High Prevalence of Anaplasma spp. in Small Ruminants in Morocco.

The prevalence of infection by Anaplasma spp. (including Anaplasma phagocytophilum) was determined using blood smear microscopy and PCR through screening of small ruminant blood samples collected from seven regions of Morocco. Co-infections of Anaplasma spp., Babesia spp, Theileria spp. and Mycoplasma spp. were investigated and risk factors for Anaplasma spp. infection assessed. A total of 422 small ruminant blood samples were randomly collected from 70 flocks. Individual animal (breed, age, tick burden and previous treatment) and flock data (GPS coordinate of farm, size of flock and livestock production system) were collected. Upon examination of blood smears, 375 blood samples (88.9%) were found to contain Anaplasma-like erythrocytic inclusion bodies. Upon screening with a large spectrum PCR targeting the Anaplasma 16S rRNA region, 303 (71%) samples were found to be positive. All 303 samples screened with the A. phagocytophilum-specific PCR, which targets the msp2 region, were found to be negative. Differences in prevalence were found to be statistically significant with regard to region, altitude, flock size, livestock production system, grazing system, presence of clinical cases and application of tick and tick-borne diseases prophylactic measures. Kappa analysis revealed a poor concordance between microscopy and PCR (k = 0.14). Agreement with PCR is improved by considering microscopy and packed cell volume (PCV) in parallel. The prevalence of double infections was found to be 1.7, 2.5 and 24% for Anaplasma-Babesia, Anaplasma-Mycoplasma and Anaplasma-Theileria, respectively. Co-infection with three or more haemoparasites was found in 1.6% of animals examined. In conclusion, we demonstrate the high burden of anaplasmosis in small ruminants in Morocco and the high prevalence of co-infections of tick-borne diseases. There is an urgent need to improve the control of this neglected group of diseases.

Molecular and serological prevalence of Anaplasma marginale in cattle of North Central Morocco.

A cross sectional study was conducted to investigate the epidemiological distribution of Anaplasma marginale in North Central Morocco. Blood samples from five provinces of Morocco were collected from apparently healthy cattle (n=668) and simultaneously analyzed by a nested polymerase chain reaction (nPCR) assay and competitive enzyme-linked immunosorbent assay (cELISA). The overall prevalence of A. marginale was 21.9% by nPCR and 16.5% by cELISA. The Kappa coefficient between nPCR and cELISA indicated a modest level of agreement (0.54). The prevalence of A. marginale varied significantly according to the province and the month of sampling. However age, gender and breed did not have a significant effect on the prevalence of this pathogen. The highest prevalence of A. marginale was found in the Gharb, a sub-humid area while the lowest was reported in the Saiss, a semi-arid area. These results indicate that an A. marginale infection are widespread in the country and suggests that either or both techniques are excellent tools for epidemiological studies and control programs.

Validation of a competitive enzyme-linked immunosorbent assay for diagnosing Babesia equi infections of Moroccan origin and its use in determining the seroprevalence of B. equi in Morocco.

A highly specific and sensitive competitive enzyme-linked immunosorbent assay for detection of specific antibody to Babesia equi in serum from equids was validated for use in Morocco. The assay is based on the specific inhibition of binding of a monoclonal antibody to a conserved epitope within a recombinant parasite peptide by serum from infected animals. The assay was compared to an established indirect immunofluorescence assay, with a concordance of 91%. The assay was used to determine seroprevalence for B. equi infections in donkeys and horses throughout Morocco. A total of 578 sera (163 horses and 415 donkeys) from 6 locations representing different bioclimatic regions were assayed. An analysis of variance, indicated no significant effect of location; however, donkeys were significantly more likely than horses to be seropositive. Management conditions contribute to greater tick infestations and thus Babesia exposure in donkeys than in horses.

Serological, clinical and histopathological changes in naturally infected dogs with Leishmania infantum in the Khemisset province, Morocco.

Canine leishmaniasis (canL) is widespread in the north of Morocco and the Leishmania infantum local strains are highly virulent. An epidemiological survey was carried out in 1993-1995 in the Khemisset province. In this region, the severity of the disease was assessed during regular visits to the identified foci by clinical examination of 323 dogs. Clinical signs were protean and occurred in various combinations. Biopsies were made on available sick dogs; the main histological changes were severe infiltration of the spleen, lymph nodes and bone marrow by mononuclear cells and hyperplasia of macrophage cells with amastigotes in their cytoplasm. The seroprevalence among 323 dog sera tested by ELISA showed a rate of 16.71%. The highest prevalence of the disease was 23.6% in the Sid El Ghandour hamlet. A comparison of the results of this study with those from the year following the first examination on the same site (Sid El Ghandour) of 67 dogs showed that the disease prevalence had not increased significantly (23.6% to 25.33%).

