Anaplasma spp. in dairy ruminants in Jordan: high individual and herd-level seroprevalence and association with abortions

Abstract

We estimated the seroprevalence of, and associated risk factors for, anaplasmosis in dairy cows, sheep, and goats in all regions of Jordan. Apparently healthy animals from 135 farms (31 cow, 68 sheep, and 36 goat farms) were tested for Anaplasma spp. antibodies by a competitive ELISA. At the farm level, 36%, 94%, and 94% of cow, sheep, and goat farms, respectively, were seropositive for Anaplasma spp. At the individual animal level, 22%, 89%, and 82% of the tested cows, sheep, and goats, respectively, were seropositive, which demonstrates widespread and high seroprevalence of Anaplasma spp. in dairy farms in Jordan. Seroprevalence did not vary between regions. Using the Fisher exact test, there was a significant association between high seroprevalence and recent abortion in small ruminants. There is an urgent need for further investigation of this disease, and the potential role of Anaplasma spp. in abortions, on dairy farms in Jordan.

Anaplasma is a genus of obligate intracellular rickettsial agents that has 3 species that can infect the red blood cells of ruminants: A. marginale and A. centrale infect cattle, andA. ovis infects small ruminants. ⁹ Generally, Anaplasma spp. are transmitted biologically by more than 20 species of ticks and mechanically by biting flies or blood-contaminated fomites such as syringes. ¹¹

In cattle, anaplasmosis is characterized by fever, progressive anemia, and abortions. ¹¹ The infection in calves (<1 y old) is usually subclinical, but cattle that are ≥1 y old are more susceptible to the development of the acute disease with mortality rates up to 50%. ¹¹ In endemic regions, cattle first become infected with A. marginale early in life, causing minimal losses. Following recovery from the infection, cattle remain carriers, serving as a source for pathogen transmission, but are generally immune to further clinical disease. ² Serious economic losses in dairy operations occur as a result of decreased body weight and milk production, and the occurrence of abortions and mortalities, especially when mature cattle with no previous exposure are moved into endemic areas (Alderink FJ, Dietrich RA. Economic and epidemiological implications of anaplasmosis in Texas cattle herds. Proc 86th Ann Meeting United States Anim Health Assoc; Nov 1982; Nashville, TN. Available from: https://oaktrust.library.tamu.edu/bitstream/handle/1969.1/150031/Bull1426a.pdf?sequence=21).

In small ruminants, A. ovis causes anemia, fever, weight loss, spontaneous abortion, jaundice, and mortality leading to economic losses. ¹⁰ The severity of the infection may be amplified in small ruminants under stressful conditions such as coinfection, vaccination, deworming, heavy tick infestation, poor health conditions, and hot weather. ¹⁴ In addition to animal infections, human infection with A. ovis, causing hepatosplenomegaly and lymphadenopathy, has been reported in Cyprus. ⁴

Anaplasmosis is distributed in cattle worldwide and listed as a notifiable animal disease by the World Organization for Animal Health (http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.04.01_bovine_anaplasmosis.pdf). Anaplasmosis in small ruminants has been reported in the United States, ⁷ in many Middle Eastern and North African countries, including Sudan and Iraq, ¹⁴ and in European countries such as Italy, ¹⁷ Hungary, ⁹ Slovakia, and the Czech Republic. ⁸ However, no studies have been conducted on this disease in dairy ruminants in many parts of the world, including Jordan, to our knowledge.

The dairy industry, which includes cow, sheep, and goat herds, is an important economic sector in Jordan.^12,13 Dairy cow production in Jordan is divided into large- and small-scale systems, with both systems based on Holstein–Friesian cows. The large-scale system is located in the east-northern area of Jordan (Al-Dulial area) and produces ~60% of the country’s milk. This system relies on zero grazing and utilizes modern management practices. The small-scale system is scattered in various regions of Jordan, mostly in the northern highland area. In this system, farmers keep cows in small brick barns under traditional management practices.

Small ruminant production systems in Jordan rely on low production technologies and can be divided into extensive and semi-extensive systems. The extensive system is primarily located in the northern highlands, which receives high rainfall, and is occupied by herders who utilize the rangelands in a constant search for grass and water. The semi-extensive system is primarily located in the eastern and southern Badia region, which is semi-arid to arid and is occupied by pastoralist or nomadic Bedouin. We determined the seroprevalence of anaplasmosis in dairy cows and small ruminants in Jordan, and any association with farming practices or other risk factors determined by interviews with farmers and herdspeople.

