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.

Hemoparasitic diseases (theileriosis, babesiosis and anaplasmosis) are serious vector-borne diseases that have a considerable impact on cattle health and the farming economy of Morocco. The aim of this study, conducted in four dairy farming regions, was to determine the prevalence and risk factors for these infections. In total 1040 cattle were examined in 96 farms. The competitive enzyme-linked immunosorbent assay (cELISA) and fluorescent antibody techniques were used to determine the seroprevalence, and Cramer’s V statistical test was used to analyze risk factors for the infection of cattle by hemoparasites. Out of the 1040 analyzed sera, 689 (66.3%) were positive. The seroprevalences varied depending on the parasite: Theileria annulata 35.4%, Anaplasma marginale 20.5 %, Babesia bigemina 13.6%, and B. bovis 12.0%. Also 155 animals (14.9%) had mixed infections with two or three hemoparasites simultaneously. The statistical analysis showed that the age (C = 0.62) and farming type (C = 0.60) had a high impact on animal infections, whereas the breed (C = 0.46) and gender (C = 0.29) had less impact. The climate (C = 0.06) did not seem to have any influence. Results of the present survey will help to understand the epizootical situation of vector-transmitted hemoparasitic diseases in cattle for the establishment of an adequate strategy for their control.

Les hémoparasitoses (theilériose, babésiose et anaplasmose) sont des maladies vectorielles graves dont l’impact économique et sur la santé des élevages bovins au Maroc est considérable. L’objectif de cette étude, réalisée dans quatre régions d’élevage bovin laitier, a été de déterminer la séroprévalence et les facteurs de risque de ces infections. Au total 1 040 bovins ont été examinés dans 96 exploitations. La technique competitive enzyme-linked immunosorbent assay (cELISA) et la réaction d’immunofluorescence indirecte ont été utilisées pour la détermination de la séroprévalence, et le test statistique V de Cramer a permis d’analyser les facteurs de risque d’infection des bovins par les hémoparasites. Sur 1 040 sérums analysés, 689 (66,3 p. 100) ont été séropositifs ; la séroprévalence a varié selon le parasite considéré : Theileria annulata (35,4 p. 100), Anaplasma marginale (20,5 p. 100), Babesia bigemina (13,6 p. 100) et B. bovis (12,0 p. 100). Par ailleurs, 155 sérums (14,9 p. 100) ont présenté des infections mixtes (deux ou trois hémoparasites simultané- ment). L’analyse statistique a montré que l’âge (C = 0,62) et le type d’élevage (C = 0,60) ont eu une forte influence sur la séroprévalence, tandis que la race (C = 0,46) et le sexe (C = 0,29) ont eu une influence moindre. Le climat (C = 0,06) n’a pas semblé avoir d’impact. Ces résultats aideront à comprendre la situation épizootique des hémoparasitoses vectorielles chez les bovins afin d’établir une stratégie adéquate pour leur contrôle.

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.

Hemoparasitosis seroprevalence in cattle from two irrigated areas in Morocco The objective of this survey was to determine in cattle the seroprevalences of Babesia bovis, Babesia bigemina, Theileria annulata and Anaplasma marginale infections in two irrigated areas of Morocco (Gharb and Doukkala). The serum antibodies against A. marginale and B. bovis were detected using specific cELISA tests and those against B. bigemina and T annulata by indirect immunofluorescence tests on 1764 animals (792 from Gharb area and 972 from Doukkala area) of different sexes, breed and ages. The overall seroprevalences against A. marginale, B. bigemina, B. bovis and T. annulata were 22.8%, 17.6%, 19.4% and 28.6%, respectively and 27.9% of infested cattle have exhibited multiple infections, involving mainly A. marginale and T annulata or B. bovis. T annulata is widely distributed in Doukkala (36.5%), while the area of Gharb is dominated by A. marginale (40.5%) as well by B. bovis (23.0%) and B. bigemina (25.0%) at a lesser extend. It was also evidenced that old animals (more than 2 years old) or those from imported breeds (or crossbreds) were significantly more often exposed to hemoparasites than young or local cattle. Furthermore, A. marginale and B. bigemina infection frequencies were significantly higher in traditional exploitations than in modern ones. These results demonstrate that bovine hemoparasitosis are highly frequent in these 2 irrigated areas probably as instable enzootic diseases but further studies are required for identifying vector ticks.

