 million animals worldwide. Species of less importance affect cattle, dogs, horses and sheep. No vaccine is available for widespread usage. This review summarises existing immunodiagnostic and immunoprophylactic methods currently available for these diseases and reports on future immunoprophylactic methods.

Anaplasma↗

Seroprevalence of antibodies against Borrelia burgdorferi and Anaplasma phagocytophilum in cats.

OBJECTIVE: To determine whether cats in the northeastern United States develop serum antibodies against antigens of Borrelia burgdorferi and Anaplasma phagocytophilum and whether coinfection with the 2 organisms occurs. SAMPLE POPULATION: Serum samples from 84 healthy cats and 9 cats with lameness, fever, anorexia, or fatigue. PROCEDURE: Serum antibodies against B. burgdorferi and A. phagocytophilum were measured with an ELISA incorporating a whole-cell preparation or purified recombinant antigens, by means of Western blot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: ELISA results indicated that 44 of 93 (47%) sera contained antibodies against > or = 3 B. burgdorferi antigens, whereas 43 (46%) were reactive to whole-cell B. burgdorferi. Serum reactivity to protein 35, VlsE, and outer surface proteins A and F was most common. Seropositivity to > or = 3 antigens occurred at the same rate (5/9) in the 9 ill cats as in the 84 healthy cats (46% [39/84]). Of 13 sera reactive to recombinant antigens, 9 were seropositive as measured by Western blot testing with whole-cell antigen. Seropositivity rates of 30% and 38% were detected for antibodies against A phagocytophilum via IFA and ELISA testing, respectively. Fifteen (16%) sera had antibodies against both pathogens. CONCLUSIONS AND CLINICAL RELEVANCE: Cats living in areas infested by Ixodes scapularis ticks are exposed to B. burgdorferi and A. phagocytophilum and, in some instances, may be coinfected. Most cats appeared healthy. An ELISA incorporating specific recombinant antigens may be used adjunctively with Western blot and other assays to confirm B. burgdorferi and A. phagocytophilum infection in cats.

Anaplasma phagocytophilum↗

Survey of tickborne infections in Denmark.

We conducted a study of the distribution and prevalence of tickborne infections in Denmark by using roe deer as sentinels. Blood samples from 237 roe deer were collected during the 2002-2003 hunting season. Overall, 36.6% of deer were Borrelia seropositive, while 95.6% were Anaplasma phagocytophilum positive; all animals were negative for Bartonella quintana and B. henselae by indirect immunofluorescence assay. When a hemagglutination-inhibition test was used, 8.7% of deer were found positive for tickborne encephalitis (TBE)-complex virus. A total of 42.6% were found positive by polymerase chain reaction (PCR) for A. phagocytophilum with significant seasonal variation. All were PCR negative for Rickettsia helvetica. PCR and sequencing also showed a novel bacterium in roe deer previously only found in ticks. The study showed that the emerging pathogen A. phagocytophilum is widely distributed and that a marked shift has occurred in the distribution of TBE-complex virus in Denmark. This finding supports studies that predict alterations in distribution due to climatic changes.

Age Factors↗

Rapid and long-term disappearance of CD4+ T lymphocyte responses specific for Anaplasma marginale major surface protein-2 (MSP2) in MSP2 vaccinates following challenge with live A. marginale.

In humans and ruminants infected with Anaplasma, the major surface protein 2 (MSP2) is immunodominant. Numerous CD4(+) T cell epitopes in the hypervariable and conserved regions of MSP2 contribute to this immunodominance. Antigenic variation in MSP2 occurs throughout acute and persistent infection, and sequentially emerging variants are thought to be controlled by variant-specific Ab. This study tested the hypothesis that challenge of cattle with Anaplasma marginale expressing MSP2 variants to which the animals had been immunized, would stimulate variant epitope-specific recall CD4(+) T cell and IgG responses and organism clearance. MSP2-specific T lymphocyte responses, determined by IFN-gamma ELISPOT and proliferation assays, were strong before and for 3 wk postchallenge. Surprisingly, these responses became undetectable by the peak of rickettsemia, composed predominantly of organisms expressing the same MSP2 variants used for immunization. Immune responsiveness remained insignificant during subsequent persistent A. marginale infection up to 1 year. The suppressed response was specific for A. marginale, as responses to Clostridium vaccine Ag were consistently observed. CD4(+)CD25(+) T cells and cytokines IL-10 and TGF-beta1 did not increase after challenge. Furthermore, a suppressive effect of nonresponding cells was not observed. Lymphocyte proliferation and viability were lost in vitro in the presence of physiologically relevant numbers of A. marginale organisms. These results suggest that loss of memory T cell responses following A. marginale infection is due to a mechanism other than induction of T regulatory cells, such as peripheral deletion of MSP2-specific T cells.

Amino Acid Sequence↗

A sero-epidemiological survey of blood parasites in cattle in the north-eastern Free State, South Africa.

A survey to determine the incidence of parasites in cattle (n = 386) was conducted in the north eastern Free State between August 1999 and July 2000. Giemsa-stained blood smears were negative for blood parasites. A total of 94% of the cattle were sero-positive for Babesia bigemina by indirect fluorescent antibody test while 87% were sero-positive for Anaplasma by enzyme-linked immunosorbent assay. The observation of negative blood smears but high incidence of positive serological results for Anaplasma and Babesia for the same group of cattle indicates that this area is endemic for these diseases but with a stable disease situation. All the animals were sero-negative for B. bovis and this is probably because the tick vector (Boophilus microplus) which transmits the disease is not present in the Free State Province. Two tick species belonging to the family ixodidae were found on cattle, namely Boophilus decoloratus and Rhipicephalus evertsi evertsi. In the present study significant differences in seasonal burdens of B. decoloratus occurred, with the highest infestations recorded from February to June. The presence of R. evertsi evertsi throughout the year without any or with small fluctuations in winter months was observed, with a peak from February to May.

Anaplasma↗