Several farms across each region of Jordan were included randomly in our cross-sectional study. The herd-level seroprevalence of Anaplasma spp. in ruminants is unknown in Jordan. At an assumed seroprevalence of 0.5, with allowable error of 0.1 at 95% confidence interval (CI), the required sample size would be 96 using the formula: n = z²p(1 – p)/d², where n is the sample size, z is the statistic for confidence level (z = 1.96 at 95% confidence level), p is the expected prevalence (p = 0.5), and d is precision (d = 0.1). However, 135 farms were included in the study to increase the power of the analysis. Specifically, 31 cow, 68 sheep, and 36 goat farms were randomly selected from different regions of Jordan from November 2015 to May 2016. The number of selected farms in each region was based on animal and farm densities as provided by the Jordan Department of Statistics and local veterinarian associations. The small ruminant farms included 45 farms in northern Jordan (Irbid, Jarash, and Ajloun), 25 farms in southern Jordan (Tafela, Ma’an, and Karak), 20 farms in the Jordan Valley, and 14 farms in the Badia (Mafraq). The dairy cow farms included 21 farms from the Al-Dulial area and 10 farms from the northern highlands. From each farm, 5 blood samples were collected randomly from clinically healthy animals by dividing the total number of animals per farm by 5, giving a value k, and every kth animal was sampled. The serum was harvested by centrifugation and stored at −20°C until analyzed; 3 serum samples from each farm were later picked from the serum bank and tested.

A questionnaire was used to collect data about possible risk factors for Anaplasma spp. seropositivity at the time of sample collection. The questionnaire was tested and revised based on a pilot sample of 5 farms. The repeatability of the questionnaire was examined by re-asking the same farmers the questionnaire by phone within 1 wk of the face-to-face questionnaire administration. The repeatability was determined by calculating the kappa scores for agreement for the answers to 3 questions: 1) How many animals are on your farm? 2) Do you purchase/add new animals to your farm? 3) How often do you clean your farm?

The serum samples were tested for antibodies against Anaplasma spp. by competitive enzyme-linked immunosorbent test (Anaplasma antibody test kit, cELISA v2; VMRD, Pullman, WA) according to the manufacturer’s instructions. The cELISA is considered more rapid, less complex, and more sensitive than the complement fixation test. ⁶ This cELISA demonstrated 100% sensitivity and 99.7% specificity in cattle using 30% inhibition as the cutoff point, ⁵ and we applied this cutoff for cattle sera. This kit is approved for use in cattle by the U.S. Department of Agriculture and has been validated and used to detect antibodies against Anaplasma spp. in goats and experimentally infected sheep because the major surface protein 5-glutathione (MSP5) epitope is conserved among the 3 Anaplasma species. ¹⁵ A cutoff of 19% inhibition was used for sheep and goats samples, based on a validation study performed on sheep sera. ¹⁵

Blood samples and questionnaire data acquisition were approved by the Institute Animal Care and Use Committee of the Jordan University of Science and Technology. Herdspeople were briefed about the study aim, assured that the study was for research purposes only, and that any generated data pertaining to their farm would be available only for them upon their request. It was emphasized to the herdspeople that their refusal to participate would not have any adverse effect on their veterinary service.

The test and questionnaire data were entered into Excel (Microsoft, Redmond, WA) and analyzed (SPSS Statistics v.21; IBM, Armonk, NY). A univariate analysis using the Fisher exact test was performed to determine the associations between Anaplasma spp. seropositivity (outcome variable) and potential associated factors. The seroprevalence at the individual animal level for each of the 3 animal groups—cows, goats, and sheep—was calculated as the total number of seropositive samples divided by the total number of all samples tested for each of the species, respectively. A herd was considered positive when at least one animal was seropositive; herd-level seroprevalence was calculated by dividing the number of seropositive herds for each animal species by the tested herds for that species.

A total of 135 dairy ruminant farms were tested. The herd-level seroprevalence in dairy cow, sheep, and goat farms was 36% (95% CI: 21–53%), 94% (95% CI: 86–98%), and 94% (95% CI: 82–98%), respectively. On an individual animal level, 22%, 89%, and 82% of the tested cows (n = 93), sheep (n = 204), and goats (n = 108) were seropositive, respectively.