Immune responses in vaccinated dogs with autoclaved Leishmania major promastigotes.

A comparative study was undertaken on the immunogen power of autoclaved Leishmania major promastigotes (ALM) vaccines given simultaneously with either BCG or saponin against canine leishmaniasis. The humoral immune response was assessed by ELISA and western blotting. The cellular immune response was evaluated by the lymphocyte transformation test. Dogs vaccinated simultaneously with ALM and saponin showed high antibody titres to crude L. infantum antigens after the first vaccine booster and reacted with several antigens, with molecular weights from 26 to 108 kDa by western blotting. However, the lymphocyte proliferation of these dogs to the crude L. infantum antigen was not significantly different from the control group. In contrast, in dogs vaccinated simultaneously with ALM and BCG, the antibody titres to crude antigen were low. Their sera reacted with the same proteins recognised by sera from dogs vaccinated simultaneously with ALM and saponin by western blotting. However, the 85-kDa protein was only identified by sera taken from dogs vaccinated simultaneously with ALM and BCG. These latter exhibited specific lymphocyte proliferation to the L. infantum antigen. This cell proliferation was observed for approximately 9 months after the first dose of the vaccine. This study indicates that a combination of ALM as the vaccine and BCG as the adjuvant, in the dog model, was successful in inducing a cell immune response, which is implicated in protection of dogs against a Leishmania infection.

Analysis of immune responses in dogs with canine visceral leishmaniasis before, and after, drug treatment.

The incidence of canine visceral leishmaniasis (CVL) is increasing in the Mediterranean region. Many drugs have been tested for treatment of CVL, but little is known regarding their effect on test immune responses. In our study, three dogs naturally infected with Leishmania infantum and five dogs experimentally infected with the same strain, were treated with dimethasulfonate pentamidine (Lomidine) and the immune response evaluated before, and after, treatment. After the last injection, animals began to gain weight and the major clinical signs disappeared. Antibody titers gradually decreased to low levels, six months after treatment. At the same time, antigen specific lymphoproliferation reappeared in the sampled animals. This study shows that, after treatment, immune cellular responses to leishmanial antigens, involved in protection against Leishmania infection, were established.

Immune response against Leishmania antigens in dogs naturally and experimentally infected with Leishmania infantum.

Cell-mediated and humoral immune response in naturally and experimentally infected dogs was studied using crude and pure antigens. Both types of infections induced severe signs of visceral disease, but the symptoms observed in natural infections were more pronounced than in experimental infections. In addition, asymptomatic infections were not observed in experimentally infected animals. Disease evolution in laboratory infections was rapid and an increase in antibody titer to crude parasite antigen was correlated with the appearance and aggravation of clinical symptoms. Peripheral blood lymphocyte proliferation to crude antigen and pure gp63 was observed early following experimental infection, but was abolished once the infected dogs began to exhibit clinical signs. A similar pattern was observed in naturally infected dogs. Serum from all patent dogs showed high antibody titers to rK39 in enzyme-linked immunosorbent assays (ELISA), and reacted by western blotting with several antigens, 12 to 120 KDa, including gp63 and gp70. In the case of asymptomatic dogs. antibody titers to crude antigen were low and only a few antigens were identified by western blotting. None of the pure proteins examined, gp63, gp70, and rK39 were recognized by western blotting or ELISA. However, asymptomatic dogs exhibited specific lymphocyte proliferation to both crude antigen and the potential vaccine candidate gp63.

Female tick Hyalomma marginatum marginatum salivary glands: preliminary study on protein changes during feeding process and antigens recognized by repeatedly infested cattle.