Cette etude epidemiologique des parasitoses caprines a ete effectuee, de septembre 1993 a aout 1994, dans la region de Chefchaouen (Rif). Les caprins conduits en elevage extensif sont exposes a un polyparasitisme interne et externe tres diversifie. La population des strongles gastro-intestinaux, constituee de sept especes, est maximale en decembre et en avril. Les strongles respiratoires composes principalement de protostrongylides montrent une forte charge parasitaire en septembre, decembre et mai. Au niveau hepatique, le trematode Fasciola hepatica manifeste un pic bicuspidal automno-hivernal. Les parasitoses de moindre importance sont la monieziose et l'oestrose. Parmi les parasitoses transmises au caprin par le chien, la cysticercose hepato-peritoneale predomine l'hydatidose. Les ectoparasites dominants sont les mallophages en hiver et les ixodides en ete. Un programme de traitement anthelminthique est propose.

Pentamidine treatment has been used successfully to establish immune cellular responses in recovered dogs. In this paper, we examined the appearance and disappearance of antibodies over time against crude Leishmania antigens and purified gp63 or gp70 proteins in sera from cured dogs using a Western blotting technique. Following the treatment, a pattern of antibody specificities to parasite antigens was observed in the sera of cured dogs. Antibodies to gp63 and gp70 were maintained after cure. In addition, the reaction with a 26 kD band observed during the clinical phase was no longer recognized by sera taken from recovery dogs. Interestingly, two proteins, 85 and 110 kD, not observed during the patent phase were detected by sera taken from treated dogs. Such patterns of antibody specificities to various parasite antigens might represent a useful parameter to determine the actual phase of the disease process.

Ehrlichiosis is an infectious disease caused by a rickettsia and transmitted by the tick Rhipicephalus sanguineus. The clinical signs can vary from a simple recurrent fever to multiple haemorrhages. In the dog, epistaxis is also a major clinical sign and often diagnostic. Although the bacteriological analysis was negative, the infection was confirmed by direct immunofluorescence. Tetracycline administration over a long period was instigated to avoid the frequent recurrences that can affect immunodepressed animals.

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.

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%).

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.

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.

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.

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.

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.

Annals of the New York Academy of SciencesVolume 849, Issue 1 p. 427-429 Seroprevalence of Bovine Anaplasmosis in Morocco† H. SAHIBI, Corresponding Author H. SAHIBI Institut Agronomique et Vétérinaire Hassan II, Département de Parsitologie, BP: 6202, Rabat, Morocco Corresponding author in Morocco. Corresponding author in USA.Search for more papers by this authorAB. RHALEM, AB. RHALEM Institut Agronomique et Vétérinaire Hassan II, Département de Parsitologie, BP: 6202, Rabat, MoroccoSearch for more papers by this authorB. BERRAG, B. BERRAG Institut Agronomique et Vétérinaire Hassan II, Département de Parsitologie, BP: 6202, Rabat, MoroccoSearch for more papers by this authorW. L. GOFF, Corresponding Author W. L. GOFF USDA-ARS, Animal Disease Research Unit, Washington State University, Pullman, Washington 99164-7030 USA Corresponding author in Morocco. Corresponding author in USA.Search for more papers by this author H. SAHIBI, Corresponding Author H. SAHIBI Institut Agronomique et Vétérinaire Hassan II, Département de Parsitologie, BP: 6202, Rabat, Morocco Corresponding author in Morocco. Corresponding author in USA.Search for more papers by this authorAB. RHALEM, AB. RHALEM Institut Agronomique et Vétérinaire Hassan II, Département de Parsitologie, BP: 6202, Rabat, MoroccoSearch for more papers by this authorB. BERRAG, B. BERRAG Institut Agronomique et Vétérinaire Hassan II, Département de Parsitologie, BP: 6202, Rabat, MoroccoSearch for more papers by this authorW. L. GOFF, Corresponding Author W. L. GOFF USDA-ARS, Animal Disease Research Unit, Washington State University, Pullman, Washington 99164-7030 USA Corresponding author in Morocco. Corresponding author in USA.Search for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb11088.xCitations: 5 † This work was supported by the USDA-FAS, ICD Project #5085348194 and accomplished in the Parasitic Diseases Laboratory of the Institut Agronomique et Veterinaire Hassan II with the collaboration of the USDA-ARS, Animal Disease Research Unit, Washington State University, Pullman, Washington, USA. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume849, Issue1TROPICAL VETERINARY MEDICINE: MOLECULAR EPIDEMIOLOGY, HEMOPARASITES AND THEIR VECTORS, AND GENERAL TOPICSJune 1998Pages 427-429 RelatedInformation

ABSTRACT: 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.

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 μl−1) and in the absolute number of macrophages (182.0 ± 82.0 cells μl−1), lymphocytes (68.5 ± 35.0 cells μl−1), eosinophils (35.3 ± 16.4 cells μl−1) and neutrophils (10.9 ± 8.7 cell μl−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 μl−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.

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.