Molecular Diagnosis of Subclinical African Trypanosoma vivax Infection and Association with Physiological Indices and Serum Metabolites in Extensively Managed Goats in the Tropics

Trypanosomosis remains a major challenge to livestock production in much of tropical Sub-Saharan Africa, while diagnosis and treatment still depend on inefficient parasitological techniques. Endemic infections depend on animal reservoirs with subclinical parasitemia. We report molecular diagnosis of subclinical Trypanosoma vivax (T. vivax) infection using polymerase chain reaction (PCR) for the first time in Nigerian goats and associate parasite presence with gross physiological traits and serum metabolites in extensively managed Nigerian goats. PCR was used to amplify a 400 bp DNA fragment of the parasite genome in 205 goats across three geographical zones of the country. Results showed a high subclinical infection rate (SCIR) of 71.7% in the total goats examined. Overall SCIRs of 71%, 75.9% and 55.6% were recorded in West African Dwarf, Red Sokoto and Sahel goats respectively, while geographical SCIRs were 71.2% (Southwest), 75% (Northwest) and 70% (Northeast). T. vivax presence had significant (P < 0.05) effect on respiratory rate and is associated with higher creatinine levels in sera. Logistic regression analyses with Hosmer-Lemeshow goodness-of-fit showed that respiratory rate is the most important predictive trait for the presence of T. vivax infection (P < 0.05). Goats appear to be a viable reservoir for T. vivax infection of other livestock. Molecular diagnosis of subclinical trypanosomosis using PCR could be useful for large scale epidemiological studies, early diagnosis of subclinical infection and treatment of the disease in extensively managed tropical goats.


Introduction
Livestock productivity in Sub-Saharan Africa suffers from high prevalence of trypanosomosis with estimated annual losses due to the direct and indirect consequences of the disease running into billions of dollars with disproportionate adverse effects in rural areas [1][2][3][4].The epidemiology of trypanosomosis and its impact on livestock production varies from one locality to another and depends largely on the level of interaction between tsetse flies, domestic and game animals [5].This protozoan disease is transmitted by the tsetse fly via the infection of animals and humans [6].
Small ruminants are renewable energy source for food, biological raw materials and play significant socioeconomic roles in the lives of rural dwellers in Africa.In Nigeria, trypanosomosis is of increasing clinical importance in small ruminants as the disease expands to previously tsetse free zones [7].Several techniques have been developed for pathogenic trypanosome identification, characterization, typing, epidemiological data collection and diagnosis [8,9].Molecular diagnosis includes the use of species-specific primers in single and nested PCR to amplify the internal transcribed spacer (ITS) regions of ribosomal DNA [10].
Epidemiological studies have shown high levels of mixed infections in tsetse flies feeding on naturally infected cattle and other livestock [11].Despite much campaign towards trypanosome eradication, there is lack of research on the molecular screening of trypanosomes in Nigeria [7].Better understanding of prevalence of subclinical infection will help with efforts to control the disease and the use of population-based screening of both animal and insect vectors.Therefore, the goal of this pioneer nationwide study was to establish the suitability of PCR-based assay in the effective diagnosis of subclinical Trypanosoma vivax infection and its association with physiological status and serum metabolites of extensively managed Nigerian goats in a tropical region.

Animals and Sampling
Two hundred and five goats (60 bucks and 145 does) representing 3 extant Nigerian breeds were sampled from across the country.The data set comprised 62 West African Dwarf (WAD), 116 Red Sokoto (RS) and 27 Sahel (SH) breeds distributed with 59 from Southwest (Ogun State), 56 from Northwest (Kano, Zaria, Sokoto and Kebbi States) and 90 from Northeast (Nassarawa, Jos, Bauchi, and Maiduguri States) zones of Nigeria respectively (Figure 1).The goats originated from different flocks and were reared under the traditional extensive system, where they grazed during the day on natural pasture containing forages such as stylo (Stylosanthes gracilis), leucaena (Leucaena leucocephala) and guinea grass (Panicum maximum), and scavenged on kitchen and household wastes whenever available.The animals' ages were assessed to be greater than 14 months by dentition (presence of 2 -8 permanent incisors) as previously described [12].