Proteins extracted from salivary glands of unfed, three days and five days fed adult Hyalomma marginatum marginatum were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We have noticed changes during the three feeding steps. Some proteins disappeared during feeding process (23, 38, 39, 40 to 50, 95 and 112 kDa), they might be proteins which were converted in other substances and are secreted. Other antigens (13 to 14, 20, 25, 29, 165 and 210 kDa) were synthesized as a result of tick attachment and feeding. They may be related to growth and development or are the ciment which fixed the adult. Also, three Holstein calves were infested five times with 100 pairs of adult ticks of the same species. The five infestations were performed two weeks from the previous infestation. The sera before infestations and after each infestation were used in western-blot analyses to identify antigens from five days salivary gland extracts of the primary infestation of ticks. Three antigens (18.7, 50 and 80 kDa) were revealed weakly after the first and the second infestations by sera samples but not at infestation onward. Others (13.5, 17 to 18.5, 25, 30, 70, 133, 176 and 193 kDa) were revealed only by sera taken after manifestation of resistance (third infestation). A 13.5 kDa antigen was particularly revealed when resistance had appeared and became more evident after the fourth and fifth infestations. The late antigens recognized might be associated with establishment of calves resistance against ticks.

Comparison of effects of low and high tick infestations on acquired cattle tick resistance: Hyalomma marginatum marginatum.

Three Holstein calves were infested with low numbers of ticks, two or three pairs of adults Hyalomma marginatum marginatum in cloth bag daily for 21 days. Infestation was carried out during tick proliferation periods. Two months later, cattle leads were challenged with 100 pairs of ticks. Another group of three Holstein calves were infested five times with 100 pairs of adult ticks of the same species. The five infestations were performed two weeks from the previous infestation. Three tick characteristics were recorded for each experiment: survival to detachment, females weight at detachment and egg mass weight. Light continuous infestations did not cause a significant change in this parameter, but every parameter declined gradually in the heavy infestations. Female and egg mass weight reached a significant difference from the first infestation by the fourth infestation. The circulating antibodies anti-salivary glands of Hyalomma m. marginatum showed that light infestation may induce like immuno-suppression. However, there is an inverse relationship between these antibodies and manifestation of resistance when calves were heavily infested. This is discussed in relation to a fraction of produced antibodies against protective antigens, and participation of another effector mechanism.

Bovine babesiosis. Seroprevalence and ticks associated with cattle from two different regions of Morocco.

A total of 475 bovine sera collected in 1995-1996 from 10 areas belonging to two different bioclimatic strata were tested for antibody activity to Babesia bigemina and Babesia bovis using indirect immunofluorescence (IIF). In the Gharb, the B. bovis seroprevalence was 21.7% and for B. bigemina, 10.8%. The infection rate for either or both species as determined microscopically with Giemsa-stained blood films was 18.9%. The Tiflet area was considered an endemic focus, and the seroprevalence was 42.2% for B. bovis and 40% for B. bigemina. The infection rate by stained blood film microscopy was 66.6%. In the Haouz region, only B. bovis was found, and the seroprevalence was 10.1% with 9.4% microscopically positive blood films. More than 80% of the cattle surveyed were infested by ticks and the mean infestation rate was 36 ticks per animal and 21 ticks per animal in the Gharb and Haouz, respectively. Six species were identified. Hyalomma marginatum, Hyalomma detritum, Hyalomma anatolicum Rhipicephalus bursa, Rhipicephalus sanguineous and Boophilus annulatus. Boophilus annulatus was found in both regions with high prevalence in the Gharb (31.3%). No further correlation was made between the identified species as vectors and the presence of B. bovis and B. bigemina in these areas.

Short report: Leishmania tropica: etiologic agent of a case of canine visceral leishmaniasis in northern Morocco.

The domestic dog has been previously demonstrated to be the reservoir of Leishmania infantum, the etiologic agent of human visceral leishmaniasis around the Mediterranean Basin. It can also be infected with L. tropica, the etiologic agent of anthroponotic cutaneous leishmaniasis in Morocco. We report a canine L. tropica visceral infection for the first time in Morocco.

Bronchoalveolar cellular responses of goats following infections with Muellerius capillaris (Protostrongylidae, Nematoda).