Anaplasma spp. seropositivity in dairy cows was not associated with, or affected by, farm location, herd size, history of abortion, adding new animals to the herd, practicing natural breeding, or frequency of farm cleaning (Supplementary Table 1). Anaplasma spp. seropositivity in small ruminants was not associated with, or affected by, the farm location, flock size, mixing animals with animals from other farms, grazing for >5 km away from the farm, frequency of farm cleaning, cleaning and sanitation of feeders, or recent mortalities (Supplementary Table 2). Although a previous history of abortion was also not associated with seropositivity, recent abortion cases in small ruminants in the last 2 seasons were significantly associated with seropositivity (p = 0.03; Supplementary Table 2).

We found that 36% of the dairy cow farms and 22% of the tested cows were seropositive for Anaplasma spp. A high seroprevalence, detected using the same serologic method (cELISA), has been reported in dairy cows in other countries such as Egypt (67%) ¹ and Costa Rica (86%). ¹⁶ We found a high seroprevalence (>90%) for sheep and goat farms, and ~80% in the tested sheep and goats. Lower, but still relatively high, prevalences using molecular methods were reported in sheep in Turkey (31%), Iraq (67%), Sudan (42%), and Portugal (82%). ¹⁴

Seroprevalence was higher in the dairy small ruminants than in dairy cows. Previous studies showed that small ruminant farmers in Jordan use open lands for grazing, whereas cows are usually housed in barns, with minimal grazing in the highlands regions and zero grazing in Al-Dulail.^12,13 The difference in management would expose small ruminants to greater tick numbers and could explain the higher seroprevalence in sheep and goats. In addition, small ruminants in Jordan are usually vaccinated for certain diseases such as brucellosis as part of the national vaccination campaigns (USDA. Emerging animal diseases in the Middle East region: potential threats to agriculture and public health. Gap Analysis Workshop report. 2016. Available from: https://go.usa.gov/xRUvc). These vaccination campaigns might explain the high seroprevalence compared to cattle, as there is the possibility of iatrogenic transmission among small ruminants by contaminated needles. In addition, the movement of small ruminants across national borders, from Syria to northern Jordan and from Saudi Arabia to southern Jordan, might impose stress given the long distances and hot conditions. Such stressful conditions, along with hot temperatures in Jordan and scarcity of water and pasture during summer, might exacerbate clinical disease associated with Anaplasma spp. infection. In addition, the high prevalence of other diseases in dairy ruminants in Jordan, such as mastitis and respiratory diseases,^12,13 might predispose to clinical disease associated with Anaplasma spp. infection. An association between poor health conditions in sheep and an increase in concurrent infections of A. ovis and A. phagocytophilum has been suggested, posing a risk to both human and animal health. ¹⁸

Seroprevalence was not affected by farm location on either dairy cattle or small ruminant farms. These findings corroborate a previous study that did not find a significant difference in the seroprevalence of A. marginale and A. ovis by location in Iran. ¹⁹ On the other hand, inter-regional differences in the prevalence of Anaplasma spp. infection have been reported in Italy. ¹⁸ Risk factors for anaplasmosis reported elsewhere, such as flock size, husbandry system, and grazing system, ¹⁷ were not associated with seropositivity in our study. Nevertheless, anaplasmosis is widespread in Jordan.

A history of recent abortion (2013–2014) was associated with Anaplasma spp. seropositivity in small ruminants but not in dairy cows in our study. Although we did not identify the species of Anaplasma, a previous study has reported that artificial infections of A. ovis can cause abortions in goats, especially in animals that develop high fever and severe anemia. ³ In particular, this earlier experimental infection study showed that Anaplasma can pass through the placenta to the red blood cells of the fetus to cause fetal death in utero. ³

There are limitations to our study. The sample size calculated for the study was based on total ruminant farms, the study tested only 3 animals per farm, and a confirmatory test was not performed. Further studies are needed to assess the importance of anaplasmosis as a cause of economic loss in dairy ruminants and abortion in small ruminants and to evaluate the impact of concurrent health problems. In those low-income countries where anaplasmosis presents a risk, a better understanding of the impact of this disease on animal productivity is recommended. In addition, the biology of the vector, the host preference, and the zoonotic potential of the various Anaplasma species need further study.

Supplemental Material

DS1_JVDI_10.1177_1040638719843171 – Supplemental material for Anaplasma spp. in dairy ruminants in Jordan: high individual and herd-level seroprevalence and association with abortions

Supplemental material, DS1_JVDI_10.1177_1040638719843171 for Anaplasma spp. in dairy ruminants in Jordan: high individual and herd-level seroprevalence and association with abortions by Mohammad M. Obaidat and Alaa E. Bani Salman in Journal of Veterinary Diagnostic Investigation