DNA Isolation and Polymerase Chain
Reaction Screening

Physiological Indices and Serum Metabolites
Digi as used to measure the body temry rate value; lue.The easured acco Measurement tal thermometer w perature via the fore limb armpit of the goats.Rectal temperature was measured with the digital thermometer placed about 2 cm in the rectum, while pulse rate was measured in beats per minute using a stethoscope placed on the 4 th half intercostal space.Respiratory rate was measured in breaths per minute by counting the number of flank movements per minute using a stop watch and stethoscope.The relationship between pulse rate and respiratory rate together with their normal average values was used to derive log transformed heat stress index (HSI) according to Olademeji et al. [15] as follows: H = (AR/AP)  (NP/NR) where H = Heat stress index; AR = Average respirato AP = Average pulse rate value; NP = Normal pulse rate value; NR = Normal respiratory rate va following serum metabolites were m rding to Jain [16]: total blood glucose (TBG), total serum protein (TSP), blood urea nitrogen (BUN), alanine amino phosphatase (ALP), bilirubin (BIL), creatinine (CRT), triglycerides (TGS), serum albumin (ALB), total cholesterol (CHOL), high density lipoproteins (HDL), low density lipoproteins (LDL), globulin (GLB), calcium (Ca 2+ ), magnesium (Mg 2+ ), chloride (Cl − ), sodium (Na + ), potassium (K + ), phosphorus    Based on presence of exp bands, infection status were categorized as either positive or negative for T. vivax.The prevalence rates of T. vivax were assessed using frequency and percentages.The effects of T. vivax infection and breed of goats on physiological and serum metabolites were estimated using PROC GLM of SAS version 9.1 [17].The linear model employed was: effect of j th breed (WAD, RS, SH); e ijk = random error associated with med to be normally, independently and identically distributed with zero mean and constant variance).
Means were separated using the Duncan Multiple range test procedure of the same statistical package.Logistic regression analyses were carried out with Hosmer-Lemeshow goodness-of-fit [18] to determine the physio-predicting the presence of T. vivax infection in Nigerian goats.This was important given that these animals are reared extensively in the hot tropics, which in turn affects the presence of the tsetse fly vector [5,6].

Prevalence of Trypanosoma vivax
Across Locatio PCR using primers specific for T. vivax showed that 147 f 205 goats were infected accounting for an av 1.7% prevalence in the total goats ble 1).Among the breeds of goats examined, 15 out of 27 (55.6%)SH, 44 out of 62 WAD (71.0%) and 88 out of 116 (75.9%)RS goats were found to be infected with the parasite.Between the sexes, the prevalence rates were 41 out of 60 bucks (68.3%) and 106 out of 145 does (73.1 %).respectively.Geographically, the prevalence rates of subclinical T. vivax infections were 42 out of 59 (71.2%) goats in Southwest, 42 out of 56 (75.0%) goats in Northwest and 63 out of 90 (70.0%) goats sampled in Northeast.

Association of T. vivax Infection Status and
Physiological Traits of Nigerian Goats.
The effect of T. vivax infection status was significant (P < 0.05) on the respiratory rate of Nigerian goats, but not ficant (P > 0.05) on the other physiological measured (Table 2).T. vivax infected goat breeds had reduced respiratory rate (48.31 ± 1.35 breaths/min) compared to their non-infected counterparts (53.69 ± 2.4 breaths/min).

Association of T. vivax Infection on Serum Metabolites of Nigerian Goats
T. vivax infection status only had significant effect (P <  0.05) on the creatinine levels of Nigerian goats (Table 3 mg/dl) compared to their non-infected counterparts (1.49 ± 0.09 mg/dl).The effect of T. vivax infection status was not significant (P > 0.05) on other serum metabolites.

Effect of Breed on Physiological Traits of
Although the mean pulse rate and respiratory rate WA 3.1 beats/min and 59.1 ± 2.59 hs/min) were high statistically (P < 0.05), this did not result in stressed condition (P > 0.05) based on their tic regression revealed that respi-5) sivel s of animal production in African countries diagnosis using PCR assay has been y to be an efficient and effective tool in mission.This was contrary to their WAD counterparts D goats (124.97 ± breat lower heat stress index compared to the SH and RS goats, respectively (Table 4).

Prediction of Subclinical Trypanosomiasis Using Physio-logical (Heat Tolerant) Traits
Across breeds, the logis ratory rate was the trait of utmost importance (P < 0.0 in predicting the presence of T. vivax infection in exten y reared Nigerian goats.Among breeds, the predic tion models showed that respiratory rate was the utmost trait of importance (P < 0.05) in predicting the presence of T. vivax infection in WAD and RS goats, respectively, while pulse rate and rectal temperature were more important (P < 0.