The development of Muellerius capillaris in the lung of goats was associated with marked tissue damage and pronounced a cellular reaction. Using broncho-alveolar lavage, the time course of the cellular responses was studied following primary and secondary infection. During the primary infection, there was a biphasic increase in total broncho-alveolar leucocytes (an average of 294.0 +/- 137.0 cells microl[-1]) and in the absolute number of macrophages (182.0 +/- 82.0 cells ul[-1]), lymphocytes (68.5 +/- 35.0 cells microl[-1]), eosinophils (35.3 +/- 16.4 cells microl[-1]) and neutrophils (10.9 +/- 8.7 cell microl[-1]). The lung tissue reaction against worms consisted of a mild infiltration of inflammatory cells. The secondary infection resulted in significant changes in the pulmonary tissue characterised by severe inflammation, leading to widespread granulomatous formation throughout the parenchyma, hyperplasia of cells Type II and a leucocytosis in the broncho-alveolar fluids, with an anamnestic-like response by all cell types. The overall average of the total leucocytes, macrophages, lymphocytes, eosinophils and neutrophils was 529.3 +/- 347.4; 265.4 +/- 148.1; 127.3 +/- 100; 125.4 +/- 100.1 and 14.0 +/- 8.7 cells microl(-1), respectively. Secondary infection also resulted in 56% reduction of worms established in the lungs and 72.3% of L1 larval production. These data suggest that the broncho-alveolar leucocyte response to infection has an immunological basis and that the alveolar macrophages, neutrophils, eosinophils, and lymphocytes may play a significant role in lung resistance against protostrongylid nematodes.

Hyalomma ticks: bovine resistance under field conditions as related to host age and breed.

Field collection of ticks from two breeds of cattle [Native breed (local) and purebred [Friesian)], showed that the number of ticks was higher on purebred than on native cattle. The susceptibility seemed to increase with the age of the animals. Furthermore, breed appeared to exert more influence than age. There was no correlation between the number of ticks associated with each animal, the antibody production and antigens recognized. The immune response under field condition looked more like immune suppression than immune protection.

Epidemiology of human visceral leishmaniasis in Taounate, a northern province of Morocco.

We report the preliminary results of an epidemiological survey carried out in the locality of Ouled Hcein, province of Taounate (North Morocco) around one visceral leishmaniasis infant case. The strain isolated from the child was characterized by three methods as Leishmania infantum and its isoenzyme pattern was indistinguishable from zymodeme MON-1. The human survey in the locality of origin of the index case did not show any additional case and the serology was negative for the whole people sampled. The canine survey showed that the highest seroprevalence of the canine disease was found in the locality of Ouled Hcein (27.1%) when compared to 4 surrounding localities. The entomological survey showed that amongst the demonstrated L. infantum vectors around the Mediterranean Basin, Phlebotomus longicuspis was the predominant species. Furthermore, the survey led to the identification of one female of P. ariasi naturally infected with L. infantum, so this species should be at least one of the vectors of this parasite in this area. Thus, this study confirms that the transmission cycle of L. infantum in North Morocco is similar to the cycle found in the remaining Mediterranean area.

Comparative immunizing power of infections, salivary extracts, and intestinal extracts of Hyalomma marginatum marginatum in cattle.

Tick concealed antigens have been successful in producing immunity that inhibits tick fertility, but require periodic revaccination and are little effective in preventing tick feeding, which is critical to stop pathogen transmission. Tick natural salivary antigens also induce important immunity, but revaccination may be unnecessary in enzootic areas. In addition these antigens may inhibit tick feeding. We immunized groups of three tick-naive calves with four prior infestations with Hyalomma marginatum marginatum, a salivary extract (SE), or an intestinal extract (IE) of the ticks. The calves were challenged with 100 pairs of homologous ticks and characteristics representing tick feeding or fertility were recorded and compared between groups. The percentage of attachment was inhibited by 46% by the infestation-generated immunity, 47% by the SE-generated immunity, and 0% by the IE-generated immunity. The percentage of engorgement was reduced 40% by the infestations, 57% by the SE, and 29% by the IE. The length of feeding was prolonged 92% by the infestations, shortened 44% by the SE, and not affected by the IE. The weight of the engorged females was decreased 67% by the infestations, 64% by the SE, and 31% by the IE. The percentage of engorged ticks that oviposited was inhibited 52% by the infestations, 27% by the SE, and 63% by the IE. The preoviposition period was prolonged 160% by the infestations, 80% by the SE, and 140% by the IE. The egg weight was reduced 60% by the infestations, 60% by the SE, and 66% by the IE. Taking into account mortality before oviposition, fertility was inhibited 88.2% by the infestations, 87.5% by SE, and 91.4% by the IE. The effect of IE immunization on tick feeding was not significant statistically.

Production and characterization of monoclonal antibodies directed against Eimeria falciformis and cross-reactive with sporozoites from two species of avian coccidia.

Monoclonal antibodies (mAbs) were obtained against the surface antigens of the Eimeria falciformis sporozoite by immunizing mice with whole homogenized sporozoite. The hybridomas were selected by their reactivities against oocyst extracts, then against glutaraldehyde-treated sporozoites. Three mAbs recognized both the surface of E. falciformis, E. tenella, and E. acervulina and their refractile bodies, whereas a fourth mAb recognized only one epitope on the refractile bodies. All mAbs bound to the same immunoaffinity-purified antigens in Western-blot analysis (P27 for E. falciformis and P25 for E. tenella and E. acervulina). Thus, the mAbs define at least two shared epitopes between sporozoite antigens from different eimerian species. Two of these mAbs are involved in the in vitro phagocytosis of E. falciformis sporozoites by macrophages and also in their lysis by neutrophils. Altogether, these properties showed that the four mAbs came from different activated B-cells. The P27 antigen recognized by our mAbs represents a major target of the in vitro destructive immune response.

Protective oral immunization of chickens against Eimeria tenella with sporozoite surface antigens.

Antigens were extracted from the surface of Eimeria tenella sporozoites with a solution containing Triton X 100 (1%), sodium dodecyl sulphate (0.5%) Na deoxycholate (1%) and EDTA (1 mM). After removal of the detergents, these surface antigen preparations conferred an immunity that protected chickens against a subsequent infection (10(4) sporulated oocysts). The best results were obtained after two 250 micrograms injections of Al(OH)3 adsorbed antigens (oocyst output per g caecal material on Day 7 post infection: 2.39 x 10(7) +/- 0.32 x 10(7) oocysts for controls and 7.37 +/- 10(6) +/- 3.19 x 10(6) oocysts for vaccinated birds) and after four gastric intubations of liposome entrapped antigens (oocysts output on Day 7 postinfection: 2.75 x 10(6) +/- 2.02 x 10(6) g-1 caecal material). These results represented respectively 70 and 88% protection indexes. Studies on the systemic and local antibody response after one or several infections of chickens with the parasite indicated at least 20 different molecules in the detergent antigens which are classified after immunoblotting according to their properties.

[Eimeria tenella and Eimeria acervulina: an antigenic stimulation in vitro of cells from immune chickens induced by adoptive transfer of protection against avian coccidiosis]. / Eimeria tenella et Eimeria acervulina: une stimulation antigénique in vitro des cellules provenant de poulets immuns induit le transfert adoptif de la protection contre les coccidioses aviaires.

The transfer of 5 x 10(7) or 10(8) spleen cells from E tenella-infected chickens to virgin animals after 12-20-h in vitro stimulation with whole sporozoite homogenates confers significant protection to recipients. The oocyst contents of ceca on d 7 post-infection with 20,000 E tenella oocysts were (1.33 +/- 1.10) x 10(6) in chickens which received 5 x 10(7) immune cells after 20-h in vitro stimulation and (4.64 +/- 2.85) x 10(6) in chickens receiving 5 x 10(7) stimulated cells from normal chickens (85% protection). Adoptive transfer by spleen cells revealed an asymmetric cross-protection between E tenella and E acervulina. Spleen cells from E tenella immune chickens protected only against a subsequent infection with the same parasite, while spleen cells from E acervulina immune chickens protected against infection with E acervulina (78%) but also against infection with E tenella (68% protection). The common antigen permits better stimulation, but common surface sporozoite antigens purified from E tenella sporozoites via anti-E acervulina biliary antibodies are capable of stimulating both types of cells without, however, changing their properties.

[Vaccination of mice against murine coccidiosis by ingestion of surface proteins of Eimeria falciformis incorporated in liposomes]. / Vaccination de la souris contre la coccidiose murine par ingestion de protéines de surface d'Eimeria falciformis incorporées dans des liposomes.

Proteins are released from the surface of sporozoites of Eimeria falciformis during their in vitro incubation in a detergent solution. Some of these proteins reacted with antibodies from infected mice and specifically stimulated the proliferation of mesenteric lymph node cells of these mice. Oral immunization of mice with liposome encapsulated sporozoite surface antigens protected mice against a challenge infection. Two proteins (M.W. 27 and 180 K) induced an antibody synthesis in these vaccinated mice.