Discussion
Animal trypanosomosis remains one of the most important constraint [19].Molecular found in this stud the diagnosis of trypanosome infection, which is consistent with the findings of earlier workers on the use of this method for the detection of various livestock diseases [20][21][22][23][24].The high prevalence rate of sub-clinical T. vivax infection was a further indication that Nigerian goats harbour this parasite and may act as a critical reservoir, similar to the findings of Ohaeri [25].The relative frequency of infections with T. vivax in the present study was higher than the 25.0% reported for goats in Ethiopia [26] and for sheep (18.2%) and goats (1.3%), respectively in Kenya [27].The unexpectedly higher incidence of T. vivax infection in RS goats found mostly in the northern parts of Nigeria (tsetse non-endemic zone), might be the result of low resistance to the parasite which could have been introduced through mechanical trans- rveillance would therefore be important to establish the the respiratory tract helps to prevent hype in these parameters are indicators of pa reports during experimental infection of T. brucei [39] rate and body temperature can be us e presence of ans w ts.
e superscript wit ow do not differ si > 0.05); WAD= an Dwarf goats; okoto goats; that have adapted to the humid tropical conditions (tsetse endemic zone) of southern Nigeria [28].Entomological ports of increased creatine levels in goats experimentally infected by Trypanosoma spp [37,38] but opposite to su correlation between the abundance period of cyclical and/or mechanical vectors (tsetse flies and haematophagous biting flies such as tabanids and stomoxys) and trypanosome infection [29].According to Migchelsen et al. [30], a common problem in non-endemic regions is an initial misdiagnosis.The chronic nature, non-specific clinical signs and symptoms, and low parasitemia may result in the disease remaining undiagnosed and unrecognized for years.In addition, a more holistic picture of trypanosomosis requires a comprehensive understanding of the epidemiology of the disease, the roles of different trypanosome species, mixed infections, and different host species [31].
In mammals, respiration is directed at the elimination of CO 2 from tissues of the body and evaporation of moisture from rthermia under high ambient temperatures [32], which is a major adaptive feature of domestic livestock in the hot humid tropics of Sub-Saharan Africa.Respiratory rate was found to be significantly affected by pathogenic T. vivax.The reduced respiratory rates of infected goats indicate that the infected animals may be more stressed than non-infected animals.This suggests that pathogenic T. vivax infection is a stressor in goats, and the measurement of respiratory rate is an indication of the physiological alterations of stressed and infected animals [33,34].This result is corroborated by earlier reports by Fajinmi, et al. [35] in cattle.Although there was a tendency towards higher heat stress in SH and RS goats found in the hotter, northern parts of the country, this was not significant because they are more adapted to the higher ambient temperatures of northern compared to southern Nigeria.
Measurement of biochemical parameters is an important and reliable way to assess the health status of animals, as changes thophysiological responses under disease assaults such as trypanosomosis [36].Only creatinine, a metabolic waste product formed in the muscle from creatinine phosphate was found to be significantly affected in this study.This effect was higher in infected than in noninfected animals.These were consistent with earlier re-and T. congolense [40] in goats.This seeming discrepancy may be explained by the different animal responses to different Trypanosoma species and the fact that many other variables were actively being manipulated under controlled conditions such as dietary energy and protein metabolism in these other studies compared to detection of subclinical levels of infection in extensively managed goats in this study.
The regression analyses indicate that respiratory rate, pulse rate and rectal temperature in goats could be used to predict existing subclinical T. vivax infection in extensively managed Nigerian goats.This result is similar to findings of earlier workers [15,41,42] that rectal temperature, respiratory ed as good assessment of heat stress in goats because they are excellent indicators of physiological response and adaptability to extremes of weather and thermal environment.The constant exposure of these animals to thermal stress brings physiological parameters into play with respect to subclinical infections.These environmental factors have direct effects on the animals and on the ecology of tsetse fly vector of the disease.The prediction models obtained in this study are simple and economical, and could be applied in the field as a first step in the determination of the health status of the animals and the putative diagnosis of subclinical trypanosomosis pending the use of laboratory assays.
In conclusion, this study has established that subclinical infections of T. vivax infection can be assessed by PCR, and this may provide preemptive ability to address disease burden in Nigerian goats.Logistic regression analysis showed that respiratory rate was the heat tolerant trait of utmost importance in predicting th T. vivax infection in extensively managed Nigerian goats.The use of PCR as a viable tool for large-scale epidemiological surveillance should be encouraged in diagnosing and identifying the prevalence of more deadly trypanosome species in Nigerian goats such as T. brucei, T. congolense kilifi, T. congolense savanna type, and T. congolense forest type in both endemic and non-endemic zones.The epidemiological data generated from preva-lence of T. vivax infection in Nigerian goats could aid policy formulation for disease control and animal management strategies.This will contribute to attenuating the rate of trypanosome infections in extensively managed goats in tropical regions where the disease is endemic.
Blood samples were collected by jugular venipuncture and genomic DNA were isolated using ZymoBead TM Genomic DNA kit (Zymo Research Corp., Irvine, CA, USA) following the manufacturer's instructions.The presence of Trypanosoma vivax (T.vivax) was determined by PCR using the T. vivax-specific primers ILO 1264/1265 forward 5'-CAGCTCGCCGAAGGCCACTT GGCTGGG-3' and reverse 5'-TCGCTACCACAGTCG CAATCGT-CGTCTCAAGG-3' to amplify a 400 bp fragment of the parasite genome as described by Masake et al.[13] and adopted by Njiru et al.[14].PCR amplifications were carried out in a Techne thermal cycler in a total reaction volume of 20 µL containing 10 -50 ng DNA and 10 pmol of each primer in AccuPower TM PCR Premix (Bioneer Corporation, Alameda, CA, USA).The thermal cycling involved 35 cycles of initial denaturation at 95˚C for 5 minutes, denaturation at 95˚C for 30 seconds, annealing at 55˚C for 120 seconds, extension at 72˚C for 120 seconds, and final extension temperature at 72˚C for 5 minutes.PCR products were separated in 1.5% agarose gel including ethidium bromide, and photographed under UV light.Confirmation by DNA sequencing of the amplified fragments was carried out using the same PCR primers with the Applied Biosystems Automated 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA) using Big Dye Terminator chemistry and AmpliTaq-FS DNA Polymerase.

Figure 1 .
Figure 1.Nigerian map showing the geographical zones of sampling.

Prevalence of T. vivax according Tabl to the breed, sex
e